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1.  Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies 
PLoS ONE  2013;8(12):e84050.
Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.
doi:10.1371/journal.pone.0084050
PMCID: PMC3873409  PMID: 24386329
2.  Toll-Like Receptor Gene Variants Associated with Bacterial Vaginosis among HIV-1 Infected Adolescents 
Bacterial vaginosis (BV) is a common vaginal disorder in women of reproductive age, especially among women with HIV-1 infection. Several bacterial products including lipopolysaccharides (LPS), lipoteichoic acids (LTA), and peptidoglycans (PGN) are stimulatory ligands for Toll-like receptors (TLRs), and recent evidence indicates the important role of variation in TLR genes for permitting overgrowth of gram negative and BV-type flora. We assessed whether genetic polymorphisms in five TLR genes (TLR1, TLR2, TLR4, TLR6, and TLR9) could be determinants of differential host immune responses to BV in 159 HIV-1-positive African American adolescents enrolled in the Reaching for Excellence in Adolescent Care and Health (REACH) study. BV was assessed biannually and diagnosed either by a Nugent Score of at least 7 of 10, or using the Amsel Criteria. Cox-proportional hazards regression models, adjusted for concurrent Chlamydia and Gonorrhea infections, douching, and absolute CD4 cell count, were used to identify host genetic factors associated with BV. Two SNPs were associated with BV as diagnosed by the Nugent Score and the combined criteria: a minor allele G of rs4986790 (frequency=0.07), which encodes a His to Tyr substitution in TLR4 (HR=1.47, 95% CI 1.15–1.87) and rs187084 (frequency=0.24) on TLR9. The minor allele of rs1898830 (frequency=0.13) was associated with an increased hazard of BV defined by the Amsel criteria (HR=1.86, 95%CI 1.17–2.95). Further studies are warranted to confirm the associations of TLR gene variants and also to understand the underlying pathways and immunogenetic correlates in the context of HIV-1 infection.
doi:10.1016/j.jri.2012.08.002
PMCID: PMC3518650  PMID: 23021866
HIV-1; Bacterial Vaginosis; Toll Like Receptors
3.  Genetic associations with 25-hydroxyvitamin D deficiency in HIV-1-infected youth: fine-mapping for the GC/DBP gene that encodes the vitamin D-binding protein 
Frontiers in Genetics  2013;4:234.
Serum 25-hydroxyvitamin D [25(OH)D] is often deficient (<12 ng/ml) or insufficient (<20 ng/ml) in youth living with human immunodeficiency virus type 1 infection (YLH). Based on evidence from multiple genome-wide association studies, we hypothesized that genetic factors associated with 25(OH)D deficiency should be readily detectable in YLH even when controlling for other known factors, including use of the antiretroviral drug efavirenz (EFV). Genotyping by bi-directional sequencing targeted 15 single nucleotide polymorphisms (SNPs) at the GC/DBP locus, with a focus on coding and regulatory variants, as well as those repeatedly reported in the literature. Three intronic SNPs (rs222016, rs222020, and rs222029) in a conserved haplotype block had unequivocal association signals (false discovery rate ≤ 0.006). In particular, the minor allele G for rs222020 was highly unfavorable among 192 YLH (99 African–Americans and 93 others), as gauged by relatively low likelihood for 25(OH)D sufficiency at enrollment (odds ratio = 0.31, p = 9.0 × 10-4). In a reduced multivariable model, race, season, latitude, body mass index, exposure to EFV, and rs222020-G were independent factors that collectively accounted for 38% of variance in the log10-transformed 25(OH)D concentration (p < 0.0001). Interaction terms were evident for rs222020-G × season (p < 0.001), latitude × season (especially fall and winter; p < 0.01), and race × EFV use (p = 0.024). Overall, variance in serum 25(OH)D is substantially attributable to multiple factors, but the exact contribution of genetic and non-genetic factors can be obscured by partial overlaps and frequent interactions.
doi:10.3389/fgene.2013.00234
PMCID: PMC3827582  PMID: 24294218
antiretroviral; genetics; HIV-1; race; youth; vitamin D
4.  Long-Term Follow-Up of HPV16-Positive Women: Persistence of the Same Genetic Variant and Low Prevalence of Variant Co-Infections 
PLoS ONE  2013;8(11):e80382.
HPV16 variants correlate with geographic origin and ethnicity. The association between infection with a specific variant and the cervical disease risk remains unclear. We studied the prevalence, persistence and association with cervical intraepithelial neoplasia (CIN) of different HPV16 variants, using cervical swabs and whole tissue sections (WTS) of biopsies from 548 women in the placebo group of a HPV16/18 vaccine trial. In HPV16-positive samples, HPV16 variants were identified by a reverse hybridization assay (RHA). Laser-capture micro-dissection (LCM) was performed for localized detection of HPV. HPV16 variants were determined in 47 women. Frequency of mixed HPV16 variant infections was lower (8.5%) than for multiple HPV genotypes (39.1%). Among 35 women having consecutive HPV16 variant-positive swabs, 32 (91.4%) had the same variant while in three (8.6%) women a change in variant(s) was observed. HPV16-positive WTS were obtained from 12 women having consecutive HPV16 variant-positive swabs. The same variant was present in WTS of 10 women, while two were negative. WTS of five women were histologically normal. A single HPV16 variant was detected in four women having CIN1-3, while additional HPV genotypes were found in three other women having CIN2 and CIN3. In the WTS of one woman with mixed genotypes, the HPV16 variant was assigned to a CIN2 lesion by LCM. HPV16 variant infections can be effectively studied in cervical swabs and tissue specimens by the HPV16 variant RHA. Multiple HPV16 variants in one woman are rare. The HPV16 genotype consistently detected in follow-up samples usually involves a persistent infection with the same variant.
doi:10.1371/journal.pone.0080382
PMCID: PMC3823622  PMID: 24244682
5.  HLA-B*57 versus HLA-B*81 in HIV-1 Infection: Slow and Steady Wins the Race? 
Journal of Virology  2013;87(7):4043-4051.
Two human leukocyte antigen (HLA) variants, HLA-B*57 and -B*81, are consistently known as favorable host factors in human immunodeficiency virus type 1 (HIV-1)-infected Africans and African-Americans. In our analyses of prospective data from 538 recent HIV-1 seroconverters and cross-sectional data from 292 subjects with unknown duration of infection, HLA-B*57 (mostly B*57:03) and -B*81 (exclusively B*81:01) had mostly discordant associations with virologic and immunologic manifestations before antiretroviral therapy. Specifically, relatively low viral load (VL) in HLA-B*57-positive subjects (P ≤ 0.03 in various models) did not translate to early advantage in CD4+ T-cell (CD4) counts (P ≥ 0.37). In contrast, individuals with HLA-B*81 showed little deviation from the normal set point VL (P > 0.18) while maintaining high CD4 count during early and chronic infection (P = 0.01). These observations suggest that discordance between VL and CD4 count can occur in the presence of certain HLA alleles and that effective control of HIV-1 viremia is not always a prerequisite for favorable prognosis (delayed immunodeficiency). Of note, steady CD4 count associated with HLA-B*81 in HIV-1-infected Africans may depend on the country of origin, as observations differed slightly between subgroups enrolled in southern Africa (Zambia) and eastern Africa (Kenya, Rwanda, and Uganda).
doi:10.1128/JVI.03302-12
PMCID: PMC3624227  PMID: 23365442
6.  THE INFLUENCE OF HLA CLASS I ALLELES AND THEIR POPULATION FREQUENCIES ON HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 CONTROL AMONG AFRICAN AMERICANS 
Human immunology  2011;72(4):312-318.
Populations of African ancestry continue to account for a disproportionate burden of human immunodeficiency virus type 1 (HIV-1) epidemic in the US. We investigated the effects of human leukocyte antigen (HLA) class I markers in association with virologic and immunologic control of HIV-1 infection among 338 HIV-1 subtype B-infected African Americans in two cohorts: REACH (Reaching for Excellence in Adolescent Care and Health) and HERS (HIV Epidemiology Research Study). One-year treatment-free interval measurements of HIV-1 RNA viral loads and CD4+ T-cells were examined both separately and combined to represent three categories of HIV-1 disease control (76 “controllers,” 169 “intermediates,” and 93 “non-controllers”). Certain previously or newly implicated HLA class I alleles (A*32, A*36, A*74, B*14, B*1510, B*3501, B*45, B*53, B*57, Cw*04, Cw*08, Cw*12, and Cw*18) were associated with one or more of the endpoints in univariate analyses. After multivariable adjustments for other genetic and non-genetic risk factors of HIV-1 progression, the subset of alleles more strongly or consistently associated with HIV-1 disease control included A*32, A*74, B*14, B*45, B*53, B*57, and Cw*08. Carriage of infrequent HLA-B but not HLA-A alleles was associated with more favorable disease outcomes. Certain HLA class I associations with control of HIV-1 infection span the boundaries of race and viral subtype; while others appear confined within one or the other of those boundaries.
doi:10.1016/j.humimm.2011.01.003
PMCID: PMC3778654  PMID: 21262311
HLA class I; Allele frequency; HIV-1 control; African American
7.  Among Patients with Sustained Viral Suppression in a Resource-Limited Setting, CD4 Gains Are Continuous Although Gender-Based Differences Occur 
PLoS ONE  2013;8(8):e73190.
Introduction
There is conflicting data on long-term CD4 immune recovery after combination antiretroviral therapy (ART) in resource-limited settings. Virologic suppression is rarely documented in cohorts from sub-Saharan Africa so objective evidence of adherence is biologically unsubstantiated. We sought to investigate long-term patterns of immune recovery in Ugandan patients on ART with sustained viral suppression.
Methods
A prospective cohort of patients starting ART between April, 2004 and April, 2005 at the Infectious Diseases Institute with sustained viral suppression (viral load ≤400 copies/ml at month 6 and 12) while on first-line ART. Propensity scores were used to adjust for treatment allocation (nevirapine or efavirenz) at ART initiation. Data were analyzed using Kaplan Meier methods and cross-sectional time series regression.
Results
Three hundred and fifty-six patients were included in the analysis.71.6% were female, 87% in WHO stage 3 or 4, median age was 37 years, (IQR:32–43), and median CD4 count was 108 cells/µL, (IQR:35–174) at ART start. At multivariable analysis, lower immune recovery (measured by change in CD4 from ART start at each time interval) was associated with male-gender (-59, 95% CI: 90, -28, P<0.001), baseline CD4 count of 101–200 cells/µL (-35, 95% CI: 62, -9, P=0.009) and >200 (-64, 95% CI: 101, -26, P=0.001), and use of AZT at baseline (-47, 95% CI: -74, -20, P=0.001). Median time to reach >400 cells/µL was longer in males (197.4 weeks, IQR:119.9–312.0), compared to females (144.7 weeks, IQR:96.6–219.7, P<0.001). The cumulative probability of attaining CD4 >400 cells/µL over 7 years was higher in females compared to males (P<0.001).
Conclusions
There was long-term, continuous, immunologic recovery up to 7 years after ART initiation in an urban Ugandan cohort. Virologically suppressed women had better sustained immune recovery than men. Men take longer to immune reconstitute and have a lower probability of reaching a CD4 cell count >400 cells/µL. The biologic mechanisms of these gender differences need further exploration.
doi:10.1371/journal.pone.0073190
PMCID: PMC3754935  PMID: 24013838
8.  Predictors of Suboptimal Virologic Response to Highly Active Antiretroviral Therapy Among Human Immunodeficiency Virus-Infected Adolescents 
Objective
To examine the prevalence and biopsychosocial predictors of sub-optimal virologic response to highly active antiretroviral therapy (HAART) among human immunodeficiency virus (HIV)-infected adolescents.
Design
Population-based cohort study.
Setting
Sixteen academic medical centers across thirteen cities in the United States.
Participants
One hundred and fifty four HIV-infected adolescents who presented for at least two consecutive visits after initiation of HAART.
Main Outcome Measures
Viral load (plasma concentration of HIV RNA), CD4+ T-lymphocyte count.
Results
Of the 154 adolescents enrolled in the study, 50 (32.5%) demonstrated early and sustained virologic suppression while receiving HAART. The remaining 104 adolescents (67.5%) had a poor virologic response. Adequate adherence (>50%) to HAART—reported by 70.8% of respondents—was associated with a 60% reduced odds of suboptimal virologic suppression in a multivariable logistic regression model (adjusted odds ratio = 0.4; 95% confidence interval : 0.2 – 1.0). Exposure to sub-optimal antiretroviral therapy (ART) prior to HAART, on the other hand, was associated with more than a two-fold increased odds of sub-optimal virologic response (adjusted odds ratio = 2.6; 95% confidence interval: 1.1 – 5.7).
Conclusions
Fully two-thirds of HIV-infected adolescents in the current study demonstrated a sub-optimal virologic response to HAART. Non-adherence and prior single or dual ART were associated with subsequent poor virologic responses to HAART. These predictors of HAART failure echo findings in pediatric and adult populations. Given the unique developmental stage of adolescence, age-specific interventions are indicated to address high rates of non-adherence and therapeutic failure.
doi:10.1001/archpediatrics.2009.204
PMCID: PMC3739292  PMID: 19996046
HIV; Adolescent; Antiretroviral Therapy; Highly Active; Adherence; Viral Load; CD4 Lymphocyte Count
9.  A Genome-wide Association Study of Host Genetic Determinants of the Antibody Response to Anthrax Vaccine Adsorbed 
Vaccine  2012;30(32):4778-4784.
Several lines of evidence have supported a host genetic contribution to vaccine response, but genome-wide assessments for specific determinants have been sparse. Here we describe a genome-wide association study (GWAS) of protective antigen-specific antibody (AbPA) responses among 726 European-Americans who received Anthrax Vaccine Adsorbed (AVA) as part of a clinical trial. After quality control, 736,996 SNPs were tested for association with the AbPA response to 3 or 4 AVA vaccinations given over a 6-month period. No SNP achieved the threshold of genome-wide significance (p=5x10−8), but suggestive associations (p<1x10−5) were observed for SNPs in or near the class II region of the major histocompatibility complex (MHC), in the promoter region of SPSB1, and adjacent to MEX3C. Multivariable regression modeling suggested that much of the association signal within the MHC corresponded to previously identified HLA DR-DQ haplotypes involving component HLA-DRB1 alleles of *15:01, *01:01, or *01:02. We estimated the proportion of additive genetic variance explained by common SNP variation for the AbPA response after the 6 month vaccination. This analysis indicated a significant, albeit imprecisely estimated, contribution of variation tagged by common polymorphisms (p=0.032). Future studies will be required to replicate these findings in European Americans and to further elucidate the host genetic factors underlying variable immune response to AVA.
doi:10.1016/j.vaccine.2012.05.032
PMCID: PMC3387748  PMID: 22658931
Anthrax vaccines; Bacillus anthracis; bacterial vaccines; vaccination; Genome-wide association study
10.  Cumulative Impact of Host and Viral Factors on HIV-1 Viral-Load Control during Early Infection 
Journal of Virology  2013;87(2):708-715.
In HIV-1 infection, the early set-point viral load strongly predicts both viral transmission and disease progression. The factors responsible for the wide spectrum of set-point viral loads are complex and likely reflect an interplay between the transmitted virus and genetically defined factors in both the transmitting source partner and the seroconverter. Indeed, analysis of 195 transmission pairs from Lusaka, Zambia, revealed that the viral loads in transmitting source partners contributed only ∼2% of the variance in early set-point viral loads of seroconverters (P = 0.046 by univariable analysis). In multivariable models, early set-point viral loads in seroconverting partners were a complex function of (i) the viral load in the source partner, (ii) the gender of the seroconverter, (iii) specific HLA class I alleles in the newly infected partner, and (iv) sharing of HLA-I alleles between partners in a transmission pair. Each of these factors significantly and independently contributed to the set-point viral load in the newly infected partner, accounting for up to 37% of the variance observed and suggesting that many factors operate in concert to define the early virological phenotype in HIV-1 infection.
doi:10.1128/JVI.02118-12
PMCID: PMC3554094  PMID: 23115285
11.  HLA-B Signal Peptide Polymorphism Influences the Rate of HIV-1 Acquisition but Not Viral Load 
The Journal of Infectious Diseases  2012;205(12):1797-1805.
Human leukocyte antigen alleles influence the immune response to HIV-1. Signal peptides cleaved from those alleles bind to HLA-E and mediate natural killer cell function. Signal peptides of HLA-A and HLA-C proteins carry methionine (Met) at anchor position 2 (P2); those of HLA-B carry Met or threonine (Thr). Different P2 residues alter HLA-E binding to its cognate receptors and may impact HIV-1 acquisition. Among Zambian couples (N = 566) serodiscordant for HIV-1, P2-Met accelerated acquisition in the HIV-1-negative partner (relative hazard [RH], 1.79). Among seroconverting Zambian (n = 240) and Rwandan (n = 64) partners, P2-Met also accelerated acquisition (RH, 1.47 and RH, 1.83 respectively). HLA-B alleles displaying the reportedly protective Bw4 epitope carry P2-Thr. Bw4/P2-Thr and Bw6/P2-Thr showed similar protective effects compared with Bw6/P2-Met. Neither motif was associated with viral load. The influence of HLA-B alleles on HIV/AIDS may derive from multiple motifs in and beyond the mature proteins.
doi:10.1093/infdis/jis275
PMCID: PMC3571229  PMID: 22492862
12.  Imputing Amino Acid Polymorphisms in Human Leukocyte Antigens 
PLoS ONE  2013;8(6):e64683.
DNA sequence variation within human leukocyte antigen (HLA) genes mediate susceptibility to a wide range of human diseases. The complex genetic structure of the major histocompatibility complex (MHC) makes it difficult, however, to collect genotyping data in large cohorts. Long-range linkage disequilibrium between HLA loci and SNP markers across the major histocompatibility complex (MHC) region offers an alternative approach through imputation to interrogate HLA variation in existing GWAS data sets. Here we describe a computational strategy, SNP2HLA, to impute classical alleles and amino acid polymorphisms at class I (HLA-A, -B, -C) and class II (-DPA1, -DPB1, -DQA1, -DQB1, and -DRB1) loci. To characterize performance of SNP2HLA, we constructed two European ancestry reference panels, one based on data collected in HapMap-CEPH pedigrees (90 individuals) and another based on data collected by the Type 1 Diabetes Genetics Consortium (T1DGC, 5,225 individuals). We imputed HLA alleles in an independent data set from the British 1958 Birth Cohort (N = 918) with gold standard four-digit HLA types and SNPs genotyped using the Affymetrix GeneChip 500 K and Illumina Immunochip microarrays. We demonstrate that the sample size of the reference panel, rather than SNP density of the genotyping platform, is critical to achieve high imputation accuracy. Using the larger T1DGC reference panel, the average accuracy at four-digit resolution is 94.7% using the low-density Affymetrix GeneChip 500 K, and 96.7% using the high-density Illumina Immunochip. For amino acid polymorphisms within HLA genes, we achieve 98.6% and 99.3% accuracy using the Affymetrix GeneChip 500 K and Illumina Immunochip, respectively. Finally, we demonstrate how imputation and association testing at amino acid resolution can facilitate fine-mapping of primary MHC association signals, giving a specific example from type 1 diabetes.
doi:10.1371/journal.pone.0064683
PMCID: PMC3675122  PMID: 23762245
13.  Clinical Impact and Cost-Effectiveness of Expanded Voluntary HIV Testing in India 
PLoS ONE  2013;8(5):e64604.
Background
Despite expanding access to antiretroviral therapy (ART), most of the estimated 2.3 to 2.5 million HIV-infected individuals in India remain undiagnosed. The questions of whom to test for HIV and at what frequency remain unclear.
Methods
We used a simulation model of HIV testing and treatment to examine alternative HIV screening strategies: 1) current practice, 2) one-time, 3) every five years, and 4) annually; and we applied these strategies to three population scenarios: 1) the general Indian population (“national population”), i.e. base case (HIV prevalence 0.29%; incidence 0.032/100 person-years [PY]); 2) high-prevalence districts (HIV prevalence 0.8%; incidence 0.088/100 PY), and 3) high-risk groups (HIV prevalence 5.0%; incidence 0.552/100 PY). Cohort characteristics reflected Indians reporting for HIV testing, with a median age of 35 years, 66% men, and a mean CD4 count of 305 cells/µl. The cost of a rapid HIV test was $3.33. Outcomes included life expectancy, HIV-related direct medical costs, incremental cost-effectiveness ratios (ICERs), and secondary transmission benefits. The threshold for “cost-effective” was defined as 3x the annual per capita GDP of India ($3,900/year of life saved [YLS]), or for “very cost-effective” was <1x the annual per capita GDP ($1,300/YLS).
Results
Compared to current practice, one-time screening was very cost-effective in the national population (ICER: $1,100/YLS), high-prevalence districts (ICER: $800/YLS), and high-risk groups (ICER: $800/YLS). Screening every five years in the national population (ICER: $1,900/YLS) and annual screening in high-prevalence districts (ICER: $1,900/YLS) and high-risk groups (ICER: $1,800/YLS) were also cost-effective. Results were most sensitive to costs of care and linkage-to-care.
Conclusions
In India, voluntary HIV screening of the national population every five years offers substantial clinical benefit and is cost-effective. Annual screening is cost-effective among high-risk groups and in high-prevalence districts nationally. Routine HIV screening in India should be implemented.
doi:10.1371/journal.pone.0064604
PMCID: PMC3669338  PMID: 23741348
14.  Genomic Copy Number Variants: Evidence for Association with Antibody Response to Anthrax Vaccine Adsorbed 
PLoS ONE  2013;8(5):e64813.
Background
Anthrax and its etiologic agent remain a biological threat. Anthrax vaccine is highly effective, but vaccine-induced IgG antibody responses vary widely following required doses of vaccinations. Such variation can be related to genetic factors, especially genomic copy number variants (CNVs) that are known to be enriched among genes with immunologic function. We have tested this hypothesis in two study populations from a clinical trial of anthrax vaccination.
Methods
We performed CNV-based genome-wide association analyses separately on 794 European Americans and 200 African-Americans. Antibodies to protective antigen were measured at week 8 (early response) and week 30 (peak response) using an enzyme-linked immunosorbent assay. We used DNA microarray data (Affymetrix 6.0) and two CNV detection algorithms, hidden markov model (PennCNV) and circular binary segmentation (GeneSpring) to determine CNVs in all individuals. Multivariable regression analyses were used to identify CNV-specific associations after adjusting for relevant non-genetic covariates.
Results
Within the 22 autosomal chromosomes, 2,943 non-overlapping CNV regions were detected by both algorithms. Genomic insertions containing HLA-DRB5, DRB1 and DQA1/DRA genes in the major histocompatibility complex (MHC) region (chromosome 6p21.3) were moderately associated with elevated early antibody response (β = 0.14, p = 1.78×10−3) among European Americans, and the strongest association was observed between peak antibody response and a segmental insertion on chromosome 1, containing NBPF4, NBPF5, STXMP3, CLCC1, and GPSM2 genes (β = 1.66, p = 6.06×10−5). For African-Americans, segmental deletions spanning PRR20, PCDH17 and PCH68 genes on chromosome 13 were associated with elevated early antibody production (β = 0.18, p = 4.47×10−5). Population-specific findings aside, one genomic insertion on chromosome 17 (containing NSF, ARL17 and LRRC37A genes) was associated with elevated peak antibody response in both populations.
Conclusion
Multiple CNV regions, including the one consisting of MHC genes that is consistent with earlier research, can be important to humoral immune responses to anthrax vaccine adsorbed.
doi:10.1371/journal.pone.0064813
PMCID: PMC3669407  PMID: 23741398
15.  Etiology of Genital Ulcer Disease in a Sexually Transmitted Infection Reference Center in Manaus, Brazilian Amazon 
PLoS ONE  2013;8(5):e63953.
Objectives
To determine the etiology and factors associated with genital ulcer disease (GUD) among patients presenting to a sexually transmitted infections clinic in Manaus, Brazil; and to compare a multiplex polymerase chain reaction (M-PCR) assay for the diagnosis of GUD with standard methods.
Methods
Ulcer swabs were collected and used for Tzanck test and processed in an M-PCR to detect herpes simplex virus (HSV-1/2), Treponema pallidum (T. pallidum), and Haemophilus ducreyi (H. ducreyi). Sera were tested for HIV and syphilis antibodies. Multivariable analysis was used to measure the association between clinical aspects and GUD. M-PCR results were compared with syphilis serology and Tzanck tests.
Results
Overall, 434 GUD samples were evaluated, 84.8% from men. DNA from HSV-2 was detected in 55.3% of GUD samples, T. pallidum in 8.3%, HSV-1 in 3.2%, and 32.5% of GUD specimens were negative for the DNA of all three pathogens. No cases of H. ducreyi were identified. HIV serology among GUD patients was 3.2%. Treponemal antibodies and Tzanck test positivity for genital herpes was detected in 25 (5.8%) and in 125 (30.3%) of GUD patients, respectively. In multivariable analysis genital herpes etiology by M-PCR was associated with the vesicular, multiple and recurrent lesions whereas T. pallidum with non-vesicular, non-recurrent lesions. Compared to M-PCR, syphilis serology was 27.8% sensitive and 96.2% specific whereas Tzanck test was 43.8% sensitive and 88.9% specific.
Conclusions
The predominance of genital herpes etiology suggests a revision of existing national syndromic treatment guidelines in Brazil to include antiherpetic treatment for all GUD patients. The use of M-PCR can significantly improve the diagnosis of GUD and provide a greater sensitivity than standard diagnostics.
doi:10.1371/journal.pone.0063953
PMCID: PMC3660360  PMID: 23704961
16.  Statin Therapy and Mortality in HIV-Infected Individuals; A Danish Nationwide Population-Based Cohort Study 
PLoS ONE  2013;8(3):e52828.
Background
Recent studies have suggested that statins possess diverse immune modulatory and anti-inflammatory properties. As statins might attenuate inflammation, statin therapy has been hypothesized to reduce mortality in HIV-infected individuals. We therefore used a Danish nationwide cohort of HIV-infected individuals to estimate the impact of statin use on mortality before and after a diagnosis of cardiovascular disease, chronic kidney disease or diabetes.
Methods
We identified all Danish HIV-infected individuals (1,738) who initiated HAART after 1 January 1998, and achieved virological suppression within 180 days. Date of first redemption of a prescription of statin was obtained from the Danish National Prescription Registry. We used Poisson regression analysis to assess adjusted mortality rate ratios (aMRR). First, time was censored at date of virological failure (VL >500 copies/ml). Second, time was not censored at virological failure. All analyses were adjusted for potential confounders.
Results
In the analyses confined to observation time without virological failure (+ censoring) statin therapy was associated with a non-statistically significant reduced rate of death (aMRR 0.75; 95% CI: 0.33–1.68). No difference was observed in the analysis with no censoring (aMRR 1.17; 95% CI: 0.66–2.07). Use of statin seemed to reduce mortality in individuals after a diagnosis of comorbidity {(+ censoring: aMRR: 0.34; 95% CI: 0.11–1.04), (−censoring: aMRR: 0.64; 95% CI: 0.32–1.29)}. No difference in rate of death could be detected before first date of diagnosis of comorbidity {(+ censoring: aMRR: 1.12; 95% CI: 0.34–3.62), (−censoring: aMRR: 0.90; 95% CI: 0.28–2.88)}.
Conclusion
Statin therapy might reduce all-cause mortality in HIV-infected individuals, but the impact on individuals with no comorbidity seems small or absent. An unambiguous proof of a causal relation can only be obtained in a randomized controlled trial, but the sample size predicted may be prohibitive for its conduct.
doi:10.1371/journal.pone.0052828
PMCID: PMC3587599  PMID: 23469159
17.  Genetic Variations and Heterosexual HIV-1 Infection: Analysis of Clustered Genes Encoding CC-motif Chemokine Ligands 
Genes and immunity  2011;13(2):202-205.
Several CC-motif chemokine ligands (CCLs) can block HIV-1 binding sites on CC-motif chemokine receptor 5 (CCR5) and inhibit viral entry. We studied single nucleotide polymorphisms (SNPs) in genes encoding three CCR5 ligands [CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES)] along with an adjacent gene encoding a CCR2 ligand [CCL2 (MCP-1)] to identify candidate markers for HIV-1 infection and pathogenesis. Analyses of 567 HIV-1 serodiscordant Zambian couples revealed that rs5029410C (in CCL3 intron 2) was associated with lower viral load (VL) in seroconverters, adjusted for gender and age (regression β=−0.57 log10, P=4×10−6). In addition, rs34171309A in CCL3 exon 3 was associated with increased risk of HIV-1 acquisition in exposed seronegatives (hazard ratio=1.52, P=0.006 when adjusted for donor VL and genital ulcer/inflammation). The CCL3 exon 3 SNP, encoding a conservative Glu-to-Asp substitution, and five neighboring SNPs in tight linkage disequilibrium all showed similar associations with HIV-1 acquisition. How these multiple CCL3 SNPs may alter the occurrence or course of HIV-1 infection remains to be determined.
doi:10.1038/gene.2011.70
PMCID: PMC3559129  PMID: 21975429
HIV-1 transmission; CCL2; CCL3; CCL4; CCL5; SNP
18.  Study of the Integrated Immune Response Induced by an Inactivated EV71 Vaccine 
PLoS ONE  2013;8(1):e54451.
Enterovirus 71 (EV71), a major causative agent of hand-foot-and-mouth disease (HFMD), causes outbreaks among children in the Asia-Pacific region. A vaccine is urgently needed. Based on successful pre-clinical work, phase I and II clinical trials of an inactivated EV71 vaccine, which included the participants of 288 and 660 respectively, have been conducted. In the present study, the immune response and the correlated modulation of gene expression in the peripheral blood mononuclear cells (PBMCs) of 30 infants (6 to 11 months) immunized with this vaccine or placebo and consented to join this study in the phase II clinical trial were analyzed. The results showed significantly greater neutralizing antibody and specific T cell responses in vaccine group after two inoculations on days 0 and 28. Additionally, more than 600 functional genes that were up- or down-regulated in PBMCs were identified by the microarray assay, and these genes included 68 genes associated with the immune response in vaccine group. These results emphasize the gene expression profile of the immune system in response to an inactivated EV71 vaccine in humans and confirmed that such an immune response was generated as the result of the positive mobilization of the immune system. Furthermore, the immune response was not accompanied by the development of a remarkable inflammatory response.
Clinical Trial Registration: NCT01391494 and NCT01512706.
doi:10.1371/journal.pone.0054451
PMCID: PMC3553120  PMID: 23372725
19.  Short-Term Mobility and the Risk of HIV Infection among Married Couples in the Fishing Communities along Lake Victoria, Kenya 
PLoS ONE  2013;8(1):e54523.
Objective
Mobility has long been associated with high HIV prevalence. We sought to assess sex differences in the relationship between mobility and risk for HIV infection among married couples in the fishing communities.
Methods
We conducted 1090 gender-matched interviews and rapid HIV testing with 545 couples proportionally representing all the different sizes of the fish-landing beaches in Kisumu County. We contacted a random sample of fishermen as our index participants and asked them to enrol in the study together with their spouses. The consenting couples were separated into different private rooms for concurrent interviews and thereafter reunited for couple rapid HIV counselling and testing. In addition to socio-economic and behavioural data, we collected information on overnight travels and divided couples in 4 groups as follows both partners not mobile, both partners mobile, only woman mobile, and only man mobile. Other than descriptive statistics, we used X2 and U tests to compare groups of variables and multivariate logistic regression to measure association between mobility and HIV infection.
Results
We found significant differences in the number of trips women travelled in the preceding month (mean 4.6, SD 7.1) compared to men (mean 3.3, SD 4.9; p<0.01) and when the women did travel, they were more likely to spend more days away from home than their male partners (mean 5.2 [SD 7.2] versus 3.4 SD 5.6; p = 0.01). With an HIV prevalence of 22.7% in women compared to 20.9% among men, mobile women who had non-mobile spouses had 2.1 times the likelihood of HIV infection compared to individuals in couples where both partners were non-mobile.
Conclusion
The mobility of fishermen’s spouses is associated with HIV infection that is not evident among fishermen themselves. Therefore, interventions in this community could be a combination of sex-specific programming that targets women and combined programming for couples.
doi:10.1371/journal.pone.0054523
PMCID: PMC3545885  PMID: 23336005
20.  Effectiveness of Integrated HIV Prevention Interventions among Chinese Men Who Have Sex with Men: Evaluation of a 16-City Public Health Program 
PLoS ONE  2012;7(12):e50873.
To examine the impacts of a multi-city HIV prevention public health program (China Global Fund Round 5 Project) on condom use and HIV infection, we analyzed four yearly cross-sectional surveys from 2006 through 2009 among 20,843 men who have sex with men (MSM) in 16 Chinese cities. Self-reported condom use at last sex with a male partner increased from 58% in 2006 to 81% in 2009 (trend test, P<0.001). HIV prevalence increased from 2.3% in 2006 to 5.3% in 2009 (P<0.001). Multivariable logistic regression analysis showed that self-reported receipt of interventions was an independent predictor of increased condom use at last sex with a male partner over time (adjusted odds ratio [aOR], 1.63 in 2006 to 2.33 in 2009; P<0.001), and lower HIV prevalence (aOR, 1.08 in 2006 to 0.45 in 2009; P<0.001). HIV prevalence increased from 2006–2009 for participants with no self-reported receipt of interventions (2.1% in 2006 to 10.3% in 2009) and less so for those with interventions (2.4% to 4.7%). This Chinese public health program had positive impacts on both behaviors and disease rate among MSM population. Escalation of the coverage and intensity of effective interventions is needed for further increasing condom use and for reversing the rising trend of HIV epidemic.
doi:10.1371/journal.pone.0050873
PMCID: PMC3534092  PMID: 23300528
21.  Taenia taeniaeformis in Rat Favors Protracted Skin Lesions Caused by Sporothrix schenckii Infection: Dectin-1 and IL-17 Are Dispensable for Clearance of This Fungus 
PLoS ONE  2012;7(12):e52514.
We occasionally found that cestode Taenia taeniaeformis in rats favored Sporothrix schenckii infection and survival, causing protracted cutaneous lesions. In this study, we compared the pathology and cytokines profile of rats co-infected with the two pathogens and infected with S. schenckii alone to explore underlying mechanisms. In the co-infection group, there was high expression of β-glucan receptor Dectin-1 in the cutaneous lesions and no multinucleated giant cells, but in the S. schenckii infection group the opposite was observed. Cytokines profiles demonstrated an expected finding that IL-4, commonly expressed in helminth and fungus infection, is undetectable in the two infection groups. In the single fungal infection group, cytokines IFN-γ, IL-10 and IL-17 kept increasing in the first few weeks of infection to a peak which was followed by gradual decrease. This study showed that Dectin-1 and IL-17, which were believed to be the major anti-fungus mechanisms, are Th2 independent and dispensable for clearance of S. schenckii infection, suggesting that S. schenckii has a different molecular recognition pattern and evokes anti-infection mechanisms other than Dectin-1 and IL-17.
doi:10.1371/journal.pone.0052514
PMCID: PMC3527553  PMID: 23285072
22.  Predictive Clinical Factors in the Diagnosis of Gastrointestinal Kaposi's Sarcoma and Its Endoscopic Severity 
PLoS ONE  2012;7(11):e46967.
Background
The diagnosis of gastrointestinal (GI) involvement in Kaposi's sarcoma (KS) is important to make because the need for treatment depends on the extent of the disease. Moreover, severe GI lesions can cause serious complications. Endoscopy with biopsy is an extremely useful method to diagnose GI-KS. However, determining the indications for endoscopy is difficult because KS can occur without GI symptoms or cutaneous KS. This study sought to clarify predictive clinical factors for GI-KS and its severity on endoscopy.
Methodology/Principal Findings
A total of 1,027 HIV-infected patients who underwent endoscopy were analyzed. Sexual behavior, CD4 count, HIV RNA, history of highly active antiretroviral therapy (HAART), GI symptoms, and cutaneous KS were assessed. Endoscopic severity including bulky tumor, ulceration, and number of lesions were evaluated. Thirty-three patients had GI-KS and 46 patients cutaneous KS. Among the GI-KS patients, 78.8% (26/33) had no GI symptoms and 24.2% (8/33) had no cutaneous KS. Univariate analysis identified men who have sex with men (MSM), CD4 <100 cells/µL, HIV RNA ≥10,000 copies/mL, no history of HAART, and cutaneous KS were significantly associated with GI-KS. Among these factors, cutaneous KS was closely related to GI-KS on multivariable analysis. Among patients without cutaneous KS, MSM and CD4 count <100 cells/µL were the only independent clinical factors related to GI-KS. Bulky tumor was significantly associated with CD4 <100 cells/µL and large number of lesions was significantly associated with HIV-RNA ≥10,000 copies/mL.
Conclusions
To diagnose GI-KS, clinical factors need to be considered before endoscopy. The presence of GI symptoms is not useful in predicting GI-KS. MSM and CD4 count <100 cells/µL are predictive factors among patients without cutaneous KS. Caution should be exercised especially in patients with low CD4 counts or high HIV viral loads as they are more likely to develop severe GI-KS lesions.
doi:10.1371/journal.pone.0046967
PMCID: PMC3511500  PMID: 23226197
23.  Allele-Level Haplotype Frequencies and Pairwise Linkage Disequilibrium for 14 KIR Loci in 506 European-American Individuals 
PLoS ONE  2012;7(11):e47491.
The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5∼KIR2DS3S5 and KIR2DS3S5∼KIR2DL1, and telomeric KIR3DL1∼KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis.
doi:10.1371/journal.pone.0047491
PMCID: PMC3489906  PMID: 23139747
24.  HIV-1 Dynamics: A Reappraisal of Host and Viral Factors, as well as Methodological Issues 
Viruses  2012;4(10):2080-2096.
The dynamics of HIV-1 viremia is a complex and evolving landscape with clinical and epidemiological (public health) implications. Most studies have relied on the use of set-point viral load (VL) as a readily available proxy of viral dynamics to assess host and viral correlates. This review highlights recent findings from population-based studies of set-point VL, focusing primarily on robust data related to host genetics. A comprehensive understanding of viral dynamics will clearly need to consider both host and viral characteristics, with close attention to (i) the timing of VL measurements, (ii) the biology of viral evolution, (iii) compartments of active viral replication, (iv) the transmission source partner as the immediate past microenvironment, and (v) proper application of statistical models.
doi:10.3390/v4102080
PMCID: PMC3497042  PMID: 23202454
Correlates; genetics; HIV-1; viral load.
25.  In vivo pharmacokinetic comparisons of ferulic acid and puerarin after oral administration of monomer, medicinal substance aqueous extract and Nao-De-Sheng to rats 
Pharmacognosy Magazine  2012;8(32):256-262.
Background:
Nao-De-Sheng decoction (NDS), a traditional Chinese medicine (TCM) prescription containing Radix puerariae lobatae, Floscarthami, Radix et Rhizoma Notoginseng, Rhizoma chuanxiong and Fructus crataegi, is effective in the treatment of cerebral arteriosclerosis, ischemic cerebral stroke and apoplexy linger effect. Ferulic acid and puerarin are the main absorbed effective ingredients of NDS.
Objective:
To assess the affection of other components in medical material and compound recipe compatibility on the pharmacokinetics of ferulaic acid and puerarin, of ferulic acid from the monomer Rhizoma chuanxiong aqueous extract and NDS were studied. And pharmacokinetics comparisons of puerarin from the monomer Radix puerariae extract and NDS decoction were investigated simultaneously.
Materials and Methods:
At respective different time points after oral administration of the monomer, medicinal substance aqueous extract and NDS at the same dose in rats, plasma concentrations of ferulic acid and puerarin in rats were determined by RP-HPLC, and the main pharmacokinetic parameters were estimated with 3P97 software.
Results:
The plasma concentration-time curves of ferulaic acid and puerarin were both best fitted with a two-compartment model. AUC0−t, AUC0→∞, Tmax, and Cmax of ferulic acid in the monomer and NDS decoction were increased significantly (P < 0.05) compared with that in Rhizoma chuanxiong aqueous extract. And statistically significant increase (P < 0.05) in pharmacokinetic parameters of puerarin including AUC0−t, AUC0→∞, CL, Tmax and Cmax were obtained after oral administration of puerarin monomer compared with Radix puerariae extract. Although the changes of AUC0−t, AUC0→∞ and CL had no statistically significant, Cmax of puerarin in NDS was increased remarkably (P < 0.05) compared with that in single puerarin.
Conclusions:
Some ingredients of Rhizoma chuanxiong and Radix puerariae may be suggested to remarkably influence plasma concentrations of ferulaic acid and puerarin. Some ingredients in NDS may increase dissolution and absorption of ferulaic acid and puerarin, delay elimination, and subsequently enhance bioavailability of ferulaic acid and puerarin in rats after compatibility.
doi:10.4103/0973-1296.103648
PMCID: PMC3785161  PMID: 24082627
Ferulic acid; medicinal substance aqueous extract; Nao-De-Sheng; pharmacokinetics comparisons; puerarin

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