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Surface enhanced Raman spectroscopy of Chlamydia trachomatis and Neisseria gonorrhoeae for diagnostics, and extra-cellular metabolomics and biochemical monitoring.

ABSTRACT: SERS spectra excited at 785?nm of the bacteria Chlamydia trahomatis (elementary bodies, EB) and Neisseria gonorrheoae, the causative pathogens for the two most common sexually transmitted diseases (STD), chlamydia and gonorrhea, respectively, are reported. Although both are Gram-negative bacteria, the SERS signatures of C. trachomatis and N. gonorrheoae are completely different. N. gonorrheoae SERS spectra are due to the starvation induced nucleotide metabolites adenine and guanine, and the surface associated co-enzyme nicotinamide adenine dinucleotide and are very similar on Au and Ag although the spectrum appears more rapidly on Ag. The C. trachomatis SERS spectrum is dominated by the vibrational features of cell surface proteins. While features attributable to specific residues and the amide backbone characterize the C. trachomatis spectrum on Ag, the corresponding SERS spectrum on Au substrates displays vibrational characteristics of aggregated proteins. The prospects for the development of a SERS based platform for rapid (

SUBMITTER: Chen Y

PROVIDER: S-EPMC5980109 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Surface enhanced Raman spectroscopy of Chlamydia trachomatis and Neisseria gonorrhoeae for diagnostics, and extra-cellular metabolomics and biochemical monitoring.

Chen Y Y Premasiri W R WR Ziegler L D LD

Scientific reports 20180326 1

SERS spectra excited at 785 nm of the bacteria Chlamydia trahomatis (elementary bodies, EB) and Neisseria gonorrheoae, the causative pathogens for the two most common sexually transmitted diseases (STD), chlamydia and gonorrhea, respectively, are reported. Although both are Gram-negative bacteria, the SERS signatures of C. trachomatis and N. gonorrheoae are completely different. N. gonorrheoae SERS spectra are due to the starvation induced nucleotide metabolites adenine and guanine, and the surf ...[more]

PMID: 29581560

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Project description:BackgroundLaboratory detection of Human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cervicovaginal cytology specimens is now based on identification of the DNA sequences unique to these infectious agents. However, current commercial test kits rely on nucleotide probe hybridization to determine DNA sequences, which may lead to diagnostic errors due to cross-reactivity. The aim of this study was to find a practical approach to perform automated Sanger DNA sequencing in clinical laboratories for validation of the DNA tests for these three infectious agents.MethodsA crude proteinase K digest of 5% of the cells collected in a liquid-based cervicovaginal cytology specimen was used for the detection of DNA molecules specific for HPV, C trachomatis and N gonorrhoeae, and for preparation of materials suitable for direct automated DNA sequencing. Several sets of commercially available polymerase chain reaction (PCR) primers were used to prepare nested PCR amplicons for direct DNA sequencing.ResultsSome variants of HPV-16 and HPV-31 were found to share an at least 34-base long sequence homology downstream of the GP5+ binding site, and all HPV-6 and HPV-11 variants shared an upstream 34-base sequence including part of the GP5+ primer. Accurate HPV genotyping frequently required more than 34-bases for sequence alignments to distinguish some of the HPV genotype variants with closely related sequences in this L1 gene hypervariable region. Using the automated Sanger DNA sequencing method for parallel comparative studies on split samples and to retest the residues of samples previously tested positive for C trachomatis and/or for N gonorrhoeae, we also found false-negative and false-positive results as reported by two commercial nucleic acid test kits.ConclusionIdentification of a signature DNA sequence by the automated Sanger method is useful for validation of HPV genotyping and for molecular testing of C trachomatis and N gonorrhoeae in liquid-based cervicovaginal Papanicolaou (Pap) cytology specimens for clinical laboratories with experience in molecular biology to increase the specificity of these DNA-based tests. However, even a highly specific test for high-risk HPV genotyping may have unacceptably low positive predictive values for precancer lesion in populations with a low cervical cancer prevalence rate.

Project description:Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) represent the most common agents of sexually transmitted rectal infections among men having sex with other men (MSM). In this study, we assessed the bacterial composition of the rectal microbiota associated with CT and/or NG infections in a cohort of men reporting unsafe rectal intercourse. A total of 125 rectal swabs were collected and four groups were compared: non-infected subjects (n = 53), patients with CT (n = 37), or NG rectal infection (n = 17) and patients with contemporary positivity for CT/NG (n = 18). CT and NG infections were detected by a real-time commercial test and the rectal microbiota composition was analyzed from rectal swabs through sequencing of the hypervariable V3-V4 regions of the 16S rRNA gene. The rectal microbiota of all subgroups was dominated by Prevotellaceae, Enterobacteriaceae, and Ruminococcaceae families. Irrespective of the analyzed subgroup, we found that the rectal environment of all the enrolled MSM was rich in Prevotella and Escherichia genera. Moreover, a shift in the bacterial composition between patients with sexually transmitted rectal infections and controls was noticed: infected patients were characterized by a depletion of Escherichia species, associated with an increase of anaerobic genera, including Peptoniphilus, Peptostreptococcus, and Parvimonas. Overall, the presence of rectal symptoms did not significantly modify the rectal microbiota profiles among the four groups of analyzed patients. We confirmed that HIV-positive patients are characterized by a lower bacterial richness than HIV-negative subjects. However, we found that the presence of HIV has a different impact on bacterial rectal communities compared to CT and NG infections, modifying the relative abundance of several genera, including Gardnerella, Lactobacillus, Corynebacterium, and Sutterella. Information about the rectal microbiota composition in CT and NG infections could shed light on the pathogenesis of these conditions and could contribute to the onset of new strategies for their control.

Project description:BackgroundAlgorithms that bridge the gap between syndromic sexually transmitted infection (STI) management and treatment based in realistic diagnostic options and local epidemiology are urgently needed across Africa. Our objective was to develop and validate a risk algorithm for Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) diagnosis among symptomatic Rwandan women and to compare risk algorithm performance to the current Rwandan National Criteria for NG/CT diagnosis.MethodsThe risk algorithm was derived in a cohort (n = 468) comprised of symptomatic women in Kigali who sought free screening and treatment for sexually transmitted infections and vaginal dysbioses at our research site. We used logistic regression to derive a risk algorithm for prediction of NG/CT infection. Ten-fold cross-validation internally validated the risk algorithm. We applied the risk algorithm to an external validation cohort also comprised of symptomatic Rwandan women (n = 305). Measures of calibration, discrimination, and screening performance of our risk algorithm compared to the current Rwandan National Criteria are presented.ResultsThe prevalence of NG/CT in the derivation cohort was 34.6%. The risk algorithm included: age < =25, having no/primary education, not having full-time employment, using condoms only sometimes, not reporting genital itching, testing negative for vaginal candida, and testing positive for bacterial vaginosis. The model was well calibrated (Hosmer-Lemeshow p = 0.831). Higher risk scores were significantly associated with increased prevalence of NG/CT infection (p < 0.001). Using a cut-point score of > = 5, the risk algorithm had a sensitivity of 81%, specificity of 54%, positive predictive value (PPV) of 48%, and negative predictive value (NPV) of 85%. Internal and external validation showed similar predictive ability of the risk algorithm, which outperformed the Rwandan National Criteria. Applying the Rwandan National Criteria cutoff of > = 2 (the current cutoff) to our derivation cohort had a sensitivity of 26%, specificity of 89%, PPV of 55%, and NPV of 69%.ConclusionsThese data support use of a locally relevant, evidence-based risk algorithm to significantly reduce the number of untreated NG/CT cases in symptomatic Rwandan women. The risk algorithm could be a cost-effective way to target treatment to those at highest NG/CT risk. The algorithm could also aid in sexually transmitted infection risk and prevention communication between providers and clients.

Dataset Information

Surface enhanced Raman spectroscopy of Chlamydia trachomatis and Neisseria gonorrhoeae for diagnostics, and extra-cellular metabolomics and biochemical monitoring.

Publications

Surface enhanced Raman spectroscopy of Chlamydia trachomatis and Neisseria gonorrhoeae for diagnostics, and extra-cellular metabolomics and biochemical monitoring.

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