Genetic diversity, haplotype analysis, and prevalence of Hepatitis B virus MHR mutations among isolates from Kenyan blood donors.

BACKGROUND: The rapid spread of HBV has resulted in the emergence of new variants. These viral genotypes and variants, in addition to carcinogenic risk, can be key predictors of therapy response and outcomes. As a result, a better knowledge of these emerging HBV traits will aid in the development of a treatment for HBV infection. However, many Sub-Saharan African nations, including Kenya, have insufficient molecular data on HBV strains circulating locally. This study conducted a population-genetics analysis to evaluate the genetic diversity of HBV among Kenyan blood donors. In addition, within the same cohort, the incidence and features of immune-associated escape mutations and stop-codons in Hepatitis B surface antigen (HBsAg) were determined. METHODS: In September 2015 to October 2016, 194 serum samples were obtained from HBsAg-positive blood donors residing in eleven different Kenyan counties: Kisumu, Machakos, Uasin Gishu, Nairobi, Nakuru, Embu, Garissa, Kisii, Mombasa, Nyeri, and Turkana. For the HBV surface (S) gene, HBV DNA was isolated, amplified, and sequenced. The sequences obtained were utilized to investigate the genetic and haplotype diversity within the S genes. RESULTS: Among the blood donors, 74.74% were male, and the overall mean age was 25.36 years. HBV genotype A1 (88.14%) was the most common, followed by genotype D (10.82%), genotype C (0.52%), and HBV genotype E (0.52%). The phylogenetic analysis revealed twelve major clades, with cluster III comprising solely of 68 blood donor isolates (68/194-35.05%). A high haplotype diversity (Hd = 0.94) and low nucleotide diversity (π = 0.02) were observed. Kisumu county had high number of haplotypes (22), but low haplotype (gene) diversity (Hd = 0.90). Generally, a total of 90 haplotypes with some consisting of more than one sequence were observed. The gene exhibited negative values for Tajima's D (-2.04, p<0.05) and Fu's Fs (-88.84). Several mutations were found in 139 isolates, either within or outside the Major Hydrophilic Area (MHR). There were 29 mutations found, with 37.9% of them situated inside the "a" determinant. The most common mutations in this research were T143M and K122R. Escape mutations linked to diagnostic failure, vaccination and immunoglobulin treatment evasion were also discovered. Also, one stop-codon, W163STP, inside the MHR, was found in one sample from genotype A. CONCLUSION: In Kenya, HBV/A1 is still the most common genotype. Despite limited genetic and nucleotide diversity, haplotype network analysis revealed haplotype variance among HBV genotypes from Kenyan blood donors. The virological properties of immune escape, which may be the source of viral replication endurance, were discovered in the viral strains studied and included immune-escape mutations and stop-codon. The discovery of HBsAg mutations in MHR in all isolates highlighted the need of monitoring MHR mutations in Kenya.

Sero-prevalence of hepatitis B virus and compliance with hepatitis B vaccination schedules among outpatient clinic attendees in Nairobi.

BACKGROUND: Hepatitis B is becoming a growing public health problem in Kenya. To combat the threat, HBV vaccination should be recommended, particularly for individuals who are not covered by the national immunization program. Vaccination provides sero-protection rates approaching 95% among healthy adults after completing the three-dose vaccination course, but decreases to 87% among those who receive only two doses, emphasizing the importance of completing the three-dose vaccination course. However, data on adult adherence to HBV multi-dose vaccines in Sub-Saharan Africa are limited, despite the fact that this information is critical for prevention. As a result, more research on HBV vaccine dose completion is required. The purpose of this study is to estimate the prevalence of hepatitis B virus infection among out-patient clinic attendees in Nairobi, Kenya, as well as to identify beneficiaries of free vaccination and barriers to completing the recommended vaccine doses. METHODS: Between July 30th and September 30th, 2015, 2644 outpatient clinic attendees aged ≥ 4 were recruited from three hospitals in Nairobi County, Kenya: Mama Lucy, Riruta, and Loco. Self-administered questionnaires were used to collect socio-demographic information, and blood samples were tested for hepatitis B surface antigen (HBsAg) using the KEMRI HEPCELL Rapid® (Hepatitis B Detection kit) test kit. Individuals who tested negative for HBsAg were given a free course of three doses of HBV vaccine. The vaccination register provided information on the number of doses administered. RESULTS: The average age of the study population was 31.4 years (range: 4-66), with females accounting for 59.2%. 1.82% (48/2644) of the participants tested positive for HBsAg. Among the 2596 individuals eligible for vaccination, 66% (1720/2596) received at least one dose, and 51.8% (1345/2596) received all three doses. Vaccination acceptance increased with age, with older patients more likely to return for subsequent dose (OR>1 for second and third dose). Unavailability and failure to contact client were cited as significant (p<0.0001) barrier to vaccination completion by 53.7% (666/1226, 95% CI 0.5-0.6) and 37% (454/1226, 95% CI 0.3-0.4) of respondents respectively. CONCLUSION: The prevalence of HBV infection among outpatient clinic attendees highlights the importance of expanding HBV immunization programs in Kenya. However, given the low vaccination completion rate, there is a need for public awareness of the vaccine's importance in preventing HBV and HBV-related complications.

Amodiaquine resistance in Plasmodium berghei is associated with PbCRT His95Pro mutation, loss of chloroquine, artemisinin and primaquine sensitivity, and high transcript levels of key transporters.

Background: The human malaria parasite Plasmodium falciparum has evolved complex drug evasion mechanisms to all available antimalarials. To date, the combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short acting, artesunate is partnered with long acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite Plasmodium berghei ANKA as a surrogate of P. falciparum to investigate the mechanisms of amodiaquine resistance. Methods: We used serial technique to select amodiaquine resistance by submitting the parasites to continuous amodiaquine pressure. We then employed the 4-Day Suppressive Test to monitor emergence of resistance and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Results: Submission of P. berghei ANKA to amodiaquine pressure yielded resistant parasite within thirty-six passages. The effective dosage that reduced 90% of parasitaemia (ED 90) of sensitive line and resistant line were 4.29mg/kg and 19.13mg/kg, respectively. After freezing at -80ºC for one month, the resistant parasite remained stable with an ED 90 of 18.22mg/kg. Amodiaquine resistant parasites are also resistant to chloroquine (6fold), artemether (10fold), primaquine (5fold), piperaquine (2fold) and lumefantrine (3fold). Sequence analysis of Plasmodium berghei chloroquine resistant transporter revealed His95Pro mutation. No variation was identified in Plasmodium berghei multidrug resistance gene-1 (Pbmdr1), Plasmodium berghei deubiquitinating enzyme-1 or Plasmodium berghei Kelch13 domain nucleotide sequences. Amodiaquine resistance is also accompanied by high mRNA transcripts of key transporters; Pbmdr1, V-type/H+ pumping pyrophosphatase-2 and sodium hydrogen ion exchanger-1 and Ca 2+/H + antiporter. Conclusions: Selection of amodiaquine resistance yielded stable "multidrug-resistant'' parasites and thus may be used to study common resistance mechanisms associated with other antimalarial drugs. Genome wide studies may elucidate other functionally important genes controlling AQ resistance in P. berghei.

Lower prevalence of Entamoeba species in children with vertically transmitted HIV infection in Western Kenya.

A cross-sectional molecular epidemiological study of Entamoeba species was conducted among asymptomatic Kenyan children with (n = 123) and without (n = 111) HIV infection. The prevalence of E. histolytica was low (0.4%). Entamoeba species infection was inversely related with HIV infection [HIV(+): 29.3% vs. HIV(-): 55.0%, P < 0.001]: multiple-species infection was related to higher CD4 T-cell counts. Thus, HIV infection is not a risk factor for amebic infection, and multiple-species infection can be an indicator of better immune status.