Serological reactivity and bacterial genotypes in Chlamydia trachomatis urogenital infections in Guadeloupe, French West Indies.

OBJECTIVES: To determine the prevalence and genotypes of Chlamydia trachomatis urogenital infection in Guadeloupe, French West Indies, and to compare C trachomatis direct detection to serological testing. METHODS: From March to November 2000, 971 consecutive patients (888 women and 83 men) who had been referred to the clinical laboratory of the Institut Pasteur de la Guadeloupe for routine testing for genital infection were recruited. Samples were subjected to a nucleic acid amplification assay (AMP CT, Gen-Probe, San Diego, California, USA). Genotypes were determined by omp1 PCR-restriction fragment length polymorphism analysis. Serological testing was carried out with the commercially available peptide-based ELISA assay (SERO-CT IgG/IgA, Savyon/BMD, Marne-La-Vallée, France). RESULTS: Positive AMP CT test results were obtained for 102 (10.5%) of the 971 samples. The prevalence of infection was 16.9% in men and 9.8% in women. The most common genotypes were E (34.3%), F (23.9%), Da (13.4%), I (9%) and Ia (7.5%). No relationship was found between genogroups and age, sex or clinical symptoms. With AMP CT used as a reference, the sensitivity, specificity, positive and negative predictive values of SERO-CT were 81.1%, 56%, 34.5% and 91.2%, respectively, for IgG and 55.4%, 76.8%, 59.4% and 85.8%, respectively, for IgA. IgG seroprevalence rates were very low (1/5, 20%) in patients infected with genotype Ia strains. CONCLUSIONS: The prevalence found in Guadeloupe did not differ significantly from that found in mainland France. The genotypes Da, F, I and Ia were more prevalent in Guadeloupe; however, the SERO-CT assay was unable to detect serum antibodies in 80% of the patients infected with genotype Ia strains.

Chlamydial infections in duck farms associated with human cases of psittacosis in France.

Five severe cases of psittacosis in individuals associated with duck farms were notified in France between January and March 2006. Diagnostic examination included serology and/or molecular detection by PCR from respiratory samples. As a consequence, we investigated all duck flocks (n=11) that were housed in the three farms where human infections occurred. While serology by complement fixation test was negative for all samples, cloacal and/or tracheal chlamydial excretion was detected by PCR in all three units. Notably, one duck flock was tested strongly positive in 2 of the 3 affected farms, and Chlamydophila (C.) psittaci strains were isolated from cloacal and/or tracheal swab samples from both farms. Human samples and duck isolates exhibited the same PCR-RFLP restriction pattern, which appeared to be an intermediate between genotypes A and B. Analysis of ompA gene sequences and comparison to those of the type strains showed that the isolates could not be strictly assigned to any of the generally accepted genotypes of C. psittaci. Further analysis by MLVA of the PCR-positive human samples revealed two distinct patterns, which were related to previously isolated C. psittaci duck strains.

Restriction endonuclease patterns of the omp1 gene of reference Chlamydia trachomatis strains and characterization of isolates from Cameroonian students.

Eighteen reference strains of Chlamydia trachomatis were differentiated by omp1 PCR- and nested PCR-based RFLP analysis, using two restriction digestions, one with AluI and the other with the three enzymes HpaII, EcoRI and HinfI. AluI digestion allowed the differentiation of 12 different profiles after CT1/CT5 PCR and 13 different profiles after the nested PCR. The triple hydrolysis permitted the identification of 15 different patterns. In all, 16/18 reference strains were clearly identified. These reference patterns were successfully used to genotype 34 of 35 (28 strains and 7 clinical specimens) samples from infected students, collected during a screening programme in Yaounde (Cameroon). Genotypes D, Da, E, F, G and J were found. The most prevalent omp1 genotype was E (n = 14; 40 %), followed by F (n = 7; 20 %). As RFLP patterns of reference strains are essential for typing clinical isolates, they will greatly facilitate C. trachomatis characterization in many resource-limited laboratories.

Swabs (dry or collected in universal transport medium) and semen can be used for the detection of Chlamydia trachomatis using the cobas 4800 system.

In this prospective study, the fully automated cobas 4800 CT/NG and the cobas TaqMan CT tests were compared for Chlamydia trachomatis detection in urine and in genital specimens collected with Copan flocked swabs in culture media. A protocol was also established for the highly sensitive detection of C. trachomatis in semen specimens using the cobas 4800 CT/NG test. A total of 708 consecutive urogenital samples (293 male urine samples and 356 vaginal, 45 cervical and 14 urethral swabs) obtained from the Bacteriology Department, as well as 100 consecutive semen samples collected from patients attending the Reproduction Biology Department, Bordeaux University Hospital, France, from July to September 2010, were analysed. Positive and negative agreements between the cobas 4800 CT/NG and cobas TaqMan CT tests were 92.7 % [95 % confidence interval (CI), 82.7-97.1 %] and 99.2 % (95 % CI, 98.2-99.7 %), respectively, with an overall agreement of 98.7 % (699/708). The clinical sensitivity of the cobas 4800 CT/NG assay for C. trachomatis ranged from 90.9 to 100 % depending on specimen type, with an overall prevalence of 7.2 % (51/708). The clinical specificity ranged from 99.1 to 100 % depending on specimen type. Dilution of 25 µl semen samples in cobas PCR medium proved to be the most sensitive protocol with the lowest inhibition rate. In conclusion, the cobas 4800 CT/NG test was found to be an effective method for detection of C. trachomatis in semen, male urine and genital swab samples collected dry or in universal transport medium.

MLVA subtyping of genovar E Chlamydia trachomatis individualizes the Swedish variant and anorectal isolates from men who have sex with men.

This study describes a new multilocus variable number tandem-repeat (VNTR) analysis (MLVA) typing system for the discrimination of Chlamydia trachomatis genovar D to K isolates or specimens. We focused our MLVA scheme on genovar E which predominates in most populations worldwide. This system does not require culture and therefore can be performed directly on DNA extracted from positive clinical specimens. Our method was based on GeneScan analysis of five VNTR loci labelled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This MLVA, called MLVA-5, was applied to a collection of 220 genovar E and 94 non-E genovar C. trachomatis isolates and specimens obtained from 251 patients and resulted in 38 MLVA-5 types. The genetic stability of the MLVA-5 scheme was assessed for results obtained both in vitro by serial passage culturing and in vivo using concomitant and sequential isolates and specimens. All anorectal genovar E isolates from men who have sex with men exhibited the same MLVA-5 type, suggesting clonal spread. In the same way, we confirmed the clonal origin of the Swedish new variant of C. trachomatis. The MLVA-5 assay was compared to three other molecular typing methods, ompA gene sequencing, multilocus sequence typing (MLST) and a previous MLVA method called MLVA-3, on 43 genovar E isolates. The discriminatory index was 0.913 for MLVA-5, 0.860 for MLST and 0.622 for MLVA-3. Among all of these genotyping methods, MLVA-5 displayed the highest discriminatory power and does not require a time-consuming sequencing step. The results indicate that MLVA-5 enables high-resolution molecular epidemiological characterisation of C. trachomatis genovars D to K infections directly from specimens.

The first performance report for the Bio-Rad Dx CT/NG/MG assay for simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in urogenital samples.

We evaluated the clinical performance of the Bio-Rad Dx CT/NG/MG assay for the detection of Chlamydia trachomatis, Mycoplasma genitalium and Neisseria gonorrhoeae in urogenital samples in comparison with the Roche COBAS® TaqMan® CT assay for C. trachomatis and an in-house TaqMan PCR assay for M. genitalium. Swab specimens were cultured for N. gonorrhoeae. In this prospective study, urogenital samples were obtained from symptomatic and asymptomatic patients attending the sexually transmitted disease clinic of Bordeaux, France, from January to April 2010. A total of 658 clinical specimens (259 male and 180 female urines, 191 vaginal, 21 endocervical and 7 urethral swabs) from 453 patients were analyzed. The prevalence of C. trachomatis and M. genitalium infections was 8.1% (21/260) and 1.9% (5/260) in men and 10.4% (20/193) and 2.1% (4/193) in women, respectively. The Bio-Rad Dx CT/NG/MG clinical sensitivity was 100% for C. trachomatis and M. genitalium in men and women. In male urine, the clinical specificity was 99.6% for C. trachomatis and 100% for M. genitalium. In women, the specificity was 99.5% for swabs and 100% for urines for detecting C. trachomatis and M. genitalium. All seven N. gonorrhoeae PCR-positive samples were also positive by culture. Patients were co-infected in 5/57 cases (8.8%), with C. trachomatis and M. genitalium in three cases, and C. trachomatis and N. gonorrhoeae in two cases. In conclusion, the Bio-Rad Dx CT/NG/MG assay can be recommended for the simultaneous detection of C. trachomatis, M. genitalium and N. gonorrhoeae in urogenital specimens of symptomatic and asymptomatic individuals.

Effects of antibiotics on Chlamydia trachomatis viability as determined by real-time quantitative PCR.

The objective of this study was to determine the effect of antibiotics on Chlamydia trachomatis viability by using a quantitative real-time PCR assay that measured DNA replication and mRNA transcription of the structural omp1 and omp2 genes, 16S rRNA and the groEL1 gene with and without antibiotics. Ofloxacin, moxifloxacin, azithromycin and doxycycline were tested against the serovar D and L2 reference strains and a derivative mutant resistant to fluoroquinolones, L2-OFXR, obtained by in vitro selection. Using DNA quantification, the antibiotic MIC was calculated when the number of DNA copies was equal to that of the chlamydial inoculum at time zero. This method allowed the easy determination of MICs by DNA quantification of the four selected genes and gave similar results to those obtained by immunofluorescence staining without biased interpretation. By using cDNA quantification, the lowest antibiotic concentration for which no RNA was transcribed corresponded to the minimum bactericidal concentration. C. trachomatis still transcribed the16S rRNA and groEL1 genes, even at concentrations well above the MIC, showing a bacteriostatic effect for all antibiotics tested. This method allows the study of antibiotic activity on growth and viability of C. trachomatis by DNA and RNA quantification at the same time without additional cell-culture passaging.

Chlamydia trachomatis infection in children: do not forget perinatal acquisition: a case report of a 7-year old girl, C. trachomatis infected, presumed sexually assaulted.

A 7-year old girl suspected of having been sexually abused owing to the presence of anal condyloma was found to be infected by Chlamydia trachomatis. Microbiological analysis and anamnesis were consistent with the infection having been acquired at birth. This case confirms that untreated infection acquired at birth can persist for months or years and highlights the value of examining those involved in the suspicion of sexual abuse of the child.

Real-time high resolution melting PCR for identification of the Swedish variant of Chlamydia trachomatis.

We developed a real-time High Resolution Melting PCR to identify the new Swedish variant of Chlamydia trachomatis ncCT. Of 1191 urogenital specimens C. trachomatis-positive by an omp1 real-time PCR, collected in France in 2007-2008, 1128 gave an interpretable profile corresponding to the wild-type strain; no nvCT was found. This test can be used on selected C. trachomatis-positive samples to monitor the nvCT spread.

Screening of volunteer students in Yaounde (Cameroon, Central Africa) for Chlamydia trachomatis infection and genotyping of isolated C. trachomatis strains.

The prevalence of Chlamydia trachomatis infection was 3.78% out of 1,277 volunteer students screened by direct fluorescence assay and Cobas Amplicor PCR. The infection was associated with the nonuse or inconsistent use of condoms in women (P = 0.026) and a previous sexually transmitted infection in men (P = 0.023). The most frequent genotypes determined by sequencing the omp1 genes of 25 clinical isolates were E (44%) and F (20%), and some strains harbored mutations, but E genotype strains did not.