Urogenital Chlamydia trachomatis serovars in men and women with a symptomatic or asymptomatic infection: an association with clinical manifestations?

To determine whether certain Chlamydia trachomatis serovars are preferentially associated with a symptomatic or an asymptomatic course of infection, C. trachomatis serovar distributions were analyzed in symptomatically and asymptomatically infected persons. Furthermore, a possible association between C. trachomatis serovars and specific clinical symptoms was investigated. C. trachomatis-positive urine specimens from 219 asymptomatically infected men and women were obtained from population-based screening programs in Amsterdam. Two hundred twenty-one C. trachomatis-positive cervical and urethral swabs from symptomatically and asymptomatically infected men and women were obtained from several hospital-based departments. Serovars were determined using PCR-based genotyping, i.e., restriction fragment length polymorphism analysis of the nested-PCR-amplified omp1 gene. The most prevalent C. trachomatis serovars, D, E, and F, showed no association with either a symptomatic or asymptomatic course of infection. The most prominent differences found were (i) the association of serovar Ga with symptoms in men (P = 0.0027), specifically, dysuria (P < 0.0001), and (ii) detection of serovar Ia more often in asymptomatically infected people (men and women) (P = 0.035). Furthermore, in women, serovar K was associated with vaginal discharge (P = 0.002) and serovar variants were found only in women (P = 0.045).     

Validation of roche COBAS Amplicor assay for detection of Chlamydia trachomatis in rectal and pharyngeal specimens by an omp1 PCR assay

Screening guidelines for men who have sex with men (MSM) recommend testing of extragenital sites (pharyngeal and rectal) for gonorrhoea and chlamydia. Testing of specimens from these sites is not validated by most commercial nucleic amplification tests, such as the COBAS Amplicor assay. To investigate the utility of the COBAS Amplicor assay for detection of Chlamydia trachomatis in extragenital specimens, this study developed and evaluated confirmatory tests using the omp1 gene as an alternative target for amplification by PCR. Of anal and throat swabs collected from men in male-only saunas, 52 swabs that tested C. trachomatis positive by COBAS Amplicor and 30 swabs that tested as negative were included for confirmatory omp1 PCR testing. A total of 49 (94%) COBAS Amplicor-positive samples were confirmed by the omp1 PCR. A substantial proportion of specimens were confirmed by using a nested omp1 PCR (27%). Not confirmed by any omp1 PCR were three anal swabs (6%). It is most probable that these samples contained lower bacterial levels that were near or below the detection level of the omp1 PCR assays. The findings of this study support the confident reporting of C. trachomatis detected by COBAS Amplicor in extragenital specimens and support the utility of this assay as a screening test for MSM.     

Evaluation of a novel PCR-based assay for detection and identification of Chlamydia trachomatis serovars in cervical specimens

The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a rural Costa Rican study population. A total of 5,828 sexually active women participating in a community-based trial in Costa Rica were tested for C. trachomatis by HC2. A sample of 1,229 specimens consisting of 100% HC2 C. trachomatis-positive specimens (n = 827) and a random sample of 8% HC2 C. trachomatis-negative specimens (n = 402) were tested with the Ct-DT assay. Agreement between the two assays was determined by the unweighted kappa statistic. Discrepant specimens were tested with a second commercially available test (COBAS TaqMan). The Ct-DT-positive specimens were further analyzed with the Ct-DT genotyping step to investigate the distribution of 14 different C. trachomatis serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). After accounting for the sampling fraction selected for Ct-DT testing, crude agreement with the HC2 assay was 98% and the kappa was 0.92 (95% confidence interval [CI], 0.89 to 0.97). The 33 discordant samples that were further analyzed with the COBAS TaqMan test showed better agreement with the Ct-DT assay (31/33, P < 0.001). Among the 806 Ct-DT-positive samples, serovar E was the most common serovar (31%), followed by serovars F and D (both 21%) and serovar I (15%). In conclusion, the novel Ct-DT assay permits reliable detection and identification of C. trachomatis serovars.     

Prevalence and serovar distribution of asymptomatic cervical Chlamydia trachomatis infections as determined by highly sensitive PCR.

The prevalence rates and serovar distributions of Chlamydia trachomatis cervical infections were investigated in two different groups of women. Group I consisted of 393 asymptomatic young women (aged 17 to 30 years) who were invited to participate in a C. trachomatis screening program. Group II consisted of 734 randomly selected patients (aged 17 to 68 years) attending an inner-city gynecological outpatient clinic. C. trachomatis was detected in cervical scrapes by PCR specific for endogenous plasmid. These plasmid PCR-positive samples were subsequently subjected to genotyping by C. trachomatis-specific omp1 PCR-based restriction fragment length polymorphism analysis (J. Lan, J. M. M. Walboomers, R. Roosendaal, G. J. van Doornum, D. M. MacLaren, C. J. L. M. Meijer, and A. J. C. van den Brule, J. Clin. Microbiol. 31:1060-1065, 1993). The overall prevalence rates of C. trachomatis found in patients younger than 30 years were 9.2 and 11.8% in groups I and II, respectively. A clear age dependency was seen in group II, with the highest prevalence rate (20%) found in patients younger than 20 years, while the rate declined significantly after 30 years of age (5.9%). In women younger than 30 years, the genotyping results showed that serovars E, I, and D (in decreasing order) were frequent in group I, while serovars F, E, and G (in decreasing order) were predominantly found in group II. The study shows that C. trachomatis infections are highly prevalent in asymptomatic young women. The different serovar distributions found most likely reflect the different compositions of the study groups, but additional analysis of the case histories of individual patients suggests that certain serovars might be associated with symptomatic (i.e., serovar G) or asymptomatic (i.e., serovars D and I) infections.     

Combination of PCR targeting the VD2 of omp1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens

In this study we developed and evaluated a new PCR-based typing assay, directed to the VD2 region of the omp1 gene, for the detection and typing of urogenital Chlamydia trachomatis infections. A nested VD2 PCR-reverse line blot (RLB) assay was developed for the typing of nine different urogenital serovars of C. trachomatis. The assay developed was tested with reference strains of C. trachomatis serovars and cervical scrapes of 86 Colombian women previously found to be positive for C. trachomatis by using plasmid PCR. Two sets of primers directed to the VD2 region of the omp1 gene of C. trachomatis were designed, and fragments of 220 and 166 bp were generated in the primary and nested PCRs, respectively. In addition, an RLB assay was developed to identify nine different urogenital serovars of C. trachomatis (Ba, D, E, F, G, H, I, J, and K) and group controls, including group B (Ba, D, and E), group C (I, J, K, and H), and an intermediate group (F and G). Using this assay, we were able to type 81 of the 86 samples (94.2%). Of these samples, 91.3% were single C. trachomatis infections, and 8.7% were multiple infections. The most common serovars identified were serovars D (22.2%), F (18.5%), G (13.6%), and E (12.3%). Of the women with multiple C. trachomatis infections, >50% contained both serovars D and E. The nested VD2 PCR-RLB developed is a simple, fast, and specific method for the identification of individual urogenital C. trachomatis serovars previously detected by using plasmid PCR. Moreover, it is an appropriate method for studying multiple C. trachomatis infections and for use in large epidemiological studies.     

External quality assessment program for Chlamydia trachomatis diagnostic testing by nucleic acid amplification assays

We report the results from 57 Australian diagnostic laboratories testing two external quality assessment panels using either the Roche Amplicor Chlamydia trachomatis test (R-PCR) or the Abbott LCx Chlamydia trachomatis assay (A-ligase chain reaction [LCR]). Panel samples were either normal urine spiked with Chlamydia trachomatis antigen or clinical urine specimens. There was no significant difference between laboratories or between assays in detection of C. trachomatis-positive clinical samples. Only at the lower limit of detection of the assays did the R-PCR demonstrate increased sensitivity over the A-LCR in the detection of C. trachomatis antigen. However, it was found that single-sample testing could lead to decreased test sensitivity. Detection of the presence of inhibitors of nucleic acid amplification differed between laboratories.     

Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars.

The amino acid sequences of major outer membrane proteins (MOMPs) from Chlamydia trachomatis serovars A, B, C, L1, and L2 are predominantly conserved but have four variable domains (VDs) in which major neutralizing and serotyping antigenic determinants are located. Because these MOMP VDs are primarily responsible for antigenic differences between serovars and are associated with important immunological and biological properties, we undertook studies focused on defining these sequences within the MOMPs of all 15 C. trachomatis serovars. We used oligonucleotide primer extension sequencing of MOMP mRNA to determine the nucleotide and deduced amino acid sequences of the four MOMP VDs of the 15 C. trachomatis serovars. Comparative amino acid sequence homologies of all four domains separated the serovars into three groups: group 1, serovars B, Ba, D, E, L1, and L2; group 2, serovars G and F; and group 3, serovars A, C, H, I, J, K, and L3. Hydrophilicity and charge values for each domain were determined. The MOMP VDs of given serovars with the greatest total hydrophilicity and charge values were found to be the location of antigenic determinants recognized by MOMP-specific monoclonal antibodies. These findings should be useful for predicting MOMP antigenic determinants and testing the antigenic properties of these VDs by using synthetic peptides corresponding to each MOMP VD. The potential usefulness of the VD sequence information is discussed in relation to the development of defined synthetic peptides and oligonucleotides that may be used to develop new serological and diagnostic assays for C. trachomatis infections.     

Bacteriology of the urethra in normal men and men with nongonococcal urethritis.

Sixty-nine Caucasian males without a previous history of urethritis and who developed nongonococcal urethritis (NGU) and 39 similar men without urethritis (NU) were cultured from the urethra for Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, aerobes, and anaerobes. C. trachomatis infection was proven by culture of serology in 26 (38%) of the NGU group and 1 (3%) of the NU group; the C. trachomatis-negative NGU group had significantly more U. urealyticum (81%) than the C. trachomatis-positive NGU group (42%) or the NU group (59%). Aerobes were isolated from significantly more NU men (91%) than from men with NGU (66%). The aerobic and anaerobic flora of the two NGU groups were similar. The NU group had significantly more aerobic lactobacilli. Haemophilus vaginalis, alpha-hemolytic streptococci (not Streptococcus faecalis), and anaerobes, predominantly Bacteroides species. This study has provided information about the prevalence and the variety of the aerobic and anaerobic microbiological flora of the anterior urethra of sexually active males. It does not implicate any bacteria other than C. trachomatis and U. urealyticum as potential causes of NGU.     

Duplex PCR system for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical specimens.

AIMS--To evaluate the use of a duplex polymerase chain reaction (PCR) assay for the simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical samples. METHODS--Genital swab specimens were obtained from both China (203 swabs) and Hong Kong (202 swabs). N gonorrhoeae and C trachomatis were detected in each specimen with a number of tests including enzyme immunoassays (IDEIA) and PCR assays using both single and double primer pairs. The primer pair for N gonorrhoeae was derived from the cppB gene on its cryptic plasmid and the PCR product was 390 base pairs long. For C trachomatis, the PCR product was 473 base pairs long, resulting from amplification of a sequence in the common 7.4 kilobase plasmid present in all serovars. For N gonorrhoeae, PCR results were also compared with those obtained by culture and Gram's smear of the discharges. RESULTS--For the 203 specimens collected in China, similar numbers of positive results (177) were obtained by both Gonozyme and duplex PCR for the detection of N gonorrhoeae. No discrepant results were found among the cultured specimens when Gonozyme and duplex PCR were compared. C trachomatis was detected in 47 specimens by duplex PCR, but was detected in only 28 by IDEIA. Of the 202 Hong Kong specimens, 46 were positive for N gonorrhoeae, detected by both Gonozyme and duplex PCR; 34 were positive for C trachomatis, 25 of which were detected by IDEIA and the remainder by duplex PCR. CONCLUSIONS--The duplex PCR assay is a satisfactory diagnostic tool for the simultaneous detection of N gonorrhoeae and C trachomatis in clinical swab samples. Further evaluation is suggested.     

Direct detection and genotyping of Chlamydia trachomatis in cervical scrapes by using polymerase chain reaction and restriction fragment length polymorphism analysis.

Detection and genotyping of Chlamydia trachomatis were optimized by using a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis performed directly with crude cells of cervical scrapes. Different PCR pretreatment methods were evaluated on samples which were positive for C. trachomatis by cell culture. In comparison with DNA extraction and different proteolytic digestion methods, a simple pretreatment of 10 min of boiling appeared to be optimal for PCR amplification. Crude samples (n = 209) were first screened for C. trachomatis by both cell culture and plasmid PCR. Subsequently, positive samples found by plasmid PCR were subjected to a direct omp1 PCR-based RFLP analysis to differentiate C. trachomatis serovars A to K, Ba, Da, and L1 to L3 and serovariant D-. All cervical scrapes that were found positive for C. trachomatis by cell culture (n = 30) were also positive by plasmid PCR and omp1 PCR and could be easily genotyped. In addition, of the culture-negative group, eight samples were found positive by plasmid PCR. Five of these eight samples were also positive by omp1 PCR; of these five, two were positive by a nested omp1 PCR. Genotyping by RFLP analysis of the 35 omp1 PCR-positive samples showed that serovars D, E, and F are the most prevalent types found in cervical scrapes, while serovariant D- was also detected. This study shows that direct PCR and PCR-based RFLP analysis are feasible for detection and genotyping of C. trachomatis in cervical scrapes and are more sensitive than culture-based serotyping.     

The role of chlamydia genus-specific and species-specific IgG antibody testing in predicting tubal disease in subfertile women

BACKGROUND: We evaluated whether measuring chlamydia genus- and species-specific immunoglobulin (Ig) G antibodies might improve the predictive value of C. trachomatis antibody testing (CAT) in screening for distal tubal pathology (DTP). METHODS: Serum of 313 subfertile women was tested for the presence of species-specific antibodies to C. trachomatis, C. pneumoniae and C. psittaci and genus-specific antibodies to chlamydia lipopolysaccharide (LPS). Only patients who had undergone a laparoscopy with tubal testing, to assess the grade of DTP, were included in this study. RESULTS: The presence of C. trachomatis antibodies was the only independent predictor for DTP. The predictive value of CAT for DTP could not be improved by adding test results of C. pneumoniae or LPS antibody testing. The role of C. psittaci could not be evaluated, due to the absence of C. psittaci-positive patients in our cohort. CONCLUSIONS: In spite of the high interspecies homology, C. pneumoniae does not contribute to the development of DTP. Anti-LPS antibodies, which are considered to be markers for ongoing infections, do not identify C. trachomatis-positive subfertile women who are at highest risk of DTP. The high prevalence of anti-LPS antibodies in C. trachomatis-positive subfertile women may suggest that C. trachomatis remains more active in the upper genital tract than currently is presumed.     

Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis

The aim of this study was to develop and evaluate multiplex and nested PCR-reverse line blot (RLB) hybridization assays for detection and serovar identification of Chlamydia trachomatis. Two sets of primers targeting the VD2 region of the omp1 gene and one set targeting the cryptic plasmid were designed for use in multiplex (both targets) and nested PCR (omp1 only). For the RLB assay, labeled omp1 amplicons were hybridized to a membrane containing probes specific for 15 C. trachomatis serovars. The assays were used to test 429 clinical specimens, which had been previously tested for C. trachomatis using the COBAS AMPLICOR system. Specimens were tested without knowledge of the COBAS AMPLICOR result. Of 205 specimens that were positive by COBAS AMPLICOR, 201 (98%) were positive by multiplex PCR-RLB and 188 (92%) were also positive by omp1 nested PCR-RLB. In addition, three of 224 COBAS AMPLICOR-negative specimens were positive by omp1 nested PCR-RLB. One hundred sixty-six of 191 (87%) specimens in which C. trachomatis serovars were identified contained only one serovar and 25 (13%) contained two or three serovars. Serovars D, E, and F were found in 31 (16%), 83 (43%), and 51 (27%) specimens, respectively. Serovar E (41%) was the most commonly identified single serovar. Serovars J and K were found alone uncommonly (<2% each), but 18 of 25 (72%) specimens with multiple C. trachomatis serovars contained one or both (10 specimens) of these serovars. The nested (ompI) PCR-RLB is a specific and sensitive method for simultaneous detection and serovar identification of C. trachomatis, which can reliably identify mixed C. trachomatis serovars. It is suitable for use in epidemiological studies.     

Chlamydia trachomatis infection in pregnancy: risk factor for an adverse outcome

A cohort of 122 pregnant women attending the hospital antenatal clinic in northern India were studied to determine the prevalence of genital chlamydial infection, and any adverse effect on the pregnancy. Endocervical swabs were taken at > 12 weeks of pregnancy and cultured for Chlamydia trachomatis. Twenty-six (21.3%) pregnant women were found to be infected with C. trachomatis. The mean age, gravidity and parity were significantly higher (25.03 vs 23.6 years, 1.88 vs 1.72 and 0.92 vs 0.68 respectively [P < 0.005]) in women from whom C. trachomatis was isolated. Follow-up was possible in 87 women who delivered in the hospital. There was increased incidence of still-birth, prematurity and low birth-weight in the C. trachomatis-positive women (16.6% vs 5.7%, 26.6% vs 18.4% and 26.6% vs 23.0%), and these differences were statistically significant (P < 0.5, P < 0.5 and P < 0.05 respectively). The results suggest a definite need for C. trachomatis screening on a wider scale, both in different risk groups of asymptomatic antenatal women and in neonates, to confirm these findings.     

Detection of Chlamydia trachomatis by the polymerase chain reaction in swabs and urine from men with non-gonococcal urethritis.

A polymerase chain reaction (PCR) was developed for Chlamydia trachomatis in which a 380 base pair DNA fragment was amplified. Amplification occurred with the DNA from the 15 serovars but not with that from other Chlamydia spp or with DNA from a variety of other organisms. Chlamydial DNA (10(-16) g) could be detected and the PCR seemed to be able to detect single organisms. Urethral swabs were obtained from 37 men with acute non-gonococcal urethritis (NGU), 18 (49%) of whom were positive for C trachomatis by MicroTrak. As a result of clinical re-examinations 65 urethral swabs were available for analysis by the PCR. In comparison with MicroTrak, PCR had a sensitivity of 95%, a specificity of 94%, a positive predictive value of 86% and a negative predictive value of 98%. The PCR was apparently less sensitive (82%) in tests on urine samples. Overall, however, values of sensitivity and specificity of the PCR compared favourably with those of MicroTrak. The PCR for C trachomatis is likely to be a valuable technique for research, but problems of DNA contamination suggest that it should not be recommended for routine diagnosis.     

Evaluation of high-resolution typing methods for Chlamydia trachomatis in samples from heterosexual couples

We aimed to compare conventional ompA typing of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). Previously used MLST and MLVA systems were compared to modified versions that used shorter target regions and nested PCR. Heterosexual couples were selected from among persons with urogenital C. trachomatis infections visiting the sexually transmitted infection outpatient clinic in Amsterdam, The Netherlands. We identified 30 couples with a total of 65 C. trachomatis-positive samples on which MLST and MLVA for eight target regions were performed. All regions were successfully sequenced in 52 samples, resulting in a complete profile for 18 couples and 12 individuals. Nine ompA genovars from D to K, with two variants of genovar G, were found. The numbers of sequence type and MLVA type profiles were 20 for MLST and 21 for MLVA, and a combination of MLST and MLVA yielded 28 profiles, with discriminatory indexes (D) ranging from 0.95 to 0.99. Partners in 17 couples shared identical profiles, while partners in 1 couple had completely different profiles. Three persons had infections at multiple anatomical locations, and within each of these three individuals, all profiles were identical. The discriminatory capacity of all MLST and MLVA methods is much higher than that of ompA genotyping (D = 0.78). No genotype variation was found within the samples of the same person or from heterosexual couples with a putative single transmission. This shows that the chlamydial genome in clinical specimens has an appropriate polymorphism to enable epidemiological cluster analysis using MLST and MLVA.     

Evaluation of serotyping using monoclonal antibodies and PCR-RFLP for Chlamydia trachomatis serotype identification

We compared genotyping by restriction fragment length polymorphism (RFLP) analysis of the amplified omp1 gene with serotyping by dot enzyme-linked immunosorbent assay (dot-ELISA) to determine the suitability of RFLP analysis for epidemiologic study. Fifteen prototypes of Chlamydia trachomatis and 30 clinical isolates were used in this study. To serotype with dot-ELISA, chlamydia antigen was spotted onto a series of replicate nitrocellulose membrane patches and reacted with 11 mAbs that distinguish the 15 known serovars of C. trachomatis. For RFLP analysis, the amplified chlamydia omp1 gene was digested with AluI to differentiate serovars A to K and L1 to L3. Serovars of C, H, I, J, and L3 were further typed by RFLP analysis after digestion with HinfI, and a combination of EcoRI and DdeI. PCR-based RFLP could identify serotype of 28 among 30 clinical isolates tested. The remaining two untypical isolates were probably due to double infections or mechanical transferring error. Serotyping of C. trachomatis isolates shows that serovars E, D, F, and H are the most prevalent types found in urogenital samples in Korea. In this study, we show that RFLP analysis of amplified omp1 gene may be useful in genotyping C. trachomatis isolates.     

Infections of the cervix uteri with human papillomavirus and Chlamydia trachomatis.

Apart from infection with human papillomavirus (HPV), other microorganisms may be involved in the development of cervical neoplasia. To study concomitant infections with HPV and Chlamydia trachomatis, cervical specimens from 4 groups of women were examined for the presence of these microorganisms by the polymerase chain reaction. The first group consisted of 143 consecutive samples from women with no cytological abnormalities who participated in a triennial screening program to prevent cervical cancer. In this group 2 samples were found positive for HPV and 2 additional samples were found positive for C. trachomatis. In the second group of 46 cytologically abnormal smears, HPV DNA was detected in 71.7% of the samples and C. tra chomatis in 4.3%. In a third group of 94 histological abnormal biopsies, the HPV prevalence ranged from 15% in mild dysplastic lesions up to 92% in invasive cervical carcinomas. Only 2 biopsies of this group (2.1%) were found positive for C. trachomatis. Finally, a group of cervical scrapes was obtained from women attending a clinic for sexually transmitted diseases. In 52 samples positive for C. trachomatis and 60 samples negative for C. trachomatis, no significant (P = 0.57) difference in the frequency of HPV infections was found (11.5% and 8.3%, respectively). The data show that in these study groups HPV and C. trachomatis are independently occurring agents.     

Distribution study of Chlamydia trachomatis serovars among high-risk women in China performed using PCR-restriction fragment length polymorphism genotyping

This was one of the first epidemiological studies in China focused on genital Chlamydia trachomatis serotype distribution in high-risk female populations using omp1 gene-based restriction fragment length polymorphism analysis. One thousand seven hundred seventy cervical swab samples from women attending sexually transmitted disease clinics and female sex workers in six cities in China (Shenzhen and Guangzhou in southern China, Nanjing and Shanghai in eastern China, and Nanning and Chengdu in southwestern China) were subjected to serovar genotyping. The proportion of omp1 genes successfully amplified in 240 C. trachomatis plasmid-positive samples was 94.2% (226/240). Serotypes E (n = 63; 27.9%), F (n = 53; 23.5%), G (n = 28; 12.4%), and D (n = 25; 11.1%) were most prevalent. Though there was no significant difference in the geographic distribution of C. trachomatis, serotype E was predominant in the South (32.1%) and East (27.1%), while serotype F was predominant in the Southwest (28.3%). Serotype F infection was associated with young age and single status. Serovar G was associated with lower abdominal pain; 47.5% of asymptomatic patients were infected with serovar E. These results provide information on distribution of genital C. trachomatis serotypes among high-risk women in China and indicate that high-risk women, including those who are asymptomatic, can be infected with multiple serovars of C. trachomatis, revealing exposure to multiple sources of infection. Although the scope for generalizations is limited by our small sample size, our results showing clinical correlations with genotypes are informative.     

PreservCyt transport medium used for the ThinPrep Pap test is a suitable medium for detection of Chlamydia trachomatis by the COBAS Amplicor CT/NG test: results of a preliminary study and future implications

The commercial COBAS Amplicor CT/NG test (Roche Diagnostic Systems, Meylan, France) is a sensitive and specific method for detection of Chlamydia trachomatis infections. This test currently consists of using a nucleic acid amplification method to detect C. trachomatis in first-void urine specimens and in endocervical swabs collected in 2-sucrose-phosphate (2SP) transport medium. We conducted a prospective study to determine whether the automated COBAS Amplicor CT/NG test can detect C. trachomatis in cervical specimens collected in PreservCyt transport medium (ThinPrep Pap Test; Cytyc Corporation, Boxborough, Mass.). PreservCyt medium is used to preserve cervical samples before the preparation of ThinPrep slides. We collected 1,000 cervical specimens from young women (age range, 15 to 25 years) during routine Pap smear tests. Only specimens with normal cytology and in which the gynecologist found no clinical evidence of urogenital infections were selected. The samples were stored in PreservCyt transport medium at 15 to 20 degrees C. C. trachomatis was detected in 22 of the 1,000 cervical specimens that had been stored in PreservCyt. To confirm the positive samples, the test was repeated on new endocervical swab specimens collected in 2SP transport medium. Only 9 of the 22 positive patients agreed to undergo this control, but all 9 retested positive. To evaluate the influence of storage conditions on the sensitivity of the C. trachomatis PCR test, all of the positive samples were stored at 15 to 20 degrees C in PreservCyt transport medium and were retested every 2 weeks for 6 weeks. C. trachomatis was successfully amplified from all 22 specimens for the whole 6-week period. The prevalence of C. trachomatis infection was 2.2% in our study population. These results demonstrate that PreservCyt transport medium is a suitable transport medium for detection of C. trachomatis by the COBAS Amplicor CT/NG test. The ThinPrep Pap Test may enable gynecologists to monitor for both cervical lesions and C. trachomatis infections with a single endocervical specimen.     

Chlamydia trachomatis serovar differentiation by direct sequence analysis of the variable segment 4 region of the major outer membrane protein gene.

The polymerase chain reaction method was used to amplify DNA from the fourth variable segment of the gene encoding the major outer membrane protein of Chlamydia trachomatis. Direct sequencing of the amplified DNA from prototype strains confirmed previously identified nucleotide sequence differences that were specific for each serovar. This analysis revealed differences in the DNA sequences of prototype strains C/UW-1 and G/IOL-238 from those of prototype strains C/TW-3 and G/UW-57, sequenced previously. This method was also used to determine the serovar types of C. trachomatis in 125 urogenital specimens from infected patients. The most common serovars were E (38%), F (17%), and G and D (14% each). Serovar D was found significantly more often in specimens from men than in specimens from women (P = 0.004). Conversely, serovar G was found significantly more often in specimens from women than in specimens from men (P = 0.026). Only two serovar G isolates gave sequences identical to that of the prototype strain G/IOL-238, suggesting that this strain may be a serovar variant. Three isolates (D+, G-, and J') gave sequences which have not been reported previously. One isolate had the same sequence as the D- serovar variant. Sequence analysis of amplified DNA reveals subtle differences between C. trachomatis strains and provides a very sensitive method for molecular epidemiological analysis.