INTERNATIONAL JOURNAL of LEPROSY Volume 72, Number 2 Printed in the U.S.A

INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) INTERNATIONAL JOURNAL OF LEPROSY and Other Mycobacterial Diseases

VOLUME 72, NUMBER 2JUNE 2004

Images from the History of Leprosy

In this issue, the JOURNAL initiates a new that the subject is important in the history of feature, “Images from the History of Lep- leprosy, and second, that the image itself is rosy.” Of all the major maladies of man- of high quality. Those who submit images kind, few have a history as extraordinary and for consideration are asked to provide as as well-documented as leprosy. We believe much documentation as possible concern- that this JOURNAL provides an appropriate ing the subject of the image, as well as doc- place in which to collect a series of images umentation about the image itself (includ- portraying the depth and richness of this his- ing the source or artist, medium, and dates tory, and issue an open invitation to mem- of creation or publication if known). If you bers of the International Leprosy Associa- would like to contribute an image for con- tion, as well as to other physicians, friends, sideration, please communicate first with and institutions who may have in their pos- the JOURNAL office ([email protected]) to describe session valuable images from this multi- the nature of the image. Please do not send faceted history. originals; we will provide contributors with Of the greatest interest for this feature are information about the preferred methods of photographs, sketches, or other images that electronic (or other) reproduction for publi- illustrate events, discoveries, institutions, cation. and ideas that have played a significant role This series begins with the haunting por- in the history of leprosy. In order to avoid trait of a 14-year-old girl published in the having this feature become only a gallery of landmark atlas of Daniellsen and Boeck in physicians and scientists who have worked 1847. The artist has carefully recorded the on leprosy, we do not wish to encourage subclinical details of the macular lesions on her mission of photographs of these individuals, cheeks, but has also captured in her eyes the believing that their contributions are more bewildered look of sadness and apprehen- appropriately recognized in other ways. sion that is familiar to generations of physi- Two major criteria will be applied for the cians who have had the task of advising selection of images for this feature: first, their patients of this diagnosis.

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72, 2 Images from the History of Leprosy 123

IMAGES FROM THE HISTORY OF LEPROSY Previous page: A young girl with macular lesions of leprosy, 1847.

Reproduced here is Planche IX from the original Atlas Colorié de Spedalskhed [Atlas of Leprosy] by D. C. Daniellsen and C. W. Boeck. This Atlas is a landmark in the medical history of leprosy, as it represents the beginning of the modern understanding and classiﬁca- tion of this disease. The Atlas was printed by Trykt i Prahls Lithographs in Bergen, Norway, in 1847. The image here is reproduced electronically from an original chromolithograph made from a painting by J. L. Losting. The lithograph measures 49.5 cm × 33.0 cm. This image and documentation were contributed by the Section of Rare Books—Library Luiza Keffer—Instituto Lauro de Souza Lima, Bauru, Brazil. This image may be viewed in color in the electronic edition of the Journal. Please visit our web-site at leprosy-ila.org, and click on the Journal icon. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) INTERNATIONAL JOURNAL OF LEPROSY and Other Mycobacterial Diseases

VOLUME 72, NUMBER 2JUNE 2004

Epidemiological Characteristics of Leprosy Reactions: 15 Years Experience from North India1

Bhushan Kumar, Sunil Dogra, and Inderjeet Kaur2 ABSTRACT A retrospective analysis of patient’s leprosy clinic records at PGIMER, Chandigarh, In- dia for the period 1983 to 1998 was undertaken to study the frequency, time of onset, and risk factors for leprosy reactions. Of the 2600 cases analyzed, 1494 were multibacillary and 1106 had paucibacillary disease. Presentation with reaction was common with 30.9% of our patients having reactions at the time of first visit. The incidence of reversal reaction (RR) was highest during 6 to 12 months after starting multi-drug therapy (MDT), thereafter de- clining gradually. Late RR occurred in 9.5% of all cases and was noted up to 7 years after treatment. Female gender, widespread disease, and multibacillary disease were identified as risk factors for RR. Erythema nodosum leprosum (ENL) reactions were noted to occur mostly during second or third year after starting MDT. Of the total number of patients who experienced ENL, 64.3% had recurrent episodes which continued for up to 8 years after the start of treatment. Lepromatous leprosy, female gender, and high Bacterial Index (≥3) were recognized as risk factors for developing ENL. Occurrence of recurrent and late reactions, even though of mild severity, highlights the importance of recognizing and treating them promptly to prevent or reduce morbidity, complications, and further deterioration in the disability status. Although it is hoped that leprosy will have been eliminated at all levels by 2005, the recognition and management of these reactions will continue to be the most essential/significant task in the post elimination era. RÉSUMÉ Une analyse rétrospective des dossiers cliniques de patients traités à la clinique contre la lèpre du PGIMER de Chandigarh aux Indes fut conduite de 1983 à 1998, afin d’étudier la fréquence, la date d’apparition et les facteurs de risque des réactions lépreuses. Parmi les 2600 cas analysés, 1494 souffraient de maladie multibacillaires et 1106 de maladie pau- cibacillaire. Les réactions étaient fréquentes au moment de la première visite, étant de 30,9%. L’incidence de réactions reverses (RR) fut la plus élevée pendant les 6 à 12 mois suivant le début de la polychimiothérapie (PCT), puis déclinèrent graduellement. Des RR tardives apparurent dans 9,5% du nombre total de cas et furent détectées jusqu’à 7 années après traitement. Le sexe féminin, maladie disséminée et maladie multibacillaires furent

1 Received for publication on 24 September 2002. Accepted for publication on 11 March 2004. 2 B. Kumar, M.D., MNAMS; S. Dogra, M.D., DNB, MNAMS; I. Kaur, M.D., MNAMS, Department of Der- matology, Venereology and Leprology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. Reprint requests to: Prof. Bhushan Kumar, Dept. of Dermatology, Venereology and Leprology, Postgraduate Institute of Medical Education and Research, Chandigarh-160 012, India. E-mail: [email protected]

125 126 International Journal of Leprosy 2004

identifiés comme des facteurs de risques de la RR. L’érythème noueux lépreux (ENL) fut principalement détecté durant les deuxième et troisième années après le début de la PCT. Parmi les patients ayant déclarés un ENL, 64,3% ont souffert d’épisodes cliniques récurrents et ce, pendant parfois plus de 8 années après le début du traitement. Une lèpre lépromateuse, un sexe féminin et un index bactérioscopique élevé (≥3) furent identifiés comme des facteurs de risque pour le développement d’un ENL. La survenue de réactions récurrentes ou tardives, même si elles sont de faible sévérité, souligne l’importance de les reconnaître et de les traiter rapidement afin de prévenir ou réduire la morbidité, les complications et une plus grande détérioration de l’état de handicap. Cependant, l’espoir de voir la lèpre éliminée à tous les niveaux vers 2005 est terni par l’enjeu essentiel, qui se devra d’être significatif, de reconnaître et de traiter ces réactions dans l’ère de l’après éradication. RESUMEN Se realizó un estudio retrospectivo de los expedientes clínicos de los pacientes con lepra en el PGIMER de Chandigarh, India, del periodo 1983 a 1998, para analizar la frecuencia, el tiempo de aparición, y los factores de riesgo de las reacciones leprosas (RL). De los 2600 casos analizados, 1494 fueron multibacilares y 1106 paucibacilares. La presencia de reac- ción leprosa fue común; 30.9% de los pacientes tuvieron RL en el tiempo de su primera visita. La incidencia de reacción reversa (RR) fue más alta entre los 6 y 12 meses después de haber iniciado el tratamiento con poliquimioterapia (PQT) y después declinó gradualmente. La RR tardía ocurrió en el 9.5% de todos los casos y se observó hasta 7 años después del tratamiento. El género femenino, la enfermedad diseminada y la enfermedad bacilar, se identificaron como factores de riesgo para la RR. Las reacciones tipo eritema nudoso leproso (ENL) se observaron más frecuentemente durante el segundo y tercer año del inicio de la PQT. Del total de los pacientes que desarrollaron ENL, el 64.3% tuvieron episodios recurrentes que continuaron hasta 8 años después de haber iniciado el tratamiento. La enfermedad lepromatosa, el género femenino y los IB altos (≥3) se reconocieron como factores de riesgo para el ENL. La ocurrencia de las reacciones recurrentes y tardías, aunque sean de severidad moderada, subraya la importancia de reconocerlas y tratarlas prontamente para prevenir o para reducir la morbilidad, las complicaciones y el progreso de las deformidades incapaci- tantes. Aunque se espera que la lepra se haya podido eliminar en todos sus niveles hacia el año 2005, el reconocimiento y manejo de las reacciones leprosas continuará siendo el reto más importante y esencial en la era de la post-eliminación de la enfermedad.

With the success of multi-drug therapy large migrant population from various (MDT) in the treatment of leprosy, attention states of the country where leprosy is en- has focused on the problem of leprosy reac- demic. In the leprosy clinic at the institute, tions, which are now the most significant is- a good record keeping system combined sue in the management of individual pa- with regular evaluation of patients has gen- tients. Despite a large burden of leprosy erated a very large and useful database for cases in India, very limited data has been retrospective analysis of reactions in lep- published on the epidemiology of reactions rosy. Systematic analysis of these records from this part of the world. The available was carried out to determine the incidence information about the epidemiology of lep- of leprosy reactions in our patients, and to rosy reactions is incomplete and scanty, de- identify risk factors if any. In this paper, we spite a growing amount of literature on its discuss the incidence and risk factors for treatment. leprosy reactions over the last 15 years. Postgraduate Institute of Medical Educa- tion and Research (PGIMER), Chandigarh, MATERIALS AND METHODS is a tertiary care institute in Northern India, A total of 2867 new, previously untreated which is a low endemic area for leprosy. patients enrolled in the leprosy clinic at Many patients with leprosy are self-referred PGIMER during the period from 1983 to and some are referred by a doctor or clinic, 1998 were included in the study. Patients citing the better quality of care available at with pure neuritic leprosy were excluded our center. In addition to the population of from this analysis. Until 1987 all patients this region, the institute also caters to a with Bacterial Index (BI) ≥2 were classified 72, 2 Kumar, et al.: Epidemiology of Leprosy Reactions 127 as multibacillary (MB), and <2 as pau- not uniform in all the patients due to chang- cibacillary (PB). From 1988 to 1998, all pa- ing definitions of PB/MB cases by the tients having positive slit skin smear were WHO; however, from a purely academic/ assigned to the MB group. Until 1994, all research point of view we have also tried to MB cases were treated with WHO MDT analyze trends of reactional events at MB regimen for a minimum period of 2 yrs presentation, during MDT, and after release or until skin smear negativity, whichever from treatment, according to Ridley Jopling occurred last. From 1994 to 1998, they classification (5) among subgroups of PB/MB were treated with fixed duration (24 cases. For validation of our supposition and months) MDT MB regimen. better understanding, we have used certain After release from treatment (RFT), pa- terminologies like “disseminated disease” tients were seen at intervals of 3 to 6 and “late ENL” because of the absence (or months for the initial 2 years and later once not very satisfactory) definitions about re- or twice a year. Apart from this, patients actions. Patients were labelled as having were instructed on features of reactions/re- “disseminated disease,” if there were ≥3 lapse, and told to attend the clinic immedi- body areas involved, 6 or more skin lesions, ately if they ever experience such symp- or involvement of at least 2 peripheral toms or any other problems. A detailed clin- nerve trunks. We have used the terminology ical examination was done on each visit and “late ENL” for occurrence of ENL reaction skin smears are taken at least once a year. 2 years after MDT completion. Details about time of onset and clinical fea- The chi square test was used to analyze tures of reactions are recorded in the pa- the difference between proportions and to tient’s record file. identify trends. The difference between two Reversal reaction (RR) involving skin unpaired sample means was tested using the was diagnosed if the patient had redness student’s t-test. The significance of various and swelling of (already existing) lesions, risk factors for developing reactions was or the appearance of a few new lesions analyzed with Cox’s proportional hazards close to the existing lesions or at distant regression, and the results were expressed sites, with or without tenderness of lesions. as rate ratios. Of the ratio, the 95% confi- Neuritis as a part of reaction was defined as dence interval is given. spontaneous pain (shooting, tingling, or burning), or tenderness of the nerves with RESULTS nerve function impairment (NFI). In most Patients. Out of these 2867 patients, 62 patients with reactions, features of con- patients were excluded, either because the comitant skin and nerve involvement were diagnosis was changed or records were not present. Erythema nodosum leprosum complete. Another 205 patients were lost to (ENL) was diagnosed if a patient developed follow-up or left the area after diagnosis. A multiple, usually small, tender, evanescent total of 2600 patients with follow-up avail- nodules, with or without ulceration, which able up to 13 yrs (at least 3 yrs) were eligi- were usually associated with constitutional ble for analysis. Average period of follow- symptoms. Wherever indicated, reactional up was 74 months (range 36 to 156 episodes were treated with oral pred- months). There were 1634 males (mean age nisolone (40–60 mg/day) tapered to a stop 36 ± 3.2 yrs) and 966 females (mean age 39 over 12 weeks. Occurrence of a RR after ± 2.3 yrs). Out of them, 1494 (57.5%) had six months of stopping MDT was labeled as MB and 1106 (42.5%) had PB disease. late RR. Recurrent episodes of RR or ENL Incidence. The incidence of reversal re- were defined as having a recurrence of actions (RR) and erythema nodosum lepro- symptoms of either type of reaction more sum (ENL), and their distribution in than six weeks after the completion of treat- PB/MB cases at the time of first presenta- ment for reaction. Recurrence of a reaction to the clinic is given in Table 1. The in- tional episode earlier than this was consid- cidence of RR was 24.1% (627/2600 pa- ered to be possibly due to inadequate or tients) and the figure for ENL in MB cases abrupt stoppage of treatment. The exact was 11.8% (177/1494 patients). Of the 627 definitions of the spectrum of the disease patients with RR, 169 (27%) had evidence and duration of treatment with MDT were of reaction in skin lesions only and the re- 128 International Journal of Leprosy 2004

TABLE 1. Prevalence of reactions at the time of ﬁrst presentation.

Reversal reaction ENL No. of Classification cases Skin Skin + Total Skin Skin + Total only nerves (%) only nerves (%) MB 1494 108 291 399 (26.7) 86 91 177 (11.8) PB 1106 61 167 228 (20.6) 0 0 170 (11.8) Total 2600 169 458 627 (24.1) 86 91 177 (11.8) maining 458 (73%) had involvement of the episodes of ENL occurred at a higher both skin and nerves, whereas in patients frequency in the second or third year after having ENL, 86 (48.6%) had only cuta- starting MDT. neous involvement and 91 (51.4%) had in- Late reactions. Late reversal reactions volvement of both skin and nerves. were seen in 4.2% (47/1106) of PB patients In the total period of observation, 858 pa- and 5.3% (79/1494 patients) with MB dis- tients experienced RRs and 337/1494 MB ease. The majority of late reactions in the cases had ENL either at the time of registra- multibacillary group were observed in pa- tion, during, or after release from treatment tients with borderline lepromatous (BL) with cumulative incidence of 33% and disease. The incidence of late RR was high- 22.5%, respectively. Altogether, there were est during the first 2 yrs after being released 1356 episodes of RR in 858 patients (1.6 re- from treatment (RFT). Even though the de- action episodes/patient) and 885 episodes cline in the incidence of RR began early af- of ENL in 337 patients (2.6 reaction ter the initiation of therapy, and most reac- episodes/patient). Of these 337 MB cases, tions occurred during the first 2 yrs, a few 203 were treated before 1994 and the rest continued to occur until 7 yrs after RFT. received fixed duration (24 months) MDT Late ENL reactions were seen in only 3% (p <0.05). (45/1494) of MB [BL and polar leproma- Time of onset. Figures about the reac- tous (LL)] patients. Late ENL, though mild tional episodes according to the period of and usually not associated with significant time they occurred are given in Table 2. A constitutional symptoms, continued to oc- great majority of RRs occurred during the cur for up to 8 yrs after completion of first 6 months after starting MDT whereas, MDT. Of these patients, 15 were treated be-

TABLE 2. Time of onset of reactional episodes.

Pauibacillary Multibacillary (N = 1494) Period BT (N = 1106) BB (N = 82) BL (N = 902) LL (N = 510) No. % No. % No. % No. % Reversal reactions At registration 228 44.5 18 27.3 379 49.0 2 40 0–6 months 144 28.1 20 30.3 191 24.7 3 60 7–12 months 65 12.6 15 22.7 114 14.7 — — 2nd year 46 9.0 13 19.7 46 5.9 — — ≥3 years 29 5.7 0 0 43 5.5 — — Total 512 10000 66 10000 773 10000 5 100

ENL Reactions At registration — — — — 35 19.4 142 00 20.1 0–6 months — — — — 19 10.5 91 00 12.9 7–12 months — — — 31 00 17.2 12300 00 17.4 2nd year — — — — 59 32.8 192 00 27.2 ≥3 years — — — — 36 20.0 157 00 21.8 Total 180 100% 705 100% 72, 2 Kumar, et al.: Epidemiology of Leprosy Reactions 129

TABLE 3. Risk factors for developing reversal reactions (RR).

Patient group Risk factors PB/MB/Both No. Rate ratio* (95%CI) p value Spectrum PB 314/1106 0.69 (0.58–0.81) p <0.01 MB 544/1494 Age (years) ≤20 Both 120/357 1.0 (0.81–1.3) p >0.1 >20 Both 738/2243 Sex Male Both 510/1634 0.80 (0.68–0.95) p <0.05 Female Both 348/966 Extent of clinical disease ≥6 skin lesions Both 544/858 2.5 (2.1–2.9) p <0.01 ≥2 nerves involved Both 523/858 2.4 (2.0–2.8) p <0.01 ≥3 Body areas involved Both 544/858 2.8 (2.2–3.1) p <0.01 *Rate ratio adjusted for the influence of age and sex fore 1994 and the remaining 30 with fixed veloping reversal reactions. The strongest duration regimen (24 months) (p <0.01). association was observed between the ex- However, there was no statistically signifi- tent of clinical disease and the risk of devel- cant difference among the number of MB oping RR. Of the 858 patients manifesting patients manifesting late RRs treated before RRs, 544 (63.4%) had ≥3 body areas in- 1994 (43/79) and those treated with fixed volved and/or ≥6 skin lesions, and 523 duration regimen (36/79) (p >0.1). (61%) had ≥2 nerve trunk involvement. Recurrent reactions. Of the 858 patients Age was not found to be a significant risk who manifested reversal reactions, 252 factor for reversal reactions (Table 3). (29.4%) had ≥2 episodes and the remaining Risk factors for developing ENL reac- 606 (70.6%) had only a single episode of tions are given in Table 4. Lepromatous lep- RR. Cumulative incidence of recurrent re- rosy, female gender and higher BI (≥3) versal reaction was 9.7% (252/2600) were significant risk factors, whereas age among all of the patients. The presence of a was not. reversal reaction at the time of initial presentation or during the first year of treat- DISCUSSION ment was more frequently associated with Incidence. Much is known about the epi- an increased risk of having another episode demiology of reactions, but their incidence occurring later on. in the period after MDT is less well docu- Out of the total patients who experienced mented because of lack of long term follow- ENL, 217/337 (64.4%) experienced more up (6). Various estimates of the frequency of than one episode. The median number of reversal reactions have been given by sev- episodes was 4 and the time between the eral authors (1, 6, 17). Published reports indi- first and last episode averaged 34 months cate that the frequency of RR at the time of (range 5 to 96 months). A considerable diagnosis varies between 2.6% and 6.4% (6) number of patients (51/217, 23.5%) had though a much higher figure of 28% was re- more than ≥4 episodes of ENL over a pe- ported in a hospital-based study from Nepal riod of observation varying from 3 years to (17). In our study, this figure was 24.1%. 8 years, with no other identified risk factor Other studies have reported relatively lower except a higher BI (BI ≥3). total figures of 9.1% from Hyderabad, India Risk factors. Female gender, multibacil- (9) and 16.5% from Ethiopia (14), probably lary disease, and widespread disease (≥3 reflecting a variable proportion of PB/MB body areas involved, ≥6 skin lesions, or ≥2 cases and the use of mixed case definitions peripheral nerve trunks involvement) were used. Figures for the percentage of patients statistically significant risk factors for de- manifesting RRs at any time vary from 130 International Journal of Leprosy 2004

TABLE 4. Risk factors for development of ENL reactions.

Factors Variables No. Rate ratio* p value Disease group BL 95/902 .12 (0.09–0.17) p <0.01 LL 242/510 Age <20 34/238 1.1 (0.75–1.6) p >0.1 >20 303/2322 Sex Male 192/1634 .75 (0.59–0.95) p <0.05 Female 145/966 BI <3 120/2109 07 (0.05–0.09) p <0.01 ≥3 217/491 *Rate ratio adjusted for the inﬂuence of age, sex and bacteriological index

3.5% among PB cases in Malawi (1) to to the hospitals for reactions. Most of our 47.5% among MB cases in Zaire (6). Due to patients were self-reporting having reac- the use of widely different case definitions, tions severe enough to force them to seek it is difficult to compare the frequencies of treatment from a hospital rather than field- RR in PB and MB patient groups in studies based clinics. This could be one reason for published from different centers. Other fac- relatively higher figures for ENL in our tors contributing to such variation could be center. the time between diagnosis and beginning In reactions (RR or ENL) involvement of MDT, duration of MDT, duration of steroid the skin and nerves occurred either singly regimen, and quality of the local leprosy or together. Of the total number of reactions control program. Overall 33% of all pa- at the time of presentation, 31.7% had only tients in our study developed RR at some cutaneous involvement, whereas 68.3% had time during treatment and follow-up, in- involvement of both skin and nerves. In a cluding those with a reaction at presenta- retrospective analysis of reversal reactions tion. in a study from Hyderabad, India, 43.1% ENL reactions were reported to occur in had only skin lesions, 31.8% had only neu- more than 50% of lepromatous leprosy ritis, and 22.7% had both skin lesions and (LL) cases and in about 25% of borderline neuritis (6). Such observations emphasize lepromatous (BL) cases in the pre-MDT era that neuritis can occur along with inflam- (7). The incidence of ENL reactions appears matory skin lesions or independently. to have fallen with the introduction of Therefore, even very mild symptoms sug- MDT, possibly due to the combined bacte- gestive of neuritis should be taken seriously ricidal effect of rifampicin and the anti- and nerves should be palpated on each visit inflammatory effect of clofazimine in sup- to detect early signs of nerve inflammation, pressing ENL (8). A hospital-based study regardless of presence or absence of reac- from Nepal reported a high frequency of tion involving skin. ENL reactions (28.6%) in LL, but only Time of onset of reactions. Signifi- 7.5% in BL cases (5). In the present study, cantly, 30.9% of our patients presented to 47.4% of LL cases and 10.5% of BL cases us because of reactions, in spite of having manifested ENL reactions. The lower inci- symptoms suggestive of leprosy for months dence of ENL among patients treated be- or years. In a hospital-based study from Hy- fore 1994 could be because of the persisting derabad, India, Lockwood, et al. (6) noted anti-inflammatory effect of clofazimine till reactions in a strikingly high percentage of bacterial clearance was achieved. Field their patients (41.3%) at the time of presen- studies have reported a rather lower inci- tation. It is obvious that many patients seek dence of ENL such as 12% in LL and 3.6% treatment only when they get frightened by in BL from Ethiopia (9), and 2.1% of all the sudden development of such lesions MB patients from Bangladesh (10). The var- particularly over face, or painful symptoms ied frequency of ENL in reported studies of neuritis due to reaction. could be due to patients in the field Although it is known that the reversal re- screened for reactions vs. patients reporting actions occur most frequently within 6 to 12 72, 2 Kumar, et al.: Epidemiology of Leprosy Reactions 131 months after starting treatment (4, 11), most from Hyderabad, India, 33% of patients previous studies did not report long term with RRs had recurrent episodes (9). The follow-up. In the AMFES data (Ethiopia), immune suppression in some of these pa- RR were reported to occur as late as 5 yrs tients may have been for of too short a du- after the start of treatment in both PB and ration, or hospitals may be more likely to MB patients (12). Our study also supports get the severely affected problem patients, the fact that though the incidence of RR which require still longer treatment with was found to decline gradually, reactions steroids or some other adjuvant therapy. continued to occur for 7 years, though in Possibly, Naafs, et al. may be right when very few patients. However, in India the they suggest that immunosuppressive treat- longest interval reported between treatment ment should continue throughout the period and reaction is 6.5 years (6). when the antigen load is sufficient to trigger The majority of ENL reactions after start- the cell mediated immune response (10, 11). ing MDT occurred in the second or third Of all patients who manifested ENL, year and the recurrence of attacks was 64.3% had recurrent episodes. In the mostly noted in the same period, though the AMFES cohort (Ethopia), 63% of all cases reactions continued to occur up to 8 yrs af- with ENL had more than one episode, and ter RFT. 31% of all ENL cases manifested 5 or more Late reactions. The percentage of pa- episodes over a period of more than 2 yrs. tients developing late RR in both PB and In general, recurrence in episodes of reac- MB groups was 4.2% and 5.3%, respec- tions is more common in ENL than in RR, tively. In a study from Thailand, 2.7% of and approximately one-third of patients PB patients and 9% of MB patients devel- with ENL reactions go on to have recurrent oped a late reversal reaction (15). In Malawi, episodes (14, 16). Patients must be warned of 3.5% of PB patients developed late reac- this possibility and be educated to return for tions during the first 4 yrs of follow-up (2). follow-up, and clinicians should also be In a cohort of MB patients (treated with aware to diagnose even very late reactions MDT for 2 yrs or longer until split-skin in post-elimination era. Though it could be smear tested negative) from Karigiri, India, in variance to other proposed definitions only 1.1% experienced reversal reactions (Transactions of 16th International Leprosy during almost 10 yrs of surveillance (18). Congress), we suggest that a patient could Late RR is known to occur mostly within be labeled as having “chronic ENL” if he the first 3 to 4 yrs after RFT (1, 15) as was needs continued antireaction treatment for a also observed by us. In the absence of a period of 6 months or more. clear definition for late RR and different Risk factors. Risk factors for RR identi- MDT regimens used in various studies, an fied to be significant in this study were exact comparison of the frequency of late female gender, and disseminated disease RRs is not possible. (extent of clinical disease measured by in- Late ENL reactions were recorded in 3% volvement of a number of body areas, of our MB cases. The majority of these nerves, and skin lesions) at the time of di- episodes consisted of a few ENL lesions, agnosis. PB patients had less risk of devel- which were mostly associated with only oping reversal reactions than MB patients. mild cutaneous and constitutional symp- Patients with three or more body areas in- toms. volved or having ≥6 skin lesions had about Recurrent reactions. Recurrent episodes twice the risk of developing RR than those of reversal reactions are an important clini- with limited disease (63.4% vs. 36.6%). cal phenomenon, which may result in con- Similar observations (>10 skin lesions) tinuing nerve damage and add on to the de- have been made by Van Brakel, et al. (17), gree and number of impairments. In our which suggested that this can be of consid- study, 9.7% patients developed recurrent erable importance for control programs, to episodes of reversal reaction. Almost half identify patients at risk of developing reac- of these episodes occurred within 3 months tions. They argued that “body area” may of stopping the course of prednisolone that just be a proxy indicator for the bacterio- had been administered for the previous re- logical index or multibacillary end of the action. Strikingly, in a hospital-based study leprosy spectrum. Like our observation, 132 International Journal of Leprosy 2004 they also noted similar association within by 2005, even those patients who have suc- the borderline tuberculoid (BT) patient sub- cessfully completed their treatment will group, indicating that body area count is continue to manifest with late or recurrent useful indicator of the risk of developing reactions in settings of poorly available ex- RR. Pregnancy and lactation are reported to pertise or services to manage these be risk factors for RR and ENL (6, 8), but the episodes. association has not been quantified and re- REFERENCES mains unclear. Leprosy lesions over the face have also been observed to develop 1. BECX-BLEUMINK, M., and BERHE, D. Occurrence RR more frequently (3). However, the rela- of reactions, their diagnosis and management in leprosy patients treated with multidrug therapy: tion between pregnancy or lactation and experience in the Leprosy Control Program of the leprosy reactions, the significance of a All Africa leprosy and Rehabilitation Training patch over the face as a risk factor for de- Center (ALERT) in Ethiopia. Int. J. Lepr. Other veloping reversal reactions, could not be Mycobact. 60 (1992) 173–184. statistically analyzed in the present study 2. BOERRIGTER, G., PONNIGHAUS, J. M., FINE, P. E. due to lack of complete information in the M., and WILSON, R. J. Four years follow-up re- records. sults of a WHO-recommended multidrug regimen For ENL, the risk factors identified in our in paucibacillary patients in Malawi. Int. J. Lepr. study were lepromatous leprosy, female gen- Other Mycobact. Dis. 59 (1991) 255–261. der, and higher bacteriological index (≥3). 3. BRITON, W. J. The management of leprosy reversal reactions (Editorial). Lepr. Rev. 69 (1998) For recurrent ENL, a number of risk factors 225–234. like age, sex, spectrum of disease were ana- 4. DE RIJK, A. J., GEBRE, S., BYASS, P., and BERHANU, T. lyzed, but only high BI (≥3) was found to be Field evaluation of WHO-MDT of fixed duration at statistically significant. In AMFES cohort ALERT, Ethiopia: the AMFES project, Part 2. Reac- (Ethopia), no specific risk factor for recur- tions and neuritis during and after MDT in PB and rent ENL could be identified except age, be- MB leprosy patients. Lepr. Rev. 65 (1994) 320–332. tween 20 and 45 yrs (14). In our study, 5. JOPLING, W. H., and MCDOUGALL, A. C. In: though the majority of cases were in this Handbook of Leprosy, 5th edn. New Delhi: CBS age range, correlation with any age group Publishers. 1996, pp. 10–47. could not be confirmed. ENL reactions are 6. LIENHARDT, C., and FINE, P. E. M. Type 1 reaction, neuritis and disability in leprosy. What is the reported to occur throughout pregnancy and current epidemiological situation? Lepr. Rev. 65 lactation, and may be severe and recurrent; (1994) 9–33. however, because of incomplete informa- 7. LOCKWOOD, D. N. J. The management of ery- tion as stated above, this relationship could thema nodosum leprosum: current and future op- not be anlyzed in our cohort. tions (Editorial). Lepr. Rev. 67 (1996) 253–259. In conclusion, this is the largest series of 8. LOCKWOOD, D. N. J., and SINHA, H. H. Pregnancy patients with long term follow-up, delineat- and leprosy: a comprehensive literature review. ing the epidemiology of reactions in leprosy Int. J. Lepr. Other Mycobact. Dis. 67 (1999) 6–12. from India. RR and ENL are common com- 9. LOCKWOOD, D. N. J., VINAYAKUMAR, S., STANLEY, plications in leprosy patients in India. Fe- J. N. A., MCADAM, P. W. J., and COLSTON, M. J. Clinical features and outcome of reversal (type 1) male gender, multibacillary leprosy, and ex- reactions in Hyderabad, India. Int. J. Lepr. Other tensive disease were found to be major risk Mycobact. Dis. 61 (1993) 8–15. factors for occurrence of RRs. The majority 10. NAAFS, B. Treatment of reactions and nerve dam- of RRs occurred within 12 months of start- age. Int. J. Lepr Other Mycobact. Dis. 64 (1996) ing MDT, and then the incidence declined S21–S28. gradually but the reactions continued to oc- 11. NAAFS, B., PEARSON, J., and WHEATE, H. Reversal cur until 7 yrs after RFT. For ENL, lepro- reaction: The prevention of permanent nerve dam- matous leprosy, female gender and higher age. Comparison of short and long term steroid bacteriological index (≥3). Significant risk treatment. Int. J. Lepr. Other Mycobact. Dis. 47 factors were approximately one-third of pa- (1979) 7–12. tients with ENL reactions go on to manifest 12. RICHARDUS, P., FINLAY, K. M., CROFT, R. P., and SMITH, W. C. Nerve Function impairment in lep- recurrent episodes spread out over a period rosy at diagnosis and at completion of MDT: a ret- of more than 2 yrs requiring specialized ex- rospective cohort study of 786 patients in pertise to manage them. Though leprosy is Bangladesh. Lepr. Rev. 67 (1996) 297–305. expected to be eliminated from all nations 13. ROSE, P., and WALTERS, M. F. Reversal reactions 72, 2 Kumar, et al.: Epidemiology of Leprosy Reactions 133

in leprosy and their management (Editorial) Lepr. 17. VAN BRAKEL, W. H., KHAWAS, I. B., and LUCAS, S. Rev. 62 (1991) 131–121. Reactions in leprosy: an epidemiological study of 14. SAUNDERSON, P., GEBRE, S., and BYASS, P. Rever- 386 patients in West Nepal. Lepr. Rev. 65 (1994) sal reactions in the skin lesions of AMFES pa- 190–203. tients: incidence and risk factors. Lepr. Rev. 71 18. VIJAYKUMARAN, V., MANIMOZHI, N., and JESU- (2000) 309–317. DASAN, K. Incidence of late lepra reaction among 15. SCHREUDER, P. A. M. The occurrence of reactions multibacillary leprosy after MDT. Int. J. Lepr. and impairments in leprosy: experience in the lep- Other Mycobact. Dis. 63 (1995) 18–22. rosy control program of three provinces in North- 19. WEMAMBU, S. N., TURK, J. L., WATERS, M. F., and eastern Thailand, 1978–1995. II. Reactions. Int. J. REES, R. J. Erythema nodosum leprosum. A clini- Lepr. Other Mycobact. Dis. 66 (1998) 159–169. cal manifestation of the Arthus phenomenon. 16. SCOLLARD, D. M., SMITH, T., BHOOPAT, L., THEETRA- Lancet 2 (1969) 933–935. NONT, C., RANGDAENG, S., and MORENS, D. M. Epi- 20. WHO EXPERT COMMITTEE ON LEPROSY. Seventh demiologic characteristics of leprosy reactions. Int. report. Geneva: World Health Organization, 1998. J. Lepr. Other Mycobact. Dis. 62 (1994) 559–567. Tech. Rep. Ser. 874. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) Neuropathic Pain in Leprosy Patients1

Patrick R. N. A. G. Stump, Rosemari Baccarelli, Lúcia H. S. C. Marciano, José R. P. Lauris, Manoel Jacobsen Teixeira, Somei Ura, and Marcos C. L. Virmond2 ABSTRACT The introduction of multidrug therapy by the World Health Organization has dramatically reduced the world prevalence of leprosy but the disease is still a public health problem in many countries, with a world prevalence of almost 600,000 cases in 2001. Damage to peripheral nerves is a key component of leprosy and the sensory and motor loss that follows is the basis for many of the classical features of this disease, such as skin wounds, cracks, plantar ulcers, clawed hands, drop foot, and incomplete closure of the eyelids. One of the most remarkable aspects of leprosy to lay persons and health care workers alike is that patients are reputed to feel no pain. However, neuropathic pain is arising as a major problem among leprosy patients. It can be nociceptive due to tissue inflammation, which mostly occurs during episodes of immune activation or neuropathic due to damage or dysfunction of the nervous system. This study, conducted among 358 leprosy patients, reveals a considerable prevalence of neuropathic pain and presents evidence that this common problem should be a high priority of those in charge of leprosy control programs. RÉSUMÉ L’introduction de la poly-chimiothérapie par l’Organisation Mondiale de la Santé a diminué de façon drastique la prévalence mondiale de la lèpre mais la maladie est encore un problème de santé publique dans plusieurs pays, avec une prévalence mondiale de presque 600 000 cas en 2001. L’atteinte des nerfs est une composante clef de la lèpre et les pertes sensorielles et motrices qui s’ensuivent forment la base des caractères classiques de cette maladie, comme les blessures cutanées, les fissures, les ulcères de la plante des pieds, les mains en crochets, les pieds tombants et la fermeture incomplète des paupières. Un des aspects les plus remarquables de la lèpre pour le grand public comme pour le prestataire de soins de santé est que les patients ont la réputation de ne pas ressentir de douleur. Cependant, les douleurs neurogènes sont en train d’émerger comme un problème majeur parmi les patients hanséniens. Elle peut être nociceptive, due à l’inflammation qui apparaît principalement durant les épisodes d’activation immunologique, ou bien neurogène, causée par une atteinte ou une dysfonction du système nerveux. Cette étude, menée chez 358 patients lépreux, révèle une prévalence considérable de douleurs neurogènes et présente des arguments afin que les responsables de santé publique et de contrôle de la lèpre considèrent ce problème comme une priorité. RESUMEN No obstante que la introducción de la poliquimioterapia por la Organización Mundial de la Salud ha reducido dramáticamente la prevalencia de la lepra a nivel mundial, la enfermedad es todavía un problema de salud pública en muchos países, con una prevalencia de casi 600,000 casos en 2001. El daño a los nervios periféricos es un componente crítico de la lepra y la pérdida sensorial y motora que le siguen es la base de muchas de las característi- cas clásicas de la enfermedad que incluyen heridas en la piel, cuarteaduras, úlceras plantares, manos en garra, pie caído y cierre incompleto de los párpados. Uno de los aspec- tos más remarcables de la lepra es la creencia general de que los pacientes no sienten dolor. Sin embargo, el dolor neuropático se está manifestando como un problema cada vez mayor entre los pacientes con lepra. El dolor puede ser enmascarado por la inflamación del tejido que ocurre principalmente durante los episodios de activación inmune o neuropática asoci-

1 Received for publication on 15 October 2003. Accepted for publication on 24 February 2004. 2 P. R. N. A. G. Stump, M.D.; R. Baccarelli, F. T., Ph.D.; Lúcia H. S. C. Marciano, O.T., M.S.; S. Ura, M.D.; M. C. L. Virmond, M.D., Ph.D., Dept. of Rehabilitation, Instituto Lauro de Souza Lima, Bauru, Brazil; J. R. P. Lauris, M.D., Ph.D., Dentistry School, U.S.P., Bauru, Brazil; M. J. Teixeira, M.D., Ph.D., School of Medicine, U.S.P., São Paulo, Brazil. Reprint requests to: Marcos Virmond, CP 3021 Bauru–SP, Brazil. 17034-971. E-mail: [email protected]

134 72, 2 Stump, et al.: Neuropathic Pain 135

ada al daño o disfunción del sistema nervioso. El presente estudio, realizado en 358 pacientes, revela una considerable prevalencia de dolor neuropático, y presenta evidencias de que este es un fenómeno común que debe ser de alta prioridad para aquellos encargados de los programas de control de la lepra.

Since the introduction of an effective during episodes of immune activation [“retreatment based upon a multi-drug therapy versal reaction” (RR) and “erythema no- (MDT) of dapsone, clofazimine, and ri- dosum leprosum” (ENL)] or neuropathic fampicin as recommended by the World due to damage or dysfunction of the ner- Health Organization (WHO), the preva- vous system. Nociceptive pain is due to lence of leprosy has dramatically decreased activation of peripheral nociceptors on (13). However, the disease is still a public A-delta and C-fibers, secondary to nerve health problem in many countries with an tissue injury during ENL or RR. There is a estimated global prevalence of near release of bradykinin, serotonin, substance 600,000 cases as for the year 2003 (14). P, histamine and prostaglandin, which facil- Damage to peripheral nerves is a key itate the transmission of pain impulses from component of leprosy and, together with the periphery to the spinal cord. typical skin lesions, accounts for the major The complex and stigmatizing burden of traditional clinical features of the disease. being diagnosed with leprosy may compel Little is known about the mechanism by patients to focus solely upon curing the dis- which the mycobacteria infect Schwann ease, which is actually accomplished with cells, but recently some evidence has the WHO drug regimen, and to accept their emerged. A glycoprotein (α-dystroglican) symptoms as an inevitable concomitant or that binds to the surface of Mycobacterium residual of the disease. However, the num- leprae also binds to a molecule on the sur- ber of cases with pain problems seemed to face of the Schwann cell surface and pro- us to be substantial (4). The aims of this vides a potential mechanism for internaliza- study, conducted in a country where leprosy tion of the bacilli by Schwann cells (1, 9). is endemic, were to estimate the prevalence The sensory and motor loss that follows of pain in patients with leprosy and to de- nerve damage in leprosy is the basis for termine the main characteristics of their many of the classical features of the disease pain. such as skin wounds, cracks, plantar ulcers, clawed hands, drop foot, and lagophtalmos. MATERIALS AND METHODS Sensory damage includes an early loss of The study was conducted at the Instituto pain and temperature perception followed Lauro de Souza Lima, Bauru, Brazil, a na- by compromise of tactile and pressure tional referral center for leprosy patients. senses. The distribution and onset of nerve Brazil has a prevalence of 77.676 cases per damage can vary according to the type of 100,000, and an important detection rate of leprosy, being more disseminated and grad- 24.1/100,000 (41.070 new cases in 2000), ual in the lepromatous cases, or localized which makes it the second largest endemic and acute in tuberculoid and borderline country for leprosy in the world after India cases. The indeterminate type is an initial (14). presentation of the disease in which major The study included 358 patients with lep- nerve damage has not yet developed. rosy who presented to the Dermatological One of the most remarkable aspects of clinic of Instituto Lauro de Souza Lima leprosy to lay persons and health care work- from October 1, 2001 to March 31, 2002. ers alike is that patients are reputed to feel Among them, 215 were male (60.1%) and no pain. This wide-spread impression is 143 were female (39.9%). The mean age rapidly changing as many patients who was 54.8 yrs (S.D. = 16 yrs), with a range have completed their WHO MDT are now of 11 to 87 yrs. reporting complaints of stimulus-independent The mean time from initial diagnosis was ongoing pain, and seeking relief. Indeed, 18.3 yrs (range, 10 months to 68 yrs, S.D. = pain in leprosy can be nociceptive due to 18.5 yrs), and 178 (49.7%) patients were di- tissue inflammation, which mostly occurs agnosed over 10 years earlier. According to 136 International Journal of Leprosy 2004 the Madrid Classification (8), 207 patients ulnar (59.2%), followed by the tibial (57.8%) were lepromatous, 92 (25.7%) bor- (30.3%), fibular (18.9%), median (4.5%), derline, 54 (15.1%) tuberculoid, and only 5 radial (2.0%), and trigeminal (1.5%). These (1.4%) were indeterminate. All cases were percentages sum to greater than 100 be- receiving treatment except for 283 (79.1%) cause patients were free to indicate pain in patients who had concluded their standard the distribution of more than one nerve. course by the time of the study. Treatment Glove (22.4%) and stocking (24.9%) distri- regimens included the WHO MDT, dapsone bution of pain were also quite common plus rifampicin, or dapsone as monotherapy (Fig. 1). The onset of episodes was reported in some previously treated cases. as abrupt by 39 patients (19.4%), as insidi- Two hundred and one (56.1%) of the pa- ous by 73 patients (36.3%), and as recurrent tients reported past or current moderate to bursts by 89 (44.3%) patients. The assess- severe chronic neuropathic pain that in- ment of quality of pain can be seen in Fig. 2. terfered with activities of daily living or In the 53 patients with pain present at the disturbed sleep. None of these cases re- time of interview, the most affected vealed signs or symptoms of RR or ENL as anatomical layer was deep in 30 (56.6%) assessed by an experienced clinician. These patients, superficial in 8 (15.1%) and mixed cases underwent clinical neurological examin 15 (28.3%). In these patients, pain was ination by trained health workers, including constant in 34 (64.2%) and episodic in 19 detailed anamnesis assessment focusing on (35.8%). Verbal ratings of present pain the occurrence of pain, its localization, dura- were severe in 29 (54.7%), moderate in 17 tion, pattern of symptoms onset, quality, and (32.1%), and mild in only 7 patients quantity. Localization of pain refers to the (13.2%). On the graphic scale, 22 (41.5%) anatomical distribution and trunk of the patients rated their pain as severe, 21 most relevant affected peripheral nerve. (39.6%) as moderate, and 10 (18.9%) as Duration of pain was classified as less or mild. These pain characteristics are summa- greater than 6 months. The pattern of symp- rized in The Table. tom onset was categorized as abrupt, insidious, or in repetitive bursts. Assessment of DISCUSSION quality of pain was based upon the Brazil- Lack of sensation is a paradigm of ian Portuguese version of the McGill Pain leprosy, and the diagnosis of this chronic Questionnaire (12). In addition, we inquired infectious disease is assured by the pres- whether present pain was experienced as ence of skin lesions (usually patchy) with superficial, deep, or mixed. Pain intensity marked sensory loss as assessed by was verbally rated by patients as mild, Semmes-Weinstein monofilaments or a moderate, or severe and was also rated on a ballpoint pen tip. Abnormalities include graphic scale (empty to full water glass). loss of touch, temperature, and pressure sensation. Although clinical consensus re- RESULTS gards leprosy as painless, in reality nerve Using the day of interview and examina- pain in leprosy is often present during neu- tion as the reference point, 148 (73.6%) pa- ritis, a feature that accompanies acute lep- tients reported episodes of pain only in the rosy reactions. These reactive episodes in- past and 53 (26.4%) had complaints at pres- clude entrapment of the nerve in selected ent. In the 148 patients with past pain only, sites (most often the ulnar canal at the el- leprosy had been diagnosed less than 10 bow) due to edema from acute and severe years earlier in 59 cases (39.8%) and more inflammation of the nerve. In such a situa- than 10 years earlier in 89 cases (60.2%). In tion, activation of the nervi nervorum may those with present pain, only 14 (26.4%) be the main contributor to pain. Another cases had been diagnosed over 10 years ear- possibility is that the acute neural inflam- lier. Pain had been present for 6 months or mation can excite and sensitize nociceptors. less in 55 cases (27.4%), whereas 141 In some cases, there is severe destruction of (70.1%) reported pain for longer than 6 nerve fibers (2) and the partial regeneration months. Only 5 patients (2.5%) could not that follows may produce discharges, estimate the duration of their pain. The diminution of stimulus thresholds, and ex- nerve most often affected by pain was the aggerated responses of nociceptors. 72, 2 Stump, et al.: Neuropathic Pain 137

THE TABLE. Some characteristics of neuropathic pain among 201 cases of leprosy with past (148) or present (53) pain.

Groups Past pain* % Past pain* % Clinical form Lepromatous 94 63 26 49 Borderline 29 20 20 38 Tuberculoid 24 16 7 13 Indeterminate 1 0.6 0 0 Onset Abrupt 30 20 9 17 Insidious 47 32 26 49 Bursts 71 48 18 34 Duration in months <6 months 49 33 6 11 >6 months 94 63 47 89 Not known 5 3 0 0 Time of leprosy diagnosis <10 years 59 40 39 74 >10 years 89 60 14 26 Treatment completion Yes 130 88 40 75 No 18 12 13 25 Pain intensity Mild — 10 19 Moderate — 21 40 Severe — 22 41 Verbal rating of pain intensity Mild — 7 13 Moderate — 17 32 Severe — 29 55 Time of worst pain Morning — 7 13 Afternoon — 9 17 Evening — 15 28 Not speciﬁc — 22 41 Anatomical layer of involvement Superﬁcial — 8 15 Deep — 30 57 Mixed — 15 28 Evolution Worsening — 20 43 Stable — 24 51 Remitting — 9 19 Character Episodic — 19 36 Constant — 34 64 *Number of patients

This study reveals that neuropathic pain and their rami may be also compromised, not directly associated with an acute reactive particularly in lepromatous and borderline episode may be present in a considerable cases in which dissemination of the disease proportion of patients with leprosy. In fact, is a characteristic feature. out of 358 patients presenting to the outpa- It is important to note that, among patient dermatological clinic for other reasons, tients with present pain, 40 patients (75.4%) 56.1% reported prior or current episodes of had completed antimicrobial treatment and, neuropathic pain. Most of these patients re- according to the present policy of leprosy ported that the intensity was severe and suffi- control, are discharged from further follow- cient to interfere with activities of daily life or up. Such patients receive little further care. sleep. Therefore, a significant number of patients According to the literature (3, 5), the most did not have ongoing access to care and common nerve affected in leprosy is the ul- could not seek assistance for relief of neu- nar nerve, and we confirmed the frequency ropathic pain and improvement of quality of this as a painful site. However, a glove of life. In addition, 130 patients (87.8%) and stocking distribution of pain was also with past pain had already completed treat- frequently reported by our patients. Al- ment by the time their pain occurred. Thus, though involvement of nerve trunks in lep- successful completion of antimicrobial rosy is common, the superficial branches treatment does not appear to prevent occur- 138 International Journal of Leprosy 2004 rence of neuropathic pain. As a matter of ies on new therapies to cope with this previ- fact, the teams caring for patients with lep- ously ignored clinical problem. rosy are not generally aware of the problem of neuropathic pain. 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ROWBOTHAM, M., HARDEN, N., STACY, B., BERN- This study reveals a considerable preva- STEIN, P., and MAGNUS-MILLER, L. Gabapentin lence of neuropathic pain among patients for the treatment of postherpetic neuralgia: a mul- with leprosy and presents evidence that this ticenter double-blind, placebo-controlled study. common problem should be a high priority JAMA 280 (1998) 1837–1842. among those in charge of leprosy control 12. TEIXIRA, M. J., and PIMENTA, C. A. M. Avaliação programs. In fact, given the present public do doente com dor. In: Dor: epidemiologia, fi- health policy of shorter regimens and imme- siopatologia, avaliação, síndromes dolorosas e diate discharge of patients after completion tratamento. São Paulo: Moreira Jr., 2001. pp. of treatment, this problem could worsen fur- 56–68. 13. WORLD HEALTH ORGANIZATION. Guide to Elimi- ther. Thus, at present there is a strong need nate Leprosy as a Public Health Problem. to review the concept of leprosy care to pro- Geneva: World Health Organization, 2000. vide adequate attention to this disabling 14. WORLD HEALTH ORGANIZATION.Weekly Epi- complication, and plenty of room for stud- demiol. Rec. 77 (2002). INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) Pinch Skin Grafting in Non-Healing Leprous Ulcers1

Elizabeth Jayaseelan and Vijay V. Aithal2 ABSTRACT Treatment of leprous ulcers has remained inadequate, owing to the fact that most of these ulcers are still being managed conservatively especially in developing nations, probably due to financial constraints. Pinch skin grafting, though obsolete now (2), tries to bridge this gap between cost and effectiveness. It is a simple office-based technique, not requiring much expertise or investment, and can be done in a simple set-up such as a side room (3). Also, pinch skin grafting has an added advantage over single grafts, in that even if one graft is rejected, there are other grafts, which successfully heal, and epidermize to the surrounding. Moreover, if the ulcer is draining, the discharge flows out in between the grafts, thus preventing the whole graft from being rejected. The only disadvantage to pinch skin grafting is the final cosmetic appearance, which might not be most pleasing. We had very good results with all four patients who underwent this procedure in our institution. The procedure and the final result are described in detail in this report. RÉSUMÉ Le traitement des ulcères lèpreux est resté insuffisant, parce que la majorité de ces ulcères est encore appréhendée de manière conservatoire, en particulier dans les pays en voie de développement, qui souffrent de contraintes financières encore importantes. La greffe de peau par pincement, quoique obsolète de nos jours (2), essaye de relier l’incompatibilité entre faible coût et bonne efficacité. C’est une technique simple de pratique courante, ne né- cessitant que peu d’expertise et d’investissement, qui peut être réalisée dans une salle de soin (3). De plus, la greffe de peau par pincement présente l’avantage par rapport à la greffe simple d’être formée de plusieurs greffes, si bien que si une d’entre elles est rejetée, la cica- trisation et l’épidermisation peut être assurée par les autres si elles réussissent. Enfin, si l’ul- cère est fistulisé dû à un drainage, celui-ci peut s’effectuer entre les greffes, évitant ainsi à toute l’aire greffée d’être rejetée. Le seul désavantage à la greffe par pincement est l’aspect cosmétique final, qui n’est pas toujours très plaisant. Nous avons eu de très bons résultats chez 4/4 patients qui ont subi cette intervention à notre institution. La procédure et le résultat final sont présentés en détail dans cet article. RESUMEN El tratamiento de las úlceras leprosas se ha mantenido inadecuado debido a que la may- oría de éstas todavía se manejan de manera conservadora especialmente en los países en vías de desarrollo, probablemente por dificultades económicas. El procedimiento de injerto de varios fragmentos de piel aunque obsoleto ahora (2), trata de cubrir este hueco entre costo y efectividad. Se trata de una técnica simple que no requiere mucha experiencia o inversión, y que puede hacerse casi en cualquier área adaptada del consultorio (3). El procedimiento de injertos múltiples también tiene la ventaja sobre la aplicación de injertos únicos, de que aun cuando uno de estos injertos se rechace, todavía quedan otros injertos que generalmente sanan exitosamente y epidermizan la zona a su alrededor. Además, si la úlcera está drenando, la descarga fluye entre los injertos, evitando así que estos sean rechazados. La única desventaja de la técnica de injertos múltiples de piel es la apariencia cosmética final, la cual puede ser no muy agradable. Nosotros hemos tenido muy buenos resultados en 4 pacientes sometidos a este procedimiento en nuestra institución. El procedimiento y el resultado final se describen en este artículo.

1 Received for publication on 19 September 2003. Accepted for publication on 14 January 2004. 2 E. Jayaseelan, DNB; and V. V. Aithal, DVD, DNB, Department of Dermatology, St. John’s Medical College and Hospital, Bangalore, India. Reprint requests to: Dr. Vijay V. Aithal, Lecturer, Department of Dermatology, St. John’s Medical College and Hospital, Sarjapur Road, Bangalore-560 034, India. E-mail : [email protected], or vijay_aithal@rediffmail .com

139 140 International Journal of Leprosy 2004

Non-healing ulcers resulting from lep- The donor area was prepared by cleaning rosy pose a great threat not only due to their the skin with Povidone-Iodine solution disability, but also their morbidity. A simple (Betadine®) and surgical spirit. The lateral technique like pinch grafting would reduce aspect of the mid-thigh was selected as the this morbidity. As compared to single sheet donor area, although abdomen and buttocks full thickness skin grafts, pinch grafts are have also been used as donor sites (5). A easy, inexpensive, and can be done in a mi- field block was given by injecting Ligno- nor operating theater, or as an office-based caine 2%, so as to cover the entire donor procedure. area, an area approximately 5 cm × 5 cm for Most pinch grafts have been described as all 10 grafts. split-thickness grafts (2), but they are full- Although various techniques have been thickness centrally and thin split-thickness described for taking pinch grafts (1, 5),we at the periphery (3). The proceedure in- recommend the shave technique using a hy- volves placing small slices of split skin over podermic needle (Fig. 2), which has been the dermabraded ulcer site, and allowing it described earlier, though some authors con- to heal by epithelialization. sider it obsolete (4). A 23 or 24 gauge needle was inserted into the skin at the donor area, MATERIALS AND METHODS and brought out 1 cm away, so that the skin This technique was done on 4 leprosy pa- taken would involve an area of 1cm2. The tients with non-healing ulcers, located at needle was pushed at a plane, as superfi- various sites and of differing duration (The cially as possible. The skin was then sliced Table). All 4 were selected from the leprosy into a thin layer using a scalpel, just below rehabilitation unit of the Dermatology de- the needle, at the approximate level of the partment at St. John’s Medical College papillary dermis. This not only ensures Hospital, Bangalore, India. minimal blood loss, but also enhances the Inclusion criteria: (i) Chronic non- chance of healing by way of epithelializa- healing ulcers due to leprosy (of more than tion. The grafts were then placed in a sterile 2 months duration); (ii) A clean ulcer bed container containing normal saline and a with healthy granulation tissue and good guaze pad. Adequate numbers of grafts vascularity. were taken so as to cover the entire ulcer. Exclusion criteria: (i) Systemic diseases The donor area was then closed with an an- not under control, such as uncontrolled ane- tibiotic-impregnated pre-sterilized gauze mia and diabetes mellitus; (ii) Ulcers not tulle (Sofratulle®), gauze pads, and an adhe- primarily due to leprosy. sive elastic plaster (Dynaplast®). Serous discharge, though copious, was The recipient area, usually anesthetic, not a contraindication. Also, those with a did not require local anesthetic infiltration. hyperkeratotic edge were taken up, after The ulcer bed was dermabraded, using a saucerizing the edge, to increase vascularity manual dermabrader. Once the area started (Fig. 1) bleeding, the grafts were placed over the re-

FIG. 1. Clean ulcer without secondary infection, FIG. 2. Shave technique of harvesting the pinch ready to be grafted. graft donor site. 72, 2 Jayaseelan and Aithal: Pinch Skin Grafting for Ulcers 141

FIG. 3. Grafts placed in the recipient ulcer site. FIG. 4. Two weeks post-surgery, inter-graft epithelialization was completed. cipient site, making sure the dermal side site, a course of antibiotics, analgesics, sup- faced downwards. The grafts were placed plementary zinc, and vitamins. as close to one another as possible (Fig. 3). After a week the dressing was removed, After the whole area was covered, it was and the grafts looked pinkish-yellow and sod- closed with an antibiotic-impregnated pre- den due to the collection of serous discharge sterilized gauze tulle (Sofratulle®), gauze underneath the dressing. It took another week pads, and an adhesive elastic plaster (Dy- or two for inter-space epithelialization to be naplast®). Joint areas and areas with a high completed (Fig. 4). After 4 to 6 weeks, the degree of mobility were immobilized in a whole ulcer area was covered by skin. splint. The dressing was kept for a week, with a change in dressing once every 3 to 4 RESULTS days. Postoperative care included immobi- In all 4 patients, the ulcers healed com- lization, avoidance of wetting the grafted pletely (The Table and Fig. 4). Two of the

THE TABLE. Details of patients who underwent Pinch skin grafting.

Site & Duration Patient Age/ Duration area of of Treatment Time for Remarks No. Sex of ulcer the ulcer leprosy received healing 1 42 year 2 months Lt. lat. 18 years Monotherapy- 1 month Immediate response F maleolus, Dapsone for 2 years not very good. 3 cm × 3 cm Antibiotics, Gradual healing in analgesic for 1 month. the ulcer. 1 2 49 year 6 months Lt. shin. 10 cm 20 years No treatment for 1 ⁄2 Osteomyelitis M × 5 cm Leprosy. months (clinically, Antibiotics, radiologically, and analgesics for culture-proved), the ulcer. treated adequately. Anemia corrected. Gradual healing in 1 1 ⁄2 months. 3 38 year 1 year Rt. ankle antr. 6 years MDT(?)-2 years. No 15 days Gradual healing M3 cm × 5 cm treatment for ulcer in 15 days 4 40 year 1 year Rt. knee antr. 10 years MDT-for 2 years. 15 days Immobilized the M4 cm × 4 cm Antibiotics, recipient area in a analgesics for Bohler’s iron the ulcer. splint. Gradual healing in 15 days. 142 International Journal of Leprosy 2004 patients had ulcers over difﬁcult sites, viz. technique can be done by anyone, with little prepatellar and anterior to the ankle. One of training and experience. (iv) The set-up re- the patients who had underlying osteo- quired is very simple and inexpensive. myelitis proven clinically and radiologi- Disadvantages (5). The only disadvan- cally, and culture-positive for aerobic and tage is that it results in cobbling, scarring, anaerobic micro-organisms, also did well and depigmentation (1). under cover of appropriate antibiotics for the osteomyelitis. CONCLUSION Pinch skin grafting is a safe, simple, and DISCUSSION inexpensive technique, which can be easily Pinch skin grafting for non-healing ulcers mastered, with minimal training and experi- is a simple procedure, which can be done ence. We recommend it to be used in all un- by any doctor with minimal training (1). To complicated leprous ulcers, which cannot the best of our knowledge, this technique be brought under control with drugs and has not been previously reported for leprous dressing alone. In the future, we plan to ex- ulcers. Single sheet split-thickness skin tend this study to include pinch grafting on grafts have been described for the treatment plantar ulcers. of non-healing ulcers; however, pinch grafting seems to have certain advantages over REFERENCES single sheet grafting. 1. HILL, T. G. Skin grafts. In: Cutaneous Surgery, 1st Advantages of pinch skin graft over edn. Philadelphia: W. B. Saunders Company, 1987. single sheet skin graft (split or full thick- pp. 587–589. 1 ness) ( ). (i) Pinch grafting can be done in 2. KRIZEK, T. J. Grafts and ﬂaps. In: Plastic Surgery: ulcers having unfavorable local factors, A core curriculum. Missouri: Mosby Year Book, such as those with copious serous dis- Inc., 1994. pp. 36–64. charge. If a single sheet of skin is placed in 3. SAVANT, S. S. Pinch skin grafting for non healing such a situation, the whole graft tends to get ulcers. In: Textbook and Atlas of Dermatosurgery thrown off, due to collection of the dis- and Cosmetology, 1st edn. Mumbai: A.S.C.A.D., charge underneath. In contrast, in pinch 1998. pp. 252–254. 4. STEGMAN, S. J., TROMOCITCH, T. A., and GLOGAN, grafts the discharge is allowed to drain from R. G. Basics of Dermatologic Surgery. Chicago: the inter-space. (ii) Even if one graft gets Year book Medical, 1982. pp. 105–106. rejected, the other grafts would still take up 5. WHEELAND, R. G. The technique and current status and the rejected area would epithelialize of pinch grafting. J. Dermat. Surg. Oncol. 13 from the adjacent grafts. (iii) This simple (1987) 873–880. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) Single Nucleotide Polymorphisms (SNPs) at -238 and -308 Positions in the TNFα Promoter: Clinical and Bacteriological Evaluation in Leprosy1

Patrícia R. Vanderborght, Haroldo J. Matos, Ana M. Salles, Sidra E. Vasconcellos, Valcemir F. Silva-Filho, Tom W. J. Huizinga, Tom H. M. Ottenhoff, Elisabeth P. Sampaio, Euzenir N. Sarno, Adalberto R. Santos, and Milton O. Moraes2 ABSTRACT Tumor necrosis factor alpha (TNFα) plays a key role in orchestrating the complex events involved in inflammation and immune response. The presence of single nucleotide polymorphisms (SNPs) within the promoter region of the TNFa gene has been associated with a number of diseases. The aim of this study was to investigate the distribution of polymorphisms at positions -238 (G/A) and -308 (G/A) at the TNFα promoter, and its association to the outcome of different clinical forms of leprosy. Furthermore, the bacteriological index (BI) was evaluated among genotyped multibacillary (MB) patients in order to investigate the possible influence of each polymorphism on the bacterial load. This study included a total of 631 leprosy patients being 401 MB and 230 paucibacillary (PB), that was further separated according to its ethnicity (Afro- and Euro-Brazilians). The combination of SNPs in haplotypes generated three different arrangements: TNFG-G, TNFG-A and TNFA-G. In spite of the marked differences observed in the frequency of the haplotypes along the ethnic groups, no statistical differences were observed in haplotype frequencies between MB and PB patients. The BI analyses showed a lower bacteriological index among the -308 carriers, while the BI of the -238 carriers was higher. Although no significance has been achieved in this analysis regarding the influence of the polymorphisms to the development of the clinical outcome, it seems that in a different stage (among the MB patients) the polymorphisms could contribute to the degree of severity observed. RÉSUMÉ Le facteur alpha de nécrose tumorale (TNFα) joue un rôle important dans l’ajustement des évènements complexes qui régulent l’inflammation et la réponse immunitaire. La présence de polymorphismes mono-nucléotidiques (SNPs) au sein de la région promotrice du gène codant pour TNFα a été associée à un certain nombre de maladies. Le but de cet article était d’explorer la distribution de polymorphismes aux positions -238 (G/A) et –308 (G/A) du promoteur de TNFα et son association aux résultats phénotypiques des différentes formes de lèpre. De plus, l’index bactérioscopique (IB) a été évalué parmi les patients multibacillaires (MB) génotypés dans le but d’évaluer la possible influence de chaque polymor- phisme sur la charge bactérienne. Cette étude a porté sur 631 lépreux comportant 401 MB et 230 PB, qui furent encore séparés par ethnie (Afro et Euro-brésiliens). La combinaison des SNPs en haplotypes a généré 3 arrangements différents : TNF-G, TNF-A et TNFA-G. En dépit de différences marquées observées dans les fréquences haplotypiques entre les groupes ethniques, aucune différence statistiquement significative ne fut observée entre les patients MB et PB. Les analyses de IB ont montré un index bactérioscopique plus faible parmi les porteurs –308, tandis que le IB des porteurs -238 était plus élevé. Bien que cette analyse de polymorphismes n’ait pas démontré de différence significative sur l’issue clinique de la

1 Received for publication on 19 November 2002. Accepted for publication on 13 January 2004. 2 P. R. Vanderborght, M.Sci., Leprosy Laboratory, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil; H. J. Matos, Ph.D., Informatica Medica, Faculty of Medical Science, State University of Rio de Janeiro, Brazil; A. M. Salles, M. D.; S. E. Vasconcellos, B.Sci; and Valcemir F. Silva-Filho, B.Sci., Leprosy Laboratory, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil; T. W. J. Huizinga, Ph.D.; and Tom H. M. Ottenhoff, Ph.D., De- partment of Rheumatology and Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Netherlands; E. P. Sampaio, M.D.; E. N. Sarno, M.D.; A. R. Santos, Ph.D.; and Milton O. Moraes, Ph.D., Leprosy Laboratory, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil. Reprint request to: Dr. Adalberto Rezende Santos, Leprosy Laboratory, Oswaldo Cruz Institute, FIOCRUZ, Av. Brasil 4365 Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil. E-mail: [email protected] 143 144 International Journal of Leprosy 2004

lèpre, il semble qu’à un certain stade de la maladie des patients multibacillaires, ces polymorphismes pourraient contribuer au degré de sévérité observé. RESUMEN El factor de necrosis tumoral alfa (TNFα) juega un papel importante en la orquestación de los complejos eventos que ocurren en la inflamación y la respuesta inmunitaria. La presencia de polimorfismos simples en polinucleótidos (SNPs) dentro de la región promotora del gene TNFα ha sido asociada a numerosas enfermedades. El objetivo del estudio fue el investigar la distribución de los polimorfismos en las posiciones -238 (G/A) y -308 (G/A) del promotor del TNFα, y su asociación con las diferentes formas clínicas de la lepra. Se evaluó, además, el índice bacteriológico entre los pacientes MB estudiados para poder investigar la posible influencia de cada polimorfismo sobre la carga bacilar. En el estudio se incluyeron 631 pacientes con lepra (401 MB y 230 PB) los cuales fueron adicionalmente separados de acuerdo a su etnicidad como Afro- y Euro-brasileños. La combinación de SNPs en los haplotipos generó 3 diferentes arreglos: TNFG-G, TNFG-A y TNFA-G. No obstante las mar- cadas diferencias en la frecuencia de los haplotipos entre los grupos étnicos, no se observaron diferencias estadísticas en las frecuencias de los haplotipos entre los pacientes MB y PB. Los análisis mostraron un menor índice bacteriológico entre los portadores-308 que entre los portadores-238 donde el IB fue mayor. Aunque en este análisis sobre la relación entre los polimorfismos y el tipo clínico de la enfermedad no se llegó a observar significancia estadística, parecería ser que al menos entre los pacientes MB los polimorfismos pueden contribuir al grado de severidad observado.

Leprosy, a chronic human disease, is the in an Indian population (21). However, in result of infection by Mycobacterium lep- Brazilian leprosy patients the association of rae. The clinical spectrum of the disease in- this polymorphism with protection has been cludes two polar (lepromatous-LL and described (23, 24, 27). A plausible explanation tuberculoid-TT) and three borderline forms for these divergent findings may involve the (tuberculoid-BT, borderline-BB, and lepro- ethnicity, where the allelic frequency of matous-BL) (19). The multibacillary (MB) TNFα -308A in leprosy patients range from form, including LL, BB, and BL, is charac- 7.0 to 10.8 in Indian and Brazilian, respec- terized by low immune responsiveness and tively (21, 24). This allelic frequency varia- high bacterial load. The paucibacillary (PB) tion is wide among populations such as form, including TT and BT, is marked by Caucasian Irish (23.0), African Zulu (22.1), strong cell-mediated immunity against the Arabian Omani (8.1), Singapore Chinese bacillus (22). Much evidence has implicated (12.0) and Mexican Mestizos (2.5) (13). cytokines in the immune response of lep- Studies at another G/A transition polymor- rosy (16), mainly the tumor necrosis factor- phism (-238) in the TNTα promoter region alpha ( TNFα), which has a beneficial func- (4) has shown that the A allele was in- tion in host defense but, if produced in high creased among patients with chronic hepati- levels, contributes to tissue damage (25). tis B and C, suggesting association to dis- The presence of single nucleotide poly- ease susceptibility (7, 8) while in cancer a morphisms (SNPs) in the TNFα promoter protective effect was observed (9). In fact, region and their association with autoim- the complex relationship between SNPs in mune and infectious diseases has been ex- the human genome and disease association tensively studied (30). The polymorphism indicates the need for the construction of detected at position -308 (G/A) within the haplotypes (specific combination of SNPs promoter region of the TNFα gene (29) was on the same chromosome) on the locus the first one found to be associated with dis- studied because they are more informative ease (18). This polymorphism has been ob- than any single SNP (1, 26). served in association with several other in- In the recent analyses from Brazilian lep- fectious diseases where excessive TNFα rosy patients we have shown in paucibacil- production seems to play a role, such as in lary forms an increased allelic frequency of cerebral malaria (12) and mucocutaneous the TNF-308A in comparison to the multi- leishmaniasis (2). An association of the -308 bacillary (0.14 and 0.09, respectively) with polymorphism with the development of lep- a borderline significance (χ2 = 3.47; p = romatous leprosy was previously reported 0.06) (24). This data did not define if TNF 72, 2 Vanderborght, et al.: SNP’s in the TNFα Promoter 145

-308A was a trend marker for protection, reaction (PCR)-restriction fragment length i.e., allele frequency in control > PB > MB polymorphism (RFLP), a single PCR step or whether TNF-308A discriminates be- and further restriction analysis (6, 29). Re- tween cases and controls only, being a restricted amplified products were visualized sistance locus of leprosy per se. Thus, the by electrophoresis in 3% agarose gel and aim of this study was to describe whether ethidium bromide staining. there was a difference between PB and MB Statistical analysis. The statistical sig- using an increased number of patients. To nificance of TNFα promoter polymorphism overcome a possible cryptic stratification distributions was analyzed by way of the χ2 that would impact the SNP frequency and test Odds-ratio (OR) and 95% confidence mask an association effect, patients were intervals (CI). Yates’ correction or Fisher’s separated according to ethnicity. Besides, exact test was used when appropriate (Epi the -308 and -238 SNPs were combined in Info 6: CDC, Atlanta, GA). The signifi- haplotypes that better analyze the TNF pro- cance level adopted was p <0.05. The hap- moter region. In addition, to understand the lotype frequencies were estimated using the impact of polymorphisms of TNFα pro- EH program (28). moter in relation to progression of the dis- Generalized additive model. the vari- ease, the comparison between SNPs in -308 ability of the bacteriological index (BI) in and -238 position and the bacteriological relation of the presence of the mutation in index (BI) was set out in MB patients. the TNFα promoter was studied through a generalized additive model (GAM). The MATERIALS AND METHODS GAM is a special regression model, in Patients. Six hundred and thirty one lep- which some of regression assumptions are rosy patients from the Leprosy Out-Patient relaxed. So, GAM models may have advan- Unit, Oswaldo Cruz Foundation (Rio de tages in many biological phenomena, and Janeiro, Brazil) were included in this study. may replace traditional linear or logistic re- They were diagnosed on the basis of clini- gression models. In mathematical terms, cal and bacteriological criteria and classi- GAM models allow predictors (or covari- fied according to the Ridley and Jopling ates) to be replaced by arbitrary smooth Scale (20). Four hundred one MB and 230 functions. For instance, B-splines (poly- PB patients were studied, including 405 nomials that can be adjusted to a set of males and 226 females. Brazilians were points) could be used as a smoothing func- classified according to their ethnic origin tion. In this context, a GAM model was after careful inspection of facial morpholog- used to study the association between the ical features, hair type and skin color. Two occurrence of the G/A substitution in the groups were ascertained: Afro-Brazilians positions -308 and -238 of the promoter and Euro-Brazilians with N = 251 and N = gene of TNFα, and the baciloscopic index, 235, respectively. Asians and Amerindians calculated as previously described. For the are not commonly represented in the popu- adjustment of the model, one should indi- lation of Rio de Janeiro and were not ob- cate the number of nodes necessary. A node served among the individuals inspected. is the number of points used for the curve to Bacteriological index determination. be adjusted. The SPLUS 2000 program, Bacteriological index (BI) was determined Professional Release 3, MathSoft, Inc. was according to Ridley, 1964 (19) among the used to perform these analyses. multibacillary patients in slit skin smears from six different anatomic sites and ranged RESULTS from 0.16 to 5.33 (mean = 2.50 ± 1.46). Analyses of TNFα haplotypes frequen- DNA extraction and SNPs genotyping. cies in leprosy patients in different Genomic DNA was prepared from frozen Brazilian ethnic groups. Haplotypes of whole blood collected with sodium citrate TNFα were estimated using a maximum- buffer by a commercially available DNAzol likelihood probability test from -308 and extraction kit (Invitrogen Life Technolo- -238 SNP genotypes of unrelated patients gies, Gaithersburg, MD, USA). Genotyping stratified in Euro- and Afro-Brazilian (The of the TNFα promoter region for analysis Table). Three differents haplotypes on the of polymorphisms at the -238 and -308 po- TNFα gene have been identified (TNFG-G, sitions was performed by polymerase chain TNFA-G, TNFG-A). No statistical differ- 146 International Journal of Leprosy 2004

THE TABLE. Haplotypes frequencies of the TNFα promoter polymorphisms among MB and PB leprosy patients within distinct ethnic groups.

Haplotypes Brazilian Euro-Brazilian Afro-Brazilian -308/-238 MB PB MB PB MB PB G-G 0.83 0.81 0.82 0.78 0.84 0.86 G-A 0.05 0.05 0.05 0.04 0.04 0.04 A-G 0.10 0.12 0.12 0.16 0.10 0.08 ences were observed in haplotypes from since they can be used as genotypic markers TNFα promoter between MB and PB pa- of specific disease phenotypes or can regu- tients irrespective to the ethnic group ana- late biological phenomena influencing lyzed. The TNFG-G haplotype in both PB mRNA expression, thereby altering mRNA and MB forms presented the highest fre- isoforms (unravelling cryptic splicing sites) quency among patients in both ethnic or modifying enzymatic activity of genes groups. Marked differences between Euro- (11). The problem is that SNP frequencies and Afro-Brazilians were observed when vary enormously among populations (13), other haplotypes were analyzed. Among PB especially Brazilians who originated from a patients, an increased frequency of the variety of ethnicities, mainly Portuguese TNFA-G haplotype in Euro-Brazilian was explorers mixed with native Amerindians detected in comparison to Afro-Brazilian and Africans (3). The outcome of this ad- (0.16 and 0.08, respectively) was detected. mixtured colonization is a dense population Analyses of the bacteriological index without a clear genetic/morphological eth- (BI) from multibacillary patients geno- nic cut off (17). However, some cryptic strat- typed for SNPs at position -308 and -238. ification may still be functioning as con- Bacteriological index (BI) variability in re- founding factors in Brazilians in popula- lation to the presence of the -238 (N = 343) tion-based studies. Indeed, the separation and -308 (N = 341) polymorphisms was an- according to the morphological features of alyzed via the GAM (Fig. 1A, B). The GAM the patients better discriminate Afro- and regression model was performed to study the Euro-Brazilians, where a difference in the association between the presence of the A -308A/-238G haplotype frequency from allele at the positions -238 and -308 of the Afro-Brazilian (9%) to Euro-Brazilian (13%) promoter gene of TNFα, as the dependent patients was observed. Still, no statistical variable, and the variability of the bacilo- differences were observed when PB and scopic index, as a predictor variable. For MB were compared, demonstrating that if the model using the substitution at position there is some cryptic stratification due to -238, a regression polynomial spline with admixture, it is not being detected by con- 1,5 nodes was used. It was observed that ventional morphological inspection in our the probability for the occurrence of the A patient population. Thus, to scrutinize the allele at the -238 position increases with stratification in Brazilian population-based higher bacteriological index (Fig. 1A). studies it would be necessary to use ge- For the model using the substitution at po- nomic controls (5). Moreover, a recent study sition -308, a regression polynomial spline performed in Gambian and Malawian with 1,1 nodes was used. In contrast to the populations studying SNPs spanning 4.4kb result obtained with -238, the probability for of the TNFα /LTα locus demonstrated the the occurence of the A allele at the -308 po- need to Type 8 in Gambians, and 7 out 12 sistion is greater with a low BI (Fig. 1B). SNPs in Malawians, to detect the haplotypic structure and informative SNPs in this DISCUSSION region due to the high frequency of recom- Studies using the frequency of single nu- bination (1). We do not have data for the cleotide polymorphisms in candidate genes Brazilians but it seems to be necessary to are interesting approaches to the investiga- enlarge the focused region of the TNF locus tion of the susceptibility and severity of dis- to capture more information about severity eases. It is believed that SNPs are relevant in leprosy. 72, 2 Vanderborght, et al.: SNP’s in the TNFα Promoter 147

data is in accordance with the literature, where it has been demonstrated that the -238A polymorphism is associated with lower levels of TNFα (10). The possibility of using slit skin smears is one of the few alternatives for in vivo determination of the bacterial load. The BI is one of the clinical parameters indicating disease progression and severity, representing a clear risk factor for the development of the acute inﬂammatory episodes in leprosy (14). Thus, by way of the adjusted model (GAM), the existence of a clinical signiﬁcance for the variability of BI in relation to the presence of polymorphisms in the TNFα promoter suggests a functional dichotomy between the -308 and -238 SNPs in relation to TNF regulation and leprosy progression.

Acknowledgment. We are grateful to the attending physicians at the Leprosy Out-Patient Unit, FIOCRUZ, to Edson Albuquerque, for his technical assistance, and to Judy Grevan for the English revision. Patrícia Van- derborght is supported by a grant from CAPES (Coor- denação de Aperfeiçoamento de Pessoal de Nível Su- perior), Brazil. This study was financially supported by the World Health Organization/World Bank/TDR, ID 930063, by the EU (INCO-DC), grant number ERBIC FIG. 1. Analyses of the bacteriological index (BI) 18CT 980377, and by The Netherlands Leprosy Foun- variability of TNFα promoter polymorphism -238 (a) dation (NLR). and -308 (b) by Generalized Additive Model (GAM) in 341 and 343 multibacillary genotyped patients, respectively. The “y” axis means the probability of “A” REFERENCES allele occurrence (homozygous or heterozygous) and 1. ACKERMAN, H., USEN, S., MOTT, R., RICHARDSON, BI at “x” axis, range from 0.16 to 5.33. Each indi- A., SISAY-JOOF, F., KATUNDU, P., TAYLOR, T., vidual BI is represented as a small bar. WARD, R., MOLYNEUX, M., PINDER, M., and KWIATKOWSKI, D. P. Haplotypic analysis of the TNF locus by association efficiency and entropy. On the other hand, the analysis of BI and Genome Biol. 4 (2003) R24. its association with polymorphisms at po- 2. CABRERA, M., SHAW, M. A., SHARPLES, C., sitions -308 and -238 in TNFα suggested WILLIAMS, H., CASTES, M., CONVIT, J., and BLACK- these polymorphisms are functionally rele- WELL, J. M. Polymorphism in tumor necrosis factor vant. We previously demonstrated that genes associated with mucocutaneous leishmania- α sis. J. Exp. Med. 182 (1995) 1259–1264. TNF -308A was associated with a 3. CARVALHO-SILVA, D. R., SANTOS, F. R., ROCHA, J., 15 stronger response in Mitsuda reaction ( ). and PENA, S. D. The phylogeography of Brazilian In this study, GAM analyses in -308A, re- Y-chromosome lineages. Am. J. Hum. Genet. 68 vealed that such patients have lower BIs. (2001) 281–286. The results of our previous findings (15) 4. D’ALFONSO, S., and RICHIARDI, P. M. A polymor- with this new data is that TNF -308A could phic variation in a putative regulation box of the be upregulating the secretion of TNFα that, TNFA promoter region. Immunogenetics 39 in turn, induces a stronger DTH skin re- (1994) 150–154. sponse in paucibacillary patients and restricts 5. DEVLIN B., and ROEDER, K. Genomic control for 4 M. leprae growth in multibacillary patients. association studies. Biometrics. (1999) 997–1004. 6. GALLAGHER, G., ESKDALE, J., OH, H. H., RICHARDS, The opposite was verified concerning the S. D., CAMPBELL, D. A., and FIELD, M. Polymor- study of -238A and BIs. In this case, the pres- phisms in the TNF gene cluster and MHC ence of the A allele was more frequent in serotypes in the West of Scotland. Immunogenet- multibacillary patients with higher BIs. This ics 45 (1997) 188–194. 148 International Journal of Leprosy 2004

7. HÖHLER, T., KRUGER, A., GERKEN, G., SCHNEIDER, P. necrosis factor-alpha promoter region polymor- M., BÜSCHENFELDE, K.-H., and RITTNER, C. phism and insulin-dependent diabetes mellitus. A tumor necrosis factor-alpha (TNFα) promoter Eur. J. Immunol. 23 (1993) 3050–3053. polymorphism is associated with chronic hepatitis B 19. RIDLEY, D. S. Bacterial indices. In: Leprosy in infection. Clin. Exp. Immunol. 111 (1998) 579–582. Theory and Practice. 2nd edn. Bristol: John 8. HÖHLER, T., KRUGER, A., GERKEN, G., SCHNEIDER, Wright, 1964, pp. 620–621. P. M., BÜSCHENFELDE, K.-H., and RITTNER, C. Tu- 20. RIDLEY, D. S., and JOPLING, W. H. Classification mor necrosis factor alpha promoter polymorphism of leprosy according to immunity: a five-group at position -238 is associated with chronic active system. Int. J. Lepr. 34 (1966) 255–273. hepatitis C infection. J. Med. Virol. 54 (1998) 21. ROY, S., MCGUIRE, W., MASCIE TAYLOR, C. G., 173–177. SAHA, B., HAZRA, S. K., HILL, A. V., and 9. JANG, W. H., YANG, Y. I., YEA, S. S., LEE, Y. J., KWIATKOWSKI, D. Tumor necrosis factor promoter CHUN, J. H., KIM, H. I., KIM, M. S., and PAIK, K. polymorphism and susceptibility to lepromatous H. The -238 tumor necrosis factor-alpha promoter leprosy. J. Infect. Dis. 176 (1997) 530–532. polymorphism is associated with decreased sus- 22. SAMPAIO, E. P, MORAES, M. O., PESSOLANI, M. C. V., ceptibility to cancers. Cancer Lett. 166 (2001) and SARNO, E. N. Role of Th1 host defenses against 41–46. Mycobacterium leprae. Cytokines and Chemokines. 10. KALUZA, W., REUSS, E., GROSSMANN, S., HUG, R., In: Infectious Diseases Handbook 1st edn. New SCHOPF, R. E., GALLE, P. R., MAERKER-HERMANN, Jersey: Humana Press, 2003, pp. 163–188. E., and HOEHLER, T. Different transcriptional ac- 23. SANTOS, A. R., ALMEIDA, A. S., SUFFYS, P. N., tivity and in vitro TNF-alpha production in psori- MORAES, M. O., FILHO, V. F., MATTOS, H. J., NERY, asis patients carrying the TNF-alpha 238A pro- J. A., CABELLO, P. H., SAMPAIO, E. P., and SARNO, moter polymorphism. J. Invest. Dermatol. 114 E. N. Tumor necrosis factor promoter polymor- (2000) 1180–1183. phism (TNF2) seems to protect against develop- 11. LAI, E. Application of SNP technologies in medi- ment of severe forms of leprosy in a pilot study in cine: lessons learned and future challenges. Brazilian patients. Int. J. Lepr. Other Mycobact. Genome Res. 11 (2001) 927–929. Dis. 68 (2000) 325–327. 12. MCGUIRE, W., HILL, A. V. S., ALLSOPP, C. E. M., 24. SANTOS, A. R., SUFFYS, P. N., VANDERBORGHT, P. R., GREENWOOD, B. M., and KWIATKOWSKI, K. Varia- MORAES, M. O., VIEIRA, L. M., CABELLO, P. H., tion in the TNFα promoter region associated with BAKKER, A. M., MATOS, H. J., HUIZINGA, T. W., OT- susceptibility to cerebral malaria. Nature 371 TENHOFF, T. H., SAMPAIO, E. P., and SARNO, E. N. (1994) 508–511. Role of tumor necrosis factor-alpha and interleukin- 13. MEENAGH, A., WILLIAMS, F., ROSS, O. A., PATTER- 10 promotor gene polymorphisms in leprosy. 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J., RAMOS, alpha promoter polymorphism TNF2 is associated F., SILVEIRA, F., and BLACKWELL, J. M. Associa- with a stronger delayed-type hypersensitivity reaction and linkage of leprosy phenotypes with HLA tion in the skin of borderline tuberculoid leprosy class II and tumour necrosis factor genes. Genes patients. Immunogenetics 53 (2001) 45–47. Immun. 2 (2001) 196–204. 16. MORAES, M. O., SARNO, E. N., ALMEIDA, A. S., 28. TERWILLIGER, J. D., and OTT, J. Handbook of Hu- SARAIVA, B. C., NERY, J. A., MARTINS, R. C., and man Genetic Linkage. Baltimore and London: The SAMPAIO, E. P. Cytokine mRNA expression in Johns Hopkins University Press, 1994, p. 307. leprosy: a possible role for interferon-gamma and 29. WILSON, A. G., DI GIOVINE, F. S, BLAKEMORE, A. I. interleukin-12 in reactions (RR and ENL). Scand. F., and DUFF, G. W. Single base polymorphism in J. Immunol. 50 (1999) 541–549. the human tumor necrosis factor (TNF) alpha gene 17. PARRA, F. C., AMADO, R. C., LAMBERTUCCI, J. R., detectable by NcoI restriction of PCR product. ROCHA, J., ANTUNES, C. M., and PENA, S. D. Hum. Mol. Gen. 1 (1992) 353. Color and genomic ancestry in Brazilians. Proc. 30. WILSON, A. G., DI GIOVINE, F. S., and DUFF, G. W. Natl. Acad. Sci. U.S.A. 100 (2003) 177–182. Genetics of tumor necrosis factor-α in autoim- 18. POCIOT, F., WILSON, A. G., NERUP, J., and DUFF, G. mune, infectious, and neoplastic diseases. J. In- W. No independent association between a tumor flam. 45 (1995) 1–12. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) Leprosy Reactions: Humoral and Cellular Immune Responses to M. leprae, 65kDa, 28kDa, and 18 kDa Antigens1

Keshar K. Mohanty, Beenu Joshi, Kiran Katoch, and Utpal Sengupta2 ABSTRACT This study examines the immune responses against some stress proteins of Mycobacte- rium leprae in leprosy patients with and without leprosy reactions. Leprosy patients showed a higher level of antibodies to all antigens compared to healthy controls. The antibody response to 18kDa antigen was significantly higher in patients with Type 1 reaction compared to those of TT or borderline patients without Type 1 reaction, or those with Type 2 reaction. Borderline (BT/BL), lepromatous (LL) and patients with reactions (Type 1 and Type 2) had higher levels of antibodies to M. leprae soluble extract (MLSE) and 65kDa than those of the tuberculoid (TT) group. LL, borderline patients, and patients with Type 1 reaction had a higher level of antibody to 28kDa than those of healthy controls. However, no significant differences could be observed in antibody response to these antigens (MLSE, 65kDa, and 28kDa) between patients with reaction and without reaction. A significant proportion of TT/BT patients showed positive lymphoproliferative response to MLSE compared to BL/LL patients. In addition, the lymphoproliferative response to MLSE was significantly greater in patients with Type 1 reaction compared to patients without reaction. No difference in proliferative response to 65kDa could be observed in any of these groups. The finding of high levels of antibodies against stress proteins in patients with Type 1 reactions, especially to 18 kDa antigen, along with a heightened lymphoproliferative response to MLSE is suggestive of a coexistence of cell mediated and humoral immunity in leprosy patients during Type 1 reactions. On the other hand, in Type 2 reactions no significant role of stress proteins could be demonstrated except a heightened lymphoproliferative response to the 28 kDa antigen. RÉSUMÉ Cette étude présente les réponses immunitaires chez les patients hanséniens et les patients souffrant de réactions, contre les protéines de stress de Mycobacterium leprae. Les patients hanséniens ont montré de plus haut niveaux d’anticorps dirigés contre tous les antigènes que les personnes témoins en bonne santé. La réponse sérique dirigée contre l’antigène de 18kDa était significativement plus élevée chez les patients souffrant de réaction de type 1 comparée à celles des patients TT ou borderline, ou celle des patients avec réaction de type 2. De plus, un plus grand pourcentage de patients avec réaction reverse avaient une réponse détectable pour cet anticorps, comparé à celui des patients sans réaction. Les patients borderline (BT/BL), lépromateux (LL) et les patients avec réactions (type 1 et type 2) présentaient de plus hauts niveaux d’anticorps dirigés contre l’extrait soluble de M. leprae (MLSE) et la protéine 65kDa que les patients tuberculoïdes (TT). Les patients LL, borderline et les patients présentant une réaction de type 1 présentaient de plus haut niveaux d’anticorps contre la protéine 28kDa par rapport aux témoins en bonne santé. Cependant, aucune différence significative ne fut observée entre les patients avec et sans réaction dans la réponse sérique contre les antigènes MLSE, 65kDa et 28kDa. Comparé aux patients BL/LL, une proportion significative de patients TT/BT montraient une réponse lymphoproliférative positive contre MLSE. De plus, comparé aux patients sans réaction en cours, les patients de type 1 montraient une réponse lymphoproliférative plus élevée contre MLSE. Aucune différence de réponse proliférative contre 65kDa ne fut observée entre les groupes. La mise en évidence de hauts niveaux d’anticorps dirigés contre les protéines de stress de M. leprae chez les pa-

1 Received for publication on 17 October 2003. Accepted for publication on 24 February 2004. 2 K. K. Mohanty, Ph.D.; Beenu Joshi, Ph.D.; Kiran Katoch, M.D., Clinical Divison; and Utpal Sengupta, Ph.D., Department of Immunology, Central JALMA Institute for Leprosy, Tajganj, Agra 282 001 (India). Reprint requests to: Utpal Sengupta, Department of Immunology, Central JALMA Institute for Leprosy, Taj- ganj, Agra 282 001 (India).

149 150 International Journal of Leprosy 2004

tients souffrant de réactions de type 1, en particuliers contre l’antigène 18kDa, accompag- nant une réponse lymphoproliférative élevée contre MLSE, suggère qu’une immunité à mé- diation cellulaire et une immunité à médiation humorale coexistent chez les patients han- séniens pendant les réactions de type 1. A l’opposé, aucun rôle significatif des protéines de stress ne fut montré pour les réactions de type 2, à l’exception d’une réponse lymphopro- liférative plus élevée contre l’antigène de 28kDa. RESUMEN En este estudio se examinó la respuesta inmunitaria contra algunas proteínas de estrés de Mycobacterium leprae en pacientes con lepra con y sin reacción leprosa. Comparados con los controles sanos, los pacientes con lepra mostraron altos niveles de anticuerpos contra todos los antígenos probados. La respuesta en anticuerpos contra el antígeno de 18 kDa fue significativamente mayor en los pacientes con reacción Tipo I que en los pacientes con lepra TT, los pacientes con lepra subpolar sin reacción Tipo 1, o los pacientes con reacción Tipo 2. Los pacientes subpolares (BT/BL), los lepromatosos (LL) y aquellos sin reacciones (tipos 1 y 2) tuvieron niveles más elevados de anticuerpos contra un extracto soluble de M. leprae (MLSE) y contra la proteína de 65 kDa, que los pacientes del grupo tuberculoide (TT). Los pacientes LL, los subpolares, y los pacientes con reacción Tipo 1 tuvieron mayores niveles de anticuerpos contra la proteína de 28 kDa que los controles sanos. Sin embargo, no se observaron diferencias significativas en la respuesta en anticuerpos contra los antígenos (MLSE, 65 Kda, y 28 kDa) entre los pacientes con reacción y aquellos sin reacción. Por otro lado, comparados con los pacientes BL/LL, una proporción significativa de los pacientes TT/BT mostraron una respuesta linfoproliferativa positiva contra el MLSE. Además, la respuesta linfoproliferativa contra el MLSE fue significativamente mayor en los pacientes con reacción Tipo 1 que en los pacientes sin reacción. En ninguno de los grupos se observó difer- encia en la respuesta proliferativa contra la proteína de 65 kDa. Así, los hallazgos de niveles elevados de anticuerpos contra las proteínas de estrés en los pacientes con reacción Tipo 1, especialmente contra la proteína de 18 kDa, junto con la elevada respuesta proliferativa contra el MLSE, sugieren la coexistencia de la inmunidad celular y la inmunidad humoral en los pacientes con lepra durante la reacción Tipo 1. En las reacciones de Tipo 2 no se pudo demostrar un papel significativo de las proteínas de estrés, excepto por la marcada respuesta linfoproliferativa contra el antígeno de 28 kDa.

Leprosy is a chronic infectious disease rise in immune complexes with their depo- caused by Mycobacterium leprae. After in- sition in tissues (40). Further, in Type 2 reac- fection the response of host immune system tions a transient rise of CMI with the ex- determines the course of the disease. On the pression of Th 1 type of cytokines has also basis of cellular immune response of the been noted (19, 35). host, a spectrum of types of leprosy has Several stress proteins of M. leprae have been described (27). Some types of leprosy been cloned (42) and are recognized by both patients suffer from immunological compli- murine and human immune cells (22, 24). M. cations known as “lepra reactions” which leprae heat shock protein (hsp) 65 kDa, ex- include both Type 1 (reversal reactions, pressed in macrophages transfected with RR) and Type 2 (erythema nodosum lepro- the mycobacterial gene, is known to be pre- sum, ENL) reactions (14). Type 1 reaction is sented to T cells in association with both characterized by episodes of increased in- MHC class I and class II (32) and also MHC flammatory activity in skin and in nerves of non-restricted manner (33). Beimnet, et al. patients with borderline leprosy [Borderline (1996) also reported the expression of Hsp tuberculoid (BT), Borderline (BB), and 60 from a human monocytic cell line in- Borderline lepromatous (BL)] (5, 31). Type 2 fected with M. leprae (2). Further, these reaction or ENL reaction is the most serious stress proteins are known to be major anti- immunological complication in BL and lep- gens of mycobacteria, which induce spe- romatous (LL) patients. Type 1 reaction is cific antibody and T cell immune response known to be due to changes in (both up and during infections (1, 6, 8,10, 11, 14, 16, 21, 28, 29, 30, 37, 41). down regulation) of cell mediated immu- Nerve and skin damage in leprosy is re- nity (CMI), whereas in Type 2, there is a ported to be associated with increased lev- 72, 2 Mohanty, et al.: Immune Response to Stress Proteins in Leprosy Reactions 151 els of intra-lesional hsp (16). The process of and 18 kDa (2µg/ml)] in carbonate bicar- reaction evokes a change in immunological bonate buffer (pH 9.2) and kept for 4 hrs at status of the host leading to stress condi- 37°C and then overnight at 4°C. The wells tions for bacteria, which might result in re- were blocked in phosphate buffered saline lease of stress proteins. However, the role (PBS) with 3% Bovine Serum Albumin of antibodies to stress proteins in Type 1 re- (BSA) for 1 hr at 37°C. One hundred µl of actions has not been elucidated so far ex- 100-fold diluted sera in PBS containing cept for the observation of Klatser, et al. 0.05% Tween 20 and 1% BSA was added in (17). To explore the immunological role of duplicate wells. After 2 hrs incubation at stress proteins of M. leprae in leprosy, 37°C, the plates were washed 3 times in analysis of circulating antibodies, and of PBS Tween (0.05%). One hundred µl of the proliferative response of peripheral 5000-fold diluted horseradish peroxidase- blood mononuclear cells (PBMC) to some conjugated anti-human IgG antibody (Sigma, of the stress proteins were performed in St. Louis) was added, and plates were incu- healthy controls, Tuberculoid (TT), Border- bated for 1 hr 30 minutes at 37°C followed line and LL patients with Type 1 or Type 2 by a final 3 washes. One hundred µl of or- reactions, and in patients without reactions. thophenylene diamine hydrochloride solution (0.5 mg/ml substrate in distilled water MATERIALS AND METHODS µ containing 30 l of 30% H2O2) was added Leprosy patients attending the out pa- to each well for development of color. The tient department of the Central JALMA In- reaction was stopped after 30 minutes by µ stitute for Leprosy (Agra, India) were in- adding 25 l of 3N H2SO4. The optical den- cluded in the study after obtaining their sity (OD) was measured in an ELISA written consent according to the guidelines reader at 492 nm. laid by the Indian Council of Medical Re- Peripheral blood mononuclear cells search, India. They were diagnosed clini- (PBMCs). Peripheral venous blood was cally and bacteriologically and were di- collected asceptically in a heparinized tube vided into five groups across the disease from 2 TT, 22 borderline (10 BT, 12 BL), 7 spectrum according to the criteria of the In- LL patients who were stable in their clinical dian association of Leprologists (12). All manifestations and 12 BL/LL patients with these patients were clinically active and Type 2, 12 BT/BL patients with Type 1, and were on multi-drug therapy (MDT) during from 12 healthy controls. PBMCs were sep- the study period. arated by ficoll hypaque density gradient Serum samples. Serum samples were centrifugation. The cells were collected obtained from 10 ml of blood drawn by an- from the interface layer and washed three tecubital venipuncture from 6 TT, 24 bor- times with RPMI 1640 and counted in a derline (13 BT, 11 BL), 8 LL patients who Neubaur’s chamber. were stable in their clinical manifestations, Proliferation of peripheral blood 21 BL/LL patients with ENL, and 29 mononuclear cells (Lymphocyte transfor- BT/BL patients with Type 1 reaction. Nine- mation test, LTT). PBMCs (2 × 106 cells/ml) teen laboratory volunteers who were hospi- were cultured in quadruplicate wells in RPMI tal contacts served as healthy controls. 1640 containing 10% human AB serum in Soluble and recombinant antigens of 96-well plates (Nunc, Rosklide, Denmark) M. leprae. M. leprae soluble extract for six days. Optimum concentrations of (MLSE) (contract No-1-A1-55262) was ob- MLSE (1 µg/ml) and different recombinant tained from Dr. P. J. Brennan, Colorado proteins [65kDa (5µg/ml), 28 kDa (10 State University, U.S.A., and the recombi- µg/ml)], were added to wells for sensitiza- nant proteins of M. leprae (ML hsp65kDa, tion of PBMCs. DNA synthesis was assayed ML 28 kDa, ML 18kDa) were gifted by Dr. by [3H] labeled thymidine incorporation M. Singh, GBH, Germany. (Amersham, U.K.). 1 µ Ci of 3H-thymidine Enzyme linked immuno sorbent assay (specific activity 5.0 Ci / m mol) was added (ELISA). Maxisorp (Nunc, Rosklide, Den- to each well on 5th day and after 18 hr cells mark) plates were coated with 100 mi- were harvested. The lymphocyte stimula- crolitre (µl) antigen solutions [MLSE (2µg/ tion index (SI) was calculated using a stan- ml), 65 kDa (1µg /ml), 28 kDa (2µg/ml) dard formula (average cpm in the presence 152 International Journal of Leprosy 2004

TABLE 1. Mean level of antibodies (with standard deviations) to antigens of M. leprae of healthy controls and leprosy patients.

HC TT Borderline LL ENL RR Antigens N = 19 N = 6 (BT/BL) N = 24 N = 8 N = 21 N = 29 MLSE 0.23 ± 0.13 0.30 ± 0.06 0.51 ± 0.36∇ 0.55 ± 0.19∇ 0.58 ± 0.37∇ 0.51 ± 0.22∇ ML65kDa 0.19 ± 0.08 0.22 ± 0.04 0.29 ± 0.13# 0.37 ± 0.16# 0.30 ± 0.13# 0.35 ± 0.22# ML28kDa 0.17 ± 0.08 0.30 ± 0.17 0.27 ± 0.13* 0.42 ± 0.25* 0.29 ± 0.13* 0.38 ± 0.19*↑ ML18kDa 0.17 ± 0.08 0.23 ± 0.06♣ 0.28 ± 0.15♣ 0.42 ± 0.19♣ 0.31 ± 0.22♣ 0.46 ± 0.35♣♠ ∇ Significantly more than HC & TT (p <0.005) # Significantly more than HC & TT (p <0.02) *Significantly more than HC (p <0.0005) ↑ Significantly more than Borderline (BT/BL) and ENL (p <0.03) ♣ Significantly more than HC (p <0.05) ♠ Significantly more than TT, Borderline (BT/BL) and ENL (p <0.005) of antigen/average cpm in the absence of ference in BL/LL patients, and those pa- antigen). tients with Type 2 reactions. Statistical analysis. Data were analyzed 65kDa. There was a higher level of anti- by Student’s t-test by using MS Excel soft- body in BT/BL, LL and also in patients ware program, Chi square test, and Fisher with reactions (ENL and RR) than in exact test. healthy controls and TT patients. No difference could be observed in antibody level RESULTS between the groups of patients with reac- Antibodies to MLSE, ML 65kDa, ML tions (Type 1 and Type 2) and without reac- 28kDa and ML 18kDa. The mean anti- tions. body response (with standard deviations) of 28 kDa. It was also observed that TT, healthy individuals, all types of leprosy pa- BT/BL and LL patients have higher level of tients is presented in Table 1. antibody to this antigen in comparison to MLSE. It can be noted that all groups of healthy controls. The highest level of anti- leprosy patients showed high antibody lev- body was detected in LL patients, followed els to MLSE compared to healthy controls. by Type 1 patients. The difference between Antibody level in BT, BL patients, and LL antibody levels in BT/BL patients as com- patients, and in patients during reactions pared to those with Type 1 reactions was (both Type 1 and Type 2) is significantly statistically significant, (p <0.03). higher than those of TT group. There was 18 kDa. The level of antibody to this no difference in antibody level between BT antigen was observed to be at a higher level and BL patients and in patients during rein all groups of leprosy patients compared versal reaction. Similarly, there was no dif- to those of healthy controls. LL patients had

TABLE 2. The percentage of seropositivity for antibodies against various antigens of M. leprae.

HC TT Borderline LL ENL RR Antigens N = 19 N = 6 (BT/BL) N = 24 N = 8 N = 21 N = 29 MLSE 0 (0) 0 (0) 10 (41.66) 5 (62.5)♦ 10 (47.61) 15 (51.72) ML65kDa 1 (5.26) 0 (0) 8 (33.33) 4 (50) 6 (28.57) 11 (37.93) ML28kDa 0 (0) 2 (33.33) 8 (33.33) 4 (50) 7 (33.33) 15 (51.72) ML18kDa 0( 0) 0 (0) 7 (29.16) 5 (62.5)♦ 5 (23.80) 16 (55.17)* Figures in parentheses ( ) show percent positive. Cut off values are mean OD +2 S.D. (MLSE = 0.49), (65kDa = 0.35), (28 kDa = 0.33), and (28 kDa = 0.33). Individuals having more OD values than cut off points are taken as positive. ♦ Signiﬁcantly more than TT (p <0.03) *Signiﬁcantly more than borderline group (p <0.06) 72, 2 Mohanty, et al.: Immune Response to Stress Proteins in Leprosy Reactions 153

TABLE 3. The positivity for lymphoproliferation in healthy controls and leprosy patients towards various antigens of M. leprae.

♦ Antigens HC TT/BT BL/LL ENL RR N = 12 N = 12 N = 19 N = 12 N = 12 MLSE 11 (91.66) 6 (50) 1 (5.2) 1 (8.33) 8 (66.66)* ML65kDa 2 (16.66) 1 (8.33) 1 (5.2) 2 (16.66) 1 (8.33) ML28 kDa 2 (16.66) 0 (0) 0 (0) 3 (25)♣ 2 (16.66) SI >2 is taken as positive response. Figures in ( ) show the percent positivity. ♦ Significantly more than BL/LL (p <0.007) *Significantly more than BT /BL (p <0.04) ♣ Significantly more than BL/LL patients (p <0.06) and BT/BL (p <0.02) significantly higher level of antibody than 1 was significantly greater compared to bor- did TT patients. The mean antibody level to derline patients without reaction (p <0.02). this antigen is highest in BT/BL patients However, there was no difference in mean during Type 1 reactions, which is signifi- proliferative response between BL/LL with- cantly different from those of TT patients, out reactions and Type 2 reaction patients. BT/BL patients without reaction and pa- Further, the mean proliferative response tients with Type 2 reaction, (p <0.005). was significantly greater in TT/BT patients The percentage of seropositivity of anti- compared to BL/LL patients (p <0.03) and bodies to these antigens is shown in Table Type 2 reaction patients (p <0.04). Signifi- 2. It was noted that significant proportion of cant proportions of TT/BT individuals LL patients showed positivity for MLSE showed lymphoproliferation when com- and 18kDa (p <0.03) when compared to pared to BL/LL patients (p <0.007). that of TT patients. Response to recombinant proteins. ML There was no significant difference in 65kDa. There was no significant difference in seropositivity of antibodies to these antigens lymphoproliferative response to 65kDa pro- amongst patients of the BT/BL groups with tein amongst these groups (Table 3 and The and without reaction. Nevertheless a signifi- Figure b). ML 28kDa. While 3/12 patients cant proportion of BT/BL patients with Type with Type 2 showed a positive response, 1 showed higher positivity to 18-kDa anti- none of the LL patients was responsive to this gens only when compared to those of bor- antigen (Table 3 and The Figure c). derline patients without reactions (p <0.06). Lymphoproliferative response to MLSE DISCUSSION and stress proteins of M. leprae (ML When infectious agents enter the host, 65kDa & ML28kDa). The positivity for they may respond to the host environment lymphoproliferation to these antigens (SI >2) by producing stress proteins. These stress in healthy controls and leprosy patients is proteins are important in eliciting immune presented in Table 3. The individual lympho- response, which can lead to pathogenesis or proliferative responses against these antigens protection in the host. Some recombinant are shown in The Figure (a, b, and c). antigens (stress proteins) have been re- MLSE. MLSE was found to be the best ported to be immunologically important inducer for proliferation of lymphocytes and induce B cell and T cell immune re- amongst all M. leprae proteins tested. All sponses in leprosy (6, 8, 16, 21, 24, 30, 37, 38, 41). Al- except one of the healthy controls showed a though these previous studies have been positive response to MLSE, whereas 1 BL conducted to analyze the immune response patient, 1 BL patient with Type 2 reaction in leprosy patients, only a few studies were and none of the LL patients, were positive carried out in leprosy patients with reac- to this antigen. 8/12 (66.6%) of Type 1 re- tions. The objective of the present study actions, and 6/12 TT/BT (50%) were posi- was to analyze the level of antibodies and tive to these antigens. The mean prolifera- immunoproliferative response of PBMCs to tive response of BT/BL patients with Type MLSE and a few stress proteins of M. lep- 154 International Journal of Leprosy 2004

THE FIGURE. Proliferative response of individuals (healthy controls and leprosy patients) to MLSE (a), 65kDa (b), 28kDa(c). PBMCs (2 × 106 cells/ml) were cultured in quadruplicate wells for six days in presence of various antigens of M. leprae (MLSE, 65kDa, 28kDa) and in absence of antigens. Thymidine [H]3 was added after 5 days and cells were harvested after 18 hrs. Proliferative response was expressed in stimulation Index (SI). SI was calculated by using the formula (Average cpm in antigen pulsed wells/average cpm in control wells). rae in patients associated with reactions, Although the mean antibody level was and to compare their levels with patients found to be highest in patients with Type 2 who are not associated with reactions. In reactions, this was not signiﬁcantly differ- addition, as controls, responses of some ent from other groups. Patients with Type 1 healthy individuals who were exposed to reactions also showed almost the same level infection in the hospital were also com- of antibody to MLSE as of borderline pa- pared with these leprosy patients. tients. The mean level of antibodies to 72, 2 Mohanty, et al.: Immune Response to Stress Proteins in Leprosy Reactions 155

MLSE was found to be significantly higher recently been suggested as a potential can- in all types of leprosy patients except TT didate antigen for initiating the Type 1 reac- patients when compared to those of healthy tion, because it has been demonstrated in individuals. Among the patient groups, TT macrophages and Schwann cells of skin patients showed lowest antibody level. The and nerve biopsies (20). Hence, our finding mean OD value gradually increased from of high antibody response may be due to the TT end to the LL end of the spectrum. A expression of this antigen by M. leprae dur- similar finding has been reported earlier by ing Type 1 reactions. Interestingly, the M. Qin-xue, et al. (26). This finding of a gradual leprae 28 kDa protein is known to have a increase of antibody level against MLSE sequence similarity with human superoxide could possibly be due to the increase in dismutase (SOD) (67%) and E. coli SOD antigenic load from the TT pole to the LL (55%) (36). The elevated antibody level to pole. 28kDa antigen in some of the LL and bor- An increased level of antibodies was seen derline patients with Type 1 reaction may to 65 kDa, 28kDa and 18 kDa stress probe attributed to the response against in- teins in all groups of leprosy patients com- creased expression of SOD in response to pared to healthy individuals, similar to pre- environmental stress during the disease vious reports (14, 16). In our study a higher process or during reaction. level of anti 65kDa antibody was observed The most interesting finding of our study in BL/LL and Type 2 patients. However, is that the antibody level against M. leprae there was no significant difference in anti- 18kDa antigen was much higher in leprosy body levels between patients with reaction patients with Type 1 reactions, although the and patients without reactions. Possibly the percent seropositivity was also high in LL 65kDa antigen induces an antibody re- patients without reactions. Our study indi- sponse in the initial phase of infection and cates that production of anti18-kDa anti- this does not change during the develop- body is a prominent event in leprosy, as all ment of various stages of disease. This ob- groups of leprosy patients except TT pa- servation would suggest that antibodies to tients had a high level of antibodies to this 65kDa do not induce any immunopatholog- antigen. Khan, et al. reported a low reactiv- ical phenomenon in patients associated with ity to this antigen in multibacillary patients reactions. Furthermore, the above finding is (15), but we observed seropositivity of consistent with the observation of Thole, et 62.5% in LL patients. Further, Roche, et al. al. (1995) who did not find any association (1991) observed a similar finding of a low with the 65kDa antigen specific responses level of anti 18kDa antibodies in pau- in BT/TT or LL types of leprosy (37). Of cibacillary (PB) patients and a high level in course, M. leprae 65 kDa has been noted to multibacillary (MB) patients (28). Many be expressed in skin and nerve of all groups other authors have described the 18kDa of leprosy patients (38) and may be pre- protein as one of the important antigens sumed to have an important role in Type 1 which produces significant B cell and T cell reaction, but it is uncertain whether this is immune response in leprosy (8, 10, 11, 24, 28). predominantly related to the initiation of However, its association with Type 1 reac- the disease or the development of disease tions has not been described previously. once the reaction has started. This protein was reported to have strikingly We observed a higher positivity for anti- similar size and sequence to a family of body responses against the 28 kDa antigen heat shock proteins (25) and is expressed in LL patients than TT patients, and this re- during heat stress (19). So, we postulate that sponse was even greater in patients with the expression of this antigen by M. leprae Type 1 reactions. Though other studies have might be increasing due to cellular resis- provided evidence of the presence of anti tance by the host, and as a result the host re- M. leprae 28 kDa antibodies in sera of lep- sponds by producing antibodies to this romatous patients (6, 16), this is the first re- stress protein. This could induce an immune port to note such a higher percentage response in the initial phase of infections (51.72%) of antibody positivity to this and during Type 1 reactions. stress protein of M. leprae in patients dur- From our observations, we conclude that ing Type 1 reactions. This antigen has circulating antibodies to some of the stress 156 International Journal of Leprosy 2004 proteins of M. leprae appear to play a role study except that of the 28 kDa antigen in Type 1 reactions. We could not observe where a significant proportion of patients any significant difference in antibody level with Type 2 reaction responded to this anti- against the recombinant proteins in LL pa- gen. While a number of studies have re- tients, nor in patients with Type 2 reaction, ported the antigenic potential of 28 kDa in though the reactivity was greater to some the humoral immune response, not much antigens in patients with Type 2 reactions information is available regarding the nathan TT patients or healthy controls. Miller, ture of cell mediated response against it. et al. (1984) have also reported that the oc- Though Wilkinson, et al. (41) have de- currence of Type 2 reaction had no signifi- scribed this as a moderate stimulator of T cant effect on the total level of IgG anti- cell responses, they did not investigate the body against arabinomannan (23). response in patients during leprosy reac- With regard to the lymphoproliferative tions (Type 1 or Type 2). The significantly responses to these antigens, all healthy indi- greater positivity in proliferative responses viduals except one responded to MLSE, but in Type 2 patients than those of BT/BL/LL none of the recombinant proteins induced a might indicate a response to the expression strong proliferative response in this group, of SOD during this reactional stress. The confirming the earlier report of Wilkinson, finding of a significantly greater number of et al. (41). Moreover, most of the patients Type 2 reaction cases responding to the 28 and all healthy controls were responsive to kDa antigen compared to BL/LL patients the purified protein derivative of M. bovis might explain their transient boost in CMI (data not included). In the present study, as reported earlier by other authors (18). TT/BT patients showed stronger lympho- Although previous workers have demon- proliferative responses than those of BL/LL strated the presence of antibodies to these patients only to MLSE and not to recombi- proteins in BT/TT and BL/LL patients, this nant proteins of M. leprae, consistent with appears to be the first study to demonstrate the study of Thole, et al. (37) Further, we ob- a high level of antibodies especially against served a significant lymphoproliferative re- 28 kDa in these patients associated with sponse to MLSE in patients with Type 1 re- Type 1 reaction. The role of M. leprae anti- action. Bjune, et al. (5) have already noted gens in Type 1 reactional pathology has this with sonicated preparations of M. lep- been noted others (17, 20, 39). Hence, the high rae in patients with Type 1 reactions. The level of antibodies observed in patients dur- finding of a significant lymphoproliferative ing Type 1 reaction may be due to the M. response to MLSE during Type 1 reactions, leprae antigens exposed in tissues during compared with borderline patients without reactions. At this moment, it is not possible reactions, clearly indicates the upregulation to conclude whether antibodies are induced of CMI in such patients. due to the development of reactional We did not observe any significant differ- pathology, or if it has been initiated due to ence in the proliferative response to 65 kDa the induction of antibodies. The cellular im- antigen among patient groups, as reported mune response associated with Type 1 reac- by others. Ilangumaran, et al. (13) reported tion is presumably due to other M. leprae that there is an inverse relationship between antigens and not due to the stress proteins cell mediated and humoral immune re- expressed by M. leprae. sponses to 65 kDa in leprosy patients. De La Barrera, et al. (7) have observed that M. Acknowledgment. Indian Council of Medical Re- leprae 65 kDa is a poor inducer of cyto- search (ICMR), Government of India, supported this toxic T lymphocyte (CTL) in MB patients, study. LEPRA grant was used for purchasing of certain but could induce proliferation and CTL in reagents, which were used in the study. We acknowl- MB patients with Type 2 reaction. edge the help of Mr. V. S. Yadav for statistical analysis (Chi square and Fisher exact test) and Mr. Hariom The finding that the 28 kDa antigen in- Agarwal for photography. We thank Mr. P. N. Sharma, duced a poor response both in TT and LL Mr. M. S. Tomar, Mr. M. Alam, and Mr. K. 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immunosorbent assay—a preliminary report. Int. J. 36. THANGARAJ, H. S., LAMB, F. I., DAVIS, E. O., JEN- Lepr. Other Mycobact. Dis. 53 (1985) 565–570. NER, P. J., JEYAKUMAR, L. H., and COLSTON, M. J. 27. RIDLEY, D.S., and JOPLING, W. H. Classification of Identification, sequencing and expression of M. leprosy according to immunity; a five-group sys- leprae superoxide dismutase, a major antigen. In- tem. Int. J. Lepr. 34 (1966) 255–273. fect. Immun. 58 (1990) 1937–1942. 28. ROCHE, P. W., PRESTIDGE, R. L., WATSON, D. J., 37. THOLE, J. E. R., JANSON, A. A. M., KIFLE, A., and BRITTON, W. J. Antibody responses to the 18- HOWE, R. C., MCLEAN, K., NURILYGN, A., FILLEY, kDa protein of M. leprae in leprosy and tuberculo- E., SHANNON, E. J., BULLA, G. J., HERMANS, J., sis patients. Int. J. Lepr. Other Mycoabct. Dis. 60 DEVRIES, R. R. P., FROMMEL, D., and RINKE DE (1991) 201–207. WIT, T. F. Analysis of T cell and B cell responses 29. ROCHE, P. W., THEUVENET, W. J., and BRITTON, W. to recombinant M. leprae antigens in leprosy pa- J. Cellular immune responses to mycobacterial tients and healthy controls: Significant T cell re- heat shock proteins in Nepali leprosy patients and sponses to antigens in M. leprae non responders. controls. Int. J. Lepr. Other Mycobact. Dis. 60 Int. J. Lepr. Other Mycobact. Dis. 63 (1995) (1991) 36–43. 369–380. 30. ROJAS, R. E., and SEGAL, E. A. Immunoglobulin 38. VAN DEN BOS, I. C., KHANOLKAR-YOUNG, S., DAS, G response against 10 kDa and 65 kDa heat shock P. K., and LOCKWOOD D. N. J. Immunohistochem- proteins in leprosy patients and their household ical detection of PGL-1, LAM, 30kDa and 65kDa contacts. FEMS Immunol. Med. Microbiol. 15 antigens in leprosy infected paraffin preserved skin (1996) 189–198. and nerve sections. Lepr. Rev. 70 (1999) 272–280. 31. ROSE, P., and WATERS, M. F. Reversal reactions in 39. VERHAGEN, C., FABER, W. R., KLATSER, P. R., leprosy and their management. Lepr. Rev. 62 BUFFING, A., NAAFS, B., and DAS, P. K. Immuno- (1991) 113–121. logical analysis of in situ expression of mycobac- 32. SILVA, C. L. M. leprae 65 hsp antigen expressed terial antigens in skin lesions of leprosy patients from a retroviral vector in a macrophage cell line across the histopathological spectrum. Association is presented to T cells in association with MHC of mycobacterial lipoarabinomanan (LAM) and class II in addition to MHC class 1. Microb. M. leprae phenolic glycolipid-1(PGL 1) with lep- Pathogen 12 (1992) 27–38. rosy reactions. Am. J. Path. 154 (1999) 33. SILVA, C. L., LUKACS, K., and LOWRIE, D. B. Ma- 1793–1804. jor histocompatibility complex non-restricted pre- 40. WEMAMBU, S. N. C., TURK, J. L., WATERS, M. F. sentation to CD4+ T lymphocytes of M. leprae R., and REES, R. J. W. Erythema nodosum lepro- heat-shock protein 65 antigen by macrophages sum: a clinical manifestations of Arthus phenome- transfected with the mycobacterial gene. Im- non. Lancet ii (1969) 933–935. munol. 78 (1993) 35–42. 41. WILKINSON, K. A., KATOCH, K., SENGUPTA, U., 34. SINHA, S., SENGUPTA, U., RAMU, G., and IVANYI, J. SINGH, M., SARIN, K. K., IVANYI, J., and WILKIN- Serological survey of leprosy and control subjects SON, R. J. Immune response to recombinant pro- by a monoclonal antibody-based immunoassay. Int. teins of M. leprae. J. Infec. Dis. 179 (1999) J. Lepr. Other Mycobact. Dis. 53 (1985) 33–38. 1034–1037. 35. SREENIVASAN, P., MISHRA, R. S., WILFRED, D., and 42. YOUNG, R. A., MEHRA, V., SWEETSER, D., NATH, I. Lepromatous leprosy patients show T BUCHANAN, T., CLARK-CURTISS, DAVIS, R. W., and helper1-like cytokine profile with differential ex- BLOOM, B. R. Genes for the major protein anti- pression of Interleukin 10 during Type 1 and Type gens of the leprosy parasite M. leprae. Nature 316 2 reactions. Immunol. 95 (1998) 529–536. (1985) 450–452. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) Axonal Spherical Bodies in the Peripheral Nerves of Leprosy Patients1

Mutsuhiro Furuta, Kentaro Hatano, Yoshiko Okano, Takanobu Matsuki, Takeshi lkeda, Kouichi Nakatani, Atsuo Sato, and Mutsue Mizushima 2

ABSTRACT Spherical bodies, roughly 10 µm in diameter, which have not been reported before, were found in the peripheral nerve axons of specimens collected during post-mortem examination of leprosy patients. These bodies were found in the fascicles of all peripheral nerves of the extremities examined (median, radial, ulnar, peroneal and sciatic nerves). Their incidence was not related to the type of leprosy. The area immediately below the thickened perineurium, a feature associated with leprosy, often showed a large number of spherical bodies. When observed under a transmission electron microscope, the spherical lesions often showed a lamellar structure, although some of them were amorphous. No structure resembling organelles was seen within the bodies. Observation with the merge technique showed a clearly lamellar structure in most of the spherical bodies. These bodies and the surrounding myelin sheaths were partially polarized. The axonal spherical bodies observed in our study seem to represent lesions gradually formed due to glycoprotein denaturation over long periods of time and to be associated with leprosy-caused thickening of the perineurium of peripheral nerves. RÉSUMÉ Des corps sphériques, mesurant environ 10 µm de diamètre, qui n’ont pas encore été rap- portés, furent trouvés dans les axones des nerfs périphériques prélevés à l’autopsie de patients lépreux. Ces corps furent retrouvés dans les faisceaux de tous les nerfs périphériques des ex- trémités examinées (nerfs médian, radial, ulnaire, péroné et sciatique). Leur incidence n’était pas liée au type de lèpre. La zone immédiatement en dessous d’un périneurium épaissi, un caractère associé à la lèpre, était fréquemment riche de ces corps sphériques. Lorsque observés au microscope électronique à transmission, ces corps sphériques montraient fréquemment une structure lamellaire, bien que certains étaient amorphes. Aucune structure ressemblant à une organelle ne fut décelée dans ces corps. L’observation par une technique de concaténation a révélé une structure clairement lamellaire dans la grande majorité des corps sphériques. Ces corps et les manchons myéliniques environnants n’étaient que partiellement polarisés. Ces corps sphériques des axones, observés dans notre étude, semblent représenter des lé- sions progressives à long terme de dénaturation des glycoprotéines et être associés aux épaississements du périneurium des nerfs périphériques causés par la lèpre. RESUMEN Se observaron cuerpos esféricos de aproximadamente 10 µm de diámetro en los axones de especimenes de nervios periféricos colectados durante el examen post-mortem de pacientes con lepra. Estos cuerpos esféricos, que no se habían descrito antes, se encontraron en los fascículos de todos los nervios de las extremidades examinados incluyendo los nervios mediano, radial, ulnar, peronal y ciático. Su incidencia no estuvo relacionada con el tipo de

1 Received for publication on 22 July 2003. Accepted for publication on 14 January 2004. 2 M. Furuta, M.D., Department of Pathology, Izumigaoka Hospital, Turuga-city, Fukui, 914-0770, Japan; K. Hatano, M.D.; Y. Okano, M.D.; T. Matsuki, M.D., National sanatorium Oku-Komyoen, Okayama, Japan; T. Ikeda, M.D.; K. Nakatani, M.D.; A. Sato, M.D., National sanatorium Minami Kyoto Hospital, Kyoto, Japan; M. Mizushima, M.D., Gifu Prefectural Tajimi Hospital, Tajimi, Japan. Reprint requests to Dr. Yoshiko Okano, Department of Ophthalmology, National sanatorium Oku-Komyoen, 6253 Mushiake, Oku-cho, Oku-gun, Okayama, 701-4593, Japan. E-mail: okano@ nsok.hosp.go. jp

159 160 International Journal of Leprosy 2004

lepra y se observaron con más frecuencia por debajo del perineurio engrosado característico de la lepra. Observados al microscopio electrónico de transmisión los cuerpos esféricos mostraron una estructura laminar, aunque algunos de ellos aparecieron amorfos. No se observaron es- tructuras sugerentes de organelos dentro de los cuerpos esféricos. Además de su estructura laminar, estos cuerpos y las capas envolventes de mielina aparecieron parcialmente polar- izados. Los cuerpos esféricos axonales observados en nuestro estudio parecen representar le- siones formadas gradualmente por la desnaturalización de glicoproteínas a lo largo de peri- odos prolongados de tiempo y parecen estar asociados con el engrosamiento del perineurio de los nervios periféricos ocasionado por la lepra.

During our 40 years of experience of post- Case 2. A 76-year-old female with tuber- mortem examinations of leprosy patients, we culoid leprosy, who was diagnosed at age have detected spherical bodies in the periph- 26 with Hansen’s disease and admitted to eral nerve axons of these patients. Many re- the sanatorium when she was 27. At age 71, ports on peripheral nerve lesions observed in she was diagnosed as having hepatocellular leprosy patients have been based on electron carcinoma during treatment for hepatitis C, microscopy examinations. These reports and in 1995, she died of rupture of the have often mentioned the presence of My- cancer-affected liver. cobacterium leprae in the axons, but none Case 3. An 89-year-old male with lepro- of them have reported the presence of ax- matous leprosy, who was diagnosed with onal spherical bodies. For the study pre- Hansen’s disease when he was 25, and was sented here, axonal spherical bodies were admitted to the sanatorium 5 yrs later. At age observed under light and electron micro- 81, he was hospitalized because of anorexia, scopes at magniﬁcations up to ×1000. and 3 yrs later in 1995, he died of exacerba- We used a new method known as merge tion of pneumonia. technique (1), which allows for simultane- Case 4. A 64-year-old male with lepro- ous viewing of an object under both a po- matous leprosy, whose diagnosis of larized microscope (PM) and a differential Hansen’s disease was established when he interference contrast microscope (DIC) was 14. The next year he was admitted to within a common visual ﬁeld. the sanatorium. At age 62, he was diagnosed with prostate cancer and began hor- MATERIALS AND METHODS mone therapy. When he was 64 years old, Subjects from which specimens were the cancer had metastasized to the stomach, obtained. Peripheral nerve specimens col- and he died of deterioration of his general lected from 6 cadavers during post-mortem condition in 1995. examination at the “Oku Komyoen” Na- Case 5. A 79-year-old male with lepro- tional Sanatorium were used for this study. matous leprosy, who was diagnosed as hav- One of the specimens was relatively old ing Hansen’s disease when he was 24, was (collected in 1983), but the other speci- admitted to the sanatorium at age 27. He mens were collected fairly recently (three was diagnosed with a urinary bladder tumor in 1995 and two in 1996). The specimens when he was 77 years old and underwent were collected from 6 cases whose history several sessions of transurethral tumor re- follows. In all cases, leprosy-related pe- section. Two years later, when he was 79, ripheral nerve disturbance was observed. he underwent a total cystectomy, but post- Case 1. An 89-year-old male with lepro- operatively developed metastasis of the matous leprosy, who was diagnosed at age cancer to the lumbar vertebrae, liver, subcu- 17 with Hansen’s disease, and was admitted taneous tissue, and other areas. He died of to the sanatorium when he was 27. At age cancer in the same year in 1996. 89, he was hospitalized because of mild Case 6. An 81-year-old male with tuber- motor paralysis of the extremities. During culoid leprosy was diagnosed at age 21 as his hospitalization, he developed fever and having Hansen’s disease and admitted to increased sputum, leading to death from the sanatorium at age 26. When he was 81 dyspnea in 1983. years old, he developed chest pain and hy- 72, 2 Furuta, et al.: Axonal Bodies in Nerves in Leprosy 161

FIG. l. Case 1. Several axonal spherical bodies are visible, some of them granular. These bodies were often detected immediately below the thickened perineurium (arrow). (×400, PAS). drothorax associated with thoracic aortic rapidly, without any distortion in the visual aneurysm, and his condition was compli- field. At room temperature and under ordi- cated by DIC, leading to his death in 1996. nary fluorescent light, polarized images Preparation of specimens. All speci- were obtained in the FL and differential- mens of the peripheral nerves were fixed in interference images in the BF mode with a 10% formalin. The specimens collected in digital camera (DP-50, Olympus). The im- 1995 and 1996 were 10 cm long were cut ages thus taken were subjected to image into sections of about 2 cm. They were sub- analysis using the Photoshop (Adobe) soft- jected to HE, PAS, Bodian, Luxol fast blue, ware package. During image analysis, po- amyloid and acid-fast bacterium staining. larizing images were overlapped with Formalin-fixed specimens that were found differential-interference images of the same to contain spherical bodies were subjected to PCR assay to determine the relationship of the bodies to Mycobacterium leprae. The specimens were also subjected to merge observation (allowing simultaneous observation under both PM and DIC in a common visual field) and electron microscopy. Merge technique. Non-stained, deparaf- finized specimens, 5–7 µm thick, were mounted on acryl-based material. The specimen can be observed simultaneously in a bright visual field under a biomicroscope (BX51, Olympus), a DIC (Olympus) and a PM (Olympus), without the need to move the stage of any of the microscopes. Micro- scopes capable of magnification up to ×1000 were used. Because DIC and PM (two systems with different properties) FIG. 2. Case 1. Two axonal spherical bodies are share the same polarizing filter (polarizer or visible, one lamellar and the other amorphous. The analyzer), switching from the DIC to the surrounding myelin sheaths are degenerative. (×1000, PM image and vice versa can be done PAS). 162 International Journal of Leprosy 2004

FIG. 3. Case 1. A spherical body is visible in the axon. The silver particles that may be something like a core are in the center. (×1000, Bodian). visual field and with the same number of detected in all peripheral nerves examined pixels. (median, radial, ulnar, femoral, perinea and Polarizing images can be used to check sciatic nerves). In Bodian-stained sections, for polarizing materials, while differential- spherical bodies are in the axons and the sil- interference images provide a view of the ver particles are in the center (like a core) entire photographed area. Merged images (Fig. 3). In Luxol fast blue-stained sections, then make it possible to determine the exact the spherical bodies did not stain at all (Fig. location of the polarizing material. 4). Their incidence did not correlate with the type of leprosy. These spherical bodies RESULTS showed no chromatic response to amyloid Spherical bodies found within periph- staining, and, when assayed by PCR, were eral nerve axons under a light micro- found to have no relationship to Mycobac- scope. The diameter of most of the axonal terium leprae (data not shown). spherical bodies was about 10 µm, with one Findings from transmission electron section usually containing 1 or 2 spherical microscopy. The spherical bodies which ap- bodies. It was rare for 3 or more bodies to peared to have no specific structure under the be observed in one section. The bodies light microscope were also amorphous when were visible in HE-stained sections, but observed under the electron microscope. The more so in PAS-stained sections (Fig. 1). bodies with a lamellar structure under the They were not stained by acid-fast staining light microscope, were found under the elec- and often had a lamellar structure, although tron microscope to contain fine powder-like some did not show any specific structure. materials with a high electron density in their The bodies were often spherical or oval, center (like a core), and show a lamellar and some of them were composed of sev- structure composed of rings with different eral small granules of irregular size. electron densities (Fig. 5). The myelin A narrow area characterized by irregular sheaths surrounding the spherical bodies swelling was often seen around the spheri- were of irregular thickness (Fig. 6). cal bodies within the axons (Fig. 2). The le- Observation with the merge technique. sions were more frequently seen immedi- A few of the spherical bodies showed par- ately below the perineurium, and they were tial polarization, while small areas within 72, 2 Furuta, et al.: Axonal Bodies in Nerves in Leprosy 163

FIG. 4. Case 1. A spherical body does not stain at all. (×1000, Luxol fast-blue). the myelin sheaths surrounding them were DISCUSSION sometimes polarized (Figs. 7, 8). Most Although a number of reports (3) have spherical bodies had clearly lamellar struc- been published concerning peripheral nerve ture. A number of minute granular or rod- lesions associated with Hansen’s disease, shaped polarized inclusions were visible in none of them have dealt with axonal spher- the histopathology specimens. The polar- ical bodies. A search of previous ﬁndings ization disappeared after treatment of the resembling the spherical bodies we detected sections with an alkaline solution. in peripheral nerve axons, revealed that the

FIG. 5. Case 1. Core-like material is visible in the FIG. 6. Case 1. An amorphous spherical body is center of the axonal spherical body showing a lamellar visible within the axon. The surrounding myelin structure. (electron micrograph). sheaths vary in thickness. (electron micrograph). 164 International Journal of Leprosy 2004

FIG. 7. Case 2. (left: DIC, middle: merge technique, right: PM) Two partially polarized axonal spherical bodies are visible, with the degenerative surrounding myelin sheaths showing some polarized spots. Degenerative connective tissue also shows wave-formed polarization. Polarization of distribution is more evident on the image taken with the merge technique. ballooning and fragmentation of axons (Bo- relation between the spherical bodies and dian stain) shown in Fig. 3 of the report the axon or myelin substance. “Pathology of Peripheral Nerve Lesion in Under the light microscope, a narrow Lepromatous Leprosy,” published in 1971 area characterized by irregular swelling was by Job (4), resembled our spherical bodies. often observed around the spherical body The spherical body and “spheroid” which within the axon. This area seemed to repre- appears pathologically at the time of sent a precursor lesion. Among the other ar- amytrophic lateral sclerosis differ from eas of the axon, without swelling, the spher- each other in the size and the location. ical body was often seen in the area where While tissue specimens, sealed in fixation the peripheral perineurium had thickened bottles, were available from not only the six and become abnormally hard. In this con- cases included in this study but also from nection, it is interesting that Kimura, et al. some earlier cases, they had been collected (2) reported that the perineurium of the pe- in various ways because post-mortem ex- ripheral nerves appears to be a target of My- aminations had been performed by several cobacterium leprae. different pathologists. These earlier speci- When observed under the transmission mens were therefore not used for our study electron microscope, the spherical lesions because it would be difficult to perform often showed a lamellar structure, although light microscopic observation of all nerve some lesions had no specific structure. The specimens under identical conditions. We lesions contained nothing resembling or- do not think that the spherical bodies result ganelles, nor showed any chromatic re- from the post mortem change, because the sponse to amyloid staining, indicating that spherical bodies can be seen in both new the lesion did not represent amyloid degen- and old specimens. The patients’ illnesses eration. The fine powder-like material with had progressed over more than 10 yrs. We a high electron density observed in the cen- did special stains (Bodian and Luxol fast- tral area and resembling a core, differed in blue stain) for the relation between these hardness from the other areas. This material spherical bodies and the axon or myelin made it difficult to slice the specimens into substance. But this special stains showed no sections suitable for electron microscopy.

FIG. 8. Case 2. (left: DIC, middle: merge technique, right: PM) A group of axonal spherical bodies with polarization mostly absent, except for some polarized spots. 72, 2 Furuta, et al.: Axonal Bodies in Nerves in Leprosy 165

When observed by the merge technique, the pathology specimens and to Mr. T. Yamada (Olympus area around the spherical bodies and the Plaza Osaka) for his help with using the merge tech- myelin sheaths surrounding them was par- nique for taking the DIC and PM pictures. The authors tially polarized in some specimens, although also wish to thank Prof. C. Ide (Department of Anatomy, Kyoto University) for the electron micro- infrequently. Most of the spherical bodies graphs he prepared for this study and to Dr. K. Saeki had a clearly lamellar structure, which is (National Sanatorium Oshima Seishoen) for the PCR consistent with the findings from HE and assay of formalin-fixed peripheral nerve specimens he PAS staining and electron microscopy. conducted for this study. The axonal spherical bodies were clearly PAS-positive, and calcification-like deposi- REFERENCES tions were occasionally seen in their center. 1. FURUTA, M., HATANO, K., MATSUKI, T., OKANO, Y., These findings, combined with that of the IKEDA, T., NAKATANI, K., and SATO,A.Observa- lamellar structure, make it appear likely tion of acid fast bacilli by merge technique of dif- that the bodies were gradually formed over ferential interference contrast and polarized micro- long periods of time. These bodies were scopes. Poster presentation at XVI International seen in cases of both lepromatous and tu- Leprosy Congress, Salvador, Brazil, 2002. berculoid leprosy. In conclusion, the axonal 2. JOB, C. K. Pathology of peripheral nerve lesions in spherical bodies observed in the study pre- lepromatous leprosy—a light and electron micro- sented here seem to represent lesions grad- scopic study. Int. J. Lepr. 29 (1971) 251–268. 3. KIMURA, T., INOUE, K., IZUMI, S., HASHIMOTO, K., ually formed due to glycoprotein denatura- and YAHARA, O. Leprous neuropathy morphologi- tion over long periods of time and are asso- cal study of biopsied peripheral nerves. Advances ciated with leprosy-caused thickening of in Neurological Sciences 143 (1999) 929–942. [in the perineurium of peripheral nerves. Japanese] 4. RIDLEY, D. S., and JOB, C. K. The pathology of lep- Acknowledgment. The authors are indebted to Mr. rosy. In: Leprosy. 1st edn. New York: Churchill K. Fukuike (technologist) for his help in preparing the Livingstone, 1985, pp. 115–117. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) INTERNATIONAL JOURNAL OF LEPROSY and Other Mycobacterial Diseases

OFFICIAL ORGAN OF THE INTERNATIONAL LEPROSY ASSOCIATION

EDITORIAL Editorial opinions expressed are those of the writers.

Classiﬁcation of Leprosy: A Full Color Spectrum, or Black and White?

Proper classification of a disease is one of leukemia or lymphoma will respond to par- the fundamental tools of modern medicine ticular medicines or combinations. Recog- in selecting treatment, evaluating prognosis, nition of this has stimulated continued, vig- measuring overall progress, and furthering orous research to refine the methods and the understanding of that disease. Classifica- concepts of classification of many malig- tion is an essential tool in our approach to a nancies. Such studies enhance the under- disease, much as a good map is necessary standing of these diseases, with implica- tool for developing and navigating a coun- tions not only for treatment but also for try. The development of a comprehensive, early detection and prevention. Today, no practical classification system for leprosy cancer researcher would consider conduct- may rate as one of the most important ac- ing a study, or publishing results of a study, complishments in the extraordinary progress using a primitive or technically outmoded against this disease in the 20th century. classification system. And no professional While the more obvious, essential accom- journal would accept such a report. plishment was the discovery of effective Yet, in current leprosy research, a dis- anti-microbial agents to treat this infection, turbing trend is to do exactly that: to aban- the availability of effective treatment does don the best classification system (one that not, by itself, guarantee success against a uses clinical assessment plus histopatho- disease. The value of classification is not logic examination of a skin biopsy), and only historical; continued application of the choose instead to group patients into only 2 best classification system is essential in the groups, multibacillary (MB) or paucibacil- efforts to better understand a disease and to lary (PB), according to their bacterial load, clearly and intelligently develop a strategy or to disregard the bacilli altogether and to combat it and, ideally, prevent it. classify according to the number of skin le- The struggles against leukemia and lym- sions on a patient’s body. This last ap- phoma provide a useful example of the con- proach, which disregards the bacteria en- tinuing value of classification. Several ef- tirely, seems highly ironic for research on fective agents were available for these ma- an infectious disease. These approaches are lignancies long before many of the current technically inferior ones that assume, and successes in treatment. Greater success has accept as satisfactory, a higher degree of in- been brought about in part by a better un- accuracy than is readily available with stan- derstanding of how to use these agents in dard technology. Such over-simplification combination, but also—and very impor- of this complex host-pathogen relationship tantly—by better classification systems that is unfortunate and unacceptable. It is as if enable physicians to know which types of some have grown intellectually weary of

166 72, 2 Editorial 167

trying to understand the full-color im- the research community should not make munopathologic spectrum, and have de- an exception and settle for this with respect cided to settle for a black and white outline. to research on leprosy. Why is this done? Two related reasons are Any classification system must be ap- generally given—cost and expertise. The plied thoughtfully, and in some circum- MB/PB categorization was promulgated by stances a simpler system truly will suffice. the World Health Organization in the global For example, in epidemiologic or imple- campaign to eliminate leprosy as a public mentation field studies, simplified classifi- health problem. For some treatment and cation may be justified. control programs, where access to expertise The diversity inherent in the immuno- and other resources is severely limited, the logic spectrum of leprosy may not be re- use of a simplified system of classification is flected in all biological parameters we set reasonable, just as paramedical workers de- out to measure. In some instances, the re- liver medical care where there are no doc- sults may reveal that patients fall into only tors. It has become too easy, however, to use 2 or 3 groups. To discover this is not a fail- this as an excuse to justify a non-critical ac- ure, nor is it wasted effort. Once such find- ceptance of oversimplification that does a ings are established, a 2- or 3-part classifi- disservice to our basic research endeavors. cation scheme for that parameter is accept- Research always requires substantial ex- able. But if the hypothesis is not first pertise and is inherently costly, and is evaluated against the full spectrum, we will nearly always conducted with specifically not know if there were more than 2 or 3 allocated research funds. Nevertheless, we groups. The burden remains on the investi- have watched with dismay as some investi- gators, however, to explain why better clas- gators and collaborative groups apply so- sification was impractical and why a simpli- phisticated molecular and immunologic fied system is actually acceptable in testing techniques to specimens from patients who their hypothesis. If we do not look, we will are classified only as MB or PB, or are clas- not know conditions as they truly exist, and sified only according to the number of skin we may thus overlook important connec- lesions. Some of these manuscripts arrive at tions and implications. our office, and some are published in other Researchers in leprosy have before them, journals. The multiple authorship and ac- at all times, one of the great immunological knowledgments of support in most of these models in nature. An essential part of the papers clearly indicate that financial re- foundation of our understanding of leprosy sources and sophisticated expertise have is the recognition that—clinically, histolog- been brought to bear, and funds have been ically and immunologically—polar lepro- allocated for expensive instruments and matous (LL) differs from borderline lepro- reagents. Experienced clinical leprologists matous (BL), and borderline tuberculoid are virtually always involved in these stud- (BT) differs from polar tuberculoid leprosy ies, implying an availability of sufficient re- (TT). From their first publication in the sources, and it is not acceptable that they do mid-1960’s, the soundness of the theoreti- not take the effort also to obtain skin biop- cal basis for this classification system (2), sies and have them examined by an experi- and the description of practical, straightfor- enced professional. ward criteria to accomplish such classifica- In some instances the pressure to publish tion anywhere in the world (1), were hailed quickly appears to play a role. The Ridley- as major accomplishments by workers Jopling classification system (1) divides pa- within and beyond the field of leprosy. Both tients into five groups, whereas MB/PB the theory and the practical criteria recog- schemes divide into only two groups. It is nize the natural diversity of the immune re- much easier (and faster) to obtain enough sponse in leprosy that has challenged im- patients for a 2-group protocol than for one munology for nearly half a century. A more with 4 or 5 groups. But is this better? Is complete understanding of the basis for this knowledge really advanced by such a sim- diversity and its underlying mechanisms plification? We are very skeptical. No self- will most probably be required before this respecting academic research advisor will disease can be eliminated (i.e., before a accept such an excuse from a student, and highly effective vaccine can be developed). 168 International Journal of Leprosy 2004

The questions posed by this complex im- entific methods available. It is common munopathologic spectrum have perplexed knowledge that funds for leprosy research more than a few great minds who have at- are in much shorter supply than they were a tempted to tackle them. Although support few years ago, and that fewer individuals for leprosy research has declined, it seems a are engaged in leprosy research. This, how- grave mistake for those of us who continue ever, is not an excuse for us to be less rigor- to work on leprosy to surrender one of our ous. To do so would be a travesty to the best scientific assets—a practical and theo- hundreds of thousands of patients still diag- retically sound classification system for lep- nosed every year, to those with lasting dis- rosy. Oversimplification fosters the illusion abilities from this disease, and to all of that this disease is simpler than it appears, those who have gone before us, who did not and easy to understand (or eliminate). In- shrink from a rigorous attempt to under- fection with Mycobacterium leprae elicits stand the complexity of leprosy even the full range of human immunologic re- though they worked without many of the sponses; this is a natural phenomenon and, technical advantages we have today. like the metastasis of cancer, it will not go away if ignored, but will be ignored at our —DMS peril. Today, although the prevalence of lep- REFERENCES rosy has declined worldwide, the number of 1. RIDLEY, D. S., and JOPLING, W. H. Classification of new cases diagnosed annually has not. This leprosy according to immunity, a five-group sys- paradox raises new, important, and interest- tem. Int. J. Lepr. 34 (1966) 255–273. ing questions. Answering these and the 2. SKINSNES, O. K. “The immunological spectrum of other still unanswered questions about lep- leprosy.” In: Leprosy in Theory and Practice. Balti- rosy will require application of the best sci- more: Williams and Wilkins, 1964, pp. 156–162. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) COMMENTARY Linkage of Leprosy Susceptibility to Parkinson’s Disease Genes ABSTRACT In early 2003, an international team of scientists conducted a genome scan in Vietnamese multiplex leprosy families and found that susceptibility to leprosy was significantly linked to region q25 on the long arm of chromosome 6. Further confirmation of the chromosome 6 locus was provided by high-resolution linkage mapping in simplex leprosy families. Now, in a continuation of these findings, the team has pinpointed the chromosome 6 susceptibility locus to the 5′ regulatory promoter region shared by both the Parkinson’s disease gene PARK2 and its co- regulated gene PACRG. The surprising discovery has important implications for the understanding of leprosy pathogenesis and for the strategy of genetic analysis of infectious diseases.

Genomic strategy to identify the chro- A higher density screening with a further 81 mosome 6 leprosy susceptibility gene. Al- SNPs in the 80 kilobase segment further though the localization of the leprosy sus- confirmed the regulatory promoter region ceptibility locus to chromosome 6q25 was and a total of 17 SNP markers were found an enormous breakthrough, the task of iden- associated with leprosy disease. By con- tifying the responsible gene still presented ducting a multivariate analysis, it could be major difficulties, since the target region shown that only 2 of the 17 SNPs captured delineated by the two markers D6S4155 the entire association between the 80 kilo- and D6S1277 covered approximately 6.4 base fragment and leprosy disease. Most million nucleotides that encoded 31 known importantly, the findings in the Vietnamese genes (2). Since the region was too large to families were confirmed in patients from a attempt straightforward comparative DNA second leprosy endemic country (3). A sub- sequencing, another strategy was needed to sequent analysis found that the SNP mark- dissect out the gene. Thus a panel of 64 ers associated with leprosy in Vietnam were single nucleotide polymorphisms (SNPs) also associated with leprosy susceptibility was selected that tagged each of the 31 in 975 unrelated individuals from Brazil. In genes in the core interval by at least one both populations, the risk alleles are associ- SNP marker (3). The SNP markers used for ated with leprosy per se, meaning that per- the Mira, et al. study were selected either sons carrying the risk alleles would be directly from the human SNP database or equally likely to develop PB as MB. There- obtained by comparative sequencing of un- fore, the cause of susceptibility is likely related Vietnamese individuals. Next, DNA to involve an early, common cellular path- samples were isolated from all members of way used by the M. leprae bacillus. While 197 Vietnamese families composed of two the study conclusively implicates PARK2 healthy parents and one leprosy-affected and/or PACRG in leprosy pathogenesis, the child, so called “simplex” families. These question if any of the leprosy-associated DNAs were genotyped for the 64 SNPs, SNPs is directly and causally involved in and systematic analysis of association with leprosy susceptibility remains unanswered. leprosy disease was performed for each PARK2 and PACRG genes reveal the SNP marker (“association scan”). Impor- function of a novel cellular pathway in sus- tantly, the phenotype employed was leprosy ceptibility to leprosy infection. The possible disease independent of specific clinical pau- identity of a “leprosy per se” pathway was cibacillary or multibacillary forms (PB or revealed through knowledge of the function MB) of the disease. The association scan of the PARK2 and PACRG genes (1, 5). showed that SNPs with strongest associa- Mutations in PARK2 are responsible for tion were located in an 80 kilobase frag- familial early-onset Parkinson disease (PD), ment that overlapped the promoter (regula- which represents approximately 3% of all tory region) of the PARK2 (parkin) and PD cases. The mutations in PARK2 in PD PACRG (parkin co-regulated gene) genes. are not found in leprosy patients. The 169 170 International Journal of Leprosy 2004

PARK2 (parkin) gene product is an ubiqui- rooted in the framework provided by the tin-protein ligase, which activates depo- Human Genome Project, the identification sition of certain proteins such as alpha- of this novel and entirely unexpected lep- synuclein in so-called intracellular Lewy rosy susceptibility locus also provides a bodies. The lack of ubiquitin ligase activity good example that recent advances in ge- in patients with PARK2 mutations causes nomics can be used for the study of dis- protein accumulation and neurodegenera- eases primarily prevalent in resource-poor tion. PACRG appears to be involved in the countries. Taken together, the papers by transport of polyubiquitylated proteins to Mira, et al. provide a general framework for the proteasome. Overall, PD is a complex the genetic analysis of complex infectious disease with both genetic and environmental diseases. Above all, it is hoped that the factors, and it has been suggested that infec- novel link of leprosy susceptibility to the tions may trigger its onset. However, neither ubiquitin proteolysis pathway will yield PARK2 or PACRG genes have yet been as- some insight to the transmission of leprosy, sociated with susceptibility to any infectious a disease which has so far evaded eradica- disease other than leprosy. One important tion despite many years of effective drugs aspect to determine then is whether the and case finding (6). PARK2 or PACRG associations are found —Ellen Buschman only in leprosy, or are associated with other —Emil Skamene1 mycobacterial diseases, such as tuberculosis, and, if the polymorphisms associated McGill Center for the Study of with leprosy also predict risk of PD. Host Resistance, In vitro experiments to determine the Montreal, Canada function of the leprosy susceptibility gene. REFERENCES The primary focus now should be to establish a connection between the risk alleles for lep- 1. KITADA, T., ASAKAWA, S., HATTORI, N., MATSUM- rosy susceptibility, the ubiquitin proteolysis INE, H., YAMAMURA, Y., MINOSHIMA, S., YOKOCHI, M., MIZUNOM Y., and SHIMIZU, N. Mutations in the pathway and the course of M. leprae infec- parkin gene cause autosomal recessive juvenile tion and growth. There are many unknowns parkinsonism. Nature 392 (1998) 605–608. to this next phase of experimentation. For 2. MIRA, M. T., ALCAIS, A., NGUYEN, V. T., MORAES, example, it is not known whether the lep- M. O., DI FLUMERI, C., VU, H. T., MAI, C. P., rosy susceptibility alleles would up or down NGUYEN, T. H., NGUYEN, N. B., PHAM, X. K., regulate the PARK2 or PACRG encoded SARNO, E. N., ALTER, A., MONTPETITM A., MORAES, proteins, or affect the cellular ubiquitin M. E., MORAES, J. R., DORE, C., GALLANT, C. J., pathway. In the context of further functional LEPAGE, P., VERNER, A., VAN DE VOSSE, E., HUD- studies, it is revealing that both PARK2 and SON, T. J., ABEL, L., and SCHURR, E. Susceptibility PACRG are expressed by Schwann cells to leprosy is associated with PARK2 and PACRG. Nature 427 (2004) 636–640. and monocyte-derived macrophages (3). 3. MIRA, M. T., ALCAIS, A., VAN THUC, N., THAI, V. Nevertheless, testing of the risk alleles in H., HUONG, N. T., BA, N. N., VERNER, A., HUDSON, patients will have to await a reliable func- T. J., ABEL, L., and SCHURR, E. Chromosome 6q25 tional assay for biological activity of M. is linked to susceptibility to leprosy in a Viet- leprae. In parallel, it is also hoped that the namese population. Nat. Genet. 33 (2003) 412–415. analysis of M. leprae and the parkin genes 4. SALGAME, P., ABRAMS, J. S., CLAYBERGER, C., GOLD- will yield information relevant to the neuro- STEIN, H., CONVIT, J., MODLIN, R. L., and BLOOM, B. logical aspects of Parkinson’s disease. R. Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science 254 CONCLUSION (1991) 279–282. 5. WEST, A. B., LOCKHART, P. J., O’FARELL, C., and The clinical spectrum of leprosy has long FARRER, M. J. Identification of a novel gene linked been recognized as an immunological to parkin via a bi-directional promoter. J. Mol. Biol. model in which various aspects of human T 326 (2003) 11–19. cell subset and cytokine function can be 6. WORLD HEALTH ORGANIZATION. Leprosy. Global characterized (4). It is fitting that leprosy situation. Weekly. Epidemiol. Rec. 77 (2002) 1–8. has proven once again to be a model for 1 Reprint requests to: Emil Skamene, Montreal Gen- molecular genomics, by providing the first eral Hospital, Room A6-149, 1650 Cedar Avenue, infectious disease locus isolated by posi- MONTREAL, Que, H3G 1A4, Canada. Email: tional cloning. Since this success is largely [email protected] INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) COMMENTARY Neuropathic Pain in Leprosy1

ABSTRACT Neuropathic pain appears to be much more common in leprosy than has been generally appreciated. Emphasis in leprosy control programs has been on the distribution of multidrug therapy, on early and better detection, and on the prevention of disability related to anesthetic limbs. Most have thus been inattentive to the problem of neuropathic pain in leprosy patients. Neuropathic pain does not respond to the usual analgesics employed for reactions, for example, and so it is important that those treating leprosy patients give this problem the special attention it requires, both in diagnosis and in treatment. RÉSUMÉ Les douleurs neurogènes de la lèpre pourraient bien être beaucoup plus fréquentes que ce qui a été considéré auparavant. L’effort des programmes de contrôle de la lèpre a été orienté vers la distribution de la polychimiothérapie, une détection meilleure et plus précoce et dans la prévention des handicaps associés à des membres anesthésiés. La plupart de ces programmes ont ainsi apporté peu d’attention au problème des douleurs neurogènes chez les patients hanséniens. Les douleurs neurogènes ne répondent pas aux analgésiques usuels em- ployés pour traiter par exemple les réactions et il est donc important que les personnes en- gagées dans le traitement des patients hanséniens considèrent particulièrement cet aspect, tant en ce qui concerne le diagnostic que le traitement. RESUMEN El dolor neuropático en la lepra parece ser mucho más común de lo que usualmente se considera. Los programas de control de la lepra han hecho mucho énfasis en la distribución de drogas para la poliquimioterapia, en la temprana y mejor detección de la enfermedad, y en la prevención de las incapacidades relacionadas con los miembros anestésicos. Muchos programas han puesto poca atención al problema del dolor neuropático en los pacientes con lepra. El dolor neuropático no response a los analgésicos usualmente empleados en las reacciones de la lepra, por ejemplo. Por esto, es muy importante que los encargados del tratamiento de los pacientes con lepra den a este problema la atención especial que requiere, tanto en el diagnóstico como en el tratamiento.

Stump, et al.’s paper, “Neuropathic pain markably few papers published on the sub- in Leprosy patients,” published in this issue ject in the world leprosy literature. Yet of the JOURNAL, is a timely and important Stump and his colleagues report that 56% contribution to the evaluation and manage- of the 358 patients assessed for neuropathic ment of leprosy sufferers. In the wider pain in his study either had experienced or medical world the management of chronic were experiencing episodes of pain of suffi- pain is developing as a specialty in its own cient intensity to interfere with activities of right complete with journals and interna- daily life or sleep. The statistics they ad- tional conferences devoted to the subject. It duce are in line with findings from the few is an interesting coincidence that a review other studies that have been carried out article on the same topic has just been pub- amongst leprosy sufferers. How could we lished in the most recent edition of Leprosy have missed it for so long? Review (1). There are several overlapping answers to In the leprosy world, we have been slow that question. For several years most pro- to catch on to the existence of chronic neu- grams, NGO and Government alike, have ropathic pain occurring in leprosy patients. simply been extremely busy and focussed Hastings’ textbook Leprosy (1995) does not on case finding and multi-drug therapy mention it at all, and there have been re- (MDT) administration. This has been ex- traordinarily successful in reducing preva- 1 Received for publication on 23 March 2004. Ac- lence and clearing the backlog of patients in cepted for publication on 14 April 2004. the community. In many places the heat has 171 172 International Journal of Leprosy 2004 now come out of that approach and perhaps Perhaps a good word to use here is demys- there is a little more space to reflect on what tify. Neuropathic pain is regarded by some our patients—including “cured” patients— doctors as a little technical, rarefied even. are actually experiencing. Another impor- The subject needs to be demystified, it tant focus in leprosy programs and in re- needs to make the jump to become the reg- search of the last decade or so has been the ular. detection and management nerve function Perhaps the situation we are in is akin to impairment (NFI) and the prevention of dis- the situation that existed a decade or so ago, ability. Both of these foci—detection and before the widespread use of corticosteroids treatment, and prevention of disability— at “field level” to treat acute NFI and reac- have had anaesthesia at the hub, since it is tions. We knew how to measure NFI, and the absence of sensation that leads both to we had an effective drug, prednisolone, but the diagnosis of leprosy (and therefore to it took a paradigm shift in thought and prac- treatment), and to the development of the tice for this technology to be widely and most damaging disabilities and consequent simply applied so that the maximum num- handicap and stigma. We have been so at- ber of people could benefit. In the same tuned to painlessness that we have missed way, it is known how to diagnose neuro- the fact that a very significant proportion of pathic pain (and it is not difficult), and at our leprosy sufferers experience pain as least one very cheap and effective drug is part of their dis-ease. Furthermore, they available (amitriptyline). A widespread ap- may continue to suffer long after they have plication of this knowledge down to the been declared “cured” and are lost to grassroots level could be of considerable follow-up. That we should have been so benefit to a large number of people. deaf and blind to this most basic of com- The current cut-and-dried WHO recom- plaints—pain—is extraordinary. mendations for the treatment of leprosy fo- As already alluded to, there has been a cuses very much on bacteriological cure paucity of studies into neuropathic pain car- with discharge after relatively short courses ried out amongst leprosy sufferers and very of treatment. It is well known that this few references to it at all in the world liter- largely ignores the existence of new nerve ature. There is a clear need for more re- damage after release from treatment, but to search into this subject and for the findings date there has been very little appreciation to be applied as rapidly as possible. How- of the way that this ignores the presence of ever, much is known already about the di- neuropathic pain among “cured” leprosy agnosis and management of neuropathic sufferers, as Stump et al. points out in his pain in general that could easily be applied conclusions. Indeed, the prevalence of neu- now. If it is as common as Stump et al. sug- ropathic pain he found is actually higher gests—and it probably is—then we should than that often quoted for NFI amongst lep- get on with it now. rosy patients. If we are to begin to help leprosy suffer- In summary, Stump’s paper both docu- ers with chronic neuropathic pain then as a ments and highlights the existence of a com- first step we must ask them about it. It mon and significant problem amongst lep- should not be left to experts in research cen- rosy patients, one that has been remarkably ters to ask the questions; it should become overlooked. There is a need to demystify the part of the routine history taking of every diagnosis and treatment of neuropathic pain paramedical worker. Before that can hap- and to develop simple strategies that will en- pen, training institutions must incorporate able the widespread application of simple this message. Leprosy workers need to un- and effective techniques for its manage- derstand the difference between nociceptive ment. and neuropathic pain, and Stump, et al. rightly draw attention to this. Crucially, it —Richard Croft should be understood that neuropathic pain will not respond to simple analgesia, but REFERENCE rather to different drugs such as tricyclics 1. HAANPÄÄ, M. LOCKWOOD, D. N. J., and HIETA- and anticonvulsants. Then, the treatment of HARJU, A. Neuropathic pain in leprosy. Lepr. Rev. neuropathic pain must become mainline. 75 (2004) 7–18. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) CORRESPONDENCE This department is for the publication of informal communications that are of interest because they are informative and stimulating, and for the discussion of controversial matters. The mandate of this JOURNAL is to disseminate information relating to leprosy in particular and also other mycobacterial diseases. Dissident comment or interpretation on published research is of course valid, but personality attacks on individuals would seem unnecessary. Political comments, valid or not, also are unwelcome. They might result in interference with the distribution of the JOURNAL and thus interfere with its prime purpose. Should Large Lesions of Leprosy Be Considered As “Multibacillary” for Treatment Purposes Even If the Total Number of Lesions Is Less Than Five?1

TO THE EDITOR: and the host response. Considering that sensory impairment and pathological hypopig- According to World Health Organization mentation in leprosy are due to this host re- (WHO) recommendations: “five, or lesser sponse by the body in the fight against the number of lesions” of leprosy should be leprosy bacilli, it is likely that, the larger the treated as paucibacillary (PB) leprosy and lesions of leprosy, the higher the number of should be given 6 months treatment with ri- bacilli that cause the pathology. A granuloma fampicin monthly and dapasone daily (5, 6). which originates due to one or more bacilli in In this type of simplified classification, the a given area can only cause a very limited size of the patches are not considered. How- spread of its effects, e.g., focal sensory loss in ever, we feel that size of the patch should be the affected area. The fact that inoculation of considered on deciding whether to treat a atypical mycobacteria causes a granuloma in case as PB with two drugs for 6 months, or the immediate vicinity of the inoculation, and as multibacillary (MB) leprosy with three that it spreads very slowly, suggests that pro- drugs for a year. Categorizing leprosy as PB liferation of bacteria are necessary to cause a or MB is particularly important in areas larger lesion. This also means that if there is where treatment is commenced without any no proliferation of bacilli in tuberculoid lep- bacteriological and histopathological confir- rosy, there can not be evolution of a small mation. Even in the time honored Ridley- patch, to become a large patch. This concept Jopling Classification and its modifications, is further supported by the fact that even in a large patches of leprosy are considered as a Type I leprosy reaction, there is no real lateral feature, more commonly found in border- spread of a leprosy lesion, though the existing line, borderline tuberculoid, or subpolar lep- lesions temporarily become inflamed. This romatous leprosy (2, 4), giving due consider- suggests that a pure immunological response ation to the size of the lesions. without an increase in bacilli is unlikely to Histopathologically, in tuberculoid leprosy cause a lesion to spread peripherally, to pro- there are tubercles composed of epithelioid duce a large hypopigmented patch. However, cells. This is due to the process of destruction it is known that lowering of one’s cell medi- of lepra bacilli by histiocytes (3). The granu- ated immunity is important in promoting the lomatous reaction thus produced is the result spread of leprosy lesions. In this situation, the of a combination of the presence of bacilli patient’s ability to destroy the multiplying lepra bacilli is impaired, allowing the le- 1 Received for publication on 23 October 2003. Ac- sion(s) to enlarge; as in the case of a tubercu- cepted for publication on 2 April 2004. Reprint requests to: S. Prarasd W. Kumarasinghe, loid leprosy (PB) lesion or lesions in an un- Consultant Dermatologist, National Skin Center, Sin- treated patient, evolving towards the “lepro- gapore, No. 1, Mandalay Road, Singapore, 308205. matous pole” (MB) over several years. E-mail: [email protected] Unless many individual cutaneous nerve 173 174 International Journal of Leprosy 2004

fibers are affected by separate bacilli, there eral instances where MB cases had been can not be sensory impairment in a large categorized and treated as PB, by others, area, involving the total area of the hy- especially by public health workers, due to popigmented macule. This is different to their strict categorization according to the distal sensory impairment due to a nerve “number of patches.” These cases were trunk involvement, for example, sensory subsequently given the MB treatment regi- impairment along ulnar nerve distribution. men. In hospital settings, facilities for Furthermore, in monitoring tuberculoid or biopsy and smears with microbiological borderline tuberculoid leprosy, peripheral evaluation are available and clinicians do extension of a lesion is considered to be a no go by the number of patches alone for feature of failure of treatment or relapse. treatment. Although relapses of leprosy after treat- Long term follow-up of patients with ment are reportedly uncommon, many au- large macules of leprosy treated with the thorities feel that they may be underesti- standard WHO treatment regimens would mated (1). If an MB case is misdiagnosed be necessary to ascertain whether relapse and treated with dapsone daily and ri- rate is higher in this group of patients. Per- fampicin monthly as a PB case, that patient sonal experience suggests this group has receives only 6 doses of the bactericidal drug more relapses or non responders. rifampicin before stopping the treatment. A consensus on the duration and type of This would be totally inadequate. Some au- treatment for large macules of leprosy thorities even believe that MB treatment would be desirable for places where should be continued for 24 months rather histopathological and microbiological facil- than the WHO recommended 12 months (1). ities are not available. It appears prudent to In countries where leprosy is still highly treat such cases with MB treatment regimen. prevalent, follow-up after discharge from active treatment is unsatisfactory. Therefore —S. Prasad W. Kumarasinghe, many relapses or suboptimal treatments may M.D., MBBS, FCCP, FAMS go unnoticed for many years. Consultant Dermatologist Considering the above facts, we feel that National Skin Center, Singapore where a large patch (more than 10 cm in diameter) of leprosy is present, irrespective of —M. P. Kumarasinghe, M.D., MBBS, the size of the other lesion or lesions, the FRCPA, Dip Cy Path, FCCP, FAMS patient should be treated as MB and given Senior Consultant Pathologist treatment at least for 12 months. Just as “5 Singapore General Hospital or less leprosy macules are considered as PB” (as recommended by the WHO) is an arbitrary limit, “the dimensions of a lesion” REFERENCES is also an arbitrary measurement, for places where microbiological and histopathologi- 1. INTERNATIONAL LEPROSY ASSOCIATION TECHNICAL cal services are unavailable. It should also FORUM. Report of the International Leprosy Associ- ation Technical Forum. Int. J. Lepr. Other My- be emphasized that counting lesions can be cobact. Dis. 70(1)(Suppl) (2002) S3–S60. erroneous if the whole body is not carefully 2. JOPLING, W. H., and MCDOUGALL, A. C., EDS. checked by the healthcare worker. A person Handbook of Leprosy, 4th edn. New Delhi: CBS may have 5 easily visible lesions, but there Publishers, 1992, pp. 22–44. may be another small lesion or lesions in 3. MEHREGAN, A. H., and HASHIMOTO, K. Pinkus’ unsuspected places such as nasal cleft, a Guide to Dermatohistopathology, 5th edn. East toe, or an elbow posteriorly. In such a situa- Norwalk: Appleton and Lange, 1991, pp. 281–290. tion, a patient would receive only PB treat- 4. RIDLEY, D. S., and JOPLING, W. H. Classification of ment. However, it is highly unlikely that a leprosy according to immunity, a five-group sys- large patch (>10 cm) of leprosy would go tem. Int. J. Lepr. 34 (1966) 255–273. 5. WHO EXPERT COMMITTEE ON LEPROSY. Seventh undetected by the patient or the clinician or report. Geneva: World Health Organization, 1998. the health care worker. Tech. Rep. Ser. 874. In our experience with cases of leprosy 6. WORLD HEALTH ORGANIZATION. Guide to Elimi- in the last two decades (mostly when work- nate Leprosy as a Public Health Problem, 1st ed. ing in Sri Lanka) we have encountered sev- Geneva: World Health Organization 2000. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) Improved Protocol for PCR Detection of Mycobacterium leprae in Buffered Formalin-Fixed Skin Biopsies1

TO THE EDITOR: Therefore, we here report the development of improved protocol for PCR diagnosis of It is often difficult to diagnose early lep- M. leprae from formalin-fixed specimens rosy by clinical criteria alone. Frequently, and report the results of a blind study using the patients’ skin smears are negative, clin- a large number of biopsies obtained from ical findings inconclusive, and history unre- both paucibacillary and multibacillary lep- liable and subjective. In many such cases, rosy patients. routine histopathology may also be nonspecific as Mycobacterium leprae cannot be MATERIALS AND METHODS demonstrated in the tissues of many early After obtaining informed consent and lesions. Therefore, the demonstration of M. using aseptic precautions and techniques leprae, or any of its components in a tissue such as cleaning of skin with iodine solu- biopsy, is crucial and important to reach a tion and alcohol, 5 mm punch biopsies were definitive diagnosis. taken from 78 patients of Indeterminate, Polymerase chain reaction (PCR) amplifi- polar tuberculoid (TT), borderline tubercu- cation has been successfully used to detect loid (BT), borderline (BB), borderline- extremely low numbers of M. leprae in fresh lepromatous (BL), or polar lepromatous unfixed human skin biopsy specimens, pro- (LL) leprosy cases from the Ghatampur viding powerful direct and unequivocal tests field area of Kanpur district, India. For for diagnosis of leprosy (1, 2, 3, 4, 5, 7, 8, 10, 11). controls, biopsies from 12 cases of other However, these studies required the speci- dermatological conditions (Pitrrysis alba, mens to be processed and analyzed promptly, Tinea versicolor, and Vitiligo) from the in addition to being properly stored and same population were also taken and ana- transported. Adoption of PCR technology lyzed using the same protocol. These biop- for detecting M. leprae and/or its compo- sies were fixed and transported in buffered nents in fixed tissues would give clinicians formalin and processed in a blind manner the option of examining biopsy specimens after 5 to 7 days. The scientists performing for the presence of M. leprae, which along the gene amplification assay were unaware with histology would help in arriving at a of the diagnosis of the case, and that biop- definite diagnosis. This PCR technology sies of other dermatological cases were would be of great help to arrive at a quick also included in the study. Each biopsy was and conclusive diagnosis, identify and treat divided into two parts: one part was used early cases of leprosy, to differentiate lep- for histopathology and the other for the rosy from non-leprosy cases and also for gene amplification assay. epidemiological purposes. Initially, the biopsies were kept in 15 ml While earlier reports have demonstrated of sterile distilled water for 8 hrs, which that buffered formalin was good for both had been determined to be optimal after histology and PCR detection, formalin fixa- testing with various time periods. The biop- tion of skin biopsy specimens for longer sies were then aseptically homogenized in 1 than 24 hr has been reported to have an in- ml of sterile T. E. buffer (Tris 10 mM, hibitory effect on PCR amplification (2). EDTA 0.1 mM, pH 8.0) using pestle and mortar in a bio-safety hood. 1 Received for publication on 19 November 2002. A technique based on the principle of a Accepted for publication on 13 January 2004. combined physiochemical approach, first Reprint requests to: Dr. Kiran Katoch, M.D. Deputy freeze-boiling and then treatment with Director (Sr. Grade) and Head, Medical Unit I, Central 7 JALMA Institute for Leprosy and Other Mycobacterial lysozyme-proteinase K ( ) was used for ex- Diseases (ICMR) Tajganj, Agra-282 001 UP–India. traction of DNA. Briefly, the homogenates E-mail: [email protected]; [email protected] in T. E. buffer were frozen, thawed, and

175 176 International Journal of Leprosy 2004 then enzymatically treated at 37°C, first THE TABLE. Positivity rates for gene with lysozyme (3 mg/ml) for 2 hr followed amplification targeting 36 kDa gene in by proteinase K (250 ug/ml) treatment for biopsies collected and fixed in buffered for- one hour at 65°C (9). After de-proteinization malin. with chloroform: isoamyl alcohol (24:1), DNA was precipitated with 0.6 volume of Type of leprosy No. No. % isopropanol. This was dissolved in T. E. tested positive positivity buffer and stored at –20°C until further use. Indeterminate 25 12 48 Primers and the gene amplification method TT/BT 47 26 55 as described by Hartskeerl, et al. (3) and as BB/BL 6 5 83 used by de Wit, et al. (1) and Singh, et al. (8) Non leprosy cases 12 0 Nil were followed for the amplification of 36kD gene of M. leprae. Primers were obtained commercially from Bioserve Biotechnolo- It would be ideal if the PCR could be per- gies (India) Ltd. In all cases, 35 cycles were formed on fixed specimens in which both used for the amplification and confirmation the detection of M. leprae and/or its compo- was done by the southern blot analysis with nents, and correlation with the histopathol- random digoxygenin (DIG) labeled ampli- ogy, can be made. Over the years, different fied 36 kDa gene fragment of M. leprae as a fixatives have been tried for histology and probe. DIG labeling and detection was done the detection of RNA/DNA of the causative by using a kit from Roche Diagnostics (Cat. organisms. Fiallo, et al. (2) and deWit, et al. No. 1093651). (1) have observed significant reduction in sensitivity of PCR for M. leprae DNA when RESULTS Zenker’s fluids (mercuric chloride) fixatives The percentage positivity of biopsy sam- are used. Although Carnoy-Lebrun’s fluid is ples for M. leprae by gene amplification is recommended for fixation of tissues for op- shown in The Table. It was observed that timal staining of glycogen and RNA, unfor- 48% of Indeterminate, 55% of TT/BT, and tunately this fixative has the potential for 83% of BB/BL were positive by gene am- chemical modification, resulting in degrada- plification. The lone BB patient who was tion and excessive depurination of the DNA negative by this assay was smear-negative due to the acidic nature of fixative. Ethanol and had taken multi-drug therapy (MDT) was reported to be a good fixative for PCR for more than a year prior to the biopsy. analysis, but this causes excessive shrinkage None of the specimens of non-leprosy cases of the tissues, and is not recommended if were positive by this method, demonstrat- the same tissue is to be used for histology ing the specificity of the method. and PCR analysis. Neutral buffered forma- For histologic investigations, tissue sam- lin fixation has been reported to be satisfac- ples are mostly stored as formalin-fixed, tory for subsequent PCR, but significant re- paraffin-embedded blocks. Widening the duction in the level of PCR signals for M. applicability of amplification techniques to leprae DNA was observed after fixation of formalin-fixed blocks could improve the 4 to 7 days (2). In the case of formalin-fixed, routine diagnosis of mycobacterial infec- paraffin-embedded specimens of smear- tions. This is particularly important in lep- positive tuberculosis, the highest sensitivity rosy as M. leprae cannot be grown in any in rates have been obtained by amplifying the vitro system. Definite diagnosis of early and highly repetitive IS 6110 insertion se- suspicious cases of leprosy is required, so quence, and the different primers tested that the patients can be treated at an early showed a sensitivity ranging from 80% to stage to prevent development of deformi- 87%, whereas a lower positivity of 47% to ties, rather than following them up closely 80% with primer targeting single copy gene and treating them when definite clinical was observed in same study (6). signs appear. While large studies are neces- In the present study, after fixation of sary to gain more confidence, published in- biopsy specimens and processing these formation shows that with PCR technology, within 5 to 7 days for PCR, 83% positivity identification of M. leprae is sensitive, as was observed in multibacillary BB/BL pa- well as and specific (1, 3, 5, 7, 8, 10). tients and 43% to 55% in paucibacillary 72, 2 Singh, et al.: M. leprae-PCR on Fixed Biopsies 177

(Indeterminate and BT). This is similar to —V. D. Sharma, Ph.D. the positivity rates of 50% to 70% for Assistant Director, ICMR smear-negative and about 100% in smear- positive cases observed with fresh frozen —D. S. Chauhan, Ph.D. biopsy specimens in earlier studies (5, 7, 8). Research Officer, ICMR Prior to this study it has been reported that formalin fixation of tissues for longer than —Mallika Lavania, M.Sc. 24 hrs is detrimental for PCR detection of Research Assistant, ICMR M. leprae. It appears that this study provides a major improvement in the ability to —Pragya Sharma, M.Sc. detect M. leprae in tissues fixed in buffered Research Assistant, ICMR formalin. This improvement is most likely due to —Mohini Sharma, M.Sc. the soaking of the tissues for 8 hrs in 15 ml Research Assistant, ICMR of sterile distilled water prior to homoge- nizing tissues, lysing and extraction of —K. Katoch M.D. DNA. In the study of Fiallo, et al., tissues Deputy Director (Sr. Grade), ICMR were soaked in HBSS solution for 30 min- Tajganj, Agra-282001 India utes. In the present study, the tissues were collected as well as processed in buffered —S. Benara, M.D. formalin which did not inhibit the PCR am- Deputy Director plification, and the results are comparable Institute for Research in Medical Statistics to the earlier reports when PCR amplifica- (ICMR) tion was done in freshly biopsied samples obtained in the outpatient clinic of the Insti- —Padam Singh, Ph.D. tute. It appears that the approach of collect- Additional Director General ing the specimens in buffered formalin will Indian Council of Medical Research, be very suitable for field situations and will Ansari Nagar, New Delhi 11002, India have the added advantage of the same specimen being used for histology, probe appli- REFERENCES cation in solution, as well as for in situ ap- 1. DE WIT, M. Y. L., FABER, W. R., KRIEG, S. R., plications. This, however, is a pilot study DOUGLAS, J. T., LUCAS, S. B., MONTREEWASUVAT, and trends need to validated by similar N., PATTYN, S. R., HUSSAIN, R., PONNIGHAUS, J. studies on a large number of specimens us- M., HARTSKEERL, R. A., and KLASTER, P. R. Ap- ing the same method by other workers. plication of polymerase chain reaction for the detection of Mycobacterium leprae in skin tissues. J. Clin. Microbiol. 29 (1991) 906–910. Acknowledgment. Authors are thankful to all tech- 2. FIALLO, P., WILLIAMS, D. L., CHAN, G. P., and nical and secretarial colleagues for support in carrying GILLIS, T. P. Effects of fixation on polymerase out this study and preparation of this manuscript. Gift chain reaction detection of Mycobacterium leprae. of reagents/plasticware by LEPRA (UK) is gratefully J. Clin. Microbiol. 30 (1992) 3095–3098. acknowledged. This study has been supported by the 3. HARTSKEERL, R. A., DE WIT, M. Y. L., and Department of Biotechnology, Govt. of India (Grant KLASTER. P. R. Polymerase chain reaction for the No. BT/IS/002/95-1998–2002). detection of Mycobacterium leprae. J. Gen. Mi- crobiol. 135 (1989) 2355–2364. —H. B. Singh, Ph.D. 4. JAMIL, S., WILSON, S. M., HACKEL, M., HUSSAIN, Research Assistant R., and STOKER, N. G. A colorimetric PCR Central JALMA Institute For Leprosy method for the detection of Mycobacterium leprae And Other Mycobacterial Diseases in skin biopsies from leprosy patients. Int. J. Lepr. (ICMR) Other Mycobact. Dis. 62 (1994) 512–526. 5. KATOCH, V. M. New investigative techniques in —V. M. Katoch, M.D. leprosy. In: Dermatology Update. Mumbai: Bha- lani Publishing House, 1998, pp. 165–174. Director, ICMR 6. MARCHETTI, G., GORI, A., CATOZZI, L., VAGO, L., —M. Natrajan, MBBS, DVD NEBULONI, M., ROSSI, M. C., ESPOSTI, A. D., BAN- DERA. A., and FRANZETTI, F. Evaluation of PCR in Deputy Director, ICMR detection of M. tuberculosis from formalin-fixed, 178 International Journal of Leprosy 2004

paraffin-embedded tissues: Comparison of four T., DALE, J., EISENACH, K. D., GICQUEL, B., HER- amplification assays. J. Clin. Microbiol. 36 (1998) MANS MARTIN, C., MEDAM, R., SHINNICK, T. M., 1512–1517. and SMALL, P. M. Strain identification of Myco- 7. SHARMA, R. K., KATOCH, K., SHIVANNAVAR, C. T., bacterium tuberculosis by DNA fingerprinting SHARMA, V. D., NATRAJAN, M., BHATIA, A. S., recommendations for standardized methodology. SAXENA, N., and KATOCH, V. M. Detection of M. J. Clin. Microbiol. 31 (1993) 406–409. leprae by gene amplification combined ethidium- 10. WILLIAMS, D. L., GILLIS, T. P., FIALLO, P., JOB, C. bromide staining and probe hybridization. Int. J. K., GELBER, R. H., HILL, C., and IZUMI, S. Detec- Lepr. Other Mycobact. Dis. 64 (1996) 409–416. tion of Mycobacterium leprae and the potential of 8. SINGH, H. B., KATOCH, K., NATRAJAN, M., monitoring anti-leprosy drug therapy directly from SHARMA, R. K., GUPTA, U. D., SHARMA, V. D., skin biopsies by polymerase chain reaction. Mol. SINGH, D., CHAUHAN, D. S., SRIVASTAVA, K., and Cell. Probes 6 (1992) 401–410. KATOCH, V. M. Effect of treatment on PCR posi- 11. WOODS, S. A., and COLE, S. T. A rapid method for tivity in multibacillary leprosy patients treated detection of potentially viable Mycobacterium with conventional and newer drugs ofloxacin and leprae in human biopsies; a novel application minocycline. Acta. Leprol. 11 (1999) 179–182. of PCR. FEMS Microbiol. Letts. 65 (1989) 9. VAN EMBDEN, J. D. A., CAVE, M. D., CRAWFORD, J. 305–310. INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) NEWS and NOTES This department furnishes information concerning institutions, organizations, and individuals engaged in work on leprosy and other mycobacterial diseases, and makes note of scientific meetings and other matters of interest.

Notice. In recognition of his life long tegration of leprosy services within the contribution for the cause of leprosy, Dr. R. general health system Ganapati, Director, Bombay Leprosy Proj- ect (BLP) and the Past President of the In- This book offers guidance to public dian Association of Leprologists (IAL) was health managers and decision-makers honored at the 24th Biennial Conference of at national and regional level faced the IAL in Haldia, West Bengalon 28th with the task of integrating leprosy February 2004. He received a memento services into the general health system. from the Member of Parliament, Mr. Lak- The guide systematically describes all shman Seth. Dr. Ganapati had also earlier the steps involved in the integration held positions of the Honorary Secretary process, from situation analysis and the and Vice President of IAL. development of a plan of action, to implementation and evaluation. It is founded on the experience of countries that have already gone through the integration process, and Notice from the ILEP. Three guides aims to help ensure that the lessons which have recently been published by learned during these experiences are ILEP on the topics of training in leprosy, in- applied more widely. tegration of leprosy services, and how to carry out a skin smear. A brief background 36 pages, 24 cm × 16 cm to each is given below. These publications ISBN 0947543279 can be ordered through [email protected]. 3. How to do a skin smear examination 1. ILEP Technical Guide: Training in for leprosy: ILEP Learning Guide Leprosy Three (available in English and French) This guide is aimed at staff who or- This guide consists of three laminated ganize, support and run leprosy train- and detachable A4 sheets, and is a ing activities at national, regional or clearly presented and durable reference district level. It offers practical guid- guide for use in the clinic or laboratory. ance on topics such as assessing train- It describes how to carry out all the ing needs, effective teaching and steps involved in taking a skin smear, learning methods, online learning, on- and is targeted largely at health workers the-job training and organizing evalu- or laboratory staff with responsibility ations. for taking and reading skin smears, as It has been developed in consultation well as laboratory technicians who may with a number of practitioners who be required to prepare the reagents. have extensive experience in leprosy 6 A4 pages in 3 detachable lami- training, and this is reﬂected in the nated sheets. many practical tools and ideas that it contains. It will be a useful guide for training managers, facilitators, trainers, supervisors and other teaching staff. × From IDEA. Leprosy and Human Rights. 64 pages, 24 cm 16 cm The ofﬁcial discussion of leprosy as a hu- ISBN 0947543260 man rights issue continued from May 29 2. ILEP Technical Guide: Facilitating the through May 31, 2004 during the 60th Ses- Integration Process—A guide to the in- sion of the UN Human Rights Commission

179 180 International Journal of Leprosy 2004 in Geneva, Switzerland. Representatives of whose lives have been challenged by lep- The Nippon Foundation, IDEA and the rosy have had their most basic human rights Sasasakawa Memorial Health Foundation denied by virtually every culture and every met with Acting UN High Commissioner major religion throughout time. Throughout Bertrand Ramcharan and also made presen- this history individuals with leprosy have tations on the denial of human rights expe- often been unjustly “blamed” for their dis- rienced by millions of individuals affected ease, with leprosy regarded as punishment by leprosy and their families. Rights that for supposed wrongdoing. Discussing lep- have been denied include but are not lim- rosy as a human rights issue shifts the bur- ited to: the Right to Education, the Right to den or responsibility for wrongdoing to so- Work, the Right to Freedom of Movement, ciety and thus provides a powerful tool for the Right to Family; the Right to Freedom eliminating the stigma and its associated from Degrading Treatment and the Right to prejudice and discrimination that have de- an Existence Worthy of Human Dignity. nied millions of individuals their rightful For over 3000 years and continuing into place in the world community. the 21st century, the stigma associated with leprosy remains the most persistent and per- “Our exclusion has been taken for vasive form of social injustice, prejudice, granted in the cultures, religions and lan- and discrimination that society has forced guages of society for generations.” upon its fellow human beings. Individuals —Arega Kassa Zelelew, IDEA Ethiopia

Calendar of Meetings and Events Day mm/yy Location Details Contact E-mail

18–23 Jul-04 Montreal 12th International Congress [email protected] of Immunology 26–30 Sep-04 Sydney Joint Meeting of the New Australian Society for Microbiol. [email protected] Zealand and Australian Societies for Microbiology 29–2 Sep/Oct-04 Tehran 7th International Congress Yahya Dowlati [email protected] of Dematology 13–8 Sep/Oct-04 Ethiopia “ALERT Training Program ALERT Training Division [email protected] Tuberculosis, Leprosy and Research Methodology” 30–4 Sep/Oct-04 Boston Infectious Disease Society Infect. Dis. Soc. of America [email protected] of America 11–22 Oct-04 Ethiopia “ALERT Training Program ALERT Training Division [email protected] in Tuberculosis, Leprosy and Research Methodology” 30–2 Oct/Nov-04 Washington, D.C. 44th Interscience Conference on Antimicrobial Agents and Chemotherapy 7–11 Nov-04 Miami 53rd Annual Meeting of the ASTMH American Society of Tropi- cal Medicine & Hygiene 17–21 Nov-2004 Florence, Italy 13th Congress of the Euro- EADV 2004 Florence pean Academy of Derma- tology and Venerology 1–5 Dec-04 Bangkok 9th Western Paciﬁc Congress Congress Secretariat [email protected] on Chemotherapy and Infectious Diseases INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916X) CURRENT LITERATURE

This department carries selected abstracts of articles published in current medical journals dealing with leprosy and other mycobacterial diseases.

General and Historical 181 Chemotherapy 187 Clinical Sciences 192 Immuno Pathology 198 Leprosy 205 Tuberculosis 207 Microbiology 219 Leprosy 224 Tuberculosis 225 Experimental Infections 230 Epidemiology 244 Other Mycobacterial Diseases 245 Molecular and Genetic Studies 254

General and Historical

Alves Moreira, T. [A panorama of Hansen’s South,whereas the rates in the North and Cen- disease: present status and perspectives]. tral East regions almost reach endemic peaks. Hist. Cienc. Saude Manguinhos. 10(Suppl. Working close to patients and ex-patients as- 1) (2003) 291–307. sociations, ﬁrst as a nurse and later in the implementation of policies for Hansen’s disease The interviewee speaks about the endemic issues, Tadiana Alves Moreira stresses the im- disease, which at present contaminates 4.4 out portance of early diagnoses, which avoid the of ten thousand inhabitants in Brazil, the physical damages and deformities that take country with the second highest number of place in the advanced stages of the disease, so patients. When Tadiana speaks about the reducing the stigma over the diseased.—Au- Brazilian participation in the program thor’s Abstract launched by the World Health Organization, she explains that WHO’s objective is to ex- tinguish Hansen’s disease on the planet until Benchimol, J. L., Sa, M. R., Alves da Cruz, 2005. However, she says that Brazilian main Mde. S., and Magalhaes de Andrade, M. goal is to reduce the occurrence of the disease Fight for survival: the life of a Hansen’s to less than one case per ten thousand people. disease sufferer through his correspon- In our country, the structure and the organiza- dence with Adolpho Lutz. Hist Cienc tion of this program, which comprehends the Saude Manguinhos. 10(Suppl. 1) (2003) education and specialization of professionals 361–396. [Article in English, Spanish] in order to guarantee early diagnoses, as well as patients’follow-up during treatment, is de- This project presents the complete set of veloped by Sistema Unico de Saude (SUS) letters between the family of a Hansen’s dis- and has been implemented in all the states of ease (leprosy) sufferer in the state of Maran- the federation. The chemotherapy treatment hao, in the Northeast of Brazil, and the doc- lasts about a year, when taken seriously. In tor and bacteriologist Adolpho Lutz. For many cases, the disease comes back a while more than twenty years Fabricio Caldas de later. Tadiana comments on the differences of Oliveira and Numa Pires de Oliveira, father the disease according to the different regions and son, exchanged a steady ﬂow of letters of the country. It has been extinguished in the with the scientist in pursuit of a cure for the

181 182 International Journal of Leprosy 2004 disease that had assailed Numa since child- the body, the stigma and the compulsive hood. The 24 letters compiled here paint a segregation, as well as the hope for a better unique portrait of the medical and social life. It is the perspective of a patient, some- drama confronted by this family, and the re- thing that is difﬁcult to ﬁnd when research- sults of the use of chaulmoogra oil and other ing the history of health. The original pub- medications in their search for alternative lication was possible thanks to the German treatments.—Authors’Abstract physician Maxime H. Kuczynski-Godard who was a German medical doctor that ar- rived in Peru in the mid 1930s and orga- Benchimol, J. L., and Romero Sa, M. nized valuable activities in the Peruvian Adolpho Lutz and controversies over the jungle as a part of an effort, which eventu- transmission of leprosy by mosquitoes. ally failed, of the Peruvian State to colo- Hist. Cienc. Saude Manguinhos. 10(Suppl. nize, or really to “civilize” the Amazon.— 1) (2003) 49–93. Authors’Abstract

During his years of study in Switzerland and Germany, Adolpho Lutz published his Levison, J. H. Beyond quarantine: a history first articles on zoology, clinical practice, of leprosy in Puerto Rico, 1898–1930s. and therapeutics. In Limeira, Sao Paulo, he Hist. Cienc. Saude Manguinhos. 10(Suppl began studies on animal and human diseases 1) (2003) 225–245. caused by germs and parasites. In 1885–1886, Lutz traveled to Hamburg to study the mor- From biblical times to the modern period, phology of germs related to skin diseases, in leprosy has been a disease associated with conjunction with Paul Gerson Unna, one of stigma. This mark of disgrace, physically Germany’s foremost dermatologists. He present in the sufferers’ sores and disfigured proposed the inclusion of Hansen’s and limbs, and embodied in the identity of a Koch’s bacilli in a new genus. In 1889, “leper,” has cast leprosy into the shadows of Unna nominated his student as physician-in- society. This paper draws on primary sources, chief of the Leper Settlement on Molokai Is- written in Spanish, to reconstruct the social land, Hawaii. From then on, Lutz sustained history of leprosy in Puerto Rico when the the theory that the disease was transmitted United States annexed this island in 1898. The by mosquitos. He conducted research to public health policies that developed over the prove this theory when he was head of the period of 1898 to the 1930s were unique to Instituto Bacteriologico de Sao Paulo Puerto Rico because of the interplay between (1893–1908) and, later, after he moved to political events, scientific developments and the Instituto Oswaldo Cruz (1908–1940). popular concerns. Puerto Rico was influenced Although this research was not successful, by the United States’ priorities for public on commissions and at congresses in which health, and the leprosy control policies that he participated until his death in October developed were superimposed on vestiges of 1940, he still held to his conviction that lep- the colonial Spanish public health system. rosy was transmitted by mosquitoes.—Au- During the United States’ initial occupation, thors’Abstract extreme segregation sacrificed the individual rights and liberties of these patients for the benefit of society. The lives of these leprosy Cueto, M., and de la Puente, J. C. Vida de sufferers were irrevocably changed as a re- leprosa: the testimony of a woman living sult.—Author’s Abstract with Hansen’s disease in the Peruvian Amazon, 1947. Hist. Cienc. Saude Man- guinhos. 10(Suppl 1) (2003) 337–360. Manton, J. Global and local contexts: the [Article in English, Spanish] Northern Ogoja Leprosy Scheme, Nige- ria, 1945–1960. Hist. Cienc. Saude Man- This is the narrative of a patient made be- guinhos. 10(Suppl 1) (2003) 209–23. fore, during and after being incarcerated in an agricultural colony in the Peruvian Ama- Deriving funding from missionary sources zon. In a vivid style, it narrates the decay of in Ireland, Britain and the USA, and from 72, 2 Current Literature, General and Historical 183

international leprosy relief organizations to the last as area was free from the isola- such as the British Empire Leprosy Relief tion policy of State in prewar days there. Association (BELRA) and drawing on de- The aim of this study will make clear the veloping capacities in international public dynamism of “The protection from the ten- health under the auspices of WHO and sion of the society of leprosy patient cur- UNICEF through the 1950s, the Roman rently persecuted” to “The defense of the Catholic Mission Ogoja Leprosy Scheme society from the leprosy patient who is a applied international expertise at a local source of infection.” In this study, explained level with ever-increasing success and cov- the factor of confusion to a National Lep- erage. This paper supplements the presen- rosarium Kuryu Rakusen-en during World tation of a successful leprosy control pro- War II and considered relation between pa- gram in missionary narratives with an tient movement and residents of Yunosawa appreciation of how international medical village at the postwar period.—Authors’ politics shaped the parameters of success Abstract and the development of therapeutic understanding in the late colonial period in Nige- ria.—Author’s Abstract Obregon, D. The anti-leprosy campaign in Colombia: the rhetoric of hygiene and science, 1920–1940. Hist. Cienc. Saude Man- Monteiro, Y. N. Prophylaxis and exclusion: guinhos. 10(Suppl 1) (2003) 179–207. compulsory isolation of Hansen’s disease patients in Sao Paulo. Hist. Cienc. Saude Since the 1920’s, the medical community Manguinhos. 10(Suppl 1) (2003) 95–121. realized that the strategy of leprosy control based on segregation and persecution of This article aims to retrieve the history of patients was inefficient and expensive. In Hansen’s disease in Brazil, analyzing the the 1930’s the new liberal government medical thinking of the time and the shaping incorporated leprosy within the general of health policies that permitted the imple- sanitary institutions, by merging the Bu- mentation, in Sao Paulo, of a prophylactic reau of Lazarettos and the National De- policy of compulsory exclusion for all partment of Hygiene. The disease-apart Hansen’s disease patients. It also analyzes approach started to be replaced by a more how the structuring and implementation of general public health strategy, which in- this policy led to a “Sao paulo model” that volved controlling other illnesses. Preven- strongly influenced the rest of the country. It tion and research played a more influential addresses the creation of the state’s network role, and the new sanitary officials saw of leper colonies, their characteristics and leprosy in the light of the economic ra- the emergence of a veritable “parallel state” tionality of expenditures, placing more that endured until 1967, with complete dis- emphasis on therapies and making them regard of all the changes taking place in both mandatory for all patients. Improvements national and international prophylactic pol- in leprosy treatment became widely known icymaking.—Author’s Abstract and available. However, the image of leprosy as a special condition and the practice of segregation were deeply entrenched Mori, S., Kato, S., Yokoyama, H., Tanaka, within the Colombian culture and institu- U., and Kaneda, S. [The history of tions. The rhetoric changed, but to break Yunosawa village and the policy of lep- with several decades of persecution was a rosy of Japan. IV]. Nihon Hansenbyo difficult task.—Author’s Abstract Gakkai Zasshi. 73(1) (2004) 47–63. [Ar- ticle in Japanese] Oliveira, M. L., Mendes, C. M., Tardin, There was a village which was called R. T., Cunha, M. D., and Arruda, A. Yunosawa, lots of leprosy patients lived, ex- Social representation of Hansen’s disease isted from 1887 to 1941, Kusatu town, thirty years after the term “leprosy” was Gunnma Prefecture, Japan. It was the only replaced in Brazil. Hist. Cienc. Saude place continued securing self-government Manguinhos. 10(Suppl 1) (2003) 41–48. 184 International Journal of Leprosy 2004

Based on the theories of social represen- Porter, J. D., Ogden, J. A., Rao, P. V., Rao, tation (SC) and Central Core (CC), a struc- V. P., Rajesh, D., Buskade, R. A., and tural study was undertaken regarding the Soutar, D. Introducing operations re- neologism hanseniase (Hansen’s disease), search into management and policy prac- the term adopted by Brazil’s Ministry of tices of a non-governmental organization Health in the 1970s. Carried out during (NGO): a partnership between an Indian 2001, this study interviewed eight hundred leprosy NGO and an international aca- housewives residing in the Rio de Janeiro demic institution. Health Policy Plan. and Duque de Caxias municipalities. It 19(2) (2004) 80–87. found that Hansen’s disease is part of a process of modernization of common think- This paper reports on a partnership being, anchored in the additional representa- tween LEPRA, a non-governmental organ- tion of leprosy. This finding is understand- ization (NGO), and the London School of able from the perspective that the central Hygiene and Tropical Medicine (LSHTM) to structure of a social representation has a his- explore the feasibility and appropriateness of torical determination, so short- and middle- incorporating operations research into the term changes are not to be expected. Fur- management and decision-making of a lep- thermore, there has been no ongoing rosy NGO. A pilot study in Orissa was used investment in social marketing to make the to determine the advantages and disadvan- new terminology more widely known. The tages of introducing operations research to authors discuss the relation between social assist in decision-making and programme im- representation and the concept of the history plementation within the organization. The re- of mentalities.—Authors’Abstract sults highlight the difficulty and complexity of the process, but point to several important themes: partnership, changing perspectives, Pandya, S. S. The first international leprosy use of time and priority-setting, identification conference, Berlin, 1897: the politics of of gaps in systems, and building institutional segregation. Hist. Cienc. Saude Man- and personal capabilities. The results of the guinhos. 10(Suppl 1) (2003) 161–177. study provide support to encourage NGOs to become actively involved in research. Be- The present paper examines the first at- cause of their work and service to local com- tempts to internationalize the problem of lep- munities, NGOs have the opportunity to col- rosy, a subject hitherto overlooked by histo- lect information about the perceptions, rians of imperialism and disease. The last resources and constraints of individuals, decade of the nineteenth century saw many families and the communities themselves in in the “civilized countries” of the imperialist accessing appropriate care. Their proximity West gripped by a paranoia about an inva- to communities gives them a feeling of re- sion of leprosy via germ-laden immigrants sponsibility for ensuring that this information and returning expatriates who had acquired is translated to the district, national and ulti- the infection in leprosy-endemic colonial mately international level. This will help to possessions. Such alarmists clamoured for ensure the creation of appropriate infectious the adoption of vigorous leper segregation disease control policies that support the needs policies in such colonies. But the conta- of patients. “Outside” academic institutions giousness of leprosy did not go unquestioned can help NGOs to facilitate this up-stream by other westerners. The convocation in flow of information from the local to the na- Berlin of the first international meeting on tional and international level, to help to en- leprosy revealed the interplay of differing sure that international disease control policies and sometimes incompatible views about the are appropriately serving local communi- containment of leprosy by segregation. The ties.—Authors’Abstract roles of officials from several countries, as well as the roles of five protagonists (Albert Ashmead, Jules Goldschmidt, Edvard Ehlers, Robertson, J. The papers of Stanley Armauer Hansen, and Phineas Abraham) in Browne: leprologist and medical mission- the shaping of the Berlin Conference are here ary (1907–1986). Hist. Cienc. Saude Man- examined.—Author’s Abstract guinhos. 10(Suppl 1) (2003) 427–433. 72, 2 Current Literature, General and Historical 185

This article elaborates a significant archival and history of Hansen’s disease in Brazil acquisition that supplement the collection told by witnesses (1960–2000). Hist. documents related to the life and work of Cienc. Saude Manguinhos. 10(Suppl 1) Stanley George Browne held at the Well- (2003) 308–335. come Library for the History and Under- standing of Medicine in London, specifically This report is a preliminary result of a sur- his work in the Belgian Congo (from 1936 to vey on memories and history of Hansen’s 1959), at Uzuakoli in Nigeria (1959 to 1966), disease, or ‘hanseniasis,’ prepared by the in London with the Leprosy Study Centre Fundacao Oswaldo Cruz (Fiocruz) and the (1966–1980), and also in his international Universidade Federal do Rio de Janeiro capacity as leprosy consultant. It also briefly (UFRJ) using statements from those who refers to an endangered collection of docu- have been afflicted by the disease or those ments, photographs, files and correspond- that have fought against it. It outlines the ence held in a small museum in Culion methodology used by the authors and gives Sanatorium, The Philippines. This research a succinct history of Hansen’s disease in is part of the International Leprosy Associ- Brazil, together wish information on the ation Global Project on the History of Lep- stage of the survey with extracts from our rosy. Its results can be accessed at the site archives of statements. The founding and http://www.leprosyhistory.org.—Author’s the role of Movement for the Reintegration Abstract of People Afflicted by Hansen’s Disease (Morhan) are explained in the testimony of Thomas Frist, a social scientist who worked Robertson, J. Leprosy and the elusive M. in Brazil in the 1970s and 1980s, when the leprae: colonial and Imperial medical ex- country’s old colonies were being restruc- changes in the nineteenth century. Hist. tured, and Cristiano Torres, a former patient Cienc. Saude Manguinhos. 10(Suppl 1) who spent time in prevention centers and (2003) 13–40. leproseries in Para state and who is now active in proposing new policy for the control In the 1800’s, humoral understandings of of Hansen’s disease.—Authors’Abstract leprosy successively give way to disease models based on morbid anatomy, phys- iopathology, and bacteriology. Linkages be- Santos, V. S. [Researching documents on the tween these disease models were reinforced history of hansen’s disease in Brazil]. Hist. by the ubiquitous seed/soil metaphor de- Cienc. Saude Manguinhos. 10(Suppl 1) ployed both before and after the identifica- (2003) 415–426. [Article in Spanish] tion of M. leprae. While this metaphor provided a continuous link between medical This article corresponds to part of the re- descriptions, Henry Vandyke Carter’s On sults of a research on leprosy-related sources Leprosy (1874) marks a convergence of dif- developed in several institutions in the city ferent models of disease. Simultaneously, of Rio de Janeiro. At Real Gabinete Por- this metaphor can be traced in popular med- tugues de Leitura, Arquivo Nacional and ical debates in the late nineteenth century, Biblioteca Nacional, banks, indexes, official accompanying fears of a resurgence of lep- documents and photos on the administration rosy in Europe. Later the mapping of the of leprosaria and articles on the treatment of genome ushers in a new model of disease the disease have been investigated. At Centro but, ironically, while leprosy research draws de Pesquisa e Documentacao de Historia Con- its logic from a view of the world in which temporanea do Brazil (CPDOC-FGV), sev- a seed and soil metaphor expresses many eral files have been researched, mainly those different aspects of the activity of the dis- with information on the health policies of the ease, the bacillus itself continues to be un- first Vargas administration (1930–1945). This receptive to cultivation.—Author’s Abstract research is part of the International Leprosy Association Global Project on the History of Leprosy. Its results can be accessed at the site Rosa Maciel, L., Oliveira, M. L., Gallo, http://www.leprosyhistory.org—Author’s M. E., and Damasco, M. S. Memories Abstract 186 International Journal of Leprosy 2004

Smith, T. H. A monument to Lazarus: the India and their position vis-à-vis leprosy or- leprosy hospital of Rio de Janeiro. Hist. ganizations, I explore some of the contexts Cienc. Saude Manguinhos 10(Suppl 1) in which leprosy patients actively manage (2003) 143–160. their own situations, often in defiance of prevailing development orthodoxies. I con- Soon after the Portuguese made landfall clude that closer observation and analysis of in 1500, Europeans and, later, African slaves the strategies patients use to manage their introduced leprosy, and Saint Lazarus, the disease status have important policy impli- patron saint of its victims, into Brazil. Social cations.—Author’s Abstract and political pressure mounted by the middle of the eighteenth century in the city of Rio de Janeiro to remove those unfortunates Taylor, G. M., Stewart, G. R., Cooke, M., from the city’s streets even before the move Chaplin, S., Ladva, S., Kirkup, J., of Brazil’s capital in 1763. Frei Antoniom Palmer, S., and Young, D. B. Koch’s the bishop of Rio, founded the venerable bacillus—a look at the first isolate of My- hospital that year in the neighborhood of cobacterium tuberculosis from a modern Sao Cristovao, He requested that the Irman- perspective. Microbiology 149(Pt 11) dade do Santissimo Sacramento da Cande- (2003) 3213–3220. laria provide oversight and administration. The brotherhood continues to honor its Using molecular methods the authors covenant of 239 years ago. The history of have studied mycobacterial DNA taken this hospital provides insight into the com- from a 19th century victim of tuberculosis. plex relationships that existed between the This was the case from which Robert Koch citizenry and church and state. Rio’s leprosy first isolated and cultured the organism re- hospital, now the Hospital Frei Antonio, had sponsible for tuberculosis. The mycobacte- an important role in the evolution of the ria were preserved within five glass culture health care professions, progress in medical tubes as abundant bacterial colonies on science, and the genesis of the hygienic slopes of a gelatinous culture medium of un- movement in Brazil. This study also con- known composition. Originally presented by tributes to the history of a disease that per- Koch to surgical laryngologist Walter Job- sists in 2002 Brazil as a public health son Horne in London in 1901, the relic has, issue.—Author’s Abstract since 1983, been in the care of the Royal College of Surgeons of England. Light and electron microscopy established the pres- Staples, J. Delineating disease: self- ence of acid-fast mycobacteria but showed management of leprosy identities in that morphological preservation was gener- South India. Med. Anthropol. 23(1) ally poor. Eleven different genomic loci (2004) 69–88. were successfully amplified by PCR. This series of experiments confirmed that the or- The national and international agencies ganisms were indeed Mycobacterium tuber- working to eliminate leprosy are also dom- culosis and further showed that the original inant in setting the boundaries of official dis- strain was in evolutionary terms similar to course on the issue. Within these boundaries ‘modern’ isolates, having undergone the TB the disease is commonly represented as a D1 deletion. Attempts to determine the medical problem with negative social con- genotypic group of the isolate were only sequences, and it is believed that both prob- partially successful, due in part to the de- lem and consequences will be resolved if graded nature of the DNA and possibly also leprosy is eliminated and its victims treated to a truncation in the katG gene, which and (if necessary) reintegrated within their formed part of the classification scheme. social groups. For those affected by leprosy Spoligotyping resulted in amplification of the issues are frequently different, elimina- DR spacers consistent with M. tuberculosis tion in some respects representing a problem but with discrepancies between independent as much as a solution. Against this back- extracts, stressing the limitations of this typ- ground, which I describe with reference to a ing method when applied to poorly pre- group of leprosy-affected people in South served material.—Authors’Abstract 72, 2 Current Literature, Chemotherapy 187

Zhang, L. Leprosy in China. Nihon Hansen- But difﬁculties as well as problems like dis- byo Gakkai Zasshi. 72(3) (2003) 209–215. abilities, discrimination, drug-resistance and dismissing of research still remain in the Leprosy has deﬁnitely been present in control of leprosy. Highly attention should China for at least 2000 years. Through be continuously paid on to attain the preva- painstaking efforts over the past half century, lence rate of less than 0.1/10,000 and the in- China has put leprosy under control and cidence rate below 0.5/100,000 in all coun- reached the WHO’s target at elimination of ties (cities) throughout the country by the leprosy at the national and subnational level. year 2010.—Author’s Abstract Chemotherapy

Ashitani, J., Yanagi, S., Arimura, Y., Sano, tion in the number of colonies on a plate as A., and Mukae, H. Acute respiratory dis- compared to that system free of the agent at tress syndrome induced by rifampicin with the same dilution of the culture suspension. high levels of neutrophil and eosinophil Ethyl 4-[[N-(2,2,4,4-tetramethylchroman-6- products in bronchoalveolar lavage fluid. yl)thiocarbamoyl]amino] benzoate (9) and Respiration 70(5) (2003) 541–543. [[(1E,3Z,5E)-1-aza-4-methyl-6-(1,2,2,4- tetramethyl(1,2-dihydroquinolyl))hexa- We reported a case with acute respiratory 1,3,5-trienyl]amino]aminomethane-1-thione distress syndrome (ARDS) caused by (10) exhibited activity at 5.0–10.0 and rifampicin during therapy for pulmonary 10.0–20.0 microg/mL, respectively, while tuberculosis. A high level of eosinophil the other examples had MIC values of 20 mi- cationic protein in bronchoalveolar lavage crog/mL or greater. The inhibitory ability of fluid (BALF) was detected as well as 8 may occur via the inhibition of mycolic interleukin-8 and neutrophil elastase. Based acid synthesis in a like manner as found with on these results together with the positive re- 7, but this requires further study. The het- sult of the drug lymphocyte-stimulating test, eroarotinoids are the first examples to exhibit we concluded that rifampicin was the inhibitory ability against the growth of My- causative drug leading to ARDS. Cortico- cobacterium bovis BCB.—Authors’Abstract steroid therapy resulted in clinical improvement and resolution of the pulmonary infil- trates on the chest radiograph without the Bucaretchi, F., Miglioli, L., Baracat, E. C., recurrence of pulmonary tuberculosis.—Au- Madureira, P. R., Capitani, E. M., and thors’Abstract Vieira, R. J. [Acute dapsone exposure and methemoglobinemia in children: treatment with multiple doses of acti- Brown, C. W., Liu, S., Klucik, J., Berlin, vated charcoal with or without the ad- K. D., Brennan, P. J., Kaur, D., and ministration of methylene blue]. J. Pedi- Benbrook, D. M. Novel heteroarotinoids atr. (Rio J). 76(4) (2000) 290–294. as potential antagonists of Mycobacte- rium bovis BCG. J. Med. Chem. 47(4) OBJECTIVE: To study the changes in (2004) 1008–1017. methemoglobinemia of 17 children admitted with acute exposure to dapsone compli- Aseries of 15 heteroarotinoids has been pre- cated by a methemoglobin concentration pared and evaluated for activity against Myco- greater than 20% of the total hemoglobin. bacterium bovis BCG with the thiourea- The children were treated with multiple containing isoxyl (7) (0.5 microg/mL) as the doses of activated charcoal with or without standard. 2,2,4-Trimethyl-2H-chromen-7-yl the administration of methylene blue. 4-(methoxycarbonyl)benzoate (8) displayed PATIENTS AND METHODS: Seventeen the most significant activity (2.0–4.0 mi- patients (ages 1–13 yrs, median 3 yrs), crog/mL) in terms of the lowest concentration were admitted 1–72 hr after the ingestion (microg/mL) (MIC, minimum inhibitory con- of 100–1200 mg (median 350 mg, 10 pa- centration) required to produce a 99% reduc- tients) or an unknown amount of dapsone 188 International Journal of Leprosy 2004

(7 patients). The methemoglobin blood con- over, the morbidity of S and the frequent centrations upon admission ranged from steroid-dependence of ENL also create real 23.5%–49.7% (median 37.8%), and the therapeutic problems. Recently, the efficacy main clinical features were cyanosis (17), of pentoxifylline (PTX), which also inhibits tachycardia (17), vomiting (11) and tachyp- in vitro and in vivo production of TNFα, has nea (8). All of the children received multi- been suggested for ENL treatment. We report ple doses of activated charcoal orally or via our experience on its use for the treatment of nasogastric tube (1g/kg, 10% solution, 4–6 15 leprosy patients suffering from a first ENL times/day, 3–16 doses with a median of 8 attack (11 cases), a chronic steroid-dependent doses). Twelve of the 14 patients with ENL (3 cases) or chronic steroid- and thalido- methemoglobin levels greater than 30% mide-dependent ENL (1 case). PTX has been were also treated with a single dose of given at 800 mg t.i.d. (2 cases), or 400 mg methylene blue (1–2% solution, 1–2 mg/kg) t.i.d. (13 cases) doses. The patients received infused IV over 5 min. RESULTS: There PTX at the initiating dosage until complete was a progressive decrease in the methe- clinical cure. At the end of ENL attacks, PTX moglobin levels after the beginning of both was either abruptly stopped or tapered down treatments (multiple doses of activated over the next 4 months. In ten of 11 patients charcoal alone or associated with methylene who developed ENL for the first time, the blue), and only one dose of methylene blue systemic symptoms and neuritic pains disap- was necessary. There were no significant peared within one week; at three weeks, half statistical differences between the results of of the patients were cured and the other half the two treatments according to the time- had striking clinical improvement; complete course decrease in methemoglobinemia (p = cure was obtained within 7 to 35 days (mean: 0.49 Wilcoxon test). CONCLUSIONS: Mul- 27 days). A relapse occurred within 2–3 tiple doses of activated charcoal given when months in the 5 patients in which PTX was methemoglobin levels were greater than abruptly stopped. In contrast, no relapse oc- 20% can be considered as a possible treat- curred in the patients who benefited from de- ment for pediatric patients, with or without creasing doses of PTX. Recurrent ENL the administration of methylene blue, after episodes also responded well to PTX. The 3 acute dapsone exposure.—Authors’Abstract patients who had chronic steroid-dependent ENL failed to show any improvement after 3 to 6 weeks of PTX. In contrast, steroid ther- De Carsalade, G. Y., Achirafi, A., and apy could be stopped in the steroid- and Flageul, B. Pentoxifylline in the treat- thalidomide-dependent patient. Our results ment of Erythema Nodosum Leprosum: confirm the action of PTX if it is slowly ta- Results of an open study. Acta Leprolog- pered down (4 months seem sufficient) and ica 12(3) 117–122. not abruptly to avoid relapses. As it is safe use, PTX could constitute the first line of Erythema nodosum leprosum (ENL) is a ENL attack treatment.—Acta Leprologica well-known immunological serious complication affecting lepromatous multibacillary leprosy patients. For a long time, ENL has Eriksson, T., Hoglund, P., Turesson, I., been regarded as an immune complex- Waage, A., Don, B. R., Vu, J., Scheffler, mediated disease or Arthus phenomenon. Re- M., and Kaysen, G. A. Pharmacokinet- cently, it has been reported that ENL was as- ics of thalidomide in patients with im- sociated with high serum tumor necrosis paired renal function and while on and off factor-alpha (TNFα) levels, suggesting that dialysis. J. Pharm. Pharmacol. 55(12) this cytokine could also play a central role in (2003) 1701–1706. the manifestations of ENL. Thalidomide (TH) and systemic steroids (S), both TNFα There is a renewed interest in thalido- production inhibitors, are the two current ef- mide for use in malignancies and systemic fective drugs for the management of ENL. inflammatory diseases. Reduced renal func- However, TH is rarely available in leprosy tion is not uncommon among patients with endemic countries, and its teratogenicity and these disease states but the pharmacokinet- neurotoxicity strongly limit its use. More- ics has not been fully investigated. The aim 72, 2 Current Literature, Chemotherapy 189 of this study was to investigate the pharma- rium avium complex selected by molec- cokinetics of thalidomide in haemodialysis ular topology: a virtual screening method. patients while on and off dialysis and in J. Antimicrob. Chemother. 53(1) (2004) myeloma patients with varying degrees of 65–73. renal function. Two studies were performed. To establish the pharmacokinetics OBJECTIVES: In order to select new of thalidomide in patients with mild to mod- drugs and to predict their in vitro activity erate renal failure, blood samples were against Mycobacterium avium complex taken over 12 weeks from 40 patients with (MAC), new quantitative structure-activity multiple myeloma. A second study was per- relationship (QSAR) models were devel- formed in six patients with end-stage renal oped. METHODS: The activities against disease both on a non-dialysis day and be- MAC of 29 structurally heterogeneous fore and during a haemodialysis session. drugs were examined by means of linear Thalidomide concentration was determined discriminant analysis (LDA) and multilinear by HPLC. A one-compartment open model regression analysis (MLRA) by using topo- with first-order absorption and elimination logical indices (TI) as structural descriptors. was used to fit total thalidomide concentra- In vitro antimycobacterial activities were de- tion to population pharmacokinetics and termined by a broth microdilution method statistical models using the NONMEM pro- with 7H9 medium. RESULTS: The topo- gram. Clearance and volumes were slightly logical model obtained successfully classi- below 10 L h-1 and 1 L kg-1, respectively, fies over 80% of compounds as active or in- in both patient groups. The inter- and intra- active; consequently, it was applied in the patient variability was low. Clearance was search for new molecules active against doubled during dialysis. There was no cor- MAC. From among the selected candidates relation between thalidomide clearance and demonstrating in vitro activity, aflatoxin B1, renal function. In conclusion, the pharma- benzalkonium chloride and pentamidine cokinetics of thalidomide in patients with stand out, with MIC50s between 4 and 32 renal failure are very similar to values re- mg/L. CONCLUSION: The method de- ported by others for patients with normal scribed in this work is able to select mole- renal function. Although clearance during cules active against MAC.—Authors’ Ab- dialysis is doubled, thalidomide dose need stract not be changed for patients with decreased kidney function. There is also no need for a supplementary dose due to haemodialy- Hansen, J. M., and Harris, C. A novel hy- sis.—Authors’Abstract pothesis for thalidomide-induced limb teratogenesis: redox misregulation of the NF-kappaB pathway. Antioxid. Redox Foroumadi, A., and Soltani, F. Antitubercu- Signal 6(1) (2004) 1–14. losis agents. IX. In vitro anti-mycobacterial activity of N-(2-phenyl-2-oxoethyl) and Several hypotheses have been proposed to N-[2-(4-fluorophenyl)-2-oxoethyl]- explain the mechanisms of thalidomide ter- ciprofloxacin derivatives against some atogenesis, although none adequately ac- drug-resistant strains of Mycobacterium counts for the observed malformations and tuberculosis and Mycobacterium avium explains the basis for species specificity. Re- isolates. Boll. Chim. Farm. 142(6) (2003) cent observations that thalidomide increases 248–250. the production of free radicals and elicits oxidative stress, coupled with new insights See Current Literature, Other Mycobac- into the redox regulation of nuclear tran- terial Diseases, p. 247. scription factors, lead to the suggestion that thalidomide may act through redox misregulation of the limb outgrowth pathways. Ox- Garcia-Garcia, A., Galvez, J., de Julian- idative stress, as marked by glutathione de- Ortiz, J. V., Garcia-Domenech, R., pletion/oxidation and a shift in intracellular Munoz, C., Guna, R., and Borras, R. redox potential toward the positive, occurs New agents active against Mycobacte- preferentially in limbs of thalidomide- 190 International Journal of Leprosy 2004 sensitive rabbits, but not in resistant rats. (TNF-alpha) production by monocytes. DNA binding of nuclear factor kappa-B However, its mechanism of action in the (NF-kappaB), a redox-sensitive transcrip- skin is not known. PURPOSE: To test our tion factor and key regulator of limb out- hypothesis that thalidomide may antago- growth, was shown to be significantly at- nize TNF-alpha production in the skin, we tenuated in rabbit limb cells and could be used a mouse model for acute ultraviolet-B restored following the addition of a free rad- (UVB) exposure, a known stimulus for in- ical spin-trapping agent, phenyl N-tert-butyl ducing this cytokine. RESULTS: A single nitrone. The inability of NF-kappaB to bind bolus dose of thalidomide (either 100 or to its DNA promoter results in the failure of 400 mg/kg) given immediately before limb cells to express fibroblast growth fac- UVB exposure (40–120 mJ/cm2) inhibited, tor (FGF)-10 and twist in the limb progress in a dose-dependent manner, sunburn cell zone (PZ) mesenchyme, which in turn at- formation (i.e., keratinocyte (KC) apopto- tenuates expression of FGF-8 in the apical sis as defined by histologic appearance and ectodermal ridge (AER). Failure to establish confirmed by terminal transferase mediated an FGF-10/FGF-8 feedback loop between biotinylated dUTP nick end labelling stain- the PZ and AER results in the truncation of ing) in mouse skin biopsy specimens. limb outgrowth. We hypothesize that species- However, this agent did not affect the for- selective alterations in redox microenviron- mation of cyclobutane pyrimidine dimers, ment caused by free radical production from a measure of UVB-induced DNA damage, thalidomide results in attenuation of the NF- which is an early event associated with kappaB-mediated gene expression that is re- apoptosis. RNase protection assays con- sponsible for limb outgrowth.—Authors’ firmed that high (400 mg/kg), but not low Abstract (100 mg/kg), doses of thalidomide inhibited the UVB-induced increase in steady- state TNF-alpha mRNA. Additionally, our Li Li, and Xue-jun Zhu. Dapsone Hyper- in vitro data using neonatal mouse KCs sensitivity Syndrome. J. Clin. Derm. showed that thalidomide prevented UVB- 32(Suppl.) (2003) s115–s117. induced cell death (JAM assay). The anti- apoptotic effects of thalidomide can be re- A 23-year-old woman with linear IgA der- versed by the addition of exogenous matosis developed dapsone hypersensitivity recombinant mouse TNF-alpha and hence syndrome (DHS) after initiation of dapsone reconstituting UVB-induced programmed therapy. She had fever, jaundice with hepatic cell death. The inhibition of sunburn cell dysfunction, lymphadenopathy, anemia and formation by low-dose thalidomide in the dermatitis. The symptoms disappeared with absence of TNF-alpha inhibition suggests methylprednisolone treatment 40 mg/day.— that other, unidentified mechanisms of Journal of Clinical Dermatology apoptosis inhibition are active. CONCLU- SIONS: These data suggest that the anti- inflammatory effects of thalidomide can af- Lu, K. Q., Brenneman, S., Burns, R. Jr., fect UVB injury, and may, in part, explain Vink, A., Gaines, E., Haake, A., and its action in photosensitivity diseases such Gaspari, A. Thalidomide inhibits UVB- as cutaneous lupus erythematosus.—Au- induced mouse keratinocyte apoptosis by thors’ Abstract both TNF-alpha-dependent and TNF- alpha-independent pathways. Photoder- matol Photoimmunol Photomed. 19(6) Lu, T., and Drlica, K. In vitro activity of (2003) 272–280. C-8-methoxy fluoroquinolones against mycobacteria when combined with anti- BACKGROUND: Thalidomide is an anti- tuberculosis agents. J. Antimicrob. Chemo- inflammatory pharmacologic agent that has ther. 52(6) (2003) 1025–1028. been utilized as a therapy for a number of dermatologic diseases. Its anti-inflammatory OBJECTIVES: To examine the effect of properties have been attributed to its ability first-line and second-line anti-tuberculosis to antagonize tumor necrosis factor-alpha agents on the ability of fluoroquinolones to 72, 2 Current Literature, Chemotherapy 191

kill mycobacteria. METHODS: A clinical oral administration of drug-loaded nanopar- isolate of Mycobacterium tuberculosis and a ticles to Mycobacterium tuberculosis-infected laboratory strain of Mycobacterium smeg- mice at every 10th day, no tubercle bacilli matis were grown in liquid medium and could be detected in the tissues after 5 oral treated with a fluoroquinolone in the pres- doses of treatment. Therefore, nanoparticle- ence or absence of anti-tuberculosis agents. based ATD therapy forms a sound basis for Bacterial survival was determined by viable reduction in dosing frequency for better colony counts on agar medium. RESULTS: management of TB.—Authors’Abstract When moxifloxacin activity was examined in two-drug combinations containing traditional anti-tuberculosis agents, activity was Selvaraj, P., Chandra, G., Kurian, S. M., greater than either compound alone with Reetha, A. M., and Narayanan, P. R. isoniazid, capreomycin and low, but not Association of Vitamin D receptor gene high, concentrations of rifampicin. Cyclo- variants of BsmI, ApaI, and FokI poly- serine contributed no additional activity, and morphisms with susceptability or resist- ethambutol interfered with the lethal action ance to pulmonary tuberculosis. Curr. of moxifloxacin and gatifloxacin. Experi- Sci. 84(12) (2003) 1564–1568. ments with M. smegmatis confirmed that both rifampicin and ethambutol reduce fluoroquinolone lethality. Moreover, ethambutol increased the recovery of fluoroquinolone- Zhu, X., Giordano, T., Yu, Q. S., Hol- resistant mutants newly created by ethyl loway, H. W., Perry, T. A., Lahiri, D. methanesulphonate treatment. CONCLU- K., Brossi, A., and Greig, N. H. Thio- SIONS: The intrinsic bactericidal activity of thalidomides: novel isosteric analogues C-8-methoxy fluoroquinolones can be ad- of thalidomide with enhanced TNF-alpha versely affected by some agents currently inhibitory activity. J. Med.Chem. 46(24) used for treatment of tuberculosis.—Au- (2003) 5222–5229. thors’Abstract Thalidomide is being increasingly used in the clinical management of a wide spectrum Pandey, R., Zahoor, A., Sharma, S., and of immunologically-mediated and infectious Khuller, G. K. Nanoparticle encapsu- diseases, and cancers. However, the mechan- lated antitubercular drugs as a potential isms underlying its pharmacological action oral drug delivery system against murine are still under investigation. In this regard, tuberculosis. Tuberculosis (Edinb). 83(6) oral thalidomide is clinically valuable in the (2003) 373–378. treatment of erythema nodosum leprosum (ENL) and multiple myeloma and effectively Patient non-compliance is the major draw- reduces tumor necrosis factor-alpha (TNF- back associated with the long-duration alpha) levels and angiogenesis in vivo. This chemotherapy of tuberculosis (TB); hence, re- contrasts with its relatively weak effects on duction in dosing frequency forms an impor- TNF-alpha and angiogenesis in in vitro stud- tant therapeutic strategy. The present study re- ies and implies that active metabolites con- ports the formulation of three frontline tribute to its in vivo pharmacologic action and antitubercular drugs (ATD), i.e., rifampicin that specific analogues would be endowed (RIF), isoniazid (INH) and pyrazinamide with potent activity. Our focus in the structural (PZA) encapsulated in poly (DL-lactide-co- modification of thalidomide is toward the dis- glycolide) (PLG) nanoparticles. Drug en- covery of novel isosteric active analogues. In capsulation efficiencies were 56.9 ± 2.7% this regard, a series of thiothalidomides and for RIF, 66.3 ± 5.8% for INH and 68 ± 5.6% analogues were synthesized and evaluated for for PZA. Following a single oral adminis- their TNF-alpha inhibitory activity against tration of these preparations to mice, the lipopolysacharide (LPS)-stimulated periph- drugs could be detected in the circulation for eral blood mononuclear cells (PBMC), This 6 days (RIF) and 9 days (INH/PZA), whereas was combined with a PBMC viability assay therapeutic concentrations in the tissues to differentiate reductions in TNF-alpha se- were maintained for 9–11 days. Further, on cretion from cellular toxicity. Two isosteric 192 International Journal of Leprosy 2004 analogues of thalidomide, compounds 15 and ity via its 3′-UTR. The potency of 19 war- 16, that mostly reflect the parent compound, rants further study and suggests that replace- together with the simple structure, dithioglu- ment of the amide carbonyl with a thiocar- tarimide 19, potently inhibited TNF-alpha se- bonyl may be beneficial for increased cretion, compared to thalidomide, 1. The TNF-alpha inhibitory action. In addition, an mechanism underpinning this most likely is intact phthalimido moiety appeared to be req- posttranscriptional, as each of these com- uisite for TNF-alpha inhibitory activity.— pounds decreased TNF-alpha mRNA stabil- Authors’Abstract Clinical Sciences Alioua, Z., Sbai, M., Elhaouri, M., Boudi, over time based on the immune status of the O., Ghfir, M., Benomar, S., and Se- individual. Immune-mediated “reactional drati, O. Histoid leprosy with erythema states” may also occur that require addi- nodosum leprosum. Acta Leprologica tional recognition and treatment. Varied in 12(3) (2004) 107–111. its manifestations, a successful treatment approach relies on proper recognition and Histoid leprosy is a particular variant of classification of disease.—Author’s Abstract lepromatous leprosy presenting as cutaneous or subcutaneous nodular and/or plaque-like lesions arising from apparently normal skin. Beyene, D., Aseffa, A., Harboe, M., Kidane, It is characterized histologically by spindle- D., Macdonald, M., Klatser, P. R., Bjune, shaped histiocytes in interlacing bundles and G. A., and Smith, W. C. Nasal carriage of whorls, containing numerous intact and rod- Mycobacterium leprae DNA in healthy in- shaped Mycobacterium leprae. It can occur dividuals in Lega Robi village, Ethiopia. de novo or secondary in patients treated for Epidemiol. Infect. 131(2) (2003) 841–848. a long course by dapsone alone. We describe a case of lepromatous leprosy treated ac- The number of registered leprosy patients cording to the national Moroccan protocol world-wide has decreased dramatically who developed histoid lesions during his after extensive application of WHO recom- treatment by dapsone. The patient re- mended Multiple Drug Therapy (MDT). sponded well to fluoroquinolone, rifampicin The annual number of new cases has, how- and clofazimine, with however, the occur- ever, been almost unchanged in several rence of erythema nodosum leprosum. populations, indicating that the infection is still present at community level. Nasal carriage of Mycobacterium leprae DNA was Bartt, R. E. Leprosy (Hansen’s Disease). studied in Lega Robi village in Ethiopia. Curr. Treat Options Neurol. 6(2) (2004) MDT had been applied for more than ten 95–103. years, and 718 residents over 5 years old were eligible for the study. During the first Leprosy (Hansen’s disease) causes the survey nasal swab samples were collected most common treatable form of neuropathy from 664 (92.5%) individuals. The results in the world. Several endemic countries ac- of a Peptide Nucleic Acid-ELISA test for count for the majority of the world’s cases M. leprae DNA interpreted by stringent sta- and most of the cases seen in the US are tistical criteria were available for 589 amongst immigrants. However, endemic (88.7%) subjects. Thirty-five (5.9%) indi- cases of leprosy occur in the US. The viduals without clinical signs of leprosy pathogen is Mycobacterium leprae, a slow- were positive for M. leprae DNA. Seven growing, obligate intracellular pathogen that PCR positive individuals lived in a house- consistently infects skin and peripheral hold where one or two other members were nerves. The clinical appearance of the skin also positive for M. leprae DNA. During a and neurologic deficits develop months to second survey 8 (46%) of 175 interpretable years after infection and are determined by PNA-ELISA tests were positive. Of 137 in- the host’s response to the infection. An indi- dividuals tested twice, only two were posi- vidual’s disease classification can change tive on both occasions whereas 10 were 72, 2 Current Literature, Clinical Sciences 193

PCR positive only once. The study confirms We evaluated a patient with disseminated the widespread distribution of M. leprae Mycobacterium tuberculosis and Mycobac- DNA in healthy individuals. The feasibility terium chelonae infection, of which he died. of curbing possible transmission of subclin- He also developed autoimmune (type I) ical infection needs further consideration. diabetes and primary hypothyroidism. His serum contained a high titer of immunoglobulin G autoantibody to interferon- Cortes, S. L., and Rodriguez, G. Leprosy gamma (IFN-gamma) capable of blocking in children: association between clinical in vitro responses to this cytokine by pe- and pathological aspects. J. Trop. Pediatr. ripheral blood mononuclear cells from nor- 50(1) (2004) 12–15. mal donors. These results suggest that autoantibodies to IFN-gamma can induce Leprosy among children is a public health susceptibility to disseminated mycobacterial problem reflecting the disease’s transmis- infection, which may be refractory to sion in the community and the efficiency of chemotherapy.—Authors’Abstract control programmes. To evaluate some clinical, epidemiological and histopathological criteria, as well as the level of agreement Fenniche, S., Ben Jennet, S., Marrak, H., between clinical and histopathological diag- Khayat, O., Zghal, M., Ben Ayed, M., noses, 207 biopsies were studied from and Mokhtar, I. [Cutaneous tuberculo- patients less than 15 years old who were sis: anatomoclinical features and clinical clinically diagnosed with leprosy between course (26 cases)]. Ann. Dermatol. March 1994 and September 2000. Leprosy Venereol. 130(11) (2003) 1021–1024. was confirmed by histopathology in 119 [Article in French] cases (57.5 percent). Forty-seven percent of children were 10 years old or more; INTRODUCTION: Despite prevention 28.5 percent shared their dwellings with lep- programs, tuberculosis is still progressing rosy patients; 35 percent had only one le- endemically in developing countries. The sion, and 43 percent were multibacillary prevalence of cutaneous tuberculosis is esti- cases. Agreement between clinical and mated as 2.1 p. 100 and represents a rare lo- histopathological classification was 36 per- calization among the extra-pulmonary cent; hypochromic chronic eczema and forms. In order to study the epidemiology, post-inflammatory incontinence of melanin the most frequent anatomoclinical forms pigment were the clinical lesions most fre- and the progressive features of cutaneous tu- quently mistaken with leprosy. Leprosy berculosis, we conducted a study in the area among children represents 7 percent of new of Tunis over a 20-year period. PATIENTS leprosy cases in Colombia and the high per- AND METHODS: All cases of cutaneous centage of multibacillary cases suggests that tuberculosis observed between 1981 and diagnosis is being made late. The disease 2000 in the dermatology department of the must be investigated in all children living Habib Thameur hospital were included in a with leprosy patients and skin biopsy is rec- retrospective study. Diagnosis of cutaneous ommended to avoid false-positive diag- tuberculosis was challenging and required noses.—Authors’Abstract the correlation of clinical, biological and progressive features. RESULTS: Twenty-six patients were observed in the study. There Doffinger, R., Helbert, M. R., Barcenas- were 12 men and 14 women with a mean Morales, G., Yang, K., Dupuis, S., age of 30.4 years (range: 6 to 74) and 20 p. Ceron-Gutierrez, L., Espitia-Pinzon, C., 100 of infantile cases. Of the various pat- Barnes, N., Bothamley, G., Casanova, J. terns of cutaneous tuberculosis seen, 11 (42 L., Longhurst, H. J., and Kumararatne, p. 100) had lupus tuberculosis, 10 (38 p. D. S. Autoantibodies to interferon-gamma 100) had scrofuloderma, 4 (15 p. 100) had in a patient with selective susceptibility to tuberculosis verrucosa cutis and 1 child had mycobacterial infection and organ-specific a perianal tubercular ulcer. The Mantoux test autoimmunity. Clin. Infect. Dis. 38(1) was positive in 20/24 patients. Histological (2004) e10–4. tuberculoid granuloma was seen in 25 cases 194 International Journal of Leprosy 2004

(96 p. 100) associated with caseating peared less common. Because of the high necrosis in 10 cases (38 p. 100). All pa- risk of neurological permanent damage, re- tients were treated successfully with triple versal reaction needs to be diagnosed and or quadruple anti-tubercular drugs for 6 to treated as soon as possible. Herein, the cur- 10 months. One patient exhibited a squa- rent antibacillary and antireactional treat- mous cell carcinoma on a lupus tuberculo- ments are being reviewed.—Bulletin de la sis scar four years later. DISCUSSION: Société de Pathologie Exotique The progression of cutaneous tuberculosis remains stable, ranging from 1.4 cases/year between 1981 and 1990 to 1.2 cases/year Jardim, M. R., Antunes, S. L. G., Santos, between 1991 and 2000. In our study, fe- A. R., Nascimento, O. J. M., Nery, J. A. males were slightly more affected than men C., Sales, A. M., Illarramendi, X., Dup- with a M/F sex ration of 0.86. Before 1984, pre, N., Chimelli, L., Sampio, E. P., scrofuloderma was the most frequent form Sarno, E. P. N. Criteria for diagnosis of among the cutaneous tuberculoses. Now pure neural leprosy. J. Neurol. 250(7) the frequency of lupus tuberculosis has (2003) 806–809.—Tropical Disease Bul- reached that of scrofuloderma, demonstrat- letin ing the increase in the incidence of clinical pattern of cutaneous tuberculosis with The clinical diagnosis of pure neural lep- strong immunity probably related to the rosy (PNL) remains a public health care improvement in health conditions and gen- problem mainly because skin lesions—the eralization of vaccination programs.—Au- cardinal features of leprosy—are always ab- thors’ Abstract sent. Moreover, the identification of the leprosy bacillus is not easily achieved even when a nerve biopsy can be performed. In Flageul, B. Prise en charge médicale actu- an attempt to reach a reliable PNL diagnosis uelle de la maladie de Hansen. Bull. Soc. in patients referred to our Leprosy Outpa- Pathol. Exot. 96(5) (2003) 357–360. [Ar- tient Clinic, this study employed a variety of ticle in French] criteria. The nerve biopsies performed on the 67 individuals whose clinical, neurolog- During the last 20 years, the global lep- ical, and electrophysiological examination rosy situation has strikingly changed with a findings strongly suggested peripheral neu- decrease of cases from 12 millions esti- ropathy were submitted to M. leprae identi- mated cases in 1982 to 600,000 registered fication via a polymerase chain reaction cases in the year 2000. However, during the (PCR). Mononeuropathy multiplex was the past 15 years, about 700,000 new cases are most frequent clinical and electrophysiolog- still detected annually. The systematic use of ical pattern of nerve dysfunction, while sen- multidrug therapy (MDT), as recommended sory impairment occurred in 89% of all by a WHO Study Group in 1982, has proven cases and motor dysfunction in 81%. Axonal its efficacy as assessed by the low reported neuropathy was the most prominent electro- relapse rate (less than 1% per year). The physiological finding, while the histopatho- initial PCT schedule has been modified sev- logical nerve study showed epithelioid gran- eral times, but this PCT remains the recom- uloma in 14% of the patients, acid fast mended chemotherapy for the great major- bacilli in 16%, and nonspecific inflamma- ity of patients. New potent antibacillary tory infiltrate and/or fibrosis in 39%. PCR drugs (ofloxacin, minocycline, clarithromy- for M. leprae was positive in 47% of the cine) have been discovered; however, their nerve biopsy samples (n =23). PCR, in con- current use is limited and should remain lim- junction with clinical and neurological ex- ited until under way trials could confirm amination results, can be a powerful tool in their efficacy. With the use of PCT, the fre- attempting to identify and confirm a PNL di- quency of immunologically mediated reac- agnosis.—Tropical Disease Bulletin tional states have changed. The occurrence of reversal reaction (type 1 reaction), has significantly increased while that of ery- Matsuo, E. [The sequelae of Hansen’s dis- thema nodosum leprosum (ENL, type 2) ap- ease. (Pathologic viewpoint of etiologies, 72, 2 Current Literature, Clinical Sciences 195

morphologies and countermeasures)]. Modi, K., Mancini, M., and Joyce, M. P. Nihon Hansenbyo Gakkai Zasshi 72(3) Lepromatous leprosy in a heart transplant (2003) 251–257. [Article in Japanese] recipient. Am. J. Transplant 3(12) (2003) 1600–1603. The proportion of glomerulonephritis, often a sequence of arteriolitis, among the Northern Louisiana is not an area for in- sequelae of Hansen’s disease after the intro- digenous cases of leprosy. Limited data are duction of chemotherapy increased available on the occurrence of leprosy in markedly in Japan and nullified that of once organ transplant recipients. No cases have prevalent tuberculosis after 1960s. How- been reported in heart transplant recipients. ever, most significant aftermath of the dis- Mr J.R. is a 68-year-old man from Shreve- ease for numbers of years in the past have port, Louisiana. He underwent orthotopic been peripheral nerve injuries worldwide for heart transplantation in March 1996. He which effective countermeasures are yet to presented in March 2000 with a macu- be developed. In this brief autopsy cases lopapular skin rash and intermittent hand study from 1960s to 1990s, we confirmed swelling for 5 months. He also complained the presence of cases in which arteriolitis of intermittent burning of his feet for a year. and resulted infarction of peripheral nerves The skin lesions were of two types—a fine and not M. leprae itself were shown to be red migratory, intermittent maculopapular the major cause of axonal damages. There rash over the upper torso and a raised, were also cases in which the accumulation larger, violaceaous lesion on his hands. of the bacilli without vascular changes did Neurological examination revealed com- not damage the axons. The cases as these plete loss of protective sensation in the right could not be solitary but should be rather foot by filamentous test and some loss in the common in this time of chemotherapy. If so, left foot. Punch skin biopsies from his right the methods to reconstruct nerves and blood arm and right chest lesion revealed abun- vessels by promoting those regeneration dant acid-fast bacilli (AFB). Histopatho- should be developed to cope with the situa- logic examination revealed perivascular, in- tion for surgeon, assisted by pathologists.— terstitial and perineural granulomatous Authors’Abstract inflammation and a large number of AFB organisms within histiocytes. Culture of the skin biopsy specimen was negative for My- Mert, A., Ozaras, R., Tabak, F., and Oz- cobacterium tuberculosis or atypical myco- turk, R. Primary tuberculosis cases pre- bacterium. Polymerase chain reaction senting with erythema nodosum. J. Der- (PCR) performed for Mycobacterium lep- matol. 31(1) (2004) 66–68. rae was positive. The patient was treated with a modified regimen consisting of dap- Erythema nodosum (EN) is seen only in sone 100 mg qd, ethionamide 250 mg qd, the primary tuberculosis (TB) form of tu- and minocycline 100 mg qd. His skin rash berculous diseases. Among the etiologies and neurological symptoms have re- of EN, TB is the most frequent disorder in solved.—Authors’Abstract developing countries. We aimed to assess our patients with EN in reference to primary TB. We evaluated 335 patients with Moses, A. E., Adelowo, K. A., and Ajayi, the diagnosis of TB during last 20 years; B. B. Prevalence of HIV-1 infection retrospectively 61 (18%) of these cases among patients with leprosy and pul- had pulmonary and 274 (82%) had extra- monary tuberculosis in a semi-arid re- pulmonary TB. Ten (16%) of the pul- gion, Nigeria. J. R. Soc. Promotion Health monary TB cases were primary. All 10 pa- 123(2) (2003) 117–119. tients with primary TB presented with EN. Among 50 patients with EN diagnosed and Much evidence exists on pulmonary tu- followed during the last 10 years, the eti- berculosis (PTB) as a presenting feature of ology was determined in 56%, and primary HIV infection or AIDS-related complex, TB was the most frequent: 20%.—Au- while few reports exist of a direct associa- thors’ Abstract tion between HIV infection and leprosy. 196 International Journal of Leprosy 2004

This study was carried out to see whether or ripheral nerves at diagnosis, correlated sta- not an association between leprosy and HIV tistically (p <0.005) with the occurrence of infection existed, similar to that of PTB in disabilities (DGBT >0). It also correlated the region of Maiduguri, Nigeria. Of 105 pa- sigiﬁcantly with the development of neuri- tients with leprosy, 11 (10.5%) were positive tis in the follow-up (average of 64.6 months for HIV antibody. Of 58 patients with sus- from diagnosis, during and after multidrug pected PTB, 11 (19%) were positive for therapy). HIV antibody. Twenty-seven (47%) of the CONCLUSIONS:We emphasize the need of 58 had active PTB, with results of sputum a good examination of peripheral nerve smear and culture positive for mycobacte- trunks in multibacillary patients at the diag- rium, and six of these (22.2%) were also nosis, in order to improve the detection of positive for HIV antibody. Odds ratios (OR) diabilities already present, and specially to obtained by conditional logistic regression prevent further disabilities. Healthy profes- (matched) analysis were 3.52 (95%, CI sionals who deal with leprosy patients must 1.03–12.07) and 2.53 (95%, CI 1.04–6.15) be aware to the initial neurological impair- for association between HIV-1 and PTB and ments because those patients are more sus- leprosy, respectively. HIV infection was ceptible to the occurrence of neuritis and more prevalent among leprosy patients aged neurological sequelae.—Anais Braseileiros under 30 years, OR = 4.25 (95%, CI de Dermatologia 1.25–14.42). The prevalence of HIV-1 infection was at borderline signiﬁcance, higher in PTB and leprosy patients than in Rodriguez, G. [Generalized adenopathy as blood donors, Fisher’s exact test (two-tailed) a manifestation of type 2 reactional lep- p = 0.07 and p = 0.05, respectively. rosy]. Biomedica 23(4) (2003) 373–387. [Article in Spanish]

Pimentel, M. I. F., Borges, E., da Costa Generalized adenopathy as a manifesta- Nery, J. A., and Gonçalves, R. R. Initial tion of type 2 reactional leprosy Leprosy pa- neurological exam of multibacillary lep- tient’s reactions are severe clinical manifes- rosy: correlation between the presence of tations of acute inflammation of chronic affected nerves and the disability present lesions, capable of producing irreversible at diagnosis and with the occurrence of and invalidating damage. We studied a 46 overt neuritis. An. bras. Dermatol., Rio de year-old man with a type 2 leprosy reaction, Janeiro, 78(5) (2003) 561–568. who presented fever, cutaneous nodules, nasal obstruction and generalized adenopa- BACKGROUND: Disabilities constitute the thy. The hemogram showed leucocytosis main problem of leprosy. It is important to with neutrophilia. None of the initial diag- identify risk factors involved, so it can be noses included leprosy. A lymph node possible the prone patients be followed-up biopsy revealed extensive necrotic areas in- more carefully. filtrated with polymorphonuclear lympho- OBJECTIVES:To determine if the presence cytes, and foamy macrophages. Eosino- of thick and/or painful peripheral nerves at phylic necrosis and thrombosis of venules diagnosis correlates with disabilities al- with lymphoid nodule depletion was also in ready present at the initial examination, as evidence. Ziehl Neelsen stain was not done, well as with subsequent development of but the Gomori stain clearly showed neuritis, during and after multidrug therapy. Hansen’s bacilli. These were were not de- METHODS: One hundred and three patients tected by the pathologist and therefore a with multibacillary forms of leprosy were final diagnosis was not provided. Twenty studied and we noted the presence of com- months later, the patient presented similar promised peripheral nerves at diagnosis, the symptoms, but with more generalized lym- disability grade before treatment (DGBT), phadenopathy and presence of cutaneous and the occurrence of neuritis episodes dur- nodules. Nodule biopsy showed leproma- ing and after multibacillary multidrug ther- tous leprosy with erythema nodusum lepro- apy. sum or type 2 reaction. Polychemotherapy RESULTS: The detection of affected pe- treatment and anti-reaction treatment with 72, 2 Current Literature, Clinical Sciences 197

thalidomide cured the patient. No sequelae fections are thought to confer protective im- were noted in 3 years following the treat- munity against exogenous reinfection. In ment. A literature review of the type 2 reac- this study, a novel polymerase chain reaction in leprosy is provided, including dis- tion method was developed to specifically cussion of risk factors, histopathology, identify M. tuberculosis strains belonging to differential diagnosis for leprosy adenopa- the Beijing and non-Beijing evolutionary thy, pathogenesis, prognosis, and treatment. lineages in sputum specimens collected Type 2 leprosy must be treated immediately from tuberculosis patients resident in an epi- upon diagnosis as it can cause serious and demiologic field site in Cape Town, South permanent tissue damage. As had occurred Africa. The sensitivity and specificity of the in the above patient, the disease can proceed polymerase chain reaction-based strain clas- with generalized and symptomatic lymph- sification method were 100% (95% confi- adenopathy.—Authors’Abstract dence interval, 85–100%) when compared with DNA fingerprinting and spacer oligo- typing (spoligotyping). Application of this Thompson, A. M., Lynn, A. A., Robson, method showed that 19% of all patients K., Joyce. M, P., Fivenson, D. P., and were simultaneously infected with Beijing Scollard, D. Lepromatous phlebitis of and non-Beijing strains, and 57% of patients the external jugular vein. J. Am. Acad. infected with a Beijing strain were also in- Dermatol. 49(6) (2003) 1180–1182. fected with a non-Beijing strain. Multiple infections were more frequent in retreatment Mycobacterium leprae (M. leprae), the cases (23%) as compared with new cases causative agent of Hansen’s disease, is en- (17%), but were not associated with sex, demic in many areas of Asia, sub-Saharan age, or smear grading. These results suggest Africa, South and Central America, the Pa- that multiple infections are frequent, imply- cific Islands, and the Philippines. The spec- ing high reinfection rates and the absence of trum of clinical disease is dependent on the efficient protective immunity conferred by patient’s cell-mediated immunity and might the initial infection. This finding could in- range from localized anesthetic patches or fluence our understanding of the epidemiol- plaques to disseminated disease. If undiag- ogy of disease in high-incidence regions and nosed, progression with damage to the in- our understanding for vaccine develop- volved sensory and motor nerves might ment.—Authors’Abstract occur. Lepromatous vasculitis occurs most commonly in patients with severe disseminated disease. Vascular disease, as the initial Yan, L., Zhang, G., Zheng, Z., Li, W., presenting sign of tuberculoid leprosy, is, Zheng, T., Watson, J. M., and Piefer, A. however, rare. We present one patient in Comprehensive treatment of complicated whom the development of Hansen’s disease plantar ulcers in leprosy. Chin. Med. J. was associated with involvement of the ex- (Engl). 116(12) (2003) 1946–1948. ternal jugular vein and was initially seen as external jugular vein fibrosis.—Authors’ OBJECTIVE: To investigate feasible treat- Abstract ment methods for plantar ulcers in leprosy patients according to the agreement between the Ministry of Health (MOH) of China and Warren, R. M., Victor, T. C., Streicher, E. the Leprosy Mission International (LMI). M., Richardson, M., Beyers, N., van METHODS: A total of 2599 complicated foot Pittius, N. C., and van Helden, P. D. Pa- ulcers in 1804 leprosy cases underwent surgic tients with active tuberculosis often have treatment. Plastic fixation and supports were different strains in the same sputum spec- used, dressings were changed regularly, and imen. Am. J. Respir. Crit. Care Med. protective footwear and modified insoles were 169(5) (2004) 610–614. provided. RESULTS: Of the 2599 foot ulcers 1446 (55.64%) healed. The cure rate of the It is generally accepted that tuberculosis patients treated in leprosy hospitals was results from a single infection with a single 71.31%, with 219 (15.15%) recurrences of Mycobacterium tuberculosis strain. Such in- foot ulcers. The recurrence rate of those who 198 International Journal of Leprosy 2004 lived at home was 18.35%. CONCLUSIONS: tance walking, intensification of education on Comprehensive treatment of foot ulcers has foot self-care and provision of financial sup- a high cure rate and a low recurrence rate. Re- port are the main measures for preventing a duction of workload, avoidance of long dis- recurrence of foot ulcers.—Authors’Abstract Immuno Pathology

Alvarez, G. R., Zwilling, B. S., and Lafuse, gamma-induced gene expression. These ﬁnd- W. P. Mycobacterium avium inhibition of ings suggest that M. avium infection of mouse IFN-gamma signaling in mouse macro- macrophages inhibits IFN-gamma signal- phages: Toll-like receptor 2 stimulation ing through a TLR2-dependent increase in increases expression of dominant-nega- STAT1beta expression by mRNAstablization tive STAT1 beta by mRNA stabilization. and a TLR2-independent inhibition of STAT1 J. Immunol. 171(12) 2003 6766–6773. tyrosine phosphorylation.—Authors’Abstract

Mycobacterial infections of macrophages have been shown to inhibit the ability of the Bisen, P. S., Garg, S. K., Tiwari, R. P., macrophage to respond to IFN-gamma. We Tagore, P. R., Chandra, R., Karnik, R., previously reported that Mycobacterium Thaker, N., Desai, N., Ghosh, P. K., avium infection of mouse macrophages de- Fraziano, M., and Colizzi, V. Analysis creases IFN-gamma-induced STAT1 tyrosine of the shotgun expression library of the phosphorylation and STAT1 DNA binding. Mycobacterium tuberculosis genome for Because macrophages respond to M. avium immunodominant polypeptides: potential through Toll-like receptor 2 (TLR2), we de- use in serodiagnosis. Clin. Diagn. Lab. termined whether TLR2 stimulation inhibits Immunol. 10(6) (2003) 1051–1058. the response to IFN-gamma. Treatment of mouse RAW264.7 macrophages with TLR2 See Current Literature, Molecular and agonists inhibited the induction of IFN- Genetic Studies, p. 255. gamma-inducible genes by IFN-gamma. In contrast to M. avium infection, TLR2 agonists did not inhibit the IFN-gamma induction of Chen, K., Lu, J., Wang, L., and Gan, Y. H. DNA-binding activity of STAT1 and the Mycobacterial heat shock protein 65 en- tyrosine phosphorylation of STAT1alpha. hances antigen cross-presentation in den- Instead, IFN-gamma induction of RAW264.7 dritic cells independent of Toll-like re- cells treated with TLR2 agonists resulted in ceptor 4 signaling. J. Leukoc. Biol. 75(2) an increase in the tyrosine phosphorylation (2004) 260–266. of the dominant-negative STAT1beta. TLR2 stimulation of RAW264.7 cells increased Heat shock proteins (HSP) have been both STAT1beta protein and mRNA shown to enhance antigen processing and expression, suggesting that the increased presentation through their association with STAT1beta phosphoylation results from in- antigenic peptides and delivery of these moi- creased STAT1beta expression. Because eties into major histocompatibility complex STAT1alpha and STAT1beta mRNA have class I pathways. In this study, mycobacterial different 3′ untranslated regions, and 3′ un- Hsp65 is demonstrated to have the ability to translated regions can regulate mRNA stabil- help cross-present an exogenous protein by ity, we examined the effects of TLR2 stimu- dendritic cells (DC) to CD8 T cells without lation on mRNA stability. TLR2 stimulation the need for complex formation between of RAW264.7 cells increased the stability of Hsp65 and the protein. This ability of Hsp65 STAT1beta mRNA, while not affecting the to enhance cross-presentation is independent stability of STAT1alpha mRNA. The ability of its weak stimulatory effect on DC, the lat- of STAT1beta to function as a dominant neg- ter seen only after prolonged incubation. ative was conﬁrmed by overexpression of When the effect of lipopolysaccharide con- STAT1beta in RAW264.7 macrophages by tamination is abrogated, Hsp65 is unable to transient transfection, which inhibited IFN- activate Toll-like receptor (TLR)4 in the 72, 2 Current Literature, Immuno Pathology 199

presence of CD14 and MD2. This accounts quickly eliminated, explaining one of the for the inability of Hsp65 to drive maturation bases of virulence.—Authors’Abstract of DC and shows that Hsp65 is not a potent stimulator of DC. Thus, Hsp65 enhances the cross-presentation of a soluble, free antigen Deng, L., Ding, W., and Granstein, R. D. by DC, independent of TLR4 signaling and Thalidomide inhibits tumor necrosis up-regulation of costimulatory molecules.— factor-alpha production and antigen pre- Authors’Abstract sentation by Langerhans cells. J. Invest. Dermatol. 121(5) (2003) 1060–1065.

Curto, M., Reali, C., Palmieri, G., Scintu, Thalidomide is an effective treatment F., Schivo, M. L., Sogos, V., Marcialis, for several inflammatory and autoimmune M. A., Ennas, M. G., Schwarz, H., disorders including erythema nodosum Pozzi, G., and Gremo, F. Inhibition of leprosum, Behcet’s syndrome, discoid cytokines expression in human microglia lupus erythematosus, and Crohn’s disease. infected by virulent and non-virulent my- Thalidomide is believed to exert its anti- cobacteria. Neurochem. Int. 44(6) (2004) inflammatory effects, at least in part, by 381–392. inhibiting tumor necrosis factor-alpha (TNF-alpha) production by monocytes. We The pathogenesis of tuberculosis (TBC) studied the effects of thalidomide on epi- meningitis is still unknown. As shown by dermal Langerhans cells (LC). LCs are previous studies, human microglia can be epidermal antigen-presenting dendritic the target of mycobacteria, but no data are cells that play important roles in skin im- available about their cellular response to inmune responses. Using the murine fection. Consequently, we studied the ex- epidermis-derived dendritic cell lines, pression of tumor necrosis factor-alpha XS106A from A/J mice and XS52 from (TNF-alpha), interleukin-1 (IL-1) and IL-10 BALB/c mice as surrogates for LC, we in human microglia pure cultures infected found that thalidomide inhibited TNF- with the two variants of Mycobacterium alpha production in a concentration- avium (domed-opaque (SmD) and transpar- dependent manner. Northern blot analysis ent (SmT)) and with Mycobacterium tuber- revealed that thalidomide significantly de- culosis. Results showed that microglia was creased the peak-induced mRNA level of productively infected by mycobacteria TNF-alpha in XS106A cells and XS52 which could grow inside the cells. Myco- cells. We then examined the effect of bacteria internalization was more rapid for thalidomide on fresh LC enriched to ap- M. avium, but M. tuberculosis infection proximately 98% using positive selection turned out to be more efficient due to the in- of Ia+ cells with antibodies conjugated to corporation of densely packed bacteria. magnetic microspheres. TNF-alpha pro- TNF-alpha expression was not affected by duction was reduced by 67.7% at a thalido- M. avium, whereas an increase followed by mide concentration of 200 microg per mL. a decrease was observed in M. tuberculosis. Thalidomide also had a profound in- Both IL-1 and IL-10 cytokine expression hibitory effect on the ability of LC to pre- was rapidly inhibited by infection with the sent antigen to a responsive TH1 clone. more virulent bacteria, whereas the non- Thalidomide inhibits TNF-alpha produc- pathogenic one had almost no effect. Also, tion and the antigen-presenting ability of the expression of the co-stimulatory mole- epidermal LCs. These mechanisms may cule CD137, a member of tumor necrosis contribute to the therapeutic effects ob- factor receptor family, was affected by served with this agent.—Authors’Abstract infection with virulent mycobacteria. Our results show that microglia response to mycobacterial infection is modulated in cor- Dugue, C., Perraut, R., Youinou, P., and relation with virulence, mainly toward inhi- Renaudineau, Y. Effects of anti- bition of inflammatory response. This endothelial cell antibodies in leprosy and observation might be one of the mechanisms malaria. Infect. Immun. 72(1) (2004) by which non-pathogenic mycobacteria are 301–309. 200 International Journal of Leprosy 2004

As a result of damaging endothelial cells study examined the differential gene ex- (ECs), Mycobacterium leprae triggers the pression pattern in alveolar macrophages of production of antibodies (Abs). These anti- patients with granulomatous disorders. The EC Abs (AECAs) can be divided into two differential mRNA regulation pattern of types. The first type nonspecifically reacts alveolar macrophages in the bronchoalveo- with components of the cytosol (CY) and lar lavage of healthy controls was compared can be detected by enzyme-linked im- to that of patients with sarcoidosis and tu- munosorbent assay (ELISA). The second berculosis by means of differential display specifically reacts with the EC membrane reverse transcription PCR. Comparative (MB) and requires fluorescence-activated analysis of 2498 PCR products in controls, cell sorter (FACS) analysis to be detected. sarcoidosis, and tuberculosis revealed a dif- The presence of both types of AECAs was ferential regulation of expressed sequence determined in 68 leprosy patients. The tags in only 6.5%. 1.8% showed a shared ex- ELISA was positive for 35 of them but also pression pattern between sarcoidosis and tu- for 30 of 34 malaria patients and 17 of 50 berculosis in contrast to the control. It can healthy African controls. However, whereas be assumed that these alterations are associ- FACS analysis showed MB reactivity in ated with common granulomatous features. only three malaria patients and four con- In contrast, 3.0% of the amplified sequence trols, this reactivity was found in 27 leprosy tags showed specific up- or downregulation patients, more of those having the leproma- in sarcoidosis and 1.6% in tuberculosis. tous than the tuberculoid form. Specificity These data indicate a significant proportion for MB, which we failed to absorb by incu- of common granuloma-associated features, bation with CY lysates, predominated over independent of the origin of the granuloma- that for CY in leprosy, unlike malaria, where tous disorder.—Authors’Abstract the EC reactivity was restricted to the CY. Western blot analysis and two-dimensional electrophoresis revealed that calreticulin, vi- Geluk, A., van Meijgaarden, K. E., mentin, tubulin, and heat shock protein 70 Franken, K. L., Wieles, B., Arend, S. were targeted by AECAs from leprosy pa- M., Faber, W. R., Naafs, B., and Otten- tients, but other proteins remained unidenti- hoff, T. H. Immunological crossreactiv- fied. These auto-Abs, but not those from ity of the Mycobacterium leprae CFP-10 malaria patients, did activate ECs, as indi- with its homologue in Mycobacterium tu- cated by the E-selectin and intercellular ad- berculosis. Scand. J. Immunol. 59(1) hesion molecule 1 upregulation, and/or in- (2004) 66–70. duced them into apoptosis, as documented by four different methods. Our findings sug- Mycobacterium tuberculosis culture fil- gest that, in some but not all leprosy pa- trate protein-10 (CFP-10) (Rv3874) is con- tients, AECAs may play a role in pathogen- sidered a promising antigen for the immuno- esis.—Authors’Abstract diagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT-6). Both ESAT-6 and CFP-10 are en- Gaede, K. I., Mamat, U., and Muller- coded by the RD1 region that is deleted Quernheim, J. Differential gene expres- from all tested M. bovis bacille Calmette- sion pattern in alveolar macrophages of Guerin (BCG) strains but present in M. lep- patients with sarcoidosis and tuberculo- rae, M. tuberculosis, M. bovis, M. kansasii, sis. J. Mol. Med. 82(3) (2004) 206–210. M. africanum and M. marinum. In this study, the homologue of CFP-10 in M. lep- Sarcoidosis is a multisystem granuloma- rae (ML0050) is identified and character- tous disorder of unknown origin characterized. Interferon-gamma production in re- ized by the presence of epithelioid granulo- sponse to this homologue by T cells from mata in the affected organs. Histological and leprosy patients, TB patients and unexposed clinical similarities between sarcoidosis and controls shows that CFP-10 of M. leprae is tuberculosis caused by M. tuberculosis sug- a potent antigen that crossreacts with CFP- gest a shared underlying pathophysiology. 10 of M. tuberculosis at the T-cell level. This However, specific markers are needed. This crossreactivity has implications for the use 72, 2 Current Literature, Immuno Pathology 201 of CFP-10 of these mycobacterial species as an exogenous lipid antigen to T cells. Na- diagnostic tool in areas endemic for both the ture Immunol. 5(2) (2004) 175–181. diseases.—Authors’Abstract

Members of the CD1 family present anti- Imai, K., Kurita-Ochiai, T., and Ochiai, K. genic lipids to T lymphocytes. CD1 mole- Mycobacterium bovis bacillus Calmette- cules survey endocytic compartments for Guerin infection promotes SOCS induc- lipid antigens that are sorted into these vesi- tion and inhibits IFN-gamma-stimulated cles after incorporation into the membrane JAK/STAT signaling in J774 macro- bilayer, and extraction from the bilayer is phages. FEMS Immunol. Med. Micro- likely to be a critical step for lipid associa- biol. 39(2) (2003) 173–180. tion. We hypothesized that lysosomal saposins, which are cofactors required for The resurgence in mycobacterial infec- sphingolipid degradation, might be involved tion worldwide has led to renewed attention in this process. Here we show that saposins, to the pathogenesis of Mycobacterium although not required for the autoreactive species. Although interferon-gamma (IFN- recognition of CD1d by natural killer T gamma) is a principal mediator of macro- cells, are indispensable for the binding of an phage activation, macrophages infected exogenous lipid antigen, α-galactosylce- with Mycobacterium are poor in response at ramide, to CD1d in the endocytic pathway. the cytokine. However, the molecular We suggest that saposins mobilize mono- mechanisms underlying mycobacterial in- meric lipids from lysosomal membranes and fection remain unclear. The purpose of this facilitate their association with CD1d.—Na- study was to elucidate the mechanism of the ture Immunology poor response to IFN-gamma in mycobacterial infection. Our data clearly demonstrate that this is due to induction of sup- Mendez-Samperio, P., Ayala, H., Trejo, A., pressor of cytokine signal (SOCS) negative and Ramirez, F. A. Differential induction regulators of IFN-gamma signal transduc- of TNF-alpha and NOS2 by mitogen- tion that closely correlates with the inhibi- activated protein kinase signaling path- tion of JAK/STAT signaling and gene ex- ways during Mycobacterium bovis infec- pression stimulated by IFN-gamma. tion. J. Infect. 48(1) (2004) 66–73. Mycobacterium bovis bacillus Calmette- Guerin infection induces the production of The role of mitogen-activated protein ki- SOCS-1 and SOCS-3 in murine J774 nase (MAPK) signaling pathways in the reg- macrophages. The level of SOCS-1 mRNA ulation of TNF-alpha and NOS2 production increased 1 h and reached a maximum 3 h by human monocytes infected with Myco- after the addition of the bacteria. SOCS-3 bacterium bovis BCG was examined. Inhi- mRNA expression appeared as early as 1 h bition studies showed that ERK1/2 and p38 after the infection. We also observed that MAPK activation were necessary for the trehalose 6,6′-dimycolate/cord factor, a monocyte response to M. bovis infection. major component of the Mycobacterium tu- Analysis of MAPK activation showed rapid berculosis cell wall, induces expression of phosphorylation of ERK1/2 and p38 in re- SOCS and inhibits IFN-gamma-stimulated sponse to M. bovis BCG. Phosphorylation phosphorylation of STAT1 extensively in was not due to an autocrine effect of TNF- the cells. The results in this study suggest alpha secretion, since an anti-TNF-alpha an- that a molecular mechanism of mycobacte- tibody had no significant effect on the levels rial infection affects the unresponsiveness of p38 phosphorylation. The inhibitor to IFN-gamma in the subsequent growth PD98059 significantly reduced M. bovis and spread of macrophages.—Authors’Ab- BCG-induced TNF-alpha production and al- stract most completely abrogated phosphorylation of ERK1/2; in addition the potent MEK inhibitor U0126 also abrogated phosphoryla- Kang, S.-J., and Cresswell, P. Saposins fa- tion. In contrast, studies using inhibitors se- cilitate CD1d-restricted presentation of lective for ERK1/2 and p38 showed that p38 202 International Journal of Leprosy 2004 plays an essential role in the induction of tion is dependent on Toll-like receptor 4 and NOS2, whereas the role of ERK1/2 was sensitive to both dexamethasone and CC minor. These results suggest that ERK1/2 chemokine-binding protein inhibition. More- and p38 kinases differentially regulate the over, by using MCP-1 neutralizing Abs and M. bovis BCG-mediated induction of TNF- genetically deficient mice we show that LPS- alpha and NOS2 in human monocytes.— and BCG-induced gammadelta T lympho- Authors’Abstract cyte influx to the pleural cavity of mice is mainly orchestrated by the CC chemokine MCP-1.—Authors’Abstract Penido, C., Vieira-de-Abreu, A., Bozza, M. T., Castro-Faria-Neto, H. C., and Bozza, P. T. Role of monocyte chemo- Ranjbar, S., Ly, N., Thim, S., Reynes, J. tactic protein-1/CC chemokine ligand 2 M., and Goldfeld, A. E. Mycobacterium on gamma delta T lymphocyte trafficking tuberculosis recall antigens suppress during inflammation induced by lipo- HIV-1 replication in anergic donor cells polysaccharide or Mycobacterium bovis via CD8+ T cell expansion and increased bacille Calmette-Guerin. J. Immunol. IL-10 levels. J. Immunol. 172(3) (2004) 171(12) (2003) 6788–6794 1953–1959.

Gammadelta T lymphocytes are involved Mycobacterium tuberculosis (MTb) is the in a great variety of inflammatory and infec- leading cause of death in the setting of tious responses. However, the mechanisms AIDS. MTb enhances the pathogenicity and by which gammadelta T lymphocytes mi- accelerates the course of HIV disease and, grate to inflamed sites are poorly understood. furthermore, infection with HIV-1 increases In this study we investigate the role of mono- the risk of reactivation or reinfection with cyte chemotactic protein (MCP)-1 in regu- MTb. In this study, we show that host- lating gammadelta T cell migration after LPS specific recall responses to one pathogen, or Mycobacterium bovis bacille Calmette- MTb, has a direct effect upon the regulation Guerin (BCG) challenge. LPS-induced gam- of a second pathogen, HIV-1. Using cells madelta T cell influx was significantly from immunocompetent former tuberculo- inhibited by either pretreatment with dexa- sis (TB) patients who displayed either a per- methasone or vaccinia virus Lister 35-kDa sistently positive (responsive) or negative chemokine binding protein, vCKBP, a CC (anergic), delayed-type hypersensitivity chemokine neutralizing protein, suggesting (DTH) reaction to intradermal injection of a role for CC chemokines in this phenome- purified protein derivative (PPD), we inves- non. LPS stimulation increased the expres- tigated the effect of recall Ags to MTb upon sion of MCP-1 mRNA and protein at the in- the replication of HIV-1 primary isolates in flammation site within 6 hr. It is noteworthy vitro. We show that HIV-1 replication of a T that LPS was unable to increase MCP-1 pro- cell-tropic isolate was significantly impaired duction or gammadelta T cell recruitment in in MTb-stimulated PBMC from PPD-anergic C3H/HeJ, indicative of the involvement of donors. Furthermore, these donors displayed Toll-like receptor 4. Gammadelta T cells ex- a significant increase in CD8(+) T cells and press MCP-1 receptor CCR2. Pretreatment IL-10 levels and lower levels of IL-2 and with anti-MCP-1 mAb drastically inhibited TNF-alpha relative to PPD-responsive LPS-induced in vivo gammadelta T cell mo- donors in response to PPD stimulation. bilization. Indeed, MCP-1 knockout mice Strikingly, CD8(+) T cell depletion and were unable to recruit gammadelta T cells to blocking of IL-10 significantly increased the pleural cavity after LPS stimulation, ef- HIV-1 replication in these PPD-anergic fect that could be restored by coadministra- donors, indicating that an immunosuppres- tion of MCP-1. In addition, BCG-induced sive response to MTb recall Ags inhibits gammadelta lymphocyte accumulation was HIV-1 replication in PPD-anergic individuals. significantly reduced in MCP-1 knockout Therefore, immunotherapeutic approaches mice when compared with wild-type mice. aimed at recapitulating Ag-specific MTb an- In conclusion, our results indicate that LPS- ergy in vivo could result in novel and effec- induced gammadelta T lymphocyte migra- tive approaches to inhibit HIV-1 disease 72, 2 Current Literature, Immuno Pathology 203 progression in MTb/HIV-1 coinfection.— rial mechanisms of infected macrophages. It Authors’Abstract has previously been reported that ATP treatment of mycobacteria-infected macrophages induces apoptosis mediated via the P2X(7) Reddy, V. M., and Suleman, F. G. Myco- pathway and that this leads to the death of bacterium avium-superoxide dismutase both the host cell and the internalized bacilli. binds to epithelial cell aldolase, glycer- We have recently identified a single nu- aldehyde-3-phosphate dehydrogenase cleotide polymorphism in the P2X7 gene and cyclophilin A. Microb. Pathog. 36(2) (1513A→C), with 1–2% prevalence in the (2004) 67–74. homozygous state, which codes for a nonfunctional receptor. IFN-gamma-primed, Mycobacterium avium complex (MAC) mycobacteria-infected macrophages from adheres, invades and multiplies inside epi- wild-type individuals were incubated with thelial cells. Earlier, we demonstrated two ATP and this induced apoptosis and reduced MAC protein adhesins, 25 and 31 kDa, mycobacterial viability by 90%. Similar binding with HEp-2 cells. The 25 kDa MAC treatment of macrophages from individuals adhesin was found to be superoxide dismu- homozygous for the 1513C polymorphism tase (SOD). In this study, epithelial cell failed to induce apoptosis and did not lead (HEp-2 and A549) ligands for MAC-SOD to mycobacterial killing via the P2X(7)- were identified by probing two-dimensional mediated pathway. These data demonstrate western blots of epithelial extracts with that a single nucleotide polymorphism in the MAC proteins followed by monoclonal anti- P2X7 gene can allow survival of mycobac- MAC-SOD antibodies. Three epithelial cell teria within infected host cells.—Authors’ proteins with molecular masses 43, 40 and Abstract 18 kDa, present in both membrane and cytosolic fractions, were found to bind with MAC-SOD. Based on the N-terminal amino Sen Gupta, R., Hillemann, D., Kubica, T., acid sequences, the 43, 40 and 18 kDa epi- Zissel, G., Muller-Quernheim, J., thelial proteins were identified as aldolase, Galle, J., Vollmer, E., and Goldmann, glyceraldehyde-3-phosphate dehydrogenase T. HOPE-fixation enables improved (GAPDH) and cyclophilin A (CypA), re- PCR-based detection and differentiation spectively. Furthermore, MAC-SOD was of Mycobacterium tuberculosis complex found to bind to purified rabbit muscle al- in paraffin-embedded tissues. Pathol. dolase, GAPDH and recombinant CypA in Res. Pract. 199(9) (2003) 619–623. western blotting.—Authors’Abstract Standard PCR-based detection of mycobacterial DNA in paraffin-embedded speci- Remus, N., Alcais, A., and Abel, L. Human mens may lack sufficient sensitivity because genetics of common mycobacterial in- of the degradation of nucleic acids caused fections. Immunol. Res. 28(2) (2003) by routinely used formalin fixation. There- 109–129. fore, we set up an approach that aimed at improving the results by applying the novel See Current Literature, Molecular and HOPE-fixative in PCR-detection of myco- Genetic Studies, p. 260. bacteria in paraffin-embedded tissues. Com- parison of PCR-results using DNA extracted from either HOPE- or formalin-fixed speci- Saunders, B. M., Fernando, S. L., Sluyter, mens in BCG-infected SCID-mice revealed R., Britton, W. J., and Wiley, J. S. A a more than 100fold enhanced sensitivity for loss-of-function polymorphism in the the HOPE-fixed material. Owing to the human P2X7 receptor abolishes ATP- preservation of DNA from degradation in mediated killing of mycobacteria. J. Im- HOPE-fixed tissues, even differentiation munol. 171(10) (2003) 5442–5446. within the M. tuberculosis complex was possible by spoligotyping. We therefore Protective immunity to mycobacterial in- conclude that the HOPE-fixative is a useful fections requires activation of the antibacte- tool for molecular pathology that enhances 204 International Journal of Leprosy 2004 the sensitivity of PCR-based methods for cell spread during infection. Here we show the detection of pathogens in paraffin- that Mycobacterium marinum, a natural embedded tissues compared to formalin- pathogen of fish and frogs and an occasional fixation. Owing to the better preserved pathogen of humans, is capable of actively DNA, improved differentiation of myco- inducing actin polymerization within mac- bacteria from archived materials is possible. rophages. M. marinum that polymerized These results promise new and a substan- actin were free in the cytoplasm and pro- tially wider range of possibilities in the field pelled by actin-based motility into adjacent of molecular pathology.—Authors’Abstract cells. Immunofluorescence demonstrated the presence of host cytoskeletal proteins, including the Arp2/3 complex and vasodilator- Siemion, I. Z., Gawlowska, M., Krajew- stimulated phosphoprotein, throughout the ski, K., Strug, I., and Wieczorek, Z. actin tails. In contrast, Wiskott-Aldrich syn- Analogs of RGDVY and GRGD peptides drome protein localized exclusively at the inhibit Mycobacterium kansasii phagocy- actin-polymerizing pole of M. marinum. tosis. Peptides 24(8) (2003) 1109–1115. These findings show that M. marinum can escape into the cytoplasm of infected Continuing our research on Mycobacteria macrophages, where it can recruit host cell kansasii phagocytosis inhibition, we have ex- cytoskeletal factors to induce actin poly- amined in that context three series of peptides merization leading to direct cell to cell derived from the RGDVY and GRGD se- spread.—Authors’Abstract quences. It was found that the levels of the inhibitory activity depend on the amino acid composition as well as on the particular pep- Weir, R. E., Black, G. F., Dockrell, H. M., tide sequence. Distinct inhibitory activity was Floyd, S., Fine, P. E., Chaguluka, S. D., found in the case of thymopentin (RKDVY), Stenson, S., King, E., Nazareth, B., the active fragment of thymopoietin. In this Warndorff, D. K., Ngwira, B., Crampin, case the Mycobacterium phagocytosis inhi- A. C., Mwaungulu, L., Sichali, L., Jar- bition should be combined with general im- man, E., Donovan, L., and Blackwell, J. munostimulatory activity of RKDVY pep- M. Mycobacterial purified protein deriv- tide. Our examination of a series of GRGDV atives stimulate innate immunity: Malaw- analogs with a successively prolonged oligo- ians show enhanced tumor necrosis fac- Gly linker inserted into the peptide chain tor alpha, interleukin-1beta (IL-1beta), showed that the distance between the Arg and and IL-10 responses compared to those of Asp residues required for such an activity adolescents in the United Kingdom. In- should be about 9A.—Authors’Abstract fect. Immun. 72(3) (2004) 1807–1811.

To investigate the role of innate immunity Stamm, L. M., Morisaki, J. H., Gao, L. Y., in variable efficacy of Mycobacterium bovis Jeng, R. L., McDonald, K. L., Roth, R., BCG vaccination in Malawi and the United Takeshita, S., Heuser, J., Welch, M. D., Kingdom, we examined 24-hr tumor necro- and Brown, E. J. Mycobacterium mar- sis factor alpha, interleukin-1beta (IL- inum escapes from phagosomes and is 1beta), and IL-10 responses to mycobacte- propelled by actin-based motility. J. Exp. rial purified protein derivatives (PPDs). The Med. 198(9) (2003) 1361–1368. rank order in stimulatory potency for different PPDs was the same for all three cy- Mycobacteria are responsible for a num- tokines. Before vaccination Malawians ber of human and animal diseases and are made higher pro- and anti-inflammatory re- classical intracellular pathogens, living in- sponses than did United Kingdom subjects. side macrophages rather than as free-living Fewer than 5% of United Kingdom subjects organisms during infection. Numerous in- made IL-10 in response to any PPD, com- tracellular pathogens, including Listeria pared to 19 to 57% responders among monocytogenes, Shigella flexneri, and Rick- Malawians. Priming for regulatory IL-10 ettsia rickettsii, exploit the host cytoskele- may contribute to the smaller increase in ton by using actin-based motility for cell to gamma interferon responses in Malawians 72, 2 Current Literature, Immuno Pathology (Leprosy) 205

compared to United Kingdom subjects fol- Fas expression increased over the course of lowing BCG vaccination.—Authors’ Ab- infection on both T cell populations, as did stract their susceptibility to the induction of apoptosis in vitro by anti-Fas mAb. Nevertheless, although the rate of apoptosis among CD4+ Zhong, J., Gilbertson, B., and Cheers, C. T cells from infected mice was reduced to Apoptosis of CD4+ and CD8+ T cells normal levels in lpr mice with a defective during experimental infection with My- Fas, CD8+ T cells were unaffected, imply- cobacterium avium is controlled by ing that Fas/FasL interaction was not im- Fas/FasL and Bcl-2-sensitive pathways, portant in these cells in vivo. Conversely, respectively. Immunol. Cell. Biol. 81(6) over-expression of B-cell lymphoma-2 (Bcl- (2003) 480–486. 2), which is known to protect T cells from apoptosis signalled through the TNF recep- Both CD4+ and CD8+ T cells from mice tor or due to the withdrawal of cytokines, to- infected with Mycobacterium avium suf- tally protected CD8+ T cells from infected fered a high rate of apoptosis, beginning mice but had no effect on CD4+. It is of in- with the onset of the immune response and terest that these two contrasting pathways of culminating in the loss of T cells from the T-cell apoptosis operate at the same time tissues and loss of IFN-gamma production. during a single infection.—Authors’Abstract

Immuno Pathology (Leprosy)

de la Barrera, S., Finiasz, M., Fink, S., response, suggesting that this cytokine affects Ilarregui, J., Aleman, M., Olivares, L., IFNgamma production by NK cells. mRNA Franco, M. C., Pizzariello, G., and del coding for IFNgamma is induced by IL-18 Carmen Sasiain, M. NK cells modulate and reduced in the presence of IL-13, when the cytotoxic activity generated by My- PBMC from N or PB patients are stimulated cobacterium leprae-hsp65 in leprosy pa- with hsp65. Neutralization of IL-13 in PBMC tients: role of IL-18 and IL-13. Clin. Exp. from multibacillary (MB) leprosy patients in- Immunol. 135(1) (2004) 105–113. duces the production of IFNgamma protein by lymphocytes. A modulatory role on the Protection against intracellular pathogens generation of hsp65 induced CTL is demon- such as Mycobacterium leprae is critically strated for IL-18 and IL-13 and this effect dependent on the function of NK cells at takes place through the production of early stages of the immune response and on IFNgamma.—Authors’Abstract Th1 cells at later stages. In the present report we evaluated the role of IL-18 and IL-13, two cytokines that can inﬂuence NK cell activity, Job, C. K. Nine-banded armadillo and lep- in the generation of M. leprae-derived hsp65- rosy research. Indian J. Pathol. Micro- cytotoxic T lymphocytes (CTL) from periph- biol. 46(4) (2003) 541–550.—Indian eral blood mononuclear cells (PBMC) of lep- Journal of Pathology and Microbiology rosy patients. We demonstrated that IL-18 modulates hsp65-induced CTL generation In this presentation an attempt has been and collaborates with IL-12 for this effect. In made to describe the nine-banded armadillo paucibacillary (PB) patients and normal con- as an animal model, probably the only one in trols (N) depletion of NK cells reduces the which lepromatous leprosy similar to that cytolytic activity. Under these conditions, IL- found in humans can be experimentally pro- 12 cannot up-regulate this CTL generation, duced. Some unique features of the physiol- while, in contrast, IL-18 increases the cyto- ogy of the animal are mentioned. The pathol- toxic activity both in the presence or absence ogy and the microbiology of leprosy in the of NK cells. IL-13 down-regulates the hsp65- armadillo are described in detail. The discov- induced CTL generation and counteracts the ery of lepromatous leprosy in the wild ar- positive effect of IL-18. The negative effect madillos in the southern parts of the United of IL-13 is observed in the early stages of the States, the transmission of disease among 206 International Journal of Leprosy 2004 them through trauma and thorn pricks and the for MCP-1, regulated upon activation normal pathogenesis of the disease are presented. The T cell expressed and secreted (RANTES) and impact of leprosy in the wild animals may IL-8 chemokines. Here, the presence of a neu- have on human leprosy is discussed.—Indian trophil chemoattractant IL-8 in leprosy le- Journal of Pathology and Microbiology sions, which do not contain neutrophils, suggests strongly a role of IL-8 as a monocyte and lymphocyte recruiter in leprosy lesions. Johansen, P., Raynaud, C., Yang, M., Col- These results suggest that the chemokines and ston, M. J., Tascon, R. E., and Lowrie, their receptors, which are known to chemo- D. B. Anti-mycobacterial immunity in- attract T lymphocytes and macrophages, are duced by a single injection of M. leprae involved in assembling the cellular inﬁltrate Hsp65-encoding plasmid DNA in bio- found in lesions across the leprosy spec- degradable microparticles. Immunol. Lett. trum.—Authors’Abstract 90(2–3) (2003) 81–85.

Leal, A. M., Magalhaes, P. K., Souza, C. See Current Literature, Experimental In- S., and Foss, N. T. Adrenocortical hor- fections, p. 235. mones and interleukin patterns in leprosy. Parasite Immunol. 25(8–9) (2003) 457–461. Kirkaldy, A. A., Musonda, A. C., Khanolkhar-Young, S., Suneetha, S., The functional status of adrenocortical and Lockwood, D. N. Expression of CC hormones and their relationship to the pat- and CXC chemokines and chemokine tern of inflammatory cytokines in the lepro- receptors in human leprosy skin lesions. matous and tuberculoid poles of leprosy Clin. Exp. Immunol. 134(3) (2003) were investigated. Interleukin (IL)-1beta, 447–453. IL-6 and tumour necrosis factor (TNF)- alpha plasma levels, C-reactive protein We have investigated the expression of (CRP) concentrations and erythrocyte sedi- chemokines and their receptors in leprosy skin mentation rates (ESR) were significantly lesions using immunohistochemistry. Skin higher in LL/BL (lepromatous) leprosy pa- biopsies from 25 leprosy patients across the tients than in control subjects. There was a leprosy spectrum, 11 patients undergoing type significant positive correlation between IL-6 I reversal reactions and four normal donors and TNF-alpha plasma levels and ESR and were immunostained by ABC peroxidase CRP concentrations. IL-1beta was posi- method using antibodies against CC and CXC tively correlated with ESR but not with chemokines and their receptors. Using an in CRP. Both baseline and stimulated adreno- situ hybridization technique we have also corticotropic hormone and cortisol plasma studied the expression of monocyte chemo- levels were not different between patients attractant protein 1 (MCP-1), RANTES and and control subjects. In contrast, adrenal an- interleukin (IL)-8 chemokines mRNA in lep- drogen dehydroepiandrosterone sulphate rosy skin lesions. Chemokines and receptor (DHEA-S) plasma levels were significantly expression was detected in all leprosy skin lower in leprosy patients than in sex- biopsies. Expression of CC chemokines matched control subjects. There was a sig- MCP-1 (p <0.01) and RANTES (p <0.01) nificant inverse correlation between DHEA-S were elevated significantly in borderline tu- and IL-6, TNF-alpha, and CRP concentra- berculoid leprosy in reversal reaction com- tions. This finding may be of pathogenetic pared to non-reactional borderline tuberculoid significance in this disease and in other in- leprosy, but there was no difference in the ex- flammatory states.—Authors’Abstract pression of IL-8 chemokine. Surprisingly, there was no significant difference in the expression of CC (CCR2 and CCR5) and CXC Sridevi, K., Neena, K., Chitralekha, K. T., (CXCR2) chemokine receptors across the lep- Arif, A. K., Tomar, D., and Rao, D. N. rosy spectrum. Similarly, there was no signif- Expression of costimulatory molecules icant difference in the expression of mRNA (CD80, CD86, CD28, CD152), accessory 72, 2 Current Literature, Immuno Pathology (Tuberculosis) 207

molecules (TCR alphabeta, TCR gam- tometric analysis. An increased surface ex- madelta) and T cell lineage molecules pression of CD80, CD86 and CD28 but de- (CD4+, CD8+) in PBMC of leprosy pa- creased CD152 expression was observed tients using Mycobacterium leprae anti- when PBMC of normal, BT/TT (tubercu- gen (MLCWA) with murabutide and T loid) and BL/LL (lepromatous) patients cell peptide of Trat protein. Int. Im- were stimulated in vitro with MLCWA+ munopharmacol. 4(1) (2004) 1–14. MDP-BE+Trat peptide using liposomal mode of antigen delivery, while opposite re- In leprosy, cell-mediated immunity (CMI) sults were obtained with the antigen alone. is more significant than humoral response to Antibody inhibition study using antihuman eliminate intracellular pathogen. T cell defect CD80 or CD86 completely abolished the T is a common feature in lepromatous leprosy cell lymphoproliferation, thereby recon- (LL) patients as compared to tuberculoid type firming the importance of these costim- (TT) patients. For efficient initiation of CD4+, ulatory molecules during T cell activa- T cell response requires T cell receptor (TCR) tion/differentiation. Though the liposome- activation and costimulation provided by mol- entrapped antigen formulation has no effect ecules on antigen-presenting cells (APC) and on expression of alphabeta/gammadelta T their counter receptors on T cells. In our pre- cell receptor, the constitutive levels of TCR vious study, the defective T cell function in gammadelta were high in lepromatous pa- LL patients was restored to a proliferating tients. Thus, TCR bearing gammadelta ap- state with the release of TH1 type cytokines pears to have a negligible regulatory role in using mycobacterial antigen(s) with two im- peripheral blood of leprosy patients. The munomodulators (Murabutide (MDP-BE) percentage of cells positive for CD4+ are and T cell epitope of Trat protein of Escheri- increased in inducible state in all the three chia coli) by presenting the antigen in partic- groups, while CD8+-positive cells were ulate form in vitro to PBMC derived from lep- decreased in LL patients, thereby recon- rosy patients. This observation prompted us to firming the fact that priming of CD4+ cells study the expression of the costimulatory mol- are necessary for producing final effector ecules (CD80, CD86, CD28, CD152), other functions. Lastly, intracellular cytokine accessory molecules (TCR alphabeta/gam- staining experiment indicated that CD4+ madelta) and T cell lineage molecules (CD4+ cells are the major producers of IFN-gamma and CD8+) during constitutive and activated but not NK cells. The study highlights the state of peripheral blood mononuclear cells reversal of T cell anergy especially in lepro- (PBMC) derived from normal and leprosy in- matous patients through the modulation of dividuals using different formulations of My- costimulatory molecule expression under cobacterium leprae total cell wall antigen the influence of Th1 cytokines, i.e., IL-2 and (MLCWA), Trat and MDP-BE using flow cy- IFNgamma.—Authors’Abstract

Immuno Pathology (Tuberculosis)

Aagaard, C., Brock, I., Olsen, A., Otten- candidate antigens (Rv2653 and Rv2654) hoff, T. H., Weldingh, K., and Ander- and investigated T cell recognition during sen, P. Mapping immune reactivity to- natural infection in humans and experimen- ward Rv2653 and Rv2654: two novel tal infection in guinea pigs. Peripheral blood low-molecular-mass antigens found mononuclear cells stimulated with peptide specifically in the Mycobacterium tuber- pools covering the full length of Rv2654 in- culosis complex. J. Infect. Dis. 189(5) duced interferon-gamma release in 10 of 19 2004 812–819. patients with TB. Neither Rv2654 single peptides nor Rv2654 pools were recognized by New tools are urgently needed for the de- bacille Calmette-Guerin-vaccinated donors. tection of latent tuberculosis (TB). We eval- However, peptides from Rv2653 were rec- uated the diagnostic potential of 2 novel My- ognized by both patients group. The cross- cobacterium tuberculosis complex-specific reactive epitope(s) in Rv2653 were located in 208 International Journal of Leprosy 2004 a 36-amino acid stretch in the center of the to suppress the antimycobacterial immune molecule. Rv2654 also induced M. tubercu- responses of humans and experimental an- losis-specific skin-test responses in 3 of 4 imals. In this study, the contributions of aerosol-infected guinea pigs. Rv2654 is a TGF-beta to cytokine production in vivo strongly recognized T cell antigen that is were investigated by using the established highly specific for TB and has potential as a guinea pig model of tuberculous pleurisy. novel cell-mediated immunity-based TB di- Mycobacterium bovis BCG-vaccinated agnostic agent.—Authors’Abstract guinea pigs were injected intrapleurally with heat-killed virulent Mycobacterium tuberculosis. Eight days following induc- Agger, E. M., Brock, I., Okkels, L. M., tion of an antigen-specific pleural effusion, Arend, S. M., Aagaard, C. S., Weldingh, guinea pigs were injected intrapleurally K. N., and Andersen, P. Human T-cell re- with anti-TGF-beta1 or isotype control an- sponses to the RD1-encoded protein tibody. The following day, pleural exudates TB27.4 (Rv3878) from Mycobacterium were removed, and the fluid volume and tuberculosis. Immunology 110(4) 2003 characteristics of the infiltrating cells were 507–512. determined. Pleural fluid was analyzed for total interferon (IFN) and tumor necrosis In recent years, there has been considerable factor (TNF) protein levels by using ap- focus on the discovery and characterization of propriate bioassays. RNA from pleural ef- proteins derived from Mycobacterium tuber- fusion cells was examined to determine culosis leading to the identification of a num- TGF-beta1, TNF-alpha, IFN-gamma, and ber of candidate antigens for use in vaccine interleukin-8 mRNA levels by using real- development or for diagnostic purposes. Pre- time PCR. Proliferative responses of pleu- vious experiments have demonstrated an im- ral effusion lymphocytes were examined in portant immunological role for proteins en- response to concanavalin A and purified coded by the RD1 region, which is absent from protein derivative (PPD) in vitro. Treat- all strains of bacillus Calmette-Guerin (BCG) ment with anti-TGF-beta1 resulted in de- but present in the genomes of virulent M. creased pleural fluid volume and decreased bovis and M. tuberculosis. Herein, we have cell numbers in the pleural space along studied human T-cell responses to the antigen with an increased percentage of lympho- encoded by the putative open reading frame cytes and a decreased percentage of neu- (rv3878) of the RD1 region. Immunoblot trophils. The bioactive TNF protein levels analysis revealed that rv3878 was expressed in pleural fluid were increased in guinea and the native protein was designated TB27.4. pigs treated with anti-TGF-beta1, while the Immunological evaluations demonstrate that bioactive IFN protein concentrations were TB27.4 elicits a prominent immune response not altered. Expression of TGF-beta1 and in human tuberculosis patients with a domi- TNF-alpha mRNA was significantly in- nant region in the C-terminal part of the mol- creased following TGF-beta1 neutraliza- ecule. In contrast, very limited responses were tion. Finally, PPD-induced proliferative re- seen in M. bovis BCG-vaccinated donors. sponses of pleural cells from anti-TGF- This study therefore emphasizes the diagnos- beta1-treated animals were significantly tic potential of proteins encoded by the RD1 enhanced. Thus, TGF-beta1 may be in- region.—Authors’Abstract volved in the resolution of this local, mycobacterial antigen-specific inflammatory response.—Authors’ Abstract Allen, S. S., Cassone, L., Lasco, T. M., and McMurray, D. N. Effect of neutralizing transforming growth factor beta1 on the Ando, M., Yoshimatsu, T., Ko, C., Con- immune response against Mycobacterium verse, P. J., and Bishai, W. R. Deletion tuberculosis in guinea pigs. Infect. of Mycobacterium tuberculosis sigma Immun. 72(3) 2004 1358–1363. factor E results in delayed time to death with bacterial persistence in the lungs of Transforming growth factor beta (TGF- aerosol-infected mice. Infect. Immun. beta) is a cytokine which has been shown 71(12) (2003) 7170–7172. 72, 2 Current Literature, Immuno Pathology (Tuberculosis) 209

See Current Literature, Experimental In- mune response. Mycobacterial cell wall fections, p. 230. components activate macrophages through Toll-like receptor (TLR) 2, suggesting that this innate immune receptor plays a role in Brookes, R. H., Pathan, A. A., McShane, the host response to M. tuberculosis infec- H., Hensmann, M., Price, D. A., and tion. After aerosol infection with either 100 Hill, A. V. CD8+ T cell-mediated sup- or 500 live mycobacteria, TLR2-deficient pression of intracellular Mycobacterium mice display reduced bacterial clearance, a tuberculosis growth in activated human defective granulomatous response, and macrophages. Eur. J. Immunol. 33(12) develop chronic pneumonia. Analysis of (2003) 3293–3302. pulmonary immune responses in TLR2- deficient mice after 500 mycobacterial Animal models of tuberculosis point to a aerosol challenge showed increased levels protective role for MHC class I-restricted of interferon-gamma, tumor necrosis factor- CD8(+) T cells, yet it is unclear how these alpha, and interleukin-12p40 as well as in- cells protect or whether such findings ex- creased numbers of CD4(+) and CD8(+) tend to humans. Here we report that macro- cells. Furthermore, TLR2-deficient mice phages infected with Mycobacterium tu- mounted elevated Ag-specific type 1 T-cell berculosis, rapidly process and present an responses that were not protective because early secreted antigenic target (ESAT-6)- all deficient mice succumb to infection specific HLA class I-restricted CD8(+) T within 5 months. Taken together, the data cell epitope. When cocultured with CD8(+) suggests that TLR2 may function as a regu- T cells restricted through classical HLA lator of inflammation, and in its absence an class I molecules the growth of bacilli exaggerated immune inflammatory response within macrophages is significantly im- develops.—Authors’Abstract paired after 7 days. This slow antimycobacterial activity did not correlate with macrophage lysis but required cell contact. Geiman, D. E., Kaushal, D., Ko, C., Tyagi, We also found that inhibitors of apoptosis S., Manabe, Y. C., Schroeder, B. G., either had no effect or augmented the CD8- Fleischmann, R. D., Morrison, N. E., mediated suppressive activity, suggesting Converse, P. J., Chen, P., and Bishai, that an activation signal might be involved. W. R. Attenuation of late-stage disease in Indeed we show that CD8(+) T cells were mice infected by the Mycobacterium tu- able to activate macrophages through re- berculosis mutant lacking the SigF alter- ceptors that include CD95 (Fas). Consistent nate sigma factor and identification of with these findings the CD8-mediated sup- SigF-dependent genes by microarray pression of mycobacterial growth was par- analysis. Infect. Immun. 72(3) (2004) tially reversed by Fas blockade. These data 1733–1745. identify a previously unrecognized CD8(+) T cell-mediated mechanism used to control The Mycobacterium tuberculosis alternate an intracellular infection of macrophages.— sigma factor, SigF, is expressed during sta- Authors’Abstract tionary growth phase and under stress conditions in vitro. To better understand the function of SigF we studied the phenotype Drennan, M. B., Nicolle, D., Quesniaux, of the M. tuberculosis DeltasigF mutant in V. J., Jacobs, M., Allie, N., Mpagi, J., vivo during mouse infection, tested the mu- Fremond, C., Wagner, H., Kirschning, tant as a vaccine in rabbits, and evaluated C., and Ryffel, B. Toll-like receptor the mutant’s microarray expression profile 2-deficient mice succumb to Mycobacte- in comparison with the wild type. In mice rium tuberculosis infection. Am. J. Pathol. the growth rates of the DeltasigF mutant and 164(1) (2004) 49–57. wild-type strains were nearly identical during the first 8 weeks after infection. At 8 Recognition of Mycobacterium tubercu- weeks, the DeltasigF mutant persisted in the losis by the innate immune system is essen- lung, while the wild type continued growing tial in the development of an adaptive im- through 20 weeks. Histopathological analy- 210 International Journal of Leprosy 2004 sis showed that both wild-type and mutant colipids purified from Mycobacterium tuber- strains had similar degrees of interstitial and culosis. The structure of this sulfoglycolipid granulomatous inflammation during the first was identified as 2-palmitoyl or 2-stearoyl-3- 12 weeks of infection. However, from 12 to hydroxyphthioceranoyl-2′-sulfate-alpha- 20 weeks the mutant strain showed smaller alpha′-d-trehalose (Ac(2)SGL). Its immuno- and fewer lesions and less inflammation in genicity is dependent on the presence of the the lungs and spleen. Intradermal vaccina- sulfate group and of the two fatty acids. tion of rabbits with the M. tuberculosis Ac(2)SGL is mainly presented by CD1b mol- DeltasigF strain, followed by aerosol chal- ecules after internalization in a cellular com- lenge, resulted in fewer tubercles than did partment with low pH. Ac(2)SGL-specific T intradermal M. bovis BCG vaccination. cells release interferon gamma, efficiently Complete genomic microarray analysis re- recognize M. tuberculosis-infected cells, and vealed that 187 genes were relatively un- kill intracellular bacteria. The presence of derexpressed in the absence of SigF in early Ac(2)SGL-responsive T cells in vivo is stationary phase, 277 in late stationary strictly dependent on previous contact with phase, and only 38 genes in exponential M. tuberculosis, but independent from the growth phase. Numerous regulatory genes development of clinically overt disease. and those involved in cell envelope synthe- These properties identify Ac(2)SGL as a sis were down-regulated in the absence of promising candidate to be tested in novel SigF; moreover, the DeltasigF mutant strain vaccines against tuberculosis.—Authors’ lacked neutral red staining, suggesting a Abstract reduction in the expression of envelope- associated sulfolipids. Examination of 5′- untranslated sequences among the down- Gold, J. A., Hoshino, Y., Tanaka, N., Rom, regulated genes revealed multiple instances W. N., Raju, B., Condos, R., and Wei- of a putative SigF consensus recognition se- den, M. D. Surfactant protein A modu- quence: GGTTTCX(18)GGGTAT. These re- lates the inflammatory response in sults indicate that in the mouse the M. tu- macrophages during tuberculosis. Infect. berculosis DeltasigF mutant strain persists Immun. 72(2) (2004) 645–650. in the lung but at lower bacterial burdens than wild type and is attenuated by Tuberculosis leads to immune activation histopathologic assessment. Microarray and increased human immunodeficiency analysis has identified SigF-dependent virus type 1 (HIV-1) replication in the lung. genes and a putative SigF consensus recog- However, in vitro models of mycobacterial nition site. infection of human macrophages do not fully reproduce these in vivo observations, suggesting that there are additional host fac- Gilleron, M., Stenger, S., Mazorra, Z., tors. Surfactant protein A (SP-A) is an im- Wittke, F., Mariotti, S., Bohmer, G., portant mediator of innate immunity in the Prandi, J., Mori, L., Puzo, G., and De lung. SP-A levels were assayed in the Libero, G. Diacylated Sulfoglycolipids human lung by using bronchoalveolar Are Novel Mycobacterial Antigens Stim- lavage (BAL). There was a threefold reduc- ulating CD1-restricted T Cells during In- tion in SP-A levels during tuberculosis only fection with Mycobacterium tuberculosis. in the radiographically involved lung seg- J. Exp. Med. 199(5) (2004) 649–659. ments, and the levels returned to normal after 1 month of treatment. The SP-A levels Mycobacterial lipids comprise a heteroge- were inversely correlated with the percent- neous group of molecules capable of inducing age of neutrophils in BAL fluid, suggesting T cell responses in humans. To identify novel that low SP-A levels were associated with antigenic lipids and increase our understand- increased inflammation in the lung. Differ- ing of lipid-mediated immune responses, we entiated THP-1 macrophages were used to established a panel of T cell clones with dif- test the effect of decreasing SP-A levels on ferent lipid specificities. Using this approach immune function. In the absence of infec- we characterized a novel lipid antigen be- tion with Mycobacterium tuberculosis, SP-A longing to the group of diacylated sulfogly- at doses ranging from 5 to 0.01 micro g/ml 72, 2 Current Literature, Immuno Pathology (Tuberculosis) 211

inhibited both interleukin-6 (IL-6) produc- Therefore, M. tuberculosis-induced factors tion and HIV-1 long terminal repeat (LTR) may inhibit in vitro HIV-1 replication in activity. In macrophages infected with M. macrophages by affecting an early postentry tuberculosis, SP-A augmented both IL-6 step in the HIV-1 cycle.—Authors’Abstract production and HIV-1 LTR activity. To better understand the effect of SP-A, we measured expression of CAAT/enhancer binding Guinn, K. M., Hickey, M. J., Mathur, S. protein beta (C/EBPbeta), a transcription K., Zakel, K. L., Grotzke, J. E., Lewin- factor central to the regulation of IL-6 and sohn, D. M., Smith, S., and Sherman, the HIV-1 LTR. In macrophages infected D. R. Individual RD1-region genes are with M. tuberculosis, SP-A reduced expres- required for export of ESAT-6/CFP-10 sion of a dominant negative isoform of and for virulence of Mycobacterium tu- C/EBPbeta. These data suggest that SP-A berculosis. Mol. Microbiol. 51(2) (2004) has pleiotropic effects even at the low con- 359–370. centrations found in tuberculosis patients. This protein augments inflammation in the The RD1 genomic region is present in vir- presence of infection and inhibits inflam- ulent strains of Mycobacterium tuberculosis mation in uninfected macrophages, protect- (MTB), missing from the vaccine strain M. ing uninvolved lung segments from the bovis BCG, and its importance to virulence deleterious effects of inflammation.—Au- has been established experimentally. Based thors’Abstract on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this re- Goletti, D., Carrara, S., Vincenti, D., Gia- gion affects virulence is unknown. Here we comini, E., Fattorini, L., Garbuglia, A. examined mutants disrupted in five indi- R., Capobianchi, M. R., Alonzi, T., vidual RD1 genes. Both in vitro and in vivo, Fimia, G. M., Federico, M., Poli, G., each mutant displayed an attenuated pheno- and Coccia, E. Inhibition of HIV-1 repli- type very similar to a mutant missing the en- cation in monocyte-derived macrophages tire RD1 region. Genetic complementation by Mycobacterium tuberculosis. J. Infect. of individual genes restored virulence. At- Dis. 189(4) (2004) 624–633. tenuated mutants could multiply within THP-1 cells, but they were unable to spread Controversial results have been obtained to uninfected macrophages. We also examin studies of the effect of Mycobacterium tu- ined export of two immunodominant RD1 berculosis on human immunodeficiency proteins, CFP-10 and ESAT-6. Export of virus type 1 (HIV-1) replication in cells of these proteins was greatly reduced or abol- the macrophage lineage. In the present study, ished in each attenuated mutant. Again, ge- monocyte-derived macrophages (MDMs), netic complementation restored a wild-type previously incubated for 2 days with heat- phenotype. Our results indicate that RD1 inactivated M. tuberculosis, were infected genes work together to form a single viru- with HIV-1. M. tuberculosis consistently in- lence determinant, and argue that RD1 en- hibited viral replication, and a similar result codes a novel specialized secretion system also was observed in the presence of super- that is required for pathogenesis of MTB.— natants from M. tuberculosis-stimulated Authors’Abstract MDMs, which indicates that this effect was mediated by soluble factors. Although CCR5-binding chemokines were induced by Harboe, M., Das, A. K., Mitra, D., Ul- M. tuberculosis stimulation, the results of vund, G., Ahmad, S., Harkness, R. E., neutralization experiments indicated that it Das, D., Mustafa, A. S., and Wiker, H. is unlikely that they were responsible for G. Immunodominant B-cell epitope in viral suppression. Inhibition occurred the Mce1A mammalian cell entry protein mainly after viral entry (demonstrated by of Mycobacterium tuberculosis cross- use of a vesicular stomatitis virus G-pseudo- reacting with glutathione S-transferase. typed HIV-1 and by analysis of HIV-1 early Scand. J. Immunol. 59(2) (2004) 190– and late reverse-transcription products). 197. 212 International Journal of Leprosy 2004

The TB1-5 76C monoclonal antibody duction by alveolar macrophages (AMs) in raised against a synthetic 60-mer peptide vitro. The cytosol fraction of live Mtb in the N-terminal part of the Mce1A mam- H37Rv induced IL-12 production by AMs in malian cell entry protein of Mycobacte- a dose-dependent manner. The addition of rium tuberculosis has previously been interferon-gamma (IFN-gamma) augmented shown to react with a linear epitope in the IL-12 production. IL-12-inducing activity KRRITPKD region, residues 131–138 in by AMs (termed as surely active keeping Mce1A, and to cross-react with Mce1F. Six rescue antigen, SAKRA) was purified by gel additional monoclonal antibodies raised filtration and ion exchange column chro- against the same peptide were also shown to matography, and the molecular weight of cross-react with Mce1F. Four of them re- SAKRA was estimated by gel filtration to be acted with a linear epitope in the same area, more than 700 kDa. SDS-polyacrylamide indicating that this area is immunodominant gel electrophoresis (PAGE) and Western but showed distinct differrences in fine blotting of SAKRA using rabbit anti- specificity. Two monoclonal antibodies did SAKRA antibody suggested that SAKRA is not react with synthetic peptides from this composed with several low molecular region on the solid phase in enzyme-linked weight proteins. Amino acids sequence immunosorbent assay, indicating greater analysis of several bands after SDS-PAGE influence of conformation on reactivity. suggested that SAKRA is a part of ribo- None of the monoclonal antibodies reacted somes. RT-PCR showed that SAKRA in- with 14-mer synthetic peptides from the duced not only expression of IL-12 p40 corresponding area in Mce2A, Mce3A, mRNA, but expression of tumor necrosis Mce4A, M. avium, M. smegmatis or M. factor (TNF)-alpha and inducible nitric leprae. The reaction pattern of the mono- oxide synthase (iNOS) mRNA at least 6 hr clonal antibodies was analysed in relation to after stimulation, suggesting that SAKRA our model of the Mce1A molecule (AK Das, activates the bactericidal activity of macro- et al. Biochem Biophys Res Commun phages. To investigate the potential use of 2003;302:442–7). The epitope is located on SAKRA as a vaccine against tuberculosis, the surface of Mce1A, at the distal beta- SAKRA was administered to BALB/c strand-loop region in the beta-domain sup- mouse that had been immunized with BCG porting its potential role in promoting up- for 18 months, and mouse were infected take of M. tuberculosis in host cells. with Mtb H37Rv via a respiratory route. Monoclonal antibody TB1-5 19C cross- Replication of Mtb in lungs and spleens was reacted with glutathione S-transferase of examined 6 weeks after infection. Admini- Schistosoma japonicum containing a PKE stration of SAKRA to BCG-vaccinated mice triplet. Monoclonal antibody TB1-5 76C significantly reduced the numbers of Mtb in gave a major band at about 44 kDa in West- lungs and spleens as compared with BCG- ern blotting of M. tuberculosis sonicate, vaccinated control mice. Taken together, whereas polyclonal rabbit anti-Mce1A pep- these results suggest that SAKRA is one of tide antibodies reacting with the extended the Mtb-derived immunomodulatory sub- TTPKNPTKRRITPKDVI area of Mce1A stances which induce IL-12 production dur- showed a distinct band above the 160 kDa ing infection and also increases mycobacte- molecular mass standard.—Authors’ Ab- ricidal activities of macrophages, and that stract SAKRA may be a promising new vaccine candidate against tuberculosis.

Higuchi, K., Sekiya, Y., and Harada, N. Characterization of M. Tuberculosis- Junqueira-Kipnis, A. P., Kipnis, A., derived IL-12-inducing material by al- Jamieson, A., Juarrero, M. G., Diefen- veolar macrophages. Vaccine 22(5–6) bach, A., Raulet, D. H., Turner, J., and (2004) 724–734. Orme, I. M. NK cells respond to pulmonary infection with Mycobacterium We have investigated the substance de- tuberculosis, but play a minimal role in rived from Mycobacterium tuberculosis protection. J. Immunol. 171(11) (2003) (Mtb) that induces interleukin (IL)-12 pro- 6039–6045. 72, 2 Current Literature, Immuno Pathology (Tuberculosis) 213

Both innate and adaptive immune systems the cavity surface: a microenvironment contribute to host defense against infection with failed immunity. Infect. Immun. with Mycobacterium tuberculosis. NK cells 71(12) (2003) 7099–7108. have been associated with early resistance against intracellular pathogens and are Protective immunity against pulmonary known to be potent producers of the cy- tuberculosis (TB) is characterized by the tokine IFN-gamma. In C57BL/6 mice information in the lungs of granulomas con- fected by aerosol exposure with M. tubercu- sisting of macrophages and activated T cells losis, NK cells increased in the lungs over producing tumor necrosis factor alpha and the first 21 days of infection. Expansion of gamma interferon, both required for the ac- the NK cell subset was associated with in- tivation of the phagocytes. In 90% of im- creased expression of activation and matu- munocompetent humans, this response con- ration markers. In addition, NK cells iso- trols the infection. To understand why lated from the infected lungs were capable immunity fails in the other 10%, we studied of producing IFN-gamma and became pos- the lungs of six patients who underwent sur- itive for perforin. In vivo depletion of NK gery for incurable TB. Histologic examina- cells using a lytic Ab had no influence on tion of different lung lesions revealed het- bacterial load within the lungs. These find- erogeneous morphology and distribution of ings indicate that NK cells can become acti- acid-fast bacilli; only at the surface of cavi- vated during the early response to pul- ties, i.e., in granulomas with a patent con- monary tuberculosis in the mouse model nection to the airways, were there numerous and are a source of IFN-gamma, but their re- bacilli. The mutation profile of the isolates moval does not substantially alter the ex- suggested that a single founder strain of My- pression of host resistance.—Authors’ Ab- cobacterium tuberculosis may undergo ge- stract netic changes during treatment, leading to acquisition of additional drug resistance independently in discrete physical locales. Ad- Kanaujia, G. V., Motzel, S., Garcia, M. A., ditional drug resistance was preferentially Andersen, P., and Gennaro, M. L. observed at the cavity surface. Cytokine Recognition of ESAT-6 sequences by an- gene expression revealed that failure to con- tibodies in sera of tuberculous nonhuman trol the bacilli was not associated with a primates. Clin. Diagn. Lab. Immunol. generalized suppression of cellular immu- 11(1) (2004) 222–226. nity, since cytokine mRNA was up regulated in all lesions tested. Rather, a selective See Current Literature, Experimental In- absence of CD4(+) and CD8(+) T cells was fections, p. 235. noted at the luminal surface of the cavity, preventing direct T-cell-macrophage interactions at this site, probably allowing lumi- Kanaujia, G. V., Garcia, M. A., Bouley, D. nal phagocytes to remain permissive for M., Peters, R., and Gennaro, M. L. bacillary growth. In contrast, in the peri- Detection of early secretory antigenic necrotic zone of the granulomas, the two target-6 antibody for diagnosis of tuber- cell types colocalized and bacillary num- culosis in non-human primates. Comp. bers were substantially lower, suggesting Med. 53(6) (2003) 602–606. that in this microenvironment an efficient bacteriostatic or bactericidal phagocyte See Current Literature, Experimental In- population was generated.—Authors’ Ab- fections, p. 235. stract

Kaplan, G., Post, F. A., Moreira, A. L., Lazarevic, V., Myers, A. J., Scanga, C. A., Wainwright, H., Kreiswirth, B. N., and Flynn, J. L. CD40, but not CD40L, Tanverdi, M., Mathema, B., Ra- is required for the optimal priming of T maswamy, S. V., Walther, G., Steyn, L. cells and control of aerosol M. tuberculo- M., Barry, C. E. 3rd, and Bekker, L. G. sis infection. Immunity. 19(6) (2003) Mycobacterium tuberculosis growth at 823–835. 214 International Journal of Leprosy 2004

CD40(–/–) mice succumbed to low-dose oratory. A devR::kan mutant of M. tubercu- aerosol infection with M. tuberculosis due to losis was constructed by allelic exchange. deficient IL-12 production leading to im- The devR mutant strain showed reduced paired priming of IFN-gamma T cell re- cell-to-cell adherence in comparison to the sponses. In contrast, CD40L(–/–) mice were parental strain in laboratory culture media. resistant to M. tuberculosis. This asymmetry This phenotype was reversed on comple- in outcome of infection between the two mentation with a wild-type copy of devR. knockout strains is likely due to the exis- The devR mutant and parental strains grew tence of an alternative ligand for CD40. at equivalent rates within human monocytes Both in vitro M. tuberculosis infection and either in the absence or in the presence of recombinant M. tuberculosis Hsp70 elicited lymphocytic cells. The expression of DevR IL-12 production from WT dendritic cells. was not modulated upon entry of M. tuber- This response was absent in both CD40(–/–) culosis into human monocytes. However, dendritic cells and CD40(–/–) mice, sug- guinea pigs infected with the mutant strain gesting that M. tuberculosis Hsp70 serves as showed a significant decrease in gross le- an alternative ligand for CD40 in vivo.— sions in lung, liver and spleen; only mild Authors’Abstract pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected Majlessi, L., Rojas, M. J., Brodin, P., and with the parental strain. Our results suggest Leclerc, C. CD8+-T-cell responses of that DevR is required for virulence in guinea Mycobacterium-infected mice to a newly pigs but is not essential for entry, survival identified major histocompatibility com- and multiplication of M. tuberculosis within plex class I-restricted epitope shared by human monocytes in vitro. The attenuation proteins of the ESAT-6 family. Infect. in virulence of the devR mutant in guinea Immun. 71(12) (2003) 7173–7177. pigs together with DevR-DevS being a bona fide signal transduction system indicates that Here we describe the identification of a new DevR plays a critical and regulatory role in CD8(+)-T-cell epitope, the GYAGTLQSL the adaptation and survival of M. tuberculo- nonamer, shared by the TB10.3 and TB10.4 sis within tissues.—Authors’Abstract proteins of the Mycobacterium tuberculosis ESAT-6 family. Cytotoxic T cells from mycobacterium-infected mice efficiently Mattow, J., Schaible, U. E., Schmidt, F., recognized this epitope. GYAGTLQSL- Hagens, K., Siejak, F., Brestrich, G., specific T-cell hybridomas, which were able Haeselbarth, G., Muller, E. C., Jung- to recognize Mycobacterium bovis BCG- blut, P. R., and Kaufmann, S. H. infected macrophages, were generated and Comparative proteome analysis of culture now allow investigation of mycobacterial- supernatant proteins from virulent Myco- antigen processing through the major histo- bacterium tuberculosis H37Rv and atten- compatibility complex class I pathway.— uated M. bovis BCG Copenhagen. Elec- Authors’Abstract trophoresis 24(19–20) (2004) 3405–3420.

A comprehensive analysis of culture Malhotra, V., Sharma, D., Ramanathan, supernatant (CSN) proteins of Mycobacte- V. D., Shakila, H., Saini, D. K., rium tuberculosis H37Rv was accomplished Chakravorty, S., Das, T. K., Li, Q., Sil- by combination of two-dimensional elec- ver, R. F., Narayanan, P. R., Tyagi, J. S. trophoresis (2-DE), mass spectrometry, and Disruption of response regulator gene, N-terminal sequencing by Edman degrada- devR, leads to attenuation in virulence of tion. Analytical 2-DE gels resolved approx- Mycobacterium tuberculosis. FEMS Mi- imately 1250 protein spots from CSN of M. crobiol Lett. 20231(2) (2004) 237–245. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or The devR-devS two-component system of Edman degradation. This study revealed 137 Mycobacterium tuberculosis was identified different proteins, 42 of which had previ- earlier and partially characterized in our lab- ously been described as secreted. Compar- 72, 2 Current Literature, Immuno Pathology (Tuberculosis) 215 ative proteome analysis of CSN from viru- cia, V. E. Expression of signaling lympho- lent M. tuberculosis H37Rv and attenuated cytic activation molecule-associated pro- Mycobacterium bovis BCG Copenhagen tein interrupts IFN-gamma production in identified 39 M. tuberculosis-specific spots human tuberculosis. J. Immunol. 172(2) containing 27 different proteins, represent- (2004) 1177–1185. ing candidate antigens for novel vaccines and diagnostics in tuberculosis. These in- Production of the Th1 cytokine IFN- cluded five proteins encoded by open read- gamma by T cells is considered crucial for ing frames absent from M. bovis BCG, e.g., immunity against Mycobacterium tubercu- early secretory antigen target (Esat6), as losis infection. We evaluated IFN-gamma well as 22 novel differential proteins, such production in tuberculosis in the context of as acetyl-CoA C-acetyltransferase (Rv0243) signaling molecules known to regulate Th1 and two putative Esat6-like proteins cytokines. Two populations of patients who (Rv1198, Rv1793).—Authors’Abstract have active tuberculosis were identified, based on their T cell responses to the bacterium. High responder tuberculosis patients McCarthy, A. A., Knijff, R., Peterson, N. displayed significant M. tuberculosis- A., and Baker, E. N. Crystallization and dependent T cell proliferation and IFN- preliminary X-ray analysis of N-acetyl-1- gamma production, whereas low responder D-myo-inosityl-2-deoxy-alpha-D- tuberculosis patients displayed weak or no T glucopyranoside deacetylase (MshB) cell responses to M. tuberculosis. The ex- from Mycobacterium tuberculosis. Acta. pression of the signaling lymphocytic acti- Crystallogr. D. Biol. Crystallogr. 59(Pt vation molecule (SLAM)-associated protein 12) (2003) 2316–2318. (SAP) on cells from tuberculosis patients was inversely correlated with IFN-gamma Mycobacteria synthesize mycothiol (MSH) production in those individuals. Moreover, as a low-molecular-weight thiol that protects patients with a nonfunctional SAP gene dis- against oxidative stress in a similar role to played immune responses to M. tuberculo- that of glutathione in many other species. sis similar to those of high responder tuber- The absence of MSH in mammals suggests culosis patients. In contrast to SAP, T cell that enzymes from its biosynthetic path- expression of SLAM was directly correlated way in Mycobacterium tuberculosis could with responsiveness to M. tuberculosis Ag. be useful targets for drug design. The gene Our data suggest that expression of SAP infor MshB (Rv1170), the enzyme that catal- terferes with Th1 responses whereas SLAM yses the second step in MSH biosynthesis expression contributes to Th1 cytokine rein M. tuberculosis, has been cloned and the sponses in tuberculosis. The study further protein has been expressed in Escherichia suggests that SAP and SLAM might be coli both in native and SeMet-substituted focal points for therapeutic modulation of T forms and crystallized in two crystal cell cytokine responses in tuberculosis.— forms. One of these, prepared in the pres- Authors’Abstract ence of beta-octylglucoside as a key additive, is suitable for high-resolution X-ray structural analysis. The crystals are or- Pereira, C. B., Palaci, M., Leite, O. H., thorhombic, with unit-cell parameters a = Duarte, A. J., and Benard, G. Mono- 71.69, b = 83.74, c = 95.65 A, space group cyte cytokine secretion in patients with P2(1)2(1)2(1) and two molecules in the pulmonary tuberculosis differs from that asymmetric unit. X-ray diffraction data to of healthy infected subjects and corre- 1.9 A resolution have been collected.— lates with clinical manifestations. Mi- Authors’ Abstract crobes Infect. 6(1) (2004) 25–33.

Cell-mediated immunity, leading to My- Pasquinelli, V., Quiroga, M. F., Martinez, cobacterium tuberculosis (Mtb)-constraining G. J., Zorrilla, L. C., Musella, R. M., granuloma formation, is the major compo- Bracco, M. M., Belmonte, L., Malbran, nent of host defense against tuberculosis and A., Fainboim, L., Sieling, P. A., and Gar- is regulated by the balance of cytokines se- 216 International Journal of Leprosy 2004 creted mostly by mononuclear phagocytes sion as a model for effects of M. tuberculo- and lymphocytes. To better understand the sis on response to IFN-gamma. We found role of monocytes in the regulation of the that IRF-1 mRNA abundance increased far immune response against pulmonary tuber- more than transcription rate in human culosis, we examined IL-10, IL-12 and monocytic THP-1 cells stimulated by IFN- TNF-alpha release by monocytes from gamma, but less than transcription rate in healthy purified protein derivative (PPD) re- cells infected by M. tuberculosis. IFN- actors and pulmonary tuberculosis patients gamma stimulation of infected cells caused with or without systemic reactions (e.g., a synergistic increase in IRF-1 transcrip- fever, weight loss, asthenia). Our study tion, yet IRF-1 mRNA abundance was sim- shows that, probably as a result of in vivo ilar in uninfected and infected cells stimu- priming by circulating antigens, monocytes lated by IFN-gamma, as was the IRF-1 from patients, especially those with sys- protein level. Comparable infection by temic manifestations, have a biased ex vivo Mycobacterium bovis bacillus Calmette- cytokine secretion, with high IL-10 and Guerin failed to induce IRF-1 expression TNF-alpha but low IL-12, in contrast with and had no effect on the response to IFN- PPD reactors. Higher spontaneous IL-10 gamma. We also examined the kinetics of and TNF-alpha release persisted when transcription, the mRNA t(1/2), and the dis- monocytes were co-cultured with autolo- tribution of IRF-1 transcripts among total gous lymphocytes. Challenge of patients’ nuclear RNA, poly(A) nuclear RNA, and monocytes with a virulent Mtb strain led to poly(A) cytoplasmic RNA pools in cells a further enhancement of IL-10 and TNF- that were infected by M. tuberculosis and/or alpha, but not of IL-12. When lymphocytes stimulated by IFN-gamma. Our data sug- were added to these cultures, IL-10 and gest that infection by M. tuberculosis in- TNF-alpha elevation persisted and, in the hibits RNA export from the nucleus. More- patients with a systemic reaction, both IL-12 over, the results indicate that regulated and IFN-gamma were significantly reduced entry of nascent transcripts into the pool of compared to PPD reactors. Intragroup com- total nuclear RNA affects IRF-1 expression parisons revealed that in the patients with sys- and that this process is stimulated by IFN- temic reactions, the lymphocyte-monocyte gamma and inhibited by M. tuberculosis. interaction resulted in a positive feedback The ability of infection by M. tuberculosis for IL-10 secretion, while in the patients to limit the increase in IRF-1 mRNA ex- without systemic reaction and PPD reactors, pression that typically follows transcrip- the feedback was positive for IL-12 secre- tional synergism may contribute to the tion. Thus, in tuberculosis, there appears to pathogenicity of M. tuberculosis.—Au- exist a relationship between the immuno- thors’Abstract logical findings and the distinct clinical manifestations.—Authors’Abstract Rousseau, C., Winter, N., Pivert, E., Bor- dat, Y., Neyrolles, O., Ave, P., Huerre, Qiao, Y., Prabhakar, S., Canova, A., M., Gicquel, B., and Jackson, M. Pro- Hoshino, Y., Weiden, M., and Pine, R. duction of phthiocerol dimycocerosates Posttranscriptional inhibition of gene ex- protects Mycobacterium tuberculosis pression by Mycobacterium tuberculosis from the cidal activity of reactive nitro- offsets transcriptional synergism with gen intermediates produced by macro- IFN-gamma and posttranscriptional up- phages and modulates the early immune regulation by IFN-gamma. J. Immunol. response to infection. Cell Microbiol. 172(5) (2004) 2935–2943. 6(3) (2004) 277–287.

Host defense against Mycobacterium tu- The growth of Mycobacterium tubercu- berculosis requires the cytokine IFN- losis mutants unable to synthesize phthio- gamma and IFN regulatory factor 1 (IRF- cerol dimycocerosates (DIMs) was recently 1), a transcription factor that is induced to shown to be impaired in mouse lungs. high levels by IFN-gamma. Therefore, we However, the precise role of these mole- chose to study regulation of IRF-1 expres- cules in the course of infection remained to 72, 2 Current Literature, Immuno Pathology (Tuberculosis) 217

be determined. Here, we provide evidence PPD-specific expression), endemic controls that the attenuation of a DIM-deficient had significantly higher responses than the strain takes place during the acute phase of patients had for TCR Vβ families 2, 3, 7, 13, infection in both lungs and spleen of mice, and 17. The majority of the patients did not and that this attenuation results in part from show usage of most of the TCR Vβ families, the increased sensitivity of the mutant to and this was attributed to T-cell downregu- the cidal activity of reactive nitrogen inter- lation. A four-way nested classification mediates released by activated mac- analysis revealed that TCR Vβ family 1, 5, rophages. We also show that the DIM- 9, 12, and 13 usage in the context of HLA deficient mutant, the growth and survival class II high-risk alleles) (DRB1*1501, of which were not impaired within resting DRB1*08, and DQB1*0601) and Mycobac- macrophages and dendritic cells, induced terium bovis BCG scar status were the de- these cells to secrete more tumour necrosis termining factors in susceptibility and re- factor (TNF)-alpha and interleukin (IL)-6 sistance to tuberculosis. The healthier status than the wild-type strain. Although purified of controls was attributed to the wider usage DIM molecules by themselves had no ef- of many TCR Vβ families readily recalled fect on the activation of macrophages and by PPD, while the disease status of the pa- dendritic cells in vitro, we found that the tients was attributed to TCR Vβ downreg- proper localization of DIMs in the cell en- ulation and the resultant T-cell (memory velope of M. tuberculosis is critical to their cell?) unresponsiveness. Host genetics biological effects. Thus, our findings sug- (HLA status) and BCG vaccination (scar gest that DIM production contributes to the status) seem to play important roles in skew- initial growth of M. tuberculosis by pro- ing the immune response in adult suscepti- tecting it from the nitric oxide-dependent bility to pulmonary tuberculosis through killing of macrophages and modulating the TCR Vβ usage.—Tropical Disease Bulletin early immune response to infection.—Au- thors’ Abstract Stanton, L. A., Fenhalls, G., Lucas, A., Gough, P., Greaves, D. R., Mahoney, J. Shanmugalakshmi, S., Dheenadhayalan, A., Helden, P., and Gordon, S. Im- V., Muthuveeralakshmi, P., Arivarig- munophenotyping of macrophages in nan, G., and Pitchappan, R. M. Myco- human pulmonary tuberculosis and sar- bacterium bovis BCG scar status and coidosis. Int. J. Exp. Pathol. 84(6) (2003) HLA class II alleles influence purified 289–304. protein derivative-specific T-cell receptor V? expression in pulmonary tuberculosis Classic studies of tuberculosis (TB) re- patients from Southern India. Infect. Im- vealed morphologic evidence of consider- munity 71(8) (2003) 4544–4553. able heterogeneity of macrophages (MOs), but the functional significance of this het- Purified protein derivative (PPD) RT23- erogeneity remains unknown. We have used recalled T-cell receptor (TCR) Vβ expres- newly available specific antibodies for se- sion was studied in the peripheral blood of lected membrane and secretory molecules to 42 pulmonary tuberculosis patients and 44 examine the phenotype of MOs in situ in a healthy controls from southern India, a re- range of South African patients with TB, gion where tuberculosis is endemic. Forty- compared with sarcoidosis. Patients were eight-hour whole-blood cultures in the pres- human immunodeficiency virus-negative ence or absence of PPD-RT23 were set up, adults and children, and the examined and at the end of the culture period total biopsy specimens included lung and lymph RNA was extracted and cDNA was synthe- nodes. Mature pulmonary MOs (alveolar, sized. Expression of various TCR Vβ fami- interstitial, epithelioid and multinucleated lies was assessed by using family-specific giant cells) selectively expressed scavenger primers. PPD-specific expression (usage) of receptor type A and a novel carboxypeptidase- TCR Vβ families 4, 6, 8 to 12, and 14 was like antigen called carboxypeptidase-related found in more controls than patients. Among vitellogenin-like MO molecule (CPVL). the responders (individuals who showed CPVL did not display enhanced expression 218 International Journal of Leprosy 2004 in sarcoidosis, vs. TB patients, as observed but not expansion, of the CD8 T cell pool with angiotensin-converting enzyme (ACE), within the aging lung.—Authors’Abstract a related molecule. Immunocytochemical studies with surfactant proteins (SP)-A and -D showed that type II alveolar cells ex- Vergne, I., Fratti, R. A., Hill, P. J., Chua, pressed these collectins, as did MOs, possi- J., Belisle, J., and Deretic, V. Mycobac- bly after binding of secreted proteins. Stud- terium tuberculosis phagosome maturation ies with an antibody specific for the arrest: mycobacterial phosphatidylinositol C-terminus of fractalkine, a tethered CX3C analog phosphatidylinositol mannoside chemokine, confirmed synthesis of this mol- stimulates early endosomal fusion. Mol. ecule by bronchiolar epithelial cells and oc- Biol. Cell. 15(2) (2004) 751–760. casional endothelial cells. These studies provide new marker antigens and extend Mycobacterium tuberculosis is a faculta- previous studies on MO differentiation, active intracellular pathogen that parasitizes tivation and local interactions in chronic macrophages by modulating properties of human granulomatous inflammation in the the Mycobacterium-containing phagosome. lung.—Authors’Abstract Mycobacterial phagosomes do not fuse with late endosomal/lysosomal organelles but re- tain access to early endosomal contents by Turner, J., and Orme, I. M. The expression an unknown mechanism. We have previ- of early resistance to an infection with ously reported that mycobacterial phos- Mycobacterium tuberculosis by old mice phatidylinositol analog lipoarabinomannan is dependent on IFN type II (IFN- (LAM) blocks a trans-Golgi network-to- gamma) but not IFN type I. Mech. Age- phagosome phosphatidylinositol 3-kinase- ing Dev. 125(1) (2004) 1–9. dependent pathway. In this work, we extend our investigations of the effects of myco- Old mice can express a transient early re- bacterial phosphoinositides on host mem- sistance to infection with M. tuberculosis brane trafficking. We present data demon- that requires the presence of CD8 T cells strating that phosphatidylinositol mannoside within the lungs. Further characterization of (PIM) specifically stimulated homotypic fu- those CD8 T cells within the aged lung es- sion of early endosomes in an ATP-, cytosol-, tablished that the majority of CD8 T cells and N-ethylmaleimide sensitive factor- from old mice expressed the IL-15 receptor dependent manner. The fusion showed (CD122) in combination with bright ex- absolute requirement for small Rab GT- pression of CD44 (CD44(hi)), and were ca- Pases, and the stimulatory effect of PIM in- pable of producing IFN-gamma after T cell creased upon partial depletion of membrane receptor cross-linking. It has been previ- Rabs with RabGDI. We found that stimula- ously described that CD8 CD44(hi) T cells tion of early endosomal fusion by PIM was proliferate in response to IFN-I, acting via higher when phosphatidylinositol 3-kinase IL-15, and therefore we determined whether was inhibited by wortmannin. PIM also IFN-I signaling could be a participant in the stimulated in vitro fusion between model response of CD8 T cells within the lungs of phagosomes and early endosomes. Finally, old mice infected with M. tuberculosis. We PIM displayed in vivo effects in macro- demonstrate here that IFN-I signaling was phages by increasing accumulation of required for the expansion of CD8 T cells plasma membrane-endosomal syntaxin 4 within the aging lung in response to infec- and transferrin receptor on PIM-coated latex tion with M. tuberculosis, but that IFN-I sig- bead phagosomes. In addition, inhibition of naling had no influence on the capacity of phagosomal acidification was detected with old mice to express early resistance to an in- PIM-coated beads. The effects of PIM, fection with M. tuberculosis. Resident CD8 along with the previously reported action of T cells were still however capable of pro- LAM, suggest that M. tuberculosis has ducing IFN-gamma, which we demonstrate evolved a two-prong strategy to modify its here to be critical in the expression of early intracellular niche: its products block acqui- resistance, suggesting that the expression of sition of late endosomal/lysosomal con- early resistance requires the participation, stituents, while facilitating fusion with early 72, 2 Current Literature, Microbiology 219

endosomal compartments.—Authors’ Ab- phage responses following Mycobacte- stract rium tuberculosis infection. FEMS Im- munol. Med. Microbiol. 39(2) (2003) 163–172. Wang, J. P., Rought, S. E., Corbeil, J., and Guiney, D. G. Gene expression pro- See Current Literature, Molecular and filing detects patterns of human macro- Genetic Studies, p. 262. Microbiology Adekambi, T., Colson, P., and Drancourt, sequence divergence from the corresponding M. rpoB-based identification of nonpig- type strain; they belonged to three taxa re- mented and late-pigmenting rapidly lated to M. mucogenicum, Mycobacterium growing mycobacteria. J. Clin. Micro- smegmatis, and Mycobacterium porcinum. biol. 41(12) 2003 5699–5708. For M. abscessus and M. mucogenicum, this partial sequence yielded a high genetic het- Nonpigmented and late-pigmenting rap- erogeneity within the clinical isolates. We idly growing mycobacteria (RGM) are in- conclude that molecular identification by creasingly isolated in clinical microbiology analysis of the 723-bp rpoB sequence is a laboratories. Their accurate identification re- rapid and accurate tool for identification of mains problematic because classification is RGM.—Authors’Abstract labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence Chattopadhyay, C., Sau, S., and Mandal, analysis underestimates RGM diversity and N. C. Cloning and characterization of the does not distinguish between all taxa. We de- promoters of temperate mycobacterio- termined the complete nucleotide sequence phage L1. J. Biochem. Mol. Biol. 36(6) of the rpoB gene, which encodes the bacterial (2003) 586–592. beta subunit of the RNA polymerase, for 20 RGM type strains. After using in-house soft- See Current Literature, Molecular and ware which analyzes and graphically repre- Genetic Studies, p. 256. sents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to Chui, L. W., King, R., Lu, P., Manninen, 97% interspecies similarity and 0 to 1.7% in- K., and Sim, J. Evaluation of four DNA traspecific divergence. Primer pair Myco-F- extraction methods for the detection of My- Myco-R was designed as a tool for both PCR cobacterium avium subsp. paratuberculo- amplification and sequencing of this region sis by polymerase chain reaction. Diagn. for molecular identification of RGM. This Microbiol. Infect. Dis. 48(1) (2004) 39–45. tool was used for identification of 63 RGM clinical isolates previously identified at the Polymerase chain reaction (PCR) has species level on the basis of phenotypic char- been widely used due to its high specificity, acteristics and by 16S rRNA gene sequence sensitivity, and rapid turn-around time. analysis. Of 63 clinical isolates, 59 (94%) ex- However, inhibitory factors may be co- hibited <2% partial rpoB gene sequence di- extracted with the target nucleic acid that vergence from 1 of 20 species under study will hinder the performance of PCR. In this and were regarded as correctly identified at study, DNA extraction methods for Myco- the species level. Mycobacterium abscessus bacterium avium subsp. paratuberculosis and Mycobacterium mucogenicum isolates were evaluated including rapid lysis, or- were clearly distinguished from Mycobacte- ganic extraction, silica-based and magnetic rium chelonae; Mycobacterium mageritense particle-based (MagaZorb) technologies on isolates were clearly distinguished from “My- bacterial cells, and spiked bovine feces. Ef- cobacterium houstonense.” Four isolates ficiency of the extraction was determined by were not identified at the species level be- PCR end point titration with primers target- cause they exhibited >3% partial rpoB gene ing the insertion sequence, IS900. Results of 220 International Journal of Leprosy 2004 the end point titrations are identical for bac- cerans produces the polyketide-derived terial cells and spiked feces. Inhibition was macrolide mycolactone, which is required observed in PCR with DNA isolated from for the immunosuppression and tissue dam- spiked feces, and a 1/100 dilution was able age which characterizes Buruli ulcer. We to alleviate this problem with DNA ex- have extracted lipids from the cell envelope tracted by MagaZorb. A 1/1000 dilution was and culture filtrate from 52 isolates of My- required for the other three methods. Mag- cobacterium species, analyzed them with aZorb proved to be more efficient at remov- thin-layer chromatography, and tested them ing inhibitory factors and required the least in a murine fibroblast cell line (L929) cyto- labor and completion time. Further evalua- toxicity assay to investigate whether these tion is required for its utilization in other mycobacterial species produce mycolac- clinical specimens.—Authors’Abstract tone. For these studies chloroform-methanol (2:1, vol/vol) extracts were prepared from representative fast- and slow-growing my- Cociorva, O. M., and Lowary, T. L. cobacterial species. Isolates tested included Synthesis of oligosaccharides as potential 16 uncharacterized, slow-growing, environ- inhibitors of mycobacterial arabinosyl- mental mycobacterial species isolated from transferases. Di- and trisaccharides con- areas in which M. ulcerans infection is en- taining C-5 modified arabinofuranosyl demic. Although several strains of myco- residues. Carbohydr. Res. 339(4) (2004) bacteria studied produced cytopathic lipids, 853–865. none of these produced a phenotype on cultured cells consistent with that produced by The synthesis of a panel of oligosac- mycolactone. Two mycobacterial species, charides containing C-5 arabinofuranosyl M. scrofulaceum and M. kansasii, and eight residues (9–20) is described. These com- of the environmental mycobacterial isolates pounds are of interest as potential inhibitors contained cell-associated lipids cytopathic of the alpha-(1→5)-arabinosyltransferase to fibroblasts at concentrations of 33 to 1000 involved in the assembly of mycobacterial microg/ml. In contrast, mycolactone pro- cell-wall arabinan. In the series of com- duces cytotoxicity at less than 2 ng/ml. pounds prepared, the 5-OH group on the Analysis of 16S rRNA sequences from the nonreducing residue(s) is replaced, inde- eight environmental isolates suggests that pendently, with an amino, azido, fluoro, or these are novel mycobacterial species. Re- methoxy functionality. The synthesis of the sults from these studies suggest that, al- target compounds involved the preparation though production of cytopathic lipids is of a series of C-5 modified arabinofuranosyl relatively common among mycobacterial thioglycosides (24–26) and their subsequent species, the production of mycolactone as a coupling to the appropriate acceptor species cell-associated or secreted molecule appears (21–23). Deprotection of the glycosylation so far to be restricted to M. ulcerans.—Au- products afforded the azido, fluoro, or thor’s Abstract methoxy analogs directly. The amino derivatives were obtained in one additional step by reduction of the azido compounds.—Au- Ergin, A., and Hascelik, G. Non-tuberculous thors’Abstract mycobacteria (NTM) in patients with underlying diseases: results obtained by using polymerase chain reaction- Daniel, A. K., Lee, R. E., Portaels, F., and restriction enzyme analysis between Small, P. L. Analysis of Mycobacterium 1997–2002. New Microbiol. 27(1) (2004) species for the presence of a macrolide 49–53. toxin, mycolactone. Infect. Immun. 72(1) (2004) 123–132. In this study, we aimed to evaluate the frequency of non-tuberculous mycobacteria Mycobacterium ulcerans is an environ- (NTM) isolated from clinical specimens mental organism which is responsible for using Polymerase Chain Reaction-Restriction the disease Buruli ulcer, a necrotizing skin Enzyme Analysis (PCR-REA) and to inves- disease emerging in west Africa. M. ul- tigate the patients who had clinically signif- 72, 2 Current Literature, Microbiology 221

icant NTM infections in our hospital mycobacterial culture collection isolates. All through the five year period from May validly described species (n = 89; up to 1997 to June 2002. A total of 364 myco- March 21, 2000) and nearly all published se- bacterial strains isolated from clinical spec- quevar variants were included. If the 16S imens which gave positive growth index in rDNA sequences were not discriminatory, the BACTEC 460 radiometric system in the internal transcribed spacer (ITS) region Hacettepe University Hospital Clinical Mi- sequences (n = 84) were also determined. crobiology Laboratory were evaluated by RESULTS: Using 5′-16S rDNA sequencing PCR-REA and clinical data were obtained a total of 64 different mycobacterial species from the patient records. Three hundred (71.9%) could be identified. With the addi- and one of the strains (82.7%) were iden- tional input of the ITS sequence, a further 16 tified as Mycobacterium tuberculosis and species or subspecies could be differenti- 63 (17.3%) were identified as nontubercu- ated. Only Mycobacterium tuberculosis lous mycobacteria. Seven (11.1%) of 63 complex species, M. marinum/M. ulcerans NTM patients were regarded as having and the M. avium subspecies could not be clinical mycobacteriosis. Chronic obstruc- differentiated using 5′-16S rDNA or ITS se- tive pulmonary disease and other pre- quencing. A total of 77 culture collection existing lung diseases were seen in 39 strain sequences, exhibiting an overlap of at (61.9%) of the patients, 11 (17.5%) of the least 80% and identical by strain number to patients had chronic renal failure. Four the isolates used in this study, were found in (6.3%) and 9 (14.3%) of them had AIDS the GenBank. Comparing these with our se- and carcinomas, respectively. PCR-REA quences revealed that an average of 4.31 nu- was found to be a reliable method for typ- cleotide differences (S.D. ± 0.57) were pres- ing of our mycobacterial isolates to the ent. CONCLUSIONS: The data from this species level. These data may shed light on analysis show that it is possible to differenti- the epidemiology of the mycobacterial ate most mycobacterial species by sequence species and help to select a proper treat- analysis of partial 16S rDNA. The high-qual- ment regimen.—Authors’ Abstract ity sequences reported here, together with ancillary information (e.g., taxonomic, medical), are available in a public database, Harmsen, D., Dostal, S., Roth, A., Nie- which is currently being expanded in the mann, S., Rothganger, J., Sammeth, RIDOM project http://www.ridom-rdna.de), M., Albert, J., Frosch, M., and Richter, for similarity searches.—Authors’Abstract E. RIDOM: Comprehensive and public sequence database for identification of Mycobacterium species. BMC Infect. Hirano, K., Aono, A., Takahashi, M., and Dis. 3(1) (2003) 26. Abe, C. Mutations including IS6110 insertion in the gene encoding the MPB64 BACKGROUND: Molecular identifica- protein of Capilia TB-negative Mycobac- tion of Mycobacterium species has two pri- terium tuberculosis isolates. J. Clin. Mi- mary advantages when compared to pheno- crobiol. 42(1) (2004) 390–392. typic identification: rapid turn-around time and improved accuracy. The information A simple immunochromatographic assay, content of the 5′ end of the 16S ribosomal Capilia TB, using anti-MPB64 monoclonal RNA gene (16S rDNA) is sufficient for antibodies, is a kit for discriminating be- identification of most bacterial species. tween the Mycobacterium tuberculosis com- However, reliable sequence-based identifi- plex and mycobacteria other than tubercle cation is hampered by many faulty and some bacilli. The sensitivity of the kit was esti- missing sequence entries in publicly acces- mated to be 99.2% (381 of 384 samples). sible databases. METHODS: In order to es- The sequencing analysis revealed that all of tablish an improved 16S rDNA sequence the Capilia TB-negative isolates had muta- database for the identification of clinical and tions within the mpb64 gene, leading to the environmental isolates, we sequenced both production of an incomplete protein as a re- strands of the 5′ end of 16S rDNA (Esche- sult of a deletion of the C-terminal region of richia coli positions 54 to 510) from 199 the protein.—Authors’Abstract 222 International Journal of Leprosy 2004

Jenkin, G. A., Stinear, T. P., Johnson, P. sociated with aquatic plants in an area of D., and Davies, J. K. Subtractive hy- the Ivory Coast where Buruli ulcer is en- bridization reveals a type I polyketide demic. Genotype analysis showed that synthase locus specific to Mycobacterium plant-associated M. ulcerans had the same ulcerans. J. Bacteriol. 185(23) (2003) profile as isolates recovered in the same re- 6870–6882. gion from both aquatic insects and clinical specimens. These observations implicate Mycobacterium ulcerans causes Buruli aquatic plants as a reservoir of M. ulcerans ulcer, the third most prevalent mycobacte- and add a new potential link in the chain of rial infection of immunocompetent humans transmission of M. ulcerans to humans.— after tuberculosis and leprosy. Recent work Authors’ Abstract has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Bu- Morita, Y. S., Patterson, J. H., Billman-Ja- ruli ulcer. To search for the genetic basis of cobe, H., and McConville, M. J. Biosyn- virulence in M. ulcerans, we took advantage thesis of mycobacterial phosphatidylinos- of the close genetic relationship between M. itol mannosides. Biochem. J. 378(Pt 2) ulcerans and Mycobacterium marinum by (2004) 589–597. performing genomic suppressive subtractive hybridization of M. ulcerans with M. mar- All mycobacterial species, including inum. We identified several DNA fragments pathogenic Mycobacterium tuberculosis, specific to M. ulcerans, in particular, a type synthesize an abundant class of phos- I polyketide synthase locus with a highly phatidylinositol mannosides (PIMs) that are repetitive modular arrangement. We postu- essential for normal growth and viability. late that this locus is responsible for the syn- These glycolipids are important cell-wall thesis of mycolactone in M. ulcerans.—Au- and/or plasma-membrane components in thors’Abstract their own right and can also be hyperglyco- sylated to form other wall components, such as lipomannan and lipoarabinomannan. We Marsollier, L., Stinear, T., Aubry, J., have investigated the steps involved in the Saint Andre, J. P., Robert, R., Legras, biosynthesis of the major PIM species in a P., Manceau, A. L., Audrain, C., Bour- new M. smegmatis cell-free system. A num- don, S., Kouakou, H., and Carbon- ber of apolar and polar PIM intermediates nelle, B. Aquatic plants stimulate the were labelled when this system was contin- growth of and biofilm formation by My- uously labelled or pulse-chase-labelled with cobacterium ulcerans in axenic culture GDP-[3H]Man, and the glycan head groups and harbor these bacteria in the environ- and the acylation states of these species ment. Appl. Environ. Microbiol. 70(2) were determined by chemical and enzymic (2004) 1097–1103. treatments and octyl-Sepharose chromatography respectively. These analyses showed Mycobacterium ulcerans is the causative that (1) the major apolar PIM species, acyl- agent of Buruli ulcer, one of the most com- PIM2, can be synthesized by at least two mon mycobacterial diseases of humans. pathways that differ in the timing of the first Recent studies have implicated aquatic acylation step, (2) early PIM intermediates insects in the transmission of this pathogen, containing a single mannose residue can be but the contributions of other elements of modified with two fatty acid residues, (3) the environment remain largely unknown. formation of polar PIM species from acyl- We report here that crude extracts from two PIM2 is amphomycin-sensitive, indicating green algae added to the BACTEC 7H12B that polyprenol phosphate-Man, rather than culture medium halved the doubling time GDP-Man, is the donor for these reactions, of M. ulcerans and promoted biofilm (4) modification of acylated PIM4 with formation. Using the 7H12B medium, alpha1-2- or alpha1-6-linked mannose modified by the addition of the algal ex- residues is probably the branch point in the tract, and immunomagnetic separation, we biosyntheses of polar PIM and lipoarabino- also demonstrate that M. ulcerans is as- mannan respectively and (5) GDP strongly 72, 2 Current Literature, Microbiology 223 inhibits the synthesis of early PIM interme- cum strain with a deletion in the pks13 gene diates and increases the turnover of poly- was shown to be deficient in mycolic acid prenol phosphate-Man. These findings are production whereas it was able to produce incorporated into a revised pathway for my- the fatty acids precursors. This mutant strain cobacterial PIM biosynthesis.—Authors’ displayed an altered cell envelope structure. Abstract We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis. A conditional M. smegmatis mutant Pina-Vaz, C., Costa-Oliveira, S., Rod- carrying its only copy of pks13 on a ther- rigues, A. G., and Salvador, A. Novel mosensitive plasmid exhibited mycolic acid method using a laser scanning cytometer biosynthesis defect if grown at nonpermis- for detection of mycobacteria in clinical sive temperature. These results indicate that samples. J. Clin. Microbiol. 42(2) (2004) Pks13 is the condensase, a promising target 906–908. for the development of new antimicrobial drugs against Corynebacterineae.—Au- In order to evaluate the capacity of laser thors’Abstract scanning cytometry (LSC) to detect acid- fast bacilli directly on clinical samples, a comparison between Kinyoun-stained Rindi, L., Bonanni, D., Lari, N., and smears analyzed under light microscopy and Garzelli, C. Most human isolates of My- propidium iodide-auramine-stained smears cobacterium avium Mav-A and Mav-B analyzed by LSC was performed. The re- are strong producers of hemolysin, a pu- sults were compared with those for culture tative virulence factor. J. Clin. Microbiol. on BACTEC MGIT 960. LSC is a new, re- 41(12) (2003) 5738–5740. liable methodology to detect MYCOBAC- TERIA.—Authors’Abstract Hemolysin was quantified in 58 isolates of Mycobacterium avium from human, animal, and environmental sources. Human Mav-A Portevin, D., De Sousa-D’Auria, C., and Mav-B isolates were the strongest pro- Houssin, C., Grimaldi, C., Chami, M., ducers; in contrast, animal and environmen- Daffe, M., and Guilhot, C. A polyketide tal Mav-A isolates and human, animal, and synthase catalyzes the last condensation environmental Mav-C organisms were low- step of mycolic acid biosynthesis in myco- level producers. Hemolysin production was bacteria and related organisms. Proc. Natl. not restricted to isolates causing invasive in- Acad. Sci. U.S.A. 101(1) (2004) 314–319. fections.—Authors’Abstract

Mycolic acids are major and specific con- stituents of the cell envelope of Corynebac- Song, T., Dove, S. L., Lee, K. H., and Hus- terineae, a suborder of bacterial species in- son, R. N. RshA, an anti-sigma factor that cluding several important human pathogens regulates the activity of the mycobacterial such as Mycobacterium tuberculosis, Myco- stress response sigma factor SigH. Mol. bacterium leprae, or Corynebacterium diph- Microbiol. 50(3) (2003) 949–959. theriae. These long-chain fatty acids are involved in the unusual architecture and See Current Literature, Molecular and impermeability of the cell envelope of these Genetic Studies, p. 261. bacteria. The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an Tortoli, E. Mycobacterium kansasii, species enigma for decades. By in silico analysis of or complex? Biomolecular and epidemio- various mycobacterial genomes, we identi- logical insights. Kekkaku. 78(11) (2003) fied a candidate enzyme, Pks13, that con- 705–709. tains the four catalytic domains required for the condensation reaction. Orthologs of this Mycobacterium kansasii is one of the best enzyme were found in other Corynebacter- known nontuberculous mycobacteria and ineae species. A Corynebacterium glutami- large awareness exists about its involvement 224 International Journal of Leprosy 2004 in diseases both of immunocompetent and such as dormant or persistent tubercle bacilli immunocompromised patients. Two pheno- and its importance are also emphasized. In typic variants within this species, which dif- order to combat the threat of drug resistant fer for the virulence in guinea pig too, have tuberculosis and to more effectively control been detected since 1962. It was however fol- the disease, an understanding of the mech- lowing recent progress in genetic studies that anisms underlying drug resistance is neces- a large variability emerged. Major contribu- sary. This knowledge could be used for the tions to the disclosure of such findings came development of molecular tests for rapid de- from the DNA probes hybridization, the nu- tection of drug resistant bacilli and future cleotide sequencing of 16 rDNA and internal anti-tuberculosis drugs.—Authors’Abstract transcribed spacer (ITS), and from the analyses of repetitive DNA sequences polymorphism. At present five subtypes of M. kansasii Xu, Z. Q., Barrow, W. W., Suling, W. J., are recognized, defined by the ITS sequence Westbrook, L., Barrow, E., Lin, Y. M., and by the polymorphism revealed by differ- and Flavin, M. T. Anti-HIV natural prod- ent restriction enzyme technologies. Such uct (+)-calanolide A is active against both variants differ from the epidemiological point drug-susceptible and drug-resistant strains of view too, with type i being isolated from of Mycobacterium tuberculosis. Bioorg. humans, type ii both from humans and envi- Med. Chem. 12(5) (2004) 1199–1207. ronment, and types iii, iv and v, from the environment only. A revision of the present tax- Naturally occurring anti-HIV-1 agent onomic status of M. kansasii and its splitting (+)-calanolide A was found to be active into different species or subspecies seems against all of the strains of Mycobacterium nowadays necessary. –Authors Abstract tuberculosis tested, including those resistant to the standard antitubercular drugs. Ef- ficacy evaluations in macrophages revealed Wade, M. M., and Zhang, Y. Mechanisms of that (+)-calanolide A significantly inhibited drug resistance in Mycobacterium tuber- intracellular replication of M. tuberculosis culosis. Front Biosci. 9 (2004) 975–994. H37Rv at concentrations below the MIC observed in vitro. Preliminary mechanistic Tuberculosis is a worldwide health prob- studies indicated that (+)-calanolide A rap- lem posing increasing threat with the spread idly inhibits RNA and DNA synthesis fol- of HIV infection and drug resistant Myco- lowed by an inhibition of protein synthesis. bacterium tuberculosis strains. Conse- Compared with known inhibitors, this sce- quently, control of this disease has become nario is more similar to effects observed a significant challenge despite the availabil- with rifampin, an inhibitor of RNA syn- ity of chemotherapy and BCG vaccine. Drug thesis. Since (+)-calanolide A was active resistance for all first-line anti-tuberculosis against a rifampin-resistant strain, it is be- agents and some second-line agents has been lieved that these two agents may involve dif- observed. Moreover, the occurrence of ferent targets. (+)-Calanolide A and its re- strains of M. tuberculosis resistant to multi- lated pyranocoumarins are the first class of ple anti-tuberculosis drugs is increasing. compounds identified to possess antimy- Mechanisms of action and resistance of cobacterial and antiretroviral activities, rep- major anti-tuberculosis drugs are reviewed. resenting a new pharmacophore for anti-TB In addition, the phenotypic drug resistance activity.—Authors’Abstract Microbiology (Leprosy)

Matsuoka, M., Zhang, L., Budiawan, T., The polymorphism of TTC repeats in My- Saeki, K., and Izumi, S. Genotyping of cobacterium leprae was examined using the Mycobacterium leprae on the basis of the bacilli obtained from residents in villages at polymorphism of TTC repeats for analy- North Maluku where M. leprae infections sis of leprosy transmission. J. Clin. Mi- are highly endemic (as well as from patients crobiol. 42(2) (2004) 741–745. at North Sulawesi of Indonesia) to elucidate 72, 2 Current Literature, Microbiology (Tuberculosis) 225 the possible mode of leprosy transmission. Genotypes of TTC repeats were found to TTC genotypes are stable for several gener- differ between a son under treatment and ations of passages in nude mice footpads two brothers. These results reveal the pos- and, hence, are feasible for the genotyping sibility that in addition to exposure via the of isolates and epidemiological analysis of presence of a leprosy patient with a multi- leprosy transmission. It was found that bacillary infection who was living with bacilli with different TTC genotypes were family members, there might have been distributed among residents at the same some infectious sources to which the resi- dwelling in villages in which leprosy is en- dents had been commonly exposed outside demic and that some household contacts the dwellings. A limited discriminative ca- harbored bacilli with a different genotype pacity of the TTC polymorphism in the epi- from that harbored by the patient. Investi- demiological analysis implies the need of gations of a father-and-son pair of patients searching other useful polymorphic loci for indicated that infections of bacilli with 10 detailed subdivision of clinical isolates.— and 18 copies, respectively, had occurred. Authors’Abstract

Microbiology (Tuberculosis)

Cheng, A. F., Yew, W. W., Chan, E. W., somal adenosine triphosphatase was atten- Chin, M. L., Hui, M. M., and Chan, R. uated in mice, and exposure to proteasomal C. Multiplex PCR amplimer conformation protease inhibitors markedly sensitized analysis for rapid detection of gyrA muta- wild-type Mtb to RNI. Thus, the mycobac- tions in ﬂuoroquinolone-resistant Myco- terial proteasome serves as a defense bacterium tuberculosis clinical isolates. against oxidative or nitrosative stress.—Au- Antimicrob. Agents Chemother. 48(2) thors’Abstract (2004) 596–601.

See Current Literature, Molecular and Ewann, F., Locht, C., and Supply, P. In- Genetic Studies, p. 256. tracellular autoregulation of the Myco- bacterium tuberculosis PrrA response regulator. Microbiology 150(Pt 1) (2004) Darwin, K. H., Ehrt, S., Gutierrez-Ramos, 241–246. J. C., Weich, N., and Nathan, C. F. The proteasome of Mycobacterium tubercu- Two-component systems are major regu- losis is required for resistance to nitric latory systems for bacterial adaptation to en- oxide. Science. 302(5652) (2003) 1963– vironmental changes. During the infectious 1996. cycle of Mycobacterium tuberculosis, adaptation to an intracellular environment is The production of nitric oxide and other critical for multiplication and survival of the reactive nitrogen intermediates (RNI) by micro-organism within the host. The M. tu- macrophages helps to control infection by berculosis prrA gene, encoding the regula- Mycobacterium tuberculosis (Mtb). How- tor of the two-component system PrrA-PrrB, ever, the protection is imperfect and infec- has been shown to be induced upon macro- tion persists. To identify genes that Mtb re- phage phagocytosis and to be transiently requires to resist RNI, we screened 10,100 quired for the early stages of macrophage in- Mtb transposon mutants for hypersuscepti- fection. In order to study the mechanisms of bility to acidified nitrite. We found 12 mu- regulation of the PrrA-PrrB two-component tants with insertions in seven genes repre- system, PrrA and the cytoplasmic part of the senting six pathways, including the repair PrrB histidine kinase were produced and pu- of DNA (uvrB) and the synthesis of a flavin rified as hexahistidine-tagged recombinant cofactor (fbiC). Five mutants had insertions proteins. Electrophoretic mobility shift as- in proteasome-associated genes. An Mtb says indicated that PrrA specifically binds to mutant deficient in a presumptive protea- the promoter of its own operon, with in- 226 International Journal of Leprosy 2004 creased affinity upon phosphorylation. Huard, R. C., Chitale, S., Leung, M., Moreover, induction of fluorescence was Lazzarini, L. C., Zhu, H., Shashkina, observed after phagocytosis of a wild-type E., Laal, S., Conde, M. B., Kritski, A. M. tuberculosis strain containing the gfp L., Belisle, J. T., Kreiswirth, B. N., reporter gene under the control of the prrA- Lapa e Silva, J. R., and Ho, J. L. The prrB promoter, while this induction was not Mycobacterium tuberculosis complex- seen in a prrA/B mutant strain containing restricted gene cfp32 encodes an ex- the same construct. These results indicate pressed protein that is detectable in tu- that the early intracellular induction of berculosis patients and is positively prrA depends on the autoregulation of this correlated with pulmonary interleukin- two-component system.—Authors’ Ab- 10. Infect. Immun. 71(12) (2003) stract 6871–6883.

Human tuberculosis (TB) is caused by the Goulding, C. W., Apostol, M. I., Gleiter, S., bacillus Mycobacterium tuberculosis, a sub- Parseghian, A., Bardwell, J., Gennaro, species of the M. tuberculosis complex M., and Eisenberg, D. Gram-positive (MTC) of mycobacteria. Postgenomic dis- DsbE proteins function differently from section of the M. tuberculosis proteome is Gram-negative DsbE homologs. A struc- ongoing and critical to furthering our under- ture to function analysis of DsbE from standing of factors mediating M. tuberculo- Mycobacterium tuberculosis. J. Biol. sis pathobiology. Towards this end, a 32-kDa Chem. 279(5) (2004) 3516–3524. putative glyoxalase in the culture filtrate (CF) of growing M. tuberculosis (originally Mycobacterium tuberculosis, a Gram- annotated as Rv0577 and hereafter desig- positive bacterium, encodes a secreted Dsb- nated CFP32) was identified, cloned, and like protein annotated as Mtb DsbE characterized. The cfp32 gene is MTC re- (Rv2878c, also known as MPT53). Because stricted, and the gene product is expressed ex Dsb proteins in Escherichia coli and other vivo as determined by the respective South- bacteria seem to catalyze proper folding dur- ern and Western blot testing of an assortment ing protein secretion and because folding of of mycobacteria. Moreover, the cfp32 gene secreted proteins is thought to be coupled to sequence is conserved within the MTC, as no disulfide oxidoreduction, the function of polymorphisms were found in the tested Mtb DsbE may be to ensure that secreted cfp32 PCR products upon sequence analysis. proteins are in their correctly folded states. Western blotting of M. tuberculosis subcel- We have determined the crystal structure of lular fractions localized CFP32 predomi- Mtb DsbE to 1.1 A resolution, which reveals nantly to the CF and cytosolic compart- a thioredoxin-like domain with a typical ments. Data to support the in vivo expression CXXC active site. These cysteines are in of CFP32 were provided by the serum recog- their reduced state. Biochemical characteri- nition of recombinant CFP32 in 32% of TB zation of Mtb DsbE reveals that this disul- patients by enzyme-linked immunosorbent fide oxidoreductase is an oxidant, unlike assay (ELISA) as well as the direct detection Gram-negative bacteria DsbE proteins, of CFP32 by ELISA in the induced sputum which have been shown to be weak reduc- samples from 56% of pulmonary TB pa- tants. In addition, the pK(a) value of the tients. Of greatest interest was the observa- active site, solvent-exposed cysteine is tion that, per sample, sputum CFP32 levels approximately 2 pH units lower than that of (a potential indicator of increasing bacterial Gram-negative DsbE homologs. Finally, the burden) correlated with levels of expression reduced form of Mtb DsbE is more stable in sputum of interleukin-10 (an immunosup- than the oxidized form, and Mtb DsbE is pressive cytokine and a putative contributing able to oxidatively fold hirudin. Structural factor to disease progression) but not levels and biochemical analysis implies that Mtb of gamma interferon (a key cytokine in the DsbE functions differently from Gram- protective immune response in TB), as mea- negative DsbE homologs, and we discuss its sured by ELISA. Combined, these data sug- possible functional role in the bacterium.— gest that CFP32 serves a necessary biologi- Authors’Abstract cal function(s) in tubercle bacilli and may 72, 2 Current Literature, Microbiology (Tuberculosis) 227

contribute to the M. tuberculosis pathogenic tracellular proteins. In pathogenic species, mechanism. Overall, CFP32 is an attractive the erp gene has been described as a viru- target for drug and vaccine design as well as lence factor. The Erp proteins comprise new diagnostic strategies.—Authors’ Ab- three domains. The N- and C-terminal do- stract mains are similar in all mycobacterial species, while the central domain consists of a repeated module that differs considerably Jaeger, T., Budde, H., Flohe, L., Menge, between species. Here we show that the Erp U., Singh, M., Trujillo, M., and Radi, protein is loosely attached to the surface and R. Multiple thioredoxin-mediated routes that the carboxy-terminal domain, which to detoxify hydroperoxides in Mycobac- displays hydrophobic features, anchors Erp terium tuberculosis. Arch. Biochem. Bio- at the surface of the bacillus. The hy- phys. 423(1) (2004) 182–191. drophobic region is not necessary for the complementation of the altered colony mor- Drug resistance and virulence of Myco- phology of a Mycobacterium smegmatis bacterium tuberculosis are in part related to erp- mutant but proved to be necessary to the pathogen’s antioxidant defense systems. achieve resistance to detergent at wild-type KatG(–) strains are resistant to the first line levels.—Authors’Abstract tuberculostatic isoniazid but need to com- pensate their catalase deficiency by alternative peroxidase systems to stay virulent. So Kotlowski, R., Shamputa, I. C., El Aila, N. far, only NADH-driven and AhpD-mediated A., Sajduda, A., Rigouts, L., van Deun, hydroperoxide reduction by AhpC has been A., and Portaels, F. PCR-based geno- implicated as such virulence-determining typing of Mycobacterium tuberculosis mechanism. We here report on two novel with new GC-rich repeated sequences pathways which underscore the importance and IS6110 inverted repeats used as of the thioredoxin system for antioxidant de- primers. J. Clin. Microbiol. 42(1) (2004) fense in M. tuberculosis: (i) NADPH-driven 372–377. hydroperoxide reduction by AhpC that is mediated by thioredoxin reductase and In the present study we attempted to thioredoxin C and (ii) hydroperoxide reduc- develop a PCR-based epidemiological tool tion by the atypical peroxiredoxin TPx that for the differentiation of Mycobacterium tu- equally depends on thioredoxin reductase berculosis isolates. Use of the designed but can use both, thioredoxin B and C. Ki- primers Mtb1 (5′-CCG-GCG-GGG-CCG- netic analyses with different hydroperoxides GCG-G) and Mtb2 (5′-CGG-CGG-CAA- including peroxynitrite qualify the redox CGG-CGG-C) targeting frequently repeated cascade comprising thioredoxin reductase, 16-bp sequences in combination with thioredoxin C, and TPx as the most efficient primers sited at the inverted repeats flanking system to protect M. tuberculosis against ox- IS6110 allowed differentiation of M. tuber- idative and nitrosative stress in situ.—Au- culosis isolates.—Authors’Abstract thors’Abstract

Morlock, G. P., Metchock, B., Sikes, D., Kocincova, D., Sonden, B., de Mendonca- Crawford, J. T., and Cooksey, R. C. Lima, L., Gicquel, B., and Reyrat, J. ethA, inhA, and katG loci of ethionamide- M. The Erp protein is anchored at the sur- resistant clinical Mycobacterium tuber- face by a carboxy-terminal hydrophobic culosis isolates. Antimicrob. Agents domain and is important for cell-wall Chemother. 47(12) (2003) 3799–3805. structure in Mycobacterium smegmatis. FEMS Microbiol. Lett. 231(2) (2004) Ethionamide (ETH) is a structural analog 191–196. of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme Erp (Exported Repetitive Protein), also involved in mycolic acid biosynthesis. INH known as P36, Pirg and Rv3810, is a mem- requires catalase-peroxidase (KatG) activa- ber of a mycobacteria-specific family of ex- tion, and mutations in katG are a major INH 228 International Journal of Leprosy 2004 resistance mechanism. Recently an enzyme nan levels were not altered. The ino1 mutant (EthA) capable of activating ETH has been was attenuated in resting macrophages and in identified. We sequenced the entire ethA SCID mice. We used site-directed mutagene- structural gene of 41 ETH-resistant Myco- sis to alter four putative active site residues; bacterium tuberculosis isolates. We also se- all four alterations resulted in a loss of activity, quenced two regions of inhA and all or part and we demonstrated that a D310N mutation of katG. The MICs of ETH and INH were caused loss of the active site Zn2+ ion and a determined in order to associate the muta- conformational change in the NAD+ cofac- tions identified with a resistance phenotype. tor.—Authors’Abstract Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were ≥50 microg/ml. The ethA mutations Parish, T. Starvation survival response of were all different, previously unreported, Mycobacterium tuberculosis. J. Bacteriol. and distributed throughout the gene. In eight 185(22) (2003) 6702–6706. of the isolates, a missense mutation in the inhA structural gene occurred. The ETH The ability of Mycobacterium tuberculo- MICs for seven of the InhA mutants were sis auxotrophs to survive long-term star- ≥100 microg/ml, and these isolates were vation was measured. Tryptophan and also resistant to ≥8 microg of INH per ml. histidine auxotrophs did not survive single- Only a single point mutation in the inhA amino-acid starvation, whereas a proline promoter was identified in 14 isolates. A auxotroph did. All three auxotrophs sur- katG mutation occurred in 15 isolates, for vived complete starvation. THP-1 cells were which the INH MICs for all but 1 were ≥32 also able to restrict the growth of the trypto- microg/ml. As expected, we found no asso- phan and histidine auxotrophs.—Author’s ciation between katG mutation and the level Abstract of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH re- Pashley, C. A., and Parish, T. Efficient sistance. Approximately 76% of isolates re- switching of mycobacteriophage L5- sistant to ≥50 microg of ETH per ml had based integrating plasmids in Mycobac- such mutations.—Authors’Abstract terium tuberculosis. FEMS Microbiol. Lett. 229(2) (2003) 211–215.

Movahedzadeh, F., Smith, D. A., Norman, We previously used a mycobacteriophage R. A., Dinadayala, P., Murray-Rust, J., L5-derived integrating vector to demon- Russell, D. G., Kendall, S. L., Rison, S. strate that glnE and aroK are essential genes C., McAlister, M. S., Bancroft, G. J., in Mycobacterium tuberculosis by showing McDonald, N. Q., Daffe, M., Av-Gay, that we were unable to excise the integrated Y., and Stoker, N. G. The Mycobacte- vector when it carried the only functional rium tuberculosis ino1 gene is essential copy of these genes. We tested three systems for growth and virulence. Mol. Micro- to replace the integrated copy with alterna- biol. 51(4) (2004) 1003–1014. tive alleles. The most efficient method was to transform the strain with a second copy of Inositol is utilized by Mycobacterium tu- the integrating vector. Excision of the resi- berculosis in the production of its major thiol dent vector and integration of the incoming and of essential cell wall lipoglycans. We have vector occurred at an extremely high effi- constructed a mutant lacking the gene encod- ciency. This technique will allow us to study ing inositol-1-phosphate synthase (ino1), the role and functionality of essential genes which catalyses the first committed step in in- in this important human pathogen.—Au- ositol synthesis. This mutant is only viable in thors’Abstract the presence of extremely high levels of inositol. Mutant bacteria cultured in inositol-free medium for four weeks showed a reduction in Shimono, N., Morici, L., Casali, N., levels of mycothiol, but phosphatidylinositol Cantrell, S., Sidders, B., Ehrt, S., and mannoside, lipomannan and lipoarabinoman- Riley, L. W. Hypervirulent mutant of 72, 2 Current Literature, Microbiology (Tuberculosis) 229

Mycobacterium tuberculosis resulting were observed in intracellular growth of two from disruption of the mce1 operon. isolates from sites of tuberculosis transmis- Proc. Natl. Acad. Sci. U.S.A. 100(26) sion, with an outbreak-associated strain (2003) 15918–15923. growing faster than a strain causing disease in only one person. Activated THP-1 cells An estimated one-third of the world’s are a suitable alternative to peripheral blood population is latently infected with Myco- monocyte models.—Authors’Abstract bacterium tuberculosis, the etiologic agent of tuberculosis. Here, we demonstrate that, unlike wild-type M. tuberculosis, a strain of M. Timm, J., Post, F. A., Bekker, L. G., tuberculosis disrupted in the mce1 operon Walther, G. B., Wainwright, H. C., was unable to enter a stable persistent state Manganelli, R., Chan, W. T., Tsenova, of infection in mouse lungs. Instead, the mu- L., Gold, B., Smith, I., Kaplan, G., and tant continued to replicate and killed the McKinney, J. D. Differential expression mice more rapidly than did the wild-type of iron-, carbon-, and oxygen-responsive strain. Histological examination of mouse mycobacterial genes in the lungs of lungs infected with the mutant strain re- chronically infected mice and tuberculo- vealed diffusely organized granulomas with sis patients. Proc. Natl. Acad. Sci. U.S.A. aberrant inflammatory cell migration. 100(24) (2003) 14321–14326. Murine macrophages infected ex vivo with the mutant strain were reduced in their Pathogenetic processes that facilitate the ability to produce tumor necrosis factor entry, replication, and persistence of Myco- alpha, IL-6, monocyte chemoattractant pro- bacterium tuberculosis (MTB) in the mam- tein 1, and nitric oxide (NO), but not IL-4. malian host likely include the regulated ex- The mce1 mutant strain complemented with pression of specific sets of genes at different the mce1 genes stimulated tumor necrosis stages of infection. Identification of genes factor alpha and NO production by murine that are differentially expressed in vivo would macrophages at levels stimulated by the provide insights into host-pathogen interac- wild-type strain. These observations indicate tions in tuberculosis (TB); this approach that the mce1 operon mutant is unable to might be particularly valuable for the study stimulate T helper 1-type immunity in mice. of human TB, where experimental opportu- The hypervirulence of the mutant strain may nities are limited. In this study, the levels of have resulted from its inability to stimulate a selected MTB mRNAs were quantified in proinflammatory response that would other- vitro in axenic culture, in vivo in the lungs of wise induce organized granuloma formation mice, and in lung specimens obtained from and control the infection without killing the TB patients with active disease. We report the organism. The mce1 operon of M. tubercu- differential expression of MTB mRNAs as- losis may be involved in modulating the host sociated with iron limitation, alternative car- inflammatory response in such a way that the bon metabolism, and cellular hypoxia, con- bacterium can enter a persistent state without ditions that are thought to exist within the being eliminated or causing disease in the granulomatous lesions of TB, in the lungs of host.—Authors’Abstract wild-type C57BL/6 mice as compared with bacteria grown in vitro. Analysis of the same set of mRNAs in lung specimens obtained Theus, S. A., Cave, M. D., and Eisenach, from TB patients revealed differences in K. D. Activated THP-1 cells: an attractive MTB gene expression in humans as com- model for the assessment of intracellular pared with mice.—Authors’Abstract growth rates of Mycobacterium tuberculosis isolates. Infect. Immun. 72(2) (2004) 1169–1173. Tufariello, J. M., Jacobs, W. R. Jr., and Chan, J. Individual Mycobacterium tu- Capacity of certain Mycobacterium tuberculosis resuscitation-promoting factor berculosis isolates to grow more rapidly in homologues are dispensable for growth human macrophages may be indicative of in vitro and in vivo. Infect. Immun. 72(1) increased virulence. Significant differences (2004) 515–526. 230 International Journal of Leprosy 2004

Mycobacterium tuberculosis possesses cobacterium tuberculosis MprAB two- five genes with significant homology to the component signal transduction system. resuscitation-promoting factor (Rpf) of Infect. Immun. 71(12) (2003) 6962–6970. Micrococcus luteus. The M. luteus Rpf is a secreted approximately 16-kDa protein The mechanisms utilized by Mycobacte- which restores active growth to cultures of rium tuberculosis to establish, maintain, or M. luteus rendered dormant by prolonged reactivate from latent infection in the host incubation in stationary phase. More re- are largely unknown but likely include cently, the Rpf-like proteins of M. tubercu- genes that mediate adaptation to conditions losis have been shown to stimulate the encountered during persistence. Previously, growth of extended-stationary-phase cul- a two-component signal transduction sys- tures of Mycobacterium bovis BCG. These tem, mprAB, was found to be required in data suggest that the Rpf proteins can influ- M. tuberculosis for establishment and ence the growth of mycobacteria; however, maintenance of persistent infection in a tis- the studies do not demonstrate specific func- sue- and stage-specific fashion. To begin to tions for the various members of this protein characterize the role of this system in M. family, nor do they assess the function of M. tuberculosis physiology and virulence, a tuberculosis Rpf homologues in vivo. To ad- functional analysis of the mprA and mprB dress these questions, we have disrupted gene products was initiated. Here, evidence each of the five rpf-like genes in M. tuber- is presented demonstrating that sensor ki- culosis Erdman, and analyzed the mutants nase MprB and response regulator MprA for their growth in vitro and in vivo. In con- function as an intact signal-transducing trast to M. luteus, for which rpf is an essen- pair in vitro and in vivo. Sensor kinase tial gene, we find that all of the M. tubercu- MprB can be autophosphorylated, can do- losis rpf deletion mutant strains are viable; nate phosphate to MprA, and can act as a in addition, all show growth kinetics similar phospho-MprA phosphatase in vitro. Cor- to Erdman wild type both in vitro and in respondingly, response regulator MprA can mouse organs following aerosol infection. accept phosphate from MprB or from small Analysis of rpf expression in M. tuberculo- phosphodonors including acetyl phosphate. sis cultures from early log phase through Mutagenesis of residues His249 in MprB late stationary phase indicates that expres- and Asp48 in MprA abolished the ability of sion of the rpf-like genes is growth phase- these proteins to be phosphorylated in dependent, and that the expression patterns vitro. Introduction of these alleles into of the five M. tuberculosis rpf genes, while Mycobacterium bovis BCG attenuated vir- overlapping to various degrees, are not uni- ulence in macrophages in vivo. Together, form. We also provide evidence that myco- these results support a role for the mprAB bacterial rpf genes are expressed in vivo in two-component system in M. tuberculosis the lungs of mice acutely infected with vir- physiology and pathogenesis. Characteriza- ulent M. tuberculosis.—Authors’Abstract tion of two-component signal transduction systems will enhance our understanding of processes regulated by M. tuberculosis dur- Zahrt, T. C., Wozniak, C., Jones, D., and ing acute and/or persistent infection in the Trevett, A. Functional analysis of the My- host.—Authors’Abstract

Experimental Infections and Prevention

Ando, M., Yoshimatsu, T., Ko, C., Con- The stress-induced extracytoplasmic verse, P. J., and Bishai, W. R. Deletion sigma factor E (SigE) of Mycobacterium tu- of Mycobacterium tuberculosis sigma berculosis shows increased expression after factor E results in delayed time to death heat shock, sodium dodecyl sulfate treat- with bacterial persistence in the lungs of ment, and oxidative stress, as well as after aerosol-infected mice. Infect. Immun. phagocytosis in macrophages. We report 71(12) (2003) 7170–7172. that deletion of sigE results in delayed 72, 2 Current Literature, Experimental Infections 231

lethality in mice without a significant re- inoculation (dpi), pigs inoculated with duction of bacterial numbers in lungs.—Au- rBCGGP5 and rBCGM developed a specific thors’Abstract humoral immune response against the viral proteins, as detected by commercial ELISA and Western blot tests, and at 60 dpi, three Arevalo, M. I., Escribano, E., Calpena, out of five animals developed neutralizing A., Domenech, J., and Queralt, J. Ther- antibodies with titers ranging from 1:4 to 1:8. mal hyperalgesia and light touch allody- At 67 dpi, an IFN-gamma response against nia after intradermal Mycobacterium BCG antigens, but not against the viral pro- butyricum administration in rat. Inflam- teins, was detected by ELISPOT in inocu- mation 27(5) (2003) 293–299. lated pigs. Following challenge with a pathogenic strain of PRRSV, pigs inoculated with We examined the time course (7 weeks) rBCG showed lower (p <0.05) temperature, of thermal hyperalgesia and light touch al- viremia and virus load in bronchial lymph lodynia in rats after intradermal administra- nodes than control animals, suggesting the tion of Mycobacterium butyricum. Nocicep- establishment of partial protection against tive thresholds to heat and light touch were PRRSV infection.—Authors’Abstract assessed. Paw edema and temperature, motor function, body weight, and propio- ception were also tested. Some rats devel- Chen, L., Wang, J., Zganiacz, A., and oped arthritis (named AA rats) but others did Xing, Z. Single intranasal mucosal My- not (named non-AA rats). Both groups were cobacterium bovis BCG vaccination con- compared with healthy animals. Persistent fers improved protection compared to hyperalgesia was found in both groups; in subcutaneous vaccination against pul- AA rats it appeared before clinical evidence monary tuberculosis. Infect. Immun. of arthritis. Transient allodynia ocurred only 72(1) (2004) 238–246. after edema development and fell when edema decreased. Motor function was im- Whether the intranasal (i.n.) route of My- paired only in AA rats. The results of this cobacterium bovis BCG vaccination pro- study demonstrate that hyperalgesia, but not vides better protection against pulmonary allodynia, appeared after Mycobacterium tuberculosis than subcutaneous (s.c.) vacci- butyricum in both groups, suggesting that nation remains an incompletely solved changes in sensitivity were not merely the issue. In the present study, we compared result of local hypersensitivity of the in- both immune responses and protection flamed tissue, but may also be due to alter- elicited by single BCG vaccinations via the ations in nociception in the central nervous i.n. or s.c. route in BALB/c mice. While both system.—Authors’Abstract i.n. and s.c. vaccination triggered comparable levels of primary immune activation in the spleen and draining lymph nodes, i.n. Bastos, R. G., Dellagostin, O. A., Barletta, vaccination led to a greater antigen-specific R. G., Doster, A. R., Nelson, E., Zuck- gamma interferon recall response in spleno- ermann, F., and Osorio, F. A. Immune cytes than s.c. vaccination upon secondary response of pigs inoculated with Myco- respiratory mycobacterial challenge, ac- bacterium bovis BCG expressing a trun- companied by an increased frequency of cated form of GP5 and M protein of antigen-specific lymphocytes. There was porcine reproductive and respiratory syn- also a quicker cellular response in the lungs drome virus. Vaccine 22(3–4) (2004) of i.n. vaccinated mice upon mycobacterial 467–474. challenge. Mice vaccinated i.n. were found to be much better protected, particularly in Pigs were immunised with recombinant the lung, than s.c. vaccinated counterparts BCG (rBCG) expressing a truncated form of against pulmonary tuberculosis at both 3 GP5 (lacking the first 30 NH(2)-terminal res- and 6 months postvaccination. These results idues) (rBCGGP5) and M protein (rBCGM) suggest that the i.n. route of vaccination im- of porcine reproductive and respiratory proves the protective effect of the current syndrome virus (PRRSV). At 30 days post- BCG vaccine.—Authors’Abstract 232 International Journal of Leprosy 2004

Chung, S. W., Choi, S. H., and Kim, T. S. cluding humans. In previous work, it was Induction of persistent in vivo resistance shown that M. bovis ATCC 35721 has a mu- to Mycobacterium avium infection in tation in its principal sigma factor gene, BALB/c mice injected with interleukin- sigA, causing a single amino acid change af- 18-secreting fibroblasts. Vaccine 22(3–4) fecting binding of SigA with the accessory (2004) 398–406 transcription factor WhiB3. ATCC 35721 is avirulent when inoculated subcutaneously Interferon-gamma (IFN-gamma) is closely into guinea pigs but can be restored to viru- associated with the generation of cell- lence by integration of wild-type sigA to mediated immunity and resistance to intracel- produce M. bovis WAg320. Subsequently, it lular parasites. Interleukin-18 (IL-18) is was surprising to discover that WAg320 was known to strongly induce IFN-gamma pro- not virulent when inoculated intratracheally duction by T cells and natural killer (NK) into the Australian brushtail possum (Tri- cells. To determine whether the paracrine chosurus vulpecula), a marsupial that is secretion of IL-18 can efficiently stimulate the normally very susceptible to infection with resistance to Mycobacterium avium complex M. bovis. In this study, an in vivo comple- (MAC) infection, 3T3 fibroblasts were stably mentation approach was used with ATCC transfected to secrete bioactive IL-18 and their 35721 to produce M. bovis WAg322, which effects on MAC infection were investigated in was virulent in possums, and to identify the genetically susceptible BALB/c mice, com- virulence-restoring gene, phoT. There are pared with that of free recombinant IL-18. Im- two point deletions in the phoT gene of munization with IL-18-secreting fibroblasts ATCC 35721 causing frameshift inactiva- (3T3/IL-18) during intranasal infection with tion, one of which is also in the phoT of MAC resulted in a significant decrease in bac- BCG. Knockout of phoT from ATCC 35723, terial load of lung during the entire 8-week a virulent strain of M. bovis, produced M. observation period, while rIL-18 reduced the bovis WAg758, which was avirulent in both bacterial load at initial 1 week but not by 8 guinea pigs and possums, confirming that weeks postinfection. Immunization with the phoT is a virulence gene. The effect on vir- 3T3/IL-18 cells induced and maintained sig- ulence of mode of infection versus animal nificantly higher levels of cytotoxic activity species susceptibility was investigated by and nitric oxide production by lung cells than inoculating all the above strains by aerosol those of rIL-18 immunization. Furthermore, into guinea pigs and mice and comparing lung cells in mice injected with the 3T3/IL-18 these to the earlier results. Characterization cells showed persistent production of IFN- of PhoT indicated that it plays a role in gamma throughout the 8-week period, sug- phosphate uptake at low phosphate concen- gesting that the 3T3/IL-18 cells induced the trations. At least in vitro, this role requires resistance to MAC infection via IFN-gamma the presence of a wild-type sigA gene and production. This work suggests that IL-18- appears separate from the ability of phoT to secreting fibroblasts may serve as a vehicle for restore virulence to ATCC 35721. This study paracrine secretion of IL-18 in immunother- shows the advantages of using different an- apy of MAC infection.—Authors’Abstract imal models as tools for the molecular biological investigation of tuberculosis virulence.—Authors’Abstract Collins, D. M., Kawakami, R. P., Buddle, B. M., Wards, B. J., and de Lisle, G. W. Dif- ferent susceptibility of two animal species Collins, D. M., Kawakami, R. P., Wards, infected with isogenic mutants of Myco- B. J., Campbell, S., and de Lisle, G. W. bacterium bovis identifies phoT as having Vaccine and skin testing properties of two roles in tuberculosis virulence and phos- avirulent Mycobacterium bovis mutants phate transport. Microbiology 149(Pt 11) with and without an additional esat-6 mu- (2003) 3203–3212. tation. Tuberculosis (Edinb). 83(6) (2003) 361–366. The Mycobacterium tuberculosis complex includes Mycobacterium bovis, which SETTING: Molecular techniques are now causes tuberculosis in most mammals, in- available to develop new live tuberculosis 72, 2 Current Literature, Experimental Infections 233

vaccines by producing avirulent strains of or with the current TB vaccine, Mycobac- the Mycobacterium tuberculosis complex terium bovis BCG, induced considerable with known genes deleted. OBJECTIVES: antituberculosis protective immunity in Determine if removal of esat-6 from new immune-deficient mice lacking CD4 cells. live tuberculosis vaccines with known at- In vaccinated CD4(–/–) animals, substan- tenuating mutations affects their vaccine ef- tially reduced bacterial burdens in organs ficacy and if it could enable the development and much improved lung pathology were of discriminating diagnostic tests. DESIGN: seen 1 month after an aerogenic M. tuber- Remove the esat-6 gene by allelic exchange culosis challenge. Importantly, the postchal- from two illegitimate mutants of Mycobac- lenge mean times to death of vaccinated terium bovis that had previously been shown CD4(–/–) mice were significantly extended to have similar vaccine efficacy to BCG in a (mean with DNA cocktail, 172 ± 7 days; guinea pig vaccination model. Determine mean with BCG, 156 ± 22 days) compared the effect this removal has on virulence, vac- to that of naive CD4(–/–) mice (33 ± 6 cine efficacy and skin test reactivity in days). Furthermore, the treatment of DNA- guinea pigs. RESULTS: Two double knock- vaccinated CD4(–/–) mice with an anti-CD8 out strains of M. bovis were produced and or anti-gamma interferon (IFN-gamma) an- their virulence and vaccine efficacy were tibody significantly reduced the effect of im- compared to their parent strains. Removal of munization, and neither IFN-gamma(–/–) the esat-6 gene had no significant effect on nor tumor necrosis factor receptor-deficient vaccine efficacy. In skin tests, animals inoc- mice were protected by DNA immunization; ulated with the double knockout strains re- therefore, the primary vaccine-induced acted to PPD but not ESAT-6, whereas those protective mechanism in these immune- inoculated with the parent strains had simi- deficient mice likely involves the secretion lar skin test reactivity to both PPD and esat-6. of cytokines from activated CD8 cells. The CONCLUSION: Removal of esat-6 from substantial CD8-mediated protective immu- new live tuberculosis vaccine candidates has nity that was generated in the absence of no significant effect on vaccine properties CD4 cells suggests that it may be possible to but does enable the use of skin tests to dis- develop effective TB vaccines for use in tinguish between vaccination and infec- HIV-infected populations.—Authors’ Ab- tion.—Authors’Abstract stract

Derrick, S. C., Repique, C., Snoy, P., Gorodezky, C., Alaez, C., Munguia, A., Yang, A. L., and Morris, S. Immuniza- Cruz, R., Vazquez, A., Camacho, A., tion with a DNA vaccine cocktail protects Flores, O., Rodriguez, M., and Ro- mice lacking CD4 cells against an aero- driguez, O. Molecular mechanisms of genic infection with Mycobacterium tu- MHC linked susceptibility in leprosy: to- berculosis. Infect. Immun. 72(3) (2004) wards the development of synthetic vac- 1685–1692. cines. Tuberculosis (Edinb). 84(1–2) (2004) 82–92. Tuberculosis (TB) is the most common opportunistic disease and a potentially fatal Tuberculoid (TT) and lepromatous lep- complication among immunocompromised rosy (LL) develop in the human host de- individuals infected with human immunode- pending on his ability to trigger a specific ficiency virus (HIV). Effective vaccination cellular immune response(CIR). Different against TB in persons with HIV has been genes have been demonstrated in suscepti- considered unlikely because of the central bility/protection and may explain the forms role that CD4 cells play in controlling tu- of leprosy. The major histocompatibility berculous infections. Here we show that the complex (MHC) play an important role. The vaccination of CD8(–/–) mice with a TB aim of the study was to explore the contri- DNA vaccine cocktail did not significantly bution of human leukocyte antigen (HLA) enhance protective responses to a Mycobac- DRB1, DQA1, DQB1 and DQ promoter terium tuberculosis infection. In contrast, genes in LL Mexican patients. Six families immunization with a DNA vaccine cocktail (26 LL, three TT patients and 27 controls) 234 International Journal of Leprosy 2004 were analyzed; 114 unrelated patients were not just those for tuberculosis, so the dis- compared with 204 controls. Class I typing covery of adjuvants that can promote the was done by the standard microlymphocy- development of cell-mediated immunity is totoxicity and class II typing using PCR- of great interest. We have previously shown SSOP. Haplotype segregation correlated that the combination of the cationic surfac- with specific CIR in vivo and in vitro using tant dimethyl dioctadecyl ammonium bro- lepromin. Haplotype sharing was significantly mide and the immunomodulator modified deviated in the affected sibs (p = 0.01). Six lipid A synergistically potentiates Th1 T-cell healthy sibs were non-responders to lepromin responses. Here we report a screening pro- and four of them were DQ1 homozgotes. gram for other adjuvants with reported Th1- DQ1 was significantly associated with LL and promoting activity and identify a second with non-responders. We set up macrophage novel adjuvant formulation that drives the activation experiments after infecting these development of Th1 responses with an ex- cells with 5 × 10(6) bacilli to demonstrate if tremely high efficacy. The combination of elimination occurred in the context or DQ1. dimethyl dioctadecyl ammonium bromide When DQ1 was present on macrophages and and the synthetic cord factor trehalose on T cells, bacteria were poorly eliminated dibehenate promotes strong protective im- from the cell (32%) while when absent, 76% mune responses, without overt toxicity, of the individuals were able to eliminate the against M. tuberculosis infection in a vacci- bacilli (p = 0.03). DRB1*1501 DQA1*0102- nation model and thus appears to be a very DQB1*0602 (DQ1 subtype) was significantly promising candidate for the development of increased in the patients, indicating its partic- human vaccines.—Authors’Abstract ipation in susceptibility. QBP 5.11/5.12 promoter present in the mentioned haplotype, and QAP 1.4, linked to DRB1*1301/02 haplo- Hsieh, M. J., Junqueira-Kipnis, A. P., types were also associated. Two mecha- Hoeffer, A., Turner, O. C., and Orme, nisms are suggested: the promoter polymor- I. M. Incorporation of CpG oligodeoxy- phisms may influence allele expression and nucleotide fails to enhance the protective thus the amount of peptides presented to the efficacy of a subunit vaccine against My- T-cell receptor, leading to a deficient CIR: cobacterium tuberculosis.Vaccine 22(5–6) HLA restriction is important for vaccine (2004) 655–659. design; the way peptides anchor the DRB1*1501 groove may be relevant to the Vaccines which offer better protection activation of TH1 cells, which contribute to than BCG are now badly needed for con- an efficient presentation of peptides induc- trolling tuberculosis infection throughout ing a protective T-cell response.—Authors’ the world. Immunological adjuvants capable Abstract of inducing a TH1 type of protective response are necessary to augment the immune response, particularly in the case of Holten-Andersen, L., Doherty, T. M., subunit vaccines. It is now well established Korsholm, K. S., and Andersen, P. that oligodeoxynucleotides (ODN) contain- Combination of the cationic surfactant di- ing cytidine phosphate guanosine (CpG) methyl dioctadecyl ammonium bromide motifs enhance cell-mediated responses in and synthetic mycobacterial cord factor vivo by increasing the production of the TH1 as an efficient adjuvant for tuberculosis cytokines IL-12 and interferon gamma subunit vaccines. Infect. Immun. 72(3) (IFNgamma). To determine if this would (2004) 1608–1617. improve subunit vaccination of mice CpG ODN were added to a subunit vaccine con- Recombinant, immunodominant antigens sisting of the culture filtrate proteins (CFP) derived from Mycobacterium tuberculosis of Mycobacterium tuberculosis H37Rv. It can be used to effectively vaccinate against was observed that although adding CpG subsequent infection. However, the efficacy ODN to the vaccines promoted substantially of these recombinant proteins is dependent increased IFNgamma production by lymph on the adjuvant used for their delivery. This node cells draining sites of inoculation, this problem affects many potential vaccines, failed to translate after aerosol challenge 72, 2 Current Literature, Experimental Infections 235

into any degree of enhancement of bacterial primates. Clin. Diagn. Lab. Immunol. clearance in the lungs, influx of IFN- 11(1) (2004) 222–226. positive T cells, or changes in histopathology. These data suggest that the vaccine en- Previous work in our laboratory showed hancing effects of CpG ODN are relatively that the ESAT-6 protein of Mycobacterium transient.—Authors’Abstract tuberculosis and Mycobacterium bovis induces strong antibody responses in a large proportion ( approximately 90%) of experi- Johansen, P., Raynaud, C., Yang, M., Col- mentally or naturally infected nonhuman ston, M. J., Tascon, R. E., and Lowrie primates. Here, the antibody response to D. B. Anti-mycobacterial immunity in- ESAT-6 in tuberculous monkeys was char- duced by a single injection of M. leprae acterized at the epitope level by measuring Hsp65-encoding plasmid DNA in antibodies to overlapping, synthetic peptides biodegradable microparticles. Immunol. spanning the ESAT-6 sequence. The anti- Lett. 90(2–3) (2003) 81–85. body response against the COOH-terminal portion of the protein was the strongest in A single sub-cutaneous injection of a plas- both experimentally and naturally infected mid DNA encoding a mycobacterial heat animals. Moreover, these antibodies became shock protein 65 (Hsp65) entrapped in detectable the earliest during experimental biodegradable poly(lactic-co-glycolic acid) infection, suggesting an ordered expansion microspheres produced high titers of anti- of ESAT-6-specific B-cell clones in the bodies, measured 5 months after the injec- course of infection. The data support use of tion in BALB/c mice. Splenocytes secreted synthetic peptides in lieu of the full-length IFN-gamma and exerted an anti-bacterial ef- ESAT-6 protein in diagnostic antibody defect on macrophages infected in vitro with tection assays.—Authors’Abstract Mycobacterium tuberculosis. The results are encouraging with regard to obtaining good compliance and vaccination coverage with Kanaujia, G. V., Garcia, M. A., Bouley, D. candidate plasmid DNA vaccines, especially M., Peters, R., and Gennaro, M. L. in developing countries.—Authors’Abstract Detection of early secretory antigenic target-6 antibody for diagnosis of tuberculosis in non-human primates. Comp. Junqueira-Kipnis, A. P., Turner, J., Med. 53(6) (2003) 602–606. Gonzalez-Juarrero, M., Turner, O. C., and Orme, I. M. Stable T-cell population Tuberculosis is one of the most economi- expressing an effector cell surface pheno- cally devastating, zoonotic infections of type in the lungs of mice chronically in- captive non-human primates. The limita- fected with Mycobacterium tuberculosis. tions of the tuberculin skin test, which is Infect. Immun. 72(1) (2004) 570–575. currently used to diagnose tuberculosis in living non-human primates, make it neces- Analysis of T-cell subsets accumulating in sary to find new, simple, and economical di- the lungs of C57BL/6 mice chronically in- agnostic methods. We describe use of an fected with Mycobacterium tuberculosis re- enzyme-linked immunoassay to detect IgG vealed that both CD4 and CD8 T-cell popu- antibodies against early secretory antigenic lations expressed a cell surface phenotype target (ESAT)-6, a small protein secreted by consistent with that of effector T cells and virulent tubercle bacilli, in paired (pre- and that a significant proportion of these cells post-outbreak) sera from 57 non-human pri- were in the process of secreting gamma in- mates involved in an outbreak of Mycobac- terferon.—Authors’Abstract terium bovis infection in a research colony. Of 25 animals with tuberculosis lesions at necropsy, 22 (88%) had high serum levels of Kanaujia, G. V., Motzel, S., Garcia, M. A., the ESAT-6 antibody. The ESAT-6 antibody Andersen, P., and Gennaro, M. L. was found in 16% (5/32) of post-outbreak Recognition of ESAT-6 sequences by an- sera from animals in which tuberculosis tibodies in sera of tuberculous nonhuman could not be confirmed at necropsy. The 236 International Journal of Leprosy 2004 strong association between the ESAT-6 an- bioinformatics tools are available, it is pos- tibody and tuberculosis in non-human pri- sible to develop new strategies for a better mates documented in this study, together and effective tuberculosis vaccine, which with the robustness of the serologic assay, can replace the traditional BCG vaccine.— make the ESAT-6 ELISA a valuable tool for Authors’Abstract diagnosis of tuberculosis in captive non- human primates.—Authors’Abstract Lasco, T. M., Yamamoto, T., Yoshimura, T., Allen, S. S., Cassone, L., and McMurray, Kumar, H., Malhotra, D., Goswami, S., D. N. Effect of Mycobacterium bovis BCG and Bamezai, R. N. How far have we vaccination on Mycobacterium-specific reached in tuberculosis vaccine develop- cellular proliferation and tumor necrosis ment? Crit. Rev. Microbiol. 29(4) (2003) factor alpha production from distinct 297–312. guinea pig leukocyte populations. Infect. Immun. 71(12) (2003) 7035–7042. Tuberculosis, a bacterial disease prevalent since ancient times, continues to cause the In this study, we focused on three most deaths globally compared with all leukocyte-rich guinea pig cell populations, other diseases. The causative agent Myco- bronchoalveolar lavage (BAL) cells, resi- bacterium tuberculosis is responsible for dent peritoneal cells (PC), and splenocytes different types of tuberculosis in humans; (SPC). BAL cells, SPC, and PC were stim- however, pulmonary tuberculosis is the ulated either with live attenuated Mycobac- most common and causes the most deaths. terium tuberculosis H37Ra or with live or Mycobacterium tuberculosis is an intracel- heat-killed virulent M. tuberculosis H37Rv lular pathogenic bacterium, which has de- (multiplicity of infection of 1:100). Each veloped sophisticated mechanisms to sur- cell population was determined to prolifer- vive inside host mononuclear phagocytes ate in response to heat-killed virulent and thus evade the host immune system. H37Rv, whereas no measurable proliferative This is attributed primarily to an inadequate response could be detected upon stimulation immune response toward infecting bacteria, with live mycobacteria. Additionally, this which results in temporary growth inhibi- proliferative capacity (in SPC and PC popu- tion rather than death and subsequently al- lations) was significantly enhanced upon lows the bacteria to multiply immensely, prior vaccination with Mycobacterium bovis leading to full-blown disease in an indi- BCG. Accordingly, in a parallel set of ex- vidual. This disease has become a challenge periments we found a strong positive corre- due to poor diagnosis, a low-efficiency tu- lation between production of antigen-specific berculosis vaccine (Mycobacterium bovis bioactive tumor necrosis factor alpha (TNF- Bacillus Calmette-Guerin [BCG]), a long- alpha) and prior vaccination with BCG. A term antibacterial chemotherapy regimen nonspecific stimulus, lipopolysaccharide, (approximately 6 months), and an emer- failed to induce this effect on BAL cells, gence of multiple drug resistant strains of SPC, and PC. These results showed that pro- Mycobacterium tuberculosis especially in duction of bioactive TNF-alpha from myco- people with human immune deficiency virus bacterium-stimulated guinea pig cell cultures (HIV) infection, for whom researchers positively correlates with the vaccination sta- worldwide must develop effective short- tus of the host and with the virulence of the term chemotherapy and an effective vac- mycobacterial strain.—Authors’Abstract cine. In this review different aspects of vaccines in tuberculosis are discussed, and these include the traditional BCG vaccine, Lasco, T. M., Turner, O. C., Cassone, L., the modern auxotrophic vaccine, the subunit Sugawara, I., Yamada, H., McMurray, or acellular vaccine; and a DNA vaccine. We D. N., and Orme, I. M. Rapid accumu- discuss also the potential of mycobacterial lation of eosinophils in lung lesions in lipids as a vaccine or as an adjuvant in the guinea pigs infected with Mycobacterium future. Since complete genome information tuberculosis. Infect. Immun. 72(2) (2004) of Mycobacterium tuberculosis H37Rv and 1147–1149. 72, 2 Current Literature, Experimental Infections 237

Guinea pig eosinophils were positively rather than being due solely to help from identiﬁed in bronchoalveolar lavage popu- CD4(+) T cells. Our study shows that gam- lations and in the lung granulomas of My- madelta T cells from pigs at early ages are cobacterium tuberculosis-infected guinea functionally enhanced by M. bovis BCG pigs. It is possible that the rapid inﬂux of vaccination and suggests an important role these cells, and their subsequent degranula- for this T-cell subset in acquired immunity tion during acute pulmonary tuberculosis, conferred by M. bovis BCG vaccination.— may play a key role in the susceptibility of Authors’Abstract this animal model.—Authors’Abstract

Lima, K. M., dos Santos, S. A., Santos, R. Lee, J., Choi, K., Olin, M. R., Cho, S. N., R., Brandao, I. T., Rodrigues, J. M. Jr., and Molitor, T. W. Gammadelta T cells and Silva, C. L. Efficacy of DNA-hsp65 in immunity induced by Mycobacterium vaccination for tuberculosis varies with bovis bacillus Calmette-Guerin vaccina- method of DNA introduction in vivo. tion. Infect. Immun. 72(3) (2004) 1504– Vaccine 22(1) (2003) 49–56. 1511. A DNA vaccine codifying the mycobac- Mycobacterium bovis bacillus Calmette- terial hsp65 can prevent infection with My- Guerin (BCG) vaccination is efficacious for cobacterium tuberculosis in a prophylactic newborns or adults with no previous expo- setting and also therapeutically reduce the sure to environmental mycobacteria. To de- number of bacteria in infected mice. The termine the relative contribution and the na- protective mechanism is thought to be re- ture of gammadelta T-cell receptor-positive lated to Th1-mediated events that result in T cells in newborns, compared to CD4(+) T bacterial killing. To determine the best cells, in immunity induced by M. bovis BCG method of hsp65 introduction for vaccina- vaccination, 4-week-old specific-pathogen- tion efficacy against tuberculosis (TB), we free pigs were vaccinated with M. bovis evaluated the immunogenicity and protec- BCG and monitored by following the gamma- tion of DNA-hsp65 administered by gene delta T-cell immune responses. A flow cy- gun bombardment or intramuscular (i.m.) tometry-based proliferation assay and intra- injection of naked DNA. Immunization by cellular staining for gamma interferon gene gun induced immune response with (IFN-gamma) were used to examine gam- plasmid doses 100-fold lower than those re- madelta T-cell responses. Pigs were found to quired for intramuscular immunization. mount Th1-like responses to M. bovis BCG However, in contrast to intramuscular im- vaccination as determined by immunopro- munization, which was protective in these liferation and IFN-gamma production. The studies, gene gun immunization did not pro- gammadelta T-cell lymphoproliferation and tect BALB/c mice against challenge infec- IFN-gamma production to stimulation with tion.—Authors’Abstract mycobacterial antigens were significantly enhanced by M. bovis BCG vaccination. The relative number of proliferating gam- Nuermberger, E. L., Yoshimatsu, T., madelta T cells after stimulating peripheral Tyagi, S., Bishai, W. R., and Grosset, J. blood mononuclear cells with Mycobacte- H. Paucibacillary tuberculosis in mice rium tuberculosis H37Rv culture filtrate after prior aerosol immunization with protein was higher than that of CD4(+) T Mycobacterium bovis BCG. Infect. cells at an early time point after M. bovis Immun. 72(2) (2004) 1065–1071. BCG vaccination, but CD4(+) T cells were found to be more abundant at a later time To develop a murine model of pau- point. Although the gammadelta T-cell re- cibacillary tuberculosis for experimental sponses were dependent on the presence of chemotherapy of latent tuberculosis infec- CD4(+) T cells for the cytokine interleukin-2, tion, mice were immunized with viable My- the enhanced gammadelta T cells were due cobacterium bovis BCG by the aerosol or to the intrinsic changes of gammadelta T intravenous route and then challenged six cells caused by M. bovis BCG vaccination weeks later with virulent Mycobacterium tu- 238 International Journal of Leprosy 2004 berculosis. The day after immunization, the was significantly better than that provided by counts were 3.71 ± 0.10 log(10) CFU in the the existing vaccine, Mycobacterium bovis lungs of aerosol-immunized mice and 3.65 bacille Calmette-Guerin (BCG), and this im- ± 0.11 and 4.93 ± 0.07 log(10) CFU in the proved protective efficacy was maintained lungs and spleens of intravenously immu- for at least 24 weeks after vaccination. Pro- nized mice, respectively. Six weeks later, the tection afforded by this attenuated strain co- lungs of all BCG-immunized mice had many incided with a number of factors that were gross lung lesions and splenomegaly; the not associated with BCG vaccination: long- counts were 5.97 ± 0.14 and 3.54 ± 0.07 term persistence of the strain within the log(10) CFU in the lungs and spleens of host, sustained and potent induction of an- aerosol-immunized mice, respectively, and timycobacterial interferon-gamma-secreting 4.36 ± 0.28 and 5.12 ± 0.23 log(10) CFU in cells equal to that induced by virulent M. tu- the lungs and spleens of intravenously im- berculosis, and elicitation of T cells recog- munized mice, respectively. Mice were then nizing dominant M. tuberculosis antigens aerosol challenged with M. tuberculosis by absent from BCG. These results suggest that implanting 2.37 ± 0.13 log(10) CFU in the the BCG vaccine may be too attenuated to lungs. Six weeks after challenge, M. tuber- afford effective protective immunity against culosis had multiplied so that the counts tuberculosis, and vaccine strains that can were 6.41 ± 0.27 and 4.44 ± 0.14 log(10) provide sustained delivery of mycobacterial CFU in the lungs and spleens of control antigens are promising antituberculosis vac- mice, respectively. Multiplication of M. tu- cine candidates.—Authors’Abstract berculosis was greatly limited in BCG- immunized mice. Six weeks after challenge, the counts were 4.76 ± 0.24 and 3.73 ± 0.34 Portaels, F., Aguiar, J., Debacker, M., log(10) CFU in the lungs of intravenously Guedenon, A., Steunou, C., Zinsou, C., immunized and aerosol-immunized mice, and Meyers, W. M. Mycobacterium respectively. In contrast to intravenously bovis BCG vaccination as prophylaxis immunized mice, there was no detectable against Mycobacterium ulcerans os- dissemination to the spleen in aerosol- teomyelitis in Buruli ulcer disease. Infect. immunized mice. Therefore, immunization Immun. 72(1) (2004) 62–65. of mice with BCG by the aerosol route prior to challenge with a low dose of M. tubercu- Mycobacterium ulcerans disease, or Bu- losis resulted in improved containment of ruli ulcer (BU), causes significant morbidity infection and a stable paucibacillary infec- in West Africa. Clinically, the disease pres- tion. This model may prove to be useful for ents in the skin as either nonulcerative or ul- evaluation of new treatments for latent tu- cerative forms and often invades bones either berculosis infection in humans. subjacent to the skin lesion (contiguous osteomyelitis) or remote from the skin lesion (metastatic osteomyelitis). Osteomyelitis Pinto, R., Saunders, B. M., Camacho, L. represents a severe form of the disease that R., Britton, W. J., Gicquel, B., and often requires numerous surgical interven- Triccas, J. A. Mycobacterium tuberculo- tions, even amputations. Surgery is accepted sis defective in phthiocerol dimyco- as the present definitive treatment for BU. In cerosate translocation provides greater the absence of an effective drug treatment, protective immunity against tuberculosis the need for the development of preventive than the existing bacille Calmette-Guerin and control strategies becomes paramount. vaccine. J. Infect. Dis. 189(1) (2004) No specific vaccine, however, is presently 105–112. available for BU. Of 372 consecutive patients in Benin presenting with BU (con- We demonstrate that Mycobacterium tu- firmed by microbiological and histopatho- berculosis that is unable to export the com- logical analyses) whose Mycobacterium plex lipid phthiocerol dimycocerosate has a bovis BCG scar statuses were known, 196 decreased capacity to replicate in mice and children (<15 years old) and 108 adults had affords sustained protective immunity neonatal BCG vaccination scars. Of 196 against M. tuberculosis infection Protection children with BCG scars, 17 (8.7%) had os- 72, 2 Current Literature, Experimental Infections 239

teomyelitis, while 7 of 28 children without monia with clusters of acid fast bacilli, BCG scars (25.0%) had osteomyelitis. Of whereas vaccinated mice showed small 108 adults with BCG scars, 17 (15.7%) had areas of damage and evidence of protection osteomyelitis, while 14 of 40 adults without subsequently. INTERPRETATION & CON- BCG scars (35.0%) had osteomyelitis. Our CLUSION: It may be concluded from the results show that effective BCG vaccination evidence presented here that mice vacci- at birth provides significant protection nated with M. habana were protected from against the development of M. ulcerans os- challenge with M. tuberculosis in both nor- teomyelitis in children and adults. Therefore, mal and immunocompromised states.—Au- health authorities should give attention to the thors’Abstract enhancement of neonatal BCG vaccination coverage in all countries of Africa where BU is endemic. Protection against severe forms Price, N. M., Gilman, R. H., Uddin, J., of BU and childhood tuberculosis would Recavarren, S., and Friedland, J. S. likewise be improved by this intervention.— Unopposed matrix metalloproteinase-9 Authors’Abstract expression in human tuberculous granuloma and the role of TNF-alpha- dependent monocyte networks. J. Im- Prem Raj, P., Srivastava, S., Jain, S. K., munol. 171(10) (2003) 5579–5586. Srivastava, B. S., and Srivastava, R. Protection by live Mycobacterium ha- Tuberculosis is characterized by granu- bana vaccine against Mycobacterium tu- loma formation and caseous necrosis, but berculosis H37Rv challenge in mice. In- the factors causing tissue destruction are dian J. Med. Res. 117 (2003) 139–145 poorly understood. Matrix metalloproteinase (MMP)-9 (92-kDa gelatinase) secretion from BACKGROUND & OBJECTIVES: In monocytes is stimulated by Mycobacterium recent years the efficacy of BCG vaccine tuberculosis (M. tb) and associated with against tuberculosis has been questioned local tissue injury in tuberculosis patients. and there is no alternative vaccine available. We demonstrate strong immunohistochem- Several strategies are being applied to get a ical MMP-9 staining in monocytic cells at satisfactory vaccine. Two approaches are the center of granuloma and adjacent to generally considered: the subunit vaccines caseous necrosis in M. tb-infected patient and the whole cell vaccines. The objective lymph nodes. Minimal tissue inhibitor of of this investigation was to evaluate an avir- MMPs-1 staining indicated that MMP-9 ac- ulent mycobacteria, Mycobacterium ha- tivity is unopposed. Because granulomas bana, as a whole cell vaccine to protect mice characteristically contain few mycobacteria, from infection of M. tuberculosis H37Rv. we investigated whether monocyte-monocyte METHODS: AKR and immunocompro- cytokine networks amplify MMP-9 se- mised SJL/J mice were immunized with live cretion. Conditioned medium from M. tb- M. habana vaccine. These mice were chal- infected primary human monocytes or lenged with M. tuberculosis H37Rv eight THP-1 cells (CoMTB) stimulated MMP-9 weeks later along with unimmunized control gene expression and a >10-fold increase in mice. Protection by M. habana vaccine was MMP-9 secretion by monocytes at 3–4 days measured through several parameters, which (p <0.009, vs controls). Although CoMTB included survival of challenged mice, dis- stimulated dose-dependent MMP-9 secretion, semination of challenge strain and histo- MMP-1 (52-kDa collagenase) was not in- pathology of lung tissues. RESULTS: M. duced. Anti-TNF-alpha Ab but not IL-1R habana vaccinated animals were healthier antagonist pretreatment decreased CoMTB- than the unvaccinated mice after challenge induced MMP-9 secretion by 50% (p = with M. tuberculosis and survived with sig- 0.0001). Anti-TNF-alpha Ab also inhibited nificant increase in mean survival time. The MMP-9 secretion from monocytic cells by viable count of challenge strain was at least 50%, 24 hr after direct M. tb infection (p = 100-fold less in vaccinated mice than the 0.0002). Conversely, TNF-alpha directly control mice. The lung tissues in unvacci- stimulated dose-dependent MMP-9 secre- nated mice showed marked bronchopneu- tion. Pertussis toxin inhibited CoMTB- 240 International Journal of Leprosy 2004 induced MMP-9 secretion and enhanced the mutations of this two-component system inhibitory effect of anti-TNF-alpha Ab (p = that were responsible for the avirulent phe- 0.05). Although chemokines bind to G pro- notype. This work identifies the PAS domain tein-linked receptors, CXCL8, CXCL10, as a possible drug target for tuberculosis and CCL2, and CCL5 did not stimulate mono- mutations in the senX3-regX signal trans- cyte MMP-9 secretion. However, the reduction system as potentially useful compo- sponse to cholera toxin confirmed that G nents of live vaccine strains.—Authors’Ab- protein signaling pathways were intact. In stract summary, MMP-9 within tuberculous granuloma is associated with tissue destruction, and TNF-alpha, critical for antimycobacte- Shim, T. S., Turner, O. C., and Orme, I. M. rial granuloma formation, is a key autocrine Toll-like receptor 4 plays no role in sus- and paracrine regulator of MMP-9 secre- ceptibility of mice to Mycobacterium tu- tion.—Authors’Abstract berculosis infection. Tuberculosis (Edinb). 83(6) (2003) 367–371

Rickman, L., Saldanha, J. W., Hunt, D. M., Although various members of the pattern Hoar, D. N., Colston, M. J., Millar, J. B., recognition Toll-like receptor (TLR) family and Buxton, R. S. A two-component have been implicated in host resistance to signal transduction system with a PAS Mycobacterium tuberculosis infection, it re- domain-containing sensor is required for mains unclear if the TLR4 receptor plays an virulence of Mycobacterium tuberculosis important role. We demonstrate here that in- in mice. Biochem. Biophys. Res. Com- fection of TRL4-competent and TLR4- mun. 314(1) (2004) 259–267. deficient mice on the C3H inbred mouse strain background had similar outcomes, Mycobacterium tuberculosis, the causative measured in terms of the course of the dis- organism of tuberculosis, encounters oxida- ease, cell accumulation patterns in the lungs, tive stress during phagocytosis by the and lung histopathology. These data argue macrophage and following macrophage ac- against a significant role for TLR4 in im- tivation during an acquired immune re- munity to tuberculosis in the mouse sponse, and also from internally generated model.—Authors’Abstract sources of radical oxygen intermediates through intermediary metabolism. We have identified the SenX3 protein, a sensor in 1 of Shirtcliffe, P. M., Goldkorn, A., Weather- the 11 complete pairs of two-component sig- all, M., Tan, P. L., and Beasley, R. Pilot nal transduction systems in M. tuberculosis, study of the safety and effect of intranasal as a possible orthologue of the Mak2p pro- delipidated acid-treated Mycobacterium tein from the fission yeast Schizosaccha- vaccae in adult asthma. Respirology 8(4) romyces pombe that is known to sense per- (2003) 497–503. oxide stress. Moreover, the SenX3-RegX3 two-component system was the top scoring OBJECTIVE: There is epidemiological hit in a homology search with the Escheri- and experimental evidence that exposure to chia coli ArcB-ArcA global control system mycobacteria has the potential to suppress the of aerobic genes. Using structural modelling development of atopy and/or asthma. Delip- techniques we have determined that SenX3 idated, deglycolipidated and arabinogalactan- contains a PAS-like domain found in a va- depleted autoclaved Mycobacterium vaccae riety of prokaryotic and eukaryotic sensors (delipidated acid-treated M. vaccae) has of oxygen and redox. Mutants with knock- been shown to suppress allergen-induced outs of senX3 or of the accompanying tran- airway eosinophilia in mice. METHODOL- scriptional regulator regX3 were constructed OGY: Thirty-seven adults with stable mod- and found to have reduced virulence in a erately severe asthma who were skin prick mouse model of tuberculosis infection, the test-positive to house dust mite were ran- mutant bacteria persisting for up to 4 months domized to receive two doses 2 weeks apart post-infection; complemented mutants had of delipidated acid-treated M. vaccae (first regained virulence confirming that it was dose 0.4 mg and second dose 0.8 mg) or 72, 2 Current Literature, Experimental Infections 241

phosphate buffered saline, given as drops in- its role in the host-pathogen interaction. In- tranasally. Safety, tolerability and markers fection of guinea pigs with the mutant strain of asthma severity (including peak flow, resulted in a 70-fold reduction in the bacil- FEV1, major and minor exacerbations, lary load of spleens in infected animals as symptom scores and beta-agonist use), and compared with the bacillary load in animals nasal symptom scores, blood eosinophil and infected with the parental strain. Upon rein- IgE levels were monitored for 8 weeks. RE- troduction of the mptpB gene into the mu- SULTS: Delipidated acid-treated M. vaccae tant strain, the complemented strain was was safe and well tolerated although there able to establish infection and survive in was an occasional mild local reaction. There guinea pigs at rates comparable to the were no statistically significant differences parental strain. These observations demon- between the treatment group and placebo for strate a role of MptpB in the pathogenesis of any of the outcome variables. CONCLU- M. tuberculosis.—Authors’Abstract SIONS: There is a requirement to elucidate the reasons why mycobacterial-based vaccines have not shown equivalent efficacy in Stanford, J., Stanford, C., and Grange, J. human trials compared with animal models. Immunotherapy with Mycobacterium The role of factors such as duration of dis- vaccae in the treatment of tuberculosis. ease, route of administration and the active Front Biosci. 9 (2004) 1701–1719. component of mycobacteria need to be ad- dressed.—Authors’Abstract All the trials of immunotherapy of tuberculosis with killed Mycobacterium vaccae, published or not, that are known to the au- Singh, R., Rao, V., Shakila, H., Gupta, R., thors are reviewed here. Following an intro- Khera, A., Dhar, N., Singh, A., Koul, A., duction giving a brief account of some ear- Singh, Y., Naseema, M., Narayanan, P. lier immunotherapies for tuberculosis, the R., Paramasivan, C. N., Ramanathan, origins of the concept of immunotherapy V. D., and Tyagi, A. K. Disruption of with M. vaccae are considered. Progress is mptpB impairs the ability of Mycobacte- traced from the early work with irradiation- rium tuberculosis to survive in guinea killed organisms in leprosy to the study in pigs. Mol. Microbiol. 50(3) (2003) 751– London of modulation of tuberculin skin- 762. test responses, and the first comparative trials in The Gambia and Kuwait. In the last of Protein tyrosine kinases and tyrosine these studies, dosages and different prepa- phosphatases from several bacterial patho- rations were compared. As a result of this gens have been shown to act as virulence subsequent studies have used 109 heat- factors by modulating the phosphorylation killed organisms, equivalent to 1mg wet- and dephosphorylation of host proteins. The weight of bacilli, as a standard dose. A series identification and characterization of two ty- of small trials in Argentina, India, Nigeria, rosine phosphatases namely MptpA and Romania, South Africa and Vietnam have MptpB from Mycobacterium tuberculosis pioneered the way forward, disclosing geo- has been reported earlier. MptpB is secreted graphic variability, with South Africa as the by M. tuberculosis into extracellular mileu only country where almost no effects were and exhibits a pH optimum of 5.6, similar to recorded. Together the studies have shown the pH of the lysosomal compartment of the that a single dose may not be sufficient. cell. To determine the role of MptpB in the These studies have confirmed the mode of pathogenesis of M. tuberculosis, we con- action of M. vaccae to be regulation of cell- structed a mptpB mutant strain by homolo- mediated immunity with enhancement of gous recombination and compared the Th1 and down-regulation of Th2, and they ability of parent and the mutant strain to sur- have shown benefits in faster bacteriologi- vive intracellularly. We show that disruption cal conversion, reduction in ESR, recovery of the mptpB gene impairs the ability of the of body weight and resolution of radiologi- mutant strain to survive in activated macro- cal opacities, leading to better recovery from phages and guinea pigs but not in resting the disease even when given to patients re- macrophages suggesting the importance of ceiving directly observed therapy, short- 242 International Journal of Leprosy 2004 course (DOTS). Three major randomised, they developed granulomatous pulmonary placebo-controlled and partly blinded trials lesions with neutrophil infiltration which have been carried out in Africa. The first, in were larger than those in wild-type (WT) South Africa showed no M. vaccae-related mice (p <0.01). The pulmonary tissue levels effects. The second trial, in Uganda, con- of mRNA for iNOS and IL-18 were slightly firmed the observations made in the earlier lower, but levels of mRNA for IL-1 beta, studies of faster sputum conversion and bet- IL-2, IL-4, IL-6, IL-10, IFN-gamma, and ter radiological clearance. The third trial, in TGF-beta were higher in MyD88 KO mice. Zambia and Malawi, showed a trend to- IFN-gamma, TNF-alpha, IL-1 beta, and IL- wards benefits in the treatment of HIV 12 also were high in the sera of MyD88 KO seronegative patients but failed to show ben- mice. There were no statistically significant eficial effects in HIV seropositive patients. differences in the expression of TNF-alpha, Studies in patients with multi-drug-resistant IL-12, and ICAM-1 mRNA between MyD88 tuberculosis have shown that multiple doses KO and WT mice. Thus, MyD88 deficiency of immunotherapy are required in most did not influence the development of murine cases, and that these markedly improve tuberculosis. NF-kappa B activity was cure-rates for these patients. This is espe- similar in the alveolar macrophages from cially so when they are also treated with the lung tissues of MyD88 KO and WT chemotherapy tailored to the resistance pat- mice. Also, there may be a TLR2-specific, tern of their infecting organisms. A small MyD88-independent IL-1 receptor/TLR- study has just commenced in which repeated mediated pathway to activate NF-kappa B in doses of M. vaccae are being administered the host defense against mycobacterial in- to a group of patients who have failed treat- fection.—Author’s Abstract ment with DOTS-Plus (directly observed therapy with drugs selected on the basis of drug susceptibility profiles). Late in the in- Tomioka, H., Shimizu, T., Sato, K., Sano, vestigation came publications from China C., Kamei, T., Emori, M., and Saito, H. supporting and confirming the data in both Comparative roles of macrophages and drug-sensitive and drug-resistant disease, by NK cells in the host innate resistance of the use of multiple injections of their own mice to Mycobacterium fortuitum infec- different preparation of M. vaccae. The trial tion. J. Infect. 48(1) (2004) 74–80. that is now beginning in Vietnam of 3 doses of M. vaccae in the treatment of newly di- OBJECTIVES: Profiles of host innate re- agnosed pulmonary tuberculosis, is accom- sistance to Mycobacterium fortuitum (MFT) panied by a chemotherapeutic regimen with infection in mice and the roles of macro- a shortened continuation phase. If this im- phages (Mphis) and NK cells in host resist- portant study is successful, immunotherapy ance to MFT infection were studied. with killed M. vaccae should be introduced METHODS: MFT-infected mice with or into the treatment regimens for tuberculosis without the treatments to reduce Mphis and worldwide. NK cells were examined for survival and the bacterial loads in the kidneys during the course of infection. RESULTS: A unique Sugawara, I., Yamada, H., Mizuno, S., profile of strain difference was found in the Takeda, K., and Akira, S. Mycobacterial innate resistance of mice to MFT. A/J, infection in MyD88-deficient mice. Mi- C3H/He and DBA/2 mice were susceptible, crobiol. Immunol. 47(11) (2003) 841–847. while BALB/c, B10A and C57BL/6 mice were resistant, in terms of survival after MyD88 is an adaptor protein that plays a MFT infection. Such profiles of host resis- major role in TLR/IL-1 receptor family sig- tance to MFT were essentially correlated naling. To understand the role of MyD88 in with the ability of individual strain mice to the development of murine tuberculosis in prevent the bacterial growth in the early pe- vivo, MyD88 knockout (KO) mice aerially riods after infection. These profiles were dif- were infected with Mycobacterium tubercu- ferent from the strain difference controlled losis. Infected MyD88 mice were not highly by Bcg gene. Studies using carrageenan, susceptible to M. tuberculosis infection, but anti-asialo GM1 antibody, and NK cell- 72, 2 Current Literature, Experimental Infections 243

deficient beige mice indicated the important that the novel findings and molecular roles of Mphis and NK cells in the host in- mechanisms associated with mycobacterial nate defense against MFT. CONCLU- infections will further strengthen the cur- SIONS: These findings suggest that Bcg rently unproved therapeutic value of im- gene does not control the host resistance to munotherapy with mycobacteria.—Au- MFT and that both Mphis and NK cells play thors’Abstract crucial roles in the host innate resistance to MFT infection.—Authors’Abstract Waters, W. R., Palmer, M. V., Nonnecke, B. J., Whipple, D. L., and Horst, R. L. Walker, C., Sawicka, E., and Rook, G. A. Mycobacterium bovis infection of vita- Immunotherapy with mycobacteria. Curr. min D-deficient NOS2–/– mice. Microb. Opin. Allergy Clin. Immunol. 3(6) (2003) Pathog. 36(1) (2004) 11–17. 481–486. Vitamin D deficiency is associated with SUMMARY: PURPOSE OF REVIEW: an increased risk for tuberculosis infec- To summarize and evaluate critically recent tion. Studies using in vitro systems indi- progress with mycobacteria as a potential cate that 1,25-dihydroxyvitamin D(3) [i.e., novel disease modifying treatment strategy 1,25(OH)(2)D(3)], the most active form of in asthma. RECENT FINDINGS: The link the vitamin, enhances mycobacterial killing between exposure to pathogenic or sapro- by increasing nitric oxide (NO) production. phytic mycobacteria and protection from To evaluate concurrently the role of allergic diseases is still controversial, and 1,25(OH)(2)D(3) and NO on the host re- recent epidemiological studies, which ad- sponse to tuberculosis infection, mice defi- dressed only exposure to Mycobacterium cient in NO synthase 2 (NOS2(–/–)) and/or tuberculosis or bacillus Calmette-Guerin, vitamin D were aerosol-challenged with did not help to clarify this issue. Moreover, Mycobacterium bovis and subsequently the clear efficacy of mycobacterial treatment evaluated for mycobacterial colonization seen in animal models has not been repro- and lesion formation. Infected NOS2(–/–) duced in human asthma, and a recent small mice developed severe necrotizing pyo- study testing the hypothesis that heat-killed granulomatous inflammation of the lungs Mycobacterium vaccae attenuates asthmatic with heavy M. bovis colonization and sys- reactions after allergen challenge did not temic dissemination of the bacillus. Colo- provide convincing results. However, it has nization and lung lesion area of NOS2(–/–) been shown that treatment of mice with M. mice exceeded that of NOS2(+/+) mice. Ad- vaccae induces the generation of allergen- ditionally, disease progression was more specific T regulatory cells capable of sup- rapid in NOS2(–/–) mice than in NOS2(+/+) pressing allergen-mediated eosinophilic mice. Lung colonization and lesion area of lung inflammation, suggesting that a general vitamin D deficient mice exceeded that of deficiency of T regulatory cell activity might vitamin D replete mice, regardless of NOS2 be responsible for the increased prevalence phenotype. However, effects of vitamin D of asthma. This hypothesis is supported by on colonization, but not lesion area, were findings that a lack of T regulatory cells, as more pronounced in NOS2(+/+) mice than found in genetic disorders of man and in NOS2(–/–) mice. These findings are con- mouse attributable to a mutation of Foxp3, sistent with the current hypothesis that a transcription factor specifically expressed 1,25(OH)(2)D(3) enhances mycobacterial by T regulatory cells, is associated with killing through a NO-dependent mecha- manifestations of severe atopy and autoim- nism. As responses of NOS2(–/–) mice were munity, precisely the spectrum of diseases affected by 1,25(OH)(2)D(3) deficiency, al- linked to the hygiene hypothesis. SUM- beit to a lesser extent than were those of MARY: Further studies on the relationship NOS2(+/+) mice, NO-independent actions between mycobacteria and atopic disorders of 1,25(OH)(2)D(3) also likely exist.—Au- are needed, but there is reason to believe thors’Abstract 244 International Journal of Leprosy 2004 Epidemiology and Prevention

Bierrenbach, A. L., Cunha, S. S., Barreto, school children. This information should be M. L., Pereira, S. M., Dourado, I., Ichi- taken into account in tuberculin skin test hara, M. Y., Brito, S. C., and Rodrigues, surveys intended to estimate M. tuberculo- L. C. Tuberculin reactivity in a population sis prevalence or to assess transmission pat- of school children with high BCG vacci- terns as well as in tuberculin skin testing of nation coverage. Pan American J. Pub. individuals used as an auxiliary tool in Health 13(5) (2003) 285–293. diagnosing tuberculosis. Taking this information into consideration is especially im- A study was conducted to investigate the portant when there is increasing BCG re- influence of BCG vaccination or revaccina- vaccination coverage. tion on tuberculin skin test reactivity, in order to guide the correct interpretation of this test in a setting of high neonatal BCG Dony, J. F., Ahmad, J., and Khen Tiong, vaccination coverage and an increasing Y. Epidemiology of tuberculosis and lep- BCG revaccination coverage at school age. rosy, Sabah, Malaysia. Tuberculosis In 1997, we conducted tuberculin skin test- (Edinb). 84(1–2) (2004) 8–18. ing and BCG scar reading in 1148 children aged 7–14 years old in the city of Salvador, The objectives in this epidemiology re- Bahia, Brazil. We measured the positive ef- view are to measure and report the extent of fect of the presence of one or two BCG scars morbidity and mortality due to tuberculosis on the proportion of tuberculin skin test re- (TB), the proportion of new sputum smear sults above different cut-off levels (indura- positive cases in districts and the status of tion sizes of ≥5 mm, ≥10 mm, and ≥15 mm) cohort analysis as of 1999. As for leprosy, and also using several ranges of induration the main objective is to determine morbid- size (0, 1–4, 5–9, 10–14, and ≥15 mm). We ity and the treatment outcomes of Multiple also measured the effects that age, gender, Drug Therapy (MDT). Based on the results and the school where the child was enrolled obtained, a comprehensive action plan for had on these proportions. The proportion of prevention, control and monitoring of tu- tuberculin results ≥10 mm was 14.2% (95% berculosis and leprosy cases and patients is confidence interval (CI) = 8.0%–20.3%) for being produced and implemented through- children with no BCG scar, 21.3% (95% CI out the state. The analysis concentrated on = 18.5%–24.1%) for children with one BCG patients diagnosed at all out-patient units scar, and 45.0% (95% CI = 32.0%–58.0%) and admitted in all of the state’s hospitals. for children with two BCG scars. There was The patient particulars were recorded using evidence for an increasing positive effect of a standardized format based on TB and Lep- the presence of one and two BCG scars on rosy Health Management Information Sys- the proportion of results ≥5 mm and ≥10 tem (TB HMIS). TB was the second highest mm. Similarly, there was evidence for an in- by notification of communicable diseases in creasing positive effect of the presence of Malaysia in 2001. 29% or about one-third of one and two scars on the proportion of tu- the national TB cases are from Sabah. How- berculin skin test results in the ranges of 5–9 ever, it has been noted that there was an av- mm and of 10–14 mm. The BCG scar effect erage decline of 2.6% in annual notification on the proportion of results ≥5 mm and ≥10 since 10 years ago to date. There was also a mm did not vary with age. There was no ev- reduction of 11.4% in 2001 as compared to idence for BCG effect on the results ≥15 annual notification in 2000. Immigrants con- mm. In Brazilian school children, BCG- tribute more than 24% in detection of new induced tuberculin reactivity is indistin- cases since 1990. Treatment success rate in guishable, for results under 15 mm, from term of completion of treatment to date is reactivity induced by Mycobacterium tuber- 82%. Mortality rate has steadily declined culosis infection. BCG revaccination at from 14 deaths to 7 deaths per 100,000 school age increases the degree of BCG- population. Leprosy in Sabah also con- induced tuberculin reactivity found among tributes to 30% of the yearly total caseload 72, 2 Current Literature, Other Mycobacterial Diseases 245

of Malaysia and has the highest notifica- corporated into the State and Districts action rate of 2 per every 100,000 population tion plans and strategies. It is also noted as compared to other states. The average that in order to translate National Plans registered leprosy cases over the past 5 and Strategies into effective action at the years are 239 cases and the prevalence rate community level, health workers need rel- is 0.7/10,000 population. The state has evant up-to-date knowledge of the pattern successfully achieved its goal to decrease of health and disease, and of their deter- leprosy as per the World Health Organiza- minants, in each district. The Sabah Health tion (WHO) goal of yearly overall preva- Department continues to organize and sup- lence rate of less than 1 case for every port programs related to management and 10,000 population. However, the districts control of tuberculosis and leprosy to pro- of Kudat, Tawau, Lahad Datu, Kota Kina- gressively reduce the incidence of these balu and Semporna are still within the diseases in the community by breaking the prevalence rate of more than one per chain of transmission of Mycobacterium 10,000 population. This review highlights tuberculosis and M. leprae, respectively.— some interesting ﬁndings which can be in- Authors’ Abstract Other Mycobacterial Diseases

Arkwright, P. D., and David, T. J. Effect ment. CONCLUSIONS: Despite a reduction of Mycobacterium vaccae on atopic der- in clinical severity associated with M. vac- matitis in children of different ages. Br. J. cae at all ages, no benefit could be found Dermatol. 149(5) (2003) 1029–1034. after administering M. vaccae to children with AD aged 2–6 years when compared BACKGROUND: Exposure to environ- with placebo. M. vaccae may offer greater mental microorganisms is associated with benefit in children over 5 years old, whose variations in the prevalence and severity of AD appears less likely to regress sponta- atopic diseases. We have previously shown neously.—Authors’Abstract that administration of a Mycobacterium vaccae suspension significantly reduced the severity of atopic dermatitis (AD) in chil- Coussens, P. M., Jeffers, A., and Colvin, dren aged 5–18 years. OBJECTIVES: This C. Rapid and transient activation of gene study aimed to extend these observations to expression in peripheral blood mononu- younger children. METHODS: Fifty-six clear cells from Johne’s disease positive children aged 2–6 years with moderate to cows exposed to Mycobacterium paratu- severe AD were enrolled in a randomized, berculosis in vitro. Microb. Pathog. 36(2) double-blind, placebo-controlled trial and (2004) 93–108. given one intradermal injection of either killed M. vaccae suspension or buffer solu- Mycobacterium avium subspecies paratu- tion (placebo). Skin surface area affected berculosis (M. paratuberculosis) is the and dermatitis severity score were assessed causative agent of Johne’s disease in rumi- before and 1, 3 and 6 months after treatment. nants. M. paratuberculosis is a slow- RESULTS: Although a 38–54% reduction in growing intracellular bacterium and infec- surface area affected by dermatitis was tions with M. paratuberculosis can persist in noted at all time points after M. vaccae ad- a subclinical state for several years. An early ministration (p = 0.005), this improvement and appropriate T cell-mediated cytotoxic was not significantly different from that ob- response (Th1-like) to M. paratuberculosis served in the placebo group. Meta-analysis infection is often replaced with an antibody of this and our previous cohort (97 children or Th 2-like response as infected animals aged 2–18 years) showed that M. vaccae move toward a progressively more clinical was associated with a significant improve- state. The reasons for this shift in immune ment in clinical severity at all ages, whereas response are unknown. Recent studies sug- within the placebo group, younger but not gest that in vitro exposure of peripheral older children showed a similar improve- blood mononuclear cells (PBMCs) from 246 International Journal of Leprosy 2004

Johne’s disease positive cows to M. paratu- logical to administer chemotherapy with both berculosis for 18–24 hr results in suppressed Rifampicin and Aminoglycosid or Fluro- expression of numerous immune cell genes. quinolon and Aminoglycosid. Surgical treat- This effect appears at odds with the notion ment depends on the size of the ulcer, as well that immune cells from infected cows would as available techniques and skills on the field. respond to M. paratuberculosis-specific Wide excision and graft are often recom- antigens in a vigorous and positive manner. mended, however limited excision followed In this report, we detail experiments de- by small islet grafts may be successful.— signed to test the hypothesis that many pos- Bulletin de la Société de Pathologie Exotique itive changes in PBMC gene expression induced by M. paratuberculosis in vitro are transient, being rapidly suppressed by as yet den Broeder, A. A., Vervoort, G., van Assen, unknown mechanisms. Our results demon- S., Verduyn Lunel, F., de Lange, W. C., strate that, indeed, in vitro stimulation with and de Sevaux, R. G. Disseminated My- M. paratuberculosis induces rapid changes cobacterium gordonae infection in a renal in infected cow PBMC gene expression transplant recipient. Transpl. Infect. Dis. (within 2–4 hr of exposure) and that many 5(3) (2003) 151–155. of these changes are no longer evident by 8–16 hr of exposure to M. paratuberculosis. The use of more intensive immunosup- Although precise mechanisms responsible pressive regimens and the increasing num- for rapid M. paratuberculosis-mediated ber of patients that are exposed to immuno- activation of PBMC gene expression and the suppressive strategies in transplantation loss thereof remain to be determined, our medicine have changed the spectrum of in- novel results suggest that PBMCs from fections that is encountered by the clinician. Johne’s disease positive cows are indeed We describe a 62-year-old female renal capable of vigorously responding to M. transplant recipient receiving immunosup- paratuberculosis and that, for many genes, pressive therapy who developed complaints this response is tempered within 8 hr of ex- of weight loss, diarrhoea, cough, and fever. posure.—Authors’Abstract Increased C-reactive protein and pancy- topenia were found. The presence of Myco- bacterium gordonae, a non-tuberculous my- Darie, H. Infection par Mycobacterium ul- cobacterium, was eventually demonstrated cerans: aspects epidemiologiques, clin- in bronchoalveolar lavage fluid, bone mar- iques et thérapeutiques. Bull. Soc. Pathol. row, spleen, and liver. Determination of the Exot. 96(5) (2003) 368–371. pathogen was accelerated using a Line Probe Assay, a reverse hybridisation tech- Mycobacteriun ulcerans causing Buruli nique using an RNA fragment specific for ulcer is an environmental mycobacteria re- different mycobacterium species. Treatment sponsible for an infectious necrotizing pan- was initiated using a combination of clarith- niculitis. The epidemiology of this disabling romycin, ethambutol, and rifampicin. The disease is strongly linked to the aquatic initial response to treatment was good, but ecosystem. Occurring mainly in children, it is splenectomy and change of immunosup- an emergent public health threat in many pressive and antimycobacterial therapy were humid rural tropical areas. Human contami- necessary for long-term control of the in- nation probably follows a direct pecutaneous fection. Problems in the diagnosis and treat- route from humid environment, but some in- ment of this uncommon pathogen are dis- sects may play a role in transmission. The cussed.—Authors’Abstract clinical features develop in three phases: pre- ulcer, ulcer with unstick margins, healing leading to functional sequelae. Treatment re- Ferrara, E., Lemire, J., Grimm, P. C., lies on antibiotics in order to sterilize the in- Reznik, V. M., Mendoza, S. A., Leake, fectious focus, together with the surgical re- J. A., and Benador, N. M. Mycobacte- pair of lost skin and joint deformities, as well rial peritonitis in pediatric peritoneal dial- as early physiotherapy. Despite uncertainties ysis patients. Pediatr. Nephrol. 19(1) of in vivo efficacy of antibiotics, it seems (2004) 114–117. 72, 2 Current Literature, Other Mycobacterial Diseases 247

Peritonitis is the most common complica- tuberculosis and Mycobacterium avium tion and the leading cause of death in pedi- isolates. Boll. Chim. Farm. 142(6) (2003) atric peritoneal dialysis (PD) patients. Ac- 248–250. cording to the most recent data available from the North American Pediatric Renal The in vitro antimycobacterial activity Transplant Cooperative Study (NAPRTCS), of two ciprofloxacin analogues (2a and 2b) approximately 25% of pediatric PD patients containing 2-phenyl-2-axoethyl and 2-(4- who die succumb to infection. There are no fluorophenyl)-2-axoethyl groups attached reported cases of Mycobacterium tubercu- to N-4 position of piperazin ring, was eval- losis (MTB) or Mycobacterium avium- uated against M. tuberculosis strains re- intracellulare peritonitis in the NAPRTCS sistant to Isoniazid (MIC 2a and 2b = 3.13 registry. With an increasing incidence of micrograms/ml), Ethambutol (MIC 2a and MTB worldwide and the impairment of cel- 2b = 1.56 micrograms/ml), Rifampin (MIC lular immunity in chronic renal failure pa- 2a and 2b = 1.56 micrograms/ml), Kana- tients, it is not surprising that mycobacte- mycin (MIC 2a and 2b = 1.56 micro- rium peritonitis can occur in PD patients. grams/ml) and ciprofloxacin (MIC 2a and We report two pediatric PD patients with 2b>25 micrograms/ml). Furthermore, the mycobacterial peritoneal infection diag- minimum bactericidal concentration (MBC) nosed over an 11-year period at our institu- of 2a and 2b was determined against M. tu- tion. One patient presented with a malfunc- berculosis H37Rv (MBC2a = 6.25 and 2b tioning Tenckhoff catheter and again 3 years = 25 micrograms/ml) and strains resistant later with hyponatremia and ascites. The to isoniazid (MBCs >25 micrograms/ml) other presented with recurrent culture- and rifampin (MBC2a = 3.13 and 2b = negative peritonitis. These cases illustrate 6.25 micrograms/ml). Also, in this study the importance of more extensive evaluation the activity of 2a and 2b was determined of PD complications, to include evaluation against a strain of M. avium (%Inhibition 2a for mycobacterium with special media or = 89 and 2b = 95%). Expanded primary peritoneal biopsy, in the above clinical set- screening was conducted for 2b (having tings if the routine work-up is unreveal- MIC <6.25 micrograms/ml) against five ing.—Authors’Abstract clinical isolates of M. avium using the MABA and BACTEC 460 systems (MIC = 2–4 micrograms/ml). The significant anti- Fogla, R., Rao, S. K., and Padmanabhan, Mycobacterium avium activity of 2b sug- P. Interface keratitis due to Mycobacte- gested further M. avium assays and so, 2b rium fortuitum following laser in situ was then retested at lower concentrations keratomileusis. Indian J. Ophthalmol. against 30 strains of M. avium including 51(3) (2003) 263–265 five strains resistant to clarithromycin (MIC = 2–16 micrograms/ml).—Authors’Ab- A case of unilateral interface keratitis due stract to Mycobacterium fortuitum following simultaneous bilateral LASIK procedure for low myopia is reported. Excimer photother- Fox, L. P., Geyer, A. S., Husain, S., Della- apeutic keratectomy was performed to the Latta, P., and Grossman, M. E. Myco- stromal bed to reduce the infective load. In- bacterium abscessus cellulitis and multi- tensive topical therapy with topical focal abscesses of the breasts in a amikacin and ciprofloxacin resulted in reso- transsexual from illicit intramammary in- lution of the keratitis.—Authors’Abstract jections of silicone. J. Am. Acad. Derma- tol. 50(3) (2004) 450–454.

Foroumadi, A., and Soltani, F. Antituber- We report the case of a 29-year-old trans- culosis agents. IX. In vitro antimycobac- sexual who developed Mycobacterium ab- terial activity of N-(2-phenyl-2-oxoethyl) scessus infection after receiving intramam- and N-[2-(4-fluorophenyl)-2- oxoethyl]- mary liquid silicone injections in the ciprofloxacin derivatives against some nonphysician office setting. Our patient rep- drug-resistant strains of Mycobacterium resents 1 of 14 confirmed and 11 suspected 248 International Journal of Leprosy 2004 cases in New York City of M. abscessus in- isolates represented a novel rapidly growing fection after illicit cosmetic procedures. As Mycobacterium species. The name “Myco- injectable cosmetic procedures are becom- bacterium hackensackense” is proposed for ing increasingly popular, dermatologists this unique strain, 147-0552(T), which was should be aware of both the common and deposited in the American Type Culture unusual complications. Furthermore, all Collection as ATCC BAA-823(T).—Au- physicians should be alerted to the current thors’Abstract cluster of M. abscessus infections after injections for cosmetic purposes by nonmed- ical practitioners in New York City.—Au- Jenkin, G. A., Stinear, T. P., Johnson, P. thors’Abstracts D., and Davies, J. K. Subtractive hybridization reveals a type I polyketide synthase locus specific to Mycobacterium Hong, H., Gates, P. J., Staunton, J., Stin- ulcerans. J. Bacteriol. 185(23) (2003) ear, T., Cole, S. T., Leadlay, P. F., and 6870–6882. Spencer, J. B. Identification using LC- MSn of co-metabolites in the biosynthe- See Current Literature, Microbiology, p. sis of the polyketide toxin mycolactone 222. by a clinical isolate of Mycobacterium ulcerans. Chem. Commun. (Camb). 22 (2003) 2822–2823. Koranyi, K. I., and Ranalli, M. A. Myco- bacterium aurum bacteremia in an im- LC-MSn analysis of mycolactone toxin munocompromised child. Pediatr. Infect. from extracts of Mycobacterium ulcerans Dis. J. 22(12) (2003) 1108–1109. has shown that minor co-metabolites, including two previously unreported, differ Mycobacterium aurum was cultured from structurally from mycolactone only in a the Broviac catheter of a 5-year-old child small portion of the polyketide side- with metastatic Wilms tumor. Removal of chain.—Authors’Abstract the catheter resulted in prompt resolution of the fever and sterilization of the blood culture. This rapidly growing mycobacterium, Hong, T., Butler, W. R., Hollis, F., Floyd, previously believed to be a commensal, can M. M., Toney, S. R., Tang, Y. W., Steele, cause disease in the immunocompromised C., and Leggiadro, R. J. Characteriza- host.—Authors’Abstract tion of a novel rapidly growing Myco- bacterium species associated with sepsis. J. Clin. Microbiol. 41(12) (2003) 5650– Marie, I., Heron, F., Lecomte, F., Jarlier, 5653. V., Truffot-Pernot, C., Laquerriere, A., Huerre, M., Levesque, H., and Cour- A rapidly growing mycobacterium was tois, H. Multiple cerebral abscesses as a isolated five times from blood cultures from complication of Mycobacterium fortui- a 6-year-old female patient with relapsed tum infection. Eur. J. Intern. Med. 14(6) pre-B-cell acute lymphocytic leukemia. All (2003) 386–389. five isolates had identical nucleotide sequences for the first 500 bp of the 16S rRNA Mycobacterium fortuitum is a rapidly gene, indicative of a single species. High- growing, nontuberculous mycobacteria that performance liquid chromatography analy- has rarely been associated with central nerv- sis of mycolic acids indicated that the ous system impairment. We describe the species was similar to Mycobacterium case of a patient who developed multiple smegmatis. Sequence analysis of the 16S cerebral abscesses revealing Mycobacterium rRNA gene (1,455 bp) for one isolate fortuitum infection. Brain biopsy specimens demonstrated that the species was closely showed suppurative, noncaseating, granulo- related to Mycobacterium diernhoferi. matous inflammation consisting of epithe- Based on the phenotypic features and phy- lioid histiocytes and multinucleated giant logenetic analysis, it was concluded that the cells. All clinical signs and CT scan cerebral 72, 2 Current Literature, Other Mycobacterial Diseases 249

lesions disappeared after institution of ap- first, but some elements develop into ab- propriate antimycobacterial therapy.—Au- scesses or ulcers that can become very thors’Abstract painful. The incidence of M. haemophilum is unknown, but cases of infection have been reported in Australia, Canada, the United Marsollier, L., Prevot, G., Honore, N., States, France, Israel, the United Kingdom Legras, P., Manceau, A. L., Payan, C., and Taiwan; to date no cases have been re- Kouakou, H., and Carbonnelle, B. Sus- ported in Italy, thus the case reported here is ceptibility of Mycobacterium ulcerans to apparently the first one observed in our a combination of amikacin/rifampicin. Int country.—Authors’Abstract J. Antimicrob. Agents. 22(6) (2003) 562– 566. Menard, A., Couppié, P., Sainte-Marie, The effectiveness of rifampicin (RIF), D., Pradinaud, R. Diagnostic par PCR amikacin (AMK) and their combination de l’infection due à Mycobacerium ul- were estimated in the treatment of mice ex- cerans: à propos de trois cas observés en perimentally infected by Mycobacterium ul- Guyane Française. Bull. Soc. Pathol. cerans and the risk of relapse after the treat- Exot. 96(5) 403–405. ment was evaluated. After 7 weeks of treatment with RIF or with the combination Mycobacterium ulcerans infection is the of AMK/RIF and 8 weeks with AMK alone, third most important mycobacterial infec- no viable bacilli were found in the infected tion in the world. It has been described in tissues and these remained uninfected dur- many different countries including French ing the following 6 months. Among the mice Guiana. The diagnosis of M. ulcerans infec- treated with AMK alone, three mice re- tion by culture is often difficult because cul- lapsed, but the minimal inhibitory concen- ture is hard to perform in endemic areas and tration of AMK for these isolates remained their sensitivity is not reliable. As a result unchanged. With RIF alone, two mice re- the diagnosis of this infection is often de- lapsed and the minimal inhibitory concen- layed. However, molecular methods are now tration of these isolated strains was higher. available to diagnose rapidly infections by However, with all the mice treated with both M. ulcerans and distinguish it from other RIF and AMK, no relapse was observed.— mycobacteria. We report three cases of skin Authors’Abstract infection due to M. ulcerans observed in French Guiana. Diagnosis was initially made by polymerase chain reaction and was Martinelli, C., Farese, A., Carocci, A., confirmed later by culture (in two patients) Giorgini, S., Tortoli, E., and Leoncini, F. and inoculation to mice (in one patient). A First case of Mycobacterium haemophilum faster diagnosis of M. ulcerans infection infection in an AIDS patient in Italy. J. Eur. should lead to a better prognosis of this in- Acad. Dermatol. Venereol. 18(1) (2004) fection.—Bulletin de la Société de Patholo- 83–85. gie Exotique

Mycobacterium haemophilum, a strongly acid- and alcohol-fast bacillus belonging to Nolt, D., Michaels, M. G., and Wald, E. R. the group of non-tuberculous mycobacteria Intrathoracic disease from nontubercu- was first described in 1978 as the cause of lous mycobacteria in children: two cases cutaneous ulcerating lesions in a woman and a review of the literature. Pediatrics with Hodgkin’s disease. Infection due to M. 112(5) (2003) e434. haemophilum is rare but increasing in prevalence in immnunosuppressed subjects, par- Pulmonary disease caused by nontuber- ticularly in patients with acquired immun- culous mycobacteria (NTM) is a relatively odeficiency syndrome (AIDS) patients. The rare occurrence in immunocompetent chil- skin is the most common site of infection dren. Two cases of endobronchial NTM in- with erythematous or violaceous papules fection in immunocompetent children are and/or nodules that are usually painless at described. In addition, 41 other children 250 International Journal of Leprosy 2004 with NTM pulmonary disease reported in against Mycobacterium ulcerans os- the English literature between 1930 and teomyelitis in Buruli ulcer disease. Infect. 2003 are reviewed. Clinical manifestations Immun. 72(1) (2004) 62–65. are either purely respiratory or respiratory with more widespread systemic symptoms. See Current Literature, Experimental In- Compared with children with only respira- fections, p. 238. tory complaints, children with constitutional symptoms from NTM pulmonary disease 1) had symptoms for a shorter period before Pumberger, W., Hallwirth, U., Pawlowsky, presentation (10 vs 28 days), 2) had more ra- J., and Pomberger, G. Cervicofacial diographic evidence of pulmonary disease, lymphadenitis due to atypical mycobacte- and 3) were treated longer with antimy- ria: a surgical disease. Pediatr. Dermatol. cobacterial agents (11.5 months vs 6 21(1) (2004) 24–29. months). The most common causative organism was Mycobacterium avium com- Despite the increasing prevalence of cervi- plex. Pediatricians should be increasingly cofacial lymphadenitis due to atypical myco- aware of NTM in the differential diagnosis bacteria (AMB) in children, the true nature of of persistent pulmonary disease in previ- AMB infection in clinical practice is poorly ously healthy children.—Authors’Abstract understood. The purpose of our study was to define the most common signs and symptoms, and to establish a workable scheme of Olivier, K. N.; NTM in CF Study Group. diagnosis and treatment. Patients fulfilling the The natural history of nontuberculous criteria of AMB infection (i.e., clinical signs, mycobacteria in patients with cystic. positive cultures or polymerase chain reac- Pediatr. Respir. Rev. 5 Suppl (2004) tion, histologic features) were included in the A:S213–A:S216. study. All children underwent a standard surgical procedure, depending on pretreatment With the increasing lifespan of persons and the course of the disease. Sixteen infants with cystic fibrosis (CF), the emergence of presented with characteristic unilateral lym- a variety of previously seldom seen phadenopathy predominantly involving the pathogens, including the nontuberculous submandibular area (13/16). Eight children mycobacteria (NTM), has been seen. Deter- had been initially treated at various institu- mining the impact of these indolent organ- tions by fine-needle puncture or incision, and isms on the natural history of cystic fibrosis 7 of the 16 patients had received antitubercu- lung disease has been difficult. We initiated lous multidrug treatment for a varying length a two-stage study in 1993 to assess the of time. Complete excision of the affected prevalence and clinical impact of these or- lymph nodes was the definitive treatment in ganisms among persons with CF in US CF all patients. Three children had transient mar- Centers. These organisms were frequently ginal mandibular nerve paralysis that re- recovered from older patients with relatively solved within a few months in all cases. mild disease. While over the short, 15- Recognition of the characteristic features of month course of follow-up no significant AMB adenitis may permit early diagnosis differences in the rate of decline of lung and appropriate surgical treatment.—Au- function attributable to NTM were seen, thors’Abstract concerning changes and progression of high-resolution computed tomography findings were seen in patients from whom these Redaelli de Zinis, L. O., Tironi, A., Nassif, organisms were repeatedly recovered.—Au- N., and Ghizzardi, D. Temporal bone in- thors’Abstract fection caused by atypical mycobacterium: case report and review of the literature. Otol. Neurotol. 24(6) (2003) 843–849. Portaels, F., Aguiar, J., Debacker, M., Guedenon, A., Steunou, C., Zinsou, C., OBJECTIVE: To discuss the clinical as- and Meyers, W. M. Mycobacterium pects and management of nontuberculous bovis BCG vaccination as prophylaxis mycobacteriosis of the temporal bone. 72, 2 Current Literature, Other Mycobacterial Diseases 251

STUDY DESIGN: Case report and review A 65-year-old woman, treated with pred- of the literature. SETTING: University hos- nisolone (5 mg daily) for rheumatoid arthritis, pital, tertiary referral center. PATIENT, visited our hospital because of right chest INTERVENTION, AND RESULTS: The pain. Chest CT showed small nodular shad- authors describe an uncommon case of non- ows in the right lung accompanied with right tuberculous mycobacteriosis of the tempo- pleural effusion. A pulmonary Mycobacte- ral bone in an immunosuppressed 62-year- rium gordonae infection was diagnosed, since old woman with facial nerve paralysis M. gordonae was identiﬁed twice from her caused by disease complication. The case sputum. She was treated with rifampicin, was cured with radical tympanomastoidec- ethambutol and streptomycin for two months, tomy and prolonged multiple antibiotic ther- and then streptomycin was replaced with clar- apy. CONCLUSIONS: Nontuberculous ithromycin. Three months after the initial mycobacteriosis should be suspected in im- treatment, M. gordonae was eradicated from munosuppressed patients with intractable her sputum. Pleural puncture revealed bloody, middle ear granulations. Cultural and histo- exudative, lymphocytotic pleural effusion, but logic examinations are the mainstay for di- no malignant cells were identiﬁed. Although agnosis. Long-standing multiantibiotic ther- pathological diagnosis by thoracoscopic pleu- apy together with aggressive surgery should ral biopsy could not be performed, it is likely be considered as appropriate manage- that the pleural effusion was associated with ment.—Authors’Abstract the pulmonary M. gordonae infection in the present case.—Authors’Abstract

Roupe, G. [Buruli ulcer—Africa’s latest mycobacterial scourge]. Lakartidningen. Saiman, L. The mycobacteriology of non- 100(45) (2003) 3596–3597. [Article in tuberculous mycobacteria. Paediatr. Respir. Swedish] Rev. 5 Suppl. (2004) A:S221– A:S223.

Buruliulcer is an extensive ulceration usu- The genus Mycobacterium consists of ally on the extremities. The ulcer can spread >50 species that have been associated with to subcutaneous fat, muscle and even bone human disease. Mycobacterium are cate- causing osteomyelitis and death. It is the the gorised into M. tuberculosis and NTM that third most common mycobacterial disease are also subdivided into rapid growers and in humans after tuberculosis and leprosy. non-rapid growers. Five major clinical syn- The bacterium grows in still standing water dromes have been described that are attrib- and infects children through small ulcera- utable to mycobacterium. These include: tions in their skin. Mycobacterium ulcerans pulmonary disease; lymphadenitis; skin, soft may also be transmitted by the bite of tissue, and skeletal infections; catheter- aquatic bugs (Naucordiae), which harbor the related blood-stream infections in immuno- bacterium in their salivary glands. The dis- compromised hosts; and disseminated disease affects poor people in rural, tropical ease in persons with AIDS. There is very areas where deforestation has led to flood- limited documentation of person-to-person ing rivers, stagnant bodies of water and transmission of NTM. Nosocomial infec- marsh. Benin, Cote d’Ivoire and Ghana in tions and outbreaks caused by inadequate West Africa are seriously hit. Skin trans- disinfection/sterilization of medical devices plantation is the treatment of choice. Treat- or environmental contamination of medica- ment with antibiotics has been disappoint- tions or medical devices are well described. ing.—Author’s Abstract Staining for AFB, culture, histopathologic, or genetic amplification technologies are used to detect and identify mycobacterium. Saeki, S., Matsuse, H., Shimoda, T., Soe- Pulsed-field gel electrophoresis is the jima, Y., Ohno, H., and Kohno, S. [A method of choice to determine strain relat- case of pulmonary Mycobacterium gor- edness. At present, susceptibility testing for donae infection with pleural effusion]. non-tuberculous mycobacteria is not fully Nihon Kokyuki Gakkai Zasshi. 42(1) standardized and has not been correlated (2004) 103–107. [Article in Japanese]. with clinical outcomes.—Authors’Abstract 252 International Journal of Leprosy 2004

Sechi, L. A., Mura, M., Tanda, E., Lissia, pulsed-field gel electrophoresis: each of the A., Fadda, G., and Zanetti, S. Mycobac- 14 patients harbored a unique strain, ruling terium avium sub. paratuberculosis in tis- out a common environmental reservoir or sue samples of Crohn’s disease patients. person-to-person transmission. Water sam- New Microbiol. 27(1) (2004) 75–77. ples collected in the cystic fibrosis center were negative for M. abscessus. This major Crohn’s disease is a non-specific chronic mycobacterial pathogen in children and transmural inflammatory disease. The dis- teenagers with cystic fibrosis does not ap- ease was associated with a frameshift mu- pear to be acquired nosocomially.—Emerg- tation in the NOD2 gene. Nevertheless, ing Infectious Diseases other researchers associated the presence of M. paratuberculosis within the intestinal tissues of patients with the disease. An adapted Shiratsuch, H., and Basson, M. D. Cas- “in situ hybridization” technique was used pase activation may be associated with to detect IS900 M. paratuberculosis DNA in Mycobacterium avium pathogenicity. paraffin embedded tissue from Crohns tissue Am. J. Surg. 186(5) (2003) 547–551. disease samples. We were able to identify M. paratuberculosis DNA in around 69% of BACKGROUND: Mycobacterium avium the paraffine embedded intestinal samples of causes disseminated infection in immuno- Crohn’s disease patients analysed. The pres- compromised patients and triggers a ence of M. paratuberculosis DNA in the in- process resembling Crohn’s disease in testinal samples analysed does not neces- goats. Colony morphotypes predict patho- sarily mean that M. paratuberculosis is genicity. Smooth-transparent (SmT) mor- responsible for Crohn’s disease. Our results photypes are more virulent and induce less support the hypothesis that infection may be interleukin (IL)-1beta and IL-18 production caused by cell wall defective M. paratuber- than avirulent smooth-domed (SmD) mor- culosis since no bacteria were detected by photypes. Caspases are essential for IL- Ziehl Neelsen stain.—Authors’Abstract 1beta and IL-18 production. METHODS: Caspase activation was examined in human monocytes after M. avium infection. RE- Sermet-Gaudelus, I., Le Bourgeouis, M., SULTS: Fresh monocytes constitutively ex- Pierre-Audigier, C., Offredo, C., Guille- pressed caspase-1 mRNA and pro-caspase- mot, D., Halley, S., Akoua-Koffi, C., 1. The M. avium infection increased Vincent, V., Sivadon-Tardy, V., Fer- monocyte caspase-1 mRNA expression. roni, A., Berche, P., Scheinmann, P., Furthermore, SmD-infected monocytes ex- Lenoir, G., and Gaillard, J.-L. Myco- pressed 2.3-fold higher levels (p <0.05, N = bacterium abcessus with Cystic Fibrosis. 3) of activated caspases than SmT-infected Emerging Infect. Dis. 9(12) (2003) 1587– monocytes. Caspase-1 inhibition signifi- 1591. cantly reduced IL-1beta production by SmT- and SmD-infected monocytes (p We prospectively studied 298 patients <0.05, N = 4). Caspase-3 inhibition inhib- with cystic fibrosis (mean age 11.3 years; ited IL-1beta production 43.5% ± 8.0% (p range 2 months to 32 years; sex ratio, 0.47) <0.02, N = 4) by SmD-infected but not for nontuberculous mycobacteria in respira- SmT-infected monocytes. CONCLU- tory samples from January 1, 1996, to De- SIONS: Decreased mature IL-1beta release cember 31, 1999. Mycobacterium abscessus by SmT-infected monocytes may reflect se- was by far the most prevalent nontubercu- lective induction of caspase-1 activity but lous mycobacterium: 15 patients (6 male, 9 not caspase-3. Differential caspase expres- female; mean age 11.9 years; range 2.5–22 sion in monocytes after infection may con- years) had at least one positive sample for tribute to M. avium pathogenicity in hu- this microorganism (versus 6 patients posi- mans.—Authors’Abstract tive for M. avium complex), including 10 with >3 positive samples (versus 3 patients for M. avium complex). The M. abscessus Sugihara, E., Hirota, N., Niizeki, T., isolates from 14 patients were typed by Tanaka, R., Nagafuchi, M., Koyanagi, 72, 2 Current Literature, Other Mycobacterial Diseases 253

T., Ono, N., Rikimaru, T., and Aizawa, were retrospectively identified from hospital H. Usefulness of bronchial lavage for the records. Between 1994 and July 2000, 136 diagnosis of pulmonary disease caused patients had been admitted for Buruli ulcer by Mycobacterium avium-intracellulare in both hospitals, and lived in areas covered complex (MAC) infection. J. Infect. in the research period. 78 (57%) Patients Chemother. 9(4) (2003) 328–332. were included in the study. Treatment and status of the patient were analyzed. Results: To evaluate the usefulness of bronchial 27 (35%) Patients were not healed. Of the 33 lavage for the diagnosis of pulmonary disease patients treated in hospital A, six (18%) due to Mycobacterium avium-intracellulare were not healed at follow-up, whereas of the complex (MAC) infection, we examined the 45 patients treated in hospital B, 21 (47%) clinical records and bacteriologic findings of were not healed. The length of stay in hos- patients admitted to our hospital between pital A was significantly longer (p = 0.002), 1999 and 2002 who fulfilled the 1997 and more operations on average were done American Thoracic Society (ATS) criteria per patient (p = 0.002). In a univariate analy- for MAC pulmonary infection. Broncho- sis, treatment in hospital A; the use of ri- scopic examinations were performed in fampicin (p = 0.013); and BCG vaccination those patients with MAC pulmonary disease status (p = 0.04) were all significantly asso- who showed negative sputum smears for ciated with ulcer healing. Using a logistic re- mycobacteria on 3 consecutive days (N = gression model for multivariate analysis, 14) or who could not expectorate sputum (n only treatment as given in hospital A, with = 2). The bronchial lavage sample was standard prctice of wide surgical excision, smear-positive for acid-fast bacilli in 8 of appeared to predict ulcer healing independ- the 16 patients (50.0%), polymerase chain ently (p = 0.02). Conclusions: This study reaction (PCR)-positive for MAC in 10 of shows large differences in treatment out- 15 (66.7%), and culture-positive for MAC come between the two hospitals; the results in 15 of 16 (93.7%). The brushing sample support the hypothesis that extent of surgical was positive for MAC in 5 of 14 patients treatment influences the chance of healing of (35.7%), and transbronchial lung biopsy Buruli ulcer.—Tropical Disease Bulletin (TBLB)-positive for MAC in 2 of 5 (40.0%). MAC was isolated by culture of bronchial lavage samples in a higher per- Ticca, F., Comparcola, D., Graziani, M. centage of patients than that in whom MAC C., Lancella, L., Marsella, P., Nicolosi, was isolated by sputum culture, and we L., Pierro, V., Rivosecchi, M. R., Ticca, could make an early diagnosis of MAC pul- C., and Tieri, L. [Otomastoiditis caused monary disease based on the smear and PCR by Mycobacterium avium: a case report]. results for bronchial lavage samples. Infez. Med. 5(2) (1997) 114–117. [Arti- Bronchial lavage is useful to screen sputum cle in Italian] smear-negative patients suspected of having MAC pulmonary disease.—Authors’ Ab- Non tuberculous Mycobacterial (NTM) stract infections mainly affect immunocompromised patients, appearing as disseminated or pulmonary disease. In immunocompetent Teelken, M. A., Stienstra, Y., Ellen, D. E., children the most common form of infection Quarshie, E., Klutse, E., van der with NTM is cervical adenitis. Ear infection Graaf, W. T. A., van der Werf, T. S. Bu- seems to be a rare disease. We present a case ruuli ulcer: differences in treatment out- of otomastoiditis caused by Mycobacterium come between two centers in Ghana. avium in a 15 months old child, immunolog- Acta Tropica 88(1) (2003) 51–56. ically normal. Patient was referred for persistent right otitis unresponsive to routine Objectives: Assess treatment effects by medical therapy. TC scan of the ear and tem- following up patients treated for Buruli ulcer poral bones revealed: soft tissue in external in two hospitals with different treatment as- auditory canal, Eustachian canal, and middle pects, including widely differing surgical ear overlying ossicles with erosion of tegmen practices. Patients/methods: Treated patients tympani. Tuberculin skin test was positive 254 International Journal of Leprosy 2004 with 5 units PPD and culture yielded M. C., Leonard, W., Wagner, J., and avium. The patient undergo timpanomas- Vugia, D. J. The clinical management toidectomy and medical therapy with antitu- and outcome of nail salon-acquired My- berculous drugs and steroids, subsequently cobacterium fortuitum skin infection. he was given Clarithromycin and Rifabutin. Clin. Infect. Dis. 38(1) (2004) 38–44. M. avium is an ubiquitous low grade pathogen found in soil, water, dust and food. Nontuberculous mycobacterial infections There is no evidence of direct transmission. are becoming more common. Recently, My- Only a few cases of otomastoiditis due to M. cobacterium fortuitum and other rapidly avium have previously been reported. The growing mycobacteria have been found to case presented underlines the importance of cause severe skin and soft-tissue infections microbiological investigations. When a NTM in association with nail salon whirlpool foot- infection is suspected surgeons and infec- baths. We recently investigated a large out- tious diseases specialists should cooperate to break of M. fortuitum furunculosis among find an optimal treatment regimen of this un- women who received pedicures at a single usual disease.—Authors’Abstract nail salon. To better define the clinical course of such infections, we collected clinical details from physicians who were treat- Toy, B. R. Foreign-body reaction with My- ing outbreak patients. We constructed mul- cobacterium abscessus superinfection. tivariable linear models to evaluate the Dermatol. Online J. 9(4) (2003) 29. effect of antibiotic treatment on disease duration. Sixty-one patients were included in Mycobacterium abscessus is a rare cause the investigation. The mean disease duration of skin and soft tissue infections that often was 170 days (range, 41–336 days). Forty- results from inoculation with contaminated eight persons received antibiotic therapy for foreign material. A 41-year-old woman is a median period of 4 months (range, 1–6 described regarding an outbreak of M. ab- months), and 13 persons were untreated. scessus following soft tissue augmentation. Isolates were most susceptible to cipro- Clinical features and treatment options are floxacin and minocycline. Early administra- reviewed.—Author’s Abstract tion of therapy was associated with shorter duration of disease only in persons with multiple boils (p <.01). One untreated, Winthrop, K. L., Albridge, K., South, D., healthy patient had lymphatic disease dis- Albrecht, P., Abrams, M., Samuel, M. semination.—Authors’Abstract

Molecular and Genetic Studies

Adekambi, T., Colson, P., and Drancourt, of Mycobacterium tuberculosis. Scand. J. M. rpoB-based identification of nonpig- Immunol. 59(1) 2004 16–24. mented and late-pigmenting rapidly growing mycobacteria. J. Clin. Micro- The secreted 24 kDa lipoprotein (LppX) biol. 41(2) 2003 5699–5708. is an antigen that is specific for Mycobacte- rium tuberculosis complex and M. leprae. See Current Literature, Microbiology, The present study was carried out to identify p. 219. the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping Al-Attiyah, R., and Mustafa, A. S. synthetic peptides (25 mers overlapping by Computer-assisted prediction of HLA- 10 residues) covering the sequence of the DR binding and experimental analysis for complete protein. The analysis of Rv2945c human promiscuous Th1-cell peptides in sequence for binding to 51 alleles of nine the 24 kDa secreted lipoprotein (LppX) serologically defined HLA-DR molecules, 72, 2 Current Literature, Molecular and Genetic Studies 255

by using a virtual matrix-based prediction morphism (RFLP) typing. Results of poly- program (propred), showed that eight of the merase chain reaction-hybridization formats 15 peptides of Rv2945c were predicted to using the non-specific region of IS6110 for bind promiscuously to ≥10 alleles from the molecular detection of mycobacteria in more than or equal to three serologically de- clinical material should be interpreted with fined HLA-DR molecules. The Th1-cell re- caution.—Authors’Abstract activity of all the peptides was assessed in antigen-induced proliferation and interferon- gamma (IFN-gamma)-secretion assays with Bisen, P. S., Garg, S. K., Tiwari, R. P., peripheral blood mononuclear cells (PBMCs) Tagore, P. R., Chandra, R., Karnik, R., from 37 bacille Calmette-Guerin (BCG)- Thaker, N., Desai, N., Ghosh, P. K., vaccinated healthy subjects. The results Fraziano, M., and Colizzi, V. Analysis showed that 17 of the 37 donors, which rep- of the shotgun expression library of the resented an HLA-DR-heterogeneous group, Mycobacterium tuberculosis genome for responded to one or more peptides of immunodominant polypeptides: potential Rv2945c in the Th1-cell assays. Although use in serodiagnosis. Clin. Diagn. Lab. each peptide stimulated PBMCs from one or Immunol. 10(6) (2003) 1051–1058. more donors in the above assays, the best positive responses (12/17 (71%) responders) A recombinant DNA strategy was applied were observed with the peptide p14 (aa to analyze and screen the shotgun expres- 196–220). This suggested a highly promis- sion library from a clinically confirmed local cuous presentation of p14 to Th1 cells. In virulent isolate of Mycobacterium tubercu- addition, the sequence of p14 is completely losis with sera from tuberculosis patients, identical among the LppX of M. tuberculo- which led to expression and purification of sis, M. bovis and M. leprae, which further highly immunoreactive and specific myco- supports the usefulness of Rv2945c and p14 bacterial antigens expressed during the in the subunit vaccine design against both course of active disease which could be of tuberculosis and leprosy.—Authors’ Ab- diagnostic significance. An enzyme-linked stract immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoex- pression library in the lambdagt11 vector. Beyene, D., Aseffa, A., Harboe, M., Ki- DNA from a virulent M. tuberculosis patient dane, D., Macdonald, M., Klatser, P. isolate (TBW-33) confirmed with the R., Bjune, G. A., and Smith, W. C. BACTEC 460 system was sheared and ex- Nasal carriage of Mycobacterium leprae pressed to generate shotgun polypeptides. DNA in healthy individuals in Lega Robi beta-Galactosidase fusion proteins capable village, Ethiopia. Epidemiol. Infect. of demarcating active tuberculosis infec- 131(2) (2003) 841–848. tions from Mycobacterium bovis BCG- vaccinated healthy subjects or people har- See Current Literature, Clinical Sciences, boring environmental mycobacteria were p. 192. selected by comparative immunoreactivity studies. Promising mycobacterial DNA cas- settes were subcloned and expressed into the Bhanu, N. V., van Soolingen, D., van glutathione S-transferase (GST) fusion vec- Embden, J. D., and Seth, P. Two Myco- tor pGEX-5X-1 with a strong tac promoter bacterium fortuitum strains isolated and were expressed in Escherichia coli from pulmonary tuberculosis patients in BL21. These fusion proteins were severed at Delhi harbor IS6110 homologue. Diagn. a built-in factor Xa recognition site to sepa- Microbiol. Infect. Dis. 48(2) (2004) rate the GST tags and were utilized in an in- 107–110. direct enzyme-linked immunoassay for serodiagnosis of patients with active tuber- We report 2 isolates of Mycobacterium culosis. The system offered a clear demar- fortuitum from patients with pulmonary tu- cation between BCG-vaccinated healthy berculosis lesions hybridizing to IS6110 subjects and patients with active tuberculo- probe in restriction fragment length poly- sis and proved to be effective in detecting 256 International Journal of Leprosy 2004 pulmonary as well as extrapulmonary tu- lates. Antimicrob. Agents Chemother. berculosis, with an overall sensitivity of 48(2) (2004) 596–601. 84.33% and an overall specificity of 93.62%.—Authors’Abstract A new strategy known as multiplex PCR amplimer conformation was developed for detection of mutation in the gyrA gene of Chattopadhyay, C., Sau, S., and Mandal, 138 clinical isolates of Mycobacterium tu- N. C. Cloning and characterization of the berculosis. The method generated a single- promoters of temperate mycobacterio- stranded and heteroduplex DNA banding phage L1. J. Biochem. Mol. Biol. 36(6) pattern of multiplex PCR amplimers of the (2003) 586–592. region of interest that was extremely sensitive to specific mutations, thus enabling Four putative promoters of the temperate much more sensitive and reliable mutation mycobacteriophage L1 were cloned by de- analysis compared to the standard single- tecting the beta-galactosidase reporter ex- stranded conformation polymorphism tech- pression in E. coli transformants that carried nique. The genetic profiles of the gyrA gene L1 specific operon-fusion library. All of the of the 138 isolates as detected by MPAC four L1 promoters were also found to ex- were confirmed by nucleotide sequencing press differentially in the homologous envi- and were found to correlate strongly with ronment of mycobacteria. Of the four pro- the in vitro susceptibilities of the mutant moters, two were suggested to be the strains to six fluoroquinolones (ofloxacin, putative early promoters of L1 since they levofloxacin, sparfloxacin, moxifloxacin, express within 0 to 10 min of the initiation gatifloxacin, and sitafloxacin). All 32 iso- of the lytic growth of L1. One of the puta- lates that contained gyrA mutations exhib- tive early promoters showed a relatively bet- ited cross-resistance to the six fluoro- ter and almost identical activity in both E. quinolones (ofloxacin MIC for 90% of coli and M. smegmatis. By a sequence strains >16 mg/liter), although moxi- analysis, we suggest that the L1 insert that floxacin, gatifloxacin, and sitafloxacin (MIC contained the stronger early promoter pos- for 90% of strains ≤4 mg/liter) were appar- sibly carries two convergent E. coli ently more active than ofloxacin, lev- sigma70-like L1 promoters, which are sep- ofloxacin, and sparfloxacin (MIC for 90% of arated from each other by about 300 nu- strains >/==″ BORDER=″0″ >16 mg/liter). cleotides. One of them is the early promoter All gyrA mutations were clustered in codons of L1 as it showed a 100% similarity with 90, 91, and 94, and aspartic acid 94 was the early Pleft promoter of the homoimmune most frequently mutated. Twenty-three iso- phage L5. The second promoter, designated lates without gyrA mutations were also P4, was suggested for its appreciable level found to exhibit reduced susceptibility to of reporter activity in the absence of the -10 ofloxacin (MIC for 90% of strains = 4 element of the Pleft equivalent of L1. By an- mg/liter), but largely remained susceptible alyzing most of the best characterized my- to other drugs (MIC for 90% of strains ≤1 cobacteriophages-specific promoters, in- mg/liter). Another 83 isolates without muta- cluding the L1 promoter P4, we suggest that tions were fully susceptible to all six fluoro- both the -10 and -35 hexamers of the my- quinolones (ofloxacin MIC for 90% of cobacteriophage promoters are highly con- strains = 1 mg/liter). In conclusion, high- served and almost similar to the consensus level phenotypic resistance to fluoro- -10 and -35 hexamers of the E. coli sigma70 quinolones among M. tuberculosis clinical promoters.—Authors’Abstract isolates, which appears to be predominantly due to gyrA mutations, may be readily detected by genotyping techniques such as Cheng, A. F., Yew, W. W., Chan, E. W., multiplex PCR amplimer conformation.— Chin, M. L., Hui, M. M., and Chan, R. Authors’Abstract C. Multiplex PCR amplimer conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-resistant Jamieson, S. E., Miller, E. N., Black, G. F., Mycobacterium tuberculosis clinical iso- Peacock, C. S., Cordell, H. J., Howson, 72, 2 Current Literature, Molecular and Genetic Studies 257

J. M., Shaw, M. A., Burgner, D., Xu, veloped for predicting whether a given nu- W., Lins-Lainson, Z., Shaw, J. J., cleotide sequence is a mycobacterial pro- Ramos, F., Silveira, F., and Blackwell, moter sequence. Owing to the high predic- J. M. Evidence for a cluster of genes on tion capability (congruent with 97%) of the chromosome 17q11-q21 controlling sus- developed network model, it has been fur- ceptibility to tuberculosis and leprosy in ther used in conjunction with the caliper ran- Brazilians. Genes Immun. 5(1) (2004) domization (CR) approach for determining 46–57. the structurally/functionally important regions in the promoter sequences. The results The region of conserved synteny on mouse obtained thereby indicate that: (i) upstream chromosome 11/human 17q11-q21 is known region of -35 box, (ii) -35 region, (iii) spacer to carry a susceptibility gene(s) for intra- region and, (iv) -10 box, are important for macrophage pathogens. The region is rich in mycobacterial promoters. The CR approach candidates including NOS2A, CCL2/MCP-1, also suggests that the -38 to -29 region plays CCL3/MIP-1alpha, CCL4/MIP-1beta,CCL5/ a significant role in determining whether a RANTES, CCR7, STAT3 and STAT5A/5B. given sequence is a mycobacterial promoter. To examine the region in man, we studied In essence, the present study establishes 92 multicase tuberculosis (627 individuals) ANNs as a tool for predicting mycobacterial and 72 multicase leprosy (372 individuals) promoter sequences and determining struc- families from Brazil. Multipoint nonpara- turally/functionally important sub-regions metric analysis (ALLEGRO) using 16 mi- therein.—Authors’Abstract crosatellites shows two peaks of linkage for leprosy at D17S250 (Z(lr) score 2.34; p = 0.01) and D17S1795 (Z(lr) 2.67; p = 0.004) Lee, C. K., Gi, H. M., Cho, Y., Kim, Y. K., and a single peak for tuberculosis at Lee, K. N., Song, K. J., Song, J. W., D17S250 (Z(lr) 2.04; p = 0.02). Combined Park, K. S., Park, E. M., Lee, H., and analysis shows significant linkage (peak Bai, G. H. The genomic heterogeneity Z(lr) 3.38) at D17S250, equivalent to an al- among Mycobacterium terrae complex lele sharing LOD score 2.48 (p = 0.0004). displayed by sequencing of 16S rRNA To determine whether one or multiple genes and hsp 65 genes. Microbiol. Immunol. contribute, 49 informative single nucleotide 48(2) (2004) 83–90. polymorphisms were typed in candidate genes. Family-based allelic association test- The species identification within Myco- ing that was robust to family clustering bacterium terrae complex has been known demonstrated significant associations with to be very difficult. In this study, the ge- tuberculosis susceptibility at four loci sepa- nomic diversity of M. terrae complex with rated by intervals (NOS2A-8.4 Mb-CCL18- eighteen clinical isolates, which were ini- 32.3 kb-CCL4-6.04 Mb-STAT5B) up to tially identified as M. terrae complex by several Mb. Stepwise conditional logistic phenotypic method, was investigated, in- regression analysis using a case/pseudo- cluding that of three type strains (M. terrae, control data set showed that the four genes M. nonchromogenicum, and M. triviale). contributed separate main effects, consistent 16S rRNA and 65-kDa heat shock protein with a cluster of susceptibility genes across (hsp 65) gene sequences of mycobacteria 17q11.2.—Authors’Abstract were determined and aligned with eleven other references for the comparison using similarity search against the GenBank and Kalate, R. N., Tambe, S. S., and Kulkarni, Ribosomal Database Project II (RDP) data- B. D. Artificial neural networks for pre- bases. 16S rRNA and hsp 65 genes of M. diction of mycobacterial promoter se- terrae complex showed genomic hetero- quences. Comput. Biol. Chem. 27(6) geneity. Amongst the eighteen clinical iso- (2003) 555–564. lates, nine were identified as M. nonchromogenicum, eight as M. terrae, one as M. A multilayered feed-forward ANN ar- mucogenicum with the molecular character- chitecture trained using the error-back- istic of rapid growth. M. nonchromogenicum propagation (EBP) algorithm has been de- could be subdivided into three subgroups, 258 International Journal of Leprosy 2004 while M. terrae could be subdivided into nificant association was observed between two subgroups using a 5 bp criterion (>1% TB and INT4 polymorphisms. In multivari- difference). Seven isolates in two subgroups ate analysis, associations of TB and D543N of M. nonchromogenicum were Mycobacte- G/A and 3′UTR TGTG+/del genotypes re- rium sp. strain MCRO 6, which was closely mained, adjusted for exposure history and related to M. nonchromogenicum. The hsp bacille Camette-Guerin immunization. Ad- 65 gene could not differentiate one M. justed OR (95% CI) was 3.04 (1.12–8.27) nonchromogenicum from M. avium or one and 2.36 (1.20–4.64), respectively. Still, no M. terrae from M. intracellulare. The nu- significant association between INT4 poly- cleotide sequence analysis of 16S rRNA and morphisms and TB was found. CONCLU- hsp 65 genes was shown to be useful in SION: Polymorphisms of D543N and identifying the M. terrae complex, but hsp 3′UTR locus in NRAMP1 gene might affect 65 was less discriminating than 16S their susceptibility to TB in Chinese Han rRNA.—Authors’Abstract population.—Authors’Abstract

Liu, W., Zhang, C. Y., Tian, L., Li, C. Z., Majeed, A. A., Ahmed, N., Rao, K. R., Wu, X. M., Zhao, Q. M., Zhang, P. H., Ghousunnissa, S., Kauser, F., Bose, B., Yang, S. M., Yang, H., and Cao, W. C. [A Nagarajaram, H. A., Katoch, V. M., case-control study on natural-resistance- Cousins, D. V., Sechi, L. A., Gilman, R. associated macrophage protein 1 gene H., and Hasnain, S. E. AmpliBASE polymorphisms and susceptibility to pul- MTTM: a Mycobacterium tuberculosis monary tuberculosis]. Zhonghua Yu Fang diversity knowledgebase. Bioinformatics Yi Xue Za Zhi. 37(6) (2003) 408–411. [Ar- 20(6) (2004) 989–992. ticle in Chinese] SUMMARY: AmpliBASE MT trade mark OBJECTIVE: To investigate association is an online databank of high-resolution DNA between the natural-resistance-associated fingerprints representing fluorescent amplified macrophage protein 1 (NRAMP1) gene fragment length polymorphism (FAFLP) pro- polymorphisms and susceptibility to pul- files or amplitypes developed for the Myco- monary tuberculosis (TB) in Chinese Han bacterium tuberculosis complex strains from population. METHODS: Hospital-based 48 different countries. AmpliBASE MT trade case-control study design was adopted. mark is based on a relational database man- Polymerase chain reaction (PCR) and re- agement system that is hyperlinked to visual- striction fragment length polymorphism ize genotyping results in the form of DNA (RFLP) technique were used to type three fingerprint images for individual strains. A NRAMP1 polymorphisms (INT4, D543N flexible search system based on systematic and 3′UTR). Information on related factors comparisons of fragment sizes in base pairs al- of tuberculosis was collected using a pre- lows inter-laboratory comparison of FAFLP tested standard questionnaire. Univariate profiles. Besides this, the database also dis- and multivariate unconditional logistic plays previously published data on IS6110 analyses were conducted using SPSS for profiles, spoligotypes, MIRU-VNTRs and window software package. Totally, 110 large sequence polymorphisms along with the cases of TB were selected during April 2001 FAFLP records that will give the overall com- to June 2002, with an average age of (27.7 ± parisons. Being the first of its kind, Am- 12.7) years. Also, 180 cases of healthy con- pliBASE MT trade mark is expected to be a trol were selected, aged (27.3 ± 9.2) years in very helpful tool in strengthening the concept average. Locus of NRAMP1 polymorphism of ‘geographic genomics’ and will be very was analysed with univariate method. RE- helpful to molecular epidemiologists and SULTS: Univariate analysis demonstrated those interested in diagnostic development for that the D543N G/A and 3′UTR TGTG+/del tuberculosis.—Authors’Abstract genotype occurred more frequently in the cases than in the controls, with crude odds ratios (OR) (95% CI) of 2.22 (1.03–4.78) Marmiesse, M., Brodin, P., Buchrieser, C, and 1.93 (1.14–3.26), respectively. No sig- Gutierrez, C., Simoes, N., Vincent, V., 72, 2 Current Literature, Molecular and Genetic Studies 259

Glaser, P., Cole, S. T., and Brosch, R. identified and further characterized during Macro-array and bioinformatic analyses this study. Together with the distribution of reveal mycobacterial ‘core’ genes, varia- a particular 6 or 7 bp micro-deletion in the tion in the ESAT-6 gene family and new gene encoding the polyketide synthase phylogenetic markers for the Mycobacte- pks15/1, these results confirm and further rium tuberculosis complex. Microbiol- extend the revised phylogenetic model for ogy. 150(Pt 2) (2004) 483–496. the M. tuberculosis complex recently presented.—Authors’Abstract To better understand the biology and the virulence determinants of the two major mycobacterial human pathogens Mycobacte- Matsuoka, M., Zhang, L., Budiawan, T., rium tuberculosis and Mycobacterium lep- Saeki, K., and Izumi, S. Genotyping of rae, their genome sequences have been Mycobacterium leprae on the basis of the determined recently. In silico comparisons polymorphism of TTC repeats for analy- revealed that among the 1439 genes com- sis of leprosy transmission. J. Clin. Mi- mon to both M. tuberculosis and M. leprae, crobiol. 42(2) (2004) 741–745. 219 genes code for proteins that show no similarity with proteins from other organ- See Current Literature, Microbiology, isms. Therefore, the latter ‘core’ genes could Leprosy, p. 224. be specific for mycobacteria or even for the intracellular mycobacterial pathogens. To obtain more information as to whether these Miller, E. N., Jamieson, S. E., Joberty, C., genes really were mycobacteria-specific, Fakiola, M., Hudson, D., Peacock, C. they were included in a focused macro- S., Cordell, H. J., Shaw, M. A., Lins- array, which also contained genes from pre- Lainson, Z., Shaw, J. J., Ramos, F., Sil- viously defined regions of difference (RD) veira, F., and Blackwell, J. M. Genome- known to be absent from Mycobacterium wide scans for leprosy and tuberculosis bovis BCG relative to M. tuberculosis. Hy- susceptibility genes in Brazilians. Genes bridization of DNA from 40 strains of the M. Immun. 5(1) (2004) 63–67. tuberculosis complex and in silico comparison of these genes with the near-complete Genome-wide scans were conducted for genome sequences from Mycobacterium tuberculosis and leprosy per se in Brazil. At avium, Mycobacterium marinum and Myco- stage 1, 405 markers (10 cM map) were bacterium smegmatis were undertaken to typed in 16 (178 individuals) tuberculosis answer this question. The results showed and 21 (173 individuals) leprosy families. that among the 219 conserved genes, very Nonparametric multipoint analysis detected few were not present in all the strains tested. 8 and 9 chromosomal regions respectively Some of these missing genes code for pro- with provisional evidence (p <0.05) for link- teins of the ESAT-6 family, a group of age. At stage 2, 58 markers from positive highly immunogenic small proteins whose regions were typed in a second set of 22 presence and number is variable among the (176 individuals) tuberculosis families, with genomically highly conserved members of 22 additional markers typed in all families; the M. tuberculosis complex. Indeed, the re- 42 positive markers in 50 (192 individuals) sults suggest that, with few exceptions, the new leprosy families, and 30 additional ‘core’ genes conserved among M. tubercu- markers in all families. Three regions losis H37Rv and M. leprae are also highly (10q26.13, 11q12.3, 20p12.1) retained sug- conserved among other mycobacterial gestive evidence (peak LOD scores 1.31, strains, which makes them interesting po- 1.85, 1.78; p = 0.007, 0.0018, 0.0021) for tential targets for developing new specific linkage to tuberculosis, 3 regions (6p21.32, anti-mycobacterial drugs. In contrast, the 17q22, 20p13) to leprosy (HLA-DQA, 3.23, genes from RD regions showed great vari- p = 5.8 × 10(–5); D17S1868, 2.38, p = ability among certain members of the M. tu- 0.0005; D20S889, 1.51, p = 0.004). The berculosis complex, and some new specific peak at D20S889 for leprosy is 3.5 Mb dis- deletions in Mycobacterium canettii, Myco- tal to that reported at D20S115 for leprosy bacterium microti and seal isolates were in India. (151 words).—Authors’Abstract 260 International Journal of Leprosy 2004

Mostowy, S., Cousins, D., and Behr, M. A. There is increasing interest in and un- Genomic interrogation of the dassie derstanding of the role of human genetic bacillus reveals it as a unique RD1 mu- factors controlling susceptibility/resistance tant within the Mycobacterium tubercu- to infectious diseases. This is of particular losis complex. J. Bacteriol. 186(1) (2004) importance for the two most common my- 104–149. cobacterial infections, tuberculosis and leprosy, because this will allow a genetic dis- Despite their remarkable genetic homol- section of antimycobacterial immunity and ogy, members of the Mycobacterium tuber- should open new fields of preventive and culosis complex express very different phe- therapeutic measures. In this review we notypes, most notably in their spectra of will initially discuss various methods of ge- clinical presentation. For example, M. tuber- netic epidemiology that have been and are culosis is regarded as pathogenic to humans, being developed to identify human genes whereas members having deleted RD1, such controlling infectious diseases, and then il- as Mycobacterium microti and Mycobacte- lustrate the findings obtained in the numer- rium bovis BCG, are not. The dassie bacillus, ous studies performed in tuberculosis and an infrequent variant of the M. tuberculosis leprosy. Although the most convincing re- complex characterized as being most similar sults were observed for HLA-DR2 and to M. microti, is the causative agent of tuber- NRAMP1 (or a closely linked gene) in pul- culosis (TB) in the dassie (Procavia capen- monary tuberculosis and leprosy subtypes sis). Intriguingly, the dassie bacillus is not and for a 10p13 locus in paucibacillary lep- pathogenic to rabbits or guinea pigs and has rosy, the molecular basis of their effects never been documented to infect humans. Al- remains to be established.—Authors’ though it was identified more than a half- Abstract century ago, the reasons behind its attenuation are unknown. Because large sequence polymorphisms have presented themselves as Sampson, S. L., Richardson, M., Van the most obvious genomic distinction among Helden, P. D., and Warren, R. M. members of the M. tuberculosis complex, the IS6110-mediated deletion polymorphism DNA content of the dassie bacillus was in isogenic strains of Mycobacterium tu- interrogated by Affymetrix GeneChip to berculosis. J. Clin. Microbiol. 42(2) identify regions that are absent from it but (2004) 895–898. present in M. tuberculosis H37Rv. Compari- son has led to the identification of nine re- Previous studies have described IS6110- gions of difference (RD), five of which are mediated polymorphism as an important shared with M. microti (RDs 3, 7, 8, 9, and driving force in Mycobacterium tuberculo- 10). Although the dassie bacillus does not sis genome evolution and have provided in- share the other documented deletions in M. direct evidence for IS6110-driven deletion microti (RD1(mic), RD5(mic), MID1, MID2, events. This study provides the first descrip- and MID3), it has endured unique deletions tion of an IS6110-mediated deletion event in in the regions of RD1, RD5, N-RD25, and truly isogenic strains. We also provide fur- Rv3081-Rv3082c (virS). RD1(das), affecting ther support for the hypothesis that the re- only Rv3874-Rv3877, is the smallest natural gion from Rv1754 to Rv1765 is a hot spot deletion of the RD1 region uncovered and for IS6110 insertion and deletion events.— points to genes within this region that are Authors’Abstract likely implicated in virulence. Newfound deletions from the dassie bacillus are discussed in relation to their evolutionary and Sen Gupta, R., Hillemann, D., Kubica, T., biological significance.—Authors’Abstract Zissel, G., Muller-Quernheim, J., Galle, J., Vollmer, E., and Goldmann, T. HOPE-fixation enables improved Remus, N., Alcais, A., and Abel, L. Human PCR-based detection and differentiation genetics of common mycobacterial in- of Mycobacterium tuberculosis complex fections. Immunol. Res. 28(2) (2003) in paraffin-embedded tissues. Pathol. 109–129. Res. Pract. 199(9) (2003) 619–623. 72, 2 Current Literature, Molecular and Genetic Studies 261

See Current Literature, Immunopathol- transporter operon (mpt), which comprises ogy, p. 203. six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics re- Song, T., Dove, S. L., Lee, K. H., and Hus- vealed that the mpt operon is flanked on one son, R. N. RshA, an anti-sigma factor that end by a fep cluster encoding proteins in- regulates the activity of the mycobacterial volved in the uptake of Fe(3+) and on the stress response sigma factor SigH. Mol. other end by a sid cluster encoding non- Microbiol. 50(3) (2003) 949–959. ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb SigH, an alternative sigma factor of My- M. avium subsp. paratuberculosis-specific cobacterium tuberculosis, is a central regu- locus flanked by an insertion sequence sim- lator of the response to oxidative and heat ilar to IS1110. Expression studies using stress. Exposure to these stresses results in Western blot analyses showed that MptC is increased expression of sigH itself, and of present in the envelope fraction of M. avium genes encoding additional regulators and ef- subsp. paratuberculosis. The MptD protein fectors of the bacterial response to these was shown to be surface exposed, using a stresses. In this work we show that RshA, a specific phage (fMptD) isolated from a protein encoded by a gene in the sigH phage-peptide library, by differential screen- operon, is an anti-sigma factor of SigH. We ing of Mycobacterium smegmatis transfor- demonstrate that RshA binds to SigH in vitro mants. The phage fMptD-derived peptide and in vivo. This protein-protein interaction, could be used in a peptide-mediated capture as well as the ability of RshA to inhibit SigH- PCR with milk from infected dairy herds, dependent transcription, is redox-dependent, thereby showing surface-exposed expres- with RshA functioning as a negative regula- sion of the MptD protein in the host. To- tor of SigH activity only under reducing con- gether, these data suggest that the 38-kb ditions. The interaction of SigH and RshA is locus constitutes an M. avium subsp. paratu- also disrupted in vitro by elevated tempera- berculosis pathogenicity island.—Authors’ ture. RshA, a protein of 101 amino acids, Abstract contains five conserved cysteine residues of which two appear to be essential for RshA to inhibit SigH activity, suggesting that these van den Braak, N., Simons, G., Gorkink, cysteines may be important for the redox R., Reijans, M., Eadie, K., Kremers, state dependence of RshA function. Our re- K., van Soolingen, D., Savelkoul, P., sults indicate that RshA is a sensor that re- Verbrugh, H., and van Belkum, A. A sponds to oxidative stress, and also to heat new high-throughput AFLP approach for stress, resulting in activation of SigH and ex- identification of new genetic polymor- pression of the SigH-dependent genes that phism in the genome of the clonal mi- allow M. tuberculosis to adapt to these croorganism Mycobacterium tuberculo- stresses.—Authors’Abstract sis. J. Microbiol. Methods 56(1) (2004) 49–62.

Stratmann, J., Strommenger, B., Goethe, We have here applied high-throughput R., Dohmann, K., Gerlach, G. F., amplified fragment length polymorphism Stevenson, K., Li, L. L., Zhang, Q., (htAFLP) analysis to strains belonging to Kapur, V., and Bull, T. J. A 38-kilobase the five classical species of the Mycobacte- pathogenicity island specific for Myco- rium tuberculosis complex. Using 20 strains, bacterium avium subsp. paratuberculosis three enzyme combinations and eight selec- encodes cell surface proteins expressed in tive amplification primer pairs, 24 AFLP re- the host. Infect. Immun. 72(3) (2004) actions were performed per strain. Overall, 1265–1274. this resulted in 480 DNA fingerprints and more than 1200 htAFLP-amplified PCR We have used representational difference fragments were visualised per strain. The analysis to identify a novel Mycobacterium cumulative dendrogram correctly clustered avium subsp. paratuberculosis-specific ABC strains from the various species, albeit 262 International Journal of Leprosy 2004 within a distance of 6.5% for most of them. major Mycobacterium species and M. tu- The single isolate of Mycobacterium canet- berculosis variants and clones.—Authors’ tii presented separately at 19% distance. All Abstract over, 169 fragments (14%) appeared to be polymorphic. Sixty-eight were specific for M. canetti and forty-five for Mycobacterium Wang, J. P., Rought, S. E., Corbeil, J., bovis. For the 10 different M. tuberculosis and Guiney, D. G. Gene expression pro- strains included in the present analysis, 56 filing detects patterns of human macro- polymorphic markers were identified. Upon phage responses following Mycobacterium sequencing 20 of these marker regions and tuberculosis infection. FEMS Immunol. comparisons with the H37Rv genome se- Med. Microbiol. 39(2) (2003) 163–172. quence, 25% appeared to share homology to members of the antigenically variable High-density oligonucleotide microarrays PE/PPE surface protein encoding gene allow simultaneous monitoring of the ex- family confirming previous findings on the pression of a large number of cellular genes. genetic heterogeneity within these genes. In Microarrays were used to screen the global addition, homologues for phage genes and human monocyte-derived macrophage tran- insertion element-encoded genes were de- scriptional response to infection with the tected. Forty-five percent of the sequences intracellular pathogen Mycobacterium tu- derived from ORFs with a currently un- berculosis. The microarray detected repro- known function, which was corroborated by ducible patterns of regulated gene expres- genome sequence comparison for the clini- sion. Analysis of the expression data showed cal M. tuberculosis CD 1551 isolate. Se- induction of cytokines and chemokines, quence variation in M. tuberculosis was as- ribosomal proteins, and the interferon- sessed in more detail for a subset of these response gene Stat1. Several changes were loci by newly designed PCR restriction frag- validated by quantitative reverse transcription ment length polymorphism (RFLP) tests and polymerase chain reaction and immunoblot direct sequencing. Fourteen novel PCR assays. Augmentation of the respiratory RFLP tests were developed and twelve burst and preservation of the response to in- novel single nucleotide polymorphisms terferon-gamma were also demonstrated. (SNPs) were identified, all suited for epi- These data supplement existing knowledge demiological analysis of M. tuberculosis. on macrophage responses to tuberculosis in- The tests allowed for identification of the fection.—Authors’Abstract INTERNATIONAL JOURNAL OF LEPROSY Volume 72, Number 2 Printed in the U.S.A. (ISSN 0148-916x) ACKNOWLEDGMENT

The Board of Directors of the INTERNATIONAL JOURNAL OF LEPROSY gratefully ac- knowledges the ﬁnancial assistance from special grantors and sustaining members which, with the special donations of certain members, has made possible the continuation of publication of the JOURNAL directly by the International Leprosy Asso- ciation. Without this assistance the ofﬁcial organ of the ILA, so essential to leprosy workers everywhere, could not be published.

SPECIAL GRANTORS

*Aide aux Lepreux Emmaus-Suisse, 9 *Le Secours aux Lépreux (Canada), 1275 Spitalgasse, CH-3011 Berne, Switzer- Rue Hodge, Bureau 12, Montreal H4N land. 3H4, Canada. *American Leprosy Missions, One ALM *Netherlands Leprosy Relief, Wibaut- Way, Greenville, South Carolina 29601, straat 137K, 1097 DN Amsterdam, The U.S.A. Netherlands. *Amici dei Lebbrosi, Fondazione Italiana *Paciﬁc Leprosy Foundation, 115 Sher- Raoul Follereau, Via Borselli 4, 40135 borne Street, Bag 4730, Christchurch, Bologna, Italy. New Zealand. Damien-Dutton Society, 616 Bedford Av- *Sasakawa Memorial Health Founda- enue, Bellmore, New York 11710, U.S.A. tion, Senpaku Shinko Bldg., 1-15-16 Toranomon, Minato-ku, Tokyo 105, *Damien Foundation (DF/APD), 16 Rue Japan. Stevin, B-1040 Bruxelles, Belgium. *Deutsches Aussatzigen-Hilfswerk e. V., Postfach 9062, D-97090 Würzburg 11, Germany.

SUSTAINING MEMBERS

c *Foundation Follereau (France), 33 Rue Japanese Leprosy Association, /o National de Dantzig, 75015 Paris, France. Institute for Leprosy Research, 2-1, 4-Chome, Aobacho, Higashimuryamashi, Tokyo 189, Japan.

* ILEP member/association.

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