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1.  Deconstructing the differences: a comparison of GBD 2010 and CHERG’s approach to estimating the mortality burden of diarrhea, pneumonia, and their etiologies 
Pneumonia and diarrhea are leading causes of death for children under five (U5). It is challenging to estimate the total number of deaths and cause-specific mortality fractions. Two major efforts, one led by the Institute for Health Metrics and Evaluation (IHME) and the other led by the World Health Organization (WHO)/Child Health Epidemiology Reference Group (CHERG) created estimates for the burden of disease due to these two syndromes, yet their estimates differed greatly for 2010.
This paper discusses three main drivers of the differences: data sources, data processing, and covariates used for modelling. The paper discusses differences in the model assumptions for etiology-specific estimates and presents recommendations for improving future models.
IHME’s Global Burden of Disease (GBD) 2010 study estimated 6.8 million U5 deaths compared to 7.6 million U5 deaths from CHERG. The proportional differences between the pneumonia and diarrhea burden estimates from the two groups are much larger; GBD 2010 estimated 0.847 million and CHERG estimated 1.396 million due to pneumonia. Compared to CHERG, GBD 2010 used broader inclusion criteria for verbal autopsy and vital registration data. GBD 2010 and CHERG used different data processing procedures and therefore attributed the causes of neonatal death differently. The major difference in pneumonia etiologies modeling approach was the inclusion of observational study data; GBD 2010 included observational studies. CHERG relied on vaccine efficacy studies.
Greater transparency in modeling methods and more timely access to data sources are needed. In October 2013, the Bill & Melinda Gates Foundation (BMGF) hosted an expert meeting to examine possible approaches for better estimation. The group recommended examining the impact of data by systematically excluding sources in their models. GBD 2.0 will use a counterfactual approach for estimating mortality from pathogens due to specific etiologies to overcome bias of the methods used in GBD 2010 going forward.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-014-0728-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4305232  PMID: 25592774
2.  HIV-1 outcompetes HIV-2 in dually infected Senegalese individuals with low CD4+ cell counts 
AIDS (London, England)  2013;27(15):2441-2450.
Dual infection with HIV-1 and HIV-2, which is not uncommon in West Africa, has implications for transmission, progression, and antiretroviral therapy (ART). Few studies have examined viral dynamics in this setting. Our objective was to directly compare HIV-1 and HIV-2 viral loads and to examine whether this relationship is associated with CD4+ cell count.
Study design
This is a retrospective analysis of data from observational cohort studies.
We compared HIV-1 and HIV-2 viral loads from 65 dually infected, ART-naive Senegalese individuals. Participants provided blood, oral fluid, and cervicovaginal lavage (CVL) or semen samples for virologic and immunologic testing. We assessed relationships between HIV-1 and HIV-2 levels using linear regression with generalized estimating equations to account for multiple study visits.
After adjusting for CD4+ cell count, age, sex, and commercial sex work, HIV-1 RNA levels were significantly higher than HIV-2 levels in semen, CVL, and oral fluids. Despite similar peripheral blood mononuclear cell DNA levels among individuals with CD4+ cell counts above 500 cells/µl, individuals with CD4+ cell counts below 500 cells/µl had higher HIV-1 and lower HIV-2 DNA levels. Individuals with high CD4+ cell counts had higher mean HIV-1 plasma RNA viral loads than HIV-2, with HIV-1 levels significantly higher and HIV-2 levels trending toward lower mean viral loads among individuals with low CD4+ cell counts.
Our data are consistent with the hypothesis that with disease progression, HIV-1 outcompetes HIV-2 in dually infected individuals. This finding helps explain differences in prevalence and outcomes between HIV-1, HIV-2, and HIV-dual infection.
PMCID: PMC4043222  PMID: 23665777
HIV dual infection; HIV viral load; HIV-1; HIV-2; Senegal
3.  A community-based cluster randomized survey of noncommunicable disease and risk factors in a peri-urban shantytown in Lima, Peru 
An estimated 863 million people–a third of the world’s urban population–live in slums, yet there is little information on the disease burden in these settings, particularly regarding chronic preventable diseases.
From March to May 2012, we conducted a cluster randomized survey to estimate the prevalence of noncommunicable diseases (NCDs) and associated risk factors in a peri-urban shantytown north of Lima, Peru. Field workers administered a questionnaire that included items from the WHO World Health Survey and the WHO STEPS survey of chronic disease risk factors. We used logistic regression to assess the associations of NCDs and related risk factors with age and gender. We accounted for sampling weights and the clustered sampling design using statistical survey methods.
A total of 142 adults were surveyed and had a weighted mean age of 36 years (range 18–81). The most prevalent diseases were depression (12%) and chronic respiratory disease (8%), while lifetime prevalence of cancer, arthritis, myocardial infarction, and diabetes were all less than 5%. Fifteen percent of respondents were hypertensive and the majority (67%) was unaware of their condition. Being overweight or obese was common for both genders (53%), but abdominal obesity was more prevalent in women (54% vs. 10% in men, p < 0.001). Thirty-five percent of men binge drank and 34% reported current smoking; these behaviors were less common among women (4% binge drank, p < 0.001; 8% smoked, p = 0.002). Increasing age was associated with an increased risk of abdominal obesity (Odds Ratio (OR) = 1.04, 95% CI = 1.01, 1.07, p = 0.02), hypertension (OR = 1.06, 95% CI = 1.02, 1.10, p = 0.006), arthritis (OR = 1.07, 95% CI = 1.03, 1.11, p < 0.001) and cancer (OR = 1.13, 95% CI = 1.07, 1.20, p < 0.001) in adjusted models. The prevalences of other NCDs and related risk factors were similar when stratified by age or gender.
This study underlines the important burden of noncommunicable disease in informal settlements in Peru and suggests that prevention and treatment interventions could be optimized according to age and gender.
PMCID: PMC4040114  PMID: 24885980
Peru; Slum; Shantytown; Noncommunicable disease
4.  HIV shedding in the oral cavity: an assessment of HIV type, immunovirologic, demographic and oral factors 
To quantify the prevalence and burden of HIV type 2 (HIV-2) and HIV-1 RNA in the oral cavity of antiretroviral therapy-naive HIV-infected Senegalese individuals and to identify correlates of oral HIV viral loads.
A cross-sectional study of 163 HIV-1 and 27 HIV-2-infected antiretroviral therapy-naive Senegalese adults.
Participants received clinical and oral exams and provided blood and oral wash samples for viral load and plasma CD4 count ascertainment. Logistic and interval regression models were used to identify univariate and multivariable associations between presence and level of oral HIV RNA and various immunovirologic, local and demographic factors.
Presence of detectable oral HIV RNA was less common in HIV-2-infected compared with HIV-1-infected study participants (33% vs 67%, OR 0.25, 95% CI 0.11 to 0.59). HIV type was no longer associated with oral shedding of HIV when plasma viral load was considered. Detection of oral HIV RNA was associated with increased plasma viral load in both HIV-1-infected and HIV-2-infected individuals (HIV-1, OR 1.89, 95% CI 1.24 to 2.61; HIV-2, OR 1.93, 95% CI 1.1 to 3.39). Oral HIV-1 detection was also associated with periodontal disease (OR 3.02, 95% CI 1.16 to 7.87).
Oral shedding of HIV-2 RNA is less common than HIV-1 RNA, a likely consequence of lower overall viral burden. Both systemic and local factors may contribute to shedding of HIV in the oral cavity.
PMCID: PMC4028315  PMID: 22250179
5.  Care-seeking and appropriate treatment for childhood acute respiratory illness: an analysis of Demographic and Health Survey and Multiple Indicators Cluster Survey datasets for high-mortality countries 
BMC Public Health  2014;14:446.
Acute lower respiratory illness (ALRI) is a major global cause of morbidity and mortality among children under 5. Antibiotic treatment for ALRI is inexpensive and decreases case fatality, but care-seeking patterns and appropriate treatment vary widely across countries. This study sought to examine patterns of appropriate treatment and estimate the burden of cases of untreated ALRI in high mortality countries.
This study used cross-sectional survey data from the Phase 5/Phase 6 DHS and MIC3/MICS4 for 39 countries. We analyzed care-seeking patterns and antibiotic treatment based on country-level trends, and estimated the burden of untreated cases using country-level predictors in a general linear model.
According to this analysis, over 66 million children were not treated with antibiotics for ALRI in 2010. Overall, African countries had a lower proportion of mothers who sought care for a recent episode of ALRI (41% to 86%) relative to Asian countries (75% to 87%). Seeking any care for ALRI was inversely related to seeking public sector care. Treatment with antibiotics ranged from 8% in Nepal to 87% in Jordan, and was significantly associated with urban residence.
Untreated ALRI remains a substantial problem in high mortality countries. In Asia, the large population numbers lead to a high burden of children with untreated ALRI. In Africa, care-seeking behaviors and access to care issues may lead to missed opportunities to treat children with antibiotics.
PMCID: PMC4024183  PMID: 24884919
Acute lower respiratory infection; Antibiotic treatment; Child health; High mortality countries
6.  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.
PMCID: PMC3966738  PMID: 24671033
7.  Association of Maternal Smoking during Pregnancy with Infant Hospitalization and Mortality Due to Infectious Diseases 
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.
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).
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.
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.
PMCID: PMC3588859  PMID: 22929173
infant; infectious disease; prenatal exposure; maternal smoking
8.  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.
PMCID: PMC3716120  PMID: 23571535
9.  Validation for Clinical Use of a Novel HIV-2 Plasma RNA Viral Load Assay Using the Abbott m2000 Platform 
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.
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.
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.
We developed and validated a highly-sensitive HIV-2 VL assay that is suitable for clinical and research use.
PMCID: PMC3444162  PMID: 22832059
HIV-2; PCR; Real-time; diagnostic; plasma viral loads
10.  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.
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)
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.
PMCID: PMC3726185  PMID: 16549974
HIV-1; HIV-2; virus shedding; semen; male; Africa; Senegal
11.  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.
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.
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.
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.
These data help in explaining the different transmission rates between HIV-1 and HIV-2 and may provide new insights regarding prevention.
PMCID: PMC3726195  PMID: 19005275
Africa; cervix; women; HIV-1; HIV-2; Senegal; vagina; virus shedding
12.  Emergence of Multiclass Drug–Resistance in HIV-2 in Antiretroviral-Treated Individuals in Senegal: Implications for HIV-2 Treatment in Resouce-Limited West Africa 
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.
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.
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).
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.
PMCID: PMC3671065  PMID: 19143530
13.  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.
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.
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.
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.
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.
PMCID: PMC3700030  PMID: 23826508
14.  Phenotypic susceptibility of HIV-2 to raltegravir: integrase mutations Q148R and N155H confer raltegravir resistance 
AIDS (London, England)  2011;25(18):2235-2241.
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.
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.
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.
PMCID: PMC3652615  PMID: 21971360
HIV-1; HIV-2; integrase; N155H; Q148R; raltegravir; resistance; Y143C
15.  Fetal head circumference, operative delivery, and fetal outcomes: a multi-ethnic population-based cohort study 
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.
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.
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.
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.
PMCID: PMC3653751  PMID: 23651454
Fetal head circumference; Cesarean section; Operative delivery; Fetal distress; Low Apgar score
16.  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.
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.
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.
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).
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.
PMCID: PMC3279625  PMID: 22209292
Human papillomavirus type 16; Vaccines; Immune memory
17.  MicroRNA expression in ovarian carcinoma and its correlation with clinicopathological features 
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.
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.
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.
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.
PMCID: PMC3449188  PMID: 22925189
miRNA; Ovarian tumor; Her2/neu; Survival
18.  Stochastic Models to Demonstrate the Effect of Motivated Testing on HIV Incidence Estimates Using the Serologic Testing Algorithm for Recent HIV Seroconversion (STARHS) 
Sexually transmitted infections  2010;86(7):506-511.
To produce valid seroincidence estimates, the serologic testing algorithm for recent HIV seroconversion (STARHS) assumes independence between infection and testing, which may be absent in clinical data. STARHS estimates are generally greater than cohort-based estimates of incidence from observable person-time and diagnosis dates. The authors constructed a series of partial stochastic models to examine whether testing motivated by suspicion of infection could bias STARHS.
One thousand Monte Carlo simulations of 10,000 men who have sex with men (MSM) were generated using parameters for HIV incidence and testing frequency from data from a clinical testing population in Seattle. In one set of simulations, infection and testing dates were independent. In another set, some intertest intervals were abbreviated to reflect the distribution of intervals between suspected HIV exposure and testing in a group of Seattle MSM recently diagnosed with HIV. Both estimation methods were applied to the simulated datasets. Both cohort-based and STARHS incidence estimates were calculated using the simulated data and compared with previously calculated, empirical cohort-based and STARHS seroincidence estimates from the clinical testing population.
Under simulated independence between infection and testing, cohort-based and STARHS incidence estimates resembled cohort estimates from the clinical dataset. Under simulated motivated testing, cohort-based estimates remained unchanged but STARHS estimates were inflated similar to empirical STARHS estimates. Varying motivation parameters appreciably affected STARHS incidence estimates, but not cohort-based estimates.
Cohort-based incidence estimates are robust against dependence between testing and acquisition of infection whereas STARHS incidence estimates are not.
PMCID: PMC3425390  PMID: 21062766
STARHS; HIV incidence; clinical populations; recency of infection; bias
19.  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.
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.
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.
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.
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.
PMCID: PMC3414255  PMID: 20576914
20.  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.
PMCID: PMC3392559  PMID: 19698077
Human papillomavirus; Prophylactic vaccines; Antibodies; Oral fluid
21.  Analysis of Tp53 Codon 72 Polymorphisms, Tp53 Mutations, and HPV Infection in Cutaneous Squamous Cell Carcinomas 
PLoS ONE  2012;7(4):e34422.
Non-melanoma skin cancers are one of the most common human malignancies accounting for 2–3% of tumors in the US and represent a significant health burden. Epidemiology studies have implicated Tp53 mutations triggered by UV exposure, and human papilloma virus (HPV) infection to be significant causes of non-melanoma skin cancer. However, the relationship between Tp53 and cutaneous HPV infection is not well understood in skin cancers. In this study we assessed the association of HPV infection and Tp53 polymorphisms and mutations in lesional specimens with squamous cell carcinomas.
We studied 55 cases of histologically confirmed cutaneous squamous cell carcinoma and 41 controls for the presence of HPV infection and Tp53 genotype (mutations and polymorphism).
We found an increased number of Tp53 mutations in the squamous cell carcinoma samples compared with perilesional or control samples. There was increased frequency of homozygous Tp53-72R polymorphism in cases with squamous cell carcinomas, while the Tp53-72P allele (Tp53-72R/P and Tp53-72P/P) was more frequent in normal control samples. Carcinoma samples positive for HPV showed a decreased frequency of Tp53 mutations compared to those without HPV infection. In addition, carcinoma samples with a Tp53-72P allele showed an increased incidence of Tp53 mutations in comparison carcinomas samples homozygous for Tp53-72R.
These studies suggest there are two separate pathways (HPV infection and Tp53 mutation) leading to cutaneous squamous cell carcinomas stratified by the Tp53 codon-72 polymorphism. The presence of a Tp53-72P allele is protective against cutaneous squamous cell carcinoma, and carcinoma specimens with Tp53-72P are more likely to have Tp53 mutations. In contrast Tp53-72R is a significant risk factor for cutaneous squamous cell carcinoma and is frequently associated with HPV infection instead of Tp53 mutations. Heterozygosity for Tp53-72R/P is protective against squamous cell carcinomas, possibly reflecting a requirement for both HPV infection and Tp53 mutations.
PMCID: PMC3335843  PMID: 22545084
22.  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.
PMCID: PMC3268673  PMID: 18185530
23.  Medical Education on the Brink 
PMCID: PMC3423297  PMID: 22949796
24.  Expression of Mir-21 and Mir-143 in Cervical Specimens Ranging from Histologically Normal through to Invasive Cervical Cancer 
PLoS ONE  2011;6(12):e28423.
MicroRNA expression is severely disrupted in carcinogenesis, however limited evidence is available validating results from cell-line models in human clinical cancer specimens. MicroRNA-21 (mir-21) and microRNA-143 (mir-143) have previously been identified as significantly deregulated in a range of cancers including cervical cancer. Our goal was to investigate the expression patterns of several well-studied microRNA species in cervical samples and compare the results to cell line samples.
Methodology/Principal Findings
We measured the expression of mir-21 and mir-143 in 142 formalin-fixed, paraffin embedded (FFPE) cervical biopsy tissue blocks, collected from Dantec Oncology Clinic, Dakar, Senegal. MicroRNA expression analysis was performed using Taqman-based real-time PCR assays. Protein immunohistochemical staining was also performed to investigate target protein expression on 72 samples. We found that mir-21 expression increased with worsening clinical diagnosis but that mir-143 was not correlated with histology. These observations were in stark contrast to previous reports involving cervical cancer cell lines in which mir-143 was consistently down-regulated but mir-21 largely unaffected. We also identified, for the first time, that cytoplasmic expression of Programmed Cell Death Protein 4 PDCD4; a known target of mir-21) was significantly lower in women with invasive cervical carcinoma (ICC) in comparison to those with cervical intraepithelial neoplasia (2–3) or carcinoma in situ (CIN2-3/CIS), although there was no significant correlation between mir-21 and PDCD4 expression, despite previous studies identifying PDCD4 transcript as a known mir-21 target.
Whilst microRNA biomarkers have a number of promising features, more studies on expression levels in histologically defined clinical specimens are required to investigate clinical relevance of discovery-based studies. Mir-21 may be of some utility in predictive screening, given that we observed a significant correlation between mir-21 expression level and worsening histological diagnosis of cervical cancer.
PMCID: PMC3237431  PMID: 22194833
25.  DNA hypermethylation of tumors from non-small cell lung cancer (NSCLC) patients is associated with gender and histologic type 
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.
We determined the DNA methylation status of 27 genes using quantitative MethyLight assays in lung tumor samples from 117 clinically well-characterized NSCLC patients.
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.
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.
PMCID: PMC2888601  PMID: 19945765
hypermethylation; lung cancer; gender; histology

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