Chlamydia trachomatis samples testing falsely negative in the Aptima Combo 2 test in Finland, 2019.
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ABSTRACT:
SUBMITTER: Rantakokko-Jalava K
PROVIDER: S-EPMC6549458 | biostudies-literature | 2019 May
REPOSITORIES: biostudies-literature
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<i>Chlamydia trachomatis</i> samples testing falsely negative in the Aptima Combo 2 test in Finland, 2019.
Rantakokko-Jalava Kaisu K Hokynar Kati K Hieta Niina N Keskitalo Anniina A Jokela Pia P Muotiala Anna A Jokiranta T Sakari TS Kuusela Rutta R Sarkkinen Hannu H Aittoniemi Janne J Vuorinen Tytti T Hakanen Antti J AJ Puolakkainen Mirja M
Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 20190501 22
Since February 2019, over 160 <i>Chlamydia trachomatis</i> (CT) cases testing negative or equivocal by Aptima Combo 2 (AC2) but positive by Aptima CT test run with Panther instruments occurred in Finland. The AC2 test targets chlamydial 23S rRNA while the CT test targets 16S rRNA. Sequencing of 10 strains revealed a nucleotide substitution in 23S rRNA. The significance of this for the failure of the AC2 test to detect the variant is not yet known. ...[more]
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Project description:The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C. trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C. trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C. trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA in conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.
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Project description:BackgroundChlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples.MethodsC. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform.ResultsFull length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies.ConclusionsThe combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.
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Project description:BackgroundInfection with Chlamydia trachomatis (Ct) is the most commonly reported sexually transmitted infection in Europe. In Germany, Ct screening is offered free of charge to pregnant women since 1995 and to women < 25 years of age since 2008. For symptomatic individuals, testing is covered by statutory health insurance. Study results have shown that repeat Ct infection occurs in 10-20% of previously infected women and men. Our aim was to describe persons tested for Ct and to investigate the determinants of (repeat) Ct infection in women and men in Germany.MethodsWe analysed Ct test results from men and women tested between 2008 and 2014 in laboratories participating in the German Chlamydia trachomatis Laboratory Sentinel surveillance. Reinfection was defined as at least 2 positive laboratory tests within more than 30 days. We performed logistic regression stratified by sex and, for women, reason for testing to determine the effect of previous test results and age group on subsequent test results.ResultsIn total, 2,574,635 Ct tests could be attributed to 1,815,494 women and 123,033 men. 5% of women and 14% of men tested positive at least once. 15-19- and 20-24-year-old women tested positive at least once respectively in 6.8 and 6.0%, while men respectively in 16.6 and 21.2%. Altogether, 23.1% of tested women and 11.9% of tested men were tested repeatedly between 2008 and 2014. Among those who previously tested positive, reinfection occurred in 2.0% of women and 6.6% of men. Likelihood to be tested Ct positive was higher in women and men with a positive Ct test in the past compared to previously tested Ct negative, odds ratios 4.7 and 2.6 (p < 0.01) respectively. Odds ratios ranged by age group and test reason.ConclusionA history of Ct infection increased the likelihood of infection with Ct in women and men taking into account the result of the previous test. Health education, safer sex and treatment of partners are necessary for women and men who have tested positive to prevent reinfection and complications and to interrupt the chain of transmission. To identify potential reinfection repeat testing after treatment should be performed.
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Project description:Background:Chlamydia trachomatis (CT), the most common bacterial sexually transmitted infection (STI), leads to pelvic inflammatory disease, infertility and chronic pelvic pain in women as well as an increased risk of vertical transmission, conjunctivitis and pneumonitis in infants. It may also be a co-factor along with human papillomavirus (HPV) in cervical cancer progression. We aimed to determine the prevalence of CT genotypes in genital specimens of women from South Khorasan, Iran and to test the association between CT and cytology statistics. MATERIALS AND METHODS:This was a cross-sectional study on 248 Pap smear samples from women who visited a gynecologist for routine Pap smear testing in South Khorasan province. Nested polymerase chain reaction (PCR) was used to test the residual fluids of Pap smears for CT-DNA after cytological examination. Direct sequencing, alignment and phylogenic analyses were performed on eight samples to identify their genotypes. RESULTS:The mean age of patients was 37.54 ± 5.21 years. Most samples had a normal cytology (214 cases, 86.29%). Overall, 31 samples were positive for CT infection (12.5%) of which 20 (9.34%) were normal and 11 (32.35%) were abnormal, with the frequency difference being significant (P=0.022). The co-infection of CT/HPV in total was identified in 14 cases (5.6%). The results of sequencing eight samples out of the 31 CT positive samples revealed the detection of genotypes D and E, each with four cases. CONCLUSION:We show that a high prevalence of genital CT infection is present in women with both normal and abnormal cytology; however, the higher prevalence among women in the abnormal group may indicate its involvement in cervical neoplasia.
| S-EPMC5767929 | biostudies-literature
Project description:BackgroundGenital chlamydia infection is the most commonly diagnosed sexually transmitted infection in the UK. C. trachomatis genital infections are usually caused by strains which fall into two pathovars: lymphogranuloma venereum (LGV) and the genitourinary genotypes D-K. Although these genotypes can be discriminated by outer membrane protein gene (ompA) sequencing or multi-locus sequence typing (MLST), neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing.Principal findingsWe evaluated variable number tandem repeat (VNTR) and ompA sequencing (now called multi-locus VNTR analysis and ompA or "MLVA-ompA") to study local epidemiology in Southampton over a period of six months. One hundred and fifty seven endocervical swabs that tested positive for C. trachomatis from both the Southampton genitourinary medicine (GUM) clinic and local GP surgeries were tested by COBAS Taqman 48 (Roche) PCR for the presence of C. trachomatis. Samples tested as positive by the commercial NAATs test were genotyped, where possible, by a MLVA-ompA sequencing technique. Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43%) were successfully recovered by repeatable passage in culture. Of the 157 samples, 93 (i.e. 59%) were fully genotyped by MLVA-ompA. Only one mixed infection (E & D) in a single sample was confirmed. There were two distinct D genotypes for the ompA gene. Most frequent ompA genotypes were D, E and F, comprising 20%, 41% and 16% of the type-able samples respectively. Within all genotypes we detected numerous MLVA sub-types.ConclusionsAmongst the common genotypes, there are a significant number of defined MLVA sub-types, which may reflect particular background demographics including age group, geography, high-risk sexual behavior, and sexual networks.
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Project description:BackgroundSeveral Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom's MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom's MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom's MLST scheme in clinical samples with low concentrations of Chlamydia DNA.ResultsIn this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom's MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv.ConclusionsThe newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.
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