Plasmodium falciparum population structure inferred by msp1 amplicon sequencing of parasites collected from febrile patients in Kenya.

BACKGROUND: Multiplicity of infection (MOI) is an important measure of Plasmodium falciparum diversity, usually derived from the highly polymorphic genes, such as msp1, msp2 and glurp as well as microsatellites. Conventional methods of deriving MOI lack fine resolution needed to discriminate minor clones. This study used amplicon sequencing (AmpliSeq) of P. falciparum msp1 ï»¿(Pfmsp1) to measure spatial and temporal genetic diversity of P. falciparum. METHODS: 264 P. falciparum positive blood samples collected from areas of differing malaria endemicities between 2010 and 2019 were used. Pfmsp1 gene was amplified and amplicon libraries sequenced on Illumina MiSeq. Sequences were aligned against a reference sequence (NC_004330.2) and clustered to detect fragment length polymorphism and amino acid variations. RESULTS: Children < 5 years had higher parasitaemia (median = 23.5 ± 5 SD, p = 0.03) than the > 5-14 (= 25.3 ± 5 SD), and those > 15 (= 25.1 ± 6 SD). Of the alleles detected, 553 (54.5%) were K1, 250 (24.7%) MAD20 and 211 (20.8%) RO33 that grouped into 19 K1 allelic families (108-270 bp), 14 MAD20 (108-216 bp) and one RO33 (153 bp). AmpliSeq revealed nucleotide polymorphisms in alleles that had similar sizes, thus increasing the K1 to 104, 58 for MAD20 and 14 for RO33. By AmpliSeq, the mean MOI was 4.8 (± 0.78, 95% CI) for the malaria endemic Lake Victoria region, 4.4 (± 1.03, 95% CI) for the epidemic prone Kisii Highland and 3.4 (± 0.62, 95% CI) for the seasonal malaria Semi-Arid region. MOI decreased with age: 4.5 (± 0.76, 95% CI) for children < 5 years, compared to 3.9 (± 0.70, 95% CI) for ages 5 to 14 and 2.7 (± 0.90, 95% CI) for those > 15. Females' MOI (4.2 ± 0.66, 95% CI) was not different from males 4.0 (± 0.61, 95% CI). In all regions, the number of alleles were high in the 2014-2015 period, more so in the Lake Victoria and the seasonal transmission arid regions. CONCLUSION: These findings highlight the added advantages of AmpliSeq in haplotype discrimination and the associated improvement in unravelling complexity of P. falciparum population structure.

Malaria Parasitemia and Parasite Density in Antiretroviral-Treated HIV-Infected Adults Following Discontinuation of Cotrimoxazole Prophylaxis.

BACKGROUND: Cotrimoxazole (CTX) discontinuation increases malaria incidence in human immunodeficiency virus (HIV)-infected individuals. Rates, quantity, and timing of parasitemia rebound following CTX remain undefined. METHODS: Serial specimens from a trial of HIV-infected individuals receiving antiretroviral treatment (ART) randomized to continue (the CTX arm) or discontinue (the STOP-CTX arm) were examined for malaria parasites by quantitative reverse transcription polymerase chain reaction (PCR). Specimens obtained at enrollment and then quarterly for 12 months and at sick visits were assessed; multiplicity of infection was evaluated by PCR that targeted the polymorphic msp-1/msp-2 alleles. RESULTS: Among 500 HIV-infected adults receiving ART (median ART duration, 4.5 years), 5% had detectable parasitemia at baseline. After randomization, parasite prevalence increased over time in the STOP-CTX arm, compared with the CTX arm, with values of 4% and <1%, respectively, at month 3, 8% and 2% at month 6, 14% and 2% at month 9, and 22% and 4% at month 12 (P = .0034). The combined mean parasite density at the various time points was higher in the STOP-CTX arm (4.42 vs 3.13 log10 parasites/mL; P < .001). The parasitemia incidence was 42.0 cases per 100 person-years in the STOP-CTX arm and 9.9 cases per 100 person-years in the CTX arm, with an incidence rate ratio of 4.3 (95% confidence interval, 2.7-7.1; P < .001). After enrollment, mixed infections (multiplicity of infection, >1) were only present in the STOP-CTX arm. CONCLUSION: Discontinuation of CTX by HIV-infected adults receiving ART resulted in progressive increases in malaria parasitemia prevalence and burden. CLINICAL TRIALS REGISTRATION: NCT01425073.

March 2019 dengue fever outbreak at the Kenyan south coast involving dengue virus serotype 3, genotypes III and V.

The first description of a disease resembling dengue fever (DF) was in the 15th century slave trade era by Spanish sailors visiting the Tanzania coast. The disease, then associated with evil spirits is now known to be caused by four serotypes of dengue virus (DENV1-4) that are transmitted by Aedes mosquitoes. Kenya has experienced multiple outbreaks, mostly associated with DENV-2. In this study, plasma samples obtained from 37 febrile patients during a DF outbreak at Kenya's south coast in March 2019 were screened for DENV. Total RNA was extracted and screened for the alpha- and flavi-viruses by real-time polymerase chain reaction (qPCR). DENV-3 was the only virus detected. Shotgun metagenomics and targeted sequencing were used to obtain DENV whole genomes and the complete envelope genes (E gene) respectively. Sequences were used to infer phylogenies and time-scaled genealogies. Following Maximum likelihood and Bayesian phylogenetic analysis, two DENV-3 genotypes (III, n = 15 and V, n = 2) were found. We determined that the two genotypes had been in circulation since 2015, and that both had been introduced independently. Genotype III's origin was estimated to have been from Pakistan. Although the origin of genotype V could not be ascertained due to rarity of these sequences globally, it was most related to a 2006 Brazilian isolate. Unlike genotype III that has been described in East and West Africa multiple times, this was the second description of genotype V in Kenya. Of note, there was marked amino acid variances in the E gene between study samples and the Thailand DENV-3 strain used in the approved Dengvaxia vaccine. It remains to be seen whether these variances negatively impact the efficacy of the Dengvaxia or future vaccines.

COVID-19 mass testing and sequencing: Experiences from a laboratory in Western Kenya.

Background: The Basic Science Laboratory (BSL) of the Kenya Medical Research Institute/Walter Reed Project in Kisumu, Kenya addressed mass testing challenges posed by the emergent coronavirus disease 2019 (COVID-19) in an environment of global supply shortages. Before COVID-19, the BSL had adequate resources for disease surveillance and was therefore designated as one of the testing centres for COVID-19. Intervention: By April 2020, the BSL had developed stringent safety procedures for receiving and mass testing potentially infectious nasal specimens. To accommodate increased demand, BSL personnel worked in units: nucleic acid extraction, polymerase chain reaction, and data and quality assurance checks. The BSL adopted procedures for tracking sample integrity and minimising cross-contamination. Lessons learnt: Between May 2020 and January 2022, the BSL tested 63 542 samples, of which 5375 (8.59%) were positive for COVID-19; 1034 genomes were generated by whole genome sequencing and deposited in the Global Initiative on Sharing All Influenza Data database to aid global tracking of viral lineages. At the height of the pandemic (August and November 2020, April and August 2021 and January 2022), the BSL was testing more than 500 samples daily, compared to 150 per month prior to COVID-19. An important lesson from the COVID-19 pandemic was the discovery of untapped resilience within BSL personnel that allowed adaptability when the situation demanded. Strict safety procedures and quality management that are often difficult to maintain became routine. Recommendations: A fundamental lesson to embrace is that there is no 'one-size-fits-all' approach and adaptability is the key to success.

Temporal lineage replacements and dominance of imported variants of concern during the COVID-19 pandemic in Kenya.

Background: Kenya's COVID-19 epidemic was seeded early in March 2020 and did not peak until early August 2020 (wave 1), late-November 2020 (wave 2), mid-April 2021 (wave 3), late August 2021 (wave 4), and mid-January 2022 (wave 5). Methods: Here, we present SARS-CoV-2 lineages associated with the five waves through analysis of 1034 genomes, which included 237 non-variants of concern and 797 variants of concern (VOC) that had increased transmissibility, disease severity or vaccine resistance. Results: In total 40 lineages were identified. The early European lineages (B.1 and B.1.1) were the first to be seeded. The B.1 lineage continued to expand and remained dominant, accounting for 60% (72/120) and 57% (45/79) in waves 1 and 2 respectively. Waves three, four and five respectively were dominated by VOCs that were distributed as follows: Alpha 58.5% (166/285), Delta 92.4% (327/354), Omicron 95.4% (188/197) and Beta at 4.2% (12/284) during wave 3 and 0.3% (1/354) during wave 4. Phylogenetic analysis suggests multiple introductions of variants from outside Kenya, more so during the first, third, fourth and fifth waves, as well as subsequent lineage diversification. Conclusions: The data highlights the importance of genome surveillance in determining circulating variants to aid interpretation of phenotypes such as transmissibility, virulence and/or resistance to therapeutics/vaccines.

The evolution of dengue-2 viruses in Malindi, Kenya and greater East Africa: Epidemiological and immunological implications.

Kenya experiences a substantial burden of dengue, yet there are very few DENV-2 sequence data available from this country and indeed the entire continent of Africa. We therefore undertook whole genome sequencing and evolutionary analysis of fourteen dengue virus (DENV)-2 strains sampled from Malindi sub-County Hospital during the 2017 DENV-2 outbreak in the Kenyan coast. We further performed an extended East African phylogenetic analysis, which leveraged 26 complete African env genes. Maximum likelihood analysis showed that the 2017 outbreak was due to the Cosmopolitan genotype, indicating that this has been the only confirmed human DENV-2 genotype circulating in Africa to date. Phylogeographic analyses indicated transmission of DENV-2 viruses between East Africa and South/South-West Asia. Time-scaled genealogies show that DENV-2 viruses shows spatial structure at the country level in Kenya, with a time-to-most-common-recent ancestor analysis indicating that these DENV-2 strains were circulating for up to 5.38 years in Kenya before detection in the 2017 Malindi outbreak. Selection pressure analyses indicated sampled Kenyan DENV strains uniquely being under positive selection at 6 sites, predominantly across the non-structural genes, and epitope prediction analyses showed that one of these sites corresponds to a putative predicted MHC-I CD8+ DENV-2 Cosmopolitan virus epitope only evident in a sampled Kenyan virus. Taken together, our findings indicate that the 2017 Malindi DENV-2 outbreak arose from a strain which had circulated for several years in Kenya before recent detection, has experienced diversifying selection pressure, and may contain new putative immunogens relevant to vaccine design. These findings prompt further genomic epidemiology studies in this and other Kenyan locations to further elucidate the transmission dynamics of DENV in this region.

Plasmodium falciparum Histidine-Rich Protein 2 and 3 Gene Deletions and Their Implications in Malaria Control.

Malaria remains the biggest threat to public health, especially among pregnant women and young children in sub-Saharan Africa. Prompt and accurate diagnosis is critical for effective case management and detection of drug resistance. Conventionally, microscopy and rapid diagnostic tests (RDTs) are the tools of choice for malaria diagnosis. RDTs are simple to use and have been extensively used in the diagnosis of malaria among travelers to malaria-endemic regions, routine case management, and surveillance studies. Most RDTs target the histidine-rich protein (PfHRP) which is exclusively found in Plasmodium falciparum and a metabolic enzyme Plasmodium lactate dehydrogenase (pLDH) which is common among all Plasmodium species. Other RDTs incorporate the enzyme aldolase that is produced by all Plasmodium species. Recently, studies have reported false-negative RDTs primarily due to the deletion of the histidine-rich protein (pfhrp2 and pfhrp3) genes in field isolates of P. falciparum. Herein, we review published literature to establish pfhrp2/pfhrp3 deletions, the extent of these deletions in different geographical regions, and the implication in malaria control. We searched for publications on pfhrp2/pfhrp3 deletions and retrieved all publications that reported on this subject. Overall, 20 publications reported on pfhrp2/pfhrp3 deletions, and most of these studies were done in Central and South America, with very few in Asia and Africa. The few studies in Africa that reported on the occurrence of pfhrp2/pfhrp3 deletions rarely evaluated deletions on the flanking genes. More studies are required to evaluate the existence and extent of these gene deletions, whose presence may lead to delayed or missed treatment. This information will guide appropriate diagnostic approaches in the respective areas.

Serological Evidence of Yersiniosis, Tick-Borne Encephalitis, West Nile, Hepatitis E, Crimean-Congo Hemorrhagic Fever, Lyme Borreliosis, and Brucellosis in Febrile Patients Presenting at Diverse Hospitals in Kenya.

Data on pathogen prevalence is crucial for informing exposure and disease risk. We evaluated serological evidence of tick-borne encephalitis (TBE), West Nile (WN), Hepatitis E virus (HEV), Crimean-Congo Hemorrhagic Fever (CCHF), Yersiniosis, Lyme Disease (LD), and brucellosis in 1033 patients presenting with acute febrile illness at 9 health care facilities from diverse ecological zones of Kenya: arid and semiarid (Garissa District Hospital, Lodwar District Hospital, Marigat District Hospital, Gilgil District Hospital), Lake Victoria basin (Kisumu District Hospital, Alupe District Hospital, Kombewa Sub-County Hospital), Kisii highland (Kisii District Hospital), and coastal (Malindi District Hospital). Epidemiological information of the patients such as geography, age, gender, and keeping animals were analyzed as potential risk factors. Of the 1033 samples, 619 (59.9%) were seropositive to at least one pathogen by IgM (current exposure), IgG/IgM (recent exposure), and IgG (past exposure). Collective seroprevalence for current, recent, and past to the pathogens was 9.4%, 5.1%, and 21.1% for LD; 3.6%, 0.5%, and 12.4% for WN; 0.9%, 0.5%, and 16.9% for HEV; 5.8%, 1.3%, and 3.9% for brucellosis; 5.7%, 0.2%, and 2.3% for yersiniosis; 1.7%, 0%, and 6.2% for TBE; and 0.4%, 0%, and 1.9% for CCHF. Brucellosis risk was higher in patients recruited at Garissa District Hospital (odds ratio [OR] = 3.41), HEV (OR = 2.45) and CCHF (OR = 5.46) in Lodwar District Hospital, LD in Alupe District Hospital (OR = 5.73), Kombewa Sub-district hospital (OR = 8.17), and Malindi District hospital (OR = 3.3). Exposure to LD was highest in the younger age group, whereas yersiniosis did not vary with age. Age was a significant risk for WN, brucellosis, CCHF, TBE, and HEV and in those aged >14 years there was an increased risk to WN (OR = 2.30, p < 0.0001), brucellosis (OR = 1.84, p = 0.005), CCHF (OR = 4.35, p = 0.001), TBE (OR = 2.78, p < 0.0001), and HEV (OR = 1.94, p = 0.0001). We conclude that LD is pervasive and constitutes a significant health burden to the study population, whereas yersiniosis and CCHF are not significant threats. Going forward, community-based studies will be needed to capture the true seroprevalence rates and the associated risk factors.

Complete Coding Sequences of Dengue Virus Type 2 Strains from Febrile Patients Seen in Malindi District Hospital, Kenya, during the 2017 Dengue Fever Outbreak.

We report here 10 complete polyprotein-coding sequences of dengue virus type 2 strains isolated from febrile patients who presented at Malindi District Hospital, Kenya, during a recent dengue fever outbreak. Phylogenetically, all the strains belonged to clonal serotype 2 of the Cosmopolitan genotype.