Cervical Dysplasia, Infection, and Phylogeny of Human Papillomavirus in HIV-Infected and HIV-Uninfected Women at a Reproductive Health Clinic in Nairobi, Kenya.

High risk human Papillomavirus (HPV) infections ultimately cause cervical cancer. Human Immunodeficiency Virus (HIV) infected women often present with multiple high-risk HPV infections and are thus at a higher risk of developing cervical cancer. However, information on the circulating high-risk HPV genotypes in Kenya in both HIV-infected and HIV-uninfected women is still scanty. This study is aimed at determining the phylogeny and the HPV genotypes in women with respect to their HIV status and at correlating this with cytology results. This study was carried out among women attending the Reproductive Health Clinic at Kenyatta National Hospital, a referral hospital in Nairobi, Kenya. A cross-sectional study recruited a total of 217 women aged 18 to 50 years. Paired blood and cervical samples were obtained from consenting participants. Blood was used for serological HIV screening while cervical smears were used for cytology followed by HPV DNA extraction, HPV DNA PCR amplification, and phylogenetic analysis. Out of 217 participants, 29 (13.4%) were HIV seropositive, while 68 (31.3%) were positive for HPV DNA. Eight (3.7%) of the participants had abnormal cervical cytology. High-risk HPV 16 was the most prevalent followed by HPV 81, 73, 35, and 52. One participant had cervical cancer, was HIV infected, and had multiple high-risk infections with HPV 26, 35, and 58. HPV 16, 6, and 81 had two variants each. HPV 16 in this study clustered with HPV from Iran and Africa. This study shows the circulation of other HPV 35, 52, 73, 81, 31, 51, 45, 58, and 26 in the Kenyan population that play important roles in cancer etiology but are not included in the HPV vaccine. Data from this study could inform vaccination strategies. Additionally, this data will be useful in future epidemiological studies of HPV in Nairobi as the introduction or development of new variants can be detected.

Partial HIV C2V3 envelope sequence analysis reveals association of coreceptor tropism, envelope glycosylation and viral genotypic variability among Kenyan patients on HAART.

BACKGROUND: HIV-1 is highly variable genetically and at protein level, a property it uses to subvert antiviral immunity and treatment. The aim of this study was to assess if HIV subtype differences were associated with variations in glycosylation patterns and co-receptor tropism among HAART patients experiencing different virologic treatment outcomes. METHODS: A total of 118 HIV env C2V3 sequence isolates generated previously from 59 Kenyan patients receiving highly active antiretroviral therapy (HAART) were examined for tropism and glycosylation patterns. For analysis of Potential N-linked glycosylation sites (PNGs), amino acid sequences generated by the NCBI's Translate tool were applied to the HIVAlign and the N-glycosite tool within the Los Alamos Database. Viral tropism was assessed using Geno2Pheno (G2P), WebPSSM and Phenoseq platforms as well as using Raymond's and Esbjörnsson's rules. Chi square test was used to determine independent variables association and ANOVA applied on scale variables. RESULTS: At respective False Positive Rate (FPR) cut-offs of 5% (p = 0.045), 10% (p = 0.016) and 20% (p = 0.005) for CXCR4 usage within the Geno2Pheno platform, HIV-1 subtype and viral tropism were significantly associated in a chi square test. Raymond's rule (p = 0.024) and WebPSSM (p = 0.05), but not Phenoseq or Esbjörnsson showed significant associations between subtype and tropism. Relative to other platforms used, Raymond's and Esbjörnsson's rules showed higher proportions of X4 variants, while WebPSSM resulted in lower proportions of X4 variants across subtypes. The mean glycosylation density differed significantly between subtypes at positions, N277 (p = 0.034), N296 (p = 0.036), N302 (p = 0.034) and N366 (p = 0.004), with HIV-1D most heavily glycosylated of the subtypes. R5 isolates had fewer PNGs than X4 isolates, but these differences were not significant except at position N262 (p = 0.040). Cell-associated isolates from virologic treatment success subjects were more glycosylated than cell-free isolates from virologic treatment failures both for the NXT (p = 0.016), and for all the patterns (p = 0.011). CONCLUSION: These data reveal significant associations of HIV-1 subtype diversity, viral co-receptor tropism, viral suppression and envelope glycosylation. These associations have important implications for designing therapy and vaccines against HIV. Heavy glycosylation and preference for CXCR4 usage of HIV-1D may explain rapid disease progression in patients infected with these strains.

Plasma nevirapine concentrations predict virological and adherence failure in Kenyan HIV-1 infected patients with extensive antiretroviral treatment exposure.

Treatment failure is a key challenge in the management of HIV-1 infection. We conducted a mixed-model survey of plasma nevirapine (NVP) concentrations (cNVP) and viral load in order to examine associations with treatment and adherence outcomes among Kenyan patients on prolonged antiretroviral therapy (ART). Blood plasma was collected at 1, 4 and 24 hours post-ART dosing from 58 subjects receiving NVP-containing ART and used to determine cNVP and viral load (VL). Median duration of treatment was 42 (range, 12-156) months, and 25 (43.1%) of the patients had virologic failure (VF). cNVP was significantly lower for VF than non- VF at 1hr (mean, 2,111ng/ml vs. 3,432ng/ml, p = 0.003) and at 4hr (mean 1,625ng/ml vs. 3,999ng/ml, p = 0.001) but not at 24hr post-ART dosing. Up to 53.4%, 24.1% and 22.4% of the subjects had good, fair and poor adherence respectively. cNVP levels peaked and were > = 3µg.ml at 4 hours in a majority of patients with good adherence and those without VF. Using a threshold of 3µg/ml for optimal therapeutic nevirapine level, 74% (43/58), 65.5% (38/58) and 86% (50/58) of all patients had sub-therapeutic cNVP at 1, 4 and 24 hours respectively. cNVP at 4 hours was associated with adherence (p = 0.05) and virologic VF (p = 0.002) in a chi-square test. These mean cNVP levels differed significantly in non-parametric tests between adherence categories at 1hr (p = 0.005) and 4hrs (p = 0.01) and between ART regimen categories at 1hr (p = 0.004) and 4hrs (p<0.0001). Moreover, cNVP levels correlated inversely with VL (p< = 0.006) and positively with adherence behavior. In multivariate tests, increased early peak NVP (cNVP4) was independently predictive of lower VL (p = 0.002), while delayed high NVP peak (cNVP24) was consistent with increased VL (p = 0.033). These data strongly assert the need to integrate plasma concentrations of NVP and that of other ART drugs into routine ART management of HIV-1 patients.

Co-Infection Burden of Hepatitis C Virus and Human Immunodeficiency Virus among Injecting Heroin Users at the Kenyan Coast.

BACKGROUND: Injection drug use is steadily rising in Kenya. We assessed the prevalence of both human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections among injecting heroin users (IHUs) at the Kenyan Coast. METHODS: A total of 186 IHUs (mean age, 33 years) from the Omari rehabilitation center program in Malindi were consented and screened for HIV-1 and HCV by serology and PCR and their CD4 T-cells enumerated by FACS. RESULTS: Prevalence of HIV-1 was 87.5%, that of HCV was 16.4%, co-infection was 17.9% and 18/152 (11.8%) were uninfected. Only 5.26% of the HIV-1 negative injectors were HCV positive. Co-infection was higher among injectors aged 30 to 40 years (20.7%) and among males (22.1%) than comparable groups. About 35% of the injectors were receiving antiretroviral treatment (ART). Co-infection was highest among injectors receiving D4T (75%) compared to those receiving AZT (21.6%) or TDF (10.5%) or those not on ART (10.5%). Mean CD4 T-cells were 404 (95% CI, 365 - 443) cells/mm3 overall, significantly lower for co-infected (mean, 146; 95% CI 114 - 179 cells/mm3) than HIV mono infected (mean, 437, 95% CI 386 - 487 cells/mm3, p<0.001) or uninfected (mean, 618, 95% CI 549 - 687 cells/mm3, p<0.001) injectors and lower for HIV mono-infected than uninfected injectors (p=0.002). By treatment arm, CD4 T-cells were lower for injectors receiving D4T (mean, 78; 95% CI, 0.4 - 156 cells/mm3) than TDF (mean 607, 95% CI, 196 - 1018 cells/mm3, p=0.005) or AZT (mean 474, 95% CI -377 - 571 cells/mm3, p=0.004). CONCLUSION: Mono and dual infections with HIV-1 and HCV is high among IHUs in Malindi, but ART coverage is low. The co-infected IHUs have elevated risk of immunodeficiency due to significantly depressed CD4 T-cell numbers. Coinfection screening, treatment-as-prevention for both HIV and HCV and harm reduction should be scaled up to alleviate infection burden.

Implementation and Operational Research: Correlates of Adherence and Treatment Failure Among Kenyan Patients on Long-term Highly Active Antiretroviral Therapy.

BACKGROUND: Universal access to highly active antiretroviral therapy (HAART) is still elusive in most developing nations. We asked whether peer support influenced adherence and treatment outcome and if a single viral load (VL) could define treatment failure in a resource-limited setting. METHODS: A multicenter longitudinal and cross-sectional survey of VL, CD4 T cells, and adherence in 546 patients receiving HAART for up to 228 months. VL and CD4 counts were determined using m2000 Abbott RealTime HIV-1 assay and FACS counters, respectively. Adherence was assessed based on pill count and on self-report. RESULTS: Of the patients, 55.8%, 22.2%, and 22% had good, fair, and poor adherence, respectively. Adherence, peer support, and regimen, but not HIV disclosure, age, or gender, independently correlated with VL and durability of treatment in a multivariate analysis (P < 0.001). Treatment failure was 35.9% using sequential VL but ranged between 27% and 35% using alternate single VL cross-sectional definitions. More patients failed stavudine (41.2%) than zidovudine (37.4%) or tenofovir (28.8%, P = 0.043) treatment arms. Peer support correlated positively with adherence (χ(2), P < 0.001), with nonadherence being highest in the stavudine arm. VL before the time of regimen switch was comparable between patients switching and not switching treatment. Moreover, 36% of those switching still failed the second-line regimen. CONCLUSION: Weak adherence support and inaccessible VL testing threaten to compromise the success of HAART scale-up in Kenya. To hasten antiretroviral therapy monitoring and decision making, we suggest strengthening patient-focused adherence programs, optimizing and aligning regimen to WHO standards, and a single point-of-care VL testing when multiple tests are unavailable.

Genotypic Variability of HIV-1 Reverse Transcriptase Gene from Long-Term Antiretroviral-Experienced Patients in Kenya.

There is continuous need to track genetic profiles of HIV strains circulating in different geographic settings to hasten vaccine discovery and inform public health and intervention policies. We partially sequenced the reverse transcriptase region of the HIV-1 pol gene from a total of 54 Kenyan patients aged 18-56 years who continued highly active antiretroviral treatment (HAART) for between 8 and 102 months. Subtyping was done using both the JPHMM tool and phylogenetic method. HIV-1 subtype A1 was the predominant strain in circulation, representing 57.4% and 70.4% of all isolates as determined by JPHMM and phylogenetic methods, respectively. Subtypes D (14.8%, 7.4%), C (5.6%, 9.3%), and A2 (0%, 5.6%) were determined at respective prevalence by both methods. JPHMM identified 22.2% of the isolates as recombinants. This surveillance focused on the RT gene and reaffirms the predominance of subtype A and an increasing proportion of recombinant strains in the Kenyan epidemic.

HIV-1 subtype diversity based on envelope C2V3 sequences from Kenyan patients on highly active antiretroviral therapy.

There is a continuous need to genetically characterize the HIV strains in circulation in order to assess interventions and inform vaccine discovery. We partially sequenced the envelope C2V3 gene from a total of 59 Kenyan patients on highly active antiretroviral treatment (HAART) and determined HIV subtypes using both the JPHMM subtyping tool and the phylogenetic method. HIV-1 subtype A1 was the predominant strain in circulation, representing 65.5% and 74.5% of all isolates as determined by JPHMM and phylogenetic methods, respectively. Subtypes C and D were the next most prevalent pure strains at 9.1% each by both methods. JPHMM identified 9.1% of the isolates as recombinant. Four isolates had short sequences not covering the entire C2V3 region and were thus not subtyped. From this study, subtype A viruses are still the predominant HIV-1 strains in local circulation in Kenya. Constant surveillance is needed to update molecular trends under continuing HAART scale-up.

Efficient monitoring of HIV-1 vertically infected children in Kenya on first-line antiretroviral therapy.

BACKGROUND: Worldwide access to antiretroviral therapy (ART) in low- and middle-income countries has significantly increased. Although this presents better treatment options for HIV-infected individuals, the challenge of monitoring ART in these settings still remains. OBJECTIVE: To investigate efficient and cost-effective criteria for assessing ART failure among HIV-1-infected children on first-line ART in resource-limited settings. STUDY DESIGN: Retrospective analysis of 75 HIV-1 vertically infected Kenyan children with a follow-up period of 24 months after initiating ART. Plasma viral load, peripheral CD4(+)T-cell counts and HIV-1 drug-resistance mutations were monitored biannually. RESULTS: Plasma viral load (VL) was suppressed to undetectable level or more than 1.5 log(10) from baseline levels in 53 (70.7%) children within 24 months. VL in the remaining 22 (29.3%) children was not suppressed significantly. Of the 22 children, 21 were infected with HIV-1 strains that developed drug-resistance mutations; 9 within 12 months and 12 between 12 and 24 months. Among the 53 who were successfully treated, VL was suppressed in 33 within 12 months and in 20 between 12 and 24 months. There was no significant difference in VL at baseline and the change of CD4(+)T-cell counts after initiating ART between those treated successfully and the failure groups. CONCLUSION: After initiating ART, children may require longer times to achieve complete viral suppression. Plasma viral load testing 24 months after initiating ART could be used to differentiate ART failures among HIV-1 vertically infected children in resource-limited settings. Additionally, drug resistance testing, if affordable, would be helpful in identifying those failing therapy and in choosing second-line regimens.

Susceptibility to Simian immunodeficiency virus ex vivo predicts outcome of a prime-boost vaccine after SIVmac239 challenge.

BACKGROUND: Efficacy assessment of AIDS vaccines relies both on preclinically challenging immunized monkeys with simian immunodeficiency virus (SIV) or monitoring infection rates in large human trials. Although conventional parameters of vaccine-induced immune responses do not completely predict outcome, existing methods for testing cellular immunity are sophisticated and difficult to establish in resource-limited settings. METHODS: We have used virus replication kinetics (VVR) on ConA-stimulated peripheral blood mononuclear cells from rhesus monkeys immunized with DNA replication-defective adenovirus vector expressing various SIV genes, as an ex vivo model, to mimic the effects of different immune effector functions on viral infection. RESULTS: VVR was attenuated by the immunization and correlated 2 weeks after first boost, with the number of interferon gamma-secreting cells and T-cell noncytotoxic antiviral responses. Importantly, VVR on the day of challenge but not interferon gamma responses correlated with viremia and with memory CD4+ T-cell measurements after SIVmac239 challenge. Similarly, T-cell noncytotoxic antiviral responses on the day of challenge correlated directly with memory CD4 T cell and inversely with plasma viremia after challenge. CONCLUSIONS: VVR thus served as a better predictor of protective capacity of the vaccine regimen in these monkeys. We suggest that VVR be considered in the evaluation of candidate AIDS vaccines in humans.

The changing trend of HIV type 1 subtypes in Nairobi.

Monitoring the distribution of HIV-1 subtypes and recombinants among infected individuals has become a priority in HIV therapy. A laboratory analysis of samples collected from HIV-positive patients attending an STI clinic in Nairobi was done between March and May 2004. PCR was carried out on pol (intergrase) and env (C2V3) regions and resulting data on the 54 samples successfully analyzed revealed the following as circulating subtypes: 35/54(65%) were A1/A1, 5/54(9%) were A/C, 4/54 (7%) were A1/D, 1/54 (2%) was C/D, 1/54 (2%) was D/D, 1/54 (2%) was A1/A2, 1/54 (2%)was G/G, 1/54 (2%) was A2/D, 1/54 (2%) was C/C, and 4/54 (7%) were CRF02_ AG. The results show an increase in HIV-1 recombinants with the emergence of A1/A2 and an increase in CRF02_AG recombinants. Subtype diversity in the advent of ARV use will impact negatively on treatment outcomes. As such, increased viral evolution and recombination will call for continuous evaluation of available anti-HIV regimens for better management of those infected with HIV-1.

Changes in the HIV type 1 envelope gene from non-subtype B HIV type 1-infected children in Kenya.

A switch of coreceptor usage from CCR5 to CXCR4 occurs in about half of HIV-1-infected individuals in the natural course of infection. To investigate whether antiretroviral therapy (ART) enhances the coreceptor switch of HIV-1, we genotypically analyzed the env-V3 amino acid sequences from 81 HIV-1-infected children in Kenya whose plasma samples were obtained between 2000 and 2007. Of 41 children on ART, 35 had HIV-1 using CCR5 as a coreceptor at baseline. In 7 (20%) of them HIV-1 switched the coreceptor usage during the follow-up period. The mean duration of ART to the time of coreceptor switch was 2.6 years (range: 0.5-5.2). Of the remaining 40 children without ART, 32 had HIV-1 using CCR5 as a coreceptor at baseline and in 3 (9.4%) HIV-1 switched the coreceptor usage. The mean age of the children with HIV-1 coreceptor switch with and without ART was 7.3 and 9.7 years, respectively. The difference in the rate and age of coreceptor switch between treated and untreated children was not significant (p = 0.38 and 0.31, respectively). Of the HIV-1-infected children, 10 started ART by the age of 5 years (rapid progressors) and 23 did not need ART by the age of 10 years (slow progressors). The rate of coreceptor switch was strongly higher in rapid progressors (40%) than slow progressors (8.7%) (p = 0.053). These results suggest that switching of coreceptor usage from CCR5 to CXCR4 among HIV-1-infected children is not influenced by ART, but by factors responsible for rapid disease progression.

Mucosal prior to systemic application of recombinant adenovirus boosting is more immunogenic than systemic application twice but confers similar protection against SIV-challenge in DNA vaccine-primed macaques.

We investigated the immunogenicity and efficacy of a bimodal prime/boost vaccine regimen given by various routes in the Simian immunodeficiency virus (SIV) rhesus monkey model for AIDS. Twelve animals were immunized with SIV DNA-vectors followed by the application of a recombinant adenovirus (rAd5) expressing the same genes either intramuscularly (i.m.) or by oropharyngeal spray. The second rAd5-application was given i.m. All vaccinees plus six controls were challenged orally with SIVmac239 12 weeks post-final immunization. Both immunization strategies induced strong SIV Gag-specific IFN-gamma and T-cell proliferation responses and mediated a conservation of CD4(+) memory T-cells and a reduction of viral load during peak viremia following infection. Interestingly, the mucosal group was superior to the systemic group regarding breadth and strength of SIV-specific T-cell responses and exhibited lower vector specific immune responses. Therefore, our data warrant the inclusion of mucosal vector application in a vaccination regimen which makes it less invasive and easier to apply.

Anti-retroviral drug resistance-associated mutations among non-subtype B HIV-1-infected Kenyan children with treatment failure.

Recently increased availability of anti-retroviral therapy (ART) has mitigated HIV-1/AIDS prognoses especially in resource poor settings. The emergence of ART resistance-associated mutations from non-suppressive ART has been implicated as a major cause of ART failure. Reverse transcriptase inhibitor (RTI)-resistance mutations among 12 non-subtype B HIV-1-infected children with treatment failure were evaluated by genotypically analyzing HIV-1 strains isolated from plasma obtained between 2001 and 2004. A region of pol-RT gene was amplified and at least five clones per sample were analyzed. Phylogenetic analysis revealed HIV-1 subtype A1 (n = 7), subtype C (n = 1), subtype D (n = 3), and CRF02_AG (n = 1). Before treatment, 4 of 12 (33.3%) children had primary RTI-resistance mutations, K103N (n = 3, ages 5-7 years) and Y181C (n = 1, age 1 year). In one child, K103N was found as a minor population (1/5 clones) before treatment and became major (7/7 clones) 8 months after RTI treatment. In 7 of 12 children, M184V appeared with one thymidine-analogue-associated mutation (TAM) as the first mutation, while the remaining 5 children had only TAMs appearing either individually (n = 2), or as TAMs 1 (M41L, L210W, and T215Y) and 2 (D67N, K70R, and K219Q/E/R) appearing together (n = 3). These results suggest that "vertically transmitted" primary RTI-resistance mutations, K103N and Y181C, can persist over the years even in the absence of drug pressure and impact RTI treatment negatively, and that appearing patterns of RTI-resistance mutations among non-subtype B HIV-1-infected children could possibly be different from those reported in subtype B-infected children.

Viral load, CD4+ T-lymphocyte counts and antibody titres in HIV-1 infected untreated children in Kenya; implication for immunodeficiency and AIDS progression.

BACKGROUND: There are limited reports on HIV-1 RNA load, CD4+ T-lymphocytes and antibody responses in relation to disease progression in HIV-1 infected untreated children in Africa. METHODS: To describe the relationships between these parameters, we conducted a longitudinal cohort study involving 51 perinatally HIV-1 infected children aged between 1 and 13 years. HIV status was determined by ELISA and confirmed by western blot and PCR. Antibodies were quantified by limiting dilution ELISA, plasma HIV-1 RNA load by RT-PCR and CD4+ T-lymphocytes by FACSCount. RESULTS: Asymptomatic and symptomatic disease had, respectively, a rise in median HIV-1 RNA load from 1,195 to 132,543 and from 42,962 to 1,109,281 copies/ml in children below 6 years. The increase in viral load was 10-fold higher for asymptomatic compared to other categories and 2-fold faster for children less than 6 years than those above. Similarly, symptomatic children below 6 years had initial median CD4+ T-lymphocyte counts of 647 (22%) cells/muL, declining to 378 (20%) while those above 6 years had initial values of below 335 (15%) but which increased to 428 (17%). Median viral load correlated significantly with median CD4+ T-lymphocyte percentage in children above 6 years (p=0.026) but not below. CONCLUSIONS: Viral load is lower in older than younger children and correlates significantly with percentage CD4+ T-lymphocytes. Survival by HIV-1 infected children requires a competent immune response early in infection to counter the rapidly replicating virus. Interventions aimed at boosting the naïve immune system may prolong survival in these children.