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

Book Reviews

Reviews of the following books: Fragile Structures: A Story of Oil Refineries, National Security and the Coast of Maine by Peter Amory Bradford; Maine Catalog: Historic American Buildings Survey, National Park Service, Department of the Interior - A List of Measured Drawings, Photographs and Written Documentation in the Survey compiled and introductory essay by Denys Peter Myers; Journals of the Gloucester Experiment by Gloucester Community Development Corporatio

The Human Papillomavirus Type 11 E1∧E4 Protein Is Phosphorylated in Genital Epithelium

AbstractThe most abundant viral transcript in human papillomavirus (HPV) 11-infected xenograft tissue has been shown to encode the E1∧E4 protein. The function of E1∧E4 protein has not been determined. Several potential phosphorylation sequence motifs were identified in the HPV 11 E1∧E4 protein, including potential sites of phosphorylation by mitogen-activated protein kinase (MAPK), cAMP-dependent protein kinase (PKA), casein kinase II, and protein kinase C. To test phosphorylation of the HPV 11 E1∧E4 protein, a soluble maltose binding protein (MBP) fusion was produced in Escherichia coli. Only MAPK and PKA phosphorylated the E1∧E4 protein. Phosphoamino acid analysis showed that one or more threonine residues were phosphorylated by MAPK, and both serine and threonine residues were phosphorylated by PKA. MBP–E1∧E4 mutant proteins were designed to delineate the E1∧E4 phosphoacceptor residues. MAPK was shown to phosphorylate E1∧E4 on threonine 53 within a MAPK consensus phorphorylation sequence motif. PKA was shown to phosphorylate E1∧E4 at two residues: threonine 36 within a consensus motif and serine 44 within a variant of the PKA consensus phosphorylation sequence motif. HPV 11-infected human genital tissue grown as a xenograft in an athymic mouse was labeled with [32P]orthophosphate. Phosphoamino acid analysis of E1∧E4 protein immunoprecipitated from 32P-labeled tissue revealed that both serine and threonine residues were phosphorylated. Analysis by liquid chromatography–mass spectrophotometry was consistent with phosphorylation of residues within the PKA and MAPK phosphorylation sequence motifs. Expression of E1∧E4 protein containing phosphorylation substitution mutations showed that the PKA mutant did not differ from wild-type E1∧E4 protein in intracellular distribution. In contrast, the MAPK mutant did not localize exclusively to the cytoplasm nor did it colocalize with wild-type E1∧E4 protein. We conclude that HPV 11 E1∧E4 protein is phosphorylated in vitro and in vivo. Our data are consistent with phosphorylation of HPV 11 E1∧E4 protein by MAPK and PKA in infected tissue

The CDC Revised Recommendations for HIV Testing: Reactions of Women Attending Community Health Clinics

The purpose of this study was to examine reactions to the Centers for Disease Control and Prevention revised recommendations for HIV testing by women attending community health clinics. A total of 30 women attending three community clinics completed semistructured individual interviews containing three questions about the recommendations. Thematic content analysis of responses was conducted. Results were that all agreed with the recommendation for universal testing. Most viewed opt-out screening as an acceptable approach to HIV testing. Many emphasized the importance of provision of explicit verbal informed consent. The majority strongly opposed the elimination of the requirement for pretest prevention counseling and spontaneously talked about the ongoing importance of posttest counseling. The conclusion was that there was strong support for universal testing of all persons 13 to 64 years old but scant support for the elimination of pretest prevention counseling. In general, respondents believed that verbal informed consent for testing as well as provision of HIV-related information before and after testing were crucial

Once Daily Valacyclovir for Reducing Viral Shedding in Subjects Newly Diagnosed with Genital Herpes

Objective. Genital herpes (GH) recurrences and viral shedding are more frequent in the first year after initial HSV-2 infection. The objective of this study was to provide the first evaluation of valacyclovir 1 g once daily compared to placebo in reducing viral shedding in subjects newly diagnosed with GH. Methods. 70 subjects were randomized to receive valacyclovir 1 g daily or placebo in a crossover design for 60 days with a 7-day washout period. A daily swab of the genital/anal-rectal area was self-collected for HSV-2 detection by PCR. Subjects attended the clinic for routine study visits and GH recurrence visits. Treatment differences were assessed using a nonparametric crossover analysis. Results. 52 subjects had at least one PCR measurement in both treatment periods and comprised the primary efficacy population. Valacyclovir significantly reduced HSV-2 shedding during all days compared to placebo (mean 2.9% versus 13.5% of all days (P < .01), a 78% reduction). Valacyclovir significantly reduced subclinical HSV-2 shedding during all days compared to placebo (mean 2.4% versus 11.0% of all days (P < .01), a 78% reduction). However, 79% of subjects had no GH recurrences while receiving valacyclovir compared to 52% of subjects receiving placebo (P < .01). Conclusion. In this study, the frequency of total and subclinical HSV-2 shedding was greater than reported in earlier studies involving subjects with a history of symptomatic genital recurrences. Our study is the first to demonstrate a significant reduction in viral shedding with valacyclovir 1 g daily compared to placebo in a population of subjects newly diagnosed with HSV-2 infection

Transient Increase in Herpes Simplex Virus Type 2 (HSV-2)-Associated Genital Ulcers Following Initiation of Antiretroviral Therapy in HIV/HSV-2-Coinfected Individuals

BACKGROUND: Immune reconstitution inflammatory syndrome (IRIS) in human immunodeficiency virus (HIV)-infected persons beginning antiretroviral therapy (ART) has been incompletely characterized for herpes simplex virus type 2 (HSV-2). METHODS: We evaluated genital ulcer disease (GUD) and HSV-2-associated GUD at quarterly visits or when spontaneously reported at monthly visits in 3381 HIV/HSV-2-coinfected individuals in a placebo-controlled trial of suppressive acyclovir therapy to prevent HIV transmission, 349 of whom initiated ART during the study. Incidence was calculated for months before and after ART initiation, and incidence rate ratios (IRRs) were calculated. RESULTS: GUD incidence increased from 15.0 episodes per 100 person-years before ART to 26.9 episodes per 100 person-years in the first full quarter after ART initiation (IRR, 1.83;P= .03), and the incidence of HSV-2-associated GUD increased from 8.1 to 19.0 episodes per 100 person-years (IRR, 2.20;P= .02). Subsequently, the incidence of GUD was similar to that before ART, although the numbers were small. Persons receiving suppressive acyclovir had fewer GUD episodes, but the IRR after beginning ART was similar in the acyclovir and placebo groups. CONCLUSIONS: Initiation of ART in HIV/HSV-2-coinfected persons is associated with a transient increase in GUD and HSV-2 GUD. Acyclovir reduces the incidence of GUD but does not prevent an increase in GUD incidence during the first quarter following initiation of ART

Imiquimod 3.75% Cream Applied Daily to Treat Anogenital Warts: Combined Results from Women in Two Randomized, Placebo-Controlled Studies

Objective. To evaluate if new imiquimod formulations using a shorter treatment duration are safe and efficacious to treat anogenital warts. Methods. In two studies 534 women ≥12 years of age (mean 33.4) with 2–30 warts (mean 7.9) and total wart area ≥10 mm2 (mean 166.3) were randomized (1 : 2 : 2) to placebo (106), imiquimod 2.5% (212) or 3.75% (216) creams applied once daily until complete clearance or a maximum of 8 weeks. Results. For placebo, imiquimod 2.5% and 3.75%, respectively, complete clearance of all warts was achieved in 14.2%, 28.3%, and 36.6% of women (intent-to-treat, P = 0.008 imiquimod 2.5%, and P < 0.001 3.75% versus placebo). Mean changes in wart counts were −10.7%, −50.9%, and −63.5% (per-protocol, P < 0.001 each active versus placebo) and safety-related discontinuation rates 0.9%, 1.4%, and 2.3%. Conclusions. Imiquimod 3.75% applied daily for up to 8 weeks was well tolerated and superior to placebo in treating women with external anogenital warts

Clinical Study Once Daily Valacyclovir for Reducing Viral Shedding in Subjects Newly Diagnosed with Genital Herpes

Objective. Genital herpes (GH) recurrences and viral shedding are more frequent in the first year after initial HSV-2 infection. The objective of this study was to provide the first evaluation of valacyclovir 1 g once daily compared to placebo in reducing viral shedding in subjects newly diagnosed with GH. Methods. 70 subjects were randomized to receive valacyclovir 1 g daily or placebo in a crossover design for 60 days with a 7-day washout period. A daily swab of the genital/anal-rectal area was self-collected for HSV-2 detection by PCR. Subjects attended the clinic for routine study visits and GH recurrence visits. Treatment differences were assessed using a nonparametric crossover analysis. Results. 52 subjects had at least one PCR measurement in both treatment periods and comprised the primary efficacy population. Valacyclovir significantly reduced HSV-2 shedding during all days compared to placebo (mean 2.9% versus 13.5% of all days (P &lt; .01), a 78% reduction). Valacyclovir significantly reduced subclinical HSV-2 shedding during all days compared to placebo (mean 2.4% versus 11.0% of all days (P &lt; .01), a 78% reduction). However, 79% of subjects had no GH recurrences while receiving valacyclovir compared to 52% of subjects receiving placebo (P &lt; .01). Conclusion. In this study, the frequency of total and subclinical HSV-2 shedding was greater than reported in earlier studies involving subjects with a history of symptomatic genital recurrences. Our study is the first to demonstrate a significant reduction in viral shedding with valacyclovir 1 g daily compared to placebo in a population of subjects newly diagnosed with HSV-2 infection

Effect of Pritelivir Compared With Valacyclovir on Genital HSV-2 Shedding in Patients With Frequent Recurrences A Randomized Clinical Trial

Importance Current therapy of herpes infections relies on nucleoside analogues. Pritelivir is a well-tolerated novel herpes simplex virus (HSV) helicase-primase inhibitor that reduced genital shedding and lesions. Objective To compare the efficacy of pritelivir with valacyclovir for suppression of genital HSV-2 infection. Design, Setting, and Participants A phase 2, randomized, double-blind, crossover clinical trial at clinical research centers in 4 US cities (October 2012-July 2013) compared daily oral doses of 100 mg of pritelivir with 500 mg of valacyclovir. The planned sample size was 98 adults, allowing for detection of a 50% reduction in viral shedding between the study treatments. Healthy adults with 4 to 9 annual genital HSV-2 recurrences were eligible. Ninety-one participants were randomized: 45 to receive pritelivir and 46 to receive valacyclovir first when the US Food and Drug Administration placed the trial on clinical hold based on findings in a concurrent nonclinical toxicity study, and the sponsor terminated the study. Interventions Participants took the first drug for 28 days followed by 28 days of washout before taking the second drug for 28 days. Throughout treatment, the participants collected genital swabs 4 times daily for testing by HSV polymerase chain reaction assays. Main Outcomes and Measures The primary end point was within-participant genital HSV shedding while receiving pritelivir compared with valacyclovir. Secondary end points included the quantity of HSV in positive swabs and the frequency of genital lesions and shedding episodes. Results Of the 91 randomized participants (median age, 48 years; 57 women [63%]), 56 had completed both treatment periods at the time of the study’s termination. In intent-to-treat analyses, HSV shedding was detected in 2.4% (173 of 7276 ) of swabs during pritelivir treatment compared with 5.3% (392 of 7453) during valacyclovir treatment (relative risk [RR], 0.42; 95% CI, 0.21 to 0.82; P = .01). In swabs with HSV, the mean quantity of HSV was 3.2 log10 copies/mL during pritelivir treatment vs 3.7 log10 copies/mL during valacyclovir treatment (difference, −0.1; 95% CI, −0.6 to 0.5; P = .83). Genital lesions were present on 1.9% of days in the pritelivir group vs 3.9% in the valacyclovir group (RR, 0.40; 95% CI, 0.17-0.96; P = .04). The frequency of shedding episodes did not differ by group, with 1.3 per person-month for pritelivir and 1.6 per person-month for valacyclovir (RR, 0.80; 95% CI, 0.52 to 1.22; P = .29). Treatment-emergent adverse events occurred in 62.3% of participants in the pritelivir group and 69.2% of participants in the valacyclovir group. Conclusions and Relevance Among adults with frequently recurring genital HSV-2, the use of pritelivir compared with valacyclovir resulted in a lower percentage of swabs with HSV detection over 28 days. Further research is needed to assess longer-term efficacy and safety