Summary of the Fourth Annual American Sexually Transmitted Diseases Association Workshop on Improving Sexually Transmitted Infection Control Efforts Through Cross-Sector Collaboration.

ABSTRACT: The American Sexually Transmitted Diseases Association has, for several years, been conducting a cross-sector workshop to bring together a variety of stakeholders to develop ideas for collaboratively improving the sexually transmitted infection control efforts in the United States. In this summary, we share the content of discussions and ideas of the fourth annual workshop for future research and potential changes to practice with a focus on diagnostic capacity.

Multicenter Comparison of Nucleic Acid Amplification Tests for the Diagnosis of Rectal and Oropharyngeal Chlamydia trachomatis and Neisseria gonorrhoeae Infections.

Research using nucleic acid amplification tests (NAATs) have repeatedly found rectal and oropharyngeal infections with Chlamydia trachomatis and Neisseria gonorrhoeae to be common and potentially more difficult to treat than genital infections. Unfortunately, public health and patient care efforts have been hampered by the lack of FDA-cleared NAATs with claims for anorectal or oropharyngeal samples. At the time of the initiation of this study, no commercially available assays had these claims. We formed a novel partnership among academic institutions and diagnostic manufacturers to address this public health need. From May 2018 through August 2019, we recruited 1108 women, 1256 men, and 26 transgender persons each of whom provided 3 anal and 3 oropharyngeal swab specimens. The 3 anal swabs were pooled into a single transport tube as were the 3 oropharyngeal swabs. The performance of each of three study assays was estimated by comparison to the composite result and relative to one another. Percent positivity for chlamydia was 5.9 and 1.2% from anal and oropharyngeal specimens, respectively, compared to 4.2 and 4.1% for gonorrhea. Sensitivity for chlamydia detection ranged from 81.0 to 95.1% and 82.8 to 100% for anal and oropharyngeal specimens, respectively. Gonorrhea sensitivity ranged from 85.9 to 99.0% and 74.0 to 100% for anal and oropharyngeal samples, respectively. Specificity estimates were ≥ 98.9% for all assays, organisms, and sample types. Although there was heterogeneity between sensitivity estimates, these assays offer better ability to detect extragenital infections than culture and potential solutions for providing appropriate sexual health care for populations in which these infections are of concern.

Comparison of Alinity m HPV and cobas HPV assays on cervical specimens in diverse storage media.

OBJECTIVE: To assess the concordance of high-risk HPV (HR-HPV) testing with the Alinity assay on cervical samples collected with diverse collection/storage protocols (ThinPrep, SurePath, Cervicollect) and to assess inter-assay concordance of HR-HPV testing of cervical cell specimens with Alinity m HR HPV assay (Alinity) vs cobas® 4800 HPV assay (cobas). METHODS: Specimens were obtained from 560 women attending a Women's Health clinic. Two specimens were obtained from each woman with combinations of two of the three collection devices and aliquots were tested by the two assays. RESULTS: Alinity showed an agreement of 93.9%, Kappa = 0.89 (263/280) between ThinPrep and SurePath specimens; 97.5%, Kappa = 0.95 (347/356) and 92.9%, Kappa = 0.85 (104/112) between ThinPrep and SurePath aliquots taken before or after cytology processing, respectively. Cervi-Collect specimens showed an agreement of 94.6%, Kappa = 0.89 (265/280) with ThinPrep specimens. Compared to cobas, Alinity showed agreements of 94.3%, Kappa = 0.88 (395/419) and 91.8%, Kappa = 0.82 (257/280) between ThinPrep and SurePath specimens, respectively. Alinity and cobas detected genotypes 16/18 and other high-risk HPV types at similar rates and showed similar correlations with cytology grades. CONCLUSIONS: Compared to cobas, Alinity performed equally well for detecting HPV in cervical specimens obtained with ThinPrep and SurePath. The Cervi-Collect device compared well to the other collection methods. Alinity is a reliable assay for simultaneous detection of HPV-16/18 and other high-risk genotypes in cervical specimens.

Multicenter clinical evaluation of alinity m HBV assay performance.

BACKGROUND: Accurate molecular methods to detect and quantify hepatitis B virus (HBV) DNA are essential to diagnose chronic infections, guide treatment decisions, assess response to treatment, and determine risk of HBV-related complications. New generations of real-time HBV DNA assay platforms provide results in less than 2-3 h, with continuous loading of specimens and true random-access capability. OBJECTIVES: We examined the clinical performance of the new Alinity m HBV assay, run on the fully automated, continuous, random-access Alinity m platform, to accurately detect and quantify HBV DNA in a large series of patient samples infected with different HBV genotypes frequently encountered in clinical practice. STUDY DESIGN: This international, multisite study assessed the precision and reproducibility of the Alinity m HBV assay and compared its performance to four HBV assays currently in clinical use. RESULTS: The Alinity m HBV assay demonstrated linear quantitation of HBV DNA in plasma samples, with high precision (coefficient of variation 4.1 %-8.8 %) and reproducibility. The Alinity m HBV assay showed excellent correlation (correlation coefficients ≥0.947) with comparator HBV assays, with an overall observed bias ranging from -0.07 to 0.17 Log10 IU/mL. 97 % of quantifiable patient results were <1 Log10 IU/mL different than the respective comparator assays, with comparable results across HBV genotypes. CONCLUSIONS: The newly developed real-time PCR-based Alinity m HBV assay is sensitive, reproducible, and accurately quantifies HBV DNA levels from HBsAg-positive patients across the full dynamic range of quantification.

Multicenter clinical evaluation of alinity m HCV assay performance.

BACKGROUND: Nucleic acid testing is essential for the detection and quantification of HCV RNA in the diagnosis of HCV infection and treatment monitoring. The Alinity m HCV assay was recently developed by Abbott Molecular for rapid detection and quantification of HCV RNA on the fully automated, continuous, random-access Alinity m analyzer. OBJECTIVES: Our study assessed the performance of the new Alinity m HCV assay for detection and quantification of HCV RNA in a large series of patient samples of various genotypes. This international, multicentric study evaluated the linearity, precision, and reproducibility of the Alinity m HCV assay and its performance in comparison to three other HCV assays currently used in clinical practice. RESULTS: The Alinity m HCV assay demonstrated high linearity (correlation coefficient r = 1.00), precision (coefficients of variation [CV] 6.6-13.5 %) and reproducibility (CV 1.7-4.3 % across three control lots). At a concentration near the lower limit of detection, the Alinity m HCV assay exhibited >98 % detectability. The Alinity m HCV assay showed excellent correlation with comparator HCV assays in serum (n = 406) and plasma (n = 1401) samples (correlation coefficients ≥0.96, bias 0.01 to 0.14 Log10 IU/mL). More than 95 % of the quantified results with the Alinity m HCV assay were less than mean bias ± 1.96 SD different from those of the comparator assays. CONCLUSIONS: The newly developed Alinity m HCV assay is sensitive, reproducible, and accurately quantifies HCV RNA levels in serum and plasma samples from patients with chronic HCV infection, with no impact of HCV genotype on assay performance.

Multicenter clinical comparative evaluation of Alinity m HIV-1 assay performance.

BACKGROUND: Accurate, rapid detection of HIV-1 RNA is critical for early diagnosis, treatment decision making, and long-term management of HIV-1 infection. OBJECTIVE: We evaluated the diagnostic performance of the Alinity m HIV-1 assay, which uses a dual target/dual probe design against highly conserved target regions of the HIV-1 genome and is run on the fully automated Alinity m platform. STUDY DESIGN: This was an international, multisite study that compared the diagnostic performance of the Alinity m HIV-1 assay to four commercially available HIV-1 assays routinely used in nine independent clinical laboratories. Alinity m HIV-1 assay precision, detectability, and reproducibility was compared across four study sites. RESULTS: The Alinity m HIV-1 assay produced comparable results to currently available HIV-1 assays (correlation coefficient >0.995), with an overall bias of -0.1 to 0.10 Log10 copies/mL. The Alinity m HIV-1 assay and its predecessor m2000 HIV-1 assay demonstrated comparable detection of 16 different HIV-1 subtypes (R2 = 0.956). A high level of agreement (>88 %) between all HIV-1 assays was seen near clinical decision points of 1.7 Log10 copies/mL (50 copies/mL) and 2.0 Log10 copies/mL (200 copies/mL). Alinity m HIV-1 assay precision was 0.08 and 0.21 Log10 copies/mL at VLs of 1000 and 50 copies/mL, respectively, with a high level of detectability (≥97 % hit rate) and reproducibility across sites. CONCLUSIONS: The Alinity m HIV-1 assay provides comparable diagnostic accuracy to current HIV-1 assays, and when run on the Alinity m system, has the capacity to shorten the time between diagnosis and treatment.

Evaluation of the Fluorescence In Situ Hybridization Test to Predict Recurrence and/or Progression of Disease after bacillus Calmette-Guérin for Primary High Grade Nonmuscle Invasive Bladder Cancer: Results from a Prospective Multicenter Trial.

PURPOSE: Single center studies have shown that positive UroVysion® fluorescence in situ hybridization results were associated with recurrence of nonmuscle invasive bladder cancer treated with intravesical bacillus Calmette-Guérin. Our goal was to validate these findings. MATERIALS AND METHODS: We performed a prospective, multicenter diagnostic trial to determine whether the fluorescence in situ hybridization test could predict recurrence or progression in patients with primary high grade nonmuscle invasive bladder cancer who were scheduled to receive bacillus Calmette-Guérin. Fluorescence in situ hybridization testing was performed prior to the first bacillus Calmette-Guérin instillation, prior to the sixth instillation and at 3-month cystoscopy. The performance of fluorescence in situ hybridization was evaluated. RESULTS: A total of 150 patients were enrolled in analysis, including 68 with Ta disease, 41 with T1 disease, 26 with carcinoma in situ alone and 15 with papillary carcinoma plus carcinoma in situ. At 9 months of followup there were 46 events, including 37 recurrences and 9 progressions. For events with positive fluorescence in situ hybridization findings the HR was 2.59 (95% CI 1.42-4.73) for the baseline test, 1.94 (95% CI 1.04-3.59) for the 6-week test and 3.22 (95% CI 1.65-6.27) at 3 months. Patients with positive results at baseline, 6 weeks and 3 months had events 55% of the time and patients with negative results at each time point had no event 76% of the time. CONCLUSIONS: The study validated that a positive UroVysion fluorescence in situ hybridization test was associated with a 3.3-fold increased risk of recurrence. The test may be useful to risk stratify patients entering clinical trials in whom induction therapy fails. However, using the test to change management decisions is limited due to the discordance between results and outcomes as well as the variance of tests results with time.

Dual regulatory roles of phosphatidylinositol 3-kinase in IFN signaling.

PI3K is activated by the type I and II IFN receptors, but its precise role in the generation of IFN responses is not well understood. In the present study we used embryonic fibroblasts from mice with targeted disruption of the genes encoding for both the p85alpha and p85beta regulatory subunits of PI3'-kinase (p85alpha(-/-)beta(-/-)) to precisely define the role of PI3K in the control of IFN-induced biological responses. Our data demonstrate that PI3K plays dual regulatory roles in the induction of IFN responses by controlling both IFN-alpha- and IFN-gamma-dependent transcriptional regulation of IFN-sensitive genes and simultaneously regulating the subsequent initiation of mRNA translation for such genes. These processes include the Isg15, Cxcl10, and/or Irf7 genes, whose functions are important in the generation of the biological effects of IFNs. Consistent with this, the induction of IFN antiviral responses is defective in double p85alpha/p85beta knockout cells. Thus, integration of signals via PI3K is a critical event during engagement of the IFN receptors that complements both the transcriptional activity of Jak-STAT pathways and controls initiation of mRNA translation.

Proteasome inhibitors uncouple rhesus TRIM5alpha restriction of HIV-1 reverse transcription and infection.

The primate TRIM5alpha proteins have recently been defined as cellular restriction factors, preventing primate infection by retroviruses from different species. For instance, rhesus TRIM5alpha (rhTRIM5alpha) restricts infection by HIV-1. Virtually all TRIM5alpha proteins block the early replication of retroviruses by preventing the accumulation of reverse transcription products, but the underlying mechanism remains unclear. In this article, we find that disrupting proteasome function alters rhTRIM5alpha localization and allows the normal generation of HIV-1 late reverse transcription products, even though HIV-1 infection and the generation of nuclear 1-LTR and 2-LTR viral cDNA forms remain impaired. This finding suggests rhTRIM5alpha restricts HIV infection in two distinct phases: (i) altering the normal passage of the reverse-transcribing viral genome to the nucleus and (ii) targeting the reverse transcription complex to be disrupted by the proteasome. Because proteasome inhibitor blocks the second phase, accumulation of a nonfunctional viral DNA genome can be readily observed. Defining each phase may reveal HIV-1 targets for future antiviral therapy in which dual blockade may be equally as effective as naturally occurring rhTRIM5alpha protein in preventing HIV-1 infection in vivo.

Nef: "necessary and enforcing factor" in HIV infection.

The Human Immunodeficiency Virus -1 (HIV-1) Nef protein that was originally identified as a viral negative factor is a 27kDa myristoylated protein. However, this so called dispensable viral protein has emerged as one of the most important proteins for viral life cycle. Nef not only establishes the host cell environment suitable for viral replication and pathogenesis but also facilitates the progression of the infection into disease. Previous efforts have been focussed to explain how Nef down modulates host cell receptors like CD4 and MHC-1 molecules, thereby helping the virus to evade host defense and to increase viral infectivity. Nef also ably modulates specific processes like apoptosis in favour of viral life cycle other than being the stimulus for cell activation and signal transduction pathways. After much maligning over its reported positive or negative functions on the HIV-1 Long Terminal Repeat (LTR) promoter, the Nef protein is now perceived to enhance viral replication and infection through a combination of different effector functions. Recent reports emphasize a role for Nef in viral gene expression and place it in a prime position to oversee and optimize viral replication. Nef may do so by enhancing Tat mediated gene expression from the LTR by activating signalling pathways that result in a concomitant increase in the activation of general transcription factors, and also by mediating translocation of repression factors from the nucleus. Thus, Nef not only enhances infection but also plays an important role in viral replication and pathogenesis.