Amalia Magaret

University of Washington Seattle, Seattle, Washington, United States

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Publications (61)411.31 Total impact

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    ABSTRACT: Genome editing by designer nucleases is a rapidly evolving technology utilized in a highly diverse set of research fields. Among all fields, the T7 endonuclease mismatch cleavage assay, or Surveyor assay, is the most commonly used tool to assess genomic editing by designer nucleases. This assay, while relatively easy to perform, provides only a semi-quantitative measure of mutation efficiency that lacks sensitivity and accuracy. We demonstrate a simple droplet digital PCR assay that quickly quantitates a range of indel mutations with detection as low as 0.02% mutant in a wild type background and precision (≤6%CV) and accuracy superior to either mismatch cleavage assay or clonal sequencing when compared to next-generation sequencing. The precision and simplicity of this assay will facilitate comparison of gene editing approaches and their optimization, accelerating progress in this rapidly-moving field.
    No preview · Article · Feb 2016 · Scientific Reports
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    ABSTRACT: Background: Clinical trial designs that include multiple treatments are currently limited to those that perform pairwise comparisons of each investigational treatment to a single control. However, there are settings, such as the recent Ebola outbreak, in which no treatment has been demonstrated to be effective; and therefore, no standard of care exists which would serve as an appropriate control. Methods/design: For illustrative purposes, we focused on the care of patients presenting in austere settings with critically ill 'sepsis-like' syndromes. Our approach involves a novel algorithm for comparing mortality among arms without requiring a single fixed control. The algorithm allows poorly-performing arms to be dropped during interim analyses. Consequently, the study may be completed earlier than planned. We used simulation to determine operating characteristics for the trial and to estimate the required sample size. Results: We present a potential study design targeting a minimal effect size of a 23% relative reduction in mortality between any pair of arms. Using estimated power and spurious significance rates from the simulated scenarios, we show that such a trial would require 2550 participants. Over a range of scenarios, our study has 80 to 99% power to select the optimal treatment. Using a fixed control design, if the control arm is least efficacious, 640 subjects would be enrolled into the least efficacious arm, while our algorithm would enroll between 170 and 430. This simulation method can be easily extended to other settings or other binary outcomes. Conclusion: Early dropping of arms is efficient and ethical when conducting clinical trials with multiple arms.
    No preview · Article · Nov 2015 · Contemporary clinical trials
  • Kurt Diem · Amalia Magaret · Alexis Klock · Lei Jin · Jia Zhu · Lawrence Corey
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    ABSTRACT: In situ detection of specific cells offers a unique perspective on the spatial interactions between host immune cells and specific viral pathogens or cancers. Most immunohistochemistry techniques require manual cell counting on biopsied and fixed tissue sections. The availability of sophisticated software packages for analyzing fluorescently labeled tissue has made it possible to quickly and accurately quantitate the number of positive cells on such slides. Manual cell counting was compared to automatic cell counting using the program CellProfiler. The two techniques were used to count CD4+and CD8+T cells in human genital skin biopsies from herpesvirus type 2 (HSV-2) infected subjects. Manual counting and CellProfiler demonstrated high correlation both in cell counting as well as detection of immune cell "clustering" in tissue, an important visceral component of localized inflammation and characteristic of most chronic infections. Overall, CellProfiler is an effective and accurate method in addition to or replacement of manual cell counting of fluorescently labeled biopsies. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · Jun 2015 · Journal of virological methods
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    ABSTRACT: Background: The live, attenuated varicella vaccine strain (vOka) is the only licensed therapeutic vaccine. Boost of varicella zoster virus (VZV)-specific cellular immunity is a likely mechanism of action. We examined memory CD4(+) T-cell responses to each VZV protein at baseline and after zoster vaccination. Methods: Serial blood samples were collected from 12 subjects vaccinated with Zostavax and immunogenicity confirmed by ex vivo VZV-specific T-cell and antibody assays. CD4(+) T-cell lines enriched for VZV specificity were generated and probed for proliferative responses to every VZV protein and selected peptide sets. Results: Zoster vaccination increased the median magnitude (2.3-fold) and breadth (4.2-fold) of VZV-specific CD4(+) T cells one month post-vaccination. Both measures declined by 6 months. The most prevalent responses at baseline included VZV open reading frames (ORFs) 68, 4, 37, and 63. After vaccination, responses to ORFs 40, 67, 9, 59, 12, 62, and 18 were also prevalent. The immunogenicity of ORF9 and ORF18 were confirmed using peptides, defining a large number of discrete CD4 T-cell epitopes. Conclusions: The breadth and magnitude of the VZV-specific CD4(+) T-cell response increase after zoster vaccination. In addition to glycoprotein E (ORF68), we identified antigenic ORFs that may be useful components of subunit vaccines.
    No preview · Article · Mar 2015 · The Journal of Infectious Diseases
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    ABSTRACT: To evaluate the utility of quantitative herpes simplex virus (HSV) polymerase chain reaction (PCR) levels for prognosis and management of neonatal HSV disease. Clinical and virologic data were abstracted by medical record review from neonatal HSV cases treated at Seattle Children's Hospital between 1993 and 2012. HSV PCR results from plasma (n = 47), cerebrospinal fluid (n = 56), or both (n = 40) at the time of diagnosis were available from 63 infants; 26 with skin-eye-mouth (SEM), 18 with central nervous system (CNS), and 19 with disseminated (DIS) disease. Plasma HSV PCR was positive in 78% of the infants with SEM, 64% with CNS and 100% with DIS disease. Mean plasma viral level was 2.8 log10 copies/mL in SEM, 2.2 log10 copies/mL in CNS, and 7.2 log10 copies/mL in DIS infants. The HSV levels were higher among infants who died compared with surviving infants, 8.1 log10 copies/mL (range 7.7-8.6) vs 3.8 log10 copies/mL (range 0.0-8.6), P = .001, however, level of HSV DNA in the cerebrospinal fluid or in plasma did not correlate with neurologic outcome. Dynamics of HSV clearance from plasma during high-dose acyclovir treatment showed single-phase exponential decay with a median viral half-life of 1.26 days (range: 0.8-1.51). Plasma HSV levels correlate with clinical presentation of neonatal HSV disease and mortality, but not neurologic outcome. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Dec 2014 · Journal of Pediatrics
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    ABSTRACT: Background: Genital HSV-2 infection causes recurrent genital lesions and frequent viral shedding; current therapies do not completely control viral replication and require daily antiviral therapy. The therapeutic vaccine GEN003 contains 2 HSV-2 proteins, gD and ICP4, and Matrix M2 adjuvant (MM) to promote both B and T cell immune responses. Methods: We conducted a randomized, placebo controlled clinical trial in which 143 participants with genital HSV-2 infection were randomized to receive 3 vaccinations with 10, 30 or 100 µg of GEN003 with or without MM, or placebo. Participants collected twice daily swabs of genital secretions to assess genital shedding, and recorded genital lesions for 28 days prior to, immediately after; and 6 and 12 months after completion of vaccinations. Safety, immunogenicity and efficacy were monitored. Results: Immediately after vaccination, viral shedding in the 30 µg and 100 µg with MM groups decreased from baseline (rate ratios [RR] 0.47 and 0.68, respectively; p<0.001 for both). Shedding was unchanged from baseline for the placebo (RR= 1.01), unadjuvanted GEN003 (RR= 1.40), and 10 µg with MM groups (RR=1.01). Shedding remained suppressed for the 30 µg with MM dose group at 6 months (RR=0.57; p<0.001). Lesion rates were reduced after vaccination for the 30 and 100 µg with MM dose groups (RR 0.42 and 0.35, respectively; p<0.001 for both) and at 6 months for the 30 µg with MM dose (RR=0.26; p<0.001). In a preliminary data review, at 12 months, lesion rates for the 30 µg with MM dose remained reduced (9.6% [n=29] at baseline versus 2.4% [n=17] at 12 months). Three participants in the 10 µg with MM group discontinued dosing due to adverse events. Injection reactions were previously reported (Wald, ICAAC 2013). Other than reactogenicity, the number (%) of participants who reported 1 or more other Grade 3 or 4 AEs considered related to vaccination by treatment group were: 10 µg with MM, 3 (10%); 30 µg with MM, 2 (7%); 100 µg with MM, 1 (4%); GEN003 without adjuvant 1 (4%); Placebo, 0 (0%). Conclusion: The antiviral effect of GEN003 persisted for at least 6 months with an effect on genital lesions up to 12 months. Immunotherapy with GEN003 may be an effective strategy, with or without traditional antiviral agents, to treat chronic genital HSV-2 infections.
    No preview · Conference Paper · Oct 2014
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    ABSTRACT: Background: Vaccines are most successful for pathogens in which wild-type infection leads to protection against homologous re-infection. For HSV-2, we have little information concerning natural protection from re-infection or the variability between strains that could modulate protection from challenge. Understanding differences in HSV-2 strains worldwide, and the potential for reinfection, is essential in development of an HSV-2 vaccine. Methods: DNA from genital HSV-2 lesions containing ≥7 log10 copies HSV DNA/swab were sequenced using the Illumina next generation sequencing (NGS) platform. Human and highly repetitive sequences were subtracted, and remaining reads were aligned to the reference HSV-2 genome. Prevalent SNPs were defined as present in >10% and <90% of the sequenced strains. Informative SNPs were identified using FastTagger. Bayesian computations were used to estimate probabilities of identifying super-infection. Results: Fifty-six samples from genital herpes lesions were sequenced. Of 49 sequences from 39 persons with adequate coverage, the HSV genome had a median of 73.9 fold coverage (range 9.8-771 fold). Sixteen persons (41%) were from Africa, 12 (31%) from Peru, and 11 (28%) from the US; one-third were HIV infected. Of 2854 loci with heterogeneity, prevalent SNPs were found at 456 (16%) loci. Using genotyping at the 6 most informative SNPs to distinguish strains, we estimate a 90% probability of correctly identifying super-infection or lack thereof. Of 10 persons with paired specimens collected a median of 2.1 years apart, 2 (20%) had super-infection; both were HIV-infected. In each case, the strains differed at 186 loci. Six of 10 remaining pairs differed at 0 loci, and 2 differed at only 1 locus, indicating minimal within host evolution and low sequencing error. Conclusion: High quality HSV-2 sequence data can be obtained directly from genital swabs, removing the need for culture which may introduce mutations. The initial phase of our study has identified super-infection in 20% of persons investigated. Further research will determine whether host immune status, gender, and sexual exposure history are correlates of super-infection.
    No preview · Conference Paper · Oct 2014
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    ABSTRACT: Background: HSV-1 infection has a wide severity spectrum in the immunocompetent host, from asymptomatic seropositivity to frequent orolabial lesions. To gain insight into virus and host genotype contributions to disease phenotype, we evaluated HSV-1 genotypes and immunity in mono- (MZ) and dizygotic (DZ) twins. Methods: HSV-1 seropositive twin pairs collected daily oral swabs for quantitative HSV-1 PCR and kept symptom diaries for 60 days. Associations of shedding rates were assessed by estimating correlations. We categorized the viral strain as identical or different in each pair with DNA available for genotyping from both. The identity and breadth of HSV-1 antigens recognized by circulating CD4 T-cells were determined using a complete HSV-1 ORF (open reading frame) set. CD4 T-cell responses were scored as present or absent to each ORF. We used a bootstrap method to estimate the distribution of agreements in ORF responses between individuals. Results: We enrolled 29 MZ and 22 DZ twin pairs. The overall shedding rate was 10.3% of days (median 9.3%; range 0-47%). There was a positive correlation between shedding rates within twin pairs (r=0.33, p=0.015) but not among unrelated individuals (r=-0.086; p=0.5). Genotyping showed that 15/14 twin pairs had the same/different HSV-1 strain, respectively. The correlation for shedding rates in all twin pairs was higher in those with the same virus (r=0.55, p=0.033) versus different (r=-0.169, p=0.56). 8 MZ pairs were analyzed for CD4 T-cell responses. The median number of ORFs recognized per person was 19 (range 6-35). The bootstrapped mean percent agreement in ORF response between unrelated pairs was 71% (5th/95th percentile, 67%/75% respectively). The percent agreement between the original 8 pairs of MZ twins was 77% (p=0.003 for difference from bootstrapped dataset). Conclusion: A relationship between HSV-1 shedding and host genotype is supported by our observation of a higher correlation in HSV-1 shedding between twin pairs than between unrelated individuals and similar CD4 T-cell responses between MZ twins. These data and the higher correlation in shedding rates among twins with the same versus different virus suggests that both viral and host genetics contribute to HSV-1 severity.
    No preview · Conference Paper · Oct 2014

  • No preview · Article · Oct 2014 · Open Forum Infectious Diseases
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    Sharanya Rajagopal · Amalia Magaret · Nelly Mugo · Anna Wald
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    ABSTRACT: The burden of HSV type 2 varies substantially by region, with the highest incidence and prevalence in sub-Saharan Africa. We undertook a systematic review to identify studies reporting prospective data on incidence rates in men and women in Africa. Of 18 eligible studies, 7 were conducted in high-risk populations. Among women, incidence rates appeared to be higher in those with high-risk sexual behavior, with rates ranging from 3 to 23 per 100 person-years. In contrast, incidence rates in men appeared to be lower, ranging from 1 to 12 per 100 person-years. Risk factors for HSV-2 in women included prevalent human immunodeficiency virus (HIV) infection, younger age at sexual initiation, and sexual activity. Among men, condom use and circumcision had a protective effect, whereas prevalent HIV increased the risk of HSV-2 acquisition. This review draws attention to the high HSV-2 acquisition rates reported in Africa, thereby identifying an efficient setting for preventative HSV-2 vaccine trials.
    Preview · Article · Sep 2014 · Open Forum Infectious Diseases
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    ABSTRACT: Maternal acquisition of genital herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) near delivery accounts for most cases of neonatal herpes, although neonatal HSV incidence has remained stable in recent decades. A decline in HSV-1, but not HSV-2, seroprevalence has been reported among reproductive-aged women. This study was designed to evaluate trends in the seroprevalence of HSV-1 and HSV-2 among parturients who delivered between 1989 and 2010. Herpes simplex virus infection status was determined using Western blot within routine prenatal tests. The proportion of patients with prenatal HSV serologic results declined from 54.7% during 1989 to 1997 to 44.8% during 1998 to 2010. Yearly changes in HSV-1 and HSV-2 seroprevalence were estimated with Poisson regression. Models were adjusted for race/ethnicity, maternal age, parity, delivery type, and insurance status. Prenatal HSV serologic results were available for 18,993 pregnancies in 15,738 women (median age, 28 years; interquartile range, 23-33 years), of whom 43% were white, 12% were black, 11% were Asian, 7% were Hispanic, and 27% were other or unreported; 26% had private insurance. Nine percent of pregnancies involved women who were seropositive for HSV-2 only, 15% for both HSV-1 and HSV-2, and 53% for HSV-1 only; 24% were seronegative for HSV. Herpes simplex virus type 1 seroprevalence decreased from 69.1% during 1989 to 1999 to 65.5% during 2000 to 2010; HSV-2 seroprevalence decreased from 30.1% to 16.3%, for a 46% relative decline. After adjustment, no significant annual trend in HSV-1 seroprevalence was noted (0.1% per year; 95% CI, 0%-0.3% per year; P = 0.13). Rates of HSV-2 seroprevalence decreased significantly by 4.8% per year (95% CI, 4.3%-5.2% per year; P < 0.001). Herpes simplex virus type 1 seroprevalence increased slightly among black women only (0.9% per year; 95% CI, 0.4%-1.3% per year; P < 0.001). Herpes simplex virus type 2 seroprevalence decreased significantly among women of all races (P < 0.001). Rates per year decreased substantially less for black women relative to white women (2.6% per year vs 5.5% per year, respectively; P < 0.001). Seroprevalence of HSV-2 among pregnant women decreased between 1989 and 2010, with the decrease particularly noted for white women. Herpes simplex virus type 1 did not decrease overall and in fact increased slightly among black women. Women who are seronegative entering pregnancy and acquire HSV during late pregnancy are at higher risk for transmission of HSV to their infants than are seropositive women. These findings offer new data on HSV seroprevalence in the pregnant population.
    No preview · Article · Aug 2014 · JAMA The Journal of the American Medical Association
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    ABSTRACT: Background: Pneumonia is the leading cause of childhood mortality globally. Respiratory syncytial virus (RSV) is the most important viral cause of pneumonia. Maternal serum antibody protects infants from RSV disease. The objective of our study was to characterize RSV antibody levels in mother-infant pairs. Methods: Serial serum samples were collected from mother-infant pairs in Bangladesh from the third trimester of pregnancy to 72 weeks postpartum and tested using an RSV antibody microneutralization assay. Serologic infection was defined as a 4-fold increase in antibody titer. Maternal antibody half-life was calculated using infant antibody titers from birth to 20 weeks. Results: The ratio of infant cord blood to maternal serum RSV antibody titers in 149 mother-infant pairs was 1.01 (95% confidence interval [CI], .99-1.03). Maternal RSV antibody titers in the third trimester and at birth were strongly correlated (R = 0.68). Antibody half-life was 38 days (95% CI, 36-42 days). Higher cord blood RSV antibody titers were associated with a lower risk of serologic infection (P = .01) and maintenance of antibody titer above a potentially protective threshold (P < .001). Conclusions: Efficient transplacental transfer of RSV-specific antibody from mother to the fetus was documented in mother-infant pairs in Asia. Higher cord blood antibody titers were associated with protection from serologic infection.
    Full-text · Article · Jun 2014 · The Journal of Infectious Diseases
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    ABSTRACT: Background Human herpesvirus 8 (HHV-8) replication increases the risk of Kaposi sarcoma (KS). Highly-active antiretroviral therapy (HAART) reduces the incidence of KS, and regimens that contain protease inhibitors (PIs) may be particularly effective. Objective To determine whether PI-based HAART regimens may more effectively inhibit HHV-8 shedding compared to regimens without PIs. Study design: Prospective, observational study of 142 HIV-1 and HHV-8 co-infected men conducted in Seattle, Washington. Quantitative HHV-8 PCR testing was performed on daily swabs of the oropharynx, the primary site of HHV-8 replication. Associations between antiretroviral regimen and detection of HHV-8 DNA in swabs were evaluated using generalized estimating equations. Results HHV-8 DNA was detected in 3,016 (26%) of 11,608 specimens collected. PI-based HAART was associated with a statistically significantly lower frequency of detection (RR 0.2; 95% CI 0.1 to 0.5) compared to ART-naïve persons, whereas HAART without a PI was not (RR 0.7; 95% CI 0.4 to 1.3). Compared to ART-naïve persons, there was also a trend toward lower quantities of HHV-8 detected during treatment with HAART regimens that contained a PI. These associations between PIs and measures of HHV-8 shedding could not be attributed to use of nelfinavir, which inhibits HHV-8 replication in vitro, and were independent of CD4 count and HIV plasma viral load (VL). Conclusions HAART regimens that contain PIs appear to decrease HHV-8 shedding compared to NNRTIs. Further study of PI-based HAART is warranted to determine the optimal regimens for prevention and treatment of KS.
    No preview · Article · Jun 2014 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
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    ABSTRACT: Human cytomegalovirus (CMV) has historically been the major infectious cause of morbidity and mortality among patients receiving hematopoietic cell or organ transplant. Standard care in a transplant setting involves frequent monitoring of CMV viral load over weeks to months to determine when antiviral treatment may be required. Quantitative PCR (qPCR) is the standard molecular diagnostic method for monitoring. Recently, digital PCR (dPCR) has shown promise in viral diagnostics, although current dPCR systems have lower throughput than qPCR systems. Here, we compare qPCR and droplet digital PCR (ddPCR) for CMV detection in patient plasma samples. Droplet digital PCR exhibits increased precision over qPCR at viral loads of ≥4 log10 with equivalent sensitivity. However, retrospective analysis of longitudinal samples from transplant patients with CMV viral loads near therapeutic thresholds did not provide evidence that the improved precision of ddPCR would be of clinical benefit. Given the throughput advantages of current qPCR systems, a widespread switch to dPCR for CMV monitoring would appear premature.
    Full-text · Article · May 2014 · Journal of Clinical Microbiology
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    ABSTRACT: Background: Despite high herpes simplex virus type 2 (HSV-2) incidence and prevalence among women in Africa, we are unaware of published neonatal herpes reports. To assess neonatal HSV transmission potential in South Africa, we investigated the frequency of the strongest risk factors: HSV acquisition in late pregnancy and HSV shedding during labor. Methods: Women admitted in early labor to a hospital in Soweto underwent HSV serologic testing and genital swab collection for HSV PCR. HSV-2 seronegative women were assessed for seroconversion 4-6 weeks after delivery. Results: Of 390 women enrolled, 229 (58.7%) were HSV-2 seropositive. Genital HSV-2 was detected in 17.2% of HSV-2 seropositive women, including 26 of 115 HIV-positive and 13 of 110 HIV-negative women (22.6% versus 11.8%; RR, 1.91; 95% CI, 1.04-3.53; P = 0.038), but in none of 161 HSV-2 seronegative women. Among the 91 HSV-2 seronegative women followed after delivery, none seroconverted. Conclusions: HSV-2 reactivation is common among South African women during labor, especially those with HIV coinfection. To determine the epidemiology of neonatal herpes in South Africa and to investigate whether the lack of reported cases is due to alterations in immune control or HSV-2 virulence, studies evaluating acutely ill neonates for HSV and studies of maternal HSV-2 shedding patterns are needed.
    Full-text · Article · May 2014 · Infectious Diseases in Obstetrics and Gynecology
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    ABSTRACT: Resiquimod, a Toll-like receptor 7 and 8 agonist, stimulates production of cytokines that promote an antigen-specific T helper type 1 acquired immune response. Animal and phase II human trials showed posttreatment efficacy in reducing recurrent herpes lesion days and/or time to first recurrence. Three phase III randomized, double-blind, vehicle-controlled trials of topical resiquimod to reduce anogenital herpes recurrences were conducted in healthy adults with ≥4 recurrences within the prior year. Participants applied resiquimod 0.01% gel or vehicle gel 2 times per week for 3 weeks to each recurrence for 12 months. Trials 1 and 2 had 2:1 resiquimod-vehicle randomization. Trial 3 had 1:1:1 randomization for resiquimod and 500 mg valacyclovir orally twice daily for 5 days (RESI-VAL), resiquimod and oral placebo (RESI-PLA), and vehicle and oral placebo (VEH-PLA). The median time to first recurrence was similar for resiquimod and vehicle (trial 1, 60 and 56 days, P = 0.7; trial 2, 54 and 48 days, P = 0.47; trial 3, 51 [RESI-VAL], 55 [RESI-PLA], and 44 [VEH-PLA] days, P = not significant [NS]). The median time to healing of initial treated recurrence was longer for resiquimod (trial 1, 18 compared to 10 days, P < 0.001; trial 2, 19 compared to 13 days, P = 0.16; trial 3, 14 [RESI-VAL], 16 [RESI-PLA], and 8 [VEH-PLA] days, P < 0.001). In trials 1 and 2, moderate to severe erythema and erosion/ulceration at the application site were more common in resiquimod recipients. In conclusion, no posttreatment efficacy of resiquimod 0.01% gel was observed. Increased application site reactions and initial recurrence healing time are consistent with resiquimod-induced cytokine effects.
    Full-text · Article · Apr 2014 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: Background. Depot medroxyprogesterone acetate (DMPA) has been linked to human immunodeficiency virus type 1 (HIV-1) acquisition. Methods. Vaginal microbiota of women using DMPA for up to 2 years were cultured. Mucosal immune cell populations were measured by immunohistological staining. Results. Over 12 months, the proportion with H2O2-positive lactobacilli decreased (n = 32; 53% vs 27%; P = .03). Median vaginal CD3+ cells also decreased (n = 15; 355 vs 237 cells/mm2; P = .03), as did CD3+CCR5+ cells (195 vs 128 cells/mm2; P = .04), HLA-DR+ cells (130 vs 96 cells/mm2; P = .27), and HLA-DR+CCR5+ cells (18 vs 10 cells/mm2; P = .33). Conclusions. DMPA contraception does not increase vaginal mucosal CCR5+ HIV target cells but does decrease CD3+ T lymphocytes and vaginal H2O2-producing lactobacilli.
    Full-text · Article · Mar 2014 · The Journal of Infectious Diseases
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    ABSTRACT: Background: Human herpesvirus 6 (HHV-6) latently infects a majority of adults. In about 1% of the population HHV-6 exists in a chromosomally integrated form (ciHHV-6) that resides in every somatic and germ cell and can be transmitted through the germ line. Patients with ciHHV-6 have been misdiagnosed and unnecessarily treated for active HHV-6 infection, sometimes with important side effects, based on results from quantitative molecular HHV-6 tests. Methods: A droplet digital PCR (ddPCR) assay was developed to identify ciHHV-6 in cellular patient samples by precisely determining the ratio of HHV-6 to cellular DNA. We validated the assay on confirmed ciHHV-6 patient samples and a cell line derived from a ciHHV-6 patient, and we analyzed hematopoietic stem cell transplant patients suspected of having ciHHV-6. We additionally evaluated whether the assay could be applied to stored plasma samples from a study of clinical correlates of HHV-6. Results: The ddPCR assay accurately identified ciHHV-6 in cellular samples (buffy coat, peripheral blood mononuclear cells), giving a ratio very close to 1 HHV-6/cell [mean (SD), 1.02 (0.03)] in fluorescence in situ hybridization-confirmed sample). In stored plasma samples, the assay performance was set by design to have 100% sensitivity, which resulted in 82% specificity for ciHHV-6. Conclusions: The possibility of ciHHV-6 is often overlooked in patients with detectable HHV-6 viral loads by quantitative PCR. Our ddPCR test provides rapid and accurate laboratory identification of ciHHV-6 from easily obtained cellular samples. In addition, the assay provides excellent sensitivity and specificity using stored plasma samples, facilitating retrospective analysis of the clinical significance of ciHHV-6.
    Preview · Article · Mar 2014 · Clinical Chemistry
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    ABSTRACT: Genital herpes simplex virus (HSV) reactivation is thought to be anatomically and temporally localized, coincident with limited ganglionic infection. Short, subclinical shedding episodes are the most common form of HSV-2 reactivation, with host clearance mechanisms leading to rapid containment. The anatomic distribution of shedding episodes has not been characterized. To precisely define patterns of anatomic reactivation, we divided the genital tract into a 22-region grid and obtained daily swabs for 20 days from each region in 28 immunocompetent, HSV-2-seropositive persons. HSV was detected via PCR, and sites of asymptomatic HSV shedding were subjected to a biopsy procedure within 24 h. CD4(+) and CD8(+) T cells were quantified by immunofluorescence, and HSV-specific CD4(+) T cells were identified by intracellular cytokine cytometry. HSV was detected in 868 (7%) of 11,603 genital swabs at a median of 12 sites per person (range, 0 to 22). Bilateral HSV detection occurred on 83 (67%) days with shedding, and the median quantity of virus detected/day was associated with the number of sites positive (P<0.001). In biopsy specimens of asymptomatic shedding sites, we found increased numbers of CD8(+) T cells compared to control tissue (27 versus 13 cells/mm(2), P = 0.03) and identified HSV-specific CD4(+) T cells. HSV reactivations emanate from widely separated anatomic regions of the genital tract and are associated with a localized cellular infiltrate that was demonstrated to be HSV specific in 3 cases. These data provide evidence that asymptomatic HSV-2 shedding contributes to chronic inflammation throughout the genital tract.
    Full-text · Article · Feb 2014 · Journal of Virology
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    ABSTRACT: Pritelivir, an inhibitor of the viral helicase-primase complex, exhibits antiviral activity in vitro and in animal models of herpes simplex virus (HSV) infection. We tested the efficacy and safety of pritelivir in otherwise healthy persons with genital HSV-2 infection. We randomly assigned 156 HSV-2-positive persons with a history of genital herpes to receive one of four doses of oral pritelivir (5, 25, or 75 mg daily, or 400 mg weekly) or placebo for 28 days. Participants obtained daily swabs from the genital area for HSV-2 testing, which was performed with a polymerase-chain-reaction assay. Participants also maintained a diary of genital signs and symptoms. The primary end point was the rate of genital HSV shedding. HSV shedding among placebo recipients was detected on 16.6% of days; shedding among pritelivir recipients was detected on 18.2% of days among those receiving 5 mg daily, 9.3% of days among those receiving 25 mg daily, 2.1% of days among those receiving 75 mg daily, and 5.3% of days among those receiving 400 mg weekly. The relative risk of viral shedding with pritelivir, as compared with placebo, was 1.11 (95% confidence interval [CI], 0.65 to 1.87) with the 5-mg daily dose, 0.57 (95% CI, 0.31 to 1.03) with the 25-mg daily dose, 0.13 (95% CI, 0.04 to 0.38) with the 75-mg daily dose, and 0.32 (95% CI, 0.17 to 0.59) with the 400-mg weekly dose. The percentage of days with genital lesions was also significantly reduced, from 9.0% in the placebo group to 1.2% in both the group receiving 75 mg of pritelivir daily (relative risk, 0.13; 95% CI, 0.02 to 0.70) and the group receiving 400 mg weekly (relative risk, 0.13; 95% CI, 0.03 to 0.52). The rate of adverse events was similar in all groups. Pritelivir reduced the rates of genital HSV shedding and days with lesions in a dose-dependent manner in otherwise healthy men and women with genital herpes. (Funded by AiCuris; ClinicalTrials.gov number, NCT01047540.).
    Preview · Article · Jan 2014 · New England Journal of Medicine

Publication Stats

1k Citations
411.31 Total Impact Points

Institutions

  • 2007-2015
    • University of Washington Seattle
      • • Department of Laboratory Medicine
      • • Department of Health Services
      Seattle, Washington, United States
  • 2012-2014
    • Fred Hutchinson Cancer Research Center
      • Division of Vaccine and Infectious Disease
      Seattle, Washington, United States