Bio

Prof. Bulimo's Bio

Is an Associate Professor of Biochemistry at the department of Biochemistry, School of Medicine in the University of Nairobi.  He is has supervised more than fifteen MSc and seven PhD students and currently supervises five PhDs in the field of Molecular virology and molecular sciences in general. He is a molecular virologist whose research work is based at the National Influenza Centre, Kenya Medical Research Institute (KEMRI) working in collaboration with the GEIS program, USAMRU-Kenya, on influenza and other respiratory pathogen surveillance in Kenya.

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Publications


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2017

Richard, K, Faith O, Margaret O, Anne N, Wallace B.  2017.  Effect of ABCB1 C3435T Polymorphism on Clinical Outcomes in Kenyan HIV Patients on Lopinavir-Based Regimens. Journal of Pharmacy and Pharmacology. 7:478-488. Abstract13-jpp2017041704.pdf

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2016

Wadegu, M, Wamunyokoli F, Osanjo G, Opanda S, Majanja J, Coldren R, Bulimo W.  2016.  Molecular Surveillance of Adamantane Resistance among Human Influenza A Viruses Isolated in Four Epidemic Seasons in Kenya. African Journal of Pharmacology and Therapeutics. 5:181-192., Number 3 Abstractwadegu_et_al_2016.pdf

Background: Adamantanes impede influenza A virus replication and are important in the treatment and prophylaxis of disease caused by these viruses. Genotypic characterization of influenza A viruses for mutations associated with resistance to adamantanes has not been fully investigated in Kenya. Objective: To characterize susceptibility of influenza A virus subtypes that circulated in Kenya from 2008-2011 to adamantanes. Methods: Archived influenza A virus strains obtained from 2008 to 2011 were propagated in MDCK cells prior to sequencing of the matrix and hemagglutinin gene segments, followed by bioinformatics analyses. Results: Ninety two virus strains consisting of 21 A/H3N2, 18 A/H1N1 and 53 A/H1N1pdm09 were analyzed. All A/H3N2 and A/H1N1pdm09 viruses displayed resistance to adamantanes due to the S31N/S31D amino acid substitution. All A/H1N1pdm09 virus strains belonged to the N-lineage characterized by S203T amino acid substitution in the HA1. All A/H1N1 viruses were sensitive to adamantane and were characterized by K140E amino acid substitution in the HA1. Conclusion: All Kenyan influenza A/H3N2 and A/H1N1pdm09 virus strains were resistant to adamantanes while seasonal A/H1N1 strains were sensitive to these drugs. During the study period, Amantadine and Rimantadine were inappropriate for prophylaxis and treatment of influenza disease caused by A/H3N2 and A/H1N1pdm09 virus subtypes in Kenya.

Mbui, FM, Achilla RA, Coldren RL, Bulimo WD.  2016.  Serotype Diversity of Respiratory Human Adenoviruses amongst Pediatric Patients from Western Kenya, 2010-2012. African Journal of Pharmacology and Therapeutics. 5(3):142-148., Number 3 Abstractmbui_et_al._2016.pdf

Background: Respiratory illnesses are common among pediatric patients in Kenya, and many are attributed to viral causes. However, there is limited knowledge of the diversity of viral etiologies associated with these illnesses. Objective: To characterize respiratory adenoviruses isolates using serological and molecular approaches. Methods: A total of 1,879 samples were collected from symptomatic pediatric patients seeking medical care at New Nyanza Provincial General Hospital during the period of June 2010 to June 2012 and screened for adenoviruses as well as other respiratory viruses. Sixteen respiratory human adenoviruses (HAdVs) were isolated in Hep 2 cell culture and characterized hem using Immunofluorescence Assay, viral DNA amplification, sequencing and phylogenomics. Results: Phylogenetic characterization of the HAdVs using the hyper variable region 7 of the hexon gene identified HAdV B and C as the major species associated with respiratory infections during the study period. Amongst these, a single B-type and four C-type serotypes were identified. The serotype distribution consisted of 31% HAdV B7, 25% HAdV C1, 25% AdV C2, 6% HAdV C5, and 13% HAdV C6. Positive selection was observed in the nucleotide sequences from HAdV B7 and HAdV C5 signaling evolution of these two serotypes. Conclusion: These finding may be useful to policy makers regarding appropriate strain selection for vaccination in Kenya.

Juma, BW, Kariuki S, Waiyaki PG, Mutugi MW, Bulimo WD.  2016.  Molecular characterization of fluoroquinolone resistance genes in isolates obtained from patients with diarrhea in Machakos District Hospital, Kenya. African Journal of Pharmacology and Therapeutics. 5:118-127., Number 3 Abstractjuma_b_et_al_2016.pdf

Background: Diarrhea caused by Enterobacteriaceae such as Shigella species and Escherichia coli (E. coli) is endemic throughout the world, and is one of the most important causes of global childhood mortality and morbidity. There is a range of antibiotics that can be used for treatment among them quinolones. However, there is emerging increase in microbial resistance to quinolones use, with E. coli and Shigellae among the species of bacteria commonly associated with quinolone resistance. Objective: To investigate the prevalence of quinolone resistance genes in Shigellae and E. coli from patients presenting with diarrhea in Machakos District Hospital. Methods: Bacteria isolates were identified to species level by biochemical methods and serology and thereafter tested for 12 different antibiotics including quinolones, cephalosporins and aminoglycosides. Those resistant to quinolones with a zone diameter of ≤20 mm were tested for the presence of quinolone resistance genes using PCR. The gyrA resistance genes were further analyzed by sequencing to determine mutations within the quinolone resistance regions. Results: There were different E. coli pathotypes and Shigellae spp. They resisted more than four antibiotics: Ciprofloxacin (4%), (Chloramphenical (28%), Cotrimoxazole (78%), Co-amoxilav (70%) Erythromycin (98%) Cefotoxime (18%) and Tetracycline (56%). Mutations responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of E. coli and Shigella spp were: gyrA (17/30, 36%) gyrB (7/30, 23.3%) topoisomerase (parC 3/30, 10%) parE (3/30, 10%). Discussion: There is an increase in fluoroquinolone resistance in Shigellae and E.coli which points to a major challenge in current treatment strategies. In addition, detection of high resistance found to commonly used antibiotics should serve as a warning call for close surveillance and understanding of the epidemiology of the resistance. Key words:

Otieno, NM, Apolot OF, Dimbuson BW, Christina M, Nkatha GA, Margaret O.  2016.  Impact of First Line Antiretroviral Therapy on Clinical Outcomes Among HIV-1 Infected Adults Attending One of the Largest HIV Care and Treatment Program in Nairobi Kenya. Journal of AIDS & Clinical Research. 7:3-7., Number 10 Abstractimpact-of-first-line-antiretroviral-therapy-on-clinical-outcomes-amonghiv1-infected-adults-attending-one-of-the-largest-hiv-care-a-2155-6113-1000615.pdf

Objective: This study evaluated the immunologic (CD4 cell count), virological (HIV RNA viral load), hepatic (alanine and aspartate aminotransferase - ALT and AST), renal (creatinine) and hematological (hemoglobin -HB, White Blood Cell - WBC, Lymphocytes - LYM and platelets - PLT) response to a six months ART treatment among HIV participants in Nairobi Kenya. Methods: Blood samples were obtained from 599 consenting HIV infected participants receiving HIV treatment in Nairobi. CD4 cell counts were measured using flow cytometer and viral load determined using real-time polymerase chain reaction. The blood hematology, liver and kidney function tests were also measured. One-way ANOVA and Linear regression analysis were conducted. Results: The median age at ART initiation was 41 years (IQR 35-47 years). The majority of participants (60.3%) were female and 56.1% started on regimens with 2 NRTIs and efavirenz based NNRTI. About 40% of the participants were failing treatment 6 month post ART initiation. The CD4 count significantly increased at the 6-month post ART initiation (301.7 ± 199.4 to 329.4 ± 305.8; P<0.05). Hepatotoxicity (ALT and AST levels >5 times the upper limit of normal - ULN) and renal abnormalities (elevated serum creatinine levels) were all high at month 6 compared to baseline; ALT (2.5 to 10.5%), AST (5.3 to 23.4%) and creatinine (63.4 to 68.84%). Fewer participants at month 6 had anemia (29.4% verses 56.4%), leucopenia (42.4% vs. 46.9%) and thrombocytopenia (6.5% vs. 84.1%) compared to baseline. In multivariable models, baseline levels of this parameter, ART regimen and duration with HIV at ART initiation were the most important determinant of month 6 levels. Conclusion: These data demonstrate sustained immunologic/virologic response to ART among participants remaining on therapy. Anemia, leucopenia and thrombocytopenia were minimized with marginal hepatotoxicity and renal impairment seen. Interventions leading to earlier HIV diagnosis and initiation of ART could substantially improve patient outcomes in Kenya.

Chebon, LJ, Ngalah BS, Ingasia LA, Juma DW, Muiruri P, Cheruiyot J, Opot B, Mbuba E, Imbuga M, Akala HM, Bulimo W, Andagalu B, Kamau E.  2016.  Genetically Determined Response to Artemisinin Treatment in Western Kenyan Plasmodium falciparum Parasites, 09. PLoS ONE. 11:1-19., Number 9: Public Library of Science Abstractchebon_et_al_2016.pdfWebsite

Genetically determined artemisinin resistance in Plasmodium falciparum has been described in Southeast Asia. The relevance of recently described Kelch 13-propeller mutations for artemisinin resistance in Sub-Saharan Africa parasites is still unknown. Southeast Asia parasites have low genetic diversity compared to Sub-Saharan Africa, where parasites are highly genetically diverse. This study attempted to elucidate whether genetics provides a basis for discovering molecular markers in response to artemisinin drug treatment in P. falciparum in Kenya. The genetic diversity of parasites collected pre- and post- introduction of artemisinin combination therapy (ACT) in western Kenya was determined. A panel of 12 microsatellites and 91 single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome were genotyped. Parasite clearance rates were obtained for the post-ACT parasites. The 12 microsatellites were highly polymorphic with post-ACT parasites being significantly more diverse compared to pre-ACT (p < 0.0001). The median clearance half-life was 2.55 hours for the post-ACT parasites. Based on SNP analysis, 15 of 90 post-ACT parasites were single-clone infections. Analysis revealed 3 SNPs that might have some causal association with parasite clearance rates. Further, genetic analysis using Bayesian tree revealed parasites with similar clearance phenotypes were more closely genetically related. With further studies, SNPs described here and genetically determined response to artemisinin treatment might be useful in tracking artemisinin resistance in Kenya.

Milanoi, S, Ongus JR, Gachara G, Coldren R, Bulimo W.  2016.  Serotype and genetic diversity of human rhinovirus strains that circulated in Kenya in 2008. Influenza and Other Respiratory Viruses. :n/a–n/a. Abstractmilanoi_et_al-2016-influenza_and_other_respiratory_viruses-2.pdfWebsite

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Gachara, G, Symekher S, Otieno M, Magana J, Opot B, Bulimo W.  2016.  Whole genome characterization of human influenza A(H1N1)pdm09 viruses isolated from Kenya during the 2009 pandemic. Infection, Genetics and Evolution. :-. Abstractwhole_genome_of_pandemic_h1n1_paper.pdfWebsite

Abstract 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. While whole genome data on new virus 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.

Juma, BW, Kariuki S, Waiyaki PG, Mutugi MM, Bulimo WD.  2016.  The prevalence of TEM and SHV genes among Extended-Spectrum Beta-Lactamase-producing Klebsiella pneumoniae and Escherichia coli.. African Journal of Pharmacology and Therapeutics . 5(1):1-7.juma_et_al_2016.pdf
Opanda, S, Wamunyokoli F, Khamadi S, Coldren R, Bulimo W.  2016.  Genotyping of enteroviruses isolated in Kenya from pediatric patients using partial VP1 region. SpringerPlus. 5:158., Number 1 Abstractopanda_et_al_2016.pdfWebsite

Enteroviruses (EV) are responsible for a wide range of clinical diseases in humans. Though studied broadly in several regions of the world, the genetic diversity of human enteroviruses (HEV) circulating in the sub-Saharan Africa remains under-documented. In the current study, we molecularly typed 61 HEV strains isolated in Kenya between 2008 and 2011 targeting the 3'-end of the VP1 gene. Viral RNA was extracted from the archived isolates and part of the VP1 gene amplified by RT-PCR, followed by sequence analysis. Twenty-two different EV types were detected. Majority (72.0%) of these belonged to Enterovirus B species followed by Enterovirus D (21.3%) and Enterovirus A (6.5%). The most frequently detected types were Enterovirus-D68 (EV-D68), followed by Coxsackievirus B2 (CV-B2), CV-B1, CV-B4 and CV-B3. Phylogenetic analyses of these viruses revealed that Kenyan CV-B1 isolates were segregated among sequences of global CV-B1 strains. Conversely, the Kenyan CV-B2, CV-B3, CV-B4 and EV-D68 strains generally grouped together with those detected from other countries. Notably, the Kenyan EV-D68 strains largely clustered with sequences of global strains obtained between 2008 and 2010 than those circulating in recent years. Overall, our results indicate that HEV strains belonging to Enterovirus D and Enterovirus B species pre-dominantly circulated and played a significant role in pediatric respiratory infection in Kenya, during the study period. The Kenyan CV-B1 strains were genetically divergent from those circulating in other countries. Phylogenetic clustering of Kenyan EV-D68 strains with sequences of global strains circulating between 2008 and 2010 than those obtained in recent years suggests a high genomic variability associated with the surface protein encoding VP1 gene in these enteroviruses.

Sipulwa, LA, Ongus JR, Coldren RL, Bulimo WD.  2016.  Molecular characterization of human coronaviruses and their circulation dynamics in Kenya, 2009-2012., 2016. Virology journal. 13(1):18. Abstractsipulwa_et_al_2016.pdf

Human Coronaviruses (HCoV) are a common cause of respiratory illnesses and are responsible for considerable morbidity and hospitalization across all age groups especially in individuals with compromised immunity. There are six known species of HCoV: HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, MERS-CoV and SARS-HCoV. Although studies have shown evidence of global distribution of HCoVs, there is limited information on their presence and distribution in Kenya.

2015

Ingasia, LA, Akala HM, Imbuga MO, Opot BH, Eyase FL, Johnson JD, Bulimo WD, Kamau E.  2015.  Molecular Characterization of the Cytochrome b Gene and In Vitro Atovaquone Susceptibility of Plasmodium falciparum Isolates from Kenya., 2015 Mar. Antimicrobial agents and chemotherapy. 59(3):1818-21. Abstractingasia_molecular_characterization_of_the_cytochrome_b_gene_and_in_vitro_atovaquone_susceptibility_of_plasmodium_falciparum_isolates_from_kenya.pdf

The prevalence of a genetic polymorphism(s) at codon 268 in the cytochrome b gene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227 Plasmodium falciparum parasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC50) and parasite genetic polymorphism; mutant alleles had higher IC50s than the wild type.

Wagaiyu, EG, Bulimo WD, Wanzala PN, Kaimenyi JT.  2015.  Interleukin - 1 Polymorphisms And Chronic Periodontitis In A Rural Kenyan Population. IOSR Journal of Dental and Medical Sciences (IOSR-JDMS). 14(2):36-43. Abstracti014213643.pdf

Abstract: Susceptibility to Chronic periodontitis (CP) has been associated with genetic polymorphisms in the proinflammatory cytokine, interleukin - 1 (IL - 1A and IL - 1B) isoforms but little is known about their role in rural Africans. Therefore, the aim of this study was to resolve the association between genetic polymorphisms in IL - 1A and IL - 1B isoforms and chronic periodontitis in the Taita people of Kenya. Methods: This was a case - control study. After informed consent, a clinical examination was then conducted which included assessment of dental plaque, gingival inflammation, pocket depths and gingival recession. Buccal swab samples were then obtained. Deoxyribonucleic acid (DNA) was isolated from the swabs using QIAamp DNA purification protocol followed by polymerase chain reaction (PCR) amplification using specific primers to IL - 1A (rs 1800587(- 889) & rs17561 (+48 45)) and IL - 1B (rs 16944 (- 511) & rs 11443624 (+3954). The amplicons were digested using Nco1, Fnu4H1, Ava1 and Taq1 respectively. Restriction fragment length polymorphisms (RFLP) were recorded. Association analyses of the RFLP and clinical data were carried out. Results: No deviation from the Hardy Weinberg equilibrium was observed. Carriage of allele2 at IL - 1B +3954 (i.e. combination of ‘2 - 2’ or‘1 - 2’ at locus +3954) was associated with CP in the Taita participants (OR = 1.94, 95%CI=1.01 - 3.70, p=0.045). There were no confounders or effect modifiers in the Taita participants and no association with severity of CP was observed in this population. None of the composite genotypes were associated with CP in the Taita participants. Haplotype 3 (i.e. allele1 at all the four loci) was significantly associated with CP amongst the Taita (O R=2.4, 95%CI=1.1 - 5.14, p=0.022). Conclusion: The significant association of allele2 at IL - 1B +3954 with CP in the Taita participants confirmed the importance of this genotype in disease pathogenesis. Keywords: chronic periodontitis, IL -1 polymorphisms, Rural Kenya, Taita

2014

B.W., J, P.G. W, W.D. B, E.K. W, M.M. M, S. K.  2014.  Molecular detection of enterotoxigenic Escherichia coli surface antigens from patients in Machakos District Hospital, Kenya.. East and Central Africa Medical Journal. 1:62-68., Number 2 Abstractmolecular_detection_of_enterotoxigenic.pdf

Introduction: Enterotoxigenic Escherichia coli (ETEC) is known for its public health importance globally, however, a protective vaccine is yet to be developed. Information regarding the immunology of ETEC’s virulence proteins that can lead to studies on vaccine development such as the heat stable toxins (ST), heat-labile toxin (LT), colonization factors (CFs) and coli surface antigens (CS) from many regions of the world is available. In Kenya, specific CFAs and CS have not been adequately characterized. This study looked at the surface antigens of diarrhoeagenic E. coli in search of indicators for vaccine materials development. Methodology: Multiplex polymerase chain reaction assay were employed to detect diarrhoeagenic E. coli pathotypes and enteroxigenic Escherichia coli surface antigens/ colonization factors antigens from 300 patients in Machakos Hospital, Kenya. Results: Enteroaggrigative Escherichia coli was the most predominant (13.7%) followed by ETEC (11%), Enteroinvesive E. coli (8.3%) and Enteropathogenic E. coli (4.3%). Among the colonization factor anti¬gens, CFAI was detected at 25 (23%), CS1, CSII 2(1.9%), CS3 1(0.9%), CS6 13(12%), CS7 2 (1.9%), CS12 1(0.9%), CS19 11 (10.25%) and those without colonization factor 37 (34.3%). Conclusions: ETEC isolates carrying ST or STLT toxins had more recoverable CFs than those with LT alone (P<0.05). The CS6 is increasing and CS19 was detected for the first time in Kenya and shown to be persistent adhesins. These may be further investigated as possible candidates for the formulation of a novel vaccine for the prevention of diarrhoea in Kenya and the region.

Omucheni, DL, Kaduki KA, Bulimo WD, Angeyo HA.  2014.  Application of principal component analysis to multispectral-multimodal optical image analysis for malaria diagnostics., 2014 Dec 11. Malaria journal. 13(1):485. Abstract1475-2875-13-485.pdf

Multispectral imaging microscopy is a novel microscopic technique that integrates spectroscopy with optical imaging to record both spectral and spatial information of a specimen. This enables acquisition of a large and more informative dataset than is achievable in conventional optical microscopy. However, such data are characterized by high signal correlation and are difficult to interpret using univariate data analysis techniques.

Wagaiyu, Evelyn, G, Bulimo WD, Wanzala, Peter N, Kaimenyi, Jacob T.  2014.  Genetic polymorphisms in IL-1A and IL-1B isoforms and their associations with chronic periodontitis in the Swahili people of Kenya, 4 November -2014. IOSR Journal of Dental and Medical Sciences. 13:07-15., Number 11 Abstractb0131120715.pdfWebsite

Genetic polymorphisms in interleukin-1 (IL-1A and IL-1B) isoforms have been associated with Chronic Periodontitis (CP) in Caucasians, Asians and Arabs but little is known about their role in Africans. Therefore, this study was to resolve the association between genetic polymorphisms in IL-1A and IL-1B isoforms and chronic periodontitis in a Kenyan community. Methods: This was a case-control study. After informed consent, a clinical examination was conducted. Buccal swab samples were then obtained. Deoxyribonucleic acid was isolated from the swabs using QIAamp DNA purification protocol followed by polymerase chain reaction amplification using specific primers to IL-1A (loci -889 & +4845) and IL-1B (loci -511 & +3954). The amplicons were digested using Nco1, Fnu4H1, Ava1 and Taq1 respectively. Restriction fragment length polymorphisms (RFLP) were recorded. Association analyses of the RFLP and clinical data were carried out. Results: After screening 523 Swahili participants from old town Mombasa, 100 cases and 100 controls were included in the study. There was more plaque present in cases than controls with OR = 9.2 (95%CI = 3.7-23.1), p<0.001. Mild Chronic Periodontitis was present in 9(9%) participants, moderate CP in 35(35%) and the severe form of CP in 56(56%). Carriage of allele 1 at IL-1A-889 amongst the Swahili participants was associated with Chronic Periodontitis (OR = 3.16, 95%CI=1.644-6.083, p<0.001). Allele 1 at locus IL-1A-889 was associated with mild, (OR=5.2, 95%CI=1.445-18.71, p=0.005), moderate (OR=4.51, 95%CI = 2.08-9.79, p<0.001) and severe disease (OR=2.19, 95%CI=1.013-4.738, p=0.042). Furthermore, plaque level was an effect modifier in the association between IL-1B-511 polymorphism and CP. Conclusions: Increased susceptibility to Chronic Periodontitis was found in Swahili participants with allele 1 at IL-1A-889.

Wangui, J, Bulimo W, Nokes J, Wurapa E, Nyaigoti C, Otieno J, Opot B, Achilla R, Coldren R.  2014.  Genotypic Diversity of HRSV-A in out-patients with Acute Respiratory Infections in Kenya 2007-2010, 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium.. , Hilton Hotel; Nairobi, Kenya Abstract

Introduction: Human Respiratory Syncytial Virus is the leading viral cause of bronchiolitis and pneumonia in children and infants. Amongst adults, the elderly and the immuno-compromised are also at risk of severe RSV. RSV is classified into two genetically distinct groups: RSV A and RSV B. In the case of RSV A, 11 genotypes have been reported to date. Amongst these are three new variants (NA1, NA2 and ON1). Appearance of new genotypes has been accompanied by rapid spread and near replacement of existing variants. There is need to constantly re-examine the nature of genotypic changes amongst circulating viruses. Objective: To assess the geographic distribution and genotypic diversity of RSV A in outpatients presenting with Influenza-Like-Illness from 2007 and 2010 across Kenya.Methods: Nasopharyngeal (NP) swabs were obtained from subjects ≥2 months of age presenting with fever ≥38ºC and cough or sore throat at district hospitals under surveillance across 5 regions of Kenya. RNA was extracted from the NP samples and screened using multiplex real-time RT-PCR. A 490bp fragment of the RSVA G-gene was amplified by nested RT-PCR and nucleotide sequencing carried out using the Sanger dideoxy termination method. The sequences were analyzed using a suite of bioinformatics software. Results: 159 NP samples were positive for RSV A, of which, 130 were genotyped; 21 (16%) were GA5, 39 (30%) GA2, 66 (50%) NA1, 1 GA1 and 3 (2%) GA2 variants closely related to NA2. GA2 and NA1 genotypes were identified in the highlands from 2007 – 2010. In 2009, NA1 was highest in Nairobi and Coast regions (11; 17%). GA2 genotype was highest in Northeastern (8; 21%) and was most prevalent in 2009 (44%). The Western region had the highest prevalence of GA5 in 2008 (6; 29%). Conclusion: Five genotypes were in circulation across the country in 2007 and of these NA1 genotype increased dominance year by year and spread to all the regions by 2009. These data provide insight into the dynamics of persistence of RSV strains within a national geographical area.

Wadegu, M, Bulimo W, Osanjo G, Wamunyokoli F, Coldren R.  2014.  Neuraminidase Inhibitor Susceptibility of Influenza A isolates obtained in Kenya, 2008-2011.. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium . , Hilton Hotel; Nairobi, Kenya Abstract

Introduction: Neuraminidase inhibitors mainly oseltamivir and zanamivir function both as prophylactic and treatment agents for influenza infections. We characterized the antiviral susceptibility of the 2008-2011 influenza A viruses circulating in Kenya by combining both the genotypic data involving known molecular markers in neuraminidase (NA) protein responsible for drug resistance and IC50 data generated from NA inhibition assays.Materials and Methods: Nasopharyngeal swab specimen from consenting outpatients of age ≥ 2 months were obtained and transported to the National Influenza Center. RT-PCR amplification of NA gene segments was performed on the virus isolates prior to nucleotide sequencing using the BigDye chemistry. Sequences were analyzed using a suite of bioinformatics tools. Drug susceptibility was determined by fluorescent enzyme inhibition assay with known NA inhibitor-resistant and inhibitor-sensitive viruses. IC50 values were determined using curve fitting software, Grafit 7.0.Results and Discussion: Out of 836 influenza A virus isolates obtained (2008-2011), 108 (13%) were analyzed for markers of resistance to NA inhibitors. 64% (7/11) of the 2008 seasonal influenza A/H1N1 isolates depicted oseltamivir resistant marker H275Y. Influenza A/H3N2 and A/ (H1N1) pdm09 isolates lacked the H275Y mutation. A total of 28 isolates were further subjected to phenotypic susceptibility assay. The mean zanamivir IC50s were 1.75nM, 2.53nM and 1.84nM for the subtypes sH1N1, pH1N1 and H3N2 respectively. Eight of the 2008-2009 sH1N1 isolates analyzed showed highly reduced sensitivity to oseltamivir with IC50s ranges from 73nM-984nM. Pandemic A/H1N1 and A/H3N2 strains obtained between 2009-2011and 2008-2011 respectively depicted normal sensitivity. The 2011, WHO range and median IC50 values for oseltamivir carboxylate were 257nM-3455nM and 458.2nM; 132nm-2179nM and 191.3nM; 23-378 and 42.3nM for the mutant subtypes sH1N1, pH1N1and sH3N2 respectively.Conclusion: Overall genotypic and phenotypic data demonstrate oseltamivir resistance in the 2008-2009, sH1N1 viruses. The H275Y mutation increased the IC50 by 50-100 fold.

Sipulwa, LA, Ongus JR, Bulimo WD.  2014.  Molecular characterization of human coronavirus circulating in Kenya, 2009-2012, 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium .. , Hilton Hotel; Nairobi, Kenya Abstract

Background: The genome of a human coronavirus (HCoV) is composed of a linear, single-stranded, non-segmented, positive-sense RNA of 27-32 kb. The pol gene of HCoV found in ORF1ab, is a good molecular chronometer for molecular characterization of HCoV types because in a region of ~900bp towards the 5’, it contains two conserved flanks with a hypervariable middle. Thus, this region of the pol gene has been used to type all known HCoVs. Thus, molecular typing using this gene segment corresponds well to the classical typing based on serological cross-reactivity which groups CoVs into four groups. HCoVs cause a variety of mild and severe respiratory tract diseases including SARS and MERS. To date there are six known types of HCoVs. Although studies have shown evidence of global distribution of HCoVs and the diseases they cause, there is limited information on their presence, distribution and genetic characteristics in Kenya.Objective: To isolate, type and infer the genetic diversity of HCoVs that circulated in Kenya from 2009-2012 using the pol gene.Methods: Archived nasopharyngeal (NP) swab specimens from consenting outpatients aged ≥2 months were screened by real-time RT-PCR using HCoV-specific primers. Positive specimens were inoculated onto LLCMK2 monolayers to isolate the virus. RNA was extracted from virus isolates followed by PCR amplification of the HCoV pol gene using gene-specific primers. Nucleotide sequencing of amplicons was carried out using the BigDye chemistry prior to analyses using a suite of bioinformatics tools.Results and discussion: 29 of the 417 (7%) NP samples tested were positive for HCoV. The highest proportion (33%) were HCoV-NL63 followed by HCoV-HKU1 (30%), HCoV-OC43 (27%) and HCoV-229E (10%) respectively. SARS-CoV and MERS-CoV were not detected. Of the 29 positive samples, 14 (47%) yielded viral isolates for nucleotide sequencing. Sequence and Phylogenetic analyses identified 4 HCoV-HKU1, 5 HCoV-NL63, 4 HCoV-OC43 and 3 HCoV-229E. Mutation analyses revealed that 2/3 of the Kenyan HCoV-229E had Y4682L and one had F4821T amino acid substitutions relative to the prototype (GenBank Acc. No. NC_002645.1) The other human coronavirus types (HKU1, NL63 & OC43) had a few disparate silent mutations and were phenotypically identical to their respective prototypes. Conclusion: Four types of HCoVs circulated in Kenya during the study period. Genetic diversity in the hypervariable region of the pol gene was only observed in the HCoV-229E.

Opanda, S, Wamunyokoli F, Khamadi S, Rodney C, Bulimo W.  2014.  Molecular typing of respiratory human enteroviruses isolated in Kenya between 2008 -2011 using the VP1 gene, 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium . , Hilton Hotel; Nairobi, Kenya Abstract

Background: Human enteroviruses (genus Enterovirus, family Picornaviridae) are common infectious agents grouped into HEV-A, HEV-B, HEV-C and HEV-D species. They comprise >100 serotypes and are responsible for a wide range of human pathologies including upper and lower respiratory tract infections. There’s scanty information about serotype diversity of HEV circulating in Kenya.Objective: To molecularly type human enteroviruses isolated in Kenya between 2008 and 2011 using hypervariable 3’-end of the VP1 gene. Methods: A total of 200 HEV isolates obtained in the country from nasopharyngeal specimens were analyzed. Viral RNA was extracted and partial VP1 gene amplified using RT-PCR followed by sequencing. The resulting VP1 sequences were evaluated by sequence homology and phylogenetic analysis relative to those of prototypes retrieved from GenBank. Results: Overall, 22 different enteroviral serotypes were detected. The majority (72%) of the serotypes were from HEV-B species (72%) followed by HEV-D (21.3%) and HEV-A (6.5%). None of the identified serotypes belonged to HEV-C species. The most frequently detected serotypes we enterovirus-68 (EV68), Coxsackie-virus types –B2, -B1, -B4 and B3. The most prevalent serotypes were enterovirus-68 (EV68), Coxsackievirus types -B2, -B1, -B4 and -B3. Conclusions: Findings from this study demonstrate the existence of high serotype diversity among HEVs that circulated in Kenya between 2008 and 2011. The viruses belonging to HEV-A, HEV-B and HEV-D species played a key role in enteroviral infections in the country during this period. The absence of HEV-C known to frequently recombine with poliovirus vaccine strains indicates a low risk of emergence of vaccine derived poliovirus (VDPV) in Kenya. Typing of HEV is important in determining temporal and spatial patterns of the circulating serotypes. This information is necessary for healthcare planning and outbreak investigation studies.

Ocholla, S, Jumba G, Bulimo W, Achilla R, Wadegu MO, Mukunzi S, Majanja JM, Opot B, Osuna F, Muthoni J, Njiri J, Mwangi J, Kibet K, Coldren R.  2014.  The Appropriateness of WHO influenza B vaccine component to Kenya in 2011-2012, 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium . , Hilton Hotel; Nairobi, Kenya Abstract

Background: In the 1980’s, influenza B viruses were discovered to belong to two evolutionary groupings (B/Victoria/2/87-like viruses and B/Yamagata/16/88-like viruses) that continue to co-circulate globally in the human population. These viruses exist as independent lineages. Information about lineage of circulating influenza B viruses in a country is important for determination of the appropriateness of either a trivalent or a quadrivalent vaccine composition for that country.Objective: To genetically analyze the HA1 of influenza B viruses isolated in Kenya during the 2011-2012 season with reference to WHO vaccine strains recommended for Kenya.Method: Nasopharyngeal swab specimens obtained from patients meeting WHO definition criterion for ILI were screened by real-time PCR for influenza B viruses. Influenza B virus positive samples were inoculated onto MDCK cells and the lineages of the isolates determined by hemagglutination inhibition assay (HAI). To confirm the lineages, HA1 gene segments of selected isolates were amplified by PCR and sequenced and analyzed using bioinformatics tools.Results: Phylogenetic analyses revealed that all the 24 influenza B viruses that circulated between 2011- 2012 were closely related to B/Brisbane/60/2008 vaccine Strain. Comparison of the HA1 amino acid sequences of influenza B viruses with the reference vaccine strain (B/Brisbane/60/2008 lineage) revealed substitutions at 19 amino acid positions. FLU-MBG-11-02-010 had R188K amino acid change in the 120-loop antigenic receptor binding site relative to B/Brisbane vaccine reference strain. FLU-MBG-12-05-011 had V124I amino acid change in the 120-loop antigenic receptor binding site relative to the vaccine reference strain. The majority (87.5%) had I146V/A amino acid change in the 150-loop antigenic receptor binding site. All the Kenyan isolates had D197N amino acid change in the 190-helix antigenic receptor site. The other fifteen positions that showed polymorphisms were outside of the antigenic sites with these mutations being randomly distributed among the isolates.Conclusion: Our study provides evidence that the WHO vaccine strain recommendations for the southern hemisphere were appropriate for use in Kenya.

Nyambura, J, Achilla R, Mitei K, Mukunzi S, Njiri J, Coldren R, Bulimo W.  2014.  Co-circulation of Human Parainfluenza viruses in Kenya, Jan 2013-Sep 2013, 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium . , Hilton Hotel; Nairobi, Kenya Abstract

Background: Human parainfluenza viruses (HPIVs) belong to the paramyxoviridae family. HPIV is the major cause of croup in which type 1 is most frequent cause, followed by type 3 and type 2 respectively. Surveillance has shown that Human Parainfluenza viruse are a major cause of respiratory infections in Kenya. In January 2013 through an existing influenza surveillance network at the Kenyan National Influenza center, we screened for parainfluenza and other non-influenza respiratory viruses. This was done within the designated Influenza network made up of eight sentinel sites. Objective: The objective of this study was to monitor and document circulation of Human parainfluenza viruses in Kenya in the period January–September 2013. Materials and Methods: Specimens were collected from the nasopharynx using a flocked swab from consenting patients meeting the WHO influenza-like-illness (ILI) case definition. Specimens were transported to the NIC while observing the cold chain and inoculated into LLCMK2 cell line. After incubation and observation for cytopathic effect, all samples were screened by direct immunofluorescence assay (IFA) using the Respiratory Panel I Viral Screening and Identification kit (Chemicon International, Inc).Results and Discussion: A total of 972 nasopharyngeal swab specimens were collected between January – September 2013. HPIVs were detected in 108 (11%) cases. Out of these, there were 36 co-infections of the parainfluenza viruses. In general, Their seasonality patterns shows two peaks; one severe one occurring in April with 40.6% and the second milder peak occurring in June with 23.1% of all the cases. There was co-circulation of HPIV sub-types throughout the year. The three subtypes circulated between January to May with a peak in April with type 1 dominating in the month of April. They formed a second peak in June with type three dominating and type three lagging behind and appearing a month later. From our analysis we found that the conditions that trigger their occurrence are the same since their peaks are synchronized.Conclusion: This study shows that parainfluenza viruses are the major contributor of influenza in Kenya.

Milanoi, S, Opanda S, Opot B, Gachara G, Ongus J, Bulimo W.  2014.  Species and Serotype Diversity of Human Rhinoviruses from patients presenting with Influenza like illness in Kenya in 2008., 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium . , Hilton Hotel; Nairobi, Kenya Abstract

tract infections, the main cause of the common cold and recently have been associated with more serious respiratory infections. There are over 100 known serotypes of HRV, classified into 3 species (A, B and C). In Kenya, there is scanty information regarding the genotypic characteristics of HRV in circulation.Objective: To determine the species and serotype diversity of HRV that circulated in Kenya in 2008 among patients presenting with ILI.Methodology: 517 nasopharyngeal samples collected from patients >2months old who attended outpatient clinics included in the USAMRU-K respiratory diseases surveillance network were used. Real time RT-PCR was employed to detect HRV followed by targeted amplification of part of the 5’ non-coding region and the VP4/VP2 gene of all HRV-positive samples. Upon nucleotide sequencing, the sequences were then compared to prototype and homotypic rhinovirus reference strains to establish serotype identities and a phylogenetic tree was constructed.Results: HRV was detected in 131 (25%) samples. Of these 33 (25%) amplified successfully which upon nucleotide sequencing 29 (87%) yielded usable sequences. Phylogenetic analysis based on VP4/VP2 genomic region revealed separation of Kenyan strains into three clusters corresponding to HRV A, B and C species. Majority (n=17) belonged to HRVA species,(serotypes: 29 [n=2], -47, -1 [n=2], -56, -49[n=2], -30, -106, -20[n=2],-45, -58 [n=2], -45 and -78) three belonged to HRV B species: (-37, -84, and -91) whereas 9 belonged to HRV C species: (-2[n=3], -14, -7, -13[n=2], -31, and -36). Conclusion: This study demonstrates circulation of Human Rhinoviruses in Kenya and indicates high genetic diversity among these viruses. The findings suggest that HRV A and C strains played a key role in human respiratory infections in Kenya in 2008. Knowledge about circulating HRV strains is important as it may help guide development of therapeutic strategies against HRV infections.

Mbui, FM, Achila RA, Opot BH, Wadegu MO, Majanja JM, Opanda SM, Ochola SA, Bulimo WD.  2014.  Phylogenetic characterization of respiratory human adenoviruses isolated from pediatrics patients in New Nyanza Provincial General Hospital; Kenya., 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium . , Hilton Hotel; Nairobi, Kenya Abstract

Background: Human adenoviruses (HAdVs) are common pathogens associated with diseases affecting the respiratory tract, gastro intestinal tract as well as various organs like the liver, kidney and the brain. Currently there are 60 human adenovirus serotypes classified into 7 species A to G on the basis of serology, genome sequencing and phylogenomics. HAdV species B, C and E are mainly implicated in respiratory tract infections whereas the other species are associated with gastrointestinal, genitourinary, and ocular infections. Globally, the respiratory HAdV species play a significant role in pediatric infections accounting for 10% of overall respiratory illnesses and 5%–11% of pneumonia cases. However the disease burden due to respiratory adenoviruses in Kenya is not well studied; there is limited data on the respiratory human adenovirus species and serotypes circulating in the country.Objective: The aim of this study was to characterize respiratory human adenoviruses using serological and molecular approaches. Specifically the study sought to determine the species and serotypes of HAdVs that were associated with pediatrics respiratory infections in New Nyanza Provincial General Hospital from June 2010 to June 2012.Methods: Respiratory human adenoviruses isolated from patient’s samples in a period of two years were characterized using serological and molecular approaches. Virus isolates in Hep-2 cell cultures were tested with specific fluorescent antibodies to confirm the presence of Human adenovirus. The loop-2 fragment of the hexon gene was PCR amplified and then sequenced using the Sanger method.Results: Phylogenomics analysis of the sequences showed that during the study period, respiratory HAdV species B and C were associated with respiratory infections accounting for approximately 1% of the overall respiratory viruses. There were no cases of infections caused by respiratory HAdV E implying that this species was not in circulation during the study period. HAdV C was the predominant species accounting for 68.75% of the reported cases with serotype distribution as HAdV C1-25%, HAdV C2-25%, HAdV C5-6.25%, and HAdV C6-12.5%. HAdV B serotype 7 was the most prevalent serotype at 31.25%. Analysis of selection pressure of the sequences revealed that HAdV C5 and HAdV B7 were under positive selection pressure indicating that these viruses are undergoing an evolutionary process which signifies instability in their genomes. Conclusions: Characterization of respiratory human adenoviruses that circulated at New Nyanza Provincial General hospital during the study period revealed that species B and C were present but not E. There was significant genetic variation in the hexon gene of the HAdVs seen at this site compared to those from other parts of the world implying continuing evolution of respiratory HAdVs. To gain a complete understanding of this evolutionary process, whole genome sequencing of these viruses is called for in order to determine genetic stability and uniqueness of these viruses.

Majanja, J, Bulimo W, Achilla R, Wadegu M, Mukunzi S, Njiri J, Mitei K, Opot B, Ocholla S, Mwangi J, Osuna F, Coldren. R.  2014.  Seasonal trend of Influenza in Kenya from January - October 2013, 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium . , Hilton Hotel; Nairobi, Kenya Abstract

Background: Influenza A and B viruses cause annual epidemics of respiratory illness. The Influenza surveillance network in Kenya through its sentinel surveillance sites located throughout the country has established that Influenza is a major cause of respiratory illness in Kenya.Objectives: The objective of the study was to determine the seasonal trend and circulation dynamics of Influenza viruses in Kenya from January to October, 2013.Methods: Nasopharyngeal swab specimens were collected from consenting patients meeting the ILI case definition and transported to the laboratory in liquid nitrogen dry shippers. RNA was extracted from the specimen using the QIAamp Viral RNA Mini Kit. Specimens were tested for influenza A and B viruses by using the Ag Path-ID One Step Real Time Reverse Transcription PCR (RT-PCR) Kit with CDC Human Influenza Virus RT-PCR Detection Panels (CDC, Atlanta, GA). Results: Of 945 specimens tested between January and October, 152 (16.1%) were positive for Influenza viruses. 38.8% tested positive for Influenza A/H3N2, 34.9% were positive for Influenza B while 26.3% were positive for the pdmH1N1 virus. Circulation of Influenza during this period was marked by three distinct peaks. The first peak was seen in the month of February at 22.7% followed by a steady decline to 5% in May. A second peak of 17.9% was seen in June and the highest circulation observed in August with a peak of 28.6%. PdmH1NI and Influenza A/H3N2 viruses co-circulated from January to April and then sharply declined in May. There were low levels of Influenza B in January which gradually increased peaking in June together with pdmH1N1. Influenza B and A/H3N2 co-circulated between July and September with peaks in August and low levels of pdmH1N1 during this period.Conclusion: Influenza viruses co-circulate throughout the year. Continuing surveillance will assist in determining their clinical and virological impact.

Jumba, G, Gachara G, Bulimo W.  2014.  Neuraminidase Inhibitor activities on Influenza B isolates obtained in Kenya, 2011-2012, 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium . , Hilton Hotel; Nairobi, Kenya Abstract

Introduction: Oseltamivir and zanamivir are neuraminidase inhibitors (NAIs) with important roles as drugs for prophylaxis and treatment of influenza. Whereas there have been reports of in vivo resistance of influenza B viruses to NAIs, currently there is lack of information regarding sensitivity or resistance to these drugs in influenza B viruses circulating in Kenya. Here, we report the isolation of influenza B viruses, phenotypic assessment to NAI activities and molecular characterization of NAI-relevant mutations in viruses that circulated in Kenya in the period 2011-2012. Materials and Methods: Influenza B viruses were isolated from patient nasopharyngeal specimens by inoculation onto MDCK monolayers. For phenotypic determination, enzyme inhibition assay using fluorescent MUNANA substrate was used. Known NA inhibitor-resistant and inhibitor-sensitive viruses were included in the assays as controls. IC50 values were determined using curve fitting implemented in Grafit version 7.0 software which is based on 50% of fitted upper asymptote. For molecular characterization of the mutations relevant in NAI resistance, RNA was extracted from the isolates followed by PCR amplification of NA gene segments using gene-specific primers. Nucleotide sequencing of the amplicons were performed using the Sanger dideoxy termination chemistry implemented using the BigDye technology prior to analyses using a suite of bioinformatics tools. Results and Discussion: Twenty four influenza B viruses were isolated and assed in this study. The mean IC50s of the isolates ranged from 17.1nM - 70.1nM for Oseltamivir and 0.0nM - 12.6nM for Zanamivir which were all within the 2011 WHO sensitive limits of 8-128nM for oseltamivir carboxylate and 0.5-12nM for Zanamivir. None of the isolates analyzed depicted oseltamivir or Zanamivir resistance at the eight amino acid positions E119, R152, D198, I222, S250, H274, R371, and G402 in the neuraminidase protein previously found to be associated with resistance or reduced susceptibility to oseltamivir and/or zanamivir. In conclusion, NAIs drugs were effective in treating influenza cause type B viruses during the 2011-2012 Kenyan seasons.

Gachara, G, Symekher S, Otieno M, Magana J, Opot B, Bulimo W.  2014.  Origins, Molecular Epidemiology and Genetic Diversity of Influenza A(H1N1)pdm09 Virus in Kenya: A Bayesian Coalescent Analysis, 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposia . , Hilton Hotel; Nairobi, Kenya Abstract

Background: An influenza pandemic caused by a swine-origin influenza virus A/H1N1 [A(H1N1)pdm09] spread worldwide in 2009 and is estimated to have caused between 151,700 and 575,400 deaths globally. The spread of influenza is tradi¬tionally tracked by epidemiological data; however, this approach gives lit¬tle insight into the different viral circulating vari¬ants. Genome sequencing is emerging as a surveillance tool for evolutionary and phylogenetic mapping, and to explore the origins, molecular epidemiology, and genetic diversity of epidemic/pandemic viruses. Objective: To determine the origins, molecular epidemiology and genetic diversity of Kenyan influenza A(H1N1)pdm09 viruses.Methodology: A total of 40 influenza A/H1N1pdm09 viruses isolated between July 2009 and August 2010 were selected. The eight segments from each isolate were amplified and directly sequenced. The resulting gene segments were concatenated and these genomes used for subsequent analysis. A Bayesian Markov chain Monte Carlo (MCMC) approach implemented in the BEAST package v1.7.4 was used to reconstruct the most recent common ancestor (MRCA) sequences, time the introduction of infection in the country, rates of substitution and estimate a time-resolved phylogeny. Results: The Kenyan complete genome sequences clustered with globally distributed clade 2 and clade 7 sequences. However, local clade 2 viruses did not circulate beyond the introductory foci while clade 7 viruses disseminated country wide and were sustained by multiple introductions generating complex spatial patterns. Often, the local isolates clustered with isolates from the United Kingdom than with isolates from other countries. The time of the most recent common ancestor was estimated between April and June 2009, two months before the first laboratory confirmed case. The complete genome had an estimated rate of nucleotide substitution of 4.9 X 10-3 substitutions/site/year and fast population growths characterized the population dynamics. Conclusions: Adaptive evolution and viral migration seem to play a vital role in shaping the evolutionary dynamics of local A(H1N1)pdm09 viruses. Continuous monitoring is thus essential.

Achilla., RA, Bulimo. WD, Majanja. JM, Wadegu. MO, Mukunzi. SO, Mwangi. J, Mwangi. JW, Njiri. JO, Opot. BH, Wurapa. EK.  2014.  Influenza surveillance in Kenya2006-2013: experiences and lessons learned , 16-17 October 20. 3rd Medical and Veterinary Virus Research in Kenya (MVVRK-3) symposium . , Hilton Hotel; Nairobi, Kenya. Abstract

Background: Influenza sentinel surveillance at the KEMRI-National Influenza Centre (NIC) was rolled out in 2006. A team of collaborators from Kenya Medical Research Unit (KEMRI), US Army Medical research unit –Kenya (USAMRU-K) and Centre for Disease Control-Kenya (CDC) came together to produce a document that was used to initiate Influenza surveillance in 8 civilian hospitals and 5 military hospitals across Kenya. Setting up the surveillance sites required a lot of input from the laboratory personnel, the principal investigator, field site coordinators and field staff including clinical/nursing officers and laboratory technologists.Methods: Prior to opening any of the sentinel sites, the field staff were brought to central Lab in Nairobi for a one week intensive training on Biosafety, Quality Assurance and all the Site Specific Standard Operating Procedures (SOPs). These included sample transportation, sample collection, Inclusion and exclusion criteria and sample rejection SOPs among others.An influenza starter pack was then sent to the respective sites including transport media, cool boxes, Dry shippers, nasopharyngeal swabs, documentation, SOPs among other Laboratory supplies Patient samples were transported weekly to the NIC in dry shippers to maintain cold chain by courier. On reception in the central laboratory a robust inventory system was put in place and tests were conducted first by real time PCR.Results: Eight Sentinel sites were activated over a period of one year. A total of 17980 samples were collected during this time period. On analysis approximately 25% of the samples showed positive results on analysis. Upper respiratory viruses discovered recently like HMPV and HCoV were later introduced to the study through student programs. By real time PCR a total of 1067 influenza A and Influenza B 879 were detected. Virus Isolation yielded 2847 Isolates. Of these the most isolated virus was adenovirus at 21% and respiratory syncitial virus at 19%.Discussion and Conclusion: Over the past eight years a lot of information was acquired through this robust surveillance program. 75% of the samples still remain undiagnosed and could be a potential source for novel viruses. Next Gen sequencing would be instrumental in detected what made the patient ill enough to have fevers of >38 degrees centigrade. This program also played a significant role during the 2009 pandemic H1N1 outbreak and still has capacity to detect MERS CoV currently circulating in the Middle East and H7N9 circulating in China. Influenza and other Upper respiratory tract viruses’ surveillance remains critical in Kenya where tourism, is a major income earner. The capacity to not only detect but disseminate information in a timely manner can help contain potential outbreaks and arrest potentially dangerous situations.

Juma, DW, Omondi AA, Ingasia L, Opot B, Cheruiyot A, Yeda R, Okudo C, Cheruiyot J, Muiruri P, Ngalah B, Chebon LJ, Eyase F, Johnson J, Bulimo WD, Akala HM, Andagalu B, Kamau E.  2014.  Trends in drug resistance codons in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in Kenyan parasites from 2008 to 2012., 2014. Malaria journal. 13:250. Abstractjuma_et_al2014.pdf

Sulphadoxine-pyrimethamine (SP), an antifolate, was replaced by artemether-lumefantrine as the first-line malaria drug treatment in Kenya in 2004 due to the wide spread of resistance. However, SP still remains the recommended drug for intermittent preventive treatment in pregnant women and infants (IPTP/I) owing to its safety profile. This study assessed the prevalence of mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes associated with SP resistance in samples collected in Kenya between 2008 and 2012.

Majanja, J, Wamunyokoli F, Mpoke S, Bulimo WD.  2014.  PGF2α Synthase-Like Proteins are Expressed in Promastigotes of Old World Leishmania Species but not in New World Species. African Journal of Pharmacology and Therapeutics. 3(2):67-73.majanja_et_al._2014.pdf
Opanda, SM, Wamunyokoli F, Khamadi S, Coldren R, Bulimo WD.  2014.  Genetic Diversity of Human Enterovirus 68 Strains Isolated in Kenya Using the Hypervariable 3′- End of VP1 Gene, 2014/07/23. PLoS ONE. 9(7):e102866-.: Public Library of Science Abstractjournal.pone_.0102866.pdfWebsite

Reports of increasing worldwide circulation of human enterovirus-68 (EV68) are well documented. Despite health concerns posed by resurgence of these viruses, little is known about EV68 strains circulating in Kenya. In this study, we characterized 13 EV68 strains isolated in Kenya between 2008 and 2011 based on the Hypervariable 3′- end of the VP1 gene. Viral RNA was extracted from the isolates and partial VP1 gene amplified by RT-PCR, followed by nucleotide sequencing. Alignment of deduced amino acid sequences revealed substitutions in Kenyan EV68 isolates absent in the prototype reference strain (Fermon). The majority of these changes were present in the BC and DE-loop regions, which are associated with viral antigenicity and virulence. The Kenyan strains exhibited high sequence homology with respect to those from other countries. Natural selection analysis based on the VP1 region showed that the Kenyan EV68 isolates were under purifying selection. Phylogenetic analysis revealed that majority (84.6%) of the Kenyan strains belonged to clade A, while a minority belonged to clades B and C. Overall, our results illustrate that although EV68 strains isolated in Kenya were genetically and antigenically divergent from the prototype strain (Fermon), they were closely related to those circulating in other countries, suggesting worldwide transmissibility. Further, the presence of shared mutations by Kenyan EV68 strains and those isolated in other countries, indicates evolution in the VP1 region may be contributing to increased worldwide detection of the viruses. This is the first study to document circulation of EV68 in Kenya.

Cheruiyot, J, Ingasia LA, Omondi AA, Juma DW, Opot BH, Ndegwa JM, Mativo J, Cheruiyot AC, Yeda R, Okudo C, Muiruri P, Bidii NS, Chebon LJ, Angienda PO, Eyase FL, Johnson JD, Bulimo WD, Andagalu B, Akala HM, Kamau E.  2014.  Polymorphisms in Pfmdr1, Pfcrt and Pfnhe1 Genes are Associated with Reduced in vitro Activities of Quinine in Plasmodium falciparum Isolates from Western Kenya., 2014 Apr 21. Antimicrobial agents and chemotherapy. Abstractzac3737.pdf

In combination with antibiotics, quinine is recommended as the second-line treatment for uncomplicated malaria, alternative first-line treatment for severe malaria and for treatment of malaria in the first trimester of pregnancy. Quinine has been shown to have frequent clinical failures and yet the mechanisms of action and resistance are not been fully elucidated. However, resistance is linked to polymorphisms in multiple genes including multidrug resistance 1 (Pfmdr1), chloroquine-resistance transporter (Pfcrt) and the sodium/hydrogen exchanger gene (Pfnhe1). Here, we investigated the association between in vitro quinine susceptibility with genetic polymorphisms in Pfmdr1codons 86 and 184, Pfcrt codon 76, and Pfnhe1 ms4760 in 88 field isolates from western Kenya. In vitro activity was assessed as the drug concentration that inhibits 50% of parasite growth (IC50) and parasite genetic polymorphisms were determined by DNA sequencing. Data revealed there was significant association between polymorphisms in Pfmdr1-86Y, -184F and Pfcrt-76T with quinine susceptibility; all with p < 0.0001. Eighty two percent of parasites resistant to quinine carried mutant alleles at these codons (Pfmdr1-86Y, -184F and Pfcrt-76T) whereas seventy four percent of parasites susceptible to quinine carried the wild type allele (Pfmdr1-N86, -Y184 and Pfcrt-K76). In addition, quinine IC50 of parasites with Pfnhe1 ms4760 3 DNNND repeats was significantly higher compared to those with 1 or 2 repeats (p = 0.033 and p = 0.0043 respectively). Clinical efficacy studies are required to confirm the validity of these markers and the importance of parasite genetic background.

2013

Eyase, FL, Akala HM, Ingasia L, Cheruiyot A, Omondi A, Okudo C, Juma D, Yeda R, Andagalu B, Wanja E, Kamau E, Schnabel D, Bulimo W, Waters NC, Walsh DS, Johnson JD.  2013.  The role of Pfmdr1 and Pfcrt in changing chloroquine, amodiaquine, mefloquine and lumefantrine susceptibility in western-Kenya P. falciparum samples during 2008-2011. PLoS One. 8:e64299., Number 5 Abstracteyase_et_al._2013.pdf

Single Nucleotide Polymorphisms (SNPs) in the Pfmdr1, and Pfcrt, genes of Plasmodium falciparum may confer resistance to a number of anti-malaria drugs. Pfmdr1 86Y and haplotypes at Pfcrt 72-76 have been linked to chloroquine (CQ) as well as amodiaquine (AQ) resistance. mefloquine (MQ) and lumefantrine (LU) sensitivities are linked to Pfmdr1 86Y. Additionally, Pfcrt K76 allele carrying parasites have shown tolerance to LU. We investigated the association between Pfmdr1 86/Pfcrt 72-76 and P. falciparum resistance to CQ, AQ, MQ and LU using field samples collected during 2008-2011 from malaria endemic sites in western Kenya. Genomic DNA from these samples was genotyped to examine SNPs and haplotypes in Pfmdr1 and Pfcrt respectively. Additionally, immediate ex vivo and in vitro drug sensitivity profiles were assessed using the malaria SYBR Green I fluorescence-based assay. We observed a rapid but steady percent increase in wild-type parasites with regard to both Pfmdr1 and Pfcrt between 2008 and 2011 (p<0.0001). Equally, a significant reciprocate decrease in AQ and CQ median IC50 values occurred (p<0.0001) during the same period. Thus, the data in this study point to a significantly rapid change in parasite response to AQ and CQ in the study period. This may be due to releasing of drug pressure on the parasite from reduced use of AQ in the face of increased Artemisinin (ART) Combination Therapy (ACT) administration following the intervention of the Global Fund in 2008. LU has been shown to select for 76K genotypes, thus the observed increase in 76K genotypes coupled with significant cross resistance between LU and MQ, may herald emergence of tolerance against both drugs in future.

Wadegu, M, Bulimo W, Achilla R, Mukunzi S, Majanja J, Opot B, Osuna F, Muthoni J, Njiri J, Mwangi J, Kibet K, Ocholla S, Wurapa E.  2013.  Characterization of neuraminidase inhibitor susceptibility of influenza A isolates obtained in Kenya, 2008-2011. Options for the Control of Influenza VII. :230., Cape Town, South Africa Abstract

Background: Vaccines and antivirals are the mainstay for mitigation and clinical management of influenza infections. However, due to the ever-changing antigenic profile, vaccine formulations are revised every year to make them efficacious. Neuraminidase (NA) inhibitors, mainly oseltamivir and zanamivir, function both as prophylactic and as treatment agents. In NA inhibition by antivirals, inhibitor molecules mimic NA’s natural substrate and bind to the active site, preventing NA from cleaving host cell receptors and releasing new virus. Currently, there exist no data on antiviral susceptibility profiles of influenza A isolates circulating within the Eastern African region. Here we characterised the antiviral susceptibility of the 2008-2011 influenza A viruses circulating in Kenya by combining both the genotypic data involving known molecular markers in NA protein responsible for drug resistance and IC50 data generated from NA inhibition assays. Materials and Methods: Nasopharyngeal swab specimens from consenting outpatients aged ≥ 2 months were obtained and transported to the National Influenza Centre and screened by real-time RT-PCR using primers targeted at the matrix and haemagglutinin genes of influenza A subtypes. Positive specimens were inoculated onto MDCK monolayers to isolate virus. RNA was extracted from virus isolates followed by PCR amplification of NA gene segments. Nucleotide sequencing was performed using the BigDye chemistry prior to analyses using a suite of bioinformatics tools. Drug susceptibility was determined by enzyme inhibition assay using fluorescent substrate with known NA inhibitor–resistant and –sensitive viruses as controls. IC50 values were determined using curve-fitting software (Grafit 7.0), which is based on 50% of fitted upper asymptote. Results: Of 836 influenza A virus isolates obtained (2008- 2011), 108 (13%) were analysed for markers of resistance to NA inhibitors: 64% (7/11) of the 2008 seasonal influenza A/H1N1 isolates analysed showed oseltamivir-resistant marker H275Y, while all 33 (100%) influenza A/H3N2 isolates obtained showed sensitivity to oseltamivir. Genetic analyses of the A (H1N1) pdm09 isolates obtained in 2009-2010 showed that all were sensitive to oseltamivir. All 14 influenza A/H3N2 isolates obtained in 2011 were also shown to be sensitive to oseltamivir. A total of 28 isolates were further subjected to phenotypic susceptibility assay. The mean zanamivir IC50 values were 1.75, 2.53 and 1.84 nM for the subtypes H1N1, pH1N1 and H3N2, respectively. Two of the 2008 sH1N1 and one of the sH1N1 obtained in 2009 showed normal sensitivity to oseltamivir in the NA inhibitor susceptibility assay (mean IC50 of 1.28 nM). The rest of the 2008-2009 sH1N1 analysed (n = 8) showed highly reduced sensitivity to oseltamivir. The IC50 values in the fluorescent assay ranged from 73 to 984 nM. Pandemic A/H1N1 strains obtained between 2009 and 2011 indicated oseltamivir IC50 values of 1.60 to 6.32 nM—categorised as normal sensitivity. All 8 candidate influenza A/H3N2 isolates obtained between 2008 and 2011 were sensitive to oseltamivir, with IC50 values ranging from 0.16 to 0.94 nM. The 2011 WHO ranges and median IC50 values for oseltamivir carboxylate were 0.4 to 10 nM and 0.5 nM, 0.1 to 5 nM and 0.2 nM, and 0.2 to 10 nM and 0.6 nM for wild-type sH1N1, sH3N2 and pH1N1, respectively. The 2011 WHO ranges and median IC50 values for oseltamivir carboxylate were 257 to 3455 nM and 458.2 nM and 132 to 2179 nM and 191.3 nM for mutant types sH1N1 and pH1N1, respectively. The WHO IC50 values for zanamivir, both for mutant and wild-type strains, ranged from 0.2 to 3 nM for all subtypes, with no significant differences between the mutant and wild-type strains for each subtype. Conclusion: Overall, our genotypic data demonstrate that there was oseltamivir resistance in seasonal influenza A (H1N1) viruses isolated in Kenya in 2008-2009. Most of the 2008-2009 sH1N1 isolates depicted highly reduced sensitivity to oseltamivir. This was due to the presence of the H275Y mutation in the NA protein sequence. H275Y mutation increased the IC50 value by 50- to 100-fold. Resistance to NA inhibitors was found to be specific to both drug and virus subtype. The drug susceptibility profile will be best informed using both elevated IC50 vales and known molecular markers of resistance.

Nzunza, R, Wurapa E, Kariuki N, Chek J, Ongus J, Bulimo W.  2013.  Epidemiological and clinical description of human metapneumovirus infectious diseases in Mbagathi District Hospital, Kenya, in 2008, 5-10 September 2. Options for the Control of Influenza VIII. :600., Cape Town, South Africa Abstract

Background: Human metapneumovirus (hMPV), a recently identified respiratory virus, is a leading cause of acute respiratory tract infection in children, the elderly, and immune-compromised persons. Studies have been done in a hospitalized pediatric population in coastal Kenya. However, there is limited information about the prevalence, epidemiology, and clinical presentation of hMPV infections in the general population. The aim of this study was to outline the epidemiologic and clinical description of human metapneumovirus infectious disease in patients attending the outpatient department of Mbagathi District Hospital, Kenya, in 2008. Methods: A retrospective study was conducted from 2008 in patients ≥ 2 months of age presenting at the outpatient department of Mbagathi District Hospital for acute respiratory infection. Nasopharyngeal swabs were systematically tested for several respiratory viruses. Epidemiologic and clinical characteristics of hMPV-infected children were compared with those of patients with respiratory syncytial virus (RSV) and other viral infections. Results: A total of 498 patients were enrolled in this study. Viral investigations detected a total of 271 viruses. Of these, 77 (15.5%) were hMPV infections, 78 (15.7%) seasonal flu A, 60 (12%) seasonal flu B, 13 (2.6%) panenterovirus, 36 (7.2%) parainfluenza viruses, and 6 (1.2%) RSV infections. Human metapneumovirus infections were higher in males (43, 55%) than in females (34, 45%), and predominantly in children ≤ 5 years (97%), only 2 (3%) were aged between 6 and 9 years. The hMPV infection had peaked in January-February, and was uncommon after March. Most of the patients infected with hMPV were < 1 year of age, and cough (100%) and difficulty in breathing (75%) were the predominant diagnosis in these patients with clinical symptoms of a lower respiratory tract infection. The severity of the disease was similar to those of RSV patients. Conclusions: These results highlight that hMPV plays an important role in seasonal acute respiratory tract infections, especially in children, with a severity similar to RSV infections. This work is ongoing to cover the wider Kenyan population.

Mukunzi, S, Bulimo W, Achilla R, Wadegu M, Majanja J, Opot B, Osuna F, Muthoni J, Njiri J, Mwangi J, Kibet K, Ochola S, Wurapa E.  2013.  Genetic variants of influenza A (H1N1) pdm09 virus circulating in Kenya, 2010 to 2011, 5-10 September 2. Options for the control of influenza VII. , Capetown, South Africa Abstract

Background: Since the emergence followed by rapid global spread to cause the 2009 pandemic, influenza A (H1N1)pdm09 viruses remained predominant among human influenza viruses that circulated in Kenya during the 2010-2011 season. In this study we sought to determine genetic variation amongst influenza A (H1N1) pdm09 viruses isolated in Kenya during the postpandemic period with reference to prototype influenza A/California/7/2009 strain and others obtained elsewhere during the same period. Materials and Methods: A total of 30 influenza A (H1N1) pdm09 isolates obtained from nasopharyngeal specimens (NP) and received from USAMRU-K’s sentinel surveillance network covering the whole of Kenya were analyzed. Virus HA1 subunit of hemagglutinin gene segment was amplified by RT-PCR and nucleotide sequences determined followed by conversion of the sequences to amino acid code before performing phylogenetic analyses. Results: The study showed all Kenyan isolates had 6 parallel amino acid substitutions when compared with the prototype A/California/07/2009 vaccine reference strain. These included P83S, D97N, S185T, S203T, I321V, and E374K. Additionally, there were further mutations that occurred stochastically in individual isolates. Most of these changes, however, did not affect virus antigenic sites; except the P83S amino acid substitution at site Cb and a few other changes at sites Ca1, Sa, and Sb in individual isolates.

Wadegu., M, Bulimo. WD, Achilla. RA, Majanja. J, Mukunzi. S, Osuna. F, Wangui. J, Mitei. K, Ocholla. S, Nyambura. J, Mwangi. J, Njiri. J, Opot. B, Wurapa. EK.  2013.  Antiviral susceptibility of influenza A viruses obtained in Kenya 2008-2011 , February 15-18, . International Meeting on Emerging Diseases and Surveillance (IMED 2013)' conference. , Vienna, Austria Abstract
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Osuna., F, Bulimo. W, Achilla. R, Majanja. J, Wadegu. M, Mukunzi. S, Njiri. J, Mwangi. J, Opot. B, Mitei. K, Nyambura. J, Wurapa. E.  2013.  Molecular Evolution of HA1 Gene of the Influenza A (H1N1) pdm09 Strain in Kenya During the 2009-2010 Period , 14th – 17th Marc. XV International Symposium on Respiratory Viral Infections. , Rotterdam, Netherlands Abstract
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