Targeted Next Generation Sequencing for malaria research in Africa:Current status and outlook

Targeted Next Generation Sequencing (TNGS) is an efficient and economical Next Generation Sequencing (NGS) platform and the preferred choice when specific genomic regions are of interest. So far, only institutions located in middle and high-income countries have developed and implemented the technology, however, the efficiency and cost savings, as opposed to more traditional sequencing methodologies (e.g. Sanger sequencing) make the approach potentially well suited for resource-constrained regions as well. In April 2018, scientists from the Plasmodium Diversity Network Africa (PDNA) and collaborators met during the 7th Pan African Multilateral Initiative of Malaria (MIM) conference held in Dakar, Senegal to explore the feasibility of applying TNGS to genetic studies and malaria surveillance in Africa. The group of scientists reviewed the current experience with TNGS platforms in sub-Saharan Africa (SSA) and identified potential roles the technology might play to accelerate malaria research, scientific discoveries and improved public health in SSA. Research funding, infrastructure and human resources were highlighted as challenges that will have to be mitigated to enable African scientists to drive the implementation of TNGS in SSA. Current roles of important stakeholders and strategies to strengthen existing networks to effectively harness this powerful technology for malaria research of public health importance were discussed

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 ( 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

Marker assisted backcross breeding to enhance drought tolerance in Kenyan chickpea (Cicer arietinum L.) germplasm

Drought is the number one constraint in chickpea production. In the past, breeding efforts to improve terminal drought tolerance have been hindered by its quantitative genetic basis and poor understanding of the physiological basis of yield in water-limited conditions

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

Performance of marker assisted backcross breeding (MABC) elite chickpea lines under drought conditions in Kenya

Drought is the most important constraint affecting production of chickpea and other crops as well. Quantitative traits like drought tolerance are multigenic and their inheritance is difficult to predict hence the need to explore more precise breeding techniques like maker assisted selection. The aim of this study was to introgress the identified root trait QTLs into Kenyan adapted cultivar to enhance drought tolerance through marker assisted backcrossing. Four varieties Chania Desi 1 (ICCV 97105), ICCV10, ICCV 92318, and Saina K1 (ICCV 95423) were selected as a recurrent parents for improvement among ten agronomically superior elite cultivars after exhibiting high polymorphism with SSR markers. Five molecular markers (CaM1903, CaM1502, TAA 170, NCPGR21 and GA11) were validated for use in MABC deployed in this study. Crosses were made between the four parents and ICC 4958 followed by marker screening of the F1 seedling progenies for the QTL of interest. Identified true heterozygotes were used as donors and backcrossed to the recurrent parent to obtain BC1F1 seeds. The process was repeated to obtain BC2F1 and finally BC3F1 with molecular marker identification of seedlings carrying the QTL region at each step. Results of evaluation in one trial site in Kenya semi-arid area (Koibatek ATC) of MABC lines for the four parents ICCV10 (24 lines), ICCV 92318 (8lines), ICCV 97105 (12 lines) and Saina K1-ICCV 95423 (10 lines) showed that the best progenies with higher levels of drought resistance and yield were ICCMABCD-21, 9, 20, 23, 15, 22, 5, 14, 16, 19 and 6 with yields > 2.5 tons/ha. The results indicated that it is possible to transfer QTL that confers drought tolerance using MABC. The best progenies are undergoing further evaluation to validate the contribution of the introgressed QTL in improving drought tolerance and yield

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

An overview of chickpea breeding programs in Kenya

Chickpea is a new crop in Kenya and its potential has not been fully utilized. The chickpea grain yields generally range between 1.2 to 3.5 tons/ha at farmers‟ fields, indicating that chickpea has a potential of becoming an important export crop in Kenya. The chickpea breeding program in Kenya is still at infant stage and being established with support from International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). Four chickpea varieties have been recently released from the breeding material supplied by ICRISAT. Efforts are being made on evaluation of germplasm and breeding lines, application of modern molecular breeding tools and techniques in chickpea breeding and establishment of effective seed system for establishing a sustainable chickpea production system in the country

Geographic-genetic analysis of Plasmodium falciparum parasite populations from surveys of primary school children in Western Kenya.

BACKGROUND: Malaria control, and finally malaria elimination, requires the identification and targeting of residual foci or hotspots of transmission. However, the level of parasite mixing within and between geographical locations is likely to impact the effectiveness and durability of control interventions and thus should be taken into consideration when developing control programs. METHODS: In order to determine the geographic-genetic patterns of Plasmodium falciparum parasite populations at a sub-national level in Kenya, we used the Sequenom platform to genotype 111 genome-wide distributed single nucleotide polymorphic (SNP) positions in 2486 isolates collected from children in 95 primary schools in western Kenya. We analysed these parasite genotypes for genetic structure using principal component analysis and assessed local and global clustering using statistical measures of spatial autocorrelation. We further examined the region for spatial barriers to parasite movement as well as directionality in the patterns of parasite movement. RESULTS: We found no evidence of population structure and little evidence of spatial autocorrelation of parasite genotypes (correlation coefficients <0.03 among parasite pairs in distance classes of 1km, 2km and 5km; p value<0.01). An analysis of the geographical distribution of allele frequencies showed weak evidence of variation in distribution of alleles, with clusters representing a higher than expected number of samples with the major allele being identified for 5 SNPs. Furthermore, we found no evidence of the existence of spatial barriers to parasite movement within the region, but observed directional movement of parasites among schools in two separate sections of the region studied. CONCLUSIONS: Our findings illustrate a pattern of high parasite mixing within the study region. If this mixing is due to rapid gene flow, then "one-off" targeted interventions may not be currently effective at the sub-national scale in Western Kenya, due to the high parasite movement that is likely to lead to re-introduction of infection from surrounding regions. However repeated targeted interventions may reduce transmission in the surrounding regions

Seroprevalence of anti-SARS-CoV-2 IgG antibodies in Kenyan blood donors.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Africa is poorly described. The first case of SARS-CoV-2 in Kenya was reported on 12 March 2020, and an overwhelming number of cases and deaths were expected, but by 31 July 2020, there were only 20,636 cases and 341 deaths. However, the extent of SARS-CoV-2 exposure in the community remains unknown. We determined the prevalence of anti-SARS-CoV-2 immunoglobulin G among blood donors in Kenya in April-June 2020. Crude seroprevalence was 5.6% (174 of 3098). Population-weighted, test-performance-adjusted national seroprevalence was 4.3% (95% confidence interval, 2.9 to 5.8%) and was highest in urban counties Mombasa (8.0%), Nairobi (7.3%), and Kisumu (5.5%). SARS-CoV-2 exposure is more extensive than indicated by case-based surveillance, and these results will help guide the pandemic response in Kenya and across Africa

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 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