A simple and rapid DNA extraction method for Chlamydia trachomatis detection from urogenital swabs.
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ABSTRACT:
SUBMITTER: Butzler MA
PROVIDER: S-EPMC5679254 | biostudies-literature | 2017 Nov
REPOSITORIES: biostudies-literature
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A simple and rapid DNA extraction method for Chlamydia trachomatis detection from urogenital swabs.
Butzler Matthew A MA Reed Jennifer L JL McFall Sally M SM
Diagnostic microbiology and infectious disease 20170817 3
A highly sensitive and specific Chlamydia trachomatis (CT) diagnostic test was developed by combining filtration isolation of nucleic acid (FINA) extraction with quantitative polymerase chain reaction including an internal control to identify test inhibition. A pilot study of 40 clinical specimens yielded 100% sensitivity and specificity. ...[more]
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Project description:BACKGROUND:Urogenital Chlamydia trachomatis infection remains prevalent and causes substantial reproductive morbidity. Recent studies have raised concern about the efficacy of azithromycin for the treatment of chlamydia infection. METHODS:We conducted a randomized trial comparing oral azithromycin with doxycycline for the treatment of urogenital chlamydia infection among adolescents in youth correctional facilities, to evaluate the noninferiority of azithromycin (1 g in one dose) to doxycycline (100 mg twice daily for 7 days). The treatment was directly observed. The primary end point was treatment failure at 28 days after treatment initiation, with treatment failure determined on the basis of nucleic acid amplification testing, sexual history, and outer membrane protein A (OmpA) genotyping of C. trachomatis strains. RESULTS:Among the 567 participants enrolled, 284 were randomly assigned to receive azithromycin, and 283 were randomly assigned to receive doxycycline. A total of 155 participants in each treatment group (65% male) made up the per-protocol population. There were no treatment failures in the doxycycline group. In the azithromycin group, treatment failure occurred in 5 participants (3.2%; 95% confidence interval, 0.4 to 7.4%). The observed difference in failure rates between the treatment groups was 3.2 percentage points, with an upper boundary of the 90% confidence interval of 5.9 percentage points, which exceeded the prespecified absolute 5-percentage-point cutoff for establishing the noninferiority of azithromycin. CONCLUSIONS:In the context of a closed population receiving directly observed treatment for urogenital chlamydia infection, the efficacy of azithromycin was 97%, and the efficacy of doxycycline was 100%. The noninferiority of azithromycin was not established in this setting. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT00980148.).
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Project description:A method for Chlamydia trachomatis restriction fragment length polymorphism (RFLP) analysis and complete sequencing of omp1 was developed for use on samples collected at home, and results were compared. Genotyping by sequencing was superior to RFLP analysis. The omp1 gene in 31 clinical strains harbored few mutations compared to the same gene in ATCC reference strains. Follow-up samples obtained during a 24-week period from 31 patients showed recurrence with the same genotype in five cases and a new genotype in one case.
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Project description:Conventional genomic DNA (gDNA) extraction methods can take hours to complete, may require fume hoods and represent the most time-consuming step in many gDNA-based molecular assays. We systematically optimized a bead bashing-based (BBB) approach for rapid gDNA extraction without the need for a fume hood. Human tissue specimens (n = 34) subjected to the 12-min BBB method yielded 0.40 ± 0.17 (mean ± SD) ?g of gDNA per milligram of tissue, sufficient for many downstream applications, and 3- and 6-min extensions resulted in an additional 0.43 ± 0.23 ?g and 0.48 ± 0.43 ?g per milligram of tissue, respectively. The BBB method provides a simple and rapid method for gDNA extraction from mammalian tissue that is applicable to time-sensitive clinical applications.
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Project description:ObjectivesTo review the literature for non-standard treatment options for uncomplicated Chlamydia trachomatis (CT) infections in adolescents and adults.DesignSystematic review.Data sourcesOvid MEDLINE/PubMed, Ovid EMBASE, Cochrane Trials & Systematic Review Databases, CINAHL Plus with Full Text, Web of Science Core Collection, Scopus, ProQuest Dissertations & Theses Global, ClinicalTrials.gov and Health Canada Trials Database were searched for studies in English or French from 1 January 2006 to 6 August 2017. Keywords included CT, anti-infective or anti-bacterial agents, therapy/pharmacotherapy/management.Review methodsIncluded were primary research studies. Outcome measures included clinical or microbiological cure, treatment failure and adverse events. We followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Studies were assessed for risk of bias using the Revised Cochrane Risk of Bias V.2.0 tool for randomised and the Newcastle-Ottawa Quality Assessment Scale for non-randomised studies.Funding sourcePublic Health Agency of Canada.ResultsOf the 6899 records identified through the database search, 11 studies were included. One randomised controlled trial reported that delayed release doxycycline was non-inferior to azithromycin. Two studies examined higher doses of azithromycin but reported no additional benefit. One study looked at a 5-day azithromycin treatment regimen and reported a high cure rate. Two studies reported efficacy of sitafloxacin, and a single study supports the use of levofloxacin. Two phase 2 studies reported efficacy of single-dose rifalazil in both men and women. Only one retrospective study was identified that examined treatment in pregnant women and reported that efficacy with single-dose azithromycin exceeded that of amoxicillin and erythromycin. A single study examining the efficacy of a beta-lactam antibiotic was stopped early due to high treatment failures.ConclusionsThe paucity of existing data highlights the need for further adequately powered studies to evaluate rifalazil, delayed release doxycycline, levofloxacin and other agents for the treatment of uncomplicated CT infections.Prospero registration numberCRD42017073096.
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Project description:Nucleic acid amplification tests (NAATs) are recommended by the CDC for detection of Chlamydia trachomatis (Ct) urogenital infections. Current commercial NAATs require technical expertise and sophisticated laboratory infrastructure, are time-consuming and expensive, and do not differentiate the lymphogranuloma venereum (LGV) strains that require a longer duration of treatment than non-LGV strains. The multiplexed microfluidic PCR-based assay presented in this work simultaneously interrogates 13 loci to detect Ct and identify LGV and non-LGV strain-types. Based on amplified fragment length polymorphisms, the assay differentiates LGV, ocular, urogenital, and proctocolitis clades, and also serovars L1, L2, and L3 within the LGV group. The assay was evaluated in a blinded fashion using 95 clinical swabs, with 76 previously reported as urogenital Ct-positive samples and typed by ompA genotyping and/or Multi-Locus Sequence Typing. Results of the 13-plex assay showed that 51 samples fell within urogenital clade 2 or 4, 24 samples showed both clade 2 and 4 signatures, indicating possible mixed infection, gene rearrangement, or inter-clade recombination, and one sample was a noninvasive trachoma biovar (either a clade 3 or 4). The remaining 19 blinded samples were correctly identified as LGV clade 1 (3), ocular clade 3 (4), or as negatives (12). To date, no NAAT assay can provide a point-of-care applicable turnaround time for Ct detection while identifying clinically significant Ct strain types to inform appropriate treatment. Coupled with rapid DNA processing of clinical swabs (approximately 60 minutes from swab-in to result-out), the assay has significant potential as a rapid POC diagnostic for Ct infections.
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Project description:The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene.
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Project description:As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry.
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Project description:BackgroundThis epidemiological study was carried out in Sfax (south of Tunisia) and focused on genital Chlamydia trachomatis (C. trachomatis) genovar distribution.MethodsOne hundred and thirty seven genital samples from 4067 patients (4.2%) attending the Habib Bourguiba University hospital of Sfax over 12 years (from 2000 to 2011) were found to be C. trachomatis PCR positive by the Cobas Amplicor system. These samples were genotyped by an in house reverse hybridization method.ResultsOne hundred and eight (78.8%) samples contained only one genovar and 29 (21.2%) samples contained two or three genovars. Genovar E was the most prevalent (70.8%) single genovar and it was detected in 90.6% of all the cases. Genovars J, C and L1-L3 were not detected in our samples whereas ocular genovars A and B were in 5 cases. All the five cases were mixed infections. Men had more mixed infections than women (p=0.02) and were more frequently infected by genovars F and K (p<0.05). No associations between current infection, infertility and the genovar distribution were observed. Patients coinfected with Neisseria gonorrhoeae were also significantly more frequently infected with mixed genovars (p=0.04).ConclusionsIn conclusion, we have reported a high prevalence of genovar E and of mixed infections in our study population. Such data could have implications for the control and vaccine development of C. trachomatis in Tunisia.
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Project description:We hypothesized that the plasmid of urogenital isolates of Chlamydia trachomatis would modulate infectivity and virulence in a mouse model. To test this hypothesis, we infected female mice in the respiratory or urogenital tract with graded doses of a human urogenital isolate of C. trachomatis, serovar F, possessing the cognate plasmid. For comparison, we inoculated mice with a plasmid-free serovar F isolate. Following urogenital inoculation, the plasmid-free isolate displayed significantly reduced infectivity compared with the wild-type strain with the latter yielding a 17-fold lower infectious dose to yield 50% infection. When inoculated via the respiratory tract, the plasmid-free isolate exhibited reduced infectivity and virulence (as measured by weight change) when compared to the wild-type isolate. Further, differences in infectivity, but not in virulence were observed in a C. trachomatis, serovar E isolate with a deletion within the plasmid coding sequence 1 when compared to a serovar E isolate with no mutations in the plasmid. We conclude that plasmid loss reduces virulence and infectivity in this mouse model. These findings further support a role for the chlamydial plasmid in infectivity and virulence in vivo.
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