Genital herpes evaluation by quantitative TaqMan PCR: correlating single detection and quantity of HSV-2 DNA in cervicovaginal lavage fluids with cross-sectional and longitudinal clinical data.

OBJECTIVE: To evaluate the utility of a single quantitative PCR (qPCR) measurement of HSV (HSV-1&2) DNA in cervicovaginal lavage (CVL) specimens collected from women with predominantly chronic HSV-2 infection in assessing genital HSV shedding and the clinical course of genital herpes (GH) within a cohort with semiannual schedule of follow up and collection of specimens. METHODS: Two previously described methods used for detection of HSV DNA in mucocutaneous swab samples were adapted for quantification of HSV DNA in CVLs. Single CVL specimens from 509 women were tested. Presence and quantity of CVL HSV DNA were explored in relation to observed cross-sectional and longitudinal clinical data. RESULTS: The PCR assay was sensitive and reproducible with a limit of quantification of ~50 copies per milliliter of CVL. Overall, 7% of the samples were positive for HSV-2 DNA with median log10 HSV-2 DNA copy number of 3.9 (IQR: 2.6-5.7). No HSV-1 was detected. Presence and quantity of HSV-2 DNA in CVL directly correlated with the clinical signs and symptoms of presence of active symptomatic disease with frequent recurrences. CONCLUSION: Single qPCR measurement of HSV DNA in CVL fluids of women with chronic HSV-2 infection provided useful information for assessing GH in the setting of infrequent sampling of specimens. Observed positive correlation of the presence and quantity of HSV-2 DNA with the presence of active and more severe course of HSV-2 infection may have clinical significance in the evaluation and management of HSV-2 infected patients.

Prevalence and correlates of Trichomonas vaginalis infection among female US federal prison inmates.

BACKGROUND: Previous studies have observed high prevalences of Trichomonas vaginalis infection among women entering US jails and state prisons (22%-47%). We sought to determine the prevalence among women incarcerated in 2 US female-only federal prisons. METHODS: Female inmates were recruited at 2 prisons (n = 624). Participants completed a self-administered questionnaire and provided self-collected first-catch urine and vaginal swab specimens. Specimens were tested for T. vaginalis DNA. RESULTS: Approximately 8.5% of participants at the first prison, and 8.3% at the second prison had a positive urine result, vaginal swab result or both, for a combined prevalence of 8.5%. Using positivity in either specimen as the reference standard, urine polymerase chain reaction had a sensitivity of 66.7% and vaginal swab polymerase chain reaction had a sensitivity of 84.4%. The only significant positive correlate of T. vaginalis infection was lower household income before arrest. Other variables nonsignificantly positively correlated with T. vaginalis were being employed at the time of arrest, having experienced sexual, physical, or emotional abuse by a family member, having a parent who had not had a drug or alcohol addiction, never exchanging sex for money or drugs, ever being pregnant, having abnormal vaginal bleeding/spotting, and having concurrent chlamydia or gonorrhea. CONCLUSIONS: Although not as high as in other studies of women entering US jails and state prisons, our observed T. vaginalis prevalence of 8.5% was much higher than in the general US population. Therefore, screening for T. vaginalis infection may be warranted at federal prison entry, as well as sexual health education during prison stay.

Use of quantitative broad-based polymerase chain reaction for detection and identification of common bacterial pathogens in cerebrospinal fluid.

BACKGROUND: Conventional laboratory diagnosis of bacterial meningitis based on microscopy followed by culture is time-consuming and has only moderate sensitivity. OBJECTIVES: The objective was to define the limit of detection (LOD), analytic specificity, and performance characteristics of a broad-based quantitative multiprobe polymerase chain reaction (PCR) assay for rapid bacterial detection and simultaneous pathogen-specific identification in patients with suspected meningitis. METHODS: A PCR algorithm consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by pathogen identification using either pathogen-specific probes or Gram-typing probes, was employed to detect pathogens. The 16S rRNA gene, which contains both conserved and variable regions, was chosen as the target. Pathogen-specific probes were designed for Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes. Gram-positive and -negative typing probes were designed based on conserved regions across all eubacteria. The LOD and time to detection were assessed by dilutional mocked-up samples. A total of 108 convenience cerebrospinal fluid (CSF) clinical samples obtained from the Johns Hopkins Hospital (JHH) microbiology laboratory were tested, and results were compared with hospital microbiologic culture reports. RESULTS: The LOD of the assay ranged from 10(1) to 10(2) colony-forming units (CFU)/mL. Pathogen-specific probes showed no cross-reactivity with other organisms. Time to detection was 3 hours. In clinical specimens, the universal probe correctly detected 16 of 22 culture-positive clinical specimens (sensitivity = 72.7%; 95% confidence interval [CI] = 49.8% to 89.3%), which were all correctly characterized by either pathogen-specific or Gram-typing probes. Adjusted sensitivity after removing probable microbiologic laboratory contaminants was 88.9% (95% CI = 65.3% to 98.6%). The universal probe was negative for 86 of 86 culture-negative specimens. CONCLUSIONS: A broad-based multiprobe PCR assay demonstrated strong analytic performance characteristics. Findings from a pilot clinical study showed promise in translation to human subjects, supporting potential utility of the assay as an adjunct to traditional diagnostics for early identification of bacterial meningitis.

Mycoplasma genitalium as a contributor to the multiple etiologies of cervicitis in women attending sexually transmitted disease clinics.

OBJECTIVES: The purpose of this study was to investigate the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium, in women attending a sexually transmitted disease (STD) clinic, as well as the frequency of coinfections, and relationship of each organism to cervicitis. METHODS: In this cross-sectional study of 324 women attending Baltimore City STD Clinics, C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium were detected using nucleic acid amplification tests. Demographic characteristics and risk factors were ascertained. RESULTS: Overall prevalence of infection with C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium was found to be 11.1%, 4.6%, 15.3%, and 19.2%, respectively. Prevalence in women with cervicitis was 15.8%, 6%, 18.9%, and 28.6% for C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium, respectively. Percentages of coinfections were high. C. trachomatis and M. genitalium were significantly associated with cervicitis in univariate analysis, but only M. genitalium was significantly associated with cervicitis (AOR: 2.5) in multiple logistic regression models. CONCLUSION: Knowledge of the statistical association of M. genitalium with cervicitis in this study increases the need for further confirmation of the etiologic significance of this organism with cervicitis in more diverse populations. The high prevalence merits more study and may have implications for diagnosis and treatment of cervicitis.

Rapid identification of biothreat and other clinically relevant bacterial species by use of universal PCR coupled with high-resolution melting analysis.

A rapid assay for eubacterial species identification is described using high-resolution melt analysis to characterize PCR products. Unique melt profiles generated from multiple hypervariable regions of the 16S rRNA gene for 100 clinically relevant bacterial pathogens, including category A and B biothreat agents and their surrogates, allowed highly specific species identification.

Performance of three nucleic acid amplification tests for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by use of self-collected vaginal swabs obtained via an Internet-based screening program.

Use of self-obtained vaginal specimens processed by nucleic acid amplification tests (NAATs) has significantly increased the utilization of nontraditional locations for Chlamydia trachomatis and Neisseria gonorrhoeae screening programs. One important emerging source of such venues includes home-based self-sampling kits available via the Internet. The objective of our study was to evaluate the performance of three commercially available NAATs (Becton-Dickinson ProbeTec SDA, Gen-Probe Aptima Combo2 TMA, and Roche Amplicor PCR) for detection of C. trachomatis and N. gonorrhoeae in vaginal samples obtained via an Internet-based screening program. From July 2004 to August 2005, 500 self-collected vaginal swabs were tested for C. trachomatis and N. gonorrhoeae by using all three NAATs. Another 500 samples were collected between August 2005 and November 2007 and tested by ProbeTec and Combo2; PCR testing was discontinued due to low specificity for N. gonorrhoeae. All tests were conducted according to the manufacturers' procedures; the "gold standard" for an infected C. trachomatis or N. gonorrhoeae patient was defined as > or = 2 positive NAAT results. Of the first 500 swabs submitted, 46 were C. trachomatis infected (9.2%) and 5 were N. gonorrhoeae infected (1.0%), and 3 of these were coinfected (0.6%). All C. trachomatis and N. gonorrhoeae Combo2-positive/ProbeTec-negative samples were confirmed as true positives by an alternative NAAT. For C. trachomatis, ProbeTec, Combo2, and PCR had sensitivities of 82.6%, 100%, and 100%, with specificities of 100%, 100%, and 99.3%, respectively. For N. gonorrhoeae, ProbeTec, Combo2, and PCR had sensitivities of 80%, 100%, and 100%, with specificities of 100%, 100%, and 98.8%, respectively. Of the total 1,000 swabs submitted, 92 were C. trachomatis infected (9.2%) and 15 were N. gonorrhoeae infected (1.5%), and 7 of these were coinfected (0.7%). There were no ProbeTec-positive/Combo2-negative samples. For C. trachomatis, ProbeTec and Combo2 had sensitivities of 81.5% and 100%, with specificities of 100% and 100%, respectively. For N. gonorrhoeae, ProbeTec and Combo2 had sensitivities of 80% and 100%, with specificities of 100% and 100%, respectively. Overall, ProbeTec had 17 C. trachomatis false-negative results (1.7%) and 3 N. gonorrhoeae false-negative results (0.3%), while Combo2 had none. Our results were consistent with the sensitivities and specificities stated by the manufacturers. NAATs perform well for detection of chlamydia and gonorrhea with self-obtained vaginal swabs shipped in a dry state to a laboratory. For 1,000 self-collected vaginal swabs tested by NAATs, the sensitivities for C. trachomatis and N. gonorrhoeae for Combo2 were 100% and 100%, while they were 81.5% and 80%, respectively, for ProbeTec. For 500 PCR samples, the C. trachomatis sensitivity was 100% and the N. gonorrhoeae sensitivity was 100%, with specificities of 99.3% and 98.8%, respectively.

Cost-effectiveness of screening strategies for Chlamydia trachomatis using cervical swabs, urine, and self-obtained vaginal swabs in a sexually transmitted disease clinic setting.

BACKGROUND: We evaluated the cost-effectiveness of Chlamydia screening strategies that use different methods of specimen collection: cervical swabs, urines, and self-obtained vaginal swabs. METHODS: A decision analysis was modeled for a hypothetical cohort of 10,000 per year of women attending sexually transmitted disease (STD) clinics. Incremental cost-effectiveness of 4 screening strategies were compared: 1) Endocervical DNA probe test (PACE2, Gen-Probe), 2) Endocervical AC2 (Aptima Combo 2, Gen-Probe), 3) Self-Obtained Vaginal AC2, and 4) Urine AC2. Sensitivities of the vaginal, urine, and cervical AC2 tests were derived from 324 women attending STD clinics. The primary outcome was cases of pelvic inflammatory disease prevented. The model incorporated programmatic screening and treatment costs and medical cost savings from sequelae prevented. RESULTS: Chlamydia prevalence in the sampled population was 11.1%. Sensitivities of vaginal, urine, and cervical AC2 were 97.2%, 91.7%, and 91.7%, respectively. The sensitivity of the DNA probe was derived from the literature and estimated at 68.8%. The self-obtained vaginal AC2 strategy was the least expensive and the most cost-effective, preventing 17 more cases of pelvic inflammatory disease than the next least expensive strategy. CONCLUSIONS: Use of a vaginal swab to detect Chlamydia in this STD clinic population was cost-saving and cost-effective.

Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents.

OBJECTIVES: To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. METHODS: The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. RESULTS: The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. CONCLUSIONS: A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.

Rapid PCR-based diagnosis of septic arthritis by early Gram-type classification and pathogen identification.

Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality. Delayed or inadequate treatment of SA can lead to irreversible joint destruction and disability. Current methods of diagnosing SA rely on synovial fluid analysis and culture which are known to be imprecise and time-consuming. We report a novel adaptation of a probe-based real-time PCR assay targeting the 16S rRNA gene for early and accurate diagnosis of bacterial SA. The assay algorithm consists of initial broad-range eubacterial detection, followed by Gram typing and species characterization of the pathogen. The platform demonstrated a high analytical sensitivity with a limit of detection of 10(1) CFU/ml with a panel of SA-related organisms. Gram typing and pathogen-specific probes correctly identified their respective targets in a mock test panel of 36 common clinically relevant pathogens. One hundred twenty-one clinical synovial fluid samples from patients presenting with suspected acute SA were tested. The sensitivity and specificity of the assay were 95% and 97%, respectively, versus synovial fluid culture results. Gram-typing probes correctly identified 100% of eubacterial positive samples as to gram-positive or gram-negative status, and pathogen-specific probes correctly identified the etiologic agent in 16/20 eubacterial positive samples. The total assay time from sample collection to result is 3 h. We have demonstrated that a real-time broad-based PCR assay has high analytical and clinical performance with an improved time to detection versus culture for SA. This assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute care setting where rapid pathogen detection and identification would assist in disposition and treatment decisions.

The microbicide tenofovir does not inhibit nucleic acid amplification tests for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine samples.

The potential inhibitory effects of tenofovir and a placebo were examined using the Becton Dickinson ProbeTec, Gen-Probe Aptima Combo 2, and Roche Amplicor tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae. Concentrations of 5% to 0% of tenofovir and the placebo were added to dilutions of C. trachomatis and N. gonorrhoeae. No appreciable inhibition was observed.

Effect of sexually transmitted disease (STD) coinfections on performance of three commercially available immunosorbent assays used for detection of herpes simplex virus type 2-specific antibody in men attending Baltimore, Maryland, STD clinics.

Two hundred seventy-nine serum samples from men attending sexually transmitted disease (STD) clinics in Baltimore, Maryland, were tested for herpes simplex virus type 2 (HSV-2)-specific antibody by three immunosorbent glycoprotein G-2-based assays (the Kalon, Focus, and Biokit assays). The results for all samples with positive results were confirmed by Western blotting (91/279; 32.6% HSV-2 seroprevalence). All patients were also tested for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, human immunodeficiency virus type 1, and hepatitis C virus. The Kalon assay performed very well with samples from this population (90.8% sensitive, 99.4% specific), whereas the Focus assay had a sensitivity (82.6%) much lower than that shown previously. For 19.7% of the samples, the Biokit assay gave an indeterminate result. It was found that the odds of a sample having a Biokit assay indeterminate result compared to that of having a definitive positive or negative results were 3.88 times greater for subjects concurrently infected with N. gonorrhoeae, after the effects of other STDs were controlled for (P = 0.001; 95% confidence interval, 1.78, 8.45). Unfortunately, we were unable to control for HSV-1 infection status in the regression model, which, on the basis of chi(2) analysis, might also affect the clarity of the Biokit test. The recommended index cutoff value of 1.1 for the Focus and Kalon assays was found to be optimal for this population.

Comparison between the Gen-Probe transcription-mediated amplification Trichomonas vaginalis research assay and real-time PCR for Trichomonas vaginalis detection using a Roche LightCycler instrument with female self-obtained vaginal swab samples and male urine samples.

This study compared two assays for Trichomonas vaginalis detection, Gen-Probe's transcription-mediated amplification (TMA) assay for Trichomonas vaginalis and BTUB FRET PCR, using self-obtained clinical samples from 611 patients. Infection status was defined as two positive results by two different tests. The initial TMA assay sensitivity was 96.7%; specificity was 97.5%. The TMA assay was comparable to BTUB FRET PCR.

Performance of the gen-probe transcription-mediated [corrected] amplification research assay compared to that of a multitarget real-time PCR for Mycoplasma genitalium detection.

Mycoplasma genitalium (MG) can cause nongonococcal urethritis and is potentially associated with urethritis, endometritis, and cervicitis. Several assays have been developed to detect MG. Molecular amplification assays for organism detection can be problematic due to the potential for false-positive and false-negative results. Confirmatory testing is often required in these situations, requiring additional time and resources. Use of multigene targets could integrate both detection and verification at lower cost. Utilizing two targets, the MgPa adhesion gene and the 16S rRNA gene, a multitarget real-time (MTRT) PCR for the detection of MG was developed. Samples from patients attending sexually transmitted disease clinics were collected in duplicate. Urine samples from males (n = 286) and self-collected vaginal swabs from females (n = 321) were analyzed by MTRT PCR for MG and the Gen-Probe transcription-mediated [corrected] amplification (TMA) assay, which targets MG rRNA for detection (TMA-MG research use only). Utilizing the criteria of any two targets being positively amplified, the MTRT PCR had a sensitivity and specificity of 91.8% (101 positive samples/110 samples tested) and 99.5% (495/497), respectively, with a positive predictive value (PPV) of 98.1% (101/103) and a negative predictive value (NPV) of 98.2% (495/504). The Gen-Probe TMA-MG assay had a sensitivity, specificity, PPV, and NPV of 98.1% (108/110), 98.1% (488/497), 92.3% (108/117), and 99.5% (488/490), respectively. Comparison between the MTRT PCR and TMA-MG assay by kappa statistic analysis indicated that an overall kappa value was 0.941 (95% confidence interval, 0.907 and 0.976). Both assays demonstrated accuracy in the detection of MG from urine samples from male patients and self-collected vaginal swabs from female patients.