Longitudinal interrelationship between HIV viral suppression, maternal weight change, breastfeeding, and length in HIV-exposed and uninfected infants participating in the Kabeho study in Kigali, Rwanda.

PURPOSE: The health of infants that are HIV-exposed and -uninfected (HEU) is a major public health concern as HIV becomes a chronic condition. We investigate the interrelationship between maternal viral suppression, maternal weight status, breastfeeding, and infants that are HEU. METHODS: The Kabeho study followed 502 HEU infants in Kigali, Rwanda, for 24 months from 2013 to 2014. We use a structural equation modeling approach to investigate the dynamic relationships between viral suppression, maternal weight change, breastfeeding, and infant length-for-age z-score (LAZ) as defined by the WHO. RESULTS: Older mothers are more likely to be virally suppressed and to breastfeed. Viral suppression and the mother being on antiretroviral treatment for longer were related to lower infant LAZ at three months. A more positive maternal weight change was related to higher infant LAZ at the end of each period. At 12 months, a higher infant LAZ was related to increased probability of continued breastfeeding. At 18 months, continued breastfeeding was related to lower LAZ, and food shortages were related to higher LAZ. CONCLUSION: There is a complex interrelationship between viral suppression, maternal weight change, breastfeeding, and infant LAZ. These relationships demonstrate the link between maternal and infant health in the context of HIV.

Predominance of the heterozygous CCR5 delta-24 deletion in African individuals resistant to HIV infection might be related to a defect in CCR5 addressing at the cell surface.

INTRODUCTION: The chemokine receptor CCR5 is the main co-receptor for R5-tropic HIV-1 variants. We have previously described a novel 24-base pair deletion in the coding region of CCR5 among individuals from Rwanda. Here, we investigated the prevalence of hCCR5Δ24 in different cohorts and its impact on CCR5 expression and HIV-1 infection in vitro. METHODS: We screened hCCR5Δ24 in a total of 3232 individuals which were either HIV-1 uninfected, high-risk HIV-1 seronegative and seropositive partners from serodiscordant couples, Long-Term Survivors, or HIV-1 infected volunteers from Africa (Rwanda, Kenya, Guinea-Conakry) and Luxembourg, using a real-time PCR assay. The role of the 24-base pair deletion on CCR5 expression and HIV infection was assessed in cell lines and PBMC using mRNA quantification, confocal analysis, flow and imaging cytometry. RESULTS AND DISCUSSION: Among the 1661 patients from Rwanda, 12 individuals were heterozygous for hCCR5Δ24 but none were homozygous. Although heterozygosity for this allele may not confer complete resistance to HIV-1 infection, the prevalence of the mutation was 2.41% (95%CI: 0.43; 8.37) in 83 Long-Term Survivors (LTS) and 0.99% (95%CI: 0.45; 2.14) in 613 HIV-1 exposed seronegative members as compared with 0.35% (95% Cl: 0.06; 1.25) in 579 HIV-1 seropositive members. The prevalence of hCCR5Δ24 was 0.55% (95%CI: 0.15; 1.69) in 547 infants from Kenya but the mutation was not detected in 224 infants from Guinea-Conakry nor in 800 Caucasian individuals from Luxembourg. Expression of hCCR5Δ24 in cell lines and PBMC showed that the hCCR5Δ24 protein is stably expressed but is not transported to the plasma membrane due to a conformational change. Instead, the mutant receptor was retained intracellularly, colocalized with an endoplasmic reticulum marker and did not mediate HIV-1 infection. Co-transfection of hCCR5Δ24 and wtCCR5 did not indicate a transdominant negative effect of CCR5Δ24 on wtCCR5. CONCLUSIONS: Our findings indicate that hCCR5Δ24 is not expressed at the cell surface. This could explain the higher prevalence of the heterozygous hCCR5Δ24 in LTS and HIV-1 exposed seronegative members from serodiscordant couples. Our data suggest an East-African localization of this deletion, which needs to be confirmed in larger cohorts from African and non-African countries.

Decreased consumption of common weaning foods is associated with poor linear growth among HIV-exposed infants participating in the Kigali antiretroviral and breastfeeding assessment for the elimination of HIV (Kabeho) study.

OBJECTIVE: The World Health Organization recommends that complementary foods that are adequate, safe, and appropriate be introduced to infants at age 6 months. Using an innovative modeling technique, we examine patterns of nutrient intake in HIV-exposed and uninfected (HEU) infants and establish their relationship with growth. METHODS: Single-day dietary recalls and anthropometrics were collected every two to 3 months from 543 infants living in Kigali, Rwanda, and attending clinics for the prevention of mother-to-child HIV transmission. A common weaning food index (CWFI) was calculated in grams and nutrient density for infants to reflect the extent to which the infants consumed the weaning foods typical of this population at ages 6 to 10, 11 to 15, and 16 to 20 months. Regressions among the CWFI, length-for-age z-scores (LAZ), and weight-for-length z-scores (WLZ) were conducted to estimate the relationship between the dietary patterns and growth. RESULTS: Mean absolute intake of zinc and calcium from complementary foods was insufficient. Increasing CWFI was related to increasing cow milk consumption. The density CWFI showed a decrease in the density of iron and folate as infants consume more of the weaning foods typical of this population. Density CWFI, breastfeeding, and caloric intake act on early LAZ and WLZ and interact with one another. Among breastfed infants, those who consume little of the common weaning foods and have a high caloric intake develop deficits in LAZ and have an elevated WLZ. CONCLUSIONS: A diet that is more dominated by the typical weaning foods of this population may support a healthy growth pattern.

Determinants of growth in HIV-exposed and HIV-uninfected infants in the Kabeho Study.

HIV-exposed and HIV-uninfected (HEU) infants may be at increased risk of poor health and growth outcomes. We characterized infant growth trajectories in a cohort of HEU infants to identify factors associated with healthy growth. HIV-positive women participating in prevention of mother-to-child HIV transmission programmes in Kigali, Rwanda, were followed until their infants were 2 years old. Infant anthropometrics were regularly collected. Latent class analysis was used to categorize infant growth trajectories. Multiple logistic regression was used to estimate the odds of infants belonging to each growth trajectory class. On average, this population of HEU infants had moderate linear growth faltering, but only modest faltering in weight, resulting in mean weight-for-length z-score (WLZ) above the World Health Organization (WHO) median. Mean WLZ was 0.53, and mean length-for-age z-score (LAZ) was -1.14 over the first 2 years of life. We identified four unique WLZ trajectories and seven trajectories in LAZ. Low neonatal weight-for-age and a high rate of illness increased the likelihood that infants were in the lightest WLZ class. Shorter mothers were more likely to have infants with linear growth faltering. Female infants who were older at the end of exclusive breastfeeding were more likely to be in the second tallest LAZ class. In conclusion, the current WHO recommendations of Option B+ and extended breastfeeding may induce higher WLZ and lower LAZ early in infancy. However, there is considerable heterogeneity in growth patterns that is obscured by simply analysing average growth trends, necessitating the analysis of growth in subpopulations.

Understanding Antiretroviral Treatment Adherence Among HIV-Positive Women at Four Postpartum Time Intervals: Qualitative Results from the Kabeho Study in Rwanda.

As lifelong antiretroviral therapy (ART) for pregnant women is implemented, it is important to understand the attitudes and norms affecting women's postpartum ART adherence. This is a qualitative cross-sectional study of HIV-positive postpartum women (n = 112) enrolled in a 2-year observational prospective cohort in Rwanda. Informed by the Theory of Reasoned Action (TRA), we conducted in-depth interviews with women whose children were 0-6, 7-12, 13-18, or 21-24 months of age to describe factors contributing to adherence and changes over time. Positive ART attitudes reported by women included mothers' health promotion, prevention of infant HIV infection, higher CD4 count, and improved physical appearance. Negative attitudes were few, but included side effects and the lifelong nature of treatment. Learning from people living with HIV (PLHIV) was identified as a norm facilitating adherence; ART adherence was inhibited by fear of disclosure or stigmatization in communities and clinics. Poor adherence behaviors were common immediately after HIV diagnosis, not necessarily during prevention of mother-to-child transmission (PMTCT). Women with older children, most of whom stopped breastfeeding by 13-18 months, reported more barriers and missed doses than women with younger children. The TRA was useful in identifying the collective influence of attitudes, norms, and intentions on behavior. Findings suggest that HIV-positive women are vulnerable to poor adherence following HIV diagnosis and around the time of breastfeeding cessation. Lifelong treatment adherence can be supported through PLHIV exemplifying long-term ART use, fewer and less stigmatizing clinic visits, and counseling messages highlighting the benefits of drugs on appearance and illness prevention and incorporating biological feedback.

24-month HIV-free survival among infants born to HIV-positive women enrolled in Option B+ program in Kigali, Rwanda: The Kabeho Study.

Lifelong antiretroviral therapy (ART) provision to all pregnant HIV-positive women ("Option B+") has been recommended by the World Health Organization since 2013, but there remain limited data on the effects of Option B+ on long-term HIV-free survival in breastfeeding HIV-exposed infants. The Kigali Antiretroviral and Breastfeeding Assessment for the Elimination of HIV (Kabeho) study enrolled HIV-positive women from the third trimester of pregnancy to 2 weeks postpartum in 14 heath facilities implementing Option B+ in Kigali, Rwanda. Mother-child pairs in the longitudinal observational cohort were followed until 24 months postpartum, with HIV diagnostic testing at 6 weeks, and 9, 18 and 24 months. The Kaplan-Meier method was used to estimate HIV transmission, survival, and HIV-free survival through 24 months. We enrolled 608 HIV-positive women in 2013-2014; birth outcome data were available for 600 women and 597 live-born infants. By 6 weeks, 11 infants had died and 3 infants had confirmed HIV infection (0.5% transmission; 95% confidence interval [CI] 0.2-1.6). At 9 months, there were 9 additional deaths and 2 new infections (cumulative transmission 0.9%, 95% CI 0.4-2.2). At 18 months, there were 6 additional deaths and no new infant infections. At 24 months, there were no additional child deaths and 1 new infection (cumulative 2.2%, 95% CI 0.7-7.0), for an overall 24-month HIV-free survival of 93.2% (95% CI 89.5-95.6). Low transmission rates and high HIV-free survival at 24 months were achieved in breastfeeding infants of HIV-positive mothers receiving universal ART in urban health facilities in Rwanda, though vigilance on maintaining viral suppression for ART-experienced women is needed.

Detectable Viral Load in Late Pregnancy among Women in the Rwanda Option B+ PMTCT Program: Enrollment Results from the Kabeho Study.

There are limited viral load (VL) data available from programs implementing "Option B+," lifelong antiretroviral treatment (ART) to all HIV-positive pregnant and postpartum women, in resource-limited settings. Extent of viral suppression from a prevention of mother-to-child transmission of HIV program in Rwanda was assessed among women enrolled in the Kigali Antiretroviral and Breastfeeding Assessment for the Elimination of HIV (Kabeho) Study. ARV drug resistance testing was conducted on women with VL>2000 copies/ml. In April 2013-January 2014, 608 pregnant or early postpartum HIV-positive women were enrolled in 14 facilities. Factors associated with detectable enrollment VL (>20 copies/ml) were examined using generalized estimating equations. The most common antiretroviral regimen (56.7%, 344/607) was tenofovir/lamivudine/efavirenz. Median ART duration was 13.5 months (IQR 3.0-48.8); 76.1% of women were on ART at first antenatal visit. Half of women (315/603) had undetectable RNA-PCR VL and 84.6% (510) had <1,000 copies/ml. Detectable VL increased among those on ART > 36 months compared to those on ART 4-36 months (72/191, 37.7% versus 56/187, 29.9%), though the difference was not significant. The odds of having detectable enrollment VL decreased significantly as duration on ART at enrollment increased (AOR = 0.99, 95% CI: 0.9857, 0.9998, p = 0.043). There was a higher likelihood of detectable VL for women with lower gravidity (AOR = 0.90, 95% CI: 0.84, 0.97, p = 0.0039), no education (AOR = 2.25, (95% CI: 1.37, 3.70, p = 0.0004), nondisclosure to partner (AOR = 1.97, 95% CI: 1.21, 3.21, p = 0.0063) and side effects (AOR = 2.63, 95% CI: 1.72, 4.03, p<0.0001). ARV drug resistance mutations were detected in all of the eleven women on ART > 36 months with genotyping available. Most women were receiving ART at first antenatal visit, with relatively high viral suppression rates. Shorter ART duration was associated with higher VL, with a concerning increasing trend for higher viremia and drug resistance among women on ART for >3 years.

Unique Insights in the Cervicovaginal Lactobacillus iners and L. crispatus Proteomes and Their Associations with Microbiota Dysbiosis.

BACKGROUND: A Lactobacillus-dominated cervicovaginal microbiota (VMB) protects women from adverse reproductive health outcomes, but the role of L. iners in the VMB is poorly understood. Our aim was to explore the association between the cervicovaginal L. iners and L. crispatus proteomes and VMB composition. METHODS: The vaginal proteomes of 50 Rwandan women at high HIV risk, grouped into four VMB groups (based on 16S rDNA microarray results), were investigated by mass spectrometry using cervicovaginal lavage (CVL) samples. Only samples with positive 16S results for L. iners and/or L. crispatus within each group were included in subsequent comparative protein analyses: Lactobacillus crispatus-dominated VMB cluster (with 16S-proven L. iners (ni) = 0, and with 16S-proven L. crispatus (nc) = 5), L. iners-dominated VMB cluster (ni = 11, nc = 4), moderate dysbiosis (ni = 12, nc = 2); and severe dysbiosis (ni = 8, nc = 2). The relative abundances of proteins that were considered specific for L. iners and L. crispatus were compared among VMB groups. RESULTS: Forty Lactobacillus proteins were identified of which 7 were specific for L. iners and 11 for L. crispatus. The relative abundances of L. iners DNA starvation/stationary phase protection protein (DPS), and the glycolysis enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate isomerase (GPI), were significantly decreased in women with L. iners-containing dysbiosis compared to women with a L. iners-dominated VMB, independent of vaginal pH and L. iners abundance. Furthermore, L. iners DPS, GAPDH, GPI, and fructose-bisphosphate aldolase (ALDO) were significantly negatively associated with vaginal pH. Glycolysis enzymes of L. crispatus showed a similar negative, but nonsignificant, trend related to dysbiosis. CONCLUSIONS: Most identified Lactobacillus proteins had conserved intracellular functions, but their high abundance in CVL supernatant might imply an additional extracellular (moonlighting) role. Our findings suggest that these proteins can be important in maintaining a Lactobacillus-dominated VMB. Functional studies are needed to investigate their roles in vaginal bacterial communities and whether they can be used to prevent vaginal dysbiosis.

A Multi-Country Cross-Sectional Study of Vaginal Carriage of Group B Streptococci (GBS) and Escherichia coli in Resource-Poor Settings: Prevalences and Risk Factors.

BACKGROUND: One million neonates die each year in low- and middle-income countries because of neonatal sepsis; group B Streptococcus (GBS) and Escherichia coli are the leading causes. In sub-Saharan Africa, epidemiological data on vaginal GBS and E. coli carriage, a prerequisite for GBS and E. coli neonatal sepsis, respectively, are scarce but necessary to design and implement prevention strategies. Therefore, we assessed vaginal GBS and E. coli carriage rates and risk factors and the GBS serotype distribution in three sub-Saharan countries. METHODS: A total of 430 women from Kenya, Rwanda and South Africa were studied cross-sectionally. Vaginal carriage of GBS and E. coli, and GBS serotype were assessed using molecular techniques. Risk factors for carriage were identified using multivariable logistic regression analysis. RESULTS: Vaginal carriage rates in reference groups from Kenya and South Africa were 20.2% (95% CI, 13.7-28.7%) and 23.1% (95% CI, 16.2-31.9%), respectively for GBS; and 25.0% (95% CI, 17.8-33.9%) and 27.1% (95% CI, 19.6-36.2%), respectively for E. coli. GBS serotypes Ia (36.8%), V (26.3%) and III (14.0%) were most prevalent. Factors independently associated with GBS and E. coli carriage were Candida albicans, an intermediate vaginal microbiome, bacterial vaginosis, recent vaginal intercourse, vaginal washing, cervical ectopy and working as a sex worker. GBS and E. coli carriage were positively associated. CONCLUSIONS: Reduced vaginal GBS carriage rates might be accomplished by advocating behavioral changes such as abstinence from sexual intercourse and by avoidance of vaginal washing during late pregnancy. It might be advisable to explore the inclusion of vaginal carriage of C. albicans, GBS, E. coli and of the presence of cervical ectopy in a risk- and/or screening-based administration of antibiotic prophylaxis. Current phase II GBS vaccines (a trivalent vaccine targeting serotypes Ia, Ib, and III, and a conjugate vaccine targeting serotype III) would not protect the majority of women against carriage in our study population.

The significance of Lactobacillus crispatus and L. vaginalis for vaginal health and the negative effect of recent sex: a cross-sectional descriptive study across groups of African women.

BACKGROUND: Women in sub-Saharan Africa are vulnerable to acquiring HIV infection and reproductive tract infections. Bacterial vaginosis (BV), a disruption of the vaginal microbiota, has been shown to be strongly associated with HIV infection. Risk factors related to potentially protective or harmful microbiota species are not known. METHODS: We present cross-sectional quantitative polymerase chain reaction data of the Lactobacillus genus, five Lactobacillus species, and three BV-related bacteria (Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia) together with Escherichia coli and Candida albicans in 426 African women across different groups at risk for HIV. We selected a reference group of adult HIV-negative women at average risk for HIV acquisition and compared species variations in subgroups of adolescents, HIV-negative pregnant women, women engaging in traditional vaginal practices, sex workers and a group of HIV-positive women on combination antiretroviral therapy. We explored the associations between presence and quantity of the bacteria with BV by Nugent score, in relation to several factors of known or theoretical importance. RESULTS: The presence of species across Kenyan, South African and Rwandan women was remarkably similar and few differences were seen between the two groups of reference women in Kenya and South Africa. The Rwandan sex workers and HIV-positive women had the highest G. vaginalis presence (p = 0.006). Pregnant women had a higher Lactobacillus genus mean log (7.01 genome equivalents (geq)/ml) compared to the reference women (6.08 geq/ml). L. vaginalis (43%) was second to L. iners (81.9%) highly present in women with a normal Nugent score. Recent sexual exposure negatively affected the presence of L. crispatus (<0.001), L. vaginalis (p = 0.001), and Lactobacillus genus (p < 0.001). Having more than one sexual partner in the last three months was associated with an increased prevalence of G. vaginalis (p = 0.044) and L. iners (p = 0.001). CONCLUSIONS: Although the composition of species across the studied African countries was similar, the presence of protective species i.e. L. crispatus and L. vaginalis in women with a normal Nugent score appeared lower compared to non-African studies. Furthermore, Lactobacillus species were negatively affected by sexual behavioural. Strategies to support protective Lactobacillus species are urgently needed. TRIAL REGISTRATION: The study is registered at the Trial Registration at the National Health Research Ethics Council South Africa with the number DOH2709103223.

The impact of hormonal contraception and pregnancy on sexually transmitted infections and on cervicovaginal microbiota in african sex workers.

BACKGROUND: The observed association between Depo-Provera injectable use and increased HIV acquisition may be caused by hormone-induced increased susceptibility to other sexually transmitted infections (STIs) or changes in the cervicovaginal microbiota (VMB), accompanied by genital immune activation and/or mucosal remodeling. METHODS: Rwandan female sex workers (n = 800) were interviewed about contraceptive use and sexual behavior and were tested for STIs, bacterial vaginosis by Nugent score and pregnancy, at baseline. A subset of 397 HIV-negative, nonpregnant women were interviewed and tested again at regular intervals for 2 years. The VMB of a subset of 174 women was characterized by phylogenetic microarray. Outcomes of STI and VMB were compared between women with hormonal exposures (reporting oral contraceptive or injectable use, or testing positive for pregnancy) and controls (not reporting hormonal contraception and not pregnant). RESULTS: Oral contraceptive use was associated with increased human papillomavirus prevalence (adjusted odds ratio [aOR], 3.10; 1.21-7.94) and Chlamydia trachomatis incidence (aOR, 6.13; 1.58-23.80), injectable use with increased herpes simplex virus-2 prevalence (aOR, 2.13; 1.26-3.59) and pregnancy with lower HIV prevalence (aOR, 0.45; 0.22-0.92) but higher candidiasis incidence (aOR, 2.14; 1.12-4.09). Hormonal status was not associated with Nugent score category or phylogenetic VMB clustering, but oral contraceptive users had lower semiquantitative vaginal abundance of Prevotella, Sneathia/Leptotrichia amnionii, and Mycoplasma species. CONCLUSIONS: Oral contraceptive and injectable use were associated with several STIs but not with VMB composition. The increased herpes simplex virus-2 prevalence among injectable users might explain the potentially higher HIV risk in these women, but more research is needed to confirm these results and elucidate biological mechanisms.

Prevalence and correlates of bacterial vaginosis in different sub-populations of women in sub-Saharan Africa: a cross-sectional study.

BACKGROUND: Clinical development of vaginally applied products aimed at reducing the transmission of HIV and other sexually transmitted infections, has highlighted the need for a better characterisation of the vaginal environment. We set out to characterise the vaginal environment in women in different settings in sub-Saharan Africa. METHODS: A longitudinal study was conducted in Kenya, Rwanda and South-Africa. Women were recruited into pre-defined study groups including adult, non-pregnant, HIV-negative women; pregnant women; adolescent girls; HIV-negative women engaging in vaginal practices; female sex workers; and HIV-positive women. Consenting women were interviewed and underwent a pelvic exam. Samples of vaginal fluid and a blood sample were taken and tested for bacterial vaginosis (BV), HIV and other reproductive tract infections (RTIs). This paper presents the cross-sectional analyses of BV Nugent scores and RTI prevalence and correlates at the screening and the enrolment visit. RESULTS: At the screening visit 38% of women had BV defined as a Nugent score of 7-10, and 64% had more than one RTI (N. gonorrhoea, C. trachomatis, T. vaginalis, syphilis) and/or Candida. At screening the likelihood of BV was lower in women using progestin-only contraception and higher in women with more than one RTI. At enrolment, BV scores were significantly associated with the presence of prostate specific antigen (PSA) in the vaginal fluid and with being a self-acknowledged sex worker. Further, sex workers were more likely to have incident BV by Nugent score at enrolment. CONCLUSIONS: Our study confirmed some of the correlates of BV that have been previously reported but the most salient finding was the association between BV and the presence of PSA in the vaginal fluid which is suggestive of recent unprotected sexual intercourse.

Lactobacillus-dominated cervicovaginal microbiota associated with reduced HIV/STI prevalence and genital HIV viral load in African women.

Cervicovaginal microbiota not dominated by lactobacilli may facilitate transmission of HIV and other sexually transmitted infections (STIs), as well as miscarriages, preterm births and sepsis in pregnant women. However, little is known about the exact nature of the microbiological changes that cause these adverse outcomes. In this study, cervical samples of 174 Rwandan female sex workers were analyzed cross-sectionally using a phylogenetic microarray. Furthermore, HIV-1 RNA concentrations were measured in cervicovaginal lavages of 58 HIV-positive women among them. We identified six microbiome clusters, representing a gradient from low semi-quantitative abundance and diversity dominated by Lactobacillus crispatus (cluster R-I, with R denoting 'Rwanda') and L. iners (R-II) to intermediate (R-V) and high abundance and diversity (R-III, R-IV and R-VI) dominated by a mixture of anaerobes, including Gardnerella, Atopobium and Prevotella species. Women in cluster R-I were less likely to have HIV (P=0.03), herpes simplex virus type 2 (HSV-2; P<0.01), and high-risk human papillomavirus (HPV; P<0.01) and had no bacterial STIs (P=0.15). Statistically significant trends in prevalence of viral STIs were found from low prevalence in cluster R-I, to higher prevalence in clusters R-II and R-V, and highest prevalence in clusters R-III/R-IV/R-VI. Furthermore, only 10% of HIV-positive women in clusters R-I/R-II, compared with 40% in cluster R-V, and 42% in clusters R-III/R-IV/R-VI had detectable cervicovaginal HIV-1 RNA (Ptrend=0.03). We conclude that L. crispatus-dominated, and to a lesser extent L. iners-dominated, cervicovaginal microbiota are associated with a lower prevalence of HIV/STIs and a lower likelihood of genital HIV-1 RNA shedding.