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.

Genomic surveillance of SARS-COV-2 reveals diverse circulating variant lineages in Nairobi and Kiambu Counties, Kenya.

Genomic surveillance and identification of COVID-19 outbreaks are important in understanding the genetic diversity, phylogeny, and lineages of SARS-CoV-2. Genomic surveillance provides insights into circulating infections, and the robustness and design of vaccines and other infection control approaches. We sequenced 57 SARS-CoV-2 isolates from a Kenyan clinical population, of which 55 passed quality checks using the Ultrafast Sample placement on the Existing tRee (UShER) workflow. Phylo-genome-temporal analyses across two regions in Kenya (Nairobi and Kiambu County) revealed that B.1.1.7 (Alpha; n = 32, 56.1%) and B.1 (n = 9, 15.8%) were the predominant lineages, exhibiting low Ct values (5-31) suggesting high infectivity, and variant mutations across the two regions. Lineages B.1.617.2, B.1.1, A.23.1, A.2.5.1, B.1.596, A, and B.1.405 were also detected across sampling sites within target populations. The lineages and genetic isolates were traced back to China (A), Costa Rica (A.2.5.1), Europe (B.1, B.1.1, A.23.1), the USA (B.1.405, B.1.596), South Africa (B.1.617.2), and the United Kingdom (B.1.1.7), indicating multiple introduction events. This study represents one of the genomic SARS-CoV-2 epidemiology studies in the Nairobi metropolitan area, and describes the importance of continued surveillance for pandemic control.

Correlation Between Malaria-Specific Antibody Profiles and Responses to Artemisinin Combination Therapy for Treatment of Uncomplicated Malaria in Western Kenya.

BACKGROUND: The impact of preexisting immunity on the efficacy of artemisinin combination therapy must be examined to monitor resistance, and for implementation of new treatment strategies. METHODS: Serum samples obtained from a clinical trial in Western Kenya randomized to receive artemether-lumefantrine (AL) or artesunate-mefloquine (ASMQ) were screened for total immunoglobulin G against preerythrocytic and erythrocytic antigens. The association and correlation between different variables, and impact of preexisting immunity on parasite slope half-life (t½) was determined. RESULTS: There was no significant difference in t½, but the number of individuals with lag phase was significantly higher in the AL than in the ASMQ arm (29 vs 13, respectively; P < .01). Circumsporozoite protein-specific antibodies correlate positively with t½ (AL, P = .03; ASMQ, P = .09), but negatively with clearance rate in both study arms (AL, P = .16; ASMQ, P = .02). The t½ correlated negatively with age in ASMQ group. When stratified based on t½, the antibody titers against circumsporozoite protein and merozoite surface protein 1 were significantly higher in participants who cleared parasites rapidly in the AL group (P = .01 and P = .02, respectively). CONCLUSION: Data presented here define immunoprofiles associated with distinct responses to 2 different antimalarial drugs, revealing impact of preexisting immunity on the efficacy of artemisinin combination therapy regimens in a malaria-holoendemic area. CLINICAL TRIALS REGISTRATION: NCT01976780.

Identification and Characterization of Orientia chuto in Trombiculid Chigger Mites Collected from Wild Rodents in Kenya.

We present data that concurs with the reported geographical expansion of scrub typhus outside the "Tsutsugamushi Triangle" and addition of Orientia chuto as a second species in the Orientia genus. Wild rodents were caught in Marigat, Baringo County, Kenya, and ectoparasites, including chiggers, were recovered. Rodent and chigger species were identified by taxonomic features. DNA was extracted from the chiggers and used to amplify and/or sequence the 47-kDa high temperature transmembrane protein (TSA47), the 56-kDa type-specific antigen (TSA56), and the 16S rRNA (rrs) Orientia genes. The main rodent hosts identified were Acomys wilsoni, Crocidura sp., and Mastomys natalensis, which accounted for 59.2% of the total collection. Of these, A. wilsoni and M. natalensis harbored most of the chiggers that belonged to the Neotrombicula and Microtrombicula genera. A pool of chiggers from one of M. natalensis was positive for Orientia by TSA47 PCR, but Orientia did not amplify with the TSA56 primers. On sequencing the 850 bp of the TSA47 gene, the closest phylogenetic relative was O. chuto, with 97.65% sequence homology compared to 84.63 to 84.76% for O. tsutsugamushi 16S rRNA deep sequencing also revealed O. chuto as the closest phylogenetic relative, with 99.75% sequence homology. These results and the existing immunological and molecular reports are strongly suggestive of the existence of Orientia species in Kenya.

Stabilization of RDT target antigens present in dried Plasmodium falciparum-infected samples for validating malaria rapid diagnostic tests at the point of care.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are a great achievement in implementation of parasite based diagnosis as recommended by World Health Organization. A major drawback of RDTs is lack of positive controls to validate different batches/lots at the point of care. Dried Plasmodium falciparum-infected samples with the RDT target antigens have been suggested as possible positive control but their utility in resource limited settings is hampered by rapid loss of activity over time. METHODS: This study evaluated the effectiveness of chemical additives to improve long term storage stability of RDT target antigens (HRP2, pLDH and aldolase) in dried P. falciparum-infected samples using parasitized whole blood and culture samples. Samples were treated with ten selected chemical additives mainly sucrose, trehalose, LDH stabilizer and their combinations. After baseline activity was established, the samples were air dried in bio-safety cabinet and stored at room temperatures (~ 25 °C). Testing of the stabilized samples using SD Bioline, BinaxNOW, CareStart, and First Response was done at intervals for 53 weeks. RESULTS: Stability of HRP2 at ambient temperature was reported at 21-24 weeks while that of PAN antigens (pLDH and aldolase) was 2-18 weeks of storage at all parasite densities. The ten chemical additives increased the percentage stability of HRP2 and PAN antigens. Sucrose alone and its combinations with Alsever's solution or biostab significantly increased stability of HRP2 by 56% at 2000 p/µL (p < 0.001). Trehalose and its combinations with biostab, sucrose or glycerol significantly increased stability of HRP2 by 57% (p < 0.001). Unlike sucrose, the stability of the HRP2 was significantly retained by trehalose at lower concentrations (500, and 200 p/µL). Trehalose in combination biostab stabilizer increased the percentage stability of PAN antigens by 42, and 32% at 2000 and 500 p/µL respectively (p < 0.01). This was also the chemical combination with the shortest reconstitution time (~ < 20 min). CONCLUSIONS: These findings confirm that stabilizing RDT target antigens in dried P. falciparum-infected samples using chemical additives provides field-stable positive controls for malaria RDTs.

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.

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya.

BACKGROUND: Early and accurate diagnosis of malaria is important in treatment as well as in the clinical evaluation of drugs and vaccines. Evaluation of Giemsa-stained smears remains the gold standard for malaria diagnosis, although diagnostic errors and potential bias estimates of protective efficacy have been reported in practice. Plasmodium genus fluorescent in situ hybridization (P-Genus FISH) is a microscopy-based method that uses fluorescent labelled oligonucleotide probes targeted to pathogen specific ribosomal RNA fragments to detect malaria parasites in whole blood. This study sought to evaluate the diagnostic performance of P-Genus FISH alongside Giemsa microscopy compared to quantitative reverse transcription polymerase chain reaction (qRT-PCR) in a clinical setting. METHOD: Five hundred study participants were recruited prospectively and screened for Plasmodium parasites by P-Genus FISH assay, and Giemsa microscopy. The microscopic methods were performed by two trained personnel and were blinded, and if the results were discordant a third reading was performed as a tie breaker. The diagnostic performance of both methods was evaluated against qRT-PCR as a more sensitive method. RESULTS: The number of Plasmodium positive cases was 26.8% by P-Genus FISH, 33.2% by Giemsa microscopy, and 51.2% by qRT-PCR. The three methods had 46.8% concordant results with 61 positive cases and 173 negative cases. Compared to qRT-PCR the sensitivity and specificity of P-Genus FISH assay was 29.3 and 75.8%, respectively, while microscopy had 58.2 and 93.0% respectively. Microscopy had a higher positive and negative predictive values (89.8 and 68.0% respectively) compared to P-Genus FISH (56.0 and 50.5%). In overall, microscopy had a good measure of agreement (76%, k = 0.51) compared to P-Genus FISH (52%, k = 0.05). CONCLUSION: The diagnostic performance of P-Genus FISH was shown to be inferior to Giemsa microscopy in the clinical samples. This hinders the possible application of the method in the field despite the many advantages of the method especially diagnosis of low parasite density infections. The P-Genus assay has great potential but application of the method in clinical setting would rely on extensive training of microscopist and continuous proficiency testing.

High seroprevalence of antibodies against spotted fever and scrub typhus bacteria in patients with febrile Illness, Kenya.

Serum samples from patients in Kenya with febrile illnesses were screened for antibodies against bacteria that cause spotted fever, typhus, and scrub typhus. Seroprevalence was 10% for spotted fever group, <1% for typhus group, and 5% for scrub typhus group. Results should help clinicians expand their list of differential diagnoses for undifferentiated fevers.

The Walter Reed Project, Kisumu Field Station: Impact of Research on Malaria Policy, Management, and Prevention.

The Walter Reed Project is a collaboration between the Walter Reed Army Institute of Research of the United States Department of Defense and the Kenya Medical Research Institute. The Kisumu field station, comprising four campuses, has until recently been devoted primarily to research on malaria countermeasures. The Kombewa Clinical Research Center is dedicated to conducting regulated clinical trials of therapeutic and vaccine candidates in development. The center's robust population-based surveillance platform, along with an active community engagement strategy, guarantees consistent recruitment and retention of participants in clinical trials. The Malaria Diagnostic Center, backed by WHO-certified microscopists and a large malaria blood film collection, champions high-quality malaria diagnosis and strict quality assurance through standardized microscopy trainings. The Malaria Drug Resistance Laboratory leverages cutting-edge technology such as real-time Polymerase Chain Reaction (qPCR) to conduct comprehensive research on resistance markers and obtain information on drug efficacy. The laboratory has been working on validating artemisinin resistance markers and improving tracking methods for current and future antimalarial compounds. Finally, the Basic Science Laboratory employs advanced genomic technology to examine endpoints such as immunogenicity and genomic fingerprinting for candidate drugs and vaccine efficacy. Herein, we examine the site's significant contributions to malaria policy, management, and prevention practices in Kenya and around the world.

Results from tandem Phase 1 studies evaluating the safety, reactogenicity and immunogenicity of the vaccine candidate antigen Plasmodium falciparum FVO merozoite surface protein-1 (MSP1(42)) administered intramuscularly with adjuvant system AS01.

BACKGROUND: The development of an asexual blood stage vaccine against Plasmodium falciparum malaria based on the major merozoite surface protein-1 (MSP1) antigen is founded on the protective efficacy observed in preclinical studies and induction of invasion and growth inhibitory antibody responses. The 42 kDa C-terminus of MSP1 has been developed as the recombinant protein vaccine antigen, and the 3D7 allotype, formulated with the Adjuvant System AS02A, has been evaluated extensively in human clinical trials. In preclinical rabbit studies, the FVO allele of MSP142 has been shown to have improved immunogenicity over the 3D7 allele, in terms of antibody titres as well as growth inhibitory activity of antibodies against both the heterologous 3D7 and homologous FVO parasites. METHODS: Two Phase 1 clinical studies were conducted to examine the safety, reactogenicity and immunogenicity of the FVO allele of MSP142 in the adjuvant system AS01 administered intramuscularly at 0-, 1-, and 2-months: one in the USA and, after evaluation of safety data results, one in Western Kenya. The US study was an open-label, dose escalation study of 10 and 50 µg doses of MSP142 in 26 adults, while the Kenya study, evaluating 30 volunteers, was a double-blind, randomized study of only the 50 µg dose with a rabies vaccine comparator. RESULTS: In these studies it was demonstrated that this vaccine formulation has an acceptable safety profile and is immunogenic in malaria-naïve and malaria-experienced populations. High titres of anti-MSP1 antibodies were induced in both study populations, although there was a limited number of volunteers whose serum demonstrated significant inhibition of blood-stage parasites as measured by growth inhibition assay. In the US volunteers, the antibodies generated exhibited better cross-reactivity to heterologous MSP1 alleles than a MSP1-based vaccine (3D7 allele) previously tested at both study sites. CONCLUSIONS: Given that the primary effector mechanism for blood stage vaccine targets is humoral, the antibody responses demonstrated to this vaccine candidate, both quantitative (total antibody titres) and qualitative (functional antibodies inhibiting parasite growth) warrant further consideration of its application in endemic settings. TRIAL REGISTRATIONS: Clinical Trials NCT00666380.

Characterization of a novel Plasmodium falciparum merozoite surface antigen and potential vaccine target.

Introduction: Detailed analyses of genetic diversity, antigenic variability, protein localization and immunological responses are vital for the prioritization of novel malaria vaccine candidates. Comprehensive approaches to determine the most appropriate antigen variants needed to provide broad protection are challenging and consequently rarely undertaken. Methods: Here, we characterized PF3D7_1136200, which we named Asparagine-Rich Merozoite Antigen (ARMA) based on the analysis of its sequence, localization and immunogenicity. We analyzed IgG and IgM responses against the common variants of ARMA in independent prospective cohort studies in Burkina Faso (N = 228), Kenya (N = 252) and Mali (N = 195) using a custom microarray, Div-KILCHIP. Results: We found a marked population structure between parasites from Africa and Asia. African isolates shared 34 common haplotypes, including a dominant pair although the overall selection pressure was directional (Tajima's D = -2.57; Fu and Li's F = -9.69; P < 0.02). ARMA was localized to the merozoite surface, IgG antibodies induced Fc-mediated degranulation of natural killer cells and strongly inhibited parasite growth in vitro. We found profound serological diversity, but IgG and IgM responses were highly correlated and a hierarchical clustering analysis identified only three major serogroups. Protective IgG and IgM antibodies appeared to target both cross-reactive and distinct epitopes across variants. However, combinations of IgG and IgM antibodies against selected variants were associated with complete protection against clinical episodes of malaria. Discussion: Our systematic strategy exploits genomic data to deduce the handful of antigen variants with the strongest potential to induce broad protection and may be broadly applicable to other complex pathogens for which effective vaccines remain elusive.

B-cell activity in children with malaria.

BACKGROUND: Recent studies implicate deficiency of red blood cell (RBC) complement regulatory proteins (CR1 and CD55) in the pathogenesis of malarial anaemia. This study explored the involvement of B cell CD21, which has an analogous role to RBC CR1. METHODS: In a case control study conducted in Kisumu District hospital, western Kenya, children with severe malaria anaemia (SMA) and those with uncomplicated malaria (UM) were assessed by flow cytometry for B cells (CD20+) numbers, expression levels of CD21 and deposition of C3dg and by ELISA for soluble CD21 (sCD21). Paired t tests were used to determine statistical significance at a = 0.05. RESULTS: Children with SMA had significantly higher lymphocyte count (9,627.7 ± 8786.1 SD vs. 5,507 ± 2436 SD, P = 0.04 in the UM group) and the computed geometric mean of mature B-cell numbers based on the absolute lymphocyte count was significantly higher for SMA group: 1,823 (1,126 to 2,982, 95% CI) and 826.6 (564 to 1,220, 95% CI)] for UM group (P = 0.003). SMA group also had a higher percentage of CD20+ B cells (26.8 ± 9.7SD vs 20.9 ± 9.01 SD in the UM) (P = 0.03), indicating considerable polyclonal B-cell activation. The CD21 median flourescence intensity was lower in the SMA (246.4 ± 87.4 SD vs 369 ± 137.7 SD) (P <0.0001), probably due to complement mediated shaving of CD21 by fixed tissue macrophages. The CD20+ B cells of SMAs had higher levels of the complement split product C3dg (18.35 ± 10 SD vs 11.5 ± 6.8 S.D), (P = 0.0002), confirming possible role of complement in CD21 removal. Unexpectedly, the SMAs had lower levels of sCD21 (226.5 ± 131.5 SD vs 341.4 ± 137.3 SD in the UM) (P < 0.0001), indicating that the shaved CD21 is not released to peripheral circulation. CONCLUSIONS: These results implicate B-cell in pathophysiology of severe malaria that involves increased B-cell proliferation, increased complement deposition and subsequent loss of membrane-bound CD21. The loss of CD21 is not by the classical enzmatic cleavage.

Marked variation in MSP-119 antibody responses to malaria in western Kenyan highlands.

BACKGROUND: Assessment of malaria endemicity at different altitudes and transmission intensities, in the era of dwindling vector densities in the highlands, will provide valuable information for malaria control and surveillance. Measurement of serum anti-malarial antibodies is a useful marker of malaria exposure that indicates long-term transmission potential. We studied the serologic evidence of malaria endemicity at two highland sites along a transmission intensity cline. An improved understanding of the micro-geographic variation in malaria exposure in the highland ecosystems will be relevant in planning effective malaria control. METHODS: Total IgG levels to Plasmodium falciparum MSP-119 were measured in an age-stratified cohort (< 5, 5-14 and ≥ 15 years) in 795 participants from an uphill and valley bottom residents during low and high malaria transmission seasons. Antibody prevalence and level was compared between different localities. Regression analysis was performed to examine the association between antibody prevalence and parasite prevalence. Age-specific MSP-119 seroprevalence data was fitted to a simple reversible catalytic model to investigate the relationship between parasite exposure and age. RESULTS: Higher MSP-119 seroprevalence and density were observed in the valley residents than in the uphill dwellers. Adults (> 15 years) recorded high and stable immune response in spite of changing seasons. Lower responses were observed in children (≤ 15 years), which, fluctuated with changing seasons particularly in the valley residents. In the uphill population, annual seroconversion rate (SCR) was 8.3% and reversion rate was 3.0%, with seroprevalence reaching a plateau of 73.3% by age of 20. Contrary, in the valley bottom population, the annual SCR was 35.8% and the annual seroreversion rate was 3.5%, and seroprevalence in the population had reached 91.2% by age 10. CONCLUSION: The study reveals the micro-geographic variation in malaria endemicity in the highland eco-system; this validates the usefulness of sero-epidemiological tools in assessing malaria endemicity in the era of decreasing sensitivity of conventional tools.

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.

Initiation of anti-retroviral/Trimethoprim-Sulfamethoxazole therapy in a longitudinal cohort of HIV-1 positive individuals in Western Kenya rapidly decreases asymptomatic malarial parasitemia.

Interactions between malaria and HIV-1 have important public health implications. Our previous cross-sectional studies showed significant associations between HIV-1 positivity and malarial parasitemia with an increased risk of gametocytemia. In this follow-up longitudinal study, we evaluated these associations to determine the magnitude of asymptomatic parasitemia over time, and to examine the effects of initiating Antiretroviral Therapy (ART) together with the broad-spectrum antibiotic Trimethoprim Sulfamethoxazole (TS) on asymptomatic parasitemia. 300 adult volunteers in a malaria holoendemic region in Western Kenya were enrolled and followed for six months. The study groups were composed of 102 HIV-1 negatives, 106 newly diagnosed HIV-1 positives and 92 HIV-1 positives who were already stable on ART/TS. Blood samples were collected monthly and asymptomatic malarial parasitemia determined using sensitive 18S qPCR. Results showed significantly higher malaria prevalence in the HIV-1 negative group (61.4%) (p=0.0001) compared to HIV-1 positives newly diagnosed (36.5%) and those stable on treatment (31.45%). Further, treatment with ART/TS had an impact on incidence of asymptomatic parasitemia. In volunteers who were malaria PCR-negative at enrollment, the median time to detectable asymptomatic infection was shorter for HIV-1 negatives (149 days) compared to the HIV-1 positives on treatment (171 days) (p=0.00136). Initiation of HIV treatment among the newly diagnosed led to a reduction in malarial parasitemia (expressed as 18S copy numbers/µl) by over 85.8% within one week of treatment and a further reduction by 96% after 2 weeks. We observed that while the impact of ART/TS on parasitemia was long term, treatment with antimalarial Artemether/Lumefantrine (AL) among the malaria RDT positives had a transient effect with individuals getting re-infected after short periods. As was expected, HIV-1 negative individuals had normal CD4+ levels throughout the study. However, CD4+ levels among HIV-1 positives who started treatment were low at enrollment but increased significantly within the first month of treatment. From our association analysis, the decline in parasitemia among the HIV-1 positives on treatment was attributed to TS treatment and not increased CD4+ levels per se. Overall, this study highlights important interactions between HIV-1 and malaria that may inform future use of TS among HIV-infected patients in malaria endemic regions.

Clinical evaluation of the BioFire Global Fever Panel for the identification of malaria, leptospirosis, chikungunya, and dengue from whole blood: a prospective, multicentre, cross-sectional diagnostic accuracy study.

BACKGROUND: Acute febrile illness is a common presentation for patients at hospitals globally. Assays that can diagnose a variety of common pathogens in blood could help to establish a diagnosis for targeted disease management. We aimed to evaluate the performance of the BioFire Global Fever Panel (GF Panel), a multiplex nucleic acid amplification test performed on whole blood specimens run on the BioFire FilmArray System, in the diagnosis of several pathogens that cause acute febrile illness. METHODS: We did a prospective, multicentre, cross-sectional diagnostic accuracy study to evaluate the GF Panel. Consenting adults and children older than 6 months presenting with fever in the previous 2 days were enrolled consecutively in sub-Saharan Africa (Ghana, Kenya, Tanzania, Uganda), southeast Asia (Cambodia, Thailand), central and South America (Honduras, Peru), and the USA (Washington, DC; St Louis, MO). We assessed the performance of six analytes (chikungunya virus, dengue virus [serotypes 1-4], Leptospira spp, Plasmodium spp, Plasmodium falciparum, and Plasmodium vivax or Plasmodium ovale) on the GF Panel. The performance of the GF Panel was assessed using comparator PCR assays with different primers followed by bidirectional sequencing on nucleic acid extracts from the same specimen. We calculated the positive percent agreement and negative percent agreement of the GF Panel with respect to the comparator assays. This study is registered with ClinicalTrials.gov, NCT02968355. FINDINGS: From March 26, 2018, to Sept 30, 2019, 1965 participants were enrolled at ten sites worldwide. Of the 1875 participants with analysable results, 980 (52·3%) were female and the median age was 22 years (range 0-100). At least one analyte was detected in 657 (35·0%) of 1875 specimens. The GF Panel had a positive percent agreement for the six analytes evaluated as follows: chikungunya virus 100% (95% CI 86·3-100), dengue virus 94·0% (90·6-96·5), Leptospira spp 93·8% (69·8-99·8), Plasmodium spp 98·3% (96·3-99·4), P falciparum 92·7% (88·8-95·6), and P vivax or P ovale 92·7% (86·7-96·6). The GF Panel had a negative percent agreement equal to or greater than 99·2% (98·6-99·6) for all analytes. INTERPRETATION: This 1 h sample-to-answer, molecular device can detect common causative agents of acute febrile illness with excellent positive percent agreement and negative percent agreement directly in whole blood. The targets of the assay are prevalent in tropical and subtropical regions globally, and the assay could help to provide both public health surveillance and individual diagnoses. FUNDING: BioFire Defense, Joint Project Manager for Medical Countermeasure Systems and US Army Medical Materiel Development Activity, and National Institute of Allergy and Infectious Diseases.

Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of plasmodium by amplifying RNA and DNA of the 18S rRNA genes.

A highly sensitive genus-specific quantitative reverse transcriptase real-time PCR (qRT-PCR) assay for detection of Plasmodium has been developed. The assay amplifies total nucleic acids (RNA and DNA) of the 18S rRNA genes with a limit of detection of 0.002 parasite/µl using cultured synchronized ring stage 3D7 parasites. Parasite densities as low as 0.000362 parasite/µl were detected when analyzing clinical samples. Analysis of clinical samples showed that detection of 18S rRNA genes from total nucleic acids increased the analytical sensitivity of the assay by more than 1 log unit compared to DNA only. When clinical samples with no parasites present by microscopy were analyzed by qRT-PCR, 90% (117 of 130) were positive for the presence of Plasmodium nucleic acids. Quantification of clinical samples by qRT-PCR using total nucleic acid versus DNA was compared to microscopy. There was a significantly greater correlation of parasite density to microscopy when DNA alone was used than with total nucleic acid. We conclude that analysis of total nucleic acids by qRT-PCR is a suitable assay for detection of low parasite levels in patients with early-stage malaria and/or submicroscopic infections and could greatly benefit malaria diagnosis, intervention trials, and malaria control and elimination efforts.

Malaria prevalence defined by microscopy, antigen detection, DNA amplification and total nucleic acid amplification in a malaria-endemic region during the peak malaria transmission season.

OBJECTIVES: To determine the malaria prevalence by microscopy, antigen detection and nucleic acid detection in a defined subpopulation in a Plasmodium falciparum-endemic region during the peak transmission season. METHODS: Blood specimens were collected in a cross-sectional study involving children aged 5-10 years (n = 195) presenting with acute fever to two clinics in Western Kenya. All specimens underwent microscopy, HRP2 and aldolase antigen detection by enzyme immunoassay (EIA), parasite-specific DNA and total nucleic acid (RNA and DNA) by real-time PCR (qPCR) and reverse-transcriptase PCR (qRT-PCR). RESULTS: Microscopy detected 65/195 cases of malaria infection [95% confidence interval (CI) 52-78]. HRP2 and aldolase EIA had similar sensitivity levels detecting antigen in 65/195 (95% CI, 52-78) and 57/195 (95% CI, 45-70) cases. Discordants in antigen detection vs. microscopy occurred at <470 parasites/µl and <4900 parasites/µl for HRP2 and aldolase, respectively. Detection of total nucleic acid allowed a 3 log lower limit of detection than just DNA detection by real-time PCR in vitro. In clinical specimens, 114/195 (95% CI, 100-127) were qPCR positive (DNA), and 187/195 (95% CI, 179-191) were qRT-PCR positive (DNA plus RNA). CONCLUSIONS: The prevalence of submicroscopic malaria infection was significantly higher when detecting total nucleic acid than just DNA in this outpatient population during the high transmission season. Defining standards for submicroscopic infection will be important for control programmes, diagnostics development efforts and molecular epidemiology studies.