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.

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.

Chlamydia trachomatis in subfertile couples undergoing an in vitro fertilization program: a prospective study.

OBJECTIVES: The objectives were to estimate the prevalence of Chlamydia trachomatis infection in subfertile couples and to study the relationship between markers of C. trachomatis infection and male infertility as well as pregnancy rates after in vitro fertilization (IVF). STUDY DESIGN: All consecutive couples consulting for infertility and IVF in Pellegrin Hospital were screened for C. trachomatis by direct (PCR test) and serological methods. RESULTS: Two hundred and seventy-seven couples were included in the study (mean age in years: 35 for men, 32 for women; mean duration of infertility: 4 years). The most frequent indication for IVF was tubal factor in 33%, endometriosis in 6%, dysovarian function in 12%, male infertility in 36% and others in 13%. C. trachomatis PCR was positive in 1.2% of men, 95% confidence interval (CI95%): (0.2%; 3.3%) and in 2.7% of women, CI95%: (1.1%; 5.5%). When combining all chlamydial markers, 17.3% of men, CI95%: (12.7%; 22.8%) and 20.4% of women, CI95%: (15.6%; 25.9%) had at least one positive marker. The presence of positive markers was not associated with altered semen characteristics. Couples with positive markers had a pregnancy rate of 23.1% (12 out of 52) compared with 20.2% (24 out of 119) among those with negative markers. CONCLUSION: In this population, the presence of past or current C. trachomatis infection was associated with neither semen characteristics nor outcome of IVF in subfertile couples.

Community epidemiology of Chlamydia and Mycoplasma pneumoniae in LRTI in France over 29 months.

BACKGROUND: The role of Chlamydia pneumoniae (CP) and Mycoplasma pneumoniae (MP) in lower respiratory tract infections (LRTI) is still little known in community settings. METHODS: In all, 3207 adult cases of LRTI (871 with pneumonia, and 2336 with acute bronchitis) were prospectively included in the ETIIC1 ETIIC : ETude de l'Incidence des Infections respiratoires basses d'origine Communautaire dues A Chlamydia pneumoniae et Mycoplasma pneumoniae (Incidence of CP and MP in LRTI in community settings) program by 303 general practitioners and 24 hospital physicians in France between September 1997 and February 2000. The polymerase chain reaction and immunoassays were used to detect CP or MP in 3198 pharyngeal specimens obtained by gargling. RESULTS: Of these 3198 patients, 232 (7.3%), were PCR-positive for CP and/or MP. Immunoassays were far less sensitive than PCRs (Se = 2 and 13% for MP and CP). Among the 2336 patients with acute bronchitis, PCR was positive for CP in 95 (4.1%), and for MP, in 54 (2.3%). Among the 671 patients with radiologically confirmed pneumonia, PCR was positive for CP in 23 (3.4%), and for MP in 49 (7.3%). CP and MP displayed significant geographic heterogeneity. Independent clinical determinants of positive PCR for CP and/or MP were age below 45 years, previous antimicrobial therapy (especially betalactams). Clinical signs were not of practical use in distinguishing accurately between etiologic diagnoses. CONCLUSIONS: CP or MP diagnosed by PCR were found in more than 7% of patients with LRTI in community settings with a significant geographical heterogeneity and significant temporal trends in the incidence.

Synthesis and antimycobacterial activity of ferrocenyl ethambutol analogues and ferrocenyl diamines.

A new series of ferrocenyl diamino alcohols and diamines were synthesized and their inhibitory potencies were probed with Mycobacterium tuberculosis. Interestingly, ferrocenyl diamines 6a and b display significant activities against M. tuberculosis H37Rv.

Fourier transform infrared spectroscopy as a new tool for characterization of mollicutes.

Fourier transform infrared (FT-IR) spectroscopy is a convenient physico-chemical technique to investigate various cell materials. Bacteria of class Mollicutes, identified by conventional methods, as Mycoplasma, Acholeplasma and Ureaplasma genera were characterized using this method. A data set of 74 independent experiments corresponding to fourteen reference strains of Mollicutes was examined by FT-IR spectroscopy to attempt a spectral characterization based on the biomolecular structures. In addition to the separation of Mollicutes within the lipidic region into five main clusters corresponding to the three phylogenetic groups tested, FT-IR spectroscopy allowed a fine discrimination between strains belonging to the same species by using selective spectral windows, particularly in the 1200-900 cm(-1) saccharide range. The results obtained by FT-IR were in good agreement with both taxonomic and phylogenetic classifications of tested strains. Thus, this technique appears to be a useful tool and an accurate mean for a rapid characterization of Mollicutes observed in humans.

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.

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.