Browsing by Author "Gachara, George"
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Item Amino acid sequence analysis and identification of mutations in the NS gene of 2009 influenza A (H1N1) isolates from Kenya.(Virus Genes, 2011-05) Gachara, George; Symekher, Samuel; Mbithi, J. N.; Simwa, James; Ng'ayo, Musa; Magana, Japheth; Bulimo, Wallace D.Although the important role of the nonstructural (NS) gene of influenza A virus in virulence and replication is well-established, the knowledge about the extent of variation in the NS gene of 2009 influenza A (H1N1) viruses in Kenya and Africa is scanty. This study analysed the NS gene of 31 isolates from Kenya in order to obtain a more detailed knowledge about the genetic variation of NS gene of 2009 influenza A (H1N1) isolates from Kenya. A comparison with the vaccine strain and viruses isolated elsewhere in Africa was also made. The amino acid sequences of the non-structural protein, NS1 of the viruses from this study and the vaccine strain revealed 18 differences. Conversely, the nuclear export protein (NEP) of the isolates in this study had 11 differences from the vaccine strain. Analysis of the NS1 protein showed only one fixed amino acid change I123V which is one of the characteristics of clade 7 viruses. In the NEP, the amino acid at position 77 was the most mutable with 9 (39%) of all mutations seen in this protein. A mutation A115T which is a characteristic of clade 5 viruses was noted in the isolates from Lagos, Nigeria. The study shows a substantial number of mutations in the NS gene that has not been reported elsewhere and gives a glimpse of the evolution of this gene in the region.Item Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene(Public Library of Science, 2020) Opanda, Silvanos; Bulimo, Wallace; Gachara, George; Ekuttan, Christopher; Amukoye, EvansInfluenza A (H1N1) pdm09 virus emerged in North America in 2009 and has been established as a seasonal strain in humans. After an antigenic stasis of about six years, new antigenically distinct variants of the virus emerged globally in 2016 necessitating a change in the vaccine formulation for the first time in 2017. Herein, we analyzed thirty-eight HA sequences of influenza A (H1N1) pdm09 strains isolated in Kenya during 2015–2018 seasons, to evaluate their antigenic and molecular properties based on the HA1 sub-unit. Our analyses revealed that the A (H1N1) pdm09 strains that circulated in Kenya during this period belonged to genetic clade 6B, subclade 6B.1 and 6B.2. The Kenyan 2015 and 2016 isolates differed from the vaccine strain A/California/07/2009 at nine and fourteen antigenic sites in the HA1 respectively. Further, those isolated in 2017 and 2018 correspondingly varied from A/Michigan/45/2015 vaccine strain at three and fifteen antigenic sites. The predicted vaccine efficacy of A/California/07/2009 against Kenyan 2015/2016 was estimated to be 32.4% while A/Michigan/45/2015 showed estimated vaccine efficacies of 39.6% - 41.8% and 32.4% - 42.1% against Kenyan 2017 and 2018 strains, respectively. Hemagglutinationinhibition (HAI) assay using ferret post-infection reference antiserum showed that the titers for the Kenyan 2015/2016 isolates were 2–8-fold lower compared to the vaccine strain. Overall, our results suggest the A (H1N1) pdm09 viruses that circulated in Kenya during 2015/2016 influenza seasons were antigenic variants of the recommended vaccine strains, denoting sub-optimal vaccine efficacy. Additionally, data generated point to a swiftly evolving influenza A (H1N1) pdm09 virus in recent post pandemic era, underscoring the need for sustained surveillance coupled with molecular and antigenic analyses, to inform appropriate and timely influenza vaccine update.Item Characterization of occult hepatitis B in highrisk populations in Kenya(PLOS ONE, 2020) Kiptoon, Beatrice Jepkemei; Missiani, Ochwoto; Swidinsky, Ken; Day, Jacqueline; Gebrebrhan, Henok; McKinnon, Lyle R.; Andonov, Anton; Oyugi, Julius; Kimani, Joshua; Gachara, George; Songok, Elijah Maritim; Osiowy, CarlaOccult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HBV) DNA in the liver or serum in the absence of detectable HBV surface antigen (HBsAg). OBI poses a risk for the development of cirrhosis and hepatocellular carcinoma. The prevalence of OBI in Kenya is unknown, thus a study was undertaken to determine the prevalence and molecular characterization of OBI in Kenyan populations at high risk of HBV infection. Sera from two Nairobi cohorts, 99 male sex workers, primarily having sex with men (MSM-SW), and 13 non-MSM men having HIV-positive partners, as well as 65 HBsAg-negative patients presenting with jaundice at Kenyan medical facilities, were tested for HBV serological markers, including HBV DNA by real-time PCR. Positive DNA samples were sequenced and MSMSW patients were further tested for hepatitis C virus (HCV) infection. Of the 166 HBsAg-negative samples tested, 31 (18.7%; 95% confidence interval [CI] 13.5–25.3) were HBV DNA positive (i.e., occult), the majority (20/31; 64.5%) of which were HBV core protein antibody positive. HCV infection was not observed in the MSM-SW participants, although the prevalence of HBsAg positivity was 10.1% (10/99; 95% CI 5.6–17.6). HBV genotype A was predominant among study cases, including both HBsAg-positive and OBI participants, although the data suggests a non-African network transmission source among MSM-SW. The high prevalence of HBV infection among MSM-SW in Kenya suggests that screening programmes be instituted among high-risk cohorts to facilitate preventative measures, such as vaccination, and establish entry to treatment and linkage to care.Item Characterization of occult hepatitis B virus infection among HIV positive patients in Cameroon(BioMed Central, 2017) Gachara, George; Magoro, Tshifhiwa; Mavhandu, Lufuno; Lum, Emmaculate; Kimbi, Helen K.; Ndip, Roland N.; Bessong, Pascal O.Purpose: Occult hepatitis B infection (OBI) among HIV positive patients varies widely in different geographic regions. We undertook a study to determine the prevalence of occult hepatitis B infection among HIV infected individuals visiting a health facility in South West Cameroon and characterized occult HBV strains based on sequence analyses. Methods: Plasma samples (n = 337), which previously tested negative for hepatitis B surface antigen (HBsAg), were screened for antibodies against hepatitis B core (anti-HBc) and surface (anti-HBs) antigens followed by DNA extraction. A 366 bp region covering the overlapping surface/polymerase gene of HBV was then amplified in a nested PCR and the amplicons sequenced using Sanger sequencing. The resulting sequences were then analyzed for genotypes and for escape and drug resistance mutations. Results: Twenty samples were HBV DNA positive and were classified as OBI giving a prevalence of 5.9%. Out of these, 9 (45%) were anti-HBs positive, while 10 (52.6%) were anti-HBc positive. Additionally, 2 had dual anti-HBs and anti-HBc reactivity, while 6 had no detectable HBV antibodies. Out of the ten samples that were successfully sequenced, nine were classified as genotype E and one as genotype A. Three sequences possessed mutations associated with lamivudine resistance. We detected a number of mutations within the major hydrophilic region of the surface gene where most immune escape mutations occur. Conclusions: Findings from this study show the presence of hepatitis B in patients without any of the HBV serological markers. Further prospective studies are required to determine the risk factors and markers of OBI. Keywords: Hepatitis B virus, Occult hepatitis B infection, HIV, CameroonItem Evaluating adherence to antiretroviral therapy using pharmacy refill records in a rural treatment site in South Africa(Hindawi Publishing Corporation, 2017) Gachara, George; Mavhandu, Lufuno G.; Rogawski, Elizabeth T.; Manhaeve, Cecile; Bessong, Pascal O.Optimal adherence to combination antiretroviral therapy (cART) is critical to maintain virologic suppression, thereby ensuring the global success of HIV treatment. We evaluated adherence to cART using pharmacy refill records and determined the adherence threshold resulting in >90% virologic suppression in a community run treatment site in South Africa. Additionally, we analysed factors associated with adherence using univariable and multivariable logistic regression models. Logistic regression was also performed to determine the relationship between adherence and virologic suppression and the adherence threshold resulting in <10% virologic failure. The overall median (interquartile range) adherence was 95% (88.6–98.4%). Out of the study participants, 210/401 (52.4%) had optimal (≥95%) adherence while only 37/401 (9.2%) had poor (≤80%) adherence. The majority (90.5%) of patients with optimal adherence had virologic suppression.Having TB at registration into care was found to be negatively associated with adherence (adjusted odds ratio [AOR], 0.382; 𝑝 ≤ .05). Compared to nonadherent individuals, optimally adherent participants were more likely to achieve virologic suppression (OR 2.92; 95% CI: 1.63–5.22). Only adherence rates above 95% were observed to lead to <10% virologic failure. cART adherence measured by pharmacy refill records could serve as a useful predictor of virologic failure; adherence rates >95% are needed to maintain optimal virologic suppressionItem Evidence in Kenya of Reassortment Between Seasonal Influenza A(H3N2) and Influenza A(H1N1)pdm09 to yield A(H3N2) Variants With the Matrix Gene Segment of A(H1N1)pdm09(Molecular Biology Reports, 2012-02) Gachara, George; Bulimo, Wallace D.; Opot, Benjamin H; Murage, Margaret W; Wurapa, Eyako KBackground: Influenza viruses evolve rapidly and undergo frequent reassortment of different gene segments leading to emergence of novel strains with new traits possessing pandemic potential. Objectives: To determine evidence of reassortment amongst A(H1N1)pdm09 and H3N2 co-circulating influenza virus subtypes and relate these to adamantine antiviral resistance. Methodology: Nasopharyngeal swabs in virus transport medium were collected from patients with influenza-like illness. The presence of influenza was determined using real-time PCR followed by culture in MDCK cells. Haemagglutination inhibition was carried out to confirm the identity of the virus. Complete haemagglutinin (HA), matrix (M) and neuraminidase (NA) genes were sequenced and analyzed using a suite of bioinformatics tools. Results: Influenza A(H3N2) was detected in 32 out of 708 samples collected between October and December 2010. Analysis of the HA gene confirmed it to be of the H3 subtype. However, analysis of the matrix gene showed that 28 of the isolates had the M gene of influenza A(H3N2) viruses while 4 had the M gene of the A(H1N1)pdm09 viruses. Discussion: Our results show that four of the 32 influenza A(H3N2) viruses isolated had acquired the M gene segment of the A(H1N1)pdm09 virus by reassortment. This has implications in their transmissibility as the M gene is implicated in the increased transmissibility of the A(H1N1)pdm09 viruses.Item Evolutionary Pattern of the Hemagglutinin Gene of Influenza B Viruses Isolated in Kenya 2005-2009(African Journal of Health Sciences, 2011-06-17) Gachara, George; Bulimo, W.; Magana, J.; Simwa, J.; Symekher, S.Background: In humans, the inability to provide lasting protection against influenza B virus infection is due, in part, to the rapid evolution of the viral surface glycoprotein, haemagglutinin (HA), which leads to a change in its antigenic nature. Therefore, the evolution of the haemagglutinin (HA) an important influenza antigen has and continues to be a subject of intensive research. In this study, we analyzed the evolution occurring in the haemagglutinin of influenza B viruses from Kenya since virological surveillance began in 2005. Methods: Thirty (30) influenza B haemagglutinin sequences of viruses isolated from different parts of the country between 2005-2009 at the NIC were analyzed. Nucleotide sequences, prediction of amino acid sequences, alignments, and phylogenetic tree construction were completed using BioEdit and MEGA® software Results: During the five year study period, the two influenza B lineages B/Yamagata and B/Victoria have co-circulated in two years (2005 and 2007) while B/Yamagata viruses exclusively circulated in 2008 and B/Victoria viruses in 2006 and 2009. The nucleotide sequence identity ranged from 92.9% – 97.0% among the B/Yamagata lineage viruses and 90.6% – 98.5% among the B/Victoria lineage viruses. There was generally noted more amino acid substitutions among the B/Yamagata lineage than the B/Victoria lineage. Substitutions were observed in all the epitope regions among B/Yamagata viruses compared to three epitopes in the B/Victoria viruses. Both lineages showed substitutions at position HA1 165. Conclusions: These results demonstrate that distinct viruses within the two lineages have been co-circulating in the country every year and that there has been a greater evolution of the B/Yamagata viruses. At the same time it is noted that like elsewhere, influenza B viruses in the country have continually been evolving by antigenic drift. In-order to understand whether reassortments have occurred, the study suggests periodic complete genomic sequencing of select.Item Human Bocavirus Infection in Children with Acute Respiratory Infection in Nairobi, Kenya(Scientific Research Publishing, 2013-12) Gachara, George; Symekher, S.L.; Simwa, J.; Gichogo, J.; Rotich, M.; Ng’ayo, M.O.; Magana, J.Background: Acute respiratory infection (ARI) is a leading cause of morbidity and mortality in children under five years of age in developing countries with viruses contributing significantly to this problem. The recently identified par-vovirus, Human Bocavirus (HBoV), has also been associated with ARI. Objective: To determine the frequency of HBoV in patients with ARI. Materials and Methods: Samples from 125 consenting patients with influenza like illness signs and symptoms were collected. DNA was extracted from these samples using the QIAamp DNA blood mini kit (Qiagen, Germany). Conventional PCR was carried out and the amplicons were examined in 2% agarose gels stained with ethidium bromide. This was followed by sequencing of the HBoV positive samples. Results: Twenty one (16.8%) patients were found to have HBoV infection. Males (n = 61.9%) were mainly infected with HBoV. Local HBoV strains had 98.9% - 100% similarities and were found to cluster together with other strains obtained elsewhere. Conclusion: These findings suggest that HBoV plays a role in respiratory tract infections in children in Kenya just like it has been found elsewhere. It also sheds light on multiple infections associated with HBoV infections in KenyaItem Impact of Influenza A(H1N1)pdm09 Virus on Circulation Dynamics of Seasonal Influenza Strains in Kenya.(The American Journal of Tropical Medicine and Hygiene, 2013-03) Gachara, George; Majanja, Janet; Njoroge, Rose N; Achilla, Rachel; Wurapa, Eyako K; Wadegu, Meshack; Mukunzi, Silvanos; Mwangi, Josephat; Njiri, James; Bulimo, Wallace D.We describe virus variations from patients with influenza-like illness before and after the appearance of influenza A(H1N1)pdm09 in Kenya during January 2008-July 2011. A total of 11,592 nasopharyngeal swabs were collected from consenting patients. Seasonal influenza B, A/H1N1, A/H3N2, A/H5N1, and influenza A(H1N1)pdm09 viruses were detected by real-time reverse transcription-polymerase chain reaction. Of patients enrolled, 2073 (17.9%) had influenza. A total of 1,524 (73.4%) of 2,073 samples were positive for influenza A virus and 549 (26.6%) were positive for influenza B virus. Influenza B virus predominated in 2008 and seasonal A(H1N1) virus predominated in the first half of 2009. Influenza A(H1N1)pdm09 virus predominated in the second half of 2009. Influenza A/H3N2 virus predominated in 2010, and co-circulation of influenza A(H1N1)pdm09 virus and influenza B virus predominated the first half of 2011. The reduction and displacement of seasonal A(H1N1) virus was the most obvious effect of the arrival of influenza A(H1N1)pdm09 virus. The decision of the World Health Organization to replace seasonal A(H1N1) virus with the pandemic virus strain for the southern hemisphere vaccine was appropriate for Kenya.Item Molecular Analysis of Human Respiratory Syncytial Virus Group B Strains Isolated in Kenya Before and During the Emergence of Pandemic Influenza A/H1N1(John Wiley & Sons, 2025-02) Wangui,Julia; Gachara, George; Mobegi,Victor; Agoti, Charles; Otieno,James; Opanda,Silvanos; Opot,Benjamin; Ngeranwa,Joseph N.; Njeru,Regina; Bulimo,WallaceBackground: We conducted a retrospective study to explore molecular insights into human respiratory syncytial virus (HRSV) group B strains among patients attending outpatient clinics at government medical facilities both prior and during the onset of Influenza A/H1N1/2009 pandemic outbreak. Methods: We screened 2300 nasopharyngeal swabs using multiplex real time reverse transcriptase polymerase chain reaction. We amplified a segment of the first and second hypervariable regions, as well as the conserved portion of the third domain of the G-gene using HRSV-B specific primers, sequenced by Sanger di-deoxy chain termination method and thereafter analyzed the sequences. Results: We characterized the circulating strains into three known genotypes: SAB4 (1.4%), BA7 (1.4%), and multiple variants of BA9 (97.2%). The majority of BA9 viruses were uniquely Kenyan with only 4% aligning with BA9 lineages found elsewhere. The mean evolutionary rate of the HRSV-B was estimated to be 3.08×10−3 substitutions per site per year. Conclusion: Our findings indicate that the circulating HRSV-B viruses in Kenya underwent a slower evolution during the pe riod of 2007–2010. Additionally, our findings reveal the existence of a unique lineage as well as new variants that have not been reported elsewhere to date.Item Potency status of oral polio virus vaccine among retrieved field samples in Kenya(Elsevier, 2014-04) Gachara, George; Hassan, J. A.; Mbugua, F.; Muchiri, J.; Symekhah, S.; Nakitari, G.; Borus, P.; Kamau, T.; Kombich, J.Background: In 1988 the World Health Organization (WHO) proposed mass immunization campaigns with the trivalent oral polio vaccine (TOPV) among children less than 5 years of age. The Vaccine Vial Monitor (VVM) is a small patch of heat-sensitive material placed on the vaccine vial to register cumulative heat exposure. A direct relationship exists between the rate at which the VVM changes colour and ambient temperature. This in turn affects the potency of the oral polio vaccine. [1] Objectives: To evaluate the status of the cold chain infrastructure in Kenya and to determine the total TOPV virus concentration of retrieved field samples. Methods & Materials: A stratified multi-stage sampling strategy was used leading to selection of 14 health centres this study. A total of 23 TOPV vial samples were collected, separated into individual serotypes generating 69 samples for the potency test. Potency of oral polio vaccine was tested using Karber's formula. This was then compared to the Vaccine Vial Monitor stage of the vials Results: Our study showed that the average potency of polio vaccine serotype1, serotype 2 and serotype 3 were as follows; Comparison between VVM1/VVM 2 and Serotype titres calculated Polio 1 standard (Mean titre) Polio 1 Test (Calculated Mean Titre) Polio 2 standard (Mean titre) Polio 2 test (Calculated mean Titre) Polio 3 Standard (Mean titre) Polio 3 test (Calculated mean Titre) VVM 106 106.05 105 104.98 105.5 105.73 1 106 106.03 105 105.08 105.5 105.35 2 Table options *The mean titre was calculated using the Karber's formula[Log CCID50 = L-d(S-0.5)]]with an allowance of + 0.5 log units Conclusion: On average the vaccine vials used in the study were potent with satisfactory VVM and mean serotype titre. We found that some OPV vials had dissatisfactory VVM stage. Vaccine potency was seen to be directly proportional to VVM stage of vaccine vials. Vaccine vials kept at temperatures below -18 °C had a better VVM leading to a better potency status. Some OPV Samples which had lower titre of serotype 2 were contributed to by the temperature of the equipment they were stored at.Item Seroprevalence and Genotypic Characterization of HBV among Low Risk Voluntary Blood Donors in Nairobi, Kenya(Springer Nature, 2020) Aluora, Patrick Okoti; Muturi, Margaret Wangui; Gachara, GeorgeBackground: Hepatitis B virus (HBV) causes signifcant morbidity and mortality globally primarily due to its ability to cause hepatitis, liver cirrhosis and hepatocellular carcinoma. The Kenya National Blood Transfusion Services screens for Hepatitis B antibodies using the chemiluminescent microparticle immunoassay method. This test does not inform on the genotypic characteristics of the virus or the actual presence of the virus in blood. This study therefore sought to determine the serologic and genotypic profles of HBV circulating among the voluntary blood donors in Nairobi. Methods: Blood samples were collected in plain and EDTA vacutainers and tested for the Hepatitis B surface antigen (HBsAg). HBV DNA was then extracted from plasma, its overlapping P/S gene amplifed and sequenced. The resulting sequences were used to analyze for the circulating genotypes and mutations within the P and S genes. Bivariate statistical analysis was used to determine the association between demographic factors and HBV infection. Results: A seroprevalence of 2.3% (n=7) was reported. The age group 19–28 years was signifcantly associated with HBV infection. Nine samples were positive for HBV DNA; these included 2 HBsAg positive samples and 7 HBsAg negative samples. Genotype A, sub genotype A1 was found to be exclusively prevalent while a number of mutations were reported in the “a” determinant segment of the major hydrophilic region of the S gene associated with antibody escape. RT mutations including mutation rt181T in the P gene conferring resistance against Lamivudine and other ʟ-nucleoside drugs were detected. Conclusion: There is a high prevalence of occult HBV infections among these blood donors and therefore the testing platform currently in use requires revision. Keywords: Occult HBV infection, Hepatitis, Liver cirrhosis, Hepatocellular carcinoma, Mutations, Escape mutations, UndetectableItem Serotype and Genetic Diversity of Human Rhinovirus Strains that Circulated in Kenya in 2008(Wiley Open Access, 2016) Milanoi, Sylvia; Ongus, Juliette R.; Gachara, George; Coldren, Rodney; Bulimo, WallaceBackground: Human Rhinoviruses (HRVs) are a well-established cause of the common cold and recent studies indicated that they may be associated with severe acute respiratory illnesses (SARIs) like pneumonia, asthma and bronchiolitis. Despite global studies on the genetic diversity of the virus, the serotype diversity of these viruses across diverse geographical regions in Kenya has not been characterized. Objectives: This study sought to characterize the serotype diversity of HRV strains that circulated in Kenya in 2008. Methods: A total of 517 archived nasopharyngeal samples collected in a previous respiratory virus surveillance program across Kenya in 2008 were selected. Participants enrolled were outpatients who presented with influenza-like (ILI) symptoms. Real time RT-PCR was employed for preliminary HRV detection. HRV positive samples were amplified using RT-PCR and thereafter the nucleotide sequences of the amplicons were determined followed by phylogenetic analysis. Results: Twenty five percent of the samples tested positive for HRV. Phylogenetic analysis revealed that the Kenyan HRVs clustered into three main species comprising HRV A (54%), HRV-B (12%) and HRV C (35%). Overall, 20 different serotypes were identified. Intra-strain sequence homology among the Kenyan strains ranged from 58% to 100% at the nucleotide level and 55% to 100% at the amino acid level. Accepted Article This article is protected by copyright. All rights reserved. Conclusion: These results show that a wide range of HRV serotypes with different levels of nucleotide variation were present in Kenya. Furthermore, our data show that HRVs contributed substantially to influenza-like illness in Kenya in 2008.Item Whole genome characterization of human influenza A(H1N1)pdm09 viruses isolated from Kenya during the 2009 pandemic(Elsevier, 2016) Gachara, George; Symekher, S.; Otieno, M.; Magana, J.; Opot, B.; Bulimo, W.An influenza pandemic caused by a novel influenza virus A(H1N1)pdm09 spread worldwide in 2009 and is estimated to have caused between 151,700 and 575,400 deaths globally.Whilewhole genome data on newvirus enables a deeper insight in the pathogenesis, epidemiology, and drug sensitivities of the circulating viruses, there are relatively limited complete genetic sequences available for this virus from African countries. We describe herein the full genome analysis of influenza A(H1N1)pdm09 viruses isolated in Kenya between June 2009 and August 2010. A total of 40 influenza A(H1N1)pdm09 viruses isolated during the pandemic were selected. The segments from each isolate were amplified and directly sequenced. The resulting sequences of individual gene segments were concatenated and used for subsequent analysis. These were used to infer phylogenetic relationships and also to reconstruct the time of most recent ancestor, time of introduction into the country, rates of substitution and to estimate a time-resolved phylogeny. The Kenyan complete genome sequences clustered with globally distributed clade 2 and clade 7 sequences but local clade 2 viruses did not circulate beyond the introductory foci while clade 7 viruses disseminated country wide. The time of the most recent common ancestor was estimated between April and June 2009, and distinct clusters circulated during the pandemic. The complete genome had an estimated rate of nucleotide substitution of 4.9 × 10−3 substitutions/site/year and greater diversity in surface expressed proteins was observed. We show that two clades of influenza A(H1N1)pdm09 virus were introduced into Kenya from the UK and the pandemic was sustained as a result of importations. Several closely related but distinct clusters co-circulated locally during the peak pandemic phase but only one cluster dominated in the late phase of the pandemic suggesting that it possessed greater adaptability