Development of a SimpleProbe real-Time PCR Assay for rapid detection and identification of the US novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC)

Urethritis, or inflammation of the urethra, is one of the most common reasons men seek clinical care. Sexually transmitted pathogens including Neisseria gonorrhoeae are responsible for over half of the symptomatic urethritis cases in U.S. men. Recently, clinics in Indianapolis, Columbus, Atlanta, and other U.S. cities began to note increasing numbers of men presenting with urethritis and Gram-negative intracellular diplococci in their urethral smears who test negative for N. gonorrhoeae. Many of these discordant cases, which have periodically reached highs of more than 25% of presumed gonococcal cases in some sexually transmitted infection clinics in the U.S. Midwest, are infected with strains in a novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC). However, no cultivation-independent tests are available for the US_NmUC strains, and prior studies relied on microbial culture and genome sequencing to identify them. Here, we describe a PCR test that can identify the US_NmUC strains and distinguish them from commensal and invasive N. meningitidis strains as well as N. gonorrhoeae. Our SimpleProbe®-based real-time PCR assay targets a conserved nucleotide substitution in a horizontally acquired region of US_NmUC strain genomes. We applied the assay to 241 urine specimens whose microbial compositions had previously been determined by deep shotgun metagenomic sequencing. The assay detected the single US_NmUC positive case in this cohort, with no false positives. Overall, our simple and readily adaptable assay could facilitate investigation of the pathogenesis and epidemiology of the US_NmUC clade

Validation of a Real Time PCR Assay for the Detection of Ureaplasma urealyticum

poster abstractUreaplasma urealyticum (UU) is a bacterium that occurs naturally in the genital flora of sexually active men and women. In high concentrations this bacterium can become pathogenic causing urethritis. Detection of UU by nucleic acid amplification is not widely practiced in the US, and culture isolation requires a specialized lab. The aim is to validate an assay utilizing a Real Time PCR (RT-PCR) for the detection of UU in our laboratory, and to determine the prevalence of UU in men and women attending a local STI clinic. A previously published RT-PCR assay was utilized to amplify a 152 base pair fragment of a highly conserved region of UU. The 152 base pair fragment of UU was amplified from an isolate and cloned into a TOPO-PCRII plasmid. The plasmid was used to develop quantitative standards of known concentrations; then to optimize and confirm the assay parameters, and determine the limit of detection for the assay. Assay performance in clinical matrix by spiked clinical specimens will be used to confirm the limit of detection when using residual samples. The UU fragment was successfully cloned and purified into a plasmid, which was verified by restriction enzyme digestion and PCR followed by gel electrophoresis. The plasmid containing the UU fragment was quantified using spectrophotometry and contained 7.27x1010 copies/μL. This plasmid was utilized in optimizing the RT-PCR assay, including primer concentrations and annealing temperature. Standards were developed through a dilution series of the purified plasmid from 1x1010-1x101 copies/μL. An increase in reaction efficiency was noted when the probe concentration was decreased from 0.2μM to 0.15 μM. Preliminary results show that the assay can detect down to ~10 copies/μl using purified plasmid. Thus far the assay has been optimized for our laboratory, and a set of standards to determine the limit of detection is being developed

Association of HPV types 6, 11, 16, and 18 DNA detection and serological response in unvaccinated adolescent women

Antibodies directed against the human papillomavirus (HPV) L1 protein are detected in approximately 70% of individuals with HPV infections. The factors associated with a serological response are not characterized. It is hypothesized that the HPV viral load, duration of detection, or both would be associated with seropositivity in adolescent women. Adolescent women (n = 117), ages 15-17 at enrolment were followed for a mean of 6.2 years. Quarterly vaginal swabs (mean 22 per participant) were used to identify HPV 6, 11, 16, or 18 DNA (Roche PCR/Linear Array). Type-specific HPV infection was defined as ≥2 positive assays. To approximate viral load, Roche PCR/Linear Array test strips were scored visually based on the strength of signal relative to beta-globin controls. Sera collected near the end of study were tested by cLIA. Regression models were fit to assess associations between strength of signal (as represented by mean and cumulative strength of signal), duration of HPV detection, seropositivity, and serotiter. Detection of HPV DNA was associated with seropositivity for four types combined and for types 6, 16, and 18. Overall, 70.1% of DNA positive episodes were associated with type-specific seropositivity. The cumulative HPV DNA signal strength during periods of HPV detection for types 6, 11, 16, and 18 combined was associated with seropositivity (OR = 1.21, 95% CI 1.02-1.44 P = 0.026). No other HPV DNA predictors were found to be associated with seropositivity or serotiter

Can Salivary Innate Immune Molecules Provide Clue on Taste Dysfunction in COVID-19?

Emerging concerns following the severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) pandemic are the long-term effects of coronavirus disease (COVID)-19. Dysgeusia in COVID-19 is supported by the abundant expression of the entry receptor, angiotensin-converting enzyme-2 (ACE2), in the oral mucosa. The invading virus perturbs the commensal biofilm and regulates the host responses that permit or suppress viral infection. We correlated the microbial recognition receptors and soluble ACE2 (sACE2) with the SARS-CoV2 measures in the saliva of COVID-19 patients. Data indicate that the toll-like receptor-4, peptidoglycan recognition protein, and sACE2 are elevated in COVID-19 saliva and correlate moderately with the viral load

Low sensitivity of the careHPV™ Assay for detection of oncogenic Human papillomavirus in cervical samples from HIV-infected and HIV-uninfected Kenyan women

Background: Human papillomavirus (HPV) infection causes cervical cancer (CC), a common malignancy among Kenyan women. New CC screening methods rely on oncogenic HPV (“highrisk”, or HR-HPV) detection, but most have not been evaluated in swabs from Kenyan women. Methods: HPV typing was performed on 155 cervical swabs from Kenyan women using the Roche Linear Array® (LA) and careHPV™ (careHPV) assays. Detection of 14 oncogenic HPV types in careHPV assay was compared to LA results. Results: Compared to LA, sensitivity and specifi city of careHPV assay was 53.0% and 80.9%, respectively. The sensitivity and specifi city of careHPV in swabs from women with cervical dysplasia was 74.1% and 65.2%, respectively. The sensitivity and specifi city of careHPV in swabs from HIV-infected women was 55.9% and of 96.4%, respectively. Overall agreements of careHPV assay with LA was substantial. Conclusion: The results for careHPV assay are promising for oncogenic HPV detection in Kenyan women. The low sensitivity of careHPV for detection of HR-HPV could limit it’s benefi t as a screening tool. Thus, a full clinical validation study is highly desirable before the careHPV assay can be accepted for cervical cancer screening

Detection of types of HPV among HIV-infected and HIV-uninfected Kenyan women undergoing cryotherapy or loop electrosurgical excision procedure

Objective: To assess the baseline types of HPV infection among HIV-positive and HIV-negative women in western Kenya undergoing cryotherapy or loop electrosurgical excision procedure (LEEP) for cervical intraepithelial neoplasia. Methods: A prospective observational study was conducted of baseline HPV characteristics of women undergoing visual inspection with acetic acid (VIA) and cryotherapy or LEEP. After a positive VIA in HIV-positive and HIV-negative women, data on demographics, CD4 count, and use of antiretroviral therapy and a cervical swab were collected. HPV typing was performed using the Roche Linear Array. Results: Of 175 participants, 86 (49.1%) were HIV-positive and had a higher prevalence of low-risk HPV types (odds ratio [OR] 5.28, P=0.005) compared with HIV-negative women. The most common high-risk (HR)-HPV types in HIV-positive women were HPV 16 (13.9%) and HPV 18 (11.1%). HIV-positive women requiring LEEP were more likely to have HR-HPV types (OR 6.67, P=0.012) and to be infected with multiple HR-HPV types (OR 7.79, P=0.024) compared to those undergoing cryotherapy. Conclusion: HIV-positive women requiring LEEP versus cryotherapy had a higher prevalence of any HR-HPV type and multiple HR-HPV types. There were no such differences in HPV types identified among HIV-negative women

Evaluation of the Performance of a Point-of-Care Test for Chlamydia and Gonorrhea

Importance: Rates of chlamydial and gonococcal infection continue to increase in the United States, as do the associated costs of untreated infections. Improved diagnostic technologies that support testing and treating in 1 clinical visit are critical to advancing efforts to control the rates of chlamydial and gonococcal infection. Objective: To evaluate the clinical performance of a point-of-care (POC) molecular diagnostic assay for the detection of chlamydia and gonorrhea. Design, setting, and participants: A noninterventional, cross-sectional clinical study was conducted from September 18, 2018, through March 13, 2019, at sexually transmitted infection (STI), HIV, family planning, and obstetrics and gynecology clinics where STI screening is routine, using a convenience sample and comparing commercially available assays with a new 30-minute POC assay. Patients included were those eligible for STI screening or diagnostic testing who had not taken antibiotics effective against chlamydia or gonorrhea within the previous 28 days. Four vaginal swab samples were collected from women and a first-catch urine sample was obtained from men. Main outcomes and measures: A composite infection status was used to classify participants as infected if 2 or more comparator results were positive, as not infected if 2 or more comparator samples were negative, and as unevaluable if 1 result was invalid and the other 2 results did not agree with each other. Results: Swab samples from 1523 women (median age, 27 years [interquartile range, 17-37 years]), 817 (53.6%) of whom presented with symptoms, and 922 men (median age, 29 years [interquartile range, 17-41 years]), 308 (33.4%) of whom were symptomatic, were tested. For chlamydia, sensitivity of the new POC assay was 96.1% (95% CI, 91.2%-98.3%) for women and 92.5% (95% CI, 86.4%-96.0%) for men. For gonorrhea, sensitivity estimates were 100.0% (95% CI, 92.1%-100.0%) for women and 97.3% (95% CI, 90.7%-99.3%) for men. For chlamydia, specificity of the new POC assay was 99.1% (95% CI, 98.4%-99.5%) for women and 99.3% (95% CI, 98.4%-99.7%) for men. For gonorrhea, specificity estimates were 99.9% (95% CI, 99.5%-100%) for women and 100% (95% CI, 95.5%-100%) for men. Non-laboratory-trained personnel performed 94.8% of all tests (2318 of 2445) during the study. Conclusions and relevance: This study suggests that self-obtained vaginal swab samples were associated with performance equivalent to laboratory-based molecular diagnostics, which can support use of this POC assay in many settings. The availability of an easy-to-use, rapid (30-minute) molecular test for accurate detection of chlamydia and gonorrhea has the power to facilitate testing and treatment in a single patient visit for these STIs

Longer duration of anti-retroviral therapy is associated with decreased risk of human papillomaviruses detection in Kenyan women living with HIV

Objective: A longitudinal study was conducted among women living with HIV in Kenya to determine if duration of anti-retroviral (ART) usage altered detection and persistence of oncogenic (high-risk) human papillomaviruses (HR-HPV). Methods: Women living with HIV without cervical dysplasia were enrolled at a cervical cancer screening clinic. Three cervical swabs, HIV viral loads, and CD4 cell counts were obtained at enrollment and at two annual visits. HPV genotyping was performed on swabs (Roche Linear Array). Linear regression models assessed effects of ART duration on HR-HPV detection and persistence. Results: Seventy-seven women, median age 38 years, completed three study visits and were included in the analysis. The mean time from HIV diagnosis to enrollment was 9.6 years (SD 3.9 years). The mean ART duration was 6.2 years (SD 3.1 years). Most women had undetectable HIV viral loads and CD4 cell counts above 500 cells/L. Each additional year of ART use reduced the likelihood of detection of HR-HPV by 10-15% and persistent detection of A9 HR-HPV by 20%. Conclusion: Among Kenyan women living with HIV, longer duration of ART use was associated with significantly reduced risk of all detection and persistent detection of HR-HPV

Decline in vaccine-type human papillomavirus prevalence in young men from a Midwest metropolitan area of the United States over the six years after vaccine introduction

Purpose: The aim of this study was to determine changes in human papillomavirus (HPV) prevalence among young men from a Midwest metropolitan area over the six years after vaccine introduction, including HPV prevalence in men overall, in vaccinated men to examine vaccine impact and in unvaccinated men to examine herd protection. An exploratory aim was to examine associations between number of vaccine doses and HPV prevalence. Methods: Men aged 14–26 years reporting male-female and/or male-male sexual contact were recruited from a primary care clinic, sexually transmitted disease clinic, and community setting during two waves of data collection: 2013–2014 (N = 400) and 2016–2017 (N = 347). Participants completed a questionnaire and were tested for penile, scrotal and anal HPV. Changes in prevalence of any (≥1 type) and vaccine-type HPV (HPV6, 11, 16, and/or 18) were examined using propensity score weighted logistic regression. Associations between number of doses and HPV infection were determined using chi-square tests and logistic regression. Results: The proportion of men with a history of ≥1 HPV vaccine doses increased from 23% to 44% (p < 0.001) from waves 1 to 2. After propensity score weighting, infection with ≥1 vaccine-type HPV significantly decreased among all men (29% to 20%; 31% decrease; odds ratio [OR] = 0.62, 95% confidence interval [CI] = 0.44–0.88) and unvaccinated men (32% to 21%; 36% decrease; OR = 0.56, 95%CI = 0.34–0.86); there was a non-significant decrease (21%) among vaccinated men. Associations between number of doses and HPV prevalence were not statistically significant. Conclusions: Prevalence of vaccine-type HPV decreased among all, vaccinated, and unvaccinated men six years after HPV vaccine recommendation, supporting vaccine impact and herd protection. Decreases in vaccine-type HPV in all men appear to be due to decreases in unvaccinated men, suggesting that the full impact of vaccination has yet to be realized. Continued monitoring and efforts to vaccinate men prior to sexual initiation are warranted