E. Van Dyck

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by enzyme immunoassay, culture, and three nucleic acid amplification tests.

ABSTRACT The purpose of this study was to evaluate and compare three commercially available nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis. Roche PCR and Becton Dickinson strand displacement amplification (SDA) were performed on 733 endocervical swab specimens from commercial sex workers. Abbott ligase chain reaction (LCR) was performed on a subset of 396 samples. Endocervical specimens from all women were also tested by culture for N. gonorrhoeae and by Syva MicroTrak enzyme immunoassay (EIA) for C. trachomatis. A positive N. gonorrhoeae result was defined as a positive result by culture or by two NAATs, and a positive C. trachomatis result was defined as a positive result by two tests. According to these definitions, the sensitivities and specificities for the subsample of 396 specimens of N. gonorrhoeae culture, PCR, SDA, and LCR were 69.8, 95.2, 88.9, and 88.9% and 100, 99.4, 100, and 99.1%, respectively; the sensitivities and specificities ofC. trachomatis EIA, PCR, SDA, and LCR were 42.0, 98.0, 94.0, and 90.0% and 100, 98.0, 100, and 98.6%, respectively. The performance characteristics of N. gonorrhoeae culture, PCR, and SDA and C. trachomatis EIA, PCR, and SDA for all 733 specimens were defined without inclusion of LCR results and by discrepant analysis after resolution of discordant N. gonorrhoeae PCR results and of discordant C. trachomatis EIA and PCR results by LCR testing. The sensitivities of N. gonorrhoeae culture, PCR, and SDA before and after LCR resolution were 67.8, 95.7, and 93.9% and 65, 95.8, and 90.0%, respectively. The sensitivities of C. trachomatis EIA, PCR, and SDA decreased from 39.4, 100, and 100% to 38.7, 98.7, and 94.7%, respectively. All three NAATs proved to be superior to N. gonorrhoeae culture and to C. trachomatis EIA. The accuracies of the different NAATs were quite similar. SDA was the only amplification assay with 100% specificity for detection of bothN. gonorrhoeae and C. trachomatis in endocervical specimens.

Diagnosing Genital Ulcer Disease in a Clinic for Sexually Transmitted Diseases in Amsterdam, The Netherlands

ABSTRACT The most common etiologic agents of genital ulcer disease (GUD) are herpes simplex virus type 1 (HSV-1), HSV-2, Treponema pallidum, and Haemophilus ducreyi. In an outpatient clinic for sexually transmitted diseases in Amsterdam, The Netherlands, specimens from 372 patients with GUD were collected from February to November 1996. Sera were collected at the time of the symptoms and, for most patients, also during follow-up visits. Swabs in viral transport medium were used for HSV culture and for detection of DNA. The most prevalent pathogen found was HSV-2, which was detected by culture in 35% of the patients and by PCR in 48% of the patients. Also, HSV-1 infection was more often detected by PCR (7.8%) than by culture (5.6%). Evidence for an active infection with T. pallidumwas found in 1.9% of the patients, using serological tests. A multiplex PCR for simultaneous T. pallidum and H. ducreyi DNA detection was positive for T. pallidum in 3.3% of the samples and for H. ducreyi in only 0.9% (3 out of 368) of the samples. The sensitivity of the PCR was superior to that of culture for HSV detection and to that of serology for T. pallidum detection. Specific H. ducreyiimmunoglobulin G antibodies were detected in sera of 5.2% of the patients, with no concordance between serology and PCR. In 37% of the cases, none of the tested microorganisms was detected. Performance of PCR in addition to conventional techniques significantly improved the diagnosis of GUD.

Comparison of culture and different PCR assays for detection of Trichomonas vaginalis in self collected vaginal swab specimens

Objectives: DNA amplification techniques have become widely used for the diagnosis of sexually transmitted infections. For the detection of Trichomonas vaginalis, PCR techniques are not yet widely used despite the publication of several assays. The sensitivity and specificity of five independent primer sets were determined on self collected vaginal specimens obtained from female commercial sex workers. Methods: Self collected specimens were obtained from symptomatic and asymptomatic women attending a female sex workers clinic in Abidjan, Côte d’Ivoire. Two vaginal specimens were collected, the first one was processed for culture and the second was processed for PCR analysis. PCR techniques for trichomonads were performed, using the primers as reported by Riley (TVA5/TVA6), Kengne (TVK3/TVK7), Madico (BTUB 9/BTUB 2), Shiao (IP1/IP2), and Mayta (TV1/TV2). An EIA amplicon detection method was designed for each of the primer sets. Results: True positive specimens were defined as culture positive and/or two positive PCR results with EIA amplicon detection in any combination. According to this definition a prevalence of 20% was obtained compared to 7% obtained by culture. The PCR primer set TVK3/TVK7 gave the highest sensitivity (89.2%). Poor sensitivities were obtained with the primer sets TV1/TV2 (60.2%) and TVA5/TVA6 (63.9%). PCR showed a sensitivity improvement of 2.4% up to 12% when EIA was used for amplicon detection. Conclusions: Overall, the sensitivities of the different PCR assays resulting from this study were lower than those previously described. These findings could be the result of the nature of the specimen population and suggests a strain variability.

Similar serological response to conventional therapy for syphilis among HIV-positive and HIV-negative women.

OBJECTIVES--To compare characteristics of syphilis serological reactivity in HIV positive (+) and HIV negative (-) female sex workers, as well as the serological response to therapy after treatment with intramuscular benzathine penicillin, 2.4 million U weekly, for three consecutive weeks. METHODS--Rapid plasma reagin (RPR) and Treponema pallidum haemagglutination assay (TPHA) results of 72 HIV-positive and 121 HIV-negative women reactive in both tests were assessed. The response to therapy was prospectively monitored with quantitative RPR serology in 47 HIV-positive and 73 HIV-negative patients. Cumulative probabilities of becoming nonreactive by RPR were compared at six months, one and two years after therapy. RESULTS--At enrolment, the geometric mean titres of RPR and TPHA were lower in HIV-positive patients (RPR, 1:2.6) than in HIV-negative patients (RPR, 1:3.8; p < 0.01). The evolution over time of RPR titres was similar among HIV-positive patients as compared to HIV-negative patients. Among patients with an initial RPR titre of < 1:8, 53% of HIV-positive and 44% of HIV-negative patients became RPR negative two years after therapy. Among patients with an RPR titre of 1:8 or greater at enrolment, 83% of HIV-positive and 90% of HIV-negative patients had reached at least a fourfold decline of RPR titres two years after therapy. CONCLUSIONS--Syphilis serology findings (both RPR and TPHA) may be altered in the presence of HIV infection, but the serological response to therapy was similar in HIV-positive and HIV-negative patients.

The vaginal microbial flora in non-specific vaginitis

The facultative and strictly anaerobic vaginal microbial flora was investigated in 40 women with non-specific vaginitis and in 40 control women seen in private gynaecological practice.Gardnerella vaginalis, anaerobic gram-negative bacilli, anaerobic gram-negative and gram-positive cocci were all associated with non-specific vaginitis (p<0.001), whereas lactobacilli occurred less frequently in non-specific vaginitis than in controls (p<0.01). The most common anaerobes wereVeillonella parvula, Bacteroides bivius, Bacteroides assaccharolyticus, Bacteroides capillosus andPeptococcus asaccharolyticus. Anaerobic gram-negative curved rods were found in 11% of cases of non-specific vaginitis. A characteristic pattern of short chain organic acids was found on gas liquid chromatographic analysis of vaginal secretions in non-specific vaginitis. A succinate/lactate peak ratio of 0.3 or more was found in 75% of women with non-specific vaginitis (p<0.001). Clue cells, a Positive amine test, a pH higher than 5.0, and the absence of lactobacilli on a Gram stained vaginal smear strongly correlated with non-specific vaginitis (p<0.001).

Effectiveness of norfloxacin and ofloxacin for treatment of gonorrhoea and decrease of in vitro susceptibility to quinolones over time in Rwanda.

OBJECTIVE--To study the effectiveness of single-dose norfloxacin and ofloxacin in the treatment of gonococcal urethritis in men, and to monitor in vitro antimicrobial susceptibility to these antibiotics over time. SETTING--Centre Médico-Social de Bilyogo, Kigali, Rwanda. The only clinic in Rwanda using quinolones for the treatment of gonorrhoea. METHODS--As part of a monitoring programme, men with gonococcal urethritis were evaluated after treatment with norfloxacin (800 mg) in 1986 and 1987, and after treatment with ofloxacin (400 mg) in 1989. RESULTS--Neisseria gonorrhoeae was eradicated from the urethra from 96.0% (189/197) and from 97.1% (166/171) men treated with norfloxacin and ofloxacin, respectively. Overall 38.2% of the pretreatment isolates produced penicillinase (PPNG isolates) and 20.4% (44/216) of the tested non-PPNG isolates were chromosomally resistant to penicillin (MIC > or = 2.0 mg/l). Resistance to tetracycline and thiamphenicol was common in both PPNG and non-PPNG and increased considerably in 1989. All isolates were susceptible to kanamycin, spectinomycin, ceftiaxone, norfloxacin, ofloxacin and ciprofloxacin. However, a higher number of isolates recovered in 1989 showed decreased susceptibility to the quinolones. Treatment failure occurred more often in subjects with isolates having MIC values > or = 0.06 mg/L of norfloxacin (p = 0.006). Seven out of 13 patients who did not respond to therapy had no signs nor symptoms of urethritis. CONCLUSION--Quinolone antibiotics are now indicated as a first line treatment of gonorrhoea in countries with a problem of antimicrobial multiresistance. However, antimicrobial susceptibility to the quinolones may decrease rapidly, and close monitoring of the in vitro susceptibility of N gonorrhoeae and the clinical effectiveness of the antibiotics is imperative.

Molecular typing of the actin gene of Trichomonas vaginalis isolates by PCR–restriction fragment length polymorphism

Human trichomoniasis, caused by the protozoan Trichomonas vaginalis, is a highly prevalent sexually transmitted infection. However, little is known about the degree of strain variability of T. vaginalis. A reliable classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, pathogenesis and transmission of T. vaginalis. A PCR-restriction fragment length polymorphism typing method was designed and evaluated using T. vaginalis isolates obtained after culture of vaginal specimens collected in the Democratic Republic of Congo and in Zambia. The variation of the actin gene of T. vaginalis was determined for three ATCC reference strains and 151 T. vaginalis isolates. Eight different types were identified, on the basis of the digestion patterns of the amplified actin gene, with each of the restriction enzymes HindII, MseI and RsaI. It was determined that the ATCC reference strains 30001, 30240 and 50141 were of actin genotypes G, H and E, respectively. The actin genotype type E was more common in the Democratic Republic of Congo, whereas type G was the commonest type in Zambia. Translation of the nucleotide sequence showed up to three amino acid substitutions. We developed a reproducible, sensitive and specific typing method for T. vaginalis, and were able to distinguish at least eight T. vaginalis actin genotypes. Further studies are needed to evaluate the method using clinical specimens and to determine the utility of the typing method for the genotypic characterization of T. vaginalis.

Ulcerative balanoposthitis associated with non-syphilitic spirochaetal infection.

Ulcerative balanoposthitis associated with non-syphilitic spirochaetal infection was found in 41 (12%) of 344 consecutive men with genital ulcer disease in two clinics in South Africa. All patients with non-syphilitic spirochaetal infection were uncircumcised. Non-syphilitic treponemes were seen in only two of 60 uncircumcised men who had urethritis without genital ulceration. In 14 patients with ulcerative balanoposthitis no cause of genital ulceration could be identified. Most patients presented with large, serpiginous, superficial, foul smelling, and tender ulcers, the base of which was purulent with undermined edges. Non-tender inguinal lymphadenopathy was present in seven of 14 patients. One female sexual partner of a patient with non-syphilitic spirochaetal infection had a vaginal infection with non-syphilitic spirochaetes.

Sexually transmitted diseases and human immunodeficiency virus infections in women attending an antenatal clinic in Abidjan, Côte d'Ivoire.

A cross-sectional survey was conducted among women attending an antenatal clinic in Abidjan to determine the prevalence of sexually transmitted diseases (STDs) and HIV infection, and to identify factors associated with the presence of gonococcal and/or chlamydial cervical infection. Among 546 women, 3.7% had a gonococcal infection and 5.5% had a chlamydial infection. The seroprevalence of syphilis and HIV was 1.1% and 16.2% respectively. Gonococcal and/or chlamydial cervical infection was associated with young age, the presence of endocervical mucopus and with more than 10 polymorphonuclear leucocytes per high power field in a vaginal smear. None of these associated factors had a large enough predictive value to allow its use as a diagnostic criterion. Sexually transmitted diseases are common in pregnant women in Abidjan. The development of rapid, inexpensive diagnostic tests for STD is a priority to improve the care of women attending antenatal clinics in the developing world.

Accuracy of two enzyme immunoassays and cell culture in the detection of Chlamydia trachomatis in low and high risk populations in Senegal.

Two enzyme immunoassays (EIAs), Chlamydiazyme (CZ; Abbott Laboratories) and Pathfinder (PF; Kallestadt), were compared with a cell culture technique in the detection of cervicalChlamydia trachomatis infection in 670 women in urban settings in Senegal (377 pregnant women and 293 prostitutes). Positive CZ and positive PF specimens were tested a second time using a monoclonal antibody blocking technique. True positive specimens were defined as those positive on culture or positive on EIA with confirmation of the result after blocking. Using this definition, the prevalence of genital chlamydial infection was 14.6 % and 14.3 % in pregnant women and prostitutes respectively. An important difference between the two populations was that the pregnant women were younger than the prostitutes, which might explain the fact that the prevalence of infection among the pregnant women was as high as that among the prostitutes, although the age-adjusted prevalence was higher among prostitutes than among pregnant women. The chlamydial detection rates of cell culture, CZ and PF were 62 % (26/42), 69 % (29/42) and 86 % (36/42) respectively in prostitutes and 76 % (42/55), 40 % (22/55) and 53 % (29/55) respectively in pregnant women. Agreement between the tests was 89 %, 85 % and 88 % for culture/CZ, culture/PF and CZ/PF respectively. However, when data were adjusted for chance agreement, kappa coefficients were 0.40 for culture/CZ, 0.34 for culture/PF and 0.48 for CZ/PF. These results indicate that the accuracy of the EIAs and cell culture may vary greatly in different populations: both EIAs showed a distinctly higher detection rate than culture in prostitutes and a significantly lower detection rate in pregnant women. Confirmation of positive EIA results with a blocking assay greatly enhanced the specificity of the antigen detection tests and should be obtained when using the EIAs in a clinical setting.