falciparum dihydrofolate reductase and

Trends in drug resistance codons in Plasmodiu

Evidence in Kenya of Reassortment Between of Reassortment BetweenSeasonal Influenza A(H3N2) and Influenza A(H1N1)pdm09 to yield A(H3N2) Variants With the Matrix Gene Segment of A(H1N1)pdm09

Influenza vaccines and antiviral drugs are the mainstay for preventing influenza and reducing the impact of influenza epidemics. Currently, there are two classes of antiviral drugs available for preventing and treating  the M gene. The neuraminidase inhibitors (NAI’s) interrupt the replication cycle by preventing virus release and allowing progeny virus to clump (Monto et al, 2002). The rapid emergence of adamantine drug resistant influenza A virus strains has limited these drugs’ clinical effectiveness.The origin and evolution of antiviral drug resistance amongst influenza viruses can occur through different molecular mechanisms that also drive the evolution of the virus. The most crucial of these mechanisms result from the segmented nature of its genome. This permits the formation of new progeny viruses with novel combinations of segments through reassortment when two or more different virus subtypes infect a single cell, a phenomenon referred to as antigenic shift. This process is capable of introducing new genes in circulating viral populations that can drastically change the biological properties of the virus. Studies have shown that reassortment led to an increase in the frequency of amantadine-resistant seasonal influenza A(H1N1) viruses since the 2005-2006 season (Yang et al, 2011). This underscores the necessity to monitor genome dynamics in circulating influenza viruses because it is through such molecular surveillance that we are able to understand the evolution and mechanisms of the emergence and spread of antiviral resistance among influenza A viruses (Boni et al, 2010).Thus, we set out to qualitatively analyze human influenza A(H3N2) viruses that circulated in Kenya in 2010, the period when influenza A(H3N2) and A(H1N1)pdm09 [previously referred to as swine flu] (WHO, 2011a) begun to co-circulate in the human population in the country, determine evidence of reassortment amongst the co-circulating subtypes and relate any such events to influenza antiviral resistance in the country. We applied the current laboratory testing protocol which involves routinely sequencing the HA, M and NA gene segments of the influenza viruses. These three gene segments were selected because they are the main antigens (NA & HA) and drug targets (M & NA) of the influenza A virus. Herein we provide evidence that indeed there was reassortment involving at least the M gene segment amongst cocirculating influenza A viruses in Kenya during this period

Analyses of selection pressure on the Hemagglutinin gene of influenza A/H3N2 Viruses circulating in Kenya 2007-2011

Background: The hemagglutinin (HA) gene of Influenza viruses, especially the HA1 portion, exhibits a rapid rate of change, largely in response to human immune surveillance in a partially immune human population. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pandemics. Objective: To determine whether the Kenyan influenza A/H3N2 viruses are undergoing adaptive evolution to become epidemic threats. Methods: Nasopharyngeal samples from patients meeting the WHO ILI case definition were collected between 2007 and 2010 from across Kenya. The detection of H3N2 virus was carried out using real-time RT-PCR. Positive samples were then cultured in MDCK cells and confirmed using the HAI assay. 156 isolates from this period were selected for amplification of the HA1 portion of the HA gene and the resulting amplicons sequenced. Global estimates, ω, of dN and dS, averaged over the entire alignment, were compared to calculate the overall strength of selection using the HyPhy 2.0 software package implemented in datamonkey. Results: Analysis of neutrality using, Kumar’s method showed that ω varied from 0.50 in 2007, 0.36 in 2008, 0.32 in 2009, 0.61 in 2010 and 0.41 in 2011. Further site by site analysis identified amino acid positions 46, 158 and 173 to be under positive selection. Analysis of differential selection showed 7 sites that harbored two or more amino acid substitutions. Amino acid positions 158, 160 and 189 had the highest number of amino acid polymorphisms. Conclusions: Overall, this study shows that local Influenza A(H3N2) viruses have been evolving via a series of ‘adaptive bursts’ characterized by positive selection occurring largely in immunological epitopes B and D. In between these bursts there is little evidence for positive selection and newly-emergent strains slowly replace the existent strains. Based on this study alone, we propose that these bursts occur after every two years. Since in a single population, dN/dS <1does not follow monotonic function, it is difficult to infer selection pressure in these results. However, by focusing on the antigenic sites we were able to observe an evolutionary pattern in the Kenyan samples. Thus evolution of Kenyan Influenza A(H3N2) is characterized by non synonymous changes followed by a period of stasis that is then followed by another period of non synonymous changes which is followed by purification selection

Were the WHO-recommended Human Influenza Vaccine Formulations Appropriate for Kenya During the 2010-2011 Season? Inferences from the HA1 Gene Analysis

Background: The knowledge of evolutionary patterns of the HA gene of the influenza virus is important in vaccine strain selection. Objective: Genetic analysis of HA1 of influenza viruses isolated in Kenya during the 2010-2011 season with reference to WHO vaccine strains. Methods: A total of twenty seven (27) influenza A (H1N1) pdm09, Nineteen (19) influenza A (H3N2) and Sixteen (16) influenza B virus isolates were analyzed. A partial HA1 gene was amplified by RT-PCR and sequenced. Results: Phylogenetic analyses revealed that influenza B viruses were closely related to B/Brisbane/60/2008 vaccine strain while A (H1N1) pdm09 viruses were genetic variants of A/California/07/2009. The Kenyan A (H1N1) pdm09 isolates had P83S, D97N, S185T, I321V and E374K amino acid substitutions. Influenza A/H3N2 isolates showed K62E, T212A and S214I simultaneous amino acid substitutions when compared to A/Perth/10/2009. The K62E change occurred at antigenic site E. Majority of the Kenyan H3N2 isolates further had S45N and K144N amino acid substitutions at sites C and A respectively, which introduced N-glycosylation motifs absent in the vaccine strain. Conclusion: The study showed that although the WHO 2010 vaccine strains recommendations for the southern hemisphere matched with influenza viruses which circulated in Kenya during the 2010-2011 season, the viruses had evolved genetically from the vaccine strains. Key words: Influenza vaccine formulations; HA1 gene; Kenya

Respiratory Adenovirus Species Circulating In Kenya from 2007-2010

Background: Human adenoviruses [HAdvs] are causative agents of several diseases including acute respiratory disease (ARD), keratoconjunctivitis, gastroenteritis, acute hemorrhagic cystitis, opportunistic infection in immuno - compromised patients and severe and potentially fatal pneumonia. Adenoviruses have been shown to affect mainly pediatric populations, military recruits and congested institutions such as hospitals and schools where they are a major cause of morbidity and mortality. In this study we examined respiratory adenovirus species and types associated with respiratory infection circulating at eight study sites in Kenya from 2007-2010. Methods: Nasopharyngeal swab samples collected in viral transport media were transported to the National Influenza Centre in dry shippers while maintaining the cold chain. Samples originated from patients with Influenza like Illness (ILI) participating in the United States Army Medical Research unit in Kenya (USAMRU-K) sentinel surveillance network across the country. The samples were inoculated into Hep2 cells and incubated at 37 °C with 5% CO2 for 14 days or until cytopathic effect was observed. Presence of HAdv in the supernatants was determined by immunofluorescence assays. To begin to analyze these Kenyan HAdvs, 10% were molecularly genotyped using HAdv type-specific primers followed by nucleotide sequencing and analysis using a suite of bioinformatics software. This work received ethical approval under the KEMRI ERC-approved protocol SSC#981. Results: A total of 12,959 samples were screened during the period. 385 (3%) of these were positive for HAdv by cell culture. Molecular characterization of ~10% of the viruses using PCR yielded 20 (45%) HAdvs of the B species and 24 (55%) HAdvs species C. We did not detect any HAdv species E during the period. Analysis of the nucleotide sequences differentiated the species into types B3 [n=5], B7 [n=15], C1 [n=6], C2 [n=13] and C5 [n=4]. Although cell culture is not the most sensitive method for screening respiratory viruses, our results showed that respiratory HAdvs contributed at least 3% of the respiratory disease burden in Kenya during this period. Furthermore, the results showed that type B7 was the most prevalent HAdv followed by type C2. HAdv type B7 has been associated with the most severe forms of respiratory infections and fatalities globally, and our results showed that a substantial burden of serious respiratory disease was due to this HAdv type. These results suggest that if a respiratory HAdv vaccine were to be introduced in Kenya, it ought to contain the HAdv types B7 and C2 components. Conclusion: We have for the first time molecularly characterized HAdv types isolated from the respiratory tract of humans in Kenya and showed that overall, types B7 and C2 substantially contribute to severe respiratory disease in Kenya. The Kenyan Ministry of Health should consider introducing a vaccine containing these two components to mitigate against respiratory illness

Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemether–lumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya

Artemether–lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995–2003) and 745 after (post-ACT; 2008–2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995–1996 to 93.2% in 2014 and 0.0% in 2002–2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use of AL in western Kenya

Selective sweeps and genetic lineages of Plasmodium falciparum multi-drug resistance (pfmdr1) gene in Kenya

Abstract Background There are concerns that resistance to artemisinin-based combination therapy might emerge in Kenya and sub-Saharan Africa (SSA) in the same pattern as was with chloroquine and sulfadoxine–pyrimethamine. Single nucleotide polymorphisms (SNPs) in critical alleles of pfmdr1 gene have been associated with resistance to artemisinin and its partner drugs. Microsatellite analysis of loci flanking genes associated with anti-malarial drug resistance has been used in defining the geographic origins, dissemination of resistant parasites and identifying regions in the genome that have been under selection. Methods This study set out to investigate evidence of selective sweep and genetic lineages in pfmdr1 genotypes associated with the use of artemether–lumefantrine (AL), as the first-line treatment in Kenya. Parasites (n = 252) from different regions in Kenya were assayed for SNPs at codons 86, 184 and 1246 and typed for 7 neutral microsatellites and 13 microsatellites loci flanking (± 99 kb) pfmdr1 in Plasmodium falciparum infections. Results The data showed differential site and region specific prevalence of SNPs associated with drug resistance in the pfmdr1 gene. The prevalence of pfmdr1 N86, 184F, and D1246 in western Kenya (Kisumu, Kericho and Kisii) compared to the coast of Kenya (Malindi) was 92.9% vs. 66.7%, 53.5% vs. to 24.2% and 96% vs. to 87.9%, respectively. The NFD haplotype which is consistent with AL selection was at 51% in western Kenya compared to 25% in coastal Kenya. Conclusion Selection pressures were observed to be different in different regions of Kenya, especially the western region compared to the coastal region. The data showed independent genetic lineages for all the pfmdr1 alleles. The evidence of soft sweeps in pfmdr1 observed varied in direction from one region to another. This is challenging for malaria control programs in SSA which clearly indicate effective malaria control policies should be based on the region and not at a country wide level

Comparative analysis of peripheral whole blood transcriptome from asymptomatic carriers reveals upregulation of subsets of surface proteins implicated in Plasmodium falciparum phenotypic plasticity

The molecular mechanism underlying Plasmodium falciparum's persistence in the asymptomatic phase of infection remains largely unknown. However, large-scale shifts in the parasites' gene expression during asymptomatic infections may enhance phenotypic plasticity, maximizing their fitness and leading to the persistence of the asymptomatic infections. To uncover these mechanisms, we aimed to identify parasite genetic factors implicated in asymptomatic infections through whole transcriptome analysis. We analyzed publicly available transcriptome datasets containing asymptomatic malaria (ASM), uncomplicated malaria (SM), and malaria-naïve (NSM) samples from 35 subjects for differentially expressed genes (DEGs) and long noncoding RNAs. Our analysis identified 755 and 1773 DEGs in ASM vs SM and NSM, respectively. These DEGs revealed sets of genes coding for proteins of unknown functions (PUFs) upregulated in ASM vs SM and ASM, suggesting their role in underlying fundamental molecular mechanisms during asymptomatic infections. Upregulated genes in ASM vs SM revealed a subset of 24 clonal variant genes (CVGs) involved in host-parasite and symbiotic interactions and modulation of the symbiont of host erythrocyte aggregation pathways. Moreover, we identified 237 differentially expressed noncoding RNAs in ASM vs SM, of which 11 were found to interact with CVGs, suggesting their possible role in regulating the expression of CVGs. Our results suggest that P. falciparum utilizes phenotypic plasticity as an adaptive mechanism during asymptomatic infections by upregulating clonal variant genes, with long noncoding RNAs possibly playing a crucial role in their regulation. Thus, our study provides insights into the parasites' genetic factors that confer a fitness advantage during asymptomatic infections