sign in sign up
<<
Home , Lymphogranuloma venereum, Neisseria gonorrhoeae

130 Genitourin Med 1992;68:130-133 Laboratory techniques in the investigation of , and donovanosis Genitourin Med: first published as 10.1136/sti.68.2.130 on 1 April 1992. Downloaded from

Eddy Van Dyck, Peter Piot

Introduction Three serovars LI, L2 and L3 are responsible Sexually transmitted diseases are not only for the vast majority of cases but other highly prevalent in many populations of the C trachomatis strains may also occasionally be developing world, but exhibit some special isolated.67 LGV is a chronic disease with features. These include a high rate of com- acute and late manifestations. A primary stage plications and sequelae as a result of delayed causing a small genital lesion is seen in a or inadequate treatment, the important minority of patients. Most common is the problem of antimicrobial resistance, par- secondary stage characterised by acute ticularly in gonorrhoeae and Haemo- inguinal (and femoral) lymphadenitis with philus ducreyi, and a high frequency of genital formation. Late complications may diseases.'2 occur in the anogenital area, such as ulcers, We briefly review three causes of genital , strictures and . ulceration which are largely confined to the Latent LGV may be reactivated in patients developing world, though all may be seen in with HIV infection with development of mul- Europe or North America as imported cases. tiple abcesses in the groins. The response to In addition, outbreaks of chancroid have been long courses of macrolides or is reported from various western countries, and poor, and the destruction of inguinal lymph- the disease has become even endemic in some nodes often results in lymphoedema of the populations of the USA.34 It therefore is not genitals, a chronic condition with persisting surprising that more research has been per- suppuration and . formed recently on chancroid than on dono- Donovanosis is a chronic infection of the vanosis and lymphogranuloma venereum genital region caused by Calymatobacterium (LGV) which are rare conditions in the granulomatis. The disease starts with a sub- western world. cutaneous nodule at the site of infection. This nodule enlarges and erodes through the skin Some clinical and epidemiological features of to reveal a red granulating ulcer. The disease http://sti.bmj.com/ tropicalgenital ulcers may spread hematogenously resulting in Genital ulcerations are complex diseases and cutaneous lesions at extragenital body sites. difficult to diagnose aetiologically on clinical grounds alone. and are Laboratory techniques common worldwide. The so called "tropical Chancroid The laboratory diagnosis of chan-

diseases" chancroid, lym- croid is based on the demonstration of the on September 30, 2021 by guest. Protected copyright. phogranuloma venereum and donovanosis causative agent, H ducreyi. Direct examination () are infrequent in the of ulcer material on Gram-stained smears may industrialised world but cause major health contribute to the diagnosis if typical small problems in many developing countries. Gram negative bacilli grouped in chains or Interest in genital ulcerations has considera- "schools offish" are observed. However, these bly increased following recognition that they typical features are infrequently seen on smears enhance the risk of transmission of the human from patients with culture-proven chancroid immunodeficiency (HIV) during sexual resulting in a sensitivity of much less than intercourse.' 50%.`10 In addition most genital ulcers har- Chancroid is caused by bour a polymicrobial flora due to secondary ducreyi. The disease starts with a painful contamination and the presence of Gram at the site of infection resulting in a negative bacilli in a smear may be misleading single or in multiple ulcers. Inguinal lym- and frequently results in a false positive diag- phadenopathy may be present in up to 50% of nosis.101' Thus, the specificity of a patients. for the diagnosis of chancroid is also less than In patients with immunosuppression caused 50%. Because of its low sensitivity and by HIV infection, extensive and persistent specificity, microscopic examination ofa Gram- Department of genital ulcers may be present without bubo stained smear is not recommended for the Microbiology, development. Despite the fact that H ducreyi diagnosis of chancroid. Institute of Tropical isolates of these patients show a normal Accurate diagnosis of chancroid depends on Medicine, Antwerp, Belgium antimicrobial sensitivity in vitro, the ulcers the ability to culture H ducreyi. Different E Van Dyck heal less frequently after short course treat- isolation media have been used with varying P Piot ment with cotrimoxazole or , success. For a review see table 1. Nutritional Correspondence to and often fail to respond to longer requirements of H ducreyi seem to be geogra- Professor P Piot antimicrobial courses. phically defined and may partly explain the Accepted for publication 1 November 1991 LGV is caused by trachomatis. variation in detection rates ofparticular culture Laboratory techniques in the investigation ofchancroid, lymphogranuloma venereum and donovanosis 131

Table 1 Comparison ofrecovery rates on dfferent culture mediafor isolation of more than 1,000 strains of H ducreyi isolated in eight countries on four continents between Total number Rate ofisolation 1978 and 1987, and also shows the mechanisms Study (reference) Site ofisolates (%) of resistance for sulfonamides, , Genitourin Med: first published as 10.1136/sti.68.2.130 on 1 April 1992. Downloaded from , streptomycin, kanamycin, Nsanze 198412 Nairobi 163 MH**: 75 GC`: 88 chloramphenicol and ampicillin. Dylewski 1986" Nairobi 75 MH*: 71 The use of non-culture techniques for diag- GC: 89 Kunimoto 1986`4 Nairobi 27 MH*: 74 nosing chancroid is very limited. Recently GC: 96 described dot immunobinding and enzyme Bieling9: 89 McDonald 1987"1 Nairobi 57 MH*: 53 immunoassays for the detection of circulating GC: 72 serum antibody show somewhat limited sen- Sheffield20: 2 Taylor 198416 Bangkok 45 MHt: 93 sitivity and IgG assays do not differentiate Heart Infusion'4: 76 between active and past infection and are Dieng Sarr 199117 Dakar 46 MHt: 100 GC: 4 currently more useful for epidemiology rather Bogaerts 198918 Kigali 38 MHZ: 76 than for diagnosis. `35 Geographical differences GC: 37 Bogaerts 1991 (unpublished) Kigali 44 MHt: 82 in the outer membrane profiles and antigenic GC: 36 composition of H ducreyi as well as the and MH*: Mueller Hinton enriched with chocolatized horse blood and Isovitalex. qualitative quantitative differences in the MHt: Mueller Hinton enriched with chocolatized horse blood, Isovitalex and 5% foetal calf human immune response to chancroid are serum. important obstacles to the development of MHt: Mueller Hinton enriched with 1% hemoglobin, 1% VX supplement and 5% foetal calf serum. diagnostic immunoassays. Monoclonal antibodies produced against outer membrane proteins and against media in different studies.'2 It has been shown react specifically with that the parallel use of two media may increase H ducreyi by using an immunofluorescence the isolation rate ofHducreyi to above 80%, for technique 36 (and E. Roggen, unpublished). cases with clinical chancroid; the two media can The performance of a monoclonal immuno- be incorporated into a single biplate to facilitate fluorescence assay directly on clinical their use.'2 13 specimens has proven to be competitive with To obtain optimal growth, plates have to be culture in sensitivity but to have a specificity of incubated at 33340C in a 5-10% CO2 and 60% only.' water saturated atmosphere for 3-4 days. DNA probes for the diagnosis of H ducreyi Colonies ofH ducreyi may vary in size depend- directly in clinical specimens have not been ing on incubation temperature and atmos- evaluated yet and the probes used for culture phere, growth medium and culture incubation confirmation are radioactively labelled with 32P, time. Colonies are nonmucoid, raised and gran- which is a handicap for routine use2324 ular and have a grayish-yellow colour and can be pushed intact across the surface of the agar Lymphogranuloma venereum The laboratory http://sti.bmj.com/ with an inoculating loop. Colonies are either diagnosis of LGV may be based on positive translucent or opaque and this variability in chlamydial , isolation of C trachomatis opacity gives the impression of a mixed non from the infected site, and histological iden- pure culture. H ducreyi is a tification ofchlamydia in infected tissue. In the with limited biochemical activity. Haemin is differential diagnosis between LGV and other required to initiate growth. Nitrate reduction possible conditions, syphilis and herpes should on September 30, 2021 by guest. Protected copyright. and alkaline phosphatase are important charac- always be considered. In serology three types of teristics. H ducreyi is oxidase positive when techniques are used: the complement-fixation tested with tetramethyl-p-phenylene-diamine. (CF) test, the single L-type immunofluores- Most isolates (nearly 100% in Africa) are ,B- cence test and the micro-immunofluorescence lactamase positive.22 A presumptive identifica- test (micro-IF). In general, a fourfold rise of tion of H ducreyi may be based on colony antibody in the course of suspected illness is characteristics, Gram stain and eventually ,B- diagnostic of active infection. However, lactamase production and oxidase.2122 Recen- seroconversion is demonstrated in only a min- tly, chromosomal DNA probes and ribosomal ority ofcases since most patients are seen by the RNA-derived oligonucleotide probes have physician after the acute stage. Moderate or been described which can be used for DNA high serum titres may also be caused by other hybridisation on bacterial cells to confirm the C trachomatis infections and may persist for identification of H ducreyi in culture.2324 many years. Single-point CF titres of > 1:64 To determine the antimicrobial suscep- are seen in the majority of patients and are tibility of H ducreyi, agar dilution, broth considered indicative for active LGV.37 Single microdilution and disk diffusion techniques L-type chlamydial fluorescence seems to be have been used but there are no recommenda- more sensitive than CF, but also broadly cross- tions for a standard medium or standard reacts with serovars of C trachomatis and, method. susceptibility varies possibly, C pneumoniae, and thus shows the geographically. Resistance has been observed same inconvenience as CF8`39 The most to sulfonamides, trimethoprim, , accurate diagnostic serologic assay is the micro- ampicillin, streptomycin, kanamycin, tetracy- IF test. During the active phase of LGV cline and chloramphenicol. H ducreyi has patients usually show high levels of IgM (> remained susceptible to erythromycin and 1:32) and IgG (> 1:512) with the type third generation . Table 2 of the infecting strain and much lower cross- summarises the antimicrobial susceptibility of reactivity with other C trachomatis strains ` 4. Piot 132 Dyck, Table 2 Antimicrobial susceptibility of clinical isolates ofH ducreyi phocytes suggests granuloma inguinale, and the use of Warthin-Starry silver impregnation Antimicrobial MIC range (mg/l)* Mechanism of resistance Molecular mass characteristic intracellular Sulfonamides 025-> 128 49 MDa stain demonstrating Ampicillin 0 03-> 128 plasmid 3.2 MDa; 5.7 MDa; organisms (Donovan bodies) is diagnostic.' 7.0 MDa Genitourin Med: first published as 10.1136/sti.68.2.130 on 1 April 1992. Downloaded from Tetracycline 0-125-128 plasmid 30 MDa; 34 MDa Kanamycin 05-128 plasmid 3-1 MDa Chloramphenicol 0-25-16 plasmid 34 MDa 1 Meheus A, De Schrijver A. Sexually transmitted diseases in Trimethoprim 0-125-32 transposon on chromosome the Third World. In: Harris JRW, Forster SM eds. Recent Erythromycin 0-0002-0-125 none Advances in Sexually Transmitted Diseases and AIDS 4, Ciproflaxcin 0-002-0-125 none 1991:201-17. Edinburgh: Churchill Livingstone. 0-001-0-06 none 2 Goeman J, Meheus A, Piot P. Epidemiologie des maladies sexuellement transmissibles dans les pays en developpe- *Source: Hammond et al,2 Sanson Le Pors et al,2 Slootmans et al,' Bilgeri et al,a Sturrn," ment a l'epoque du SIDA. Ann Soc Belge Med Trop Taylor et al,3" Bowmer et al," Dangor," Morse.33 1991;71:81-1 13. 3 Blackmore CA, Limpakarujarat K, Rigau-Perez JG, Albrit- ton WL, Greenwood JR. An outbreak of chancroid in Orange County, California: descriptive epidemiology and disease control measures. J Infect Dis 1985;151:840-44. 4 Schmid GP, Sanders LL, Blount JH, Alexander ER. A major disadvantage of the micro-IF is that Chancroid in the United States. Reestablishment of an old commercially ready to use reagents are not disease. JAMA 1987;258:3265-8. 5 Piot P, Laga M. Genital ulcers, other sexually transmitted available and it therefore is primarily used in a diseases, and the sexual transmission of HIV. BMJ few specialised reference laboratories. Cell cul- 1989;298:623-4. of 6 Schachter J, Meyer KF. Lymphogranuloma venereum. ture is another method used for the diagnosis Characterization of some recently isolated strains. J LGV. C trachomatis can be isolated from bubo Bacteriol 1969;99:636-8. 7 Piot P, Ballard RC, Fehler HG, Van Dyck E, Ursi JP, , genital ulcer and rectal tissue on cyclo- Meheus AZ. Isolation of from heximide treated McCoy cells or DEAE genital.ulcerations in Southern Africa. In: Mardh PA, Holmes KK, Oriel JD, Piot P, Schachter J eds. treated Hela 229 cells, but the recovery rate Chlamydial Infections 1982:115-8. Amsterdam: Elsevier seems to be lower than 50%.' Material from Biomedical Press. 8 Choudhary BP, Kumari S, Bhati R, Agarwal DS. Bac- the genital ulcer may be inoculated directly into teriological study of chancroid. Ind J Med Res 1982; cell tissue culture but bubo pus and tissues 76:379-85. to be in tissue culture 9 Coovadia YM, Kharsany A, Hoosen A. The microbial have homogenized aetiology of genital ulcers in black men in Durban, South medium to obtain a 10-20% (W/V) suspension, Africa. Genitourin Med 1985;61:266-9. 10 Sturm AW, Stolting GJ, Cormane RH, Zanen HC. Clinical and 10-11 and 102 dilutions are inoculated into and microbiological evaluation of 46 episodes of genital tissue culture. This is necessary to reduce a ulceration. Genitourin Med 1987;63:98-101. 11 Chapel TA, Brown WJ, Jeffris C, Stewart JA. The microbial toxic effect of the pus on the culture cells. flora of penile ulcerations. J Infect Dis 1979;137:50-6. 12 Nsanze H, Plummer FA, Maggwa ABN, Maitha G, Dylew- ski J, Piot P, Ronald AR. Comparison of media for the Donovanosis The causative agent Calymato- primary isolation of Haemophilus ducreyi. Sex Transm Dis bacterium granulomatis appears in vacuoles in 1984;11:6-9. 13 Dylewski J, Nsanze H, Maitha G, Ronald A. Laboratory the cytoplasma of large histiocytes and diagnosis of Haemophilus ducreyi: Sensitivity of culture occasionally in plasma cells and polymorpho- media. Diagn Microbiol Infect Dis 1986;4:241-5. 14 Kunimoto DY, Slaney L, Koss J, et al. Field testing of nuclear leukocytes. The intracellular organ- media for the isolation of modified Bieling Haemophilus http://sti.bmj.com/ isms are called Donovan bodies and have a ducreyi in Kenya. Eur J Clin Microbiol 1986;5:673-5. 15 MacDonald K, Cameron D, Irungu G, et al. Comparison of prominent clear capsule when mature. Culture Sheffield media with standard media in the isolation of ofthe bacterium in the chicken embryonic yolk Haemophilus ducreyi. Sex Transm Dis 1989;16:88-90. 16 Taylor DN, Dyangmani C, Suvongse C, et al. The role of sac has been reported, but is unsuccessful on Haemophilus ducreyi in penile ulcers in Bangkok, artificial culture media.4142 Thailand. Sex Transm Dis 1984;11:148-51. 17 Dieng Sarr A, Diouf G, Counillon E, et al. Diagnostic au The clinical manifestation is highly sugges- laboratoire du mou du bacille de Ducreyi:

tive of granuloma inguinale and may be con- Experience du Senegal. Seventh African Union against on September 30, 2021 by guest. Protected copyright. venereal Diseases and Treponematoses Regional Con- firmed by Giemsa's, Leishman's or Wright's ference; Lusaka, Zambia, 1991. Abstract 142. stain of a smear prepared from the lesion. A 18 Bogaerts J, Alvarez Ricart C, Van Dyck E, Piot P. The etiology of genital ulceration in Rwanda. Sex Transm Dis piece of clean granulation tissue is removed 1989;16:123-6. with a thin scalpel and crushed and spread on a 19 Sturm AW, Zanen HC. Characteristics of Haemophilus ducreyi in culture. J Clin Microbiol 1984;19:672-4. slide and the impression obtained is air dried 20 Hafiz S, McEntegart MG, Kinghorn GR. Sheffield medium and stained. Deep coloured ovoid with for the cultivation of Haemophilus ducreyi. Br J Venereal Dis 1984;60:196-8. or without capsule and with a closed safety-pin 21 Sottnek FO, Biddle JW, Kraus SJ, Weaver RE, Stewart JA. appearance are typical. Recently, a simple and Isolation and identification of Haemophilus ducreyi in a clinical study. J Clin Microbiol 1980;12:170-4. more rapid diagnostic method has been des- 22 Lubwoma SW, Plummer FA, Ndinya-Achola J, Nsanze H, cribed using a cotton swab for specimen collec- Namaara W. Isolation and identification of Haemophilus ducreyi in a clinical laboratory. J Med Microbiol tion and a one minute rapid differentiation 1986;22: 175-8. staining technique, using eosin and thiazine 23 Parsons LM, Shayegani M, Waring AL, Bopp LH. DNA probes for the identification ofHaemophilusducreyi. J Clin dye solutions. As compared with a classic stain, Microbiol 1989;27:1441-5. no discrepant results were obtained.43 24 Rossau R, Duhamel M, Jannes G, Decourt JL, Van Heuverswyn H. The development of specific rRNA- Donovanosis is likely to be confused with a derived oligonucleotide probes for Haemophilus ducreyi, number ofdiseases affecting the genital region. the causative agent of chancroid. J Gen Microbiol 1991;137:277-85. Perianal lesions may simulate condylomata 25 Hammond GW, Lian CJ, Wilt JC, Ronald AR. lata. Dark field microscopy and serology may Antimicrobial susceptibility of Haemophilus ducreyi. Antimicrob Agents Chemother 1978;7:39-43. exclude syphilis. Appropriate culture may help 26 Sanson Le Pors MJ, Casin IM,ThebaultMC,ArletG,Perol differentiate chancroid. Other diseases which Y. In vitro activities ofU-63366, a spectinomycin analog; roxithromycin (RU-28965), a new macrolide antibiotic; should be excluded are tuberculosis, and five quinolone derivates against Haemophilus ducreyi. amoebiasis, schistosomiasis and carcinoma. Antimicrob Agents Chemother 1986;30:512-3. may 27 Slootmnans L, Vanden Berghe DA, Van Dyck E, Piot P. Histological aspects of a biopsy specimen Susceptibility of 40 Haemophilus ducreyi strains to 34 be helpful: an ulcer with a mixed inflammatory antimicrobial products. Antimicrob Agents Chemother and 1983;24:564-7. infiltrate of plasma cells, his- 28 Bilgeri YR, Ballard RC, Duncan MD, MauffAC, Koornhof tiocytes, with a conspicuous absence of lym- HJ. Antimicrobial susceptibility of 103 strains of Haemo- Laboratory techniques in the investigation of chancroid, lymphogranuloma venereum and donovanosis 133

philus ducreyi isolated in Johannesburg. Antimicrob Agents tions using monoclonal antibody: a preliminary evalua- Chemother 1982;22:686-8. tion. Genitouin Med 1989;65:361-5. 29 Sturm AW. Comparison of antimicrobial susceptibility 37 Schachter J. Lymphogranuloma venereum and other non- patterns of fifty-seven strains of Haemophilus ducreyi ocular Chlamydia trachomatis infections. In: Hobson D, isolated in Amsterdam from 1978 to 1985. J Antimicrob Holmes KK eds. Nongonococcal and Related Chemother 1987;19:187-91. Infections 1977:91-7. Washington: American Society for

30 Taylor DN, Echeverria P, Hanchalay S, Pitarangsi C, Microbiology. Genitourin Med: first published as 10.1136/sti.68.2.130 on 1 April 1992. Downloaded from Slootmans L, Piot P. Antimicrobial susceptibility and 38 Treharne JD, Forsey T. Chlamydial serology. Br Med Bull characterization of outer membrane proteins of Haemo- 1983;39:194-200. philus ducreyi isolated in Thailand. J Clin Microbiol 39 Wang SP, Grayston JT. Micro immunofluorescence 1985j21:442-4. antibody responses in Chlamydia trachomatis infection, a 31 Bowmer MI, Nsanze H, D'Costa LJ, Dylewski J, Fransen L, review. In: Mardh PA, Holmes KK, Oriel JD, Piot P, Piot P, Ronald AR. Single dose ceftriaxone for chancroid. Schachter J eds. Chlamydial Infections 1982:301-16. Antimirob Agents Chemother 1987;31:67-9. Amsterdam: Elsevier Biomedical Press. 32 Dangor Y, Miller SD, Exposto F da L, Koornhof HJ. 40 Perine PL, Osoba AO. Lymphogranuloma venereum. In: Antimicrobial susceptibilities of Southern African Holmes KK, Mardh PA, Sparling PF, Wiesner PJ, Cates Isolates of Haemophilus ducreyi. Antimicrob Agents W, Lemon SM, Stamm WE eds. Sexually Transmitted Chemother 1988;32:1458-60. Diseases 1990:195-204. New York: McGraw-Hill 33 Morse SA. Chancroid and Haemophilus ducreyi. Clin Book Co. Microbiol Rev 1989;2:137-57. 41 Anderson K, De Monbreun WA, Goodpasture EW. An 34 Shalla WO, Sanders LL, Schmid GP, Tam MR, Morse SA. etiologic consideration of Donovania granulomatis cul- Use of dot immunobinding and immunofluorescence tivated from granuloma inguinale (three cases) in assays to investigate clinically suspected cases of chan- embryonic yolk. JExp Med 1945;81:25-39. croid. J Infect Dis 1986;153:879-87. 42 Sehgal VN, Shyam Prasad AL. Donovanosis. Current 35 Museyi K, Van DyckE, Vervoort T, Taylor D, Hoge C, Piot Concepts. Int J Dermatol 1986j25:8-16. P. Use of an enzyme immunoassay to detect serum IgG 43 O'Farrell N, Hoosen AA, Coetzee K, Van den Ende J. A antibodies to Haemophilus ducreyi. J Infect Dis 1988; rapid stain for the diagnosis of granuloma inguinale. 157:1039-43. Genitourin Med 1990;66:200-1. 36 Karim QN, Finn GY, Easmon CSF, et al. Rapid detection of 44 Freinkel AL. Histological aspects of sexually transmitted Haemophilus ducreyi in clinical and experimental infec- genital lesions. Histopathology 1987;11:819-31. http://sti.bmj.com/ on September 30, 2021 by guest. Protected copyright.