Development of immunoassays for detection of Human Immunodeficiency Virus based on Consensus env gp41 Immunodominant Region Peptide from HIV-1 infections in Kenya

Background: Human Immunodeficiency Virus (HIV) is characterized by high rates of genetic variability in vivo that could affect the performance of the HIV antibody-based detection kits. Objective: This study aimed at developing immunoassays for HIV based on Consensus env gp41 Immunodominant region (IDR) from HIV infections in Kenya. Methods: HIV RNA was extracted from 91 samples collected from 5 regional blood transfusion centers in Kenya. The RNA was reverse transcribed, sequenced in the env gp41-Immunodominant Region (IDR) and the Consensus sequence generated used to synthesize corresponding peptide. The Global HIV envgp41-IDR Consensus peptide was obtained from the literature and also synthesized. The two peptides were used to separately develop HIV immunoassays based on Enzyme-linked Immunosorbent Assay (ELISA) and Lateral Flow Assay (LFA) platforms and the performance of developed assays was evaluated. The same HIV env gp41 IDR peptides were used to develop ELISA-based immunoassays for determination HIV Incidence / Recency. Results: The study did not find significant difference between the performance of the immunoassays that were developed with Consensus env gp41-IDR peptide (Kenya) and those developed using Consensus env gp41-IDR peptide (Global). However, the study found a significant difference between the performance of HIV ELISA for HIV Incidence testing that was developed with Consensus envgp41-IDR peptide (Kenya) and that which was developed using Consensus envgp41-IDR peptide (Global) with the former displaying superior performance. Conclusions: The developed immunoassays demonstrated that both Consensus env gp41-IDR peptides (Kenya and Global) could be used to develop HIV immunoassays but Consensus env gp41-IDR peptide (Kenya) could be more suitable for development of HIV Incidence assays in Kenya. Keywords: HIV, Consensus sequence, env gp41-Immunodominant Region, Immunoassay

Performance of selected HIV testing centers in a HIV Proficiency Testing Scheme in Kenya: a case study

Background: The Proficiency Testing (PT) for Human Immunodeficiency Virus (HIV) using Lateral flow assays provides an avenue for participating institutions/individuals to assess their technical competence in testing for HIV using LFAs that are recommended in the National HIV Testing Algorithm (NHTA) in Kenya. It also provides confidence to the participating institutions and potential users of their services besides giving the institutions an opportunity for improvement. Objective: To determine the performance of selected HIV testing centers in a HIV PT Scheme in Kenya Methods: Fifty one participants (51) in Kenya were selected from 7 sites (Kisumu, Mombasa, Kilifi, Nairobi and Malindi) to participate in this PT round. The sites comprised both private sector and institutions that do not participate in the National HIV referral Lab-PT scheme. They were provided with panels containing six samples to analyze using the current NHTA in Kenya. Obtained results were sent to our laboratory electronically. Results: Eighty nine percent (89.0%) of the panels were correctly identified by the participants as positive or negative. Of the 11.0% errors, 74.2% were committed in one or more test result obtained while 12.9% committed in failure to follow NHTA. Two minor errors repeated by participants were; failure to record the final results in spite of obtaining correct tests and correct reactive results with the first and second test kits but in conclusion the participant recorded negative (12.9%). Root cause analysis revealed that the error committed by participants were as a result of failure to observe the kit manufactures’ instructions and NHTA guidelines. Conclusion: The results of this PT Scheme enhance the need for constant training of personnel conducting HIV testing and Counseling in Kenya on proper techniques of carrying out HIV testing using Lateral flow assays in the NHTA. Key words: HIV, Proficiency Testing, errors, false negative, false positive

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

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

The Potential for DPPIV/CD26 usage as a surrogate marker for Antiretroviral Therapy Efficacy in HIV Infected populations

Background: Human Immunodeficiency Virus (HIV) viral load and CD4+ cell counts are the most commonly used markers for monitoring efficacy of anti-retroviral therapy (ART) in HIV infected individuals. The high cost of viral load monitoring limits its usage in resource limited countries, often leaving the use of CD4+ T cell counts as the only alternative. Though cheaper and more readily available, CD4+ cell counts as a measure of detecting treatment failure, is an unreliable predictor of disease progression. Hence, there is a need for more sensitive alternative, but less costly techniques for detecting treatment failure which can be used in resource limited settings. Objective: To evaluate the feasibility of using plasma CD26/Dipeptidyl peptidase IV (DPPIV) as a novel marker for clinical evaluation of treatment efficacy in HIV infected children. Method: Blood samples collected from HIV+ children (n=76) before and after initiation on ART, were assessed for HIV RNA (viral load), CD4+ T-cell count and DPPIV/CD26 levels. Viral load levels were analyzed using Roche Amplicor HIV-1 Monitor Test kit; CD4+ T-Cell Counts were analyzed using BD FACS Calibur flow cytometer while DPPIV/CD 26 levels were analyzed using Human DPPIV/CD26 Quantikine ELISA kit (R&D Systems, Minneapolis MN). Results: The plasma DPPIV/CD26 levels increased significantly in children after ART initiation (p = 0.017), while the viral load levels declined after ART initiation with subsequent CD4+ cell counts increase. The DPPIV/CD 26 increase positively correlated with viral load decrease while negatively correlating to the CD4+ cell count increase. Conclusion: These findings demonstrate an inverse relationship between DPPIV/CD26 levels and HIV viral load and the direct proportionality of CD4+ Cell counts and DPPIV/CD26 levels, suggesting potential for use of DPPIV/CD26 as a surrogate marker for evaluating HIV disease progression in children receiving anti-retroviral therapy. Key words: CD26/Dipeptidyl peptidase IV (DPPIV), ELISA, Surrogate marker, Viral Load, CD4 Count, antiretroviral

Spatial distribution, prevalence and potential risk factors of Tungiasis in Vihiga County, Kenya.

BACKGROUND:Tungiasis is a parasitic disease caused by the sand flea Tunga penetrans also known as jigger flea. Communities living in precarious conditions in tropical and sub tropical countries bear the brunt of the infection. The main objective of this study was to determine the burden of Tungiasis in Vihiga County in Kenya. METHODS:This was a cross-sectional study conducted in 21 villages in 3 Sub-locations in Vihiga County, western Kenya. A total of 437 participants, 5 years old and above were clinically examined for the presence of tungiasis after consenting to take part in the study. Diagnosis was made following standard methods. A semi- structured questionnaire was administered to assess socio-demographic factors, housing, presence and ownership of animals, knowledge and practice related to tungiasis. Data were analyzed using bivariate and multivariate regression analysis. GIS was used to map the geographic distribution of tungiasis in the area. RESULTS:The overall prevalence was found to be (21.5%; 95% CI: 17.7-25.3%). The cases were analysed and visualized in a map form. Multivariate analysis suggested that the occurrence of tungiasis was associated with variables that indicated low economic status (like a monthly income of Ksh ≤ 1000 (adjusted odds ratio 27.85; 95% CI: 4.13-187.59), earthen floor (0.36; 0.13-1.024) and lack of toilet facilities (4.27; 0.82-22.34), age of participant ≤14 (27.414; 10.02-74.99), no regular use of closed footwear (1.98; 0.987-3.97) and common resting place inside the house (1.93; 0.96-3.89). CONCLUSIONS:Tungiasis is an important health problem in Vihiga County occasioned by the low economic status of the people affected. Factors that point to poverty contribute to the occurrence of tungiasis. These findings suggest a need to design control strategies for tungiasis that are cost effective and easily accessible

Prevalence, awareness and risk factors associated with Hepatitis B infection among pregnant women attending the antenatal clinic at Mbagathi District Hospital in Nairobi, Kenya

Introduction: hepatitis B Viral Infection (HBV) remains one of the leading cause of morbidity and mortality globally accounting for 38-53% of chronic liver diseases and about 686,000 deaths annually. The prevalence of HBV is 9-20% in Sub-Saharan Africa, and in Kenya it is 5-30% among the general population and 9.4% among pregnant women. This study was aimed at identifying the prevalence, awareness and risk factors associated with HBV infections among pregnant women attending Antenatal clinic (ANC) at Mbagathi District hospital, Nairobi. Methods: this was a cross-sectional study involving 287 pregnant women enrolled for three months (September to December 2014) from Nairobi and neighbouring counties. A structured questionnaire that captured social, demographic and explanatory variables was administered to the study participants. Blood samples were also drawn from the participants and tested for HBV using Enzyme-Linked Immunosorbent Assay (ELISA) system. Results: the study established that the prevalence of HBV infections among pregnant women attending antenatal clinic at Mbagathi District Hospital was 3.8% with highest infection rate among the 20-24 years age group. Seventy six (60.8 %) of the participants reported sexual encounters in less than a month before the interview of which 5 (7.6%) reported encounters involving other partners apart from their spouses.HBV awareness among the study participants was 12.2%. Before the interview, those with at least tertiary education (Mean =1.33, SD = 1.131), were more informed about HBV infection as compared to those with primary and secondary education (Mean = 0.63, SD = 0.722; (Mean =0.31, SD= 0.664). In regards to assessment of the risk factors; type of family (χ² =19.753 df2 p<0.01), parity (χ² =7.128 df2 p<0.01), History of abortions (χ²=9.094 df1 p<0.01), early age (11-15 years) at first sexual encounter (χ² =8.185 df1 p<0.01) were significantly associated with HBV positivity. Conclusion: the prevalence of HBV infection among pregnant women attending Antenatal clinic (ANC) at Mbagathi District hospital, Nairobi was lower (3.8%) than the prevalence among pregnant women nationally (9.4%). These women also showed a low level of HBV awareness (12.2%.).The Pan African Medical Journal 2016;2

Anti-bacterial efficacy of alcoholic hand rubs in the Kenyan market, 2015

Abstract Background Hand hygiene is known to be effective in preventing hospital and community-acquired infections. The increasing number of hand sanitizer brands in Kenyan hospitals and consumer outlets is of concern. Thus the main aim of this study was to evaluate the anti-bacterial efficacy and organoleptic properties of these hand sanitizers in Kenya. Methods This was an experimental, laboratory-based study of 14 different brands of hand sanitizers (coded HS1-14) available in various retail outlets and hospitals in Kenya. Efficacy was evaluated using standard non-pathogenic Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) as per the European Standard (EN). The logarithmic reduction factors (RF) were assessed at baseline and after treatment, and log reduction then calculated. Ten and 25 healthy volunteers participated in the efficacy and organoleptic studies respectively. Results Four (28.6%) hand sanitizers (HS12, HS9, HS13 and HS14) showed a 5.9 reduction factor on all the three bacteria strains. Seven (50%) hand sanitizers had efficacies of <3 against all the three bacteria strains used. Efficacy on E. Coli was higher compared to the other pathogens. Three hand sanitizers were efficacious on one of the pathogens and not the other. In terms of organoleptic properties, gel-based formulations were rated far higher than the liquid based formulations brands. Conclusion Fifty percent (50%) of the selected hand sanitizers in the Kenyan market have efficacy that falls below the World Health Organization (WHO) and DIN EN 1500:2013. Of the 14 hand sanitizers found in the Kenyan market, only four showed efficacies that were comparable to the WHO-formulation. There is a need to evaluate how many of these products with <3 efficacy that have been incorporated into the health system for hand hygiene and the country\u2019s policy on regulations on their usage

Secondary bacterial infections and antibiotic resistance among tungiasis patients in Western, Kenya.

Tungiasis or jigger infestation is a parasitic disease caused by the female sand flea Tunga penetrans. Secondary infection of the lesions caused by this flea is common in endemic communities. This study sought to shed light on the bacterial pathogens causing secondary infections in tungiasis lesions and their susceptibility profiles to commonly prescribed antibiotics. Participants were recruited with the help of Community Health Workers. Swabs were taken from lesions which showed signs of secondary infection. Identification of suspected bacteria colonies was done by colony morphology, Gram staining, and biochemical tests. The Kirby Bauer disc diffusion test was used to determine the drug susceptibility profiles. Out of 37 participants, from whom swabs were collected, specimen were positive in 29 and 8 had no growth. From these, 10 different strains of bacteria were isolated. Two were Gram positive bacteria and they were, Staphylococcus epidermidis (38.3%) and Staphylococcus aureus (21.3%). Eight were Gram negative namely Enterobacter cloacae (8.5%), Proteus species (8.5%), Klebsiellla species (6.4%), Aeromonas sobria (4.3%), Citrobacter species (4.3%), Proteus mirabillis(4.3%), Enterobacter amnigenus (2.1%) and Klebsiella pneumoniae (2.1%). The methicillin resistant S. aureus (MRSA) isolated were also resistant to clindamycin, kanamycin, erythromycin, nalidixic acid, trimethorprim sulfamethoxazole and tetracycline. All the Gram negative and Gram positive bacteria isolates were sensitive to gentamicin and norfloxacin drugs. Results from this study confirms the presence of resistant bacteria in tungiasis lesions hence highlighting the significance of secondary infection of the lesions in endemic communties. This therefore suggests that antimicrobial susceptibility testing may be considered to guide in identification of appropriate antibiotics and treatment therapy among tungiasis patients

Cytoarchitecture of ex vivo midgut cultures of unfed Ixodes scapularis infected with a tick-borne flavivirus

A bite from an infected tick is the primary means of transmission for tick-borne flaviviruses (TBFV). Ticks ingest the virus while feeding on infected blood. The traditional view is that the virus first replicates in and transits the tick midgut prior to dissemination to other organs, including salivary glands. Thus, understanding TBFV infection in the tick midgut is a key first step in identifying potential countermeasures against infection. Ex vivo midgut cultures prepared from unfed adult female Ixodes scapularis ticks were viable and remained morphologically intact for more than 8 days. The midgut consisted of two clearly defined cell layers separated by a basement membrane: an exterior network of smooth muscle cells and an internal epithelium composed of digestive generative cells. The smooth muscle cells were arranged in a stellate circumferential pattern spaced at regular intervals along the long axis of midgut diverticula. When the cultures were infected with the TBFV Langat virus (LGTV), virus production increased by two logs with a peak at 96 hours post-infection. Infected cells were readily identified by immunofluorescence staining for the viral envelope protein, nonstructural protein 3 (NS3) and dsRNA. Microscopy of the stained cultures suggested that generative cells were the primary target for virus infection in the midgut. Infected cells exhibited an expansion of membranes derived from the endoplasmic reticulum; a finding consistent with TBFV infected cell cultures. Electron microscopy of infected cultures revealed virus particles in the basolateral region between epithelial cells. These results demonstrated LGTV replication in midgut generative cells of artificially infected, ex vivo cultures of unfed adult female I. scapularis ticks