Genetic Epidemiology of <i>Plasmodium falciparum</i> Asymptomatic Infections and Antimalarial Drug-resistance Markers in Kilifi, Kenya.

Asymptomatic infections and anti-malarial drug resistance are impediments to malaria elimination. Markedly, asymptomatic infections often go undetected and untreated and this creates a parasite reservoir that fuels malaria transmission as well as being a risk factor for febrile malaria. On the other hand, drug-resistance renders antimalarial drugs ineffective and has been associated with increased morbidity and mortality in the past. Using longitudinal malaria monitoring data and Plasmodium falciparum positive samples from Kilifi, Kenya, (i) an evaluation was conducted to assess the impact of age and malaria transmission intensity on the risk of developing febrile malaria in individuals harbouring asymptomatic infections, (ii) amplicon deep-sequencing was used to evaluate P. falciparum genetic diversity in asymptomatic and febrile infections and (iii) the diversity of twelve drug-resistance markers (crt, mdr1, dhps, nfs, k13, ap2mu, falcipain-2a, ubp-1, as well as four artemisinin resistance predisposing mutations; arps10 codon V127M, crt codon I356T, fd codon D193Y and mdr2 codon T484I) in Kilifi was evaluated using Sanger sequencing, including a neutral marker (serine-tRNA ligase) that is not under drug-pressure. Analyses of the data revealed that in the moderate and high transmission intensity settings, asymptomatic infections were associated with a reduced risk of febrile malaria in older children (>3 years), while in the lower transmission setting, asymptomatic infections were associated with an increased risk of febrile malaria in children of all ages. Amplicon deep-sequencing revealed that P. falciparum genetic diversity in asymptomatic and febrile infections differ significantly, similar to previous reports. Also, a majority of the febrile cases (86%) were due to the introduction of P. falciparum clones that were not detected in the preceding asymptomatic episode. Regarding the analysis of antimalarial drug resistance markers, none of kelch 13 (k13) validated markers of artemisinin resistance were detected in the population, nonetheless, a single k13 allele, K189T, was maintained at a stable high frequency (>10%) over time. There was a distinct shift from chloroquine-resistant transporter (crt)-76, multi-drug resistant gene 1 (mdr1)-86 and mdr1-1246 chloroquine (CQ) resistance alleles to a 99% prevalence of CQ sensitive alleles in the population, following the withdrawal of CQ from routine use. In contrast, the dihydropteroate synthetase (dhps) double mutant (437G and 540E) associated with sulfadoxine-pyrimethamine (SP) resistance was maintained at a high frequency (>75%), after a change from SP to artemisinin combination therapies (ACTs). The novel cysteine desulfurase (nfs) K65 allele, implicated in resistance to lumefantrine in a West African study, showed a gradual significant decline in allele frequency pre- and post-ACT introduction (from 38% to 20%), suggesting evidence of directional selection in Kenya, potentially not due to lumefantrine. The frequency of AP-2 complex subunit mu (ap2-mu) S160N allele, a mutation that has been associated with directional selection after artemisinin combination therapy, was stable over time indicating that it is not under drug-pressure. On the other hand, the ubiquitin carboxyl-terminal hydrolase 1 (ubp-1) mutation at codon E1528D, also associated with directional selection after artemisinin combination therapy, was not detected. The S69Stop mutation in falcipain-2a that has been associated with artemisinin resistance, in vitro, was not detected and none of the artemisinin resistance predisposing mutations were identified. Asymptomatic infections were found to be modified by transmission intensity and age, altering the risk of developing febrile episodes and this suggested that host immunity plays a prominent role in mediating this process. The differences in P. falciparum genetic diversity between asymptomatic and febrile malaria infections can be attributed to the broader spectrum of immunological memory in the form of antibodies that has been found to be higher in asymptomatic infections compared to febrile malaria infections. While evidence of apparent protection from developing febrile episodes was observed in children with asymptomatic infections, amplicon deep-sequencing revealed that this protection was offset when a new infection occurred. Lastly, due to lack of the validated molecular markers of artemisinin resistance, there appears to be no problem of resistance in the population, however, continued surveillance remains a requirement. To conclude, P. falciparum genetic epidemiology revealed the ability to characterise P. falciparum complexity of infection (COI), a proxy that can be used to characterise malaria transmission intensity. Additionally, frequent sampling of P. falciparum positive samples from a hospital setting coupled with the molecular genotyping of drug resistance markers revealed the utility of P. falciparum genetic epidemiology in surveillance of antimalarial drug resistance

The Plasmodium falciparum Rh5 invasion protein complex reveals an excess of rare variant mutations.

BACKGROUND: The invasion of the red blood cells by Plasmodium falciparum merozoites involves the interplay of several proteins that are also targets for vaccine development. The proteins PfRh5-PfRipr-PfCyRPA-Pfp113 assemble into a complex at the apical end of the merozoite and are together essential for erythrocyte invasion. They have also been shown to induce neutralizing antibodies and appear to be less polymorphic than other invasion-associated proteins, making them high priority blood-stage vaccine candidates. Using available whole genome sequencing data (WGS) and new capillary sequencing data (CS), this study describes the genetic polymorphism in the Rh5 complex in P. falciparum isolates obtained from Kilifi, Kenya. METHODS: 162 samples collected in 2013 and 2014 were genotyped by capillary sequencing (CS) and re-analysed WGS from 68 culture-adapted P. falciparum samples obtained from a drug trial conducted from 2005 to 2007. The frequency of polymorphisms in the merozoite invasion proteins, PfRh5, PfRipr, PfCyRPA and PfP113 were examined and where possible polymorphisms co-occurring in the same isolates. RESULTS: From a total 70 variants, including 2 indels, 19 SNPs [27.1%] were identified by both CS and WGS, while an additional 15 [21.4%] and 36 [51.4%] SNPs were identified only by either CS or WGS, respectively. All the SNPs identified by CS were non-synonymous, whereas WGS identified 8 synonymous and 47 non-synonymous SNPs. CS identified indels in repeat regions in the p113 gene in codons 275 and 859 that were not identified in the WGS data. The minor allele frequencies of the SNPs ranged between 0.7 and 34.9% for WGS and 1.1-29.6% for CS. Collectively, 12 high frequency SNPs (> 5%) were identified: four in Rh5 codon 147, 148, 203 and 429, two in p113 at codons 7 and 267 and six in Ripr codons 190, 259, 524, 985, 1003 and 1039. CONCLUSION: This study reveals that the majority of the polymorphisms are rare variants and confirms a low level of genetic polymorphisms in all proteins within the Rh5 complex

No evidence of P. falciparum K13 artemisinin conferring mutations over a 24-year analysis in Coastal Kenya, but a near complete reversion to chloroquine wild type parasites.

Antimalarial drug resistance is a substantial impediment to malaria control. The spread of resistance has been described using genetic markers which are important epidemiological tools. We carried out a temporal analysis of changes in allele frequencies of 12 drug resistance markers over two decades of changing antimalarial drug policy in Kenya. We did not detect any of the validated kelch 13 (k13) artemisinin resistance markers, nonetheless, a single k13 allele, K189T, was maintained at a stable high frequency (>10%) over time. There was a distinct shift from chloroquine resistant transporter (crt)-76, multi-drug resistant gene 1 (mdr1)-86 and mdr1-1246 chloroquine (CQ) resistance alleles to a 99% prevalence of CQ sensitive alleles in the population, following the withdrawal of CQ from routine use. In contrast, the dihydropteroate synthetase (dhps) double mutant (437G and 540E) associated with sulfadoxine-pyrimethamine (SP) resistance was maintained at a high frequency (>75%), after a change from SP to artemisinin combination therapies (ACTs). The novel cysteine desulfurase (nfs) K65 allele, implicated in resistance to lumefantrine in a West African study, showed a gradual significant decline in allele frequency pre- and post-ACT introduction (from 38% to 20%), suggesting evidence of directional selection in Kenya, potentially not due to lumefantrine. The high frequency of CQ-sensitive parasites circulating in the population suggests that the re-introduction of CQ in combination therapy for the treatment of malaria can be considered in the future. However, the risk of a re-emergence of CQ resistant parasites circulating below detectable levels or being reintroduced from other regions remains

A review of the frequencies of Plasmodium falciparum Kelch 13 artemisinin resistance mutations in Africa.

Artemisinin resistance (AR) emerged in South East Asia 13 years ago and the identification of the resistance conferring molecular marker, Plasmodium falciparum Kelch 13 (Pfk13), 7 years ago has provided an invaluable tool for monitoring AR in malaria endemic countries. Molecular Pfk13 surveillance revealed the resistance foci in the Greater Mekong Subregion, an independent emergence in Guyana, South America, and a low frequency of mutations in Africa. The recent identification of the R561H Pfk13 AR associated mutation in Tanzania, Uganda and in Rwanda, where it has been associated with delayed parasite clearance, should be a concern for the continent. In this review, we provide a summary of Pfk13 resistance associated propeller domain mutation frequencies across Africa from 2012 to 2020, to examine how many other countries have identified these mutations. Only four African countries reported a recent identification of the M476I, P553L, R561H, P574L, C580Y and A675V Pfk13 mutations at low frequencies and with no reports of clinical treatment failure, except for Rwanda. These mutations present a threat to malaria control across the continent, since the greatest burden of malaria remains in Africa. A rise in the frequency of these mutations and their spread would reverse the gains made in the reduction of malaria over the last 20 years, given the lack of new antimalarial treatments in the event artemisinin-based combination therapies fail. The review highlights the frequency of Pfk13 propeller domain mutations across Africa, providing an up-to-date perspective of Pfk13 mutations, and appeals for an urgent and concerted effort to monitoring antimalarial resistance markers in Africa and the efficacy of antimalarials by re-establishing sentinel surveillance systems

Epidemiology of COVID-19 infections on routine polymerase chain reaction (PCR) and serology testing in Coastal Kenya [version 1; peer review: 2 approved]

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 17th March 2020 and 30th 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

Comprehensive transcriptome of the maize stalk borer, Busseola fusca, from multiple tissue types, developmental stages, and parasitoid wasp exposures

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Once a year school-based deworming with praziquantel and albendazole combination may not be adequate for control of urogenital schistosomiasis and hookworm infection in Matuga District, Kwale County, Kenya

Background Neglected tropical diseases (NTDs) predominantly occur in resource poor settings where they often present a serious public health burden. Sustained global advocacy has been important in raising awareness of NTDs and the relatively low cost for control of helminthic NTDs using preventive chemotherapy. This enthusiasm was boosted at the London declaration on NTDs in 2012 through commitments by different partners to avail resources required for control of NTDs particularly those that employ preventive chemotherapy as the major intervention strategy. Subsequently, national NTD programmes are responding to these new opportunities by implementing preventive chemotherapy including school-based deworming (SBD). Further, with the availability of increased resources, both financial and pharma, the optimal strategies for implementing preventive chemotherapy in highly endemic settings are under debate and this paper goes some way to addressing this issue in a specific setting in coastal Kenya. Methods We conducted a repeated cross-sectional study in Matuga District, Kwale County, Kenya to evaluate the effect of school-based co-administration of praziquantel and albendazole against urogenital schistosomiasis and soil-transmitted helminth (STH) infections. A total of 1022 school children in 5 study schools were tested for the infections in urine and stool samples during a baseline survey in September 2009. The presence of Schistosoma haematobium infection was determined by the urine filtration method while STH infections were determined by Kato-Katz technique. Results Urogenital schistosomiasis and hookworm infection were the major parasitic infections among the children in the study area. There was significant decrease in both prevalence and intensity of S. haematobium infection after treatment but varying levels of rebound were observed during the period between the treatments. The school-based treatment, however, did not have any significant effect on both the prevalence and intensity of hookworm infection. Conclusions Once per year SBD programmes may not be adequate for controlling hookworm infection and urogenital schistosomiasis in rural areas of Kwale County. There is a need to consider expanded preventive chemotherapy strategies that will allow inclusion of the adult populations. Community-based health education campaigns focusing on increasing household latrine ownership and use, as a complementary measure to control STH and urogenital schistosomiasis in similar settings, may also be useful

Asymptomatic Parasitemia and Risk of Febrile Malaria (Kilifi)

The data is based on 3 cohorts in Kilifi of varying malaria transmission intensities, comprising Ngerenya (low transmission), Junju (moderate to high transmission), and Chonyi (high transmission). The data were prospectively collected between 1998 and 2014 for Ngerenya, 2005 and 2010 for Junju, and 1999 and 2001 for Chonyi. In these cohorts, children were recruited at birth for weekly clinical malaria monitoring until the age of 15 years.</p

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

Replication Data for: The emergence of an indigenous artemisinin-resistant Plasmodium falciparum in Africa

A summary of mutations in the Plasmodium falciparum kelch 13 across sub-Saharan Africa from 2012-2020 (2020-11-26