International Journal of STD & AIDS >
Volume 19, Number 12 >
Pp. 851-855
Original research articles |
* Institute for Health and Human Development, University of East London, Arthur Edwards Building, Romford Road, London;
Department of Reproductive Health and Research, World Health Organization, Geneva, Switzerland;
Sexually Transmitted Bacteria Reference Laboratory, Health Protection Agency for Infections, 61 Colindale Avenue, London;
Department of Epidemiology and Social Medicine, University of Antwerp, Belgium;
** Moscow Gabrichevsky Institute of Microbiology, City Department of Health, Moscow;
Gamalea Institute of Epidemiology and Microbiology, Russian Academy of Science, Moscow, Russian Federation;
Division of Medicine, Imperial College London, St Mary's Campus, Paddington, London, UK
Correspondence to: Professor Adrian Renton, Institute for Health and Human Development, University of East London, Arthur Edwards Building, Romford Road, London E15 4LZ, UK Email: a.renton{at}uel.ac.uk
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Key Words: Chlamydia trachomatis • diagnosis • fluorescent antibody test • ligase chain reaction • Russian clinics
| INTRODUCTION |
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Infections with Chlamydia trachomatis are the most common acquired STIs in developed countries, and often lead to serious sequelae including pelvic inflammatory disease and tubal infertility.7,8 The majority of recently acquired urogenital infections in both men and women are asymptomatic.9
Development of laboratory tests for C. trachomatis that are affordable and have adequate performance for routine diagnostic or screening use has been proved to be difficult. While culture was traditionally considered the gold-standard test, studies conducted during the 1980s suggested that direct microscopic identification of C. trachomatis elementary bodies in genital tract smears using fluorescent antibody in a direct fluorescent antibody (DFA) test10 was more sensitive in expert hands. However, DFA tests are time-consuming, technically demanding and performance may vary with operator and antibody preparation used. Despite these problems, and because labour supply within STD services in Russia has been abundant, DFA tests using Russian-manufactured antibody preparations have, to some extent, remained the standard for the diagnosis of genital chlamydial infection.
A major advance over recent years has been the commercial introduction and widespread adoption in Europe and North America of nucleic acid amplification tests (NAATs) for C. trachomatis, including the polymerase chain reaction (PCR, Roche Amplicor, Roche Diagnostics), ligase chain reaction (LCR, Abbott LCx, Maidenhead, UK), transcription-mediated amplification (TMA, GenProbe AmpCT and APTIMA [Gen Probe San Diego, CA, USA]) and strand displacement amplification (SDA, Becton Dickinson ProbeTec [NJ, USA]). These tests have shown high sensitivity and specificity when used to examine swabs from urethral and cervical epithelium as well as vaginal and urine specimens, despite relatively low organism load in the latter samples11,12 and are delivered in batch-processing formats suitable for large-scale diagnostic or screening use.
Given the importance of achieving STD control, both for itself, and for the prevention of generalized HIV epidemics, we assessed the performance in routine practice of DFA testing carried out on genital specimens in three Russian STD clinics compared with that of a NAAT (LCR).
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We recruited men and women over the age of 18 years attending three STD clinics, designated A, B and C, in the Moscow Oblast between March 2000 and March 2001 who were assessed to be at high risk for chlamydial and gonococcal infections. They were tested for C. trachomatis by both a DFA test and a NAAT (LCR). The protocol, instruments and all subsequent amendments were reviewed and ratified by a District Health Authority Ethics Review Board in London, UK, and by the Ethics Review Board of the Central Institute of Skin and Venereal Diseases, Moscow, Russian Federation.
A detailed operations manual for the study was prepared and provided to each participating clinic, and staff were given training. Subjects were included who tested negative for gonorrhoea on an initial Gram-stained genital smear and who either had an abnormal clinical examination (e.g. vaginal discharge), were the sexual partners of patients with gonorrhoea, or, in a smear, had evidence of polymorphonuclear leucocytosis, or Gram-negative organisms which were extracellular, or intracellular but of atypical morphology. Subjects were recruited as part of a wider trial of the performance of gonococcal diagnostic tests.
Patients meeting the inclusion criteria were invited to participate by the study doctor, provided with a written information sheet, and asked to sign a consent form indicating their willingness to participate. Consenting patients were then allocated a study number. All subjects were interviewed by the study doctor using a standard proforma to obtain their characteristics and medical and sexual history, and these and the results of the previous and a further clinical examination were recorded on the proforma. Then cervical swabs were obtained from women and urethral swabs from men for a C. trachomatis DFA test, culture for Neisseria gonorrhoeae and LCR assay. All subjects who were regarded as C. trachomatis-positive by the local DFA test were advised to abstain from sexual activity and were treated with doxycycline 100 mg b.i.d. for seven days and further managed according to standard clinical guidelines. Subjects who were found to be Gram-stain or culture-positive for N. gonorrhoeae after collection of specimens for the study were given ciprofloxacin 500 mg orally and doxycycline 200 mg b.i.d. for one week.
Specimen collection, processing and microbiological methods
Cervical or urethral specimens were obtained from each subject at the recruitment visit. A disposable loop specimen for a DFA test was applied to a slide and the smear stained and read in Moscow according to the manufacturer's protocol. A specimen for the LCR assay, to be carried out in London, was obtained using a thin LCR swab, placed in transport medium, and refrigerated immediately at +4°C. Such specimens in transport medium were taken on ice, on the same day, to the microbiology laboratory of the Central Institute of Skin and Venereal Diseases. On arrival, these specimens were placed immediately at –80°C. Before transportation to London all specimens were thawed, mixed thoroughly and then the swab squeezed against the side of the tube and discarded. The sample was divided into two equal volumes in 2 mL screw-capped vials and the aliquots refrozen at –80°C. One aliquot was left frozen at –80°C and the other was transported to London. Aliquots were packed in boxes of 100. The vials were kept cold but they thawed during transport. They were, however, refrozen at –80°C within six hours of leaving Russia. All specimens obtained were tested for C. trachomatis using the LCR assay (Abbott Laboratories) according to the manufacturer's instructions. Specimens were categorized as positive, negative or equivocal according to the manufacturer's cut-off value. Specimens providing positive results were retested at a dilution of 1:4 as were those for which there were equivocal results. After this, any equivocal results were considered negative. Box records were kept for all specimens to check for evidence of cross-contamination in transport, but none was found.
Some specimens taken from patients attending the STD clinic of St Mary's Hospital, London, were also examined in London using C. trachomatis antibody that was part of a Russian DFA test.
Data were analysed using SAS version 8.2. Concordant and discordant positive and negative results were cross-tabulated and sensitivities and specificities calculated. Exact 95% CI on sensitivities and specificities were calculated from exact binomial distributions.
| RESULTS |
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Two hundred and four women and 232 men were recruited to the study. Of the women, 108 (53%) met a single inclusion criterion (6% complaining of an abnormal vaginal discharge, 21% being contacts of men with gonorrhoea and 26% having an abnormal cervical smear); 96 (47%) met two or more inclusion criteria. Of these, 21% were aged 18–19 years; 28% aged 20–24 years and 51% were 25 years or more. Ten women had taken antibiotics in the previous month and four had performed vaginal cleaning in the morning prior to clinic attendance. Seventy-four percent of women complained of genital symptoms and 79% had evidence of an abnormal vaginal discharge on examination. Forty-three percent of women reported their last vaginal sexual intercourse as being within the previous week, and 24% between eight and 14 days prior to recruitment. Twenty percent of women reported more than one sexual partner in the previous week and 77% more than one partner in the previous month. The influence of these factors on the odds of chlamydia-positivity by the LCR among 176 women for whom LCR results were available (prevalence by LCR = 19%; n = 34) was examined by both univariate and multivariate (logistic regression) analysis. Having had more than one partner in the previous week was the only factor significantly associated (adjusted OR = 5.2; 95% CI = 1.5–18.7; P = 0.012) on multivariate analysis.
A total of 232 men were recruited to the study. Of these, 146 (63%) met a single inclusion criterion (9% complaining of an abnormal urethral discharge, 14% being contacts of women with gonorrhoea and 40% having an abnormal Gram-stained urethral smear); 86 (37%) met two or more inclusion criteria. There were 15% aged 18–19 years, 29% aged 20–24 years and 66% were 25 years or more. Seven had taken antibiotics in the previous month. Seventy-eight percent of men complained of genital symptoms and 78% had evidence of an abnormal urethral discharge on examination. Thirty-four percent reported last vaginal sexual intercourse within the previous week, and 19% between eight and 14 days prior to recruitment. None reported having more than one new sexual partner in the previous month. The influence of these factors on the odds of chlamydia-positivity by the LCR among 201 men for whom LCR results were available (prevalence by LCR = 33%; n = 66) was examined by both univariate and multivariate (logistic regression) analysis. Recruitment at clinic C was the only factor significantly associated (adjusted OR = 3.1; 95% CI = 1.2–8.1; P = 0.0282) on multivariate analysis.
Performance of DFA tests compared with LCR: patient-based analysis
Specimens for both DFA tests and the LCR assay were available from 171 women and 201 men. Among women the prevalence of C. trachomatis as judged by the LCR assay was 19% (n = 33), and among men 33% (n = 66). All LCR-positive results were ones confirmed at a 1:4 dilution of the original specimen. Numbers of concordant and discordant tests and sensitivities and specificities for DFA tests compared with the LCR (gold standard) are shown in Table 1, separately for each clinic and for all clinics taken together.
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The sensitivity and specificity of DFA tests among men were: 13% (95% CI: 0–35) and 91% (95% CI: 81–100), respectively, at clinic A; 8% (95% CI: 0–19) and 88% (95% CI: 79–96) at clinic B, and 9% (95% CI: 2–16) and 90% (95% CI: 83–94) at clinic C. Among men recruited from all clinics, the overall sensitivity of DFA tests was 9% (95% CI: 2–16) and the specificity was 90% (95% CI: 83–94).
We compared the distribution of LCR optical density (OD) readings between LCR-positive results that were DFA-positive (concordant positives) and LCR-positive results that were DFA-negative (discordant positives) separately for men and women. For both sexes, concordant-positive results had a higher median OD reading than discordant-positives, and concordant-negatives had a lower median OD reading than discordant-negatives, but the differences were not statistically significant by Kruskall-Wallis 
2 analysis. We also examined differences in subject characteristics according to test concordance separately for men and women. No statistically significant differences were found by either univariate or multivariate analysis between concordant- and discordant-positives, or between concordant- and discordant-negatives for either sex. Among 31 women who had positive LCR results but negative DFA results, LCR results for a second specimen were available in 30 cases and, of these, 22 (78%) were positive and eight were negative. Among 60 men who had positive LCR results but negative DFA results, LCR results for a second specimen were available in 45 cases and, of these, 41 (91%) were positive and four were negative.
We calculated predictive values of positive and negative tests for different levels of true chlamydial prevalence among groups of women to be tested and 95% CI for these (Figure 1 – top panel). We also calculated the expected numbers of true- and false-positives and -negatives per 100 women tested, at different group prevalences (Figure 1 – bottom panel). The prevalence of chlamydial infection may be expected to be around 10% in sexually active groups of young women. At a prevalence of 11%, the DFA test would yield one true-positive, seven false-positive, 82 true-negative and 10 false-negative results per 100 tested. At a prevalence of 41%, that might be expected in high-risk groups such as commercial sex workers, the DFA test would yield two true-positive, five false-positive, 54 true-negative and 43 false-negative results per 100 tested.
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Genital tract specimens taken from patients in London were used. Thirty specimens that were shown to be C. trachomatis-positive by both the LCR assay and the MicroTrak DFA test were examined by a DFA test using a Russian propriety C. trachomatis antibody. Because of intense non-specific background staining, it was not possible to detect typical fluorescing elementary bodies with any certainty.
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Secondly, and probably the main reason for the discrepancy between the LCR assay and DFA test results is the poor specificity of the locally produced antibody preparation. This was amply demonstrated in London where smears stained with a Russian proprietary antibody preparation were very difficult to read because of high levels of background staining. Undoubtedly, this must have hampered staff in the Russian Federation, particularly where specimens contained small numbers of chlamydial elementary bodies. Suffice to say, the performance of the DFA test was consistently poor in the three clinics studied. Estimates of sensitivity and specificity make this test inadequate for screening either low prevalence groups, where the vast majority of positive results will be false and 90% of true-positive results will be missed, or high prevalence groups, where almost 50% of negative results will be false. Thus, the test fails to detect the majority of cases of true infection, and wrongly identifies many individuals to be infected, who are actually not. It is interesting to know that others in Russia (St. Petersburg) have been disappointed with the sensitivity of Russian DFA tests when compared with an in-house PCR.14
We used the LCR assay because it was in routine use in London at the time and was a test with which we were familiar. The fact that the LCR assay is no longer commercially available does not diminish the significance of the results. A similar outcome would have been seen if another NAAT, currently available, of similar high sensitivity had been used. As far as the DFA test is concerned, it is not the one to be used for routine testing on a large scale, although it may still be valuable in situations where small numbers of specimens are to be tested. This being so and given the major epidemics of STDs and HIV that are currently occurring in the former Soviet Union, the importance of chlamydial infection in the pathogenesis of female infertility, and the concerns within Russia about the decline in fertility, there is a need for the Russian Ministry of Health to require registration of DFA tests based on systematic evaluation of performance, restrict use of unregistered tests, and for Russian manufacturers to seek ways of improving test performance. This is a plea endorsed by other Russian investigators14 and not to be seen as encouragement for widespread use of DFA testing. Indeed, the results overwhelmingly indicate that the focus of attention should be on the use of NAATs.
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