An in vitro evaluation of drugs used in the Kenyan ART program

The majority of anti-HIV drug susceptibility tests have been performed on subtype B HIV-1 strains, since these are the most prevalent in countries designing, testing, and manufacturing the current anti-HIV agents. The increasing global spread of HIV subtype highlights the need to determine the activity of anti-HIV drugs against subtypes of HIV other than subtype B. Furthermore an increasing number of individuals infected with many of the non subtype B virus strains now receive antiretroviral therapy because of rollout programs in developing countries as well as increasing migration to the developed world. The phenotypic susceptibility of two laboratory strains HIV-1JFRL and HIV-1IIIB (representing subtype B) and two clinical isolates HIV-104RTA and HIV-1025RTA (representing subtypes A and D respectively) was determined. The in vitro drug susceptibility testing of the isolates was carried out in C8166 cell line and in peripheral blood mononuclear cells (PBMCs). The study revealed that the drugs used in the Kenyan national ART program inhibited HIV-1 replication in-vitro as their inhibitory concentrations (IC50) compared well with the standard Inhibitory concentration values. The results also suggest a biochemical similarity of the reverse transcriptase (RT) and protease enzymes from these subtypes despite the divergence at the genetic level. The findings suggest that similar clinical benefits of antiviral therapy obtain in persons infected with other subtypes of HIV-1other than subtype B and that the generic drugs used in the national ART program in Kenya are as efficacious as branded drugs in inhibiting HIV replication in vitro despite the limited number of the viruses studied.Pan African Medical Journal 2016; 2

Prevalence and Associated Risk Factors of Hepatitis B Virus Infections Among HIV-1 Infected Patients Attending the Comprehensive Care Clinic in Malindi Sub-County Hospital in Kenya

Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV) co-infections are common all over the world. Infection with HIV increases rates of HBV chronicity, prolong the time the HBV stays in circulation and increase liver-related morbidity. Factors such as intravenous drug use, multiple blood transfusions, presence of tattoos, unsafe sexual practices and being health workers have been implicated as drivers of infection & transmission of HBV & HIV. This study aimed to determine the prevalence and genotypes of HBV associated risk factors among HIV infected patients in a descriptive cross-sectional study. Malindi was chosen as a suitable study site because of the high numbers of residents involved in sex tourism as well as intravenous drug use. A structured questionnaire was used to capture social demographic data such as age, gender, employment status, occupation, the level of education and marital status, clinical history information such as duration since diagnosis with HIV, ART drug history, duration taking ARVs and baseline CD4 count and risk factors associated with HBV infections such as intravenous drug use, history of blood transfusion, tattooing/scarification, and the sexual history from 446 consenting randomly selected HIV infected participants. Five millilitres of whole blood was obtained from each participant, 50µl of which was used for CD4 cell counts using a flow cytometer. HBsAg serology was done using Diaspot® rapid diagnostic test and confirmed by Hepanostika® HBsAg Ultra ELISA kit (BioMérieux SA) and HBV DNA was extracted from all HBsAg positive samples. Nested polymerise chain (PCR) reaction and sequencing of the Pre S1 region was done. Sample sequences were compared with published HBV genotypes sequences from GenBank and Phylogenetic trees were constructed using the NJ Plot software using a PHB file created through DNA Database of Japan (DDBJ) to determine the HBV genotypes. Out of the 446 HIV positive participants, 126 (28.3%) were males and 320 (71.7%) females. Only 19/446 (4.26%) participants were positive for HBV based on rapid strip test while 22/446 (4.93%) participants had HBV based on ELISA. Twelve of the 22 ELISA positive samples were successfully amplified by PCR. Out of the 12 PCR positive samples 10 were successfully sequenced. Phylogenetic analysis revealed that 9/10 (90%) samples belonged to genotype A while 1/10 (10%) belonged to genotype E. Males (p=0.028) and intravenous drug use (p= 0.08) were significantly associated HBV infections. The high prevalence (4.9%) of HBV among HIV patients attending Malindi Sub-county hospital is most likely highly driven by intravenous drug use and multiple sexual partners among the male gender and is predominantly genotypes A and E which is similar to the general population. Keywords: Hepatitis B virus, HIV, Co-infection, HBsAg, genotypes, intravenous drug us

Mutations in the “a” Determinant Region of Hepatitis B Virus Genotype A among Voluntary Kenyan Blood Donors

Background: Occurrence of mutations within the major antigenic alpha determinant region of hepatitis B surface antigen (HBsAg can alter HBV antigenicity resulting in   failures in diagnosis, vaccine and hepatitis B immunoglobulin therapy. Objective: This study aimed at detection of mutations in the “a” determinant region of HBV surface antigen among voluntary blood donors in Kenya. Design: A cross sectional study involving serology and molecular techniques Settings: This study involved analysis of samples from blood transfusion centers Subjects: A total of 301 blood samples from donor blood were collected for the study. Methods: Sero-status for HBsAg was determined using Enzyme-Linked Immunosorbent Assay (ELISA). A fragment of the S gene including the "a" determinant was amplified by PCR from the HBsAg positive samples and sequenced for mutation analysis. Mutations and phylogenetic analyses were performed using Mega 6 software, Bioedit software and GENETYX® software version 9.1.0. Results: Out of the 301 samples tested 69/301 (22.9%) were Polymerase Chain Reaction (PCR) positive including 2/69(2.9%) were sero-negative for HBsAg. All isolates were genotype A, sub-genotype A1. A total of 29 mutations were observed of which 37.9% were located within the “a” determinant. Mutations T143M and K122R were the most frequent in this study. Escape mutations associated with diagnostic failure, vaccine and immunoglobulin therapy escape were also identified. Conclusions: These findings are important for policies related to vaccine implementation and therapeutic and diagnostic guidelines. Keywords: Escape mutants, genotype, hepatitis B virus, antigenic determinant, surface antigen

Human Immunodeficiency Virus -1 and Hepatitis B Virus Co-Infections among Injecting Drug Users in Malindi, Kenya

Currently no published data addressing the burden of Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV) co-infection among injecting drug users (IDUs) in Kenya exists. These two viruses share similar routes of transmission, with illicit drug use by injection being the major route of infection. Injecting drug use is a rapidly growing problem in coastal towns of Kenya and the problem is aggravated by sex tourism.This study aimed at determining the prevalence of HBV in HIV positive IDUs and correlating the findings with socio-demographic factors of the study population.A cross-sectional study was conducted using structured questionnaires and laboratory testing of blood samples. Surface antigens for HBV (HBsAg) and anti-HIV antibodies were screened using rapid kits followed by Enzyme Linked Immunosorbent assay tests on positive samples using Hepanostika and Vironostika test kits, for HIV and HBV, respectively. The CD4+ T-cell count was determined by flow cytometry.The prevalence of HIV/HBV co-infection was 14.3% (13/91) with a mean age of 33.2 (SD ± 8.1) years. The mean CD4+ cell count in the HIV/HBV co-infected individuals was significantly lower than HIV mono-infection. Needle sharing and duration of active injection of drugs were significantly associated with HIV/HBV co-infections.This study concludes a potentially high prevalence of HBV/ HIV co-infection in injecting drug users in Malindi, Kenya. With limited evidence on IDU prevalence and its consequences in sub-Saharan Africa, the results of this study highlight the need for a more refined policy on HIV treatment strategy among IDUs. There is a further need for triple testing for HIV, HBV and HCV among suspected IDUs and other associated risk groups like the commercial sex workers before commencement of treatment. Keywords: Injecting drug users, HIV-1, HBV, viral co-infection, Malindi, Keny

Mutations in the “a” Determinant Region of Hepatitis B Virus Genotype A among Voluntary Kenyan Blood Donors

Occurrence of mutations within the major antigenic alpha determinant region of hepatitis B surface antigen (HBsAg can alter HBV antigenicity resulting in   failures in diagnosis, vaccine and hepatitis B immunoglobulin therapy. This study aimed at detection of mutations in the “a” determinant region of HBV surface antigen among voluntary blood donors in Kenya. This was a cross sectional study involving serology and molecular techniques. This study involved analysis of samples from blood transfusion centers. A total of 301 blood samples from donor blood were collected for the study.  Sero-status for HBsAg was determined using Enzyme-Linked Immunosorbent Assay (ELISA). A fragment of the S gene including the "a" determinant was amplified by PCR from the HBsAg positive samples and sequenced for mutation analysis. Mutations and phylogenetic analyses were performed using Mega 6 software, Bioedit software and GENETYX® software version 9.1.0. Out of the 301 samples tested 69/301 (22.9%) were Polymerase Chain Reaction (PCR) positive including 2/69(2.9%) were sero-negative for HBsAg. All isolates were genotype A, sub-genotype A1. A total of 29 mutations were observed of which 37.9% were located within the “a” determinant. Mutations T143M and K122R were the most frequent in this study. Escape mutations associated with diagnostic failure, vaccine and immunoglobulin therapy escape were also identified. These findings are important for policies related to vaccine implementation and therapeutic and diagnostic guidelines. Keywords: Escape mutants, genotype, hepatitis B virus, antigenic determinant, surface antige

Detection and typing of Human Papillomavirus in urine from patients attending a sexually transmitted infections clinic in Nairobi County, Kenya

Human papillomavirus (HPV) is a common sexually transmitted infection (STI) that has been etiologically linked to cervical cancer. Different types of samples can be used for cervical screening, including Pap test or biopsy and Liquid Based Cytology, visual inspection using acetic acid or Lugol’s iodine, and HPV testing. These methods are invasive. The use of urine as an alternative specimen may be more widely accepted since it is non-invasive and the sample is readily available. The study aimed at detecting and genotyping HPV in urine from patients attending a sexually transmitted infections clinic in Nairobi County. It also aimed at assessing the factors associated with HPV infection. In this cross-sectional study, a structured ‘risk factor’ questionnaire was administered and HPV from urine specimen was genotyped using the L1 gene. Phylogenetic and molecular evolutionary analyses were conducted. Bivariate analysis and Pearson’s chi square (χ2) tests were used to determine the association between HPV infection and factors associated with HPV. A total of 222 adults (45 males and 177 females) aged 18-49 years were recruited. The prevalence of HPV among males and females was 22.2% (10/45) and 32.8% (58/177) respectively. The prevalence of high-risk types among males and females was 25% (1/4) and 27.5% (11/40) respectively. The high risk HPV genotypes detected among females were: HPV-16 (10%), -66 (7.5%), and -70 (7.5%) while low risk types were HPV 6 (27.5%), followed by -81 (25%), -83 (10%), -11 (7.5%), and -54 (2.5%) respectively. The prevalence of low risk types among males and females was 75% (3/4) and 72.5% (29/40) respectively. The prevalent low-risk HPV type detected in males was HPV type 6 (75%) while HPV-58 (25%) was the only high risk type in males. History of sexually transmitted infections was significantly associated with HPV infection among females (P=0.002). There was also significant association between marital status among males (p=0.046), how often one had used the contraceptives among females (p=0.038) and HPV genotypes at bivariate level. The results indicate high HPV prevalence, high risk and low risk HPVs could be detected in urine from the two populations. Therefore; molecular testing of HPV on urine samples is a method that utilizes a non-invasive technique that may increase screening coverage as it is easy to obtain. Key words: urine, Human papillomavirus, HPV genotypes, PCR, cervical cancer

Assessment of Risk Predisposition to Human Papilloma Virus through Cervical Infections Screening of Women Attending an Outpatient Health Facility in Nairobi, Kenya

There is limited data on comparative disposition to cervical cancer among HPV infected women in Kenya. We aimed to determine the distribution of HPV infection, cervical abnormalities and infections commonly reported on cervical pap smears among both HIV positive and HIV negative women attending a reproductive health clinic at the largest national hospital in Kenya. A total of 187 women aged 18 to 50 years attending the reproductive clinic at Kenyatta National Referral Hospital in Nairobi were recruited into the study. All consenting subjects were screened for HIV by serology and their cervical smears taken and immediately fixed on slides for Papanicolaou (Pap) staining. A second endocervical swab was collected in the same sitting for HPV DNA extraction and PCR amplification of the HPV LI region.  Of the 187 women studied, 27 (14.4 %) were positive for HIV and 90 (48.1%) had one or more infection associated with bacterial vaginosis, candidiasis, cervicitis or inflammation of the cervix of unknown cause.  Eight (4.3%) women had abnormal cervix, 3/8 being of high grade squamous intraepithelial lesions (HSIL), 1/8 of low grade squamous intraepithelial lesions (LSIL), 1/8 had adenocarcinoma while the remaining 3 had atypical squamous cells of undetermined significance (ASC-US). The remaining 89/187 (47.6%) women had normal smears with no infection. Of the 89 women with normal smears, 82 (92.1%) were HIV negative.  A total of 66 (35.3%) women were positive for HPV L1 DNA by PCR and included 30 of the 89 women with normal cytology. Of the 27 HIV positive women, 14 (51.9%) were also positive for HPV LI DNA. 52 of the 160 (32.5%) HIV negative women were positive for HPV L1 DNA. We report more cases of cervical intraepithelial lesions among HIV positive than HIV negative women. Similarly, the other infections commonly found on Pap smear tests were higher among HIV negative than HIV positive women. HPV prevalence among these clinic-attending women was higher in those with normal cytology, indicating an increased underlying risk of cervical cancer in a setting where routine diagnostic screening is limited or non-existent. Keywords: cervical cancer, HIV, HPV, cervical cytolog

Comparison of HIV-1 nef and gag variations and host HLA characteristics as determinants of disease progression among HIV-1 vertically infected Kenyan children

Objectives: Disease progression varies among HIV-1-infected individuals. The present study aimed to explore possible viral and host factors affecting disease progression in HIV-1-infected children. Methods: Since 2000, 102 HIV-1 vertically-infected children have been followed-up in Kenya. Here we studied 29 children (15 male/14 female) who started antiretroviral treatment at 10 years of age (slow progressors; SP). Sequence variations in the HIV-1 gag and nef genes and the HLA class I-related epitopes were compared between the two groups. Results: Based on nef sequences, HIV-1 subtypes A1/D were detected in 62.5%/12.5% of RP and 66.7%/20%of SP, with no significant difference in subtype distribution between groups (p = 0.8). In the ten Nef functional domains, only the PxxP3 region showed significantly greater variation in RP (33.3%) than SP (7.7%, p = 0.048). Gag sequences did not significantly differ between groups. The reportedly protective HLA-A alleles, A∗74:01, A∗32:01 and A∗26, were more commonly observed in SP (50.0%) than RP (11.1%, p = 0.010), whereas the reportedly disease-susceptible HLA-B∗45:01 was more common in RP (33.3%) than SP (7.4%, p = 0.045). Compared to RP, SP showed a significantly higher median number of predicted HLA-B-related 12-mer epitopes in Nef (3 vs. 2, p = 0.037), HLA-B-related 11-mer epitopes in Gag (2 vs. 1, p = 0.029), and HLA-A-related 9-mer epitopes in Gag (4 vs. 1, p = 0.051). SP also had fewer HLA-C-related epitopes in Nef (median 4 vs. 5, p = 0.046) and HLA-C-related 11-mer epitopes in Gag (median 1 vs. 1.5, p = 0.044) than RP. Conclusions: Compared to rapid progressors, slow progressors had more protective HLA-A alleles and more HLA-B-related epitopes in both the Nef and Gag proteins. These results suggest that the host factor HLA plays a stronger role in disease progression than the Nef and Gag sequence variations in HIV-1-infected Kenyan children. © 2015 Saina et al

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 log10 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. © 2011 Elsevier B.V. All rights reserved