October-December 2007 INDIAN JOURNAL OF 451 MEDICAL MICROBIOLOGY (OfÞ cial publication of Indian Association of Medical Microbiologists, Published quarterly in January, April, July and October) Indexed in Index Medicus/MEDLINE/PubMed, ‘Elsevier Science - EMBASE’, ‘IndMED’ EDITORIAL BOARD EDITOR Dr. SAVITRI SHARMA L V Prasad Eye Institute Bhubaneswar - 751 024, India ASSOCIATE EDITOR ASSISTANT EDITOR Dr. Shobha Broor Dr. V Lakshmi Professor, Department of Microbiology Professor and Head, Dept. of Microbiology All India Institute of Medical Sciences Nizam’s Institute of Medical Sciences New Delhi - 110 029, India Punjagutta, Hyderabad - 500 082, India ASSISTANT EDITOR ASSISTANT EDITOR Dr. P Sugandhi Rao Dr. Reba Kanungo Professor Professor and Head Department of Microbiology Department of Microbiology, Perunthalaivar Kamaraj Kasturba Medical College Medical College and Research Institute, Kadhirkamam, Manipal - 576 119, India Puducherry - 605 009, India MEMBERS

National International Dr. Arora DR (Rohtak) Dr. Arseculeratne SN (Srilanka) Dr. Arunaloke Chakrabarthi (Chandigarh) Dr. Arvind A Padhye (USA) Dr. Camilla Rodrigues (Mumbai) Dr. Chinnaswamy Jagannath (USA) Dr. Chaturvedi UC (Lucknow) Dr. Christian L Coles (USA) Dr. Hemashettar BM (Belgaum) Dr. David WG Brown (UK) Dr. Katoch VM (Agra) Dr. Diane G Schwartz (USA) Dr. Madhavan HN (Chennai) Dr. Govinda S Visveswara (USA) Dr. Mahajan RC (Chandigarh) Dr. Kailash C Chadha (USA) Dr. Mary Jesudasan (Thrissur) Dr. Madhavan Nair P (USA) Dr. Meenakshi Mathur (Mumbai) Dr. Madhukar Pai (Canada) Dr. Nancy Malla (Chandigarh) Dr. Mohan Sopori (USA) Dr. Philip A Thomas (Tiruchirapally) Dr. Paul R Klatser (Netherlands) Dr. Ragini Macaden (Bangalore) Dr. Vishwanath P Kurup (USA) Dr. Ramesh K Aggarwal (Hyderabad) Dr. Renu Bhardwaj (Pune) Dr. Sarman Singh (New Delhi) Dr. Seyed E Hasnain (Hyderabad) Dr. Sitaram Kumar M (Hyderabad) Dr. Sridharan G (Vellore) Dr. Sritharan V (Hyderabad) Dr. Subhas C Parija (Pondicherry)

ADVISORY BOARD Dr. KB Sharma (New Delhi), Dr. NK Ganguly (New Delhi), Dr. SP Thyagarajan (Chennai), Dr. R Sambasiva Rao (New Delhi), Dr. MK Lalitha (Chennai), Dr. PG Shivananda (Manipal) Annual Subscription Rs 2,000/- US $ 150 Single Copy Rs 600/- US $ 75 Editorial OfÞ ce: LV Prasad Eye Institute, Patia, Bhubaneswar - 751 024, Orissa, India Ph: (+91)-0674-3987 209, 099370 37298, Fax: (+91)-0674-3987 130, E-mail: [email protected], Website: www.ijmm.org Published by MEDKNOW PUBLICATIONS A-109, Kanara Business Center, Off Link Rd, Ghatkopar (E), Mumbai - 400075, INDIA Phone: 91-22-6649 1818/1816, Fax: 91-22-6649 1817 • E-mail: [email protected], Web: www.medknow.com

The journalwww.ijmm.org is printed on acid free paper. 452 Indian Journal of Medical Microbiology vol. 25, No. 4

INDIAN JOURNAL OF MEDICAL MICROBIOLOGY

(Publication of Indian Association of Medical Microbiologists)

ISSN 0255-0857 Volume 25 Number 4 October-December, 2007

CONTENTS Page No. Guest Editorial The Need for Control of Viral Illnesses in India: A Call for Action ...... 309 C Lahariya, UK Baveja Review Article Immunobiology of Human ImmunodeÞ ciency Virus Infection ...... 311 P Tripathi, S Agrawal Special Articles Serum Levels of Bcl-2 and Cellular Oxidative Stress in Patients with Viral Hepatitis ...... 323 HG Osman, OM Gabr, S Lotfy, S Gabr Rapid IdentiÞ cation of Non-sporing Anaerobes using Nuclear Magnetic Resonance Spectroscopy and an IdentiÞ cation Strategy ...... 330 S Menon, R Bharadwaj, AS Chowdhary, DV Kaundinya, DA Palande Original Articles Species Distribution and Physiological Characterization of Acinetobacter Genospecies from Healthy Human Skin of Tribal Population in India ...... 336 SP Yavankar, KR Pardesi, BA Chopade Extended-spectrum Beta-lactamases in Ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae Isolates in Turkish Hospitals ...... 346 S Hoşoğlu, S Gündeş, F Kolaylõ, A Karadenizli, K Demirdağ, M Günaydõn, M Altõndis, R Çaylan, H Ucmak Typhoid Myopathy or Typhoid Hepatitis: A Matter of Debate ...... 351 M Mirsadraee, A Shirdel, F Roknee Correlation Between in Vitro Susceptibility and Treatment Outcome with Azithromycin in Gonorrhoea: A Prospective Study ...... 354 P Khaki, P Bhalla, A Sharma, V Kumar Comparison of Radiorespirometric Buddemeyer Assay with ATP Assay and Mouse Foot Pad Test in Detecting Viable leprae from Clinical Samples ...... 358 VP Agrawal, VP Shetty Detection of Species in Cell Culture by PCR And RFLP Based Method: Effect of BM-cyclin to Cure Infections ...... 364 V Gopalkrishna, H Verma, NS Kumbhar, RS Tomar, PR Patil

www.ijmm.org October-December 2007 453

Virulence Factors and Drug Resistance in Escherichia coli Isolated from Extraintestinal Infections ...... 369 S Sharma, GK Bhat, S Shenoy Antimicrobial Susceptibility Testing of Helicobacter pylori to Selected Agents by Agar Dilution Method in Shiraz-iran ...... 374 J Kohanteb, A Bazargani, M Saberi-Firoozi, A Mobasser Outbreak of Acute Viral Hepatitis due to Hepatitis E virus in Hyderabad ...... 378 P Sarguna, A Rao, KN Sudha Ramana A Comparative Study for the Detection of Mycobacteria by BACTEC MGIT 960, Lowenstein Jensen Media and Direct AFB Smear Examination ...... 383 S Rishi, P Sinha, B Malhotra, N Pal Cytokine Levels in Patients with and their Relations with the Treatment ...... 387

H Akbulut, I Celik, A Akbulut Brief Communications Rapid Detection of Non-enterobacteriaceae Directly from Positive Blood Culture using Fluorescent In Situ Hybridization ...... 391 EH Wong, G Subramaniam, P Navaratnam, SD Sekaran Latex Particle Agglutination Test as an Adjunct to the Diagnosis of Bacterial Meningitis ...... 395 K Surinder, K Bineeta, M Megha Helminthic Infestation in Children of Kupwara District: A Prospective Study ...... 398 SA Wani, F Ahmad, SA Zargar, BA Fomda, Z Ahmad, P Ahmad Clinical and Mycological ProÞ le of Cryptococcosis in a Tertiary Care Hospital ...... 401 MR Capoor, D Nair, M Deb, B Gupta, P Aggarwal Candida spp. other than Candida albicans: A Major Cause of Fungaemia in a Tertiary Care Centre ...... 405 S Shivaprakasha, K Radhakrishnan, PMS Karim Case Reports Enterobacter sakazakii in Infants: Novel Phenomenon in India ...... 408 P Ray, A Das, V Gautam, N Jain, A Narang, M Sharma Ocular Toxocariasis in a Child: A Case Report from Kashmir, North India ...... 411 BA Fomda, Z Ahmad, NN Khan, S Tanveer, SA Wani Cutaneous : A Rare Case ...... 413 SC Metgud, H Sumati, P Sheetal Fatal Haemophagocytic Syndrome and Hepatitis Associated with Visceral Leishmaniasis ...... 416 P Mathur, JC Samantaray, P Samanta A Rare Case of Mucormycosis of Median Sternotomy Wound Caused by Rhizopus arrhizus ...... 419 R Chawla, S Sehgal, S Ravindra Kumar, B Mishra Keratitis ...... 422 C Sanghvi Correspondence Prevention of Parent-to-Child Transmission of HIV: An Experience in Rural Population ...... 425 N Nagdeo, VR Thombare www.ijmm.org 454 Indian Journal of Medical Microbiology vol. 25, No. 4

Combining Vital Staining with Fast Plaque: TB Assay ...... 426 D Rawat, MR Capoor, A Hasan, D Nair, M Deb, P Aggarwal Disseminated Histoplasmosis ...... 427 PK Maiti, MS Mathews Authors’ Reply ...... 428 RS Bharadwaj Microwave Disinfection of Gauze Contaminated with and Fungi ...... 428 VH Cardoso, DL Gonçalves, E Angioletto, F Dal-Pizzol, EL Streck Endoscope Reprocessing: Stand up and Take Notice! ...... 429 A Das, P Ray, M Sharma Prevalence of Toxoplasma gondii Infection amongst Pregnant Women in Assam, India ...... 431 BJ Borkakoty, AK Borthakur, M Gohain Evaluation of Glucose-Methylene-Blue-Mueller-Hinton Agar for E-Test Minimum Inhibitory Concentration Determination in Candida spp...... 432 MR Capoor, D Rawat, D Nair, M Deb, P Aggarwal Resurgence of in the Vaccination Era ...... 434 N Khan, J Shastri, U Aigal, B Doctor A Report of Pseudomonas aeruginosa Antibiotic Resistance from a Multicenter Study in Iran ...... 435 MA Boroumand, P Esfahanifard, S Saadat, M Sheihkvatan, S Hekmatyazdi, M Saremi, L Nazemi Trends of Antibiotic Resistance in Salmonella enterica Serovar Typhi Isolated from Hospitalized Patients from 1997 to 2004 in Lagos, Nigeria ...... 436 KO Akinyemi, AO Coker Book Review Hospital-Acquired Infections: Power Strategies for Clinical Practice ...... 438 Reba Kanungo

Title Index, 2007 ...... 440 Author Index, 2007 ...... 442 Scienti fi c Reviewers, 2007 ...... 446

The copies of the journal to members of the association are sent by ordinary post. The editorial board, association or publisher will not be responsible for non-receipt of copies. If any of the members wish to receive the copies by registered post or courier, kindly contact the journal’s / publisher’s offi ce. If a copy returns due to incomplete, incorrect or changed address of a member on two consecutive occasions, the names of such members will be deleted from the mailing list of the journal. Providing complete, correct and up-to-date address is the responsibility of the members. Copies are sent to subscribers and members directly from the publisher’s address; it is illegal to acquire copies from any other source. If a copy is received for personal use as a member of the association/society, one cannot resale or give- away the copy for commercial or library use.

www.ijmm.org Indian358 Journal of Medical Microbiology, (2007) 25(4):Indian 358-63 Journal of Medical Microbiology vol. 25, No. 4 Original Article

COMPARISON OF RADIORESPIROMETRIC BUDDEMEYER ASSAY WITH ATP ASSAY AND MOUSE FOOT PAD TEST IN DETECTING VIABLE FROM CLINICAL SAMPLES VP Agrawal, *VP Shetty Abstract

Purpose: This study compares the results of radiorespirometric Buddemeyer assay with adenosine triphosphate (ATP) assay and mouse foot pad (MFP) test to validate the sensitivity of Buddemeyer assay in detecting viable M. leprae in clinical samples. Methods: Viability was assessed using all the three methods in 60 skin biopsy specimens, including 20 untreated lepromatous (BL-LL), 13 treated BL-LL, 12 untreated borderline tuberculoid to mid borderline (BT- BB) and 15 treated BT-BB cases. Results: Of the 20 untreated BL-LL cases tested, positivity indicating the presence of viable M. leprae was detected in 85, 60 and 85% with Buddemeyer, ATP and MFP test, respectively. Among the 13 treated BL-LL cases, scores were 61, 54 and 0%; among the 12 untreated BT-BB cases, the scores were 58, 16 and 16% and among the 15 treated BT-BB cases, the scores were 46, 20, 0%, respectively. Conclusion: The detection sensitivity (positive scores) with three tests were closely comparable in the two untreated groups of cases. On the other hand, in the two treated groups, a good proportion of cases scored positive in the in vitro tests but none in the MFP test. Among the two in vitro methods, the Buddemeyer assay emerged as a better test, in terms of sensitivity and speciÞ city.

Key words: adenosine triphosphate assay, M. leprae, mouse foot pad test, radiorespirometric Buddemeyer assay, viability

There are no easy tools to determine the viability samples. The results indicated good applicability particularly of Mycobacterium leprae. Mouse foot pad (MFP) test in low bacteriological index (BI), i.e., tuberculoid cases.[13] developed by Shephard1,2 is still considered a gold standard To further validate the assay system, three tests, i.e., MFP for the detection of viable M. leprae, but is laborious, takes test, Buddemeyer and ATP assay were compared. The results 6 months to yield results and involves usage of animals. thus obtained in 60 clinical samples are presented and The morphological index (MI) is widely used as an index discussed in this paper. of M. leprae viability in Þ eld conditions but besides its poor sensitivity, the method is highly subjective.3 Materials and Methods

Among the various in vitro methods available, adenosine Lesional skin biopsies obtained from 60 cases, including tri-phosphate (ATP) bioluminescence assay has been 20 untreated borderline lepromatous to shown to be more sensitive than MFP test in the hands of (BL-LL), 13 treated BL-LL, 12 untreated borderline some investigators.4-6 Jagannathan and Mahadevan7 showed tuberculoid to mid-borderline (BT-BB) and 15 treated a good correlation between ATP and Fc receptor assay. BT-BB were studied using all the three methods. The Katoch et al.,8 found ATP, an easy and a better tool for treated cases under study had received 12 months of WHO viability determination than morphological index (MI) and multibacillary multidrug therapy (MB-MDT) and the ß uorescence di-acetate (FDA-EB) staining. Gupta et al.,6 biopsies were collected after 4-6 months of completion found a good correlation with gene ampliÞ cation assay. of treatment. The classiÞ cations of cases were done using Ridley-Jopling scale.14 In each case, approximately 0.15 g of 14 The Buddemeyer assay based on evolution of CO2 the biopsy tissue was homogenized using a constant weight/ from 1-14C palmitic acid by viable cells has been used to volume ratio (i.e., 1 mL/0.1 g wt of tissue). Bacterial count/ determine the viability and efÞ cacy of antileprosy drugs mL of tissue, BI and MI were determined using the standard using M. leprae derived from nude mice and armadillos.9- protocol.15 The homogenate thus obtained was divided in 12 We standardized this assay system in our laboratory and to three parts and used with in 48 h, for Buddemeyer assay, applied it to determine the viability of M. leprae in clinical ATP assay and MFP test, respectively.

Radiorespirometric Buddemeyer assay *Corresponding author (email:< [email protected]>) The Foundation for Medical Research, Worli, Mumbai-400 018, Palmitic acid oxidation assay described by Buddemeyer 16 India et al., was standardized in our laboratory with slight Received: 22-12-06 modiÞ cations. In brief, glass Þ bre Þ lter papers were soaked Accepted: 18-5-07 in liquid scintillation ß uid, dried and overlaid with 2 N

www.ijmm.org October-December 2007 Agrawal and Shetty - Detection of Viable M. leprae by Buddemeyer Assay 359 methylated NaOH. They were then rolled and inserted into Statistical methods scintillation vials. For each sample, in a glass roller tube, 3.5 mL of Dubos [with 10% fetal calf serum (FCS) and The statistical signiÞ cance of the difference between the without glycerol] containing 0.1 µCi/mL of 1-14C palmitic results of all the three methods was determined by using acid [Board of Radiation and Isotope Technology (BRIT), Fisher-exact test. The statistical tests were performed using speciÞ c activity - 20.06 mCi/mmol] was taken and 0.3 mL the programme InStat 3.06 (GraphPad, Inc.). P ≤ 0.05 was of homogenate sample (neat), regardless of bacterial considered statistically signiÞ cant. count, was added. After thorough mixing, 1 mL each was Ethical approval dispensed into three plastic vials (triplicates) of 4 mL capacity and each vial was then gently placed (without cap) The study had a clearance from the institutional 14 within the scintillation vial. The readings of CO2 evolution ethics committee. All the biopsies were done using local were recorded at an interval of 24-48 h for 3 weeks using a anaesthesia, with informed consent from the patients and liquid scintillation counter (Wallac 1409 DSA). The vials controls before recruitment. Committee for the Purpose were maintained at 33 °C and were checked for sterility of Control and Supervision of Experiments on Animals at the end, using nutrient broth, Sabouraud broth and (CPCSEA) cleared the use of animals in the study. The Loweinstein Jensen slants. If found contaminated, the test Foundation for Medical Research is registered with CPCSEA results were not considered for analysis. and has a valid registration number 424/01/a/CPCSEA).

Normal skin biopsy samples obtained from 15 donor non- Results leprosy cases were processed in a similar manner. The cut- off counts per minute (CPM) value for Buddemeyer assay In case of Buddemeyer assay, the average CPM value for was determined using these samples. the 15 non-leprous skin biopsy samples was 300.17 ± 175.09 (results not shown). Applying 2SE, any value above 390.1 ATP assay CPM at the peak interval was considered as an indication Adenosine triphosphate estimation was carried out using of positive metabolic activity and hence presence of viable Dhople’s method.5 Brieß y, 0.3 mL of homogenate was M. leprae. centrifuged, bacillary pellet obtained was decontaminated It was generally noted that, an initial lag phase was with 2% NaOH, followed by treatment with 0.4% w/v followed by a log phase with peak CPM output, followed trypsin, 0.1% w/v triton x-100 and 0.34% ATPase. After by a plateau. The peak CPM output was observed between each step, the pellet was washed with 0.05 M phosphate 11 and 14 days. The results of three representative cases are buffer. ATP was extracted by adding Tris EDTA-HCl buffer depicted in the Figure. and boiling it in water bath for 10 min. The amount of ATP was estimated using ATP monitoring kit (Biothema). The The results of the three tests in four groups of cases are statistical signiÞ cance was determined using unpaired t test given in Tables 1-4, while the comparison between the three by comparing test readings with the blank readings. methods with respect to positivity is given in Table 5.

Mouse foot pad inoculation and harvesting of M. leprae Comparison of results between Buddemeyer assay and ATP assay The hind foot pads of 8-10 Swiss white (S/W) mice per sample were inoculated with 0.03 mL of appropriately Buddemeyer assay showed better sensitivity than ATP diluted tissue homogenates containing ≤1 × 104 M. leprae assay in all the four groups (Tables 1-5), but the differences cells. All the mice were fed with standard mouse diet and kept in a 12 hourly light-dark cycle in an air-conditioned room. Foot pads were harvested at 6, 7 and/or 8 and 12 5000 4500 months post-inoculation using Shepherd’s method.1 Brieß y, 4000 3500 3000 foot pads were disinfected, cut and collected in sterile 1 2500 cups containing sterile saline (0.5 mL/foot pad). This was CPM 2000 2 1500 3 then electrically homogenized and the bacterial counts 1000 were taken by Ziehl Neelsen Carbol Fuchsin staining on a 500 0 spot slide using the standard method. A minimum of 200 1 2 3 4 5 6 7 8 9 10111213141516 microscopic Þ elds/sample was covered for counting acid No. of days fast bacilli (AFB). The lower limit of detectability by this method is 1 × 104 M. leprae per foot pad. A minimum of Figure: Depiction of CPM output against the time interval in the two counts per foot pad per interval was obtained at 6, 7 Buddemeyer assay in three representative BL-LL cases with BI and MI as given below. Note that the initial lag phase of ~7 days is and 8 months. The remaining mice were harvested at the followed by a log and plateau phase. Peak is observed between 11 and 5 twelfth month. Any foot pad count exceeding 1 × 10 was 14 days. 1. 3.4 × 106/mL BI = 3+, MI = 15%; 2. 8.8 × 106/mL BI = 3+, considered as signiÞ cant increase. MI = 3.3%; 3. 2.5 × 105/mL BI = 3+, MI = 1.5%

www.ijmm.org 360 Indian Journal of Medical Microbiology vol. 25, No. 4

Table 1: Peak counts per minute output in Buddemeyer assay and the corresponding adenosine triphosphate assay (expressed in pmol) and mouse foot pad test results in untreated BL-LL cases (n = 20) Patient Count/mL BIa MIb Buddemeyer ATP in pmold MFPe code (%) c(CPM) (P-value) KB 3.9 × 108 5+ 8.9 2523 0.0124 (0.014) +ve RW 2.7 × 108 5+ 6 9836 0.016 (0.0091) +ve AW 2.5 × 108 5+ 7.5 5240 0.011 (0.022) +ve MC 9 × 107 4+ 2 225 0.175 (0.038) +ve JM 8.8 × 106 3+ 3.3 4205 0.0021 (0.643) –ve MJ 5 × 106 3+ 0 644 0.0067 (0.58) +ve NS 1.5 × 107 3+ 5 673 0.0032 (0.022) +ve SU 2.1 × 108 4+ 6 736 0.0022 (0.091) +ve RA 2.3 × 108 4+ 3.3 225 0.002 (0.257) –ve MR 1.7 × 105 1+ 0 2140 0.039 (0.04) +ve SG 2.4 × 108 4+ 4 6432 0.12 (0.0013) +ve SK 1.1 × 106 5+ 4.4 2351 0.057 (0.028) +ve JJ 2.14 × 106 2+ 3.7 5812 0.012 (<0.0001) +ve GI 2.8 × 107 3+ 15 9282 0.040 (0.0037) +ve IB 7.08 × 106 3+ 18.6 1734 0.027 (0.406) +ve AL 1.1 × 108 4+ 4.3 6255 0.027 (0.365) +ve RH 2.5 × 107 4+ 1.4 126 0.0017 (0.08) –ve JB 6.7 × 107 4+ 4 677 0.0042 (0.015) +ve MN 1.7 × 108 4+ 4.7 3420 0.0019 (0.67) +ve RB 1.52 × 106 2+ 0.6 1549 0.001 (0.028) +ve Total positives 20/20 17/20 12/20 17/20 aSemiquantitative estimation of the density of AFB present in smear. b% of solidly stained AFB representing viable bacillary population in smear. c>390 CPM, considered as positive metabolic activity. dP < 0.05 considered signiÞ cant. e+ve - >1 × 105/foot pad and –ve - <1 × 104/foot pad

Table 2: Peak counts per minute output in Buddemeyer assay and the corresponding adenosine triphosphate assay (expressed in pmol) and mouse foot pad test results in treated BL-LL cases (n = 13) Patient Count/mL BIa MIb Buddemeyerc ATP in pmold MFPe code (%) (CPM) (P-value) SP <1 × 104 0 0 270 0.253 (0.45) –ve AR 2.4 × 107 4+ 1.2 279 0.191 (0.807) –ve SM <1 × 104 0 0 88 0.140 (0.028) –ve RA 1.2 × 106 2+ 1.6 1644 0.80 (0.129) –ve IB 3.1 × 104 1+ 0 1975 0 (0.311) –ve SE 6.2 × 106 3+ 0 325 0.0001 (0.022) –ve SS 8.7 × 106 3+ 1 1247 0.00019 (0.012) –ve UK 2.09 × 104 1+ 0 500 0.065 (0.127) –ve RM 5.8 × 105 4+ 6 1522 0.022 (0.032) –ve DC <1 × 104 0 0 141 0.0003 (<0.0001) –ve GI 1.6 × 105 2+ 0 664 0.014 (0.019) –ve RM <1 × 104 0 0 950 0.019 (0.029) –ve SK <1 × 104 0 0 407 0.011 (0.69) –ve Total positives 8/13 8/13 7/13 0/13 aSemiquantitative estimation of the density of AFB present in smear. b% of solidly stained AFB representing viable bacillary population in smear. c>390 CPM, considered as positive metabolic activity. dP < 0.05 considered signiÞ cant. e+ve - >1 × 105/foot pad and –ve - <1 × 104/foot pad were not statistically signiÞ cant. One-to-one concordance positive in ATP assay but negative in Buddemeyer assay. between the two tests was seen in 33/60 (55%) cases. Of the Number of cases positive in Buddemeyer assay was more remaining 27 cases, 21 (35%) were positive in Buddemeyer in the discordance group, indicating better sensitivity of this assay but negative in ATP assay, while 6/60 (10%) were detection method.

www.ijmm.org October-December 2007 Agrawal and Shetty - Detection of Viable M. leprae by Buddemeyer Assay 361

Table 3: Peak counts per minute output in Buddemeyer assay and the corresponding adenosine triphosphate assay (expressed in pmol) and mouse foot pad test results in untreated BT-BB cases (n = 12) Patient Count/mL BIa MIb Buddemeyerc ATP in pmold MFPe code (%) (CPM) (P-value) HD <1 × 104 0 0 3056 0.00053 (0.56) +ve SV <1 × 104 0 0 175 0.00092 (0.66) –ve NA <1 × 104 0 0 2205 0.0024 (0.492) –ve SY 9.2 × 104 1+ 14 959 0.0001 (0.381) –ve MA <1 × 104 0 0 310 0.14 (0.15) –ve RC 7.9 × 103 1+ 0 3958 0.008 (0.365) –ve AN <1 × 104 0 0 938 0.0053 (0.61) +ve NJ <1 × 104 0 0 419 0.012 (0.01) –ve LK 2.3 × 105 1+ 11 235 0.000072 (0.09) –ve MZ <1 × 104 0 0 2114 0.00028 (0.32) –ve SN <1 × 104 0 0 116 0.0067 (0.013) –ve KI <1 × 104 0 0 137 0.0064 (0.051) –ve Total positives 3/12 7/12 2/12 2/12 aSemiquantitative estimation of the density of AFB present in smear. b% of solidly stained AFB representing viable bacillary population in smear. c>390 CPM, considered as positive metabolic activity. dP < 0.05 considered signiÞ cant. e+ve - >1 × 105/foot pad and –ve - <1 × 104/footpad

Table 4: Peak counts per minute output in Buddemeyer assay and the corresponding ATP assay (expressed in pmol) and MFP test results in treated BT-BB cases (n = 15) Patient Count/mL BIa MIb Buddemeyerc ATP in pmold MFPe code (%) (CPM) (P-value) DP <1 × 104 0 0 311 0.285 (0.08) –ve PP <1 × 104 0 0 824 0.48 (0.047) –ve RS <1 × 104 0 0 209 0.123 (0.063) –ve KS <1 × 104 0 0 1105 0.1 (0.009) –ve AK <1 × 104 0 0 224 0.045 (0.061) –ve PB <1 × 104 0 0 420 0.250 (0.311) –ve AY <1 × 104 0 0 367 0.3 9 (0.191) –ve RM <1 × 104 0 0 119 0 (0.311) –ve YP <1 × 104 0 0 446 0.000064 (0.06) –ve SG <1 × 104 0 0 143 0.004 (0.121) –ve LK <1 × 104 0 0 578 0.11 (0.056) –ve DC <1 × 104 0 0 718 0.057 (0.105) –ve MN <1 × 104 0 0 3027 0.24 (0.11) –ve SK <1 × 104 0 0 211 0.2 (0.15) –ve NS <1 × 104 0 0 207 0.075 (0.02) –ve Total positives 0/15 7/15 3/15 0/15 aSemiquantitative estimation of the density of AFB present in smear. b% of solidly stained AFB representing viable bacillary population in smear. c>390 CPM, considered as positive metabolic activity. dP < 0.05 considered signiÞ cant. e+ve - >1 × 105/foot pad and –ve - <1 × 104/foot pad

Table 5: Summary data showing no. and (%) of cases detected as positive (indicating presence of viable M. leprae) by ATP, Buddemeyer and MFP test in four groups of cases Groups of cases Buddemeyer assay* (%) ATP assay** (%) MFP test (%) Total no. of cases tested Untreated BL-LL 17 (85) 12 (60) 17 (85) 20 Treated BL-LL 8 (61) 7 (54) 0 (0) 13 Untreated BT-BB 7 (58) 2 (16) 2 (16) 12 Treated BT-BB 7 (46) 3 (20) 0 (0) 15 Positivity scores are comparable in all the three tests in untreated groups (1 and 3). However in treated groups (2 and 4), both the in vitro tests showed positivity in a good proportion of cases but none in MFP test. *Results of Buddemeyer assay vs. MFP test in groups 2 and 4 statistically signiÞ cant (P < 0.05). **Results of ATP assay vs. MFP test in group 2 statistically signiÞ cant (P < 0.05)

www.ijmm.org 362 Indian Journal of Medical Microbiology vol. 25, No. 4

The differences in the results of Buddemeyer assay and a longitudinal study and comparison with another proved MFP test were not signiÞ cant in untreated groups (Tables sensitive method, probably molecular method based on RNA 1 and 3), indicating good agreement between the two. In may help in further validating the assay system. treated groups (Tables 2 and 4), Buddemeyer assay showed better sensitivity and the differences were statistically Presence of viable M. leprae after MB-MDT treatment 17-20 signiÞ cant (P < 0.05). Overall one-to-one concordance has been cited in the literature. Poor sensitivity of MFP between the results of two tests was observed in 38/60 (63%) test as a viability detection method in treated cases is also cases. Remaining 21 cases (35%) showed discordance, of well recognized. It is known that the multiplication of few which 20 (33%) were positive in Buddemeyer assay but viable organisms among mostly dead cells is hampered due 14 negative in the MFP test. One case (1.6%) was positive in to immune response mounted by the normal mice and is MFP test that was negative in Buddemeyer assay (Table 1). the reason why immunocompromised mice are preferred in the detection of persisters.21 Needless to say that the latter is In treated BL-LL cases, the ATP assay showed a more difÞ cult to maintain besides the cost. signiÞ cant difference when compared with MFP test results (P < 0.05), whereas in the other three groups the results were It was observed that there was no linear relationship comparable (Table 5). One-to-one concordance between two between the CPM output and BI and MI that is the tests was observed in 41 (68%) cases. Remaining 19 (32%) microscopic hallmark of infectivity and viability of bacteria. cases showed discordance, of which 12 (20%) were positive Similar lack of correlation between BI/MI and other in vitro in ATP assay but negative in MFP test and 7 (11.6%) were viability detection methods like FDA-EB staining, thymidine 4,8 negative in ATP assay but positive in MFP test. uptake and ATP assay have been reported. Moreover in this study, in few particularly high BI cases, the cpm output was Sensitivity and speciÞ city of both the in vitro methods very inconsistent with BI (Table 1). Two likely explanations were determined using MFP test as gold standard in could be inhibition of metabolic activity by host tissue the untreated cases only. The calculated sensitivity and or suppression of metabolic activity due to competitive speciÞ city with Buddemeyer assay was 75 and 87.5% and inhibition in the medium. Although all the earlier studies with ATP assay it was 85.7 and 61%, respectively. used puriÞ ed M. leprae, in this study no puriÞ cation steps were applied to remove host tissue to prevent the loss of Discussion bacterial cells. However, a constant weight to volume ratio during homogenization, i.e., 0.1 g/mL was maintained Overall, both the in vitro assays showed a better sensitivity throughout and a Þ xed volume of homogenate, i.e., 0.3 mL, as compared to MFP test in detecting viable M. leprae in was used for the Buddemeyer assay regardless of the clinical samples. Among the two in vitro methods, Buddemeyer bacterial count. A further investigation along this line may assay emerged as more sensitive in all four groups of cases be in order that may help in improving the sensitivity and (Tables 1-5). In the untreated group of cases, the differences in speciÞ city of the assay system. the results were not statistically signiÞ cant when compared to MFP test and ATP assay indicating a good agreement between The usefulness of Buddemeyer assay system in the three tests. Moreover, in untreated BT-BB group of cases, 9 determining the efÞ cacy of various antileprosy agents has been out of 12 were negative for AFB, but Buddemeyer assay scored established.11,12 Radiorespirometric method has also been used positive in 7 (58%) cases as against two cases (16%) positive for detecting M. .22,23 In a parallel study on many by both ATP assay and MFP test. Considering the limitation of clinical samples, we record the sensitivity and speciÞ city the microscopic method where in the lower limit of sensitivity of Buddemeyer assay as 75% and 74.2% respectively, using is 1 × 104 cells/mL, both the in vitro assay systems have MFP test as gold standard.[13] The results of that study also detected viable cells in a good proportion of BT-BB cases. indicated its better utility in treated cases, the fact that has been reiterated in this study. Notably, in the Buddemeyer assay, a good proportion of cases scored positive in both the treated groups (P < 0.05) To conclude, among the two in vitro tests, Buddemeyer while in the ATP assay signiÞ cant positivity was recorded assay emerged as a better and a more sensitive method. Our only in treated BL-LL group (P < 0.05). On the other hand, Þ ndings suggest that it will be a good tool in day-to-day none of the treated BT-BB and BL-LL cases scored positive in patient care in detecting persisters, relapses and determining the MFP test. This Þ nding in turn could be interpreted in two the efÞ cacy of chemotherapy but we hasten to add that further ways. Firstly, there are a good proportion of treated cases that veriÞ cation to rule out false positivity would be needed. still harbour viable M. leprae, if proven true will have a great implication on treatment efÞ cacy. Secondly, it can be argued Acknowledgements that in the in vitro tests, some of the results obtained could be We gratefully thank Dr Sunil Ghate, Medical OfÞ cer, The false positives. It should be noted that in the untreated group Foundation for Medical Research (FMR), for performing the of cases, the Buddemeyer assay has shown a speciÞ city of biopsies, Mrs Anju Wakade, Research assistant and Fatema 87.5%, which is within the acceptable limits. Never the less Khambati, Msc Research student, FMR for technical help. This

www.ijmm.org October-December 2007 Agrawal and Shetty - Detection of Viable M. leprae by Buddemeyer Assay 363 study was supported by a grant (5/8/3(6)/2001-ECD-1) from the 13. Agrawal VP & Shetty, VP. Comparison of the mouse foot Indian council of Medical Research (ICMR). pad test with a Buddemeyer type radiorespirometric assay in detecting viable Mycobacterium leprae in human lesional References biopsies. Ind J Dermatol Venereol Leprol 2007;73:384-8.

1. Shephard CC. The experimental disease that follows the 14. Ridley DS, Jopling WH. ClassiÞ cation of leprosy according to infections of human leprosy bacilli into the foot pads of mice. immunity: A Þ ve group system. Int J Lepr Other Mycobact Dis J Exp Med 1960;112:445-54. 1966;34:255-73.

2. Shepard CC. A survey of drugs with activity against M. leprae. 15. World Health Organization. Laboratory techniques for leprosy. Int J Lepr Other Mycobact Dis 1971;39:340-8. WHO/CDS/LEP/86.4. World Health Organization: Geneva; 1987. 3. Desikan KV. Correlation of morphology with viability of Mycobacterium leprae. Lepr India 1976;48:391-7. 16. Buddemeyer E, Hutchinson R, Cooper M. Automatic quantitative radiometric assay of bacterial metabolism. Clin 4. In vitro methods for rapid monitoring of drug therapy and drug Chem 1976;22:4159-64. resistance in leprosy. ICMR Bull 2001;31:73-84. 17. Gupta UD, Katoch K, Singh HB, Natrajan M, Sharma VD, 5. Dhople AM. Adenosine triphosphate content of Mycobacterium Katoch VM. Detection of viable organisms in leprosy patients leprae from leprosy patients. Int J Lepr Other Mycobact Dis treated with MDT. Acta Leprol 1999;11:89-92. 1984;52:183-8. 18. Chae GT, Kim MJ, Kang TJ, Lee SB, Shin HK, Kim JP, 6. Gupta UD, Katoch K, Sharma RK, Singh HB, Natragan et al. DNA-PCR and RNA-PCR for the 18-kDa gene of M, Singh D, et al. Analysis of quantitative relationship Mycobacterium leprae to assess the efÞ cacy of multi-drug between viability determination in leprosy by MFP, ATP therapy for leprosy. J Med Microbiol 2002;51:417-22. bioluminescence and gene ampliÞ cation assay. Int J Lepr Other Mycobact Dis 2001;69:328-34. 19. Ebenezer GJ, Daniel S, Norman G, Daniel E, Job CK. Are viable Mycobacterium leprae present in lepromatous patients 7. Jagannathan R, Mahadevan PR. Concurrence of the in vitro after completion of 12 months’ and 24 months’ multi-drug methods of Fc receptor assay and ATP estimation for viability therapy? Indian J Lepr 2004;76:199-206. of Mycobacterium leprae. Med Sci Res 1987;15:1377-8. 20. Shetty VP, Suchitra K, Uplekar MW, Antia NH. Higher 8. Katoch VM, Katoch K, Ramu G, Sharma VD, Datta AK, incidence of viable Mycobacterium leprae with in nerve Shivannavar CT, et al. In vitro methods for determination as compared to skin among multibacillary leprosy patients of viability of mycobacteria: Comparison of ATP content, released from multidrug therapy. Lepr Rev 1997;68:131-8. morphological index and FDA-EB ß uorescent staining in 21. Shetty VP, Suchitra K, Uplekar MW, Antia NH. Persistence Mycobacteria leprae. Lepr Rev 1988;59:137-43. of Mycobacterium leprae in the peripheral nerve as compared 9. Franzblau SG. Oxidation of palmitic acid by Mycobacterium to the skin of multidrug-treated leprosy patients. Lepr Rev leprae in an axenic medium. J Clin Microbiol 1988;26:18-21. 1992;63:329-36.

10. Truman RW, Krahenuhl JL. Viable M. leprae as a research 22. Shah DH, Deodhar MN, Ganatra RD, Narkar AA, Buddemeyer reagent. Int J lepr Other Mycobact Dis 2001;69:1-12. EU. A rapid radiometric method for detection of M. tuberculosis: Optimization of experimental conditions. Int J 11. Franzblau SG, Biswas AN, Jenner P, Colston MJ. Double- Nucl Med Biol 1984;11:283-6. blind evaluation of BACTEC and Buddemeyer-type radiorespirometric assays for in vitro screening of antileprosy 23. Shah DH, Devdhar MN, Ganatra RD, Kale PN, Virdi SS, agents. Lepr Rev 1992;63:125-33. Deshmukh MD. ModiÞ ed rapid radiometric method for detection of Mycobacterium tuberculosis from sputum samples. 12. Chan GP, Garcia-Ignacio BY, Chavez VE, Livelo JB, Int J Nucl Med Biol 1985;12:333-5. Jimenez CL, Parilla ML, et al. Clinical trial of clarithromycin for lepromatous leprosy. Antimicrob Agents Chemother Source of Support: Nil, Conß ict of Interest: None declared. 1994;38:515-7.

www.ijmm.org