Midtrimester Cervicovaginal Microbiota: Identification of Microbial Variations Associated with Puerperal Infection at Term.

Objective This study aims to evaluate differences in the midtrimester cervicovaginal microbiota between women who developed puerperal infections at term and those who did not, and whether obesity modulates this relationship. Methods Previously, cervicovaginal swabs were collected at 21 to 25 weeks gestation (stored at -80°C). Samples were identified from Black women with normal vaginal flora (Nugent score: 0-2) delivering term singletons. Patients were in one of four equally sized groups (total n = 120) characterized by absence or presence of puerperal infection and maternal obesity. Samples were thawed, DNA extracted, and polymerase chain reaction with primers targeting the 16S rDNA V4 region was used to prepare an amplicon library sequenced and analyzed using Quantitative Insights into Microbial Ecology (QIIME) suite. Microbiota differences were assessed using permutation-based anodis over three ß-diversity measures; Kruskal-Wallis test was used for taxa level analysis. Results After quality control measures, 113 samples were analyzed. Overall, there was significant clustering by puerperal infection (p = 0.03), but not by obesity (p > 0.05). Detailed taxa level analysis revealed approximately 66% less Proteobacteria phylum and 400% more BVAB1 genera in the second-trimester microbiota of women who had puerperal infections at term (p < 0.05). Conclusion Women who develop puerperal infections at term have a significantly altered midtrimester cervicovaginal microbiome with less Proteobacteria and greater BVAB1. This finding may represent a potential method to identify women at an increased risk of puerperal infection.

Quantitative Polymerase Chain Reaction to Assess Response to Treatment of Bacterial Vaginosis and Risk of Preterm Birth.

OBJECTIVE: The aim of this study was to determine whether quantitative polymerase chain reaction (qPCR) bacterial load measurement is a valid method to assess response to treatment of bacterial vaginosis and risk of preterm birth in pregnant women. STUDY DESIGN: Secondary analysis by utilizing stored vaginal samples obtained during a previous randomized controlled trial studying the effect of antibiotics on preterm birth (PTB). All women had risk factors for PTB: (1) positive fetal fibronectin (n=146), (2) bacterial vaginosis (BV) and a prior PTB (n=43), or (3) BV and a prepregnancy weight<50 kg (n=54). Total and several individual BV-related bacteria loads were measured using qPCR for 16S rRNA. Loads were correlated with Nugent scores (Spearman correlation coefficients). Loads were compared pre- and posttreatment with Wilcoxon rank-sum test. Individual patient differences were examined with Wilcoxon signed-rank test. RESULTS: A total of 243 paired vaginal samples were available for analysis: 123 antibiotics and 120 placebo. Groups did not differ by risk factors for PTB. For all samples, bacterial loads were correlated with Nugent score and each of its specific bacterial components (all p<0.01). Baseline total bacterial load did not differ by treatment group (p=0.87). Posttreatment total bacterial load was significantly lower in the antibiotics group than the placebo group (p<0.01). Individual patient total bacterial load decreased significantly posttreatment in the antibiotics group (p<0.01), but not in the placebo group (p=0.12). The rate of PTB did not differ between groups (p=0.24). PTB relative risks calculated for BV positive versus BV negative women and women with the highest quartile total and individual bacterial loads were not statistically significant. CONCLUSION: qPCR correlates with Nugent score and demonstrates decreased bacterial load after antibiotic treatment. Therefore, it is a valid method of vaginal flora assessment in pregnant women who are at high risk for PTB.

Human leukocyte antigen class I supertypes and HIV-1 control in African Americans.

The role of human leukocyte antigen (HLA) class I supertypes in controlling human immunodeficiency virus type 1 (HIV-1) infection in African Americans has not been established. We examined the effects of the HLA-A and HLA-B alleles and supertypes on the outcomes of HIV-1 clade B infection among 338 African American women and adolescents. HLA-B58 and -B62 supertypes (B58s and B62s) were associated with favorable HIV-1 disease control (proportional odds ratio [POR] of 0.33 and 95% confidence interval [95% CI] of 0.21 to 0.52 for the former and POR of 0.26 and 95% CI of 0.09 to 0.73 for the latter); B7s and B44s were associated with unfavorable disease control (POR of 2.39 and 95% CI of 1.54 to 3.73 for the former and POR of 1.63 and 95% CI of 1.08 to 2.47 for the latter). In general, individual alleles within specific B supertypes exerted relatively homogeneous effects. A notable exception was B27s, whose protective influence (POR, 0.58; 95% CI, 0.35 to 0.94) was masked by the opposing effect of its member allele B*1510. The associations of most B supertypes (e.g., B58s and B7s) were largely explained either by well-known effects of constituent B alleles or by effects of previously unimplicated B alleles aggregated into a particular supertype (e.g., B44s and B62s). A higher frequency of HLA-B genotypic supertypes correlated with a higher mean viral load (VL) and lower mean CD4 count (Pearson's r = 0.63 and 0.62, respectively; P = 0.03). Among the genotypic supertypes, B58s and its member allele B*57 contributed disproportionately to the explainable VL variation. The study demonstrated the dominant role of HLA-B supertypes in HIV-1 clade B-infected African Americans and further dissected the contributions of individual class I alleles and their population frequencies to the supertype effects.

Midtrimester microbial DNA variations in maternal serum of women who experience spontaneous preterm birth.

Objectives: To evaluate if midtrimester maternal serum contains microbial DNA and whether it differs between women with spontaneous preterm birth (SPTB) and those delivering at term.Study design: In this retrospective case-control study, we identified 20 healthy nulliparas with SPTB at 24-33 weeks of a nonanomalous singleton in 2014. Each case was matched by race/ethnicity to a control delivering at 39-40 weeks. Serum samples, collected at 15-20 weeks and stored at -80 C, were thawed and DNA extracted. PCR with primers targeting the 16S rDNA V4 region were used to prepare an amplicon library, sequenced using Illumina MiSeq, and analyzed using quantitative insight into microbial ecology (QIIME). Taxonomy was assigned using Ribosomal Database program (RDP) Classifier (threshold 0.8) against a modified Greengenes database. Differences in number of observed species, microbial alpha-diversity and beta-diversity, and taxa level analyses were undertaken.Results: All 40 samples were included. Women with SPTB had more unique observed species (p = .046) and higher mean alpha-diversity by Shannon index (but not Chao1 or Simpson) (p = .024). Microbial composition was different between groups by Bray-Curtis clustering (p = .03) but not by weighted (p = .13) or unweighted Unifrac (p = .11). Numerous taxa in the Firmicutes, Proteobacteria, and Actinobacteria phyla differed between groups (p < .05).Conclusions: SPTB is associated with distinct microbial DNA changes detected in midtrimester maternal serum.

Positive and negative associations of human leukocyte antigen variants with the onset and prognosis of adult glioblastoma multiforme.

Associations of genetic factors with malignant gliomas have been modest. We examined the relationships of human leukocyte antigen (HLA) and related polymorphisms to glioblastoma multiforme in adult Caucasians (non-Hispanic Whites) from the San Francisco Bay area. For 155 glioblastoma multiforme patients and 157 control subjects closely matched by ethnicity, age, and gender, PCR-based techniques resolved alleles at HLA-A, -B, -C, and -DRB1 loci along with short tandem repeat polymorphisms of MICA exon 5 and TNFb. By multivariable logistic regression, B*13 and the B*07-Cw*07 haplotype were positively associated with glioblastoma multiforme (P=0.01 and <0.001, respectively), whereas Cw*01 was the only variant showing a negative association (P=0.05). Among glioblastoma multiforme patients, progression to death after diagnosis was slower in those with A*32 (relative hazard, 0.45; P<0.01) and faster in those with B*55 (relative hazard, 2.27; P<0.01). Thus, both the occurrence and the prognosis of glioblastoma multiforme could be associated with specific but different HLA genotypes. B*07 and the B*07-Cw*07 haplotype are much more common in Caucasians than other ethnic groups in the U.S., which may partially explain the higher incidence of glioblastoma multiforme in Caucasians.

Transmission of HIV-1 and HLA-B allele-sharing within serodiscordant heterosexual Zambian couples.

Factors that might increase risk of HIV-1 transmission include age, sex, and amount of HIV-1 RNA in plasma, but findings for HLA allele-sharing are not in agreement. We tested the hypothesis that allele sharing at HLA loci is associated with increased risk of transmission of HIV-1 infection in cohabiting heterosexual Zambian couples. We studied 125 initially serodiscordant partners with sequence-confirmed interpartner HIV-1 transmission and 104 couples who were persistently serodiscordant, and we analysed relations with molecularly typed HLA-A, B, and C alleles by survival techniques. After adjustment for other genetic and non-genetic risk factors seen with heterosexual transmission of HIV-1 in this cohort, sharing of HLA-B alleles was independently associated with accelerated intracouple transmission (relative hazard 2.23, 95% CI 1.52-3.26, p<0.0001). Selective pressure by HLA-B alleles on transmitted viruses accords with current understanding of the effect of B locus polymorphism in HIV-1 and perhaps other infections.

Host genetic profiles predict virological and immunological control of HIV-1 infection in adolescents.

OBJECTIVE: To evaluate the correlation between host genetic profiles and virological and immunological outcomes among HIV-1-seropositive participants from the Reaching for Excellence in Adolescent Care and Health (REACH) cohort. METHODS: HLA class I and chemokine coreceptor (CCR) alleles and haplotypes were resolved in 227 HIV-1-seropositive adolescents (ages 13-18 years; 75% females; 71% African-Americans) and 183 HIV-seronegative individuals, with quarterly follow-up visits between 1996 and 2000. Each HLA and CCR variant with consistent risk and protective effect on HIV-1 pathogenesis was assigned a score of -1 and +1, respectively. All individual markers and genetic scores were analyzed in relation to plasma viral load (VL) and CD4 T lymphocytes during a 6-12-month interval when no antiretroviral therapy was taken. RESULTS: HLA-B*57 alone was a strong predictor of VL (P < 0.0001), but composite genetic profiles found in over 50% of patients consistently outperformed the individual component markers in multivariable analyses with or without adjustment for gender, race, age, and membership of clinical patient groups. Adolescents (n = 37) with a favorable combination of VL (< 1000 copies/ml) and CD4 T cell counts (> 450 x 10(6) cells/l) consistently had more positive (+1 to +2) than negative (-1 to -4) HLA and CCR scores compared with those (n = 56) with an unfavorable combination (VL > 16,000 copies/ml and CD4 cells < 450 x 10(6) cells/l) or the remainder (n = 134) of the cohort (overall P < 0.0001). CONCLUSION: A generalizable genetic scoring algorithm based on seven HLA class I and CCR markers is highly predictive of viremia and immunodeficiency in HIV-1-infected adolescents.

HLA allele sharing and HIV type 1 viremia in seroconverting Zambians with known transmitting partners.

Rapid HIV type 1 (HIV-1) mutation coupled with immune evasion poses a major obstacle to effective interventions. In particular, transmission of HIV-1 from a donor partner (transmitter) to a recipient (seroconverter) with similar antigen-presenting molecules (i.e., human leukocyte antigens, HLA) may favor or expedite viral adaptation to host immune responses. Our PCR-based HLA-A, HLA-B, and HLA-DRB1 genotyping for 115 Zambian couples with documented intracouple HIV-1 (mostly clade C) transmission revealed that single-locus HLA allele sharing ranged from 28 to 36%. Different degrees of allele sharing, at single or multiple HLA loci between donor-recipient pairs, were associated with only modest increases in seroconverter RNA level (+0.04 to + 0.24 log(10) copies/mL, p > 0.25). Thus, partial HLA allele sharing commonly seen in Zambian couples did not appear to confer unequivocal early advantage for viral replication in the newly seroconverting subjects. However, correlation of virus loads in seroconverters with those of their known index partners (adjusted Pearson r = 0.21, p = 0.03) did imply that viral characteristics can independently contribute to variability in plasma virus load.

The influence of human leukocyte antigen class I alleles and their population frequencies on human immunodeficiency virus type 1 control among African Americans.

Populations of African ancestry continue to account for a disproportionate burden of the human immunodeficiency virus type 1 (HIV-1) epidemic in the United States. 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 2 cohorts: Reaching for Excellence in Adolescent Care and Health (REACH) and HIV Epidemiology Research Study (HERS). One-year treatment-free interval measurements of HIV-1 RNA viral loads and CD4(+) T cells were examined both separately and combined to represent 3 categories of HIV-1 disease control (76 controllers, 169 intermediates, and 93 noncontrollers). 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 1 or more of the endpoints in univariate analyses. After multivariable adjustments for other genetic and nongenetic 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 cross the boundaries of race and viral subtype, whereas others appear confined within one or the other of those boundaries.

Human leukocyte antigen B58 supertype and human immunodeficiency virus type 1 infection in native Africans.

Human leukocyte antigen (HLA) class I alleles can be grouped into supertypes according to their shared peptide binding properties. We examined alleles of the HLA-B58 supertype (B58s) in treatment-naïve human immunodeficiency virus type 1 (HIV-1)-seropositive Africans (423 Zambians and 202 Rwandans). HLA-B and HLA-C alleles were resolved to four digits by a combination of molecular methods, and their respective associations with outcomes of HIV-1 infection were analyzed by statistical procedures appropriate for continuous or categorical data. The effects of the individual alleles on natural HIV-1 infection were heterogeneous. In HIV-1 subtype C-infected Zambians, the mean viral load (VL) was lower among B*5703 (P = 0.01) or B*5703-Cw*18 (P < 0.001) haplotype carriers and higher among B*5802 (P = 0.02) or B*5802-Cw*0602 (P = 0.03) carriers. The B*5801-Cw*03 haplotype showed an association with low VL (P = 0.05), whereas B*5801 as a whole did not. Rwandans with HIV-1 subtype A infection showed associations of B*5703 and B*5802 with slow (P = 0.06) and rapid (P = 0.003) disease progression, respectively. In neither population were B*1516-B*1517 alleles associated with more favorable responses. Overall, B58s alleles, individually or as part of an HLA-B-HLA-C haplotype, appeared to have a distinctive impact on HIV-1 infection among native Africans. As presently defined, B58s alleles cannot be considered uniformly protective against HIV/AIDS in every population.

HLA-B, -DRB1/3/4/5, and -DQB1 gene polymorphisms in human immunodeficiency virus-related Kaposi's sarcoma.

Polymorphisms of genes in the human leukocyte antigen (HLA) complex, particularly those encoding HLA-DR, have been suggested as markers of susceptibility to Kaposi's sarcoma (KS). We conducted a case-control study comparing 147 homosexual men who developed KS after infection by human immunodeficiency virus-1 (HIV-1) and human herpes virus 8 (HHV8) with 147 matched dually infected men without HIV-associated KS (HIV-KS) from the Multicenter AIDS Cohort Study. HLA-B, DRB1, DRB3, DRB4, DRB5, and DQB1 polymorphisms were examined by high-resolution DNA-based methods. Differences in distributions of genetic variants were tested by conditional logistic regression. Previously reported relationships with HLA-DRB1 alleles could not be confirmed. Instead, other associations were observed. In univariate analysis, KS was weakly associated with B*2702/5 (odds ratio (OR)=0.40, 95% confidence interval (CI)=0.18-0.91). Similar or stronger associations, positive or negative, were seen for haplotypes containing class II alleles: DRB1*1302-DQB1*0604 (OR=3.67, 95% CI=1.02-13.1), DRB4 (DR53) haplotype family members [OR=0.52, 95% CI=0.32-0.85], and DRB3 (DR52) haplotype family members (OR=1.69, 95% CI=1.07-2.67). The B*1402-DRB1*0102 haplotype, which invariably contains the V281L mutation in the 21-hydroxylase gene governing adrenal steroid biosynthesis, occurred in five cases and one control (OR=5.0, 95% CI=0.58-42.8). In a final multivariable analysis, only DRB1*1302-DQB1*0604 (OR=6.43, 95% CI=1.28-32.3, P=0.02) remained significantly associated with KS. Associations of HLA-DRB families with HIV-KS could reflect underlying immune dysregulation. The HLA B*1402-DRB1*0102 haplotype associated with increased risk of KS might represent an antigen-presenting pathway unfavorable for immune response to HHV8. Alternatively, the relationship might hold a clue to the predilection of KS for men because that haplotype harbors the mutant form of the 21-hydroxylase gene.

HLA-DRB1 and -DQB1 alleles and haplotypes in Zambian couples and their associations with heterosexual transmission of HIV type 1.

In 292 initially human immunodeficiency virus (HIV)-1-serodiscordant and cohabiting Zambian couples, HLA-DRB1 and -DQB1 variants were associated with HIV-1 transmission events during a 7-year follow-up period. Initially seronegative partners with either DRB1*0301-DQB1*0201 (relative hazard [RH], 1.60; P=.009) or DRB1*1503-DQB1*0602 (RH, 1.67; P=.03) showed accelerated seroconversion. Carriage of DRB1*1301 in initially seropositive partners led to delayed transmission of HIV to their spouses (RH, 0.54; P=.05). The combined groups of seroprevalent and seroincident partners (n=433) also differed from those who remained seronegative (n=151), with regard to 2 common haplotypes, DRB1*1302-DQB1*0604 (relative odds [RO], 0.28; P=.003) and DRB1*1503-DQB1*0602 (RO, 1.81; P=.02). Statistical adjustments for other host factors (age, sex, genital ulcer, and index partner's virus load) known to influence transmission of HIV-1 seldom altered the genetic relationships. Overall, associations of HLA class II polymorphisms with both HIV transmission and acquisition are not as readily interpretable as are effects reported for other loci.

HLA and cytokine gene polymorphisms are independently associated with responses to hepatitis B vaccination.

Variable immune responses to hepatitis B virus (HBV) infection and recombinant HBV vaccines have been associated with polymorphisms in several genes within the human leukocyte antigen (HLA) complex. Analyses of polymerase chain reaction (PCR)-based genotyping data from 164 North American adolescents vaccinated with recombinant HBV products confirmed that HLA-DRB1*07 (relative odds [RO] = 5.18, P <.0001) and human immunodeficiency virus type 1 (HIV-1) infection (RO = 3.91, P <.001) were both associated with nonresponse to full-dose vaccination. Further associations were observed with single nucleotide polymorphisms (SNPs) at the IL2 and IL4 loci along with insertion/deletion variants at the IL12B locus (P =.003-.01). Host genetic associations were independent of one another as well as other HLA (A, B, C, and DQB1) and cytokine gene (IL4R, IL6, IL10, and TNF) variants. Statistical adjustments for nongenetic factors (gender, ethnicity, age, HIV-1 infection, and vaccination protocols) did not substantially alter the strengths of the genetic relationships. The overall distribution pattern of genetic variations was similar between the analyzed vaccinees and additional adolescents (n = 292) from the same cohort. In conclusion, DRB1*07 (or a closely linked allele) and immunoregulatory cytokine gene polymorphisms correlate with variable immune response to recombinant HBV vaccines.

Cytokine and chemokine gene polymorphisms among ethnically diverse North Americans with HIV-1 infection.

Twenty-four common single nucleotide polymorphisms (SNPs) in 10 cytokine and chemokine genes were defined in 579 North Americans at high risk of HIV-1 infection due to sexual behavior and injection drug use. Among the 3 major ethnic (African-American, Hispanic/Latino, and other) groups involved, HIV-1-seropositive individuals differed significantly from ethnically matched HIV-1-seronegative individuals (odds ratios = 2.13-4.82; P = 0.003-0.05) for several SNPs and haplotypes defined at the IL4, IL4R, IL6, IL10, CCL5 (RANTES), and CXCL12 (SDF1) loci. In addition, the homozygous IL4-590T/T genotype was associated with higher (+87-131 cells/microL) CD4 T-cell counts in HIV-1-infected and AIDS-free adolescents not receiving antiretroviral therapy (adjusted P = 0.004). No SNPs at IFNG, IL2, IL12B, TNF, or CCL2 (MCP1) showed any association with HIV-related outcomes. Additional typing for IL1A, IL1B, IL1R1, IL1RN, and TGFB1 SNPs also failed to demonstrate any influence on HIV-1 infection or virologic/immunologic control in more selected patient groups. Coupled with previous findings, our data suggest that heritable IL4 and IL10 variations may contribute to the acquisition or progression of HIV infection and that the effects of other targeted loci in the cytokine and chemokine system cannot be established unequivocally in the study populations.

Favorable and unfavorable HLA class I alleles and haplotypes in Zambians predominantly infected with clade C human immunodeficiency virus type 1.

The setpoint of viral RNA concentration (viral load [VL]) during chronic human immunodeficiency virus type 1 (HIV-1) infection reflects a virus-host equilibration closely related to CD8(+) cytotoxic T-lymphocyte (CTL) responses, which rely heavily on antigen presentation by the human major histocompatibility complex (MHC) (i.e., HLA) class I molecules. Differences in HIV-1 VL among 259 mostly clade C virus-infected individuals (137 females and 122 males) in the Zambia-UAB HIV Research Project (ZUHRP) were associated with several HLA class I alleles and haplotypes. In particular, general linear model analyses revealed lower log(10) VL among those with HLA allele B*57 (P = 0.002 [without correction]) previously implicated in favorable response and in those with HLA B*39 and A*30-Cw*03 (P = 0.002 to 0.016); the same analyses also demonstrated higher log(10) VL among individuals with A*02-Cw*16, A*23-B*14, and A*23-Cw*07 (P = 0.010 to 0.033). These HLA effects remained strong (P = 0.0002 to 0.075) after adjustment for age, gender, and duration of infection and persisted across three orders of VL categories (P = 0.001 to 0.084). In contrast, neither B*35 (n = 15) nor B*53 (n = 53) showed a clear disadvantage such as that reported elsewhere for these closely related alleles. Other HLA associations with unusually high (A*68, B*41, B*45, and Cw*16) or low (B*13, Cw*12, and Cw*18) VL were either unstable or reflected their tight linkage respecting disequilibria with other class I variants. The three consistently favorable HLA class I variants retained in multivariable models and in alternative analyses were present in 30.9% of subjects with the lowest (<10,000 copies per ml) and 3.1% of those with the highest (>100,000) VL. Clear differential distribution of HLA profiles according to level of viremia suggests important host genetic contribution to the pattern of immune control and escape during HIV-1 infection.