Distinct transcriptomic signatures define febrile malaria depending on initial infective states, asymptomatic or uninfected.

BACKGROUND: Cumulative malaria parasite exposure in endemic regions often results in the acquisition of partial immunity and asymptomatic infections. There is limited information on how host-parasite interactions mediate the maintenance of chronic symptomless infections that sustain malaria transmission. METHODS: Here, we determined the gene expression profiles of the parasite population and the corresponding host peripheral blood mononuclear cells (PBMCs) from 21 children (< 15 years). We compared children who were defined as uninfected, asymptomatic and those with febrile malaria. RESULTS: Children with asymptomatic infections had a parasite transcriptional profile characterized by a bias toward trophozoite stage (~ 12 h-post invasion) parasites and low parasite levels, while early ring stage parasites were characteristic of febrile malaria. The host response of asymptomatic children was characterized by downregulated transcription of genes associated with inflammatory responses, compared with children with febrile malaria,. Interestingly, the host responses during febrile infections that followed an asymptomatic infection featured stronger inflammatory responses, whereas the febrile host responses from previously uninfected children featured increased humoral immune responses. CONCLUSIONS: The priming effect of prior asymptomatic infection may explain the blunted acquisition of antibody responses seen to malaria antigens following natural exposure or vaccination in malaria endemic areas.

Extracellular vesicles could be a putative posttranscriptional regulatory mechanism that shapes intracellular RNA levels in Plasmodium falciparum.

Plasmodium falciparum secretes extracellular vesicles (PfEVs) that contain parasite-derived RNA. However, the significance of the secreted RNA remains unexplored. Here, we compare secreted and intracellular RNA from asexual cultures of six P. falciparum lines. We find that secretion of RNA via extracellular vesicles is not only periodic throughout the asexual intraerythrocytic developmental cycle but is also highly conserved across P. falciparum isolates. We further demonstrate that the phases of RNA secreted via extracellular vesicles are discernibly shifted compared to those of the intracellular RNA within the secreting whole parasite. Finally, transcripts of genes with no known function during the asexual intraerythrocytic developmental cycle are enriched in PfEVs compared to the whole parasite. We conclude that the secretion of extracellular vesicles could be a putative posttranscriptional RNA regulation mechanism that is part of or synergise the classic RNA decay processes to maintain intracellular RNA levels in P. falciparum.

New SARS-CoV-2 Omicron Variant with Spike Protein Mutation Y451H, Kilifi, Kenya, March-May 2023.

We report a newly emerged SARS-CoV-2 Omicron subvariant FY.4 that has mutations Y451H in spike and P42L in open reading frame 3a proteins. FY.4 emergence coincided with increased SARS-CoV-2 cases in coastal Kenya during April-May 2023. Continued SARS-CoV-2 genomic surveillance is needed to identify new lineages to inform COVID-19 outbreak prevention.

SARS-CoV-2 seroprevalence and implications for population immunity: Evidence from two Health and Demographic Surveillance System sites in Kenya, February-December 2022.

BACKGROUND: We sought to estimate SARS-CoV-2 antibody seroprevalence within representative samples of the Kenyan population during the third year of the COVID-19 pandemic and the second year of COVID-19 vaccine use. METHODS: We conducted cross-sectional serosurveys among randomly selected, age-stratified samples of Health and Demographic Surveillance System (HDSS) residents in Kilifi and Nairobi. Anti-spike (anti-S) immunoglobulin G (IgG) serostatus was measured using a validated in-house ELISA and antibody concentrations estimated with reference to the WHO International Standard for anti-SARS-CoV-2 immunoglobulin. RESULTS: HDSS residents were sampled in February-June 2022 (Kilifi HDSS N = 852; Nairobi Urban HDSS N = 851) and in August-December 2022 (N = 850 for both sites). Population-weighted coverage for ≥1 doses of COVID-19 vaccine were 11.1% (9.1-13.2%) among Kilifi HDSS residents by November 2022 and 34.2% (30.7-37.6%) among Nairobi Urban HDSS residents by December 2022. Population-weighted anti-S IgG seroprevalence among Kilifi HDSS residents increased from 69.1% (65.8-72.3%) by May 2022 to 77.4% (74.4-80.2%) by November 2022. Within the Nairobi Urban HDSS, seroprevalence by June 2022 was 88.5% (86.1-90.6%), comparable with seroprevalence by December 2022 (92.2%; 90.2-93.9%). For both surveys, seroprevalence was significantly lower among Kilifi HDSS residents than among Nairobi Urban HDSS residents, as were antibody concentrations (p < 0.001). CONCLUSION: More than 70% of Kilifi residents and 90% of Nairobi residents were seropositive for anti-S IgG by the end of 2022. There is a potential immunity gap in rural Kenya; implementation of interventions to improve COVID-19 vaccine uptake among sub-groups at increased risk of severe COVID-19 in rural settings is recommended.

Symptom prevalence and secondary attack rate of SARS-CoV-2 in rural Kenyan households: A prospective cohort study.

BACKGROUND: We estimated the secondary attack rate of SARS-CoV-2 among household contacts of PCR-confirmed cases of COVID-19 in rural Kenya and analysed risk factors for transmission. METHODS: We enrolled incident PCR-confirmed cases and their household members. At baseline, a questionnaire, a blood sample, and naso-oropharyngeal swabs were collected. Household members were followed 4, 7, 10, 14, 21 and 28 days after the date of the first PCR-positive in the household; naso-oropharyngeal swabs were collected at each visit and used to define secondary cases. Blood samples were collected every 1-2 weeks. Symptoms were collected in a daily symptom diary. We used binomial regression to estimate secondary attack rates and survival analysis to analyse risk factors for transmission. RESULTS: A total of 119 households with at least one positive household member were enrolled between October 2020 and September 2022, comprising 503 household members; 226 remained in follow-up at day 14 (45%). A total of 43 secondary cases arose within 14 days of identification of the primary case, and 81 household members remained negative. The 7-day secondary attack rate was 4% (95% CI 1%-10%), the 14-day secondary attack rate was 28% (95% CI 17%-40%). Of 38 secondary cases with data, eight reported symptoms (21%, 95% CI 8%-34%). Antibody to SARS-CoV-2 spike protein at enrolment was not associated with risk of becoming a secondary case. CONCLUSION: Households in our setting experienced a lower 7-day attack rate than a recent meta-analysis indicated as the global average (23%-43% depending on variant), and infection is mostly asymptomatic in our setting.

Validation of saline, PBS and a locally produced VTM at varying storage conditions to detect the SARS-CoV-2 virus by qRT-PCR.

Coronavirus Disease-2019 tests require a Nasopharyngeal (NP) and/or Oropharyngeal (OP) specimen from the upper airway, from which virus RNA is extracted and detected through quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR). The viability of the virus is maintained after collection by storing the NP/OP swabs in Viral Transport Media (VTM). We evaluated the performance of four transport media: locally manufactured ("REVITAL") Viral Transport Media (RVTM), Standard Universal Transport Media (SUTM), PBS and 0.9% (w/v) NaCl (normal saline). We used laboratory cultured virus to evaluate: i) viral recovery and maintaining integrity at different time periods and temperatures; ii) stability in yielding detectable RNA consistently for all time points and conditions; and iii) their overall accuracy. Four vials of SARS-CoV-2 cultured virus (2 high and 2 low concentration samples) and 1 negative control sample were prepared for each media type (SUTM, RVTM, PBS and normal saline) and stored at the following temperatures, -80°C, 4°C, 25°C and 37°C for 7 days. Viral RNA extractions and qRT-PCR were performed at 1, 2, 3, 4 and 7 days after inoculation with the cultured virus to assess virus stability and viral recovery. Ct values fell over time at 25°C and 37°C, but normal saline, PBS, RVTM and SUTM all showed comparable performance in maintaining virus integrity and stability allowing for the detection of SARS-CoV-2 RNA. Overall, this study demonstrated that normal saline, PBS and the locally manufactured VTM can be used for COVID-19 sample collection and testing, thus expanding the range of SARS-CoV-2 viral collection media.

Serum immunoglobulin G and mucosal immunoglobulin A antibodies from prepandemic samples collected in Kilifi, Kenya, neutralize SARS-CoV-2 in vitro.

OBJECTIVES: Many regions of Africa have experienced lower COVID-19 morbidity and mortality than Europe. Pre-existing humoral responses to endemic human coronaviruses (HCoV) may cross-protect against SARS-CoV-2. We investigated the neutralizing capacity of SARS-CoV-2 spike reactive and nonreactive immunoglobulin (Ig)G and IgA antibodies in prepandemic samples. METHODS: To investigate the presence of pre-existing immunity, we performed enzyme-linked immunosorbent assay using spike antigens from reference SARS-CoV-2, HCoV HKU1, OC43, NL63, and 229E using prepandemic samples from Kilifi in coastal Kenya. In addition, we performed neutralization assays using pseudotyped reference SARS-CoV-2 to determine the functionality of the identified reactive antibodies. RESULTS: We demonstrate the presence of HCoV serum IgG and mucosal IgA antibodies, which cross-react with the SARS-CoV-2 spike. We show pseudotyped reference SARS-CoV-2 neutralization by prepandemic serum, with a mean infective dose 50 of 1: 251, which is 10-fold less than that of the pooled convalescent sera from patients with COVID-19 but still within predicted protection levels. The prepandemic naso-oropharyngeal fluid neutralized pseudo-SARS-CoV-2 at a mean infective dose 50 of 1: 5.9 in the neutralization assay. CONCLUSION: Our data provide evidence for pre-existing functional humoral responses to SARS-CoV-2 in Kilifi, coastal Kenya and adds to data showing pre-existing immunity for COVID-19 from other regions.

Safety and immunogenicity of ChAdOx1 nCoV-19 (AZD1222) vaccine in adults in Kenya: a phase 1/2 single-blind, randomised controlled trial.

Background: There are limited data on the immunogenicity of coronavirus disease 2019 (COVID-19) vaccines in African populations. Here we report the immunogenicity and safety of the ChAdOx1 nCoV-19 (AZD1222) vaccine from a phase 1/2 single-blind, randomised, controlled trial among adults in Kenya conducted as part of the early studies assessing vaccine performance in different geographical settings to inform Emergency Use Authorisation. Methods: We recruited and randomly assigned (1:1) 400 healthy adults aged ≥18 years in Kenya to receive ChAdOx1 nCoV-19 or control rabies vaccine, each as a two-dose schedule with a 3-month interval. The co-primary outcomes were safety, and immunogenicity assessed using total IgG enzyme-linked immunosorbent assay (ELISA) against SARS-CoV-2 spike protein 28 days after the second vaccination. Results: Between 28 th October 2020 and 19 th August 2021, 400 participants were enrolled and assigned to receive ChAdOx1 nCoV-19 (n=200) or rabies vaccine (n=200). Local and systemic adverse events were self-limiting and mild or moderate in nature. Three serious adverse events were reported but these were deemed unrelated to vaccination. The geometric mean anti-spike IgG titres 28 days after second dose vaccination were higher in the ChAdOx1 group (2773 ELISA units [EU], 95% CI 2447, 3142) than in the rabies vaccine group (61 EU, 95% CI 45, 81) and persisted over the 12 months follow-up. We did not identify any symptomatic infections or hospital admissions with respiratory illness and so vaccine efficacy against clinically apparent infection could not be measured. Vaccine efficacy against asymptomatic SARS-CoV-2 infection was 38.4% (95% CI -26.8%, 70.1%; p=0.188). Conclusions: The safety, immunogenicity and efficacy against asymptomatic infection of ChAdOx1 nCoV-19 among Kenyan adults was similar to that observed elsewhere in the world, but efficacy against symptomatic infection or severe disease could not be measured in this cohort. Pan-African Clinical Trials Registration: PACTR202005681895696 (11/05/2020).

Low frequency of Plasmodium falciparum hrp2/3 deletions from symptomatic infections at a primary healthcare facility in Kilifi, Kenya.

There is a growing concern for malaria control in the Horn of Africa region due to the spread and rise in the frequency of Plasmodium falciparum Histidine-rich Protein (hrp) 2 and 3 deletions. Parasites containing these gene deletions escape detection by the major PfHRP2-based rapid diagnostic test. In this study, the presence of Pfhrp2/3 deletions was examined in uncomplicated malaria patients in Kilifi County, from a region of moderate-high malaria transmission. 345 samples were collected from the Pingilikani dispensary in 2019/2020 during routine malaria care for patients attending this primary health care facility. The Carestart™ RDT and microscopy were used to test for malaria. In addition, qPCR was used to confirm the presence of parasites. In total, 249 individuals tested positive for malaria by RDT, 242 by qPCR, and 170 by microscopy. 11 samples that were RDT-negative and microscopy positive and 25 samples that were qPCR-positive and RDT-negative were considered false negative tests and were examined further for Pfhrp2/3 deletions. Pfhrp2/3-negative PCR samples were further genotyped at the dihydrofolate reductase (Pfdhfr) gene which served to further confirm that parasite DNA was present in the samples. The 242 qPCR-positive samples (confirmed the presence of DNA) were also selected for Pfhrp2/3 genotyping. To determine the frequency of false negative results in low parasitemia samples, the RDT- and qPCR-negative samples were genotyped for Pfdhfr before testing for Pfhrp2/3. There were no Pfhrp2 and Pfhrp3 negative but positive for dhfr parasites in the 11 (RDT negative and microscopy positive) and 25 samples (qPCR-positive and RDT-negative). In the larger qPCR-positive sample set, only 5 samples (2.1%) were negative for both hrp2 and hrp3, but positive for dhfr. Of the 5 samples, there were 4 with more than 100 parasites/µl, suggesting true hrp2/3 deletions. These findings revealed that there is currently a low prevalence of Pfhrp2 and Pfhrp3 deletions in the health facility in Kilifi. However, routine monitoring in other primary health care facilities across the different malaria endemicities in Kenya is urgently required to ensure appropriate use of malaria RDTs.

Epidemiological impact and cost-effectiveness analysis of COVID-19 vaccination in Kenya.

BACKGROUND: A few studies have assessed the epidemiological impact and the cost-effectiveness of COVID-19 vaccines in settings where most of the population had been exposed to SARS-CoV-2 infection. METHODS: We conducted a cost-effectiveness analysis of COVID-19 vaccine in Kenya from a societal perspective over a 1.5-year time frame. An age-structured transmission model assumed at least 80% of the population to have prior natural immunity when an immune escape variant was introduced. We examine the effect of slow (18 months) or rapid (6 months) vaccine roll-out with vaccine coverage of 30%, 50% or 70% of the adult (>18 years) population prioritising roll-out in those over 50-years (80% uptake in all scenarios). Cost data were obtained from primary analyses. We assumed vaccine procurement at US$7 per dose and vaccine delivery costs of US$3.90-US$6.11 per dose. The cost-effectiveness threshold was US$919.11. FINDINGS: Slow roll-out at 30% coverage largely targets those over 50 years and resulted in 54% fewer deaths (8132 (7914-8373)) than no vaccination and was cost saving (incremental cost-effectiveness ratio, ICER=US$-1343 (US$-1345 to US$-1341) per disability-adjusted life-year, DALY averted). Increasing coverage to 50% and 70%, further reduced deaths by 12% (810 (757-872) and 5% (282 (251-317) but was not cost-effective, using Kenya's cost-effectiveness threshold (US$919.11). Rapid roll-out with 30% coverage averted 63% more deaths and was more cost-saving (ICER=US$-1607 (US$-1609 to US$-1604) per DALY averted) compared with slow roll-out at the same coverage level, but 50% and 70% coverage scenarios were not cost-effective. INTERPRETATION: With prior exposure partially protecting much of the Kenyan population, vaccination of young adults may no longer be cost-effective.

Maintenance of high temporal Plasmodium falciparum genetic diversity and complexity of infection in asymptomatic and symptomatic infections in Kilifi, Kenya from 2007 to 2018.

BACKGROUND: High levels of genetic diversity are common characteristics of Plasmodium falciparum parasite populations in high malaria transmission regions. There has been a decline in malaria transmission intensity over 12 years of surveillance in the community in Kilifi, Kenya. This study sought to investigate whether there was a corresponding reduction in P. falciparum genetic diversity, using msp2 as a genetic marker. METHODS: Blood samples were obtained from children (< 15 years) enrolled into a cohort with active weekly surveillance between 2007 and 2018 in Kilifi, Kenya. Asymptomatic infections were defined during the annual cross-sectional blood survey and the first-febrile malaria episode was detected during the weekly follow-up. Parasite DNA was extracted and successfully genotyped using allele-specific nested polymerase chain reactions for msp2 and capillary electrophoresis fragment analysis. RESULTS: Based on cross-sectional surveys conducted in 2007-2018, there was a significant reduction in malaria prevalence (16.2-5.5%: P-value < 0.001), however msp2 genetic diversity remained high. A high heterozygosity index (He) (> 0.95) was observed in both asymptomatic infections and febrile malaria over time. About 281 (68.5%) asymptomatic infections were polyclonal (> 2 variants per infection) compared to 46 (56%) polyclonal first-febrile infections. There was significant difference in complexity of infection (COI) between asymptomatic 2.3 [95% confidence interval (CI) 2.2-2.5] and febrile infections 2.0 (95% CI 1.7-2.3) (P = 0.016). Majority of asymptomatic infections (44.2%) carried mixed alleles (i.e., both FC27 and IC/3D7), while FC27 alleles were more frequent (53.3%) among the first-febrile infections. CONCLUSIONS: Plasmodium falciparum infections in Kilifi are still highly diverse and polyclonal, despite the reduction in malaria transmission in the community.

Genomic Epidemiology of SARS-CoV-2 in Seychelles, 2020-2021.

Seychelles, an archipelago of 155 islands in the Indian Ocean, had confirmed 24,788 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the 31st of December 2021. The first SARS-CoV-2 cases in Seychelles were reported on the 14th of March 2020, but cases remained low until January 2021, when a surge was observed. Here, we investigated the potential drivers of the surge by genomic analysis of 1056 SARS-CoV-2 positive samples collected in Seychelles between 14 March 2020 and 31 December 2021. The Seychelles genomes were classified into 32 Pango lineages, 1042 of which fell within four variants of concern, i.e., Alpha, Beta, Delta and Omicron. Sporadic cases of SARS-CoV-2 detected in Seychelles in 2020 were mainly of lineage B.1 (lineage predominantly observed in Europe) but this lineage was rapidly replaced by Beta variant starting January 2021, and which was also subsequently replaced by the Delta variant in May 2021 that dominated till November 2021 when Omicron cases were identified. Using the ancestral state reconstruction approach, we estimated that at least 78 independent SARS-CoV-2 introduction events occurred in Seychelles during the study period. The majority of viral introductions into Seychelles occurred in 2021, despite substantial COVID-19 restrictions in place during this period. We conclude that the surge of SARS-CoV-2 cases in Seychelles in January 2021 was primarily due to the introduction of more transmissible SARS-CoV-2 variants into the islands.

Transmission networks of SARS-CoV-2 in Coastal Kenya during the first two waves: A retrospective genomic study.

Background: Detailed understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) regional transmission networks within sub-Saharan Africa is key for guiding local public health interventions against the pandemic. Methods: Here, we analysed 1139 SARS-CoV-2 genomes from positive samples collected between March 2020 and February 2021 across six counties of Coastal Kenya (Mombasa, Kilifi, Taita Taveta, Kwale, Tana River, and Lamu) to infer virus introductions and local transmission patterns during the first two waves of infections. Virus importations were inferred using ancestral state reconstruction, and virus dispersal between counties was estimated using discrete phylogeographic analysis. Results: During Wave 1, 23 distinct Pango lineages were detected across the six counties, while during Wave 2, 29 lineages were detected; 9 of which occurred in both waves and 4 seemed to be Kenya specific (B.1.530, B.1.549, B.1.596.1, and N.8). Most of the sequenced infections belonged to lineage B.1 (n = 723, 63%), which predominated in both Wave 1 (73%, followed by lineages N.8 [6%] and B.1.1 [6%]) and Wave 2 (56%, followed by lineages B.1.549 [21%] and B.1.530 [5%]). Over the study period, we estimated 280 SARS-CoV-2 virus importations into Coastal Kenya. Mombasa City, a vital tourist and commercial centre for the region, was a major route for virus imports, most of which occurred during Wave 1, when many Coronavirus Disease 2019 (COVID-19) government restrictions were still in force. In Wave 2, inter-county transmission predominated, resulting in the emergence of local transmission chains and diversity. Conclusions: Our analysis supports moving COVID-19 control strategies in the region from a focus on international travel to strategies that will reduce local transmission. Funding: This work was funded by The Wellcome (grant numbers: 220985, 203077/Z/16/Z, 220977/Z/20/Z, and 222574/Z/21/Z) and the National Institute for Health and Care Research (NIHR), project references: 17/63/and 16/136/33 using UK Aid from the UK government to support global health research, The UK Foreign, Commonwealth and Development Office. The views expressed in this publication are those of the author(s) and not necessarily those of the funding agencies.

Epidemiology of COVID-19 infections on routine polymerase chain reaction (PCR) and serology testing in Coastal Kenya.

Background: There are limited studies in Africa describing the epidemiology, clinical characteristics and serostatus of individuals tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We tested routine samples from the Coastal part of Kenya between 17 th March 2020 and 30 th June 2021. Methods: SARS-CoV-2 infections identified using reverse transcription polymerase chain reaction (RT-PCR) and clinical surveillance data at the point of sample collection were used to classify as either symptomatic or asymptomatic. IgG antibodies were measured in sera samples, using a well validated in-house enzyme-linked immunosorbent assay (ELISA). Results: Mombasa accounted for 56.2% of all the 99,694 naso-pharyngeal/oro-pharyngeal swabs tested, and males constituted the majority tested (73.4%). A total of 7737 (7.7%) individuals were SARS-CoV-2 positive by RT-PCR. The majority (i.e., 92.4%) of the RT-PCR positive individuals were asymptomatic. Testing was dominated by mass screening and travellers, and even at health facility level 91.6% of tests were from individuals without symptoms. Out of the 97,124 tests from asymptomatic individuals 7,149 (7%) were positive and of the 2,568 symptomatic individuals 588 (23%) were positive. In total, 2458 serum samples were submitted with paired naso-pharyngeal/oro-pharyngeal samples and 45% of the RT-PCR positive samples and 20% of the RT-PCR negative samples were paired with positive serum samples. Symptomatic individuals had significantly higher antibody levels than asymptomatic individuals and become RT-PCR negative on repeat testing earlier than asymptomatic individuals. Conclusions: In conclusion, the majority of SARS-CoV-2 infections identified by routine testing in Coastal Kenya were asymptomatic. This reflects the testing practice of health services in Kenya, but also implies that asymptomatic infection is very common in the population. Symptomatic infection may be less common, or it may be that individuals do not present for testing when they have symptoms.

Amplicon Sequencing as a Potential Surveillance Tool for Complexity of Infection and Drug Resistance Markers in Plasmodium falciparum Asymptomatic Infections.

BACKGROUND: Genotyping Plasmodium falciparum subpopulations in malaria infections is an important aspect of malaria molecular epidemiology to understand within-host diversity and the frequency of drug resistance markers. METHODS: We characterized P. falciparum genetic diversity in asymptomatic infections and subsequent first febrile infections using amplicon sequencing (AmpSeq) of ama1 in Coastal Kenya. We also examined temporal changes in haplotype frequencies of mdr1, a drug-resistant marker. RESULTS: We found >60% of the infections were polyclonal (complexity of infection [COI] >1) and there was a reduction in COI over time. Asymptomatic infections had a significantly higher mean COI than febrile infections based on ama1 sequences (2.7 [95% confidence interval {CI}, 2.65-2.77] vs 2.22 [95% CI, 2.17-2.29], respectively). Moreover, an analysis of 30 paired asymptomatic and first febrile infections revealed that many first febrile infections (91%) were due to the presence of new ama1 haplotypes. The mdr1-YY haplotype, associated with chloroquine and amodiaquine resistance, decreased over time, while the NY (wild type) and the NF (modulates response to lumefantrine) haplotypes increased. CONCLUSIONS: This study emphasizes the utility of AmpSeq in characterizing parasite diversity as it can determine relative proportions of clones and detect minority clones. The usefulness of AmpSeq in antimalarial drug resistance surveillance is also highlighted.

Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol.

INTRODUCTION: The ARTIC Network's primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol. An update to the V3 primer set was released on 18th June 2021 to address amplicon drop-off observed among the Delta variant of concern. Here, we report on an in-house optimization of a modified version of the ARTIC Network V4 protocol that improves SARS-CoV-2 genome recovery in instances where the original V4 pooling strategy was characterized by amplicon drop-offs. METHODS: We utilized a matched set of 43 clinical samples and serially diluted positive controls that were amplified by ARTIC V3, V4 and optimized V4 primers and sequenced using GridION from the Oxford Nanopore Technologies'. RESULTS: We observed a 0.5% to 46% increase in genome recovery in 67% of the samples when using the original V4 pooling strategy compared to the V3 primers. Amplicon drop-offs at primer positions 23 and 90 were observed for all variants and positive controls. When using the optimized protocol, we observed a 60% improvement in genome recovery across all samples and an increase in the average depth in amplicon 23 and 90. Consequently, ≥95% of the genome was recovered in 72% (n = 31) of the samples. However, only 60-70% of the genomes could be recovered in samples that had <28% genome coverage with the ARTIC V3 primers. There was no statistically significant (p > 0.05) correlation between Ct value and genome recovery. CONCLUSION: Utilizing the ARTIC V4 primers, while increasing the primer concentrations for amplicons with drop-offs or low average read-depth, greatly improves genome recovery of Alpha, Beta, Delta, Eta and non-VOC/non-VOI SARS-CoV-2 variants.

Targeted Amplicon Deep Sequencing for Monitoring Antimalarial Resistance Markers in Western Kenya.

Molecular surveillance of Plasmodium falciparum parasites is important to track emerging and new mutations and trends in established mutations and should serve as an early warning system for antimalarial resistance. Dried blood spots were obtained from a Plasmodium falciparum malaria survey in school children conducted across eight counties in western Kenya in 2019. Real-time PCR identified 500 P. falciparum-positive samples that were amplified at five drug resistance loci for targeted amplicon deep sequencing (TADS). The absence of important kelch 13 mutations was similar to previous findings in Kenya pre-2019, and low-frequency mutations were observed in codons 569 and 578. The chloroquine resistance transporter gene codons 76 and 145 were wild type, indicating that the parasites were chloroquine and piperaquine sensitive, respectively. The multidrug resistance gene 1 haplotypes based on codons 86, 184, and 199 were predominantly present in mixed infections with haplotypes NYT and NFT, driven by the absence of chloroquine pressure and the use of lumefantrine, respectively. The sulfadoxine-pyrimethamine resistance profile was a "superresistant" combination of triple mutations in both Pfdhfr (51I 59R 108N) and Pfdhps (436H 437G 540E), rendering sulfadoxine-pyrimethamine ineffective. TADS highlighted the low-frequency variants, allowing the early identification of new mutations, Pfmdr1 codon 199S and Pfdhfr codon 85I and emerging 164L mutations. The added value of TADS is its accuracy in identifying mixed-genotype infections and for high-throughput monitoring of antimalarial resistance markers.

Comparative performance of WANTAI ELISA for total immunoglobulin to receptor binding protein and an ELISA for IgG to spike protein in detecting SARS-CoV-2 antibodies in Kenyan populations.

Many SARS-CoV-2 antibody detection assays have been developed but their differential performance is not well described. In this study we compared an in-house (KWTRP) ELISA which has been used extensively to estimate seroprevalence in the Kenyan population with WANTAI, an ELISA which has been approved for widespread use by the WHO. Using a wide variety of sample sets including pre-pandemic samples (negative gold standard), SARS-CoV-2 PCR positive samples (positive gold standard) and COVID-19 test samples from different periods (unknowns), we compared performance characteristics of the two assays. The overall concordance between WANTAI and KWTRP was 0.97 (95% CI, 0.95-0.98). For WANTAI and KWTRP, sensitivity was 0.95 (95% CI 0.90-0.98) and 0.93 (95% CI 0.87-0.96), respectively. Specificity for WANTAI was 0.98 (95% CI, 0.96-0.99) and 0.99 (95% CI 0.96-1.00) while KWTRP specificity was 0.99 (95% CI, 0.98-1.00) and 1.00 using pre-pandemic blood donors and pre-pandemic malaria cross-sectional survey samples respectively. Both assays show excellent characteristics to detect SARS-CoV-2 antibodies.

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial.

Introduction: Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 ( ama1), and multidrug resistance gene 1 ( mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods: P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results: The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions: This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.