The L2b real-time PCR targeting the pmpH gene of Chlamydia trachomatis used for the diagnosis of lymphogranuloma venereum is not specific to L2b strains (original) (raw)
Related papers
Molecular and Cellular Probes, 2006
Lymphogranuloma venereum (LGV) is caused by a rare form of Chlamydia trachomatis that is difficult to diagnose, since culture is not readily available, and since other methods are not reliable or lack sensitivity. We report here a rapid, sensitive, and specific real-time multiplex polymerase chain reaction (PCR) assay capable of detecting C. trachomatis and identifying serovar L-2 in the same reaction, directly from rectal swabs. The analytical sensitivity of the assay was 25 genome copies for C. trachomatis, and 50 genome copies for L-2. The analytical specificity was 100%, as demonstrated with a diverse range of C. trachomatis serovars and other site-specific bacterial pathogens. With the use of a rapid DNA extraction method, a blinded validation of spiked rectal swabs correctly identified 30 samples containing C. trachomatis cells, L-2 DNA, or negative samples. The multiplexed PCR assay also identified serovar L-2 in 13 of 70 rectal swab samples taken from symptomatic patients. Twelve additional samples were positive for C. trachomatis only, and omp1 sequencing determined these samples as either serovar D, E, G, J, or K. This assay represents the first real-time PCR method capable of detecting C. trachomatis DNA, and of simultaneously identifying C. trachomatis infection as serovar L-2.
BMC Infectious Diseases, 2008
Background: Infection due to Chlamydia trachomatis is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men. Results: The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (omp-1)-gene and a L-serovar-specific region of the polymorphic protein H (pmp-H)-gene for the detection of Chlamydia trachomatis is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2-and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific omp-1-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5-95,2%. In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for C. trachomatis we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male. Conclusion: This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies.
Typing of Lymphogranuloma VenereumChlamydia trachomatisStrains
Emerging Infectious Diseases, 2010
We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s, including a variant from Europe.
PLOS ONE, 2015
The evolution of Chlamydia trachomatis is mainly driven by recombination events. This fact can be fuelled by the coincidence in several European regions of the high prevalence of non-invasive urogenital genotypes and lymphogranuloma venereum (LGV) outbreaks. This scenario could modify the local epidemiology and favor the selection of new C. trachomatis variants. Quantifying the prevalence of co-infection could help to predict the potential risk in the selection of new variants with unpredictable results in pathogenesis or transmissibility. In the 2009-2013 period, 287 clinical samples with demonstrated presence of C. trachomatis were selected. They were divided in two groups. The first group was constituted by 137 samples with C. trachomatis of the LGV genotypes, and the second by the remaining 150 samples in which the presence of LGV genotypes was previously excluded. They were analyzed to detect the simultaneous presence of non-LGV genotypes based on pmpH and ompA genes. In the first group, co-infections were detected in 10.9% of the cases whereas in the second group the prevalence was 14.6%, which is the highest percentage ever described among European countries. Moreover, bioinformatic analyses suggested the presence among men who have sex with men of a pmpH-recombinant variant, similar to strains described in Seattle in 2002. This variant was the result of genetic exchange between genotypes belonging to LGV and members of G-genotype. Sequencing of other genes, phylogenetically related to pathotype, confirmed that the putative recombinant found in Madrid could have a common origin with the strains described in Seattle. Countries with a high prevalence of co-infections and high migration flows should enhance surveillance programs in at least their vulnerable population. Fig 3. Maximum likelihood analysis of concatenated rs2-tarP-incEF-pmpH genes. Red letters correspond to putative pmpH-recombinant variants described in this study. Bold letters correspond to genotype G strains described in Seattle. The general time reversible plus proportion of invariable sites and gamma distribution model (GTR+ I+G) implemented in PhyML software was used. Numbers indicate bootstrap values based on 1000 replicates (%).
Journal of Clinical Microbiology, 2008
The clinical performance of two nucleic acid amplification assays targeting the cryptic plasmid and two assays targeting rRNA molecules in Chlamydia trachomatis was examined. First-catch urine samples from Malmoe, Sweden, were tested for C. trachomatis with the Abbott real-time PCR assay m2000 and an in-house PCR for the new variant strain of C. trachomatis with a deletion in the cryptic plasmid. Aliquots of the urine samples were sent to Aarhus, Denmark, and further examined with the Roche COBAS Amplicor CT (RCA) PCR, the Gen-Probe Aptima Combo 2 assay (AC2) targeting the C. trachomatis 23S rRNA, and the Aptima C. trachomatis assay (ACT) targeting the 16S rRNA molecule. A positive prevalence of 9% (163/1,808 urine samples examined) was detected according to the combined reference standard. The clinical sensitivity and specificity of the four assays were as follows: for ACT, 100% (163/163) and 99.9% (1,643/1,645), respectively; for AC2, 100% (163/163) and 99.6% (1,640/1,645); for m2000, 68.7% (112/163) and 99.9% (1,644/1,645); for RCA, 63.8% (104/163) and 99. . The two Gen-Probe assays detected all mutant strains characterized by the in-house PCR as having the deletion in the cryptic plasmid, whereas the Roche and the Abbott PCRs targeting the plasmid were both unable to detect the plasmid mutant. The difference in clinical sensitivity between the plasmid PCR assays m2000 and RCA, on the one hand, and the rRNA assays AC2 and ACT, on the other, could be attributed almost exclusively to the presence of the plasmid mutant in about one-quarter of the Chlamydia-positive samples examined.
Detection by multiplex polymerase chain reaction and typing of Chlamydia trachomatis isolates
FEMS Microbiology Letters, 1995
The multiplex polymerase chain reaction (PCR) was applied for the detection of the Chlumydia trachomatis chromosome and plasmid. The multiplex PCR demonstrated a sensitivity of 0.8 fg of chlamydial DNA, corresponding to the detection of about 5 copies of the plasmid. Analysis of 195 genital specimens collected randomly from a female population, showed that the multiplex PCR is more sensitive and rapid than culturing for detecting Chlamydia trachomatis. Moreover, sequencing of the II variable domain of the ompl gene, directly from DNA of the clinical specimens, appears to be a simple and rapid method for determining serovar isolates.
mBio, 2011
Chlamydia trachomatis is an obligate intracellular bacterium that causes a diversity of severe and debilitating diseases worldwide. Sporadic and ongoing outbreaks of lymphogranuloma venereum (LGV) strains among men who have sex with men (MSM) support the need for research on virulence factors associated with these organisms. Previous analyses have been limited to single genes or genomes of laboratory-adapted reference strain L 2 /434 and outbreak strain L 2 b/UCH-1/proctitis. We characterized an unusual LGV strain, termed L 2 c, isolated from an MSM with severe hemorrhagic proctitis. L 2 c developed nonfusing, grape-like inclusions and a cytotoxic phenotype in culture, unlike the LGV strains described to date. Deep genome sequencing revealed that L 2 c was a recombinant of L 2 and D strains with conserved clustered regions of genetic exchange, including a 78-kb region and a partial, yet functional, toxin gene that was lost with prolonged culture. Indels (insertions/deletions) were discovered in an ftsK gene promoter and in the tarp and hctB genes, which encode key proteins involved in replication, inclusion formation, and histone H1-like protein activity, respectively. Analyses suggest that these indels affect gene and/or protein function, supporting the in vitro and disease phenotypes. While recombination has been known to occur for C. trachomatis based on gene sequence analyses, we provide the first whole-genome evidence for recombination between a virulent, invasive LGV strain and a noninvasive common urogenital strain. Given the lack of a genetic system for producing stable C. trachomatis mutants, identifying naturally occurring recombinants can clarify gene function and provide opportunities for discovering avenues for genomic manipulation.