Evidence of circulation of Orthobunyaviruses in diverse mosquito species in Kwale County, Kenya.

BACKGROUND: Arbovirus surveillance and recurrence of outbreaks in Kenya continues to reveal the re-emergence of viruses of public health importance. This calls for sustained efforts in early detection and characterization of these agents to avert future potential outbreaks. METHODS: A larval survey was carried out in three different sites in Kwale County, Vanga, Jego and Lunga Lunga. All containers in every accessible household and compound were sampled for immature mosquitoes. In addition, adult mosquitoes were also sampled using CO2-baited CDC light traps and BG-Sentinel traps in the three sites and also in Tsuini. The mosquitoes were knocked down using trimethylamine and stored in a liquid nitrogen shipper for transportation to the laboratory where they were identified to species, pooled and homogenized ready for testing. RESULTS: A total of 366 houses and 1730 containers were inspected. The House Index (HI), Container Index (CI) and Breateau Index (BI) for Vanga Island were (3%: 0.66: 3.66) respectively. In Jego, a rural site, the HI, CI and BI were (2.4%: 0.48: 2.4) respectively. In Lunga Lunga, a site in an urban area, the HI, CI and BI were (22.03%: 3.97: 29.7) respectively. The indices suggest that this region is at risk of arbovirus transmission given they were above the WHO threshold (CI > 1, HI > 1% and BI > 5). The most productive containers were the concrete tanks (44.4%), plastic tank (22.2%), claypot (13.3%), plastic drums (8.9%), plastic basins (4%), jerricans (1.2%) and buckets (0.3%). Over 20,200 adult mosquitoes were collected using CDC light traps, and over 9,200 using BG- sentinel traps. These mosquitoes were screened for viruses by inoculating in Vero cells. Eleven Orthobunyavirus isolates were obtained from pools of Ae. pembaensis (4), Ae. tricholabis (1), Cx. quinquefasciatus (3), Culex spp. (1) and Cx. zombaensis (2). Five of the Orthobunyaviruses were sequenced and four of these were determined to be Bunyamwera viruses while one isolate was found to be Nyando virus. One isolate remained unidentified. CONCLUSIONS: These results indicate circulation of Orthobunyaviruses known to cause diverse grades of febrile illness with rash in humans in this region and highlights the need for continued monitoring and surveillance to avert outbreaks.

Plasmid mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates from Kenya.

BACKGROUND: Treatment of gonorrhea is complicated by the development of antimicrobial resistance in Neisseria gonorrhoeae (GC) to the antibiotics recommended for treatment. Knowledge on types of plasmids and the antibiotic resistance genes they harbor is useful in monitoring the emergence and spread of bacterial antibiotic resistance. In Kenya, studies on gonococcal antimicrobial resistance are few and data on plasmid mediated drug resistance is limited. The present study characterizes plasmid mediated resistance in N. gonorrhoeae isolates recovered from Kenya between 2013 and 2018. METHODS: DNA was extracted from 36 sub-cultured GC isolates exhibiting varying drug resistance profiles. Whole genome sequencing was done on Illumina MiSeq platform and reads assembled de-novo using CLC Genomics Workbench. Genome annotation was performed using Rapid Annotation Subsystem Technology. Comparisons in identified antimicrobial resistance determinants were done using Bioedit sequence alignment editor. RESULTS: Twenty-four (66.7%) isolates had both ß-lactamase (TEM) and TetM encoding plasmids. 8.3% of the isolates lacked both TEM and TetM plasmids and had intermediate to susceptible penicillin and tetracycline MICs. Twenty-six (72%) isolates harbored TEM encoding plasmids. 25 of the TEM plasmids were of African type while one was an Asian type. Of the 36 isolates, 31 (86.1%) had TetM encoding plasmids, 30 of which harbored American TetM, whereas 1 carried a Dutch TetM. All analyzed isolates had non-mosaic penA alleles. All the isolates expressing TetM were tetracycline resistant (MIC> 1 mg/L) and had increased doxycycline MICs (up to 96 mg/L). All the isolates had S10 ribosomal protein V57M amino acid substitution associated with tetracycline resistance. No relation was observed between PenB and MtrR alterations and penicillin and tetracycline MICs. CONCLUSION: High-level gonococcal penicillin and tetracycline resistance in the sampled Kenyan regions was found to be mediated by plasmid borne blaTEM and tetM genes. While the African TEM plasmid, TEM1 and American TetM are the dominant genotypes, Asian TEM plasmid, a new TEM239 and Dutch TetM have emerged in the regions.

gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae isolates from Kenya.

BACKGROUND: Phenotypic fluoroquinolone resistance was first reported in Western Kenya in 2009 and later in Coastal Kenya and Nairobi. Until recently gonococcal fluoroquinolone resistance mechanisms in Kenya had not been elucidated. The aim of this paper is to analyze mutations in both gyrA and parC responsible for elevated fluoroquinolone Minimum Inhibitory Concentrations (MICs) in Neisseria gonorrhoeae (GC) isolated from heterosexual individuals from different locations in Kenya between 2013 and 2017. METHODS: Antimicrobial Susceptibility Tests were done on 84 GC in an ongoing Sexually Transmitted Infections (STI) surveillance program. Of the 84 isolates, 22 resistant to two or more classes of antimicrobials were chosen for analysis. Antimicrobial susceptibility tests were done using E-test (BioMerieux) and the results were interpreted with reference to European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. The isolates were sub-cultured, and whole genomes were sequenced using Illumina platform. Reads were assembled de novo using Velvet, and mutations in the GC Quinolone Resistant Determining Regions identified using Bioedit sequence alignment editor. Single Nucleotide Polymorphism based phylogeny was inferred using RaxML. RESULTS: Double GyrA amino acid substitutions; S91F and D95G/D95A were identified in 20 isolates. Of these 20 isolates, 14 had an additional E91G ParC substitution and significantly higher ciprofloxacin MICs (p = 0.0044*). On the contrary, norfloxacin MICs of isolates expressing both GyrA and ParC QRDR amino acid changes were not significantly high (p = 0.82) compared to MICs of isolates expressing GyrA substitutions alone. No single GyrA substitution was found in the analyzed isolates, and no isolate contained a ParC substitution without the simultaneous presence of double GyrA substitutions. Maximum likelihood tree clustered the 22 isolates into 6 distinct clades. CONCLUSION: Simultaneous presence of amino acid substitutions in ParC and GyrA has been reported to increase gonococcal fluoroquinolone resistance from different regions in the world. Our findings indicate that GyrA S91F, D95G/D95A and ParC E91G amino acid substitutions mediate high fluoroquinolone resistance in the analyzed Kenyan GC.

Detection of dengue virus serotypes 1, 2 and 3 in selected regions of Kenya: 2011-2014.

BACKGROUND: Dengue fever, a mosquito-borne disease, is associated with illness of varying severity in countries in the tropics and sub tropics. Dengue cases continue to be detected more frequently and its geographic range continues to expand. We report the largest documented laboratory confirmed circulation of dengue virus in parts of Kenya since 1982. METHODS: From September 2011 to December 2014, 868 samples from febrile patients were received from hospitals in Nairobi, northern and coastal Kenya. The immunoglobulin M enzyme linked immunosorbent assay (IgM ELISA) was used to test for the presence of IgM antibodies against dengue, yellow fever, West Nile and Zika. Reverse transcription polymerase chain reaction (RT-PCR) utilizing flavivirus family, yellow fever, West Nile, consensus and sero type dengue primers were used to detect acute arbovirus infections and determine the infecting serotypes. Representative samples of PCR positive samples for each of the three dengue serotypes detected were sequenced to confirm circulation of the various dengue serotypes. RESULTS: Forty percent (345/868) of the samples tested positive for dengue by either IgM ELISA (14.6 %) or by RT-PCR (25.1 %). Three dengue serotypes 1-3 (DENV1-3) were detected by serotype specific RT-PCR and sequencing with their numbers varying from year to year and by region. The overall predominant serotype detected from 2011-2014 was DENV1 accounting for 44 % (96/218) of all the serotypes detected, followed by DENV2 accounting for 38.5 % (84/218) and then DENV3 which accounted for 17.4 % (38/218). Yellow fever, West Nile and Zika was not detected in any of the samples tested. CONCLUSION: From 2011-2014 serotypes 1, 2 and 3 were detected in the Northern and Coastal parts of Kenya. This confirmed the occurrence of cases and active circulation of dengue in parts of Kenya. These results have documented three circulating serotypes and highlight the need for the establishment of active dengue surveillance to continuously detect cases, circulating serotypes, and determine dengue fever disease burden in the country and region.

Molecular characterization of the cytochrome b gene and in vitro atovaquone susceptibility of Plasmodium falciparum isolates from Kenya.

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.

Reduced in vitro doxycycline susceptibility in plasmodium falciparum field isolates from Kenya is associated with PfTetQ KYNNNN sequence polymorphism.

Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pfmdt) and P. falciparum GTPase tetQ (PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC50s) of ≥35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC50s of >35,000 nM relative to those with IC50s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3; P value of <0.0002). Isolates with 1 copy of the Pfmdt gene had a median IC50 of 6,971 nM, whereas those with a Pfmdt copy number of >1 had a median IC50 of 9,912 nM (P = 0.0245). Isolates with 1 copy of PftetQ had a median IC50 of 6,370 nM, whereas isolates with a PftetQ copy number of >1 had a median IC50 of 3,422 nM (P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC50 of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC50 of 3,352 nM (P = 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing.

Polymorphisms in Pfmdr1, Pfcrt, and Pfnhe1 genes are associated with reduced in vitro activities of quinine in Plasmodium falciparum isolates from western Kenya.

In combination with antibiotics, quinine is recommended as the second-line treatment for uncomplicated malaria, an 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 have not been fully elucidated. However, resistance is linked to polymorphisms in multiple genes, including multidrug resistance 1 (Pfmdr1), the chloroquine resistance transporter (Pfcrt), and the sodium/hydrogen exchanger gene (Pfnhe1). Here, we investigated the association between in vitro quinine susceptibility and 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 based on the drug concentration that inhibited 50% of parasite growth (the IC50), and parasite genetic polymorphisms were determined from DNA sequencing. Data revealed there were significant associations between polymorphism in Pfmdr1-86Y, Pfmdr1-184F, or Pfcrt-76T and quinine susceptibility (P < 0.0001 for all three associations). Eighty-two percent of parasites resistant to quinine carried mutant alleles at these codons (Pfmdr1-86Y, Pfmdr1-184F, and Pfcrt-76T), whereas 74% of parasites susceptible to quinine carried the wild-type allele (Pfmdr1-N86, Pfmdr1-Y184, and Pfcrt-K76, respectively). In addition, quinine IC50 values for parasites with Pfnhe1 ms4760 3 DNNND repeats were significantly higher than for those with 1 or 2 repeats (P = 0.033 and P = 0.0043, respectively). Clinical efficacy studies are now required to confirm the validity of these markers and the importance of parasite genetic background.

Efficacy and safety of artemether-lumefantrine and dihydroartemisinin-piperaquine in the treatment of uncomplicated Plasmodium falciparum malaria in Kenyan children aged less than five years: results of an open-label, randomized, single-centre study.

BACKGROUND: This open-label, randomized study evaluated efficacy and safety of artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) in treatment of uncomplicated falciparum malaria in children below five years of age, to build evidence on use of AL as first-line treatment and DP as second-line treatment in Kenya. METHODS: A total of 454 children aged six to 59 months with uncomplicated falciparum malaria were randomized (1:1) to receive AL dispersible or DP paediatric tablets and followed up for 42 days. Primary efficacy variable was corrected adequate clinical and parasitological response (ACPR) rate on day 28. Secondary variables included corrected (day 14, 28 and 42), uncorrected (day 3, 14, 28 and 42) cure rates, parasitological failure at days 3, 14 and 42. Acceptability and tolerability of both drugs were assessed by caregiver questionnaire. RESULTS: On day 28, corrected ACPR rates for AL dispersible and DP paediatric were 97.8% (95% CI: 94.9-99.3) and 99.1% (95% CI: 96.8-99.9), respectively, in intention-to-treat population, with no significant treatment differences noted between AL dispersible and DP paediatric arms. Additionally, no significant differences were observed for PCR corrected cure rates on days 14 and ACPR on day 42 for AL dispersible (100%; 96.8%) and DP paediatric (100%; 98.7%). Similarly, for PCR uncorrected cure rates, no significant differences were seen on days 3, 14, 28, and 42 for AL dispersible (99.1%; 98.7%; 81.1%; 67.8%) and DP paediatric (100%; 100%; 87.7%; 70.5%). Parasite clearance was rapid, with approximately 90% clearance achieved in 40 hours in both treatment arms. Incidence of adverse events was related to underlying disease; malaria being reported in both treatment arms. One serious adverse event was noted in AL dispersible (0.42%) arm, not related to study drug. Adherence to treatment regimen was higher for children treated with AL dispersible (93.6%) compared to DP paediatric (85.6%). Acceptability of AL dispersible regimen was assessed as being significantly better than DP paediatric. CONCLUSIONS: AL and DP were both efficacious and well tolerated, and had similar effects at day 42 on risk of recurrent malaria. No signs of Plasmodium falciparum tolerance to artemisinins were noted. TRIAL REGISTRATION: PACTR201111000316370.

Trends in drug resistance codons in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in Kenyan parasites from 2008 to 2012.

BACKGROUND: 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. METHODS: Field isolates collected from Kisumu, Kisii, Kericho and Malindi district hospitals were assessed for genetic polymorphism at various loci within Pfdhfr and Pfdhps genes by sequencing. RESULTS: Among the Pfdhfr mutations, codons N51I, C59R, S108N showed highest prevalence in all the field sites at 95.5%, 84.1% and 98.6% respectively. Pfdhfr S108N prevalence was highest in Kisii at 100%. A temporal trend analysis showed steady prevalence of mutations over time except for codon Pfdhps 581 which showed an increase in mixed genotypes. Triple Pfdhfr N51I/C59R/S108N and double Pfdhps A437G/ K540E had high prevalence rates of 86.6% and 87.9% respectively. The Pfdhfr/Pfdhps quintuple, N51I/C59R/S108N/A437G/K540E mutant which has been shown to be the most clinically relevant marker for SP resistance was observed in 75.7% of the samples. CONCLUSION: SP resistance is still persistently high in western Kenya, which is likely due to fixation of key mutations in the Pfdhfr and Pfdhps genes as well as drug pressure from other antifolate drugs being used for the treatment of malaria and other infections. In addition, there is emergence and increasing prevalence of new mutations in Kenyan parasite population. Since SP is used for IPTP/I, molecular surveillance and in vitro susceptibility assays must be sustained to provide information on the emergence and spread of SP resistance.

Detection of pathogenic bacteria in ticks from Isiolo and Kwale counties of Kenya using metagenomics.

Ticks are arachnid ectoparasites that rank second only to mosquitoes in the transmission of human diseases including bacteria responsible for anaplasmosis, ehrlichiosis, spotted fevers, and Lyme disease among other febrile illnesses. Due to the paucity of data on bacteria transmitted by ticks in Kenya, this study undertook a bacterial metagenomic-based characterization of ticks collected from Isiolo, a semi-arid pastoralist County in Eastern Kenya, and Kwale, a coastal County with a monsoon climate in the southern Kenyan border with Tanzania. A total of 2,918 ticks belonging to 3 genera and 10 species were pooled and screened in this study. Tick identification was confirmed through the sequencing of the Cytochrome C Oxidase Subunit 1 (COI) gene. Bacterial 16S rRNA gene PCR amplicons obtained from the above samples were sequenced using the MinION (Oxford Nanopore Technologies) platform. The resulting reads were demultiplexed in Porechop, followed by trimming and filtering in Trimmomatic before clustering using Qiime2-VSearch. A SILVA database pretrained naïve Bayes classifier was used to classify the Operational Taxonomic Units (OTUs) taxonomically. The bacteria of clinical interest detected in pooled tick assays were as follows: Rickettsia spp. 59.43% of pools, Coxiella burnetii 37.88%, Proteus mirabilis 5.08%, Cutibacterium acnes 6.08%, and Corynebacterium ulcerans 2.43%. These bacteria are responsible for spotted fevers, query fever (Q-fever), urinary tract infections, skin and soft tissue infections, eye infections, and diphtheria-like infections in humans, respectively. P. mirabilis, C. acnes, and C. ulcerans were detected only in Isiolo. Additionally, COI sequences allowed for the identification of Rickettsia and Coxiella species to strain levels in some of the pools. Diversity analysis revealed that the tick genera had high levels of Alpha diversity but the differences between the microbiomes of the three tick genera studied were not significant. The detection of C. acnes, commonly associated with human skin flora suggests that the ticks may have contact with humans potentially exposing them to bacterial infections. The findings in this study highlight the need for further investigation into the viability of these bacteria and the competency of ticks to transmit them. Clinicians in these high-risk areas also need to be appraised for them to include Rickettsial diseases and Q-fever as part of their differential diagnosis.

Isolation and Growth Kinetics of Bogoria Virus from Phlebotomine Sandflies Sampled in Baringo, Kenya.

Phleboviruses are an emerging threat to public health. Recent surveillance efforts in Kenya have unveiled novel phleboviruses. Despite these efforts, there remain knowledge gaps. This study tested female sandflies from diverse ecological settings in Kenya for arboviruses. Sandfly pools were cultured in Vero-CCL cells. Pools showing reproducible cytopathic effects were subjected to next-generation sequencing, followed by phylogenetic analysis. In vitro, cell kinetics analysis was performed using both Vero-E6 cells and C6/36 mosquito cells. One pool from Baringo, Kenya, tested positive for Bogoria virus (BOGV). The BOGV genome clustered in a single clade with previously obtained BOGV genomes. No significant differences were observed between Vero and C6/36 cell growth kinetics. This study has confirmed the presence of BOGV among sandflies in Baringo Kenya and demonstrated growth in mosquito cells.

Genome sequences of Phasi Charoen-like phasivirus and Fako virus from Aedes aegypti mosquitoes collected in coastal Kenya.

We report the sequencing of two viruses, Phasi Charoen-like phasivirus (PCLV) and Fako virus (FAKV), which were detected in a pool of Aedes aegypti from Kenya. Analysis showed a high similarity of PCLV to publicly available PCLV genomes from Kenya. FAKV showed a high genetic divergence from publicly available FAKV genomes.

Antiplasmodial quinones from Pentas longiflora and Pentas lanceolata.

The dichloromethane/methanol (1:1) extracts of the roots of Pentas longiflora and Pentas lanceolata showed low micromolar (IC(50) = 0.9-3 µg/mL) IN VITRO antiplasmodial activity against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of PLASMODIUM FALCIPARUM. Chromatographic separation of the extract of PENTAS LONGIFLORA led to the isolation of the pyranonaphthoquinones pentalongin (1) and psychorubrin (2) with IC(50) values below 1 µg/mL and the naphthalene derivative mollugin (3), which showed marginal activity. Similar treatment of Pentas lanceolata led to the isolation of eight anthraquinones ( 4-11, IC(50) = 5-31 µg/mL) of which one is new (5,6-dihydroxydamnacanthol, 11), while three--nordamnacanthal (7), lucidin-ω-methyl ether (9), and damnacanthol (10)--are reported here for the first time from the genus Pentas. The compounds were identified by NMR and mass spectroscopic techniques.

Profiling of RNA Viruses in Biting Midges (Ceratopogonidae) and Related Diptera from Kenya Using Metagenomics and Metabarcoding Analysis.

Vector-borne diseases (VBDs) cause enormous health burden worldwide, as they account for more than 17% of all infectious diseases and over 700,000 deaths each year. A significant number of these VBDs are caused by RNA virus pathogens. Here, we used metagenomics and metabarcoding analysis to characterize RNA viruses and their insect hosts among biting midges from Kenya. We identified a total of 15 phylogenetically distinct insect-specific viruses. These viruses fall into six families, with one virus falling in the recently proposed negevirus taxon. The six virus families include Partitiviridae, Iflaviridae, Tombusviridae, Solemoviridae, Totiviridae, and Chuviridae. In addition, we identified many insect species that were possibly associated with the identified viruses. Ceratopogonidae was the most common family of midges identified. Others included Chironomidae and Cecidomyiidae. Our findings reveal a diverse RNA virome among Kenyan midges that includes previously unknown viruses. Further, metabarcoding analysis based on COI (cytochrome c oxidase subunit 1 mitochondrial gene) barcodes reveal a diverse array of midge species among the insects used in the study. Successful application of metagenomics and metabarcoding methods to characterize RNA viruses and their insect hosts in this study highlights a possible simultaneous application of these two methods as cost-effective approaches to virus surveillance and host characterization. IMPORTANCE The majority of the viruses that currently cause diseases in humans and animals are RNA viruses, and more specifically arthropod-transmitted viruses. They cause diseases such as dengue, West Nile infection, bluetongue disease, Schmallenberg disease, and yellow fever, among others. Several sequencing investigations have shown us that a diverse array of RNA viruses among insect vectors remain unknown. Some of these could be ancient lineages that could aid in comprehensive studies on RNA virus evolution. Such studies may provide us with insights into the evolution of the currently pathogenic viruses. Here, we applied metagenomics to field-collected midges and we managed to characterize several RNA viruses, where we recovered complete and nearly complete genomes of these viruses. We also characterized the insect host species that are associated with these viruses. These results add to the currently known diversity of RNA viruses among biting midges as well as their associated insect hosts.

Probable contribution of Culex quinquefasciatus mosquitoes to the circulation of chikungunya virus during an outbreak in Mombasa County, Kenya, 2017-2018.

BACKGROUND: Chikungunya virus is an alphavirus, primarily transmitted by Aedes aegypti and Ae. albopictus. In late 2017-2018, an outbreak of chikungunya occurred in Mombasa county, Kenya, and investigations were conducted to establish associated entomological risk factors. METHODS: Homes were stratified and water-filled containers inspected for immature Ae. aegypti, and larval indices were calculated. Adult mosquitoes were collected in the same homesteads using BG-Sentinel and CDC light traps and screened for chikungunya virus. Experiments were also conducted to determine the ability of Culex quinquefasciatus to transmit chikungunya virus. RESULTS: One hundred thirty-one houses and 1637 containers were inspected; 48 and 128 of them, respectively, were positive for immature Ae. aegypti, with the house index (36.60), container index (7.82) and Breteau index (97.71) recorded. Jerry cans (n = 1232; 72.26%) and clay pots (n = 2; 0.12%) were the most and least inspected containers, respectively, while drums, the second most commonly sampled (n = 249; 15.21%), were highly positive (65.63%) and productive (60%). Tires and jerry cans demonstrated the highest and lowest breeding preference ratios, 11.36 and 0.2, respectively. Over 6900 adult mosquitoes were collected and identified into 15 species comprising Cx. quinquefasciatus (n = 4492; 65.04%), Aedes vittatus (n = 1137; 16.46%) and Ae. aegypti (n = 911; 13.19%) and 2 species groups. Simpson's dominance and Shannon-Wiener diversity indices of 0.4388 and 1.1942 were recorded, respectively. Chikungunya virus was isolated from pools of Ae. aegypti (1) and Cx. quinquefasciatus (4), two of which were males. Minimum infection rates of 3.0 and 0.8 were observed for female Ae. aegypti and Cx. quinquefasciatus, respectively. Between 25 and 31.3% of exposed mosquitoes became infected with CHIKV 7, 14 and 21 days post-exposure. For the experimentally infected Cx. quinquefasciatus mosquitoes, between 13 and 40% had the virus disseminated, with 100% transmission being observed among those with disseminated infection. CONCLUSIONS: These results demonstrated high risk of chikungunya transmission for residents in the sampled areas of Mombasa. Transmission data confirmed the probable role played by Cx. quinquefasciatus in the outbreak while the role of Ae. vittatus in the transmission of chikungunya virus remains unknown.

Increased prevalence of the pfdhfr/phdhps quintuple mutant and rapid emergence of pfdhps resistance mutations at codons 581 and 613 in Kisumu, Kenya.

BACKGROUND: Anti-malarial drug resistance in Kenya prompted two drug policy changes within a decade: sulphadoxine-pyrimethamine (SP) replaced chloroquine (CQ) as the first-line anti-malarial in 1998 and artemether-lumefantrine (AL) replaced SP in 2004. Two cross-sectional studies were conducted to monitor changes in the prevalence of molecular markers of drug resistance over the period in which SP was used as the first-line anti-malarial. The baseline study was carried out from 1999-2000, shortly after implementation of SP, and the follow-up study occurred from 2003-2005, during the transition to AL. MATERIALS AND METHODS: Blood was collected from malaria smear-positive, symptomatic patients presenting to outpatient centers in Kisumu, Kenya, during the baseline and follow-up studies. Isolates were genotyped at codons associated with SP and CQ resistance. In vitro IC50 values for antifolates and quinolones were determined for isolates from the follow-up study. RESULTS: The prevalence of isolates containing the pfdhfr N51I/C59R/S108N/pfdhps A437G/K540E quintuple mutant associated with SP-resistance rose from 21% in the baseline study to 53% in the follow-up study (p < 0.001). Isolates containing the pfdhfr I164L mutation were absent from both studies. The pfdhps mutations A581G and A613S/T were absent from the baseline study but were present in 85% and 61%, respectively, of isolates from the follow-up study. At follow-up, parasites with mutations at five pfdhps codons, 436, 437, 540, 581, and 613, accounted for 39% of isolates. The CQ resistance-associated mutations pfcrt K76T and pfmdr1 N86Y rose from 82% to 97% (p = 0.001) and 44% to 76% (p < 0.001), respectively, from baseline to follow-up. CONCLUSIONS: During the period in which SP was the first-line anti-malarial in Kenya, highly SP-resistant parasites emerged, including isolates harboring pfdhps mutations not previously observed there. SP continues to be widely used in Kenya; however, given the highly resistant genotypes observed in this study, its use as a first-line anti-malarial should be discouraged, particularly for populations without acquired immunity to malaria. The increase in the pfcrt K76T prevalence, despite efforts to reduce CQ use, suggests that either these efforts are not adequate to alleviate CQ pressure in Kisumu, or that drug pressure is derived from another source, such as the second-line anti-malarial amodiaquine.

Emergence of a novel chikungunya virus strain bearing the E1:V80A substitution, out of the Mombasa, Kenya 2017-2018 outbreak.

Between late 2017 and mid-2018, a chikungunya fever outbreak occurred in Mombasa, Kenya that followed an earlier outbreak in mid-2016 in Mandera County on the border with Somalia. Using targeted Next Generation Sequencing, we obtained genomes from clinical samples collected during the 2017/2018 Mombasa outbreak. We compared data from the 2016 Mandera outbreak with the 2017/2018 Mombasa outbreak, and found that both had the Aedes aegypti adapting mutations, E1:K211E and E2:V264A. Further to the above two mutations, 11 of 15 CHIKV genomes from the Mombasa outbreak showed a novel triple mutation signature of E1:V80A, E1:T82I and E1:V84D. These novel mutations are estimated to have arisen in Mombasa by mid-2017 (2017.58, 95% HPD: 2017.23, 2017.84). The MRCA for the Mombasa outbreak genomes is estimated to have been present in early 2017 (2017.22, 95% HPD: 2016.68, 2017.63). Interestingly some of the earliest genomes from the Mombasa outbreak lacked the E1:V80A, E1:T82I and E1:V84D substitutions. Previous laboratory experiments have indicated that a substitution at position E1:80 in the CHIKV genome may lead to increased CHIKV transmissibility by Ae. albopictus. Genbank investigation of all available CHIKV genomes revealed that E1:V80A was not present; therefore, our data constitutes the first report of the E1:V80A mutation occurring in nature. To date, chikungunya outbreaks in the Northern and Western Hemispheres have occurred in Ae. aegypti inhabited tropical regions. Notwithstanding, it has been suggested that an Ae. albopictus adaptable ECSA or IOL strain could easily be introduced in these regions leading to a new wave of outbreaks. Our data on the recent Mombasa CHIKV outbreak has shown that a potential Ae. albopictus adapting mutation may be evolving within the East African region. It is even more worrisome that there exists potential for emergence of a CHIKV strain more adapted to efficient transmission by both Ae. albopictus and Ae.aegypti simultaneously. In view of the present data and history of chikungunya outbreaks, pandemic potential for such a strain is now a likely possibility in the future. Thus, continued surveillance of chikungunya backed by molecular epidemiologic capacity should be sustained to understand the evolving public health threat and inform prevention and control measures including the ongoing vaccine development efforts.

Origin and evolution of dengue virus type 2 causing outbreaks in Kenya: Evidence of circulation of two cosmopolitan genotype lineages.

Dengue fever (DF) is an arboviral disease caused by dengue virus serotypes 1-4 (DENV 1-4). Globally, DF incidence and disease burden have increased in the recent past. Initially implicated in a 1982 outbreak, DENV-2 recently reemerged in Kenya causing outbreaks between 2011 and 2014 and more recently 2017-8. The origin and the evolutionary patterns that may explain the epidemiological expansion and increasing impact of DENV-2 in Kenya remain poorly understood. Using whole-genome sequencing, samples collected during the 2011-4 and 2017-8 dengue outbreaks were analyzed. Additional DENV-2 genomes were downloaded and pooled together with the fourteen genomes generated in this study. Bioinformatic methods were used to analyze phylogenetic relationships and evolutionary patterns of DENV-2 causing outbreaks in Kenya. The findings from this study have shown the first evidence of circulation of two different Cosmopolitan genotype lineages of DENV-2; Cosmopolitan-I (C-I) and Cosmopolitan-II (C-II), in Kenya. Our results put the origin location of C-I lineage in India in 2011, and C-II lineage in Burkina Faso between 1979 and 2013. C-I lineage was the most isolated during recent outbreaks, thus showing the contribution of this newly emerged strain to the increased DENV epidemics in the region. Our findings, backed by evidence of recent local epidemics that have been associated with C-I in Kenya and C-II in Burkina Faso, add to the growing evidence of expanding circulation and the impact of multiple strains of DENV in the region as well as globally. Thus, continued surveillance efforts on DENV activity and its evolutionary trends in the region, would contribute toward effective control and the current vaccine development efforts.

Global Outbreaks and Origins of a Chikungunya Virus Variant Carrying Mutations Which May Increase Fitness for Aedes aegypti: Revelations from the 2016 Mandera, Kenya Outbreak.

In 2016, a chikungunya virus (CHIKV) outbreak was reported in Mandera, Kenya. This was the first major CHIKV outbreak in the country since the global reemergence of this virus in Kenya in 2004. We collected samples and sequenced viral genomes from this outbreak. All Kenyan genomes contained two mutations, E1:K211E and E2:V264A, recently reported to have an association with increased infectivity, dissemination, and transmission in the Aedes aegypti vector. Phylogeographic inference of temporal and spatial virus relationships showed that this variant emerged within the East, Central, and South African lineage between 2005 and 2008, most probably in India. It was also in India where the first large outbreak caused by this virus appeared, in New Delhi, 2010. More importantly, our results also showed that this variant is no longer contained to India. We found it present in several major outbreaks, including the 2016 outbreaks in Pakistan and Kenya, and the 2017 outbreak in Bangladesh. Thus, this variant may have a capability of driving large CHIKV outbreaks in different regions of the world. Our results point to the importance of continued genomic-based surveillance and prompt urgent vector competence studies to assess the level of vector susceptibility and virus transmission, and the impact this might have on this variant's epidemic potential and global spread.

First report of Entamoeba moshkovskii in human stool samples from symptomatic and asymptomatic participants in Kenya.

Entamoeba moshkovskii is a member of the Entamoeba complex and a colonizer of the human gut. We used nested polymerase chain reaction (PCR) to differentiate Entamoeba species in stool samples that had previously been screened by microscopy. Forty-six samples were tested, 23 of which had previously been identified as Entamoeba complex positive by microscopy. Of the 46 specimens tested, we identified nine (19.5%) as E. moshkovskii-positive. In seven of these nine E. moshkovskii-positive samples, either E. dispar or E. histolytica (or both) were also identified, suggesting that co-infections may be common. E. moshkovskii was also detected in both symptomatic and asymptomatic participants. To the best of our knowledge, this is the first report of E. moshkovskii in Kenya.