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1.  Association of Maternal Smoking during Pregnancy with Infant Hospitalization and Mortality Due to Infectious Diseases 
Background
Maternal smoking is associated with infant respiratory infections and with increased risk of low birthweight (LBW) infants and preterm birth. This study assesses the association of maternal smoking during pregnancy with both respiratory and non-respiratory infectious disease (ID) morbidity and mortality in infants.
Methods
We conducted two retrospective case-control analyses of infants born in Washington State from 1987–2004 using linked birth certificate, death certificate, and hospital discharge records. One assessed morbidity—infants hospitalized due to ID within one year of birth (47,404 cases/48,233 controls). The second assessed mortality—infants who died within one year due to ID (627 cases/2,730 controls).
Results
Maternal smoking was associated with both hospitalization (Adjusted Odds Ratio (AOR)=1.52; 95%CI: 1.46, 1.58) and mortality (AOR=1.51; 95%CI: 1.17, 1.96) due to any ID. In subgroup analyses, maternal smoking was associated with hospitalization due to a broad range of ID including both respiratory (AOR=1.69; 95%CI: 1.63, 1.76) and non-respiratory ID (AOR=1.27; 95%CI: 1.20, 1.34). Further stratification by birthweight and gestational age did not appreciably change these estimates. In contrast, there was no association of maternal smoking with ID infant mortality when only LBW infants were considered.
Conclusions
Maternal smoking was associated with a broad range of both respiratory and non-respiratory ID outcomes. Despite attenuation of the mortality association among LBW infants, ID hospitalization was found to be independent of both birthweight and gestational age. These findings suggest that full-term infants of normal weight whose mothers smoked may suffer an increased risk of serious ID morbidity and mortality.
doi:10.1097/INF.0b013e3182704bb5
PMCID: PMC3588859  PMID: 22929173
infant; infectious disease; prenatal exposure; maternal smoking
2.  Technologies for Detecting Falsified and Substandard Drugs in Low and Middle-Income Countries 
PLoS ONE  2014;9(3):e90601.
Falsified and substandard drugs are a global health problem, particularly in low- and middle-income countries (LMIC) that have weak pharmacovigilance and drug regulatory systems. Poor quality medicines have important health consequences, including the potential for treatment failure, development of antimicrobial resistance, and serious adverse drug reactions, increasing healthcare costs and undermining the public's confidence in healthcare systems. This article presents a review of the methods employed for the analysis of pharmaceutical formulations. Technologies for detecting substandard and falsified drugs were identified primarily through literature reviews. Key-informant interviews with experts augmented our methods when warranted. In order to aid comparisons, technologies were assigned a suitability score for use in LMIC ranging from 0–8. Scores measured the need for electricity, need for sample preparation, need for reagents, portability, level of training required, and speed of analysis. Technologies with higher scores were deemed the most feasible in LMICs. We categorized technologies that cost $10,000 USD or less as low cost, $10,000–100,000 USD as medium cost and those greater than $100,000 USD as high cost technologies (all prices are 2013 USD). This search strategy yielded information on 42 unique technologies. Five technologies were deemed both low cost and had feasibility scores between 6–8, and an additional four technologies had medium cost and high feasibility. Twelve technologies were deemed portable and therefore could be used in the field. Many technologies can aid in the detection of substandard and falsified drugs that vary from the simplest of checklists for packaging to the most complex mass spectrometry analyses. Although there is no single technology that can serve all the requirements of detecting falsified and substandard drugs, there is an opportunity to bifurcate the technologies into specific niches to address specific sections within the workflow process of detecting products.
doi:10.1371/journal.pone.0090601
PMCID: PMC3966738  PMID: 24671033
3.  Complex Patterns of Protease Inhibitor Resistance among Antiretroviral Treatment-Experienced HIV-2 Patients from Senegal: Implications for Second-Line Therapy 
Protease inhibitor (PI)-based antiretroviral therapy (ART) can effectively suppress HIV-2 plasma load and increase CD4 counts; however, not all PIs are equally active against HIV-2, and few data exist to support second-line therapy decisions. To identify therapeutic options for HIV-2 patients failing ART, we evaluated the frequency of PI resistance-associated amino acid changes in HIV-2 sequences from a cohort of 43 Senegalese individuals receiving unboosted indinavir (n = 18 subjects)-, lopinavir/ritonavir (n = 4)-, or indinavir and then lopinavir/ritonavir (n = 21)-containing ART. Common protease substitutions included V10I, V47A, I54M, V71I, I82F, I84V, L90M, and L99F, and most patients harbored viruses containing multiple changes. Based on genotypic data, we constructed a panel of 15 site-directed mutants of HIV-2ROD9 containing single- or multiple-treatment-associated amino acid changes in the protease-encoding region of pol. We then quantified the susceptibilities of the mutants to the HIV-2 “active” PIs saquinavir, lopinavir, and darunavir using a single-cycle assay. Relative to wild-type HIV-2, the V47A mutant was resistant to lopinavir (6.3-fold increase in the mean 50% effective concentration [EC50]), the I54M variant was resistant to darunavir and lopinavir (6.2- and 2.7-fold increases, respectively), and the L90M mutant was resistant to saquinavir (3.6-fold increase). In addition, the triple mutant that included I54M plus I84V plus L90M was resistant to all three PIs (31-, 10-, and 3.8-fold increases in the mean EC50 for darunavir, saquinavir, and lopinavir, respectively). Taken together, our data demonstrate that PI-treated HIV-2 patients frequently harbor viruses that exhibit complex patterns of PI cross-resistance. These findings suggest that sequential PI-based regimens for HIV-2 treatment may be ineffective.
doi:10.1128/AAC.00405-13
PMCID: PMC3716120  PMID: 23571535
4.  Validation for Clinical Use of a Novel HIV-2 Plasma RNA Viral Load Assay Using the Abbott m2000 Platform 
Background
Optimal care of persons infected with human immunodeficiency virus type 2 (HIV-2) requires an accurate assessment of HIV-2 plasma viral load (VL), but no clinically-approved quantitative HIV-2 RNA VL assay exists.
Objectives
To validate a novel quantitative HIV-2 RNA assay for clinical and research use.
Study Design
The Abbott m2000sp/rt platform was adapted for quantification of HIV-2 RNA in plasma. Amplification targeted a region of the long terminal repeat conserved in Group A and B HIV-2. Electron microscopy-counted-HIV-2 standards, the WHO/NIBSC HIV-2 International Standard and clinical specimens (N=162) were used to determine the precision, sensitivity, specificity, linear range, accuracy, and clinical performance of the assay.
Results
The quantitative linear range of the HIV-2 RNA assay was 10–1,000,000 copies/mL (R2 >0.99), with a limit of detection of 8 copies/mL (95% CI, 5–18 copies/ml). The assay did not cross-react with HIV-1, and quantification of HIV-2 RNA was not affected by the presence of >5 log10 HIV-1 RNA copies/mL. The total standard deviation (SD) and intra- and inter- run SD were 0.095, 0.093 and 0.162, respectively, at nominal inputs of 3.7, 1.7 and 1.0 log10 HIV-2 RNA copies/mL. The HIV-2 WHO/NIBSC International Standard (1000 IU) was shown to contain 152 RNA copies/mL (95% CI 141–163). Overall, HIV-2 RNA was quantified at ≥ 10 copies/mL from 86 (53%) clinical specimens (median, 2.24 log10copies/mL; range 10–16870), and nine specimens (6%) had HIV-2 RNA detected at <10 copies/mL.
Conclusions
We developed and validated a highly-sensitive HIV-2 VL assay that is suitable for clinical and research use.
doi:10.1016/j.jcv.2012.06.024
PMCID: PMC3444162  PMID: 22832059
HIV-2; PCR; Real-time; diagnostic; plasma viral loads
5.  Lower levels of HIV RNA in semen in HIV-2 compared with HIV-1 infection: implications for differences in transmission 
AIDS (London, England)  2006;20(6):895-900.
Background and objectives
HIV-2 infection, in comparison with HIV-1, is characterized by lower plasma viral loads, slower CD4 cell count decline, decreased AIDS-related mortality, and lower rates of mother-to-child and sexual transmission. To gain further insight into why HIV-1 is more readily transmitted as compared with HIV-2, we analyzed semen and plasma HIV RNA levels in HIV-1 and HIV-2-positive men from Senegal.
Design and methods
Twenty-two HIV-1 and 10 HIV-2-infected subjects from the University of Dakar donated semen and blood samples for this analysis. HIV-1 and HIV-2 viral loads in semen and plasma were quantified using type-specific polymerase chain reaction assays.
Results
The mean age of the subjects was 37 and 40 years; mean CD4 cell count was 222 and 276 cells/µl and the mean plasma viral load was 4.7 and 3.0 log10 copies/ml for HIV-1 and HIV-2, respectively (P = 0.002). HIV RNA was detected in semen in 21 of 22 (95%) of HIV-1 and seven of 10 (70%) of HIV-2-infected subjects; P = 0.07). However, the levels of HIV RNA present in semen were markedly different between those with HIV-1 and HIV-2, with a mean of 4.4 log10 copies/ml among those with HIV-1 and a mean of 2.6 log10 copies/ml among those with HIV-2 (P < 0.001). In multivariate analysis, plasma viral load and HIV type, but not CD4 cell count, were independently predictive of semen viral load (P = 0.03, 0.05, 0.48, respectively)
Conclusions
These data suggest that differences in semen viral load between HIV-1 and HIV-2 may be in part responsible for the markedly different transmission rates of these two viruses. In addition, risk of male genital tract shedding strongly correlates with plasma viral loads. Interventions that decrease viral load may help decrease transmission of both HIV-1 and HIV-2.
doi:10.1097/01.aids.0000218554.59531.80
PMCID: PMC3726185  PMID: 16549974
HIV-1; HIV-2; virus shedding; semen; male; Africa; Senegal
6.  Lower levels of HIV-2 than HIV-1 in the female genital tract: correlates and longitudinal assessment of viral shedding 
AIDS (London, England)  2008;22(18):2517-2525.
Background
The differing magnitude of the HIV-1 and HIV-2 epidemics is likely a consequence of differing transmission rates between the two viruses. Similar to other sexually transmitted pathogens, risk of HIV-1 and HIV-2 transmission is likely associated with the presence and amount of HIV in the genital tract. Thus, understanding patterns of, and risk factors for HIV genital tract shedding is critical to effective control of HIV transmission.
Methods
We evaluated HIV DNA and RNA detection in cervicovaginal specimens among 168 HIV-1 and 50 HIV-2-infected women in Senegal, West Africa. In a subset of 31 women (20 with HIV-1, 11 with HIV-2), we conducted a prospective study in which cervicovaginal specimens were taken at 3-day intervals over a 6-week period.
Results
We found significantly lower rates and levels of HIV-2 RNA (58% shedding; 13% with >1000 copies/ml) in the female genital tract than HIV-1 RNA (78% shedding; 40% with >1000 copies/ml) (P =0.005 and 0.005, respectively), and shedding correlated with plasma viral load irrespective of virus type (odds ratio =1.9, 95% confidence interval =1.3–2.8 for each log10 increase in HIV viral RNA). Plasma viral load, not HIV type, was the strongest predictor of genital viral load. Over 80% of closely monitored women, regardless of HIV type, had at least intermittent HIV RNA detection during every 3-day sampling over a 6-week time period.
Conclusion
These data help in explaining the different transmission rates between HIV-1 and HIV-2 and may provide new insights regarding prevention.
doi:10.1097/QAD.0b013e328315cdbc
PMCID: PMC3726195  PMID: 19005275
Africa; cervix; women; HIV-1; HIV-2; Senegal; vagina; virus shedding
7.  Emergence of Multiclass Drug–Resistance in HIV-2 in Antiretroviral-Treated Individuals in Senegal: Implications for HIV-2 Treatment in Resouce-Limited West Africa 
Background
The efficacy of various antiretroviral (ARV) therapy regimens for human immunodeficiency virus type 2 (HIV-2) infection remains unclear. HIV-2 is intrinsically resistant to the nonnucleoside reverse-transcriptase inhibitors and to enfuvirtide and may also be less susceptible than HIV-1 to some protease inhibitors (PIs). However, the mutations in HIV-2 that confer ARV resistance are not well characterized.
Methods
Twenty-three patients were studied as part of an ongoing prospective longitudinal cohort study of ARV therapy for HIV-2 infection in Senegal. Patients were treated with nucleoside reverse-transcriptase inhibitor (NRTI)– and PI (indinavir)–based regimens. HIV-2 pol genes from these patients were genotyped, and the mutations predictive of resistance in HIV-2 were assessed. Correlates of ARV resistance were analyzed.
Results
Multiclass drug–resistance mutations (NRTI and PI) were detected in strains in 30% of patients; 52% had evidence of resistance to at least 1 ARV class. The reverse-transcriptase mutations M184V and K65R, which confer high-level resistance to lamivudine and emtricitabine in HIV-2, were found in strains from 43% and 9% of patients, respectively. The Q151M mutation, which confers multinucleoside resistance in HIV-2, emerged in strains from 9% of patients. HIV-1–associated thymidine analogue mutations (M41L, D67N, K70R, L210W, and T215Y/F) were not observed, with the exception of K70R, which was present together with K65R and Q151M in a strain from 1 patient. Eight patients had HIV-2 with PI mutations associated with indinavir resistance, including K7R, I54M, V62A, I82F, L90M, L99F; 4 patients had strains with multiple PI resistance–associated mutations. The duration of ARV therapy was positively associated with the development of drug resistance (P = .02). Nine (82%) of 11 patients with HIV-2 with detectable ARV resistance had undetectable plasma HIV-2 RNA loads (<1.4 log10 copies/mL), compared with 3 (25%) of 12 patients with HIV-2 with detectable ARV resistance (P = .009). Patients with ARV-resistant virus had higher plasma HIV-2 RNA loads, compared with those with non–ARV-resistant virus (median, 1.7 log10 copies/mL [range, <1.4 to 2.6 log10 copies/mL] vs. <1.4 log10 copies/mL [range, <1.4 to 1.6 log10 copies/mL]; P = .003).
Conclusions
HIV-2–infected individuals treated with ARV therapy in Senegal commonly have HIV-2 mutations consistent with multiclass drug resistance. Additional clinical studies are required to improve the efficacy of primary and salvage treatment regimens for treating HIV-2 infection.
doi:10.1086/596504
PMCID: PMC3671065  PMID: 19143530
8.  Scaling up access to oral rehydration solution for diarrhea: Learning from historical experience in low– and high–performing countries 
Journal of Global Health  2013;3(1):010404.
Aim
This paper aims to identify factors that systematically predict why some countries that have tried to scale up oral rehydration solution (ORS) have succeeded, and others have not.
Methods
We examined ORS coverage over time, across countries, and through case studies. We conducted expert interviews and literature and data searches to better understand the history of ORS scale–up efforts and why they failed or succeeded in nine countries. We used qualitative, pairwise (or three–country) comparisons of geographically or otherwise similar countries that had different outcomes in terms of ORS scale–up. An algorithm was developed which scored country performance across key supply, demand and financing activities to quantitatively assess the scale–up efforts in each country.
Results
The vast majority of countries have neither particularly low nor encouragingly high ORS use rates. We observed three clearly identifiable contrasts between countries that achieved and sustained high ORS coverage and those that did not. Key partners across sectors have critical roles to play to effectively address supply– and demand–side barriers. Efforts must synchronize demand generation, private provider outreach and public sector work. Many donor funds are either suspended or redirected in the event of political instability, exacerbating the health challenges faced by countries in these contexts. We found little information on the cost of scale–up efforts.
Conclusions
We identified a number of characteristics of successful ORS scale–up programs, including involvement of a broad range of key players, addressing supply and demand generation together, and working with both public and private sectors. Dedicated efforts are needed to launch and sustain success, including monitoring and evaluation plans to track program costs and impacts. These case studies were designed to inform programmatic decision–making; thus, rigorous academic methods to qualitatively and quantitatively evaluate country ORS scale–up programs might yield additional, critical insights and confirm our conclusions.
doi:10.7189/jogh.03.010404
PMCID: PMC3700030  PMID: 23826508
9.  Phenotypic susceptibility of HIV-2 to raltegravir: integrase mutations Q148R and N155H confer raltegravir resistance 
AIDS (London, England)  2011;25(18):2235-2241.
Objectives
Raltegravir is the first integrase strand transfer inhibitor approved for treating HIV-1 infection. Although emerging data suggest that raltegravir may also be useful for HIV-2 treatment, studies addressing the in-vitro susceptibility of HIV-2 to raltegravir are scarce, and the genetic pathways leading to raltegravir resistance in HIV-2 have not been adequately characterized. Our objectives were to directly compare the susceptibilities of HIV-1 and HIV-2 to raltegravir and to examine the role of mutations in HIV-2 integrase in emergent raltegravir resistance.
Materials and methods
Single-cycle and spreading infection assays were used to quantify the sensitivities of wild-type HIV-1 and HIV-2 strains to raltegravir. HIV-2 integrase mutants were constructed by site-directed mutagenesis, and the replication capacities and raltegravir susceptibilities of the resultant variants were analyzed in single-cycle assays.
Results
Raltegravir showed comparable activity against wild-type HIV-1 and HIV-2 in both single-cycle and spreading infections, with EC50 values in the low nanomolar range. Amino acid changes Q148R and N155H individually conferred resistance to raltegravir (14-fold and seven-fold, respectively), whereas the Y143C replacement had no statistically significant effect on raltegravir sensitivity. The combination of Q148R with N155H resulted in high-level raltegravir resistance (>1000-fold). In addition, all HIV-2 integrase variants tested showed impairments in replication capacity.
Conclusion
Our data support clinical studies of raltegravir for treating HIV-2 infection and show that the Q148R and N155H changes alone are sufficient for raltegravir resistance in HIV-2. Further efforts are needed to improve access to HIV-2-active antiretrovirals, including raltegravir, in resource-limited areas where HIV-2 is endemic.
doi:10.1097/QAD.0b013e32834d8e52
PMCID: PMC3652615  PMID: 21971360
HIV-1; HIV-2; integrase; N155H; Q148R; raltegravir; resistance; Y143C
10.  Fetal head circumference, operative delivery, and fetal outcomes: a multi-ethnic population-based cohort study 
Background
Operative delivery procedures, such as primary cesarean section, vacuum-assisted, and forceps-assisted vaginal delivery increase maternal and fetal morbidity, and the cost of care. We evaluated whether large fetal head circumference (FHC) independently increases risk of such interventions, as well as fetal distress or low Apgar score, in anatomically normal infants.
Methods
We conducted a population-based retrospective cohort study using Washington State birth certificate data. We included singleton, term infants born to nulliparous mothers from 2003–2009. We compared mode of delivery and fetal outcomes in 10,750 large-FHC (37-41 cm) infants relative to 10,750 average-FHC (34 cm) infants, frequency matched by birth-year.
Results
Large-FHC infants were nearly twice as likely to be delivered by primary cesarean section as average-FHC infants (unadjusted relative risk [RR] 1.84, 95% confidence interval [CI]: 1.77, 1.92). The RR for primary cesarean section associated with large-FHC was largest for mothers aged 19 years or less (RR 2.28; 95% CI: 1.99, 2.61), and smallest for mothers aged 35 years or greater (RR 1.51; 95% CI: 1.37, 1.66) [test of homogeneity, p < 0.001]. Large-FHC infants were at increased risk of vacuum-assisted vaginal delivery (RR 1.55; 95% CI: 1.43, 1.69), and forceps-assisted vaginal delivery (RR 1.61; 95% CI: 1.32, 1.97). There was no difference in risk of fetal distress (RR 0.97; 95% CI: 0.89, 1.07) for large-FHC versus average-FHC infants. Risk estimates were unaffected by adjustment for potential confounders.
Conclusions
Nulliparous mothers of large-FHC infants are at increased risk of primary cesarean section, vacuum-assisted and forceps-assisted vaginal delivery relative to mothers of average-FHC infants. Maternal age modifies the association between FHC and primary cesarean section.
doi:10.1186/1471-2393-13-106
PMCID: PMC3653751  PMID: 23651454
Fetal head circumference; Cesarean section; Operative delivery; Fetal distress; Low Apgar score
11.  Evidence of immune memory 8.5 years following administration of a prophylactic human papillomavirus type 16 vaccine 
Journal of Clinical Virology  2011;53(3):239-243.
Background
The duration of protection conferred by prophylactic human papillomavirus (HPV) L1 virus-like particle vaccines is a critical determinant of their public health impact. A feature of vaccines that confer long-term immunity is their ability to induce immune memory.
Objectives
We evaluated antibody responses against HPV types 6, 11, 16 and 18 following administration of the quadrivalent HPV-6/11/16/18 vaccine to women who had previously received a monovalent HPV-16 vaccine.
Study design
As part of an extended follow-up study conducted between 2006 and 2009 in Seattle, Washington, we administered the quadrivalent HPV-6/11/16/18 vaccine to 52 women (19 vaccine and 33 placebo recipients) who had participated in a monovalent HPV-16 vaccine trial 8.5 years earlier. Serum samples were tested for anti-HPV antibodies using competitive Luminex immunoassay.
Results
Following administration of the first dose of the quadrivalent HPV-6/11/16/18 vaccine, the anti-HPV-16 geometric mean titer among monovalent HPV-16 vaccine recipients (GMT = 5024.0 milli-Merck units per milliliter [mMU/mL]; 95% confidence interval [CI]: 2710.1, 9313.6 mMU/mL) substantially exceeded that among the placebo recipients (GMT = 136.1; 95% CI: 78.5, 235.8 mMU/mL; p < 0.01) and their own highest anti-HPV-16 response observed during the original trial (GMT at month 7 of the original trial = 1552.7 mMU/mL; 95% CI: 1072.6, 2247.7 mMU/mL; p < 0.01).
Conclusions
The findings suggest that the administration of the three-dose regimen of the monovalent HPV-16 vaccine had produced memory lymphocytes, characterized by a heightened immune response following administration of the quadrivalent HPV-6/11/16/18 vaccine that effectively served as an antigen challenge.
doi:10.1016/j.jcv.2011.12.009
PMCID: PMC3279625  PMID: 22209292
Human papillomavirus type 16; Vaccines; Immune memory
12.  Pregnancy outcomes in women infected with Chlamydia trachomatis: a population‐based cohort study in Washington State 
Sexually Transmitted Infections  2007;83(4):314-318.
Objectives
To measure the risk of preterm delivery, premature rupture of membranes, infant low birth weight and infant mortality, by a population‐based retrospective cohort study using Washington State birth certificate data.
Methods
All women diagnosed with Chlamydia trachomatis infection (n = 851), noted with a check box on the birth certificate from 2003, and a randomly selected sample of women not diagnosed with C trachomatis (n = 3404) were identified. To assess the RR between chlamydia infection and pregnancy outcomes, multivariable logistic regression analysis was used.
Results
Women with chlamydia infection were younger, more likely to be non‐white and had less years of education compared with women without chlamydia. Additionally, they were more likely to have inadequate prenatal care and coinfections with other sexually transmitted infections. After adjusting for age and education, chlamydia‐infected women were at an increased risk of preterm delivery (RR 1.46, 95% CI 1.08 to 1.99) and premature rupture of membranes (RR 1.50, 95% CI 1.03 to 2.17) compared with non‐infected women. However, no increased risk of infant death (RR 1.02, 95% CI 0.37 to 2.80) or low birth weight (RR 1.12, 95% CI 0.74 to 1.68) associated with chlamydia infection was observed.
Conclusion
This study suggests that C trachomatis is associated with an increased risk of preterm delivery and premature rupture of membranes, but not with infant death and low birth weight. Routine screening and opportune treatment for C trachomatis should be considered a necessary part of prenatal care to reduce these adverse pregnancy outcomes.
doi:10.1136/sti.2006.022665
PMCID: PMC2598687  PMID: 17344249
13.  MicroRNA expression in ovarian carcinoma and its correlation with clinicopathological features 
Background
MicroRNA (miRNA) expression is known to be deregulated in ovarian carcinomas. However, limited data is available about the miRNA expression pattern for the benign or borderline ovarian tumors as well as differential miRNA expression pattern associated with histological types, grades or clinical stages in ovarian carcinomas. We defined patterns of microRNA expression in tissues from normal, benign, borderline, and malignant ovarian tumors and explored the relationship between frequently deregulated miRNAs and clinicopathologic findings, response to therapy, survival, and association with Her-2/neu status in ovarian carcinomas.
Methods
We measured the expression of nine miRNAs (miR-181d, miR-30a-3p, miR-30c, miR-30d, miR-30e-3p, miR-368, miR-370, miR-493-5p, miR-532-5p) in 171 formalin-fixed, paraffin-embedded ovarian tissue blocks as well as six normal human ovarian surface epithelial (HOSE) cell lines using Taqman-based real-time PCR assays. Her-2/neu overexpression was assessed in ovarian carcinomas (n = 109 cases) by immunohistochemistry analysis.
Results
Expression of four miRNAs (miR-30c, miR-30d, miR-30e-3p, miR-370) was significantly different between carcinomas and benign ovarian tissues as well as between carcinoma and borderline tissues. An additional three miRNAs (miR-181d, miR-30a-3p, miR-532-5p) were significantly different between borderline and carcinoma tissues. Expression of miR-532-5p was significantly lower in borderline than in benign tissues. Among ovarian carcinomas, expression of four miRNAs (miR-30a-3p, miR-30c, miR-30d, miR-30e-3p) was lowest in mucinous and highest in clear cell samples. Expression of miR-30a-3p was higher in well-differentiated compared to poorly differentiated tumors (P = 0.02), and expression of miR-370 was higher in stage I/II compared to stage III/IV samples (P = 0.03). In multivariate analyses, higher expression of miR-181d, miR-30c, miR-30d, and miR-30e-3p was associated with significantly better disease-free or overall survival. Finally, lower expression of miR-30c, miR-30d, miR-30e-3p and miR-532-5p was significantly associated with overexpression of Her-2/neu.
Conclusions
Aberrant expression of miRNAs is common in ovarian tumor suggesting involvement of miRNA in ovarian tumorigenesis. They are associated with histology, clinical stage, survival and oncogene expression in ovarian carcinoma.
doi:10.1186/1477-7819-10-174
PMCID: PMC3449188  PMID: 22925189
miRNA; Ovarian tumor; Her2/neu; Survival
14.  Interpopulation variation in HIV testing promptness may introduce bias in HIV incidence estimates using the serologic testing algorithm for recent HIV seroconversion 
Sexually transmitted infections  2010;86(4):254-257.
Objectives
The serologic testing algorithm for recent HIV seroconversion (STARHS) calculates incidence using the proportion of testers who produce a level of HIV antibody high enough to be detected by ELISA but low enough to suggest recent infection. The validity of STARHS relies on independence between dates of HIV infection and dates of antibody testing. When subjects choose the time of their own test, testing may be motivated by risky behaviour or symptoms of infection and the criterion may not be met. This analysis was conducted to ascertain whether estimates of incidence derived using STARHS were consistent with estimates derived using a method more robust against motivated testing.
Methods
A cohort-based incidence estimator and two STARHS methods were applied to identical populations (n=3821) tested for HIV antibody at publicly funded sites in Seattle. Overall seroincidence estimates, demographically stratified estimates and incidence rate ratios were compared across methods. The proportion of low-antibody testers among HIV-infected individuals was compared with the proportion expected given their testing histories.
Results
STARHS estimates generally exceeded cohort-based estimates. Incidence ratios derived using STARHS between demographic strata were not consistent across methods. The proportion of HIV-infected individuals with lower antibody levels exceeded that which would be expected under independence between infection and testing.
Conclusions
Incidence estimates and incidence rate ratios derived using methods that rely on the changing antibody level over the course of HIV infection may be vulnerable to bias when applied to populations who choose the time of their own testing.
doi:10.1136/sti.2009.037291
PMCID: PMC3414255  PMID: 20576914
15.  Antibody responses in oral fluid following administration of prophylactic human papillomavirus vaccines 
The Journal of Infectious Diseases  2009;200(9):1452-1455.
We sought to determine whether oral fluid can be used to assess serum human papillomavirus (HPV) antibody status by enrolling women who had received a prophylactic HPV-16 vaccine in a new follow-up study. After the prophylactic HPV-6/11/16/18 vaccine was licensed in the United States, we administered it to consenting participants. The sensitivity of oral fluid, treating serology as the gold standard, before and after administration of the quadrivalent vaccine was 49.6% (95% confidence interval [CI]: 42.0%–57.3%) and 100% (95% CI: 92.0%–100%), respectively. Oral fluid may have the potential to be used for monitoring of prophylactic HPV vaccines in the future.
doi:10.1086/606026
PMCID: PMC3392559  PMID: 19698077
Human papillomavirus; Prophylactic vaccines; Antibodies; Oral fluid
16.  Detection of Human Papillomavirus DNA in Cutaneous Squamous Cell Carcinoma among Immunocompetent Individuals 
The presence of certain types of human papillomavirus (HPV) is a known risk factor for the development of anogenital squamous cell carcinomas (SCCs). A similar association has been hypothesized for cutaneous SCCs, although, to our knowledge, no studies to date have combined sensitive HPV DNA detection techniques with epidemiologic data controlling for known risk factors to explore the association. We designed a case–control study examining HPV prevalence using highly sensitive PCR-detection assays in tissue samples from 85 immunocompetent patients with histologically confirmed SCCs and 95 age-matched individuals without a prior history of skin cancer. A standardized interview was administered to all study subjects to collect information pertaining to potential confounding variables. The overall detection rate of HPV DNA was high in case lesions (54%) and perilesions (50%) and in both sun-exposed normal tissue (59%) and non-sun-exposed normal tissue (49%) from controls. In comparing case tissue to control tissue, there was no differential detection of HPV DNA across various HPV species. However, HPV DNA from β-papillomavirus species 2 was more likely to be identified in tumors than in adjacent healthy tissue among cases (paired analysis, odds ratio = 4.0, confidence interval = 1.3–12.0). The high prevalence of HPV DNA detected among controls suggests that HPV DNA is widely distributed among the general population. However, the differential detection of HPV β-papillomavirus species in tumors among cases suggests that certain HPV types may be involved in the progression of cutaneous SCCs.
doi:10.1038/sj.jid.5701227
PMCID: PMC3268673  PMID: 18185530
17.  DNA hypermethylation of tumors from non-small cell lung cancer (NSCLC) patients is associated with gender and histologic type 
Background
We previously identified a number of genes which were methylated significantly more frequently in the tumor compared to the non-cancerous lung tissues from non-small cell lung cancer (NSCLC) patients. Detection of methylation profiles of genes in NSCLC could provide insight into differential pathways to malignancy and lead to strategies for better treatment of individuals with NSCLC.
Methods
We determined the DNA methylation status of 27 genes using quantitative MethyLight assays in lung tumor samples from 117 clinically well-characterized NSCLC patients.
Results
Hypermethylation was detected in one of more of the genes in 106 (91%) of 117 cases and was detected at high levels (Percentage of Methylation Reference (PMR)≥4%) in 79% of NSCLC cases. Methylation of APC, CCND2, KCNH5 and, RUNX was significantly more frequent in adenocarcinomas compared to squamous cell carcinomas (SCC), while methylation of CDKN2A was more common in SCC. Hypermethylation of KCNH5, KCNH8, and RARB was more frequent in females compared to males. Hypermethylation of APC and CCND2 was inversely associated with proliferation score assessed by Ki-67 level.
Conclusions
Our findings of differential gene hypermethylation frequencies in tumor tissues from patients with adenocarcinoma or squamous cell cancers and in females compared to males suggests that further investigation is warranted in order to more fully understand the potential disparate pathways and/or risk factors for NSCLC associated with histologic type and gender.
doi:10.1016/j.lungcan.2009.11.002
PMCID: PMC2888601  PMID: 19945765
hypermethylation; lung cancer; gender; histology
18.  HIV-2 Integrase Variation in Integrase Inhibitor-Naïve Adults in Senegal, West Africa 
PLoS ONE  2011;6(7):e22204.
Background
Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance.
Methods
We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2–infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1.
Results
No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155). Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein.
Conclusion
Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at “secondary” HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2–infected patients.
doi:10.1371/journal.pone.0022204
PMCID: PMC3134476  PMID: 21765953
19.  Why the Wait? Delayed HIV Diagnosis among Men Who Have Sex with Men 
We sought to identify factors associated with delayed diagnosis of human immunodeficiency virus (HIV; testing HIV-seropositive 6 months or more after HIV seroconversion), by comparing delayed testers to non-delayed testers (persons who were diagnosed within 6 months of HIV seroconversion), in King County, Washington among men who have sex with men (MSM). Participants were recruited from HIV testing sites in the Seattle area. Delayed testing status was determined by the Serologic Testing Algorithm for Recent HIV Seroconversion or a self-reported previous HIV-negative test. Quantitative data on sociodemographic characteristics, health history, and drug-use and sexual behaviors were collected via computer-assisted self-interviews. Qualitative semi-structured interviews regarding testing and risk behaviors were also conducted. Multivariate analysis was used to identify factors associated with delayed diagnosis. Content analysis was used to establish themes in the qualitative data. Out of the 77 HIV-seropositive MSM in this sample, 39 (51%) had evidence of delayed diagnosis. Factors associated with delayed testing included being African-American, homeless, “out” to 50% or less people about male-male sex, and having only one sex partner in the past 6 months. Delayed testers often cited HIV-related sickness as their reason for testing and fear and wanting to be in denial of their HIV status as reasons for not testing. Delayed testers frequently did not identify as part of the MSM community, did not recognize that they were at risk for HIV acquisition, and did not feel a responsibility to themselves or others to disclose their HIV status. This study illustrates the need to further explore circumstances around delayed diagnosis in MSM and develop outreach methods and prevention messages targeted specifically to this potentially highly marginalized population in order to detect HIV infections earlier, provide HIV care, and prevent new infections.
doi:10.1007/s11524-010-9434-8
PMCID: PMC2900570  PMID: 20186493
MSM; HIV/AIDS; HIV testing; Delayed diagnosis; Delayed testing
20.  Hypermethylation of CCND2 May Reflect a Smoking-Induced Precancerous Change in the Lung 
Journal of Oncology  2011;2011:950140.
It remains unknown whether tobacco smoke induces DNA hypermethylation as an early event in carcinogenesis or as a late event, specific to overt cancer tissue. Using MethyLight assays, we analyzed 316 lung tissue samples from 151 cancer-free subjects (121 ever-smokers and 30 never-smokers) for hypermethylation of 19 genes previously observed to be hypermethylated in nonsmall cell lung cancers. Only APC (39%), CCND2 (21%), CDH1 (7%), and RARB (4%) were hypermethylated in >2% of these cancer-free subjects. CCND2 was hypermethylated more frequently in ever-smokers (26%) than in never-smokers (3%). CCND2 hypermethylation was also associated with increased age and upper lobe sample location. APC was frequently hypermethylated in both ever-smokers (41%) and never-smokers (30%). BVES, CDH13, CDKN2A (p16), CDKN2B, DAPK1, IGFBP3, IGSF4, KCNH5, KCNH8, MGMT, OPCML, PCSK6, RASSF1, RUNX, and TMS1 were rarely hypermethylated (<2%) in all subjects. Hypermethylation of CCND2 may reflect a smoking-induced precancerous change in the lung.
doi:10.1155/2011/950140
PMCID: PMC3090638  PMID: 21577262
21.  Evaluation of Transported Dry and Wet Cervical Exfoliated Samples for Detection of Human Papillomavirus Infection▿  
Journal of Clinical Microbiology  2010;48(9):3068-3072.
We determined the feasibility of human papillomavirus (HPV) detection in cervical exfoliated cells collected as dry swab samples. Both dry cervical swab and specimen transport medium (STM) cervical swab samples were collected from 135 patients attending either colposcopy or women's clinics in Guayaquil, Ecuador, who had a cytology diagnosis within 6 months. HPV was detected by dot blot hybridization and genotyped by the liquid bead microarray assay (LBMA). Overall, 23.1% of dry samples were positive for any high-risk HPV types, and 24.6% of STM samples were positive for any high-risk HPV types. Of 125 paired samples, the type-specific high-risk HPV proportion positive agreement was 60.7% (kappa, 0.69; 95% confidence interval [CI], 0.53 to 0.82). Of six women with cytological evidence of invasive cervical cancer, high-risk HPV DNA was detected in three of their STM samples and in five of their dry samples. Dry samples were more likely to be insufficient for HPV testing than STM samples. Consistent with this observation, the amount of genomic DNA quantitated with the β-actin gene was almost 20 times lower in dry samples than in STM samples when detected by the real-time TaqMan assay; however, HPV DNA viral loads in dry samples were only 1.6 times lower than those in matched STM samples. We concluded that exfoliated cervical cells could be collected as dry swab samples for HPV detection.
doi:10.1128/JCM.00736-10
PMCID: PMC2937663  PMID: 20610686
22.  Is there a role for abstinence only programmes for HIV prevention in high income countries? 
BMJ : British Medical Journal  2007;335(7613):217-218.
A robust systematic review finds no evidence that such programmes reduce risky sexual behaviours, incidence of sexually transmitted infections, or pregnancy
doi:10.1136/bmj.39287.463889.80
PMCID: PMC1939748  PMID: 17673733
23.  DNA Methylation in Tumor and Matched Normal Tissues from Non-Small Cell Lung Cancer Patients 
We used MethyLight assays to analyze DNA methylation status of 27 genes on 49 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients who underwent surgical resection. Seven genes (RARB, BVES, CDKN2A, KCNH5, RASSF1, CDH13, and RUNX) were found to be methylated significantly more frequently in tumor tissues than in noncancerous tissues. Only methylation of CCND2 and APC was frequently detected in both cancerous and noncancerous tissues, supporting the hypothesis that the methylation of these two genes is a preneoplastic change and may be associated with tobacco smoking exposure. Methylation of any one of eight genes (RASSF1, DAPK1, BVES, CDH13, MGMT, KCNH5, RARB, or CDH1) was present in 80% of NSCLC tissues but only in 14% of noncancerous tissues. Detection of methylation of these genes in blood might have utility in monitoring and detecting tumor recurrence in early-stage NSCLC after curative surgical resection.
doi:10.1158/1055-9965.EPI-07-2518
PMCID: PMC2798850  PMID: 18349282
24.  DNA Hypermethylation, Her-2/neu Overexpression and p53 Mutations in Ovarian Carcinoma 
Gynecologic oncology  2008;111(2):320-329.
Objectives
To define patterns of aberrant DNA methylation, p53 mutation and Her-2/neu overexpression in tissues from benign (N=29), malignant (N=100), and border line malignant ovaries (N=10), as compared to normal (N=68) ovarian tissues. Further, to explore the relationship between the presence of genetic and epigenetic abnormalities in ovarian cancers, and assess the association between epigenetic changes and clinical stage of malignancy at presentation and response to therapy.
Methods
The methylation status of 23 genes that were previously reported associated with various epithelial malignancies was assessed in normal and abnormal ovarian tissues by methylation specific PCR. The presence of p53 mutation (N=82 cases) and Her-2/neu overexpression (N=51 cases) were assessed by DNA sequencing and immunohistochemistry, respectively.
Results
Methylation of four genes (MINT31, HIC1, RASSF1, and CABIN1) was significantly associated with ovarian cancer but not other ovarian pathology. Her-2/neu overexpression was associated with aberrant methylation of three genes (MINT31, RASSF1, and CDH13), although aberrant methylation was not associated with p53 mutations. Methylation of RASSF1 and HIC1 was more frequent in early compared to late stage ovarian cancer, while methylation of CABIN1 and RASSF1 was associated with response to chemotherapy.
Conclusion
DNA methylation of tumor suppressor genes is a frequent event in ovarian cancer, and in some cases is associated with Her-2/neu overexpression. Methylation of CABIN1 and RASSF1 may have the utility to predict response to therapy.
doi:10.1016/j.ygyno.2008.07.036
PMCID: PMC2642648  PMID: 18757082
hypermethylation; Her-2/neu overexpression; p53; ovarian cancer
25.  Development and Evaluation of a Liquid Bead Microarray Assay for Genotyping Genital Human Papillomaviruses▿ †  
Journal of Clinical Microbiology  2009;47(3):547-553.
We developed a liquid bead microarray (LBMA) assay for genotyping genital human papillomaviruses (HPVs) based on the MY09-MY11-HMB01 PCR system and the reverse line blot (RLB) assay probe sequences. Using individual HPV plasmids, we were able to detect as few as 50 copies per reaction. In two separate retrospective studies, the LBMA assay was compared to the RLB assay and to the Hybrid Capture II (hc2) assay. Testing was performed without knowledge of other assay results. In the first study, 614 cervical swab samples (enriched for HPV infection) from 160 young women were tested for HPV DNA, and 360 (74.8%) type-specific HPV infections were detected by both assays, 71 (14.8%) by the LBMA assay only, and 50 (10.4%) by the RLB assay only. Type-specific agreement for the two assays was excellent (99.1%; kappa = 0.85; 95% confidence interval [95% CI], 0.82 to 0.88). Samples with discrepant LBMA and RLB test results tended to have low viral loads by a quantitative type-specific PCR assay. In the second study, cervical swab samples from 452 women (including 54 women with histologically confirmed cervical-intraepithelial neoplasia grade 2 or worse [≥CIN2]) were tested initially by the hc2 and subsequently by the LBMA assay. The estimated sensitivities for ≥CIN2 were similar for the LBMA and hc2 assays (98.4% [95% CI, 95.0 to 100%] and 95.6% [95% CI, 89.2 to 100%], respectively). The percentages of negative results among 398 women without ≥CIN2 were similar for the LBMA and hc2 assays (45% and 50%, respectively). The repeat test reproducibility for 100 samples was 99.1% (kappa = 0.92; 95% CI, 0.90 to 0.95). We conclude that the new LBMA assay will be useful for clinical and epidemiological research.
doi:10.1128/JCM.01707-08
PMCID: PMC2650937  PMID: 19144800

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