Africa in the era of pathogen genomics: Unlocking data barriers.

Rapid expansion of pathogen sequencing capacity in Africa has led to a paradigm shift from relying on others to locally generating genomic data and sharing it with the global community. However, several barriers remain to be unlocked for timely processing, analysis, dissemination, and effective use of pathogen sequence data for pandemic prevention, preparedness, and response.

Clade I mpox virus genomic diversity in the Democratic Republic of the Congo, 2018-2024: Predominance of zoonotic transmission.

Recent reports raise concerns on the changing epidemiology of mpox in the Democratic Republic of the Congo (DRC). High-quality genomes were generated for 337 patients from 14/26 provinces to document whether the increase in number of cases is due to zoonotic spillover events or viral evolution, with enrichment of APOBEC3 mutations linked to human adaptation. Our study highlights two patterns of transmission contributing to the source of human cases. All new sequences from the eastern South Kivu province (n = 17; 4.8%) corresponded to the recently described clade Ib, associated with sexual contact and sustained human-to-human transmission. By contrast, all other genomes are clade Ia, which exhibits high genetic diversity with low numbers of APOBEC3 mutations compared with clade Ib, suggesting multiple zoonotic introductions. The presence of multiple clade I variants in urban areas highlights the need for coordinated international response efforts and more studies on the transmission and the reservoir of mpox.

Africa Needs a New Public Health Order to Tackle Infectious Disease Threats.

The SARS-CoV-2 pandemic has revealed that Africa needs a new public health order to be resilient, to adapt, and to cope with 21st-century disease threats. The new order will need strengthened continental and national public health institutions; local manufacturing of vaccines, therapeutics, and diagnostics; attraction, training, and retention of a public health workforce; and fostering of respectful local and international partnerships.

Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus.

We propose a novel, non-discriminatory classification of monkeypox virus diversity. Together with the World Health Organization, we named three clades (I, IIa and IIb) in order of detection. Within IIb, the cause of the current global outbreak, we identified multiple lineages (A.1, A.2, A.1.1 and B.1) to support real-time genomic surveillance.

Co-circulation of monkeypox virus subclades Ia and Ib in Kinshasa Province, Democratic Republic of the Congo, July to August 2024.

Between January and August 2024, mpox cases have been reported in nearly all provinces of the Democratic Republic of the Congo (DRC). Monkeypox virus genome sequences were obtained from 11 mpox cases' samples, collected in July-August 2024 in several health zones of Kinshasa. Characterisation of the sequences showed subclades Ia and Ib co-circulating in the Limete health zone, while phylogenetic analyses suggested multiple introductions of the two subclades in Kinshasa. This illustrates the growing complexity of Clade I mpox outbreaks in DRC.

Amplicon deep sequencing of five highly polymorphic markers of Plasmodium falciparum reveals high parasite genetic diversity and moderate population structure in Ethiopia.

BACKGROUND: Plasmodium falciparum genetic diversity can add information on transmission intensity and can be used to track control and elimination interventions. METHODS: Dried blood spots (DBS) were collected from patients who were recruited for a P. falciparum malaria therapeutic efficacy trial in three malaria endemic sites in Ethiopia from October to December 2015, and November to December 2019. qPCR-confirmed infections were subject to amplicon sequencing of polymorphic markers ama1-D3, csp, cpp, cpmp, msp7. Genetic diversity, the proportion of multiclonal infections, multiplicity of infection, and population structure were analysed. RESULTS: Among 198 samples selected for sequencing, data was obtained for 181 samples. Mean MOI was 1.38 (95% CI 1.24-1.53) and 17% (31/181) of infections were polyclonal. Mean He across all markers was 0.730. Population structure was moderate; populations from Metema and Metehara 2015 were very similar to each other, but distinct from Wondogent 2015 and Metehara 2019. CONCLUSION: The high level of parasite genetic diversity and moderate population structure in this study suggests frequent gene flow of parasites among sites. The results obtained can be used as a baseline for additional parasite genetic diversity and structure studies, aiding in the formulation of appropriate control strategies in Ethiopia.

Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome.

BACKGROUND: Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. METHODS: A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls. RESULTS: Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes. CONCLUSIONS: The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high-throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

Effectiveness of reactive focal mass drug administration and reactive focal vector control to reduce malaria transmission in the low malaria-endemic setting of Namibia: a cluster-randomised controlled, open-label, two-by-two factorial design trial.

BACKGROUND: In low malaria-endemic settings, screening and treatment of individuals in close proximity to index cases, also known as reactive case detection (RACD), is practised for surveillance and response. However, other approaches could be more effective for reducing transmission. We aimed to evaluate the effectiveness of reactive focal mass drug administration (rfMDA) and reactive focal vector control (RAVC) in the low malaria-endemic setting of Zambezi (Namibia). METHODS: We did a cluster-randomised controlled, open-label trial using a two-by-two factorial design of 56 enumeration area clusters in the low malaria-endemic setting of Zambezi (Namibia). We randomly assigned these clusters using restricted randomisation to four groups: RACD only, rfMDA only, RAVC plus RACD, or rfMDA plus RAVC. RACD involved rapid diagnostic testing and treatment with artemether-lumefantrine and single-dose primaquine, rfMDA involved presumptive treatment with artemether-lumefantrine, and RAVC involved indoor residual spraying with pirimiphos-methyl. Interventions were administered within 500 m of index cases. To evaluate the effectiveness of interventions targeting the parasite reservoir in humans (rfMDA vs RACD), in mosquitoes (RAVC vs no RAVC), and in both humans and mosquitoes (rfMDA plus RAVC vs RACD only), an intention-to-treat analysis was done. For each of the three comparisons, the primary outcome was the cumulative incidence of locally acquired malaria cases. This trial is registered with ClinicalTrials.gov, number NCT02610400. FINDINGS: Between Jan 1, 2017, and Dec 31, 2017, 55 enumeration area clusters had 1118 eligible index cases that led to 342 interventions covering 8948 individuals. The cumulative incidence of locally acquired malaria was 30·8 per 1000 person-years (95% CI 12·8-48·7) in the clusters that received rfMDA versus 38·3 per 1000 person-years (23·0-53·6) in the clusters that received RACD; 30·2 per 1000 person-years (15·0-45·5) in the clusters that received RAVC versus 38·9 per 1000 person-years (20·7-57·1) in the clusters that did not receive RAVC; and 25·0 per 1000 person-years (5·2-44·7) in the clusters that received rfMDA plus RAVC versus 41·4 per 1000 person-years (21·5-61·2) in the clusters that received RACD only. After adjusting for imbalances in baseline and implementation factors, the incidence of malaria was lower in clusters receiving rfMDA than in those receiving RACD (adjusted incidence rate ratio 0·52 [95% CI 0·16-0·88], p=0·009), lower in clusters receiving RAVC than in those that did not (0·48 [0·16-0·80], p=0·002), and lower in clusters that received rfMDA plus RAVC than in those receiving RACD only (0·26 [0·10-0·68], p=0·006). No serious adverse events were reported. INTERPRETATION: In a low malaria-endemic setting, rfMDA and RAVC, implemented alone and in combination, reduced malaria transmission and should be considered as alternatives to RACD for elimination of malaria. FUNDING: Novartis Foundation, Bill & Melinda Gates Foundation, and Horchow Family Fund.

Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples.

BACKGROUND: Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. The effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification were evaluated on the quality and fidelity of WGS data recovered from DBS. METHODS: Low parasite density mock DBS samples were created, extracted either with Tween-Chelex or QIAamp, treated with or without McrBC, and amplified with one of three different amplification techniques (two sWGA primer sets and one rWGA). Extraction conditions were evaluated on performance of sequencing depth, percentiles of coverage, and expected SNP concordance. RESULTS: At 100 parasites/µL, Chelex-Tween-McrBC samples had higher coverage (5 × depth = 93% genome) than QIAamp extracted samples (5 × depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. CONCLUSIONS: Overall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

Parasite genetic diversity reflects continued residual malaria transmission in Vhembe District, a hotspot in the Limpopo Province of South Africa.

BACKGROUND: South Africa aims to eliminate malaria transmission by 2023. However, despite sustained vector control efforts and case management interventions, the Vhembe District remains a malaria transmission hotspot. To better understand Plasmodium falciparum transmission dynamics in the area, this study characterized the genetic diversity of parasites circulating within the Vhembe District. METHODS: A total of 1153 falciparum-positive rapid diagnostic tests (RDTs) were randomly collected from seven clinics within the district, over three consecutive years (2016, 2017 and 2018) during the wet and dry malaria transmission seasons. Using 26 neutral microsatellite markers, differences in genetic diversity were described using a multiparameter scale of multiplicity of infection (MOI), inbreeding metric (Fws), number of unique alleles (A), expected heterozygosity (He), multilocus linkage disequilibrium (LD) and genetic differentiation, and were associated with temporal and geospatial variances. RESULTS: A total of 747 (65%) samples were successfully genotyped. Moderate to high genetic diversity (mean He = 0.74 ± 0.03) was observed in the parasite population. This was ascribed to high allelic richness (mean A = 12.2 ± 1.2). The majority of samples (99%) had unique multi-locus genotypes, indicating high genetic diversity in the sample set. Complex infections were observed in 66% of samples (mean MOI = 2.13 ± 0.04), with 33% of infections showing high within-host diversity as described by the Fws metric. Low, but significant LD (standardised index of association, ISA = 0.08, P < 0.001) was observed that indicates recombination of distinct clones. Limited impact of temporal (FST range - 0.00005 to 0.0003) and spatial (FST = - 0.028 to 0.023) variation on genetic diversity existed during the sampling timeframe and study sites respectively. CONCLUSIONS: Consistent with the Vhembe District's classification as a 'high' transmission setting within South Africa, P. falciparum diversity in the area was moderate to high and complex. This study showed that genetic diversity within the parasite population reflects the continued residual transmission observed in the Vhembe District. This data can be used as a reference point for the assessment of the effectiveness of on-going interventions over time, the identification of imported cases and/or outbreaks, as well as monitoring for the potential spread of anti-malarial drug resistance.

Within-household clustering of genetically related Plasmodium falciparum infections in a moderate transmission area of Uganda.

BACKGROUND: Evaluation of genetic relatedness of malaria parasites is a useful tool for understanding transmission patterns, but patterns are not easily detectable in areas with moderate to high malaria transmission. To evaluate the feasibility of detecting genetic relatedness in a moderate malaria transmission setting, relatedness of Plasmodium falciparum infections was measured in cohort participants from randomly selected households in the Kihihi sub-county of Uganda (annual entomological inoculation rate of 27 infectious bites per person). METHODS: All infections detected via microscopy or Plasmodium-specific loop mediated isothermal amplification from passive and active case detection during August 2011-March 2012 were genotyped at 26 microsatellite loci, providing data for 349 samples from 230 participants living in 80 households. Pairwise genetic relatedness was calculated using identity by state (IBS). RESULTS: As expected, genetic diversity was high (mean heterozygosity [He] = 0.73), and the majority (76.5 %) of samples were polyclonal. Despite the high genetic diversity, fine-scale population structure was detectable, with significant spatiotemporal clustering of highly related infections. Although the difference in malaria incidence between households at higher (mean 1127 metres) versus lower elevation (mean 1015 metres) was modest (1.4 malaria cases per person-year vs. 1.9 per person-year, respectively), there was a significant difference in multiplicity of infection (2.2 vs. 2.6, p = 0.008) and, more strikingly, a higher proportion of highly related infections within households (6.3 % vs. 0.9 %, p = 0.0005) at higher elevation compared to lower elevation. CONCLUSIONS: Genetic data from a relatively small number of diverse, multiallelic loci reflected fine scale patterns of malaria transmission. Given the increasing interest in applying genetic data to augment malaria surveillance, this study provides evidence that genetic data can be used to inform transmission patterns at local spatial scales even in moderate transmission areas.

Confirmation of the absence of local transmission and geographic assignment of imported falciparum malaria cases to China using microsatellite panel.

BACKGROUND: Current methods to classify local and imported malaria infections depend primarily on patient travel history, which can have limited accuracy. Genotyping has been investigated as a complementary approach to track the spread of malaria and identify the origin of imported infections. METHODS: An extended panel of 26 microsatellites (16 new microsatellites) for Plasmodium falciparum was evaluated in 602 imported infections from 26 sub-Saharan African countries to the Jiangsu Province of People's Republic of China. The potential of the 26 microsatellite markers to assign imported parasites to their geographic origin was assessed using a Bayesian method with Markov Chain Monte Carlo (MCMC) as implemented in the program Smoothed and Continuous Assignments (SCAT) with a modification to incorporate haploid genotype data. RESULTS: The newly designed microsatellites were polymorphic and are not in linkage disequilibrium with the existing microsatellites, supporting previous findings of high rate of recombination in sub-Saharan Africa. Consistent with epidemiology inferred from patients' travel history, no evidence for local transmission was found; nearly all genetically related infections were identified in people who travelled to the same country near the same time. The smoothing assignment method assigned imported cases to their likely geographic origin with an accuracy (Angola: 59%; Nigeria: 51%; Equatorial Guinea: 40%) higher than would be achieved at random, reaching statistical significance for Angola and Equatorial Guinea. CONCLUSIONS: Genotyping using an extended microsatellite panel is valuable for malaria case classification and programme evaluation in an elimination setting. A Bayesian method for assigning geographic origin of mammals based on genetic data was adapted for malaria and showed potential for identification of the origin of imported infections.

High levels of imported asymptomatic malaria but limited local transmission in KwaZulu-Natal, a South African malaria-endemic province nearing malaria elimination.

BACKGROUND: KwaZulu-Natal, one of South Africa's three malaria endemic provinces, is nearing malaria elimination, reporting fewer than 100 locally-acquired cases annually since 2010. Despite sustained implementation of essential interventions, including annual indoor residual spraying, prompt case detection using malaria rapid diagnostics tests and treatment with effective artemisinin-based combination therapy, low-level focal transmission persists in the province. This malaria prevalence and entomological survey was therefore undertaken to identify the drivers of this residual transmission. METHODS: Malaria prevalence as well as malaria knowledge, attitudes and practices among community members and mobile migrant populations within uMkhanyakude district, KwaZulu-Natal were assessed during a community-based malaria prevalence survey. All consenting participants were tested for malaria by both conventional and highly-sensitive falciparum-specific rapid diagnostic tests. Finger-prick filter-paper blood spots were also collected from all participants for downstream parasite genotyping analysis. Entomological investigations were conducted around the surveyed households, with potential breeding sites geolocated and larvae collected for species identification and insecticide susceptibility testing. A random selection of households were assessed for indoor residual spray quality by cone bioassay. RESULTS: A low malaria prevalence was confirmed in the study area, with only 2% (67/2979) of the participants found to be malaria positive by both conventional and highly-sensitive falciparum-specific rapid diagnostic tests. Malaria prevalence however differed markedly between the border market and community (p < 0001), with the majority of the detected malaria carriers (65/67) identified as asymptomatic Mozambican nationals transiting through the informal border market from Mozambique to economic hubs within South Africa. Genomic analysis of the malaria isolates revealed a high degree of heterozygosity and limited genetic relatedness between the isolates supporting the hypothesis of limited local malaria transmission within the province. New potential vector breeding sites, potential vector populations with reduced insecticide susceptibility and areas with sub-optimal vector intervention coverage were identified during the entomological investigations. CONCLUSION: If KwaZulu-Natal is to successfully halt local malaria transmission and prevent the re-introduction of malaria, greater efforts need to be placed on detecting and treating malaria carriers at both formal and informal border crossings with transmission blocking anti-malarials, while ensuring optimal coverage of vector control interventions is achieved.

Genetic Evidence of Focal Plasmodium falciparum Transmission in a Pre-elimination Setting in Southern Province, Zambia.

BACKGROUND: Southern Province, Zambia has experienced a dramatic decline in Plasmodium falciparum malaria transmission in the past decade and is targeted for elimination. Zambia's National Malaria Elimination Program recommends reactive case detection (RCD) within 140 m of index households to enhance surveillance and eliminate remaining transmission foci. METHODS: To evaluate whether RCD captures local transmission, we genotyped 26 microsatellites from 106 samples collected from index (n = 27) and secondary (n = 79) cases detected through RCD in the Macha Hospital catchment area between January 2015 and April 2016. RESULTS: Participants from the same RCD event harbored more genetically related parasites than those from different RCD events, suggesting that RCD captures, at least in part, infections related through local transmission. Related parasites clustered in space and time, up to at least 250 m from index households. Spatial analysis identified a putative focal transmission hotspot. CONCLUSIONS: The current RCD strategy detects focal transmission events, although programmatic guidelines to screen within 140 m of index households may fail to capture all secondary cases. This study highlights the utility of parasite genetic data in assessing programmatic interventions, and similar approaches may be useful to malaria elimination programs seeking to tailor intervention strategies to the underlying transmission epidemiology.

High Genetic Diversity of Plasmodium falciparum in the Low-Transmission Setting of the Kingdom of Eswatini.

BACKGROUND: To better understand transmission dynamics, we characterized Plasmodium falciparum genetic diversity in Eswatini, where transmission is low and sustained by importation. METHODS: Twenty-six P. falciparum microsatellites were genotyped in 66% of confirmed cases (2014-2016; N = 582). Population and within-host diversity were used to characterize differences between imported and locally acquired infections. Logistic regression was used to assess the added value of diversity metrics to classify imported and local infections beyond epidemiology data alone. RESULTS: Parasite population in Eswatini was highly diverse (expected heterozygosity [HE] = 0.75) and complex: 67% polyclonal infections, mean multiplicity of infection (MOI) 2.2, and mean within-host infection fixation index (FWS) 0.84. Imported cases had comparable diversity to local cases but exhibited higher MOI (2.4 vs 2.0; P = .004) and lower mean FWS (0.82 vs 0.85; P = .03). Addition of MOI and FWS to multivariate analyses did not increase discrimination between imported and local infections. CONCLUSIONS: In contrast to the common perception that P. falciparum diversity declines with decreasing transmission intensity, Eswatini isolates exhibited high parasite diversity consistent with high rates of malaria importation and limited local transmission. Estimates of malaria transmission intensity from genetic data need to consider the effect of importation, especially as countries near elimination.

Applying next-generation sequencing to track falciparum malaria in sub-Saharan Africa.

Next-generation sequencing (NGS) technologies are increasingly being used to address a diverse range of biological and epidemiological questions. The current understanding of malaria transmission dynamics and parasite movement mainly relies on the analyses of epidemiologic data, e.g. case counts and self-reported travel history data. However, travel history data are often not routinely collected or are incomplete, lacking the necessary level of accuracy. Although genetic data from routinely collected field samples provides an unprecedented opportunity to track the spread of malaria parasites, it remains an underutilized resource for surveillance due to lack of local awareness and capacity, limited access to sensitive laboratory methods and associated computational tools and difficulty in interpreting genetic epidemiology data. In this review, the potential roles of NGS in better understanding of transmission patterns, accurately tracking parasite movement and addressing the emerging challenges of imported malaria in low transmission settings of sub-Saharan Africa are discussed. Furthermore, this review highlights the insights gained from malaria genomic research and challenges associated with integrating malaria genomics into existing surveillance tools to inform control and elimination strategies.

Drug-Resistance and Population Structure of Plasmodium falciparum Across the Democratic Republic of Congo Using High-Throughput Molecular Inversion Probes.

A better understanding of the drivers of the spread of malaria parasites and drug resistance across space and time is needed. These drivers can be elucidated using genetic tools. Here, a novel molecular inversion probe (MIP) panel targeting all major drug-resistance mutations and a set of microsatellites was used to genotype Plasmodium falciparum infections of 552 children from the 2013-2014 Demographic and Health Survey conducted in the Democratic Republic of the Congo (DRC). Microsatellite-based analysis of population structure suggests that parasites within the DRC form a homogeneous population. In contrast, sulfadoxine-resistance markers in dihydropteroate synthase show marked spatial structure with ongoing spread of double and triple mutants compared with 2007. These findings suggest that parasites in the DRC remain panmictic despite rapidly spreading antimalarial-resistance mutations. Moreover, highly multiplexed targeted sequencing using MIPs emerges as a cost-effective method for elucidating pathogen genetics in complex infections in large cohorts.

Antibodies to Intercellular Adhesion Molecule 1-Binding Plasmodium falciparum Erythrocyte Membrane Protein 1-DBLß Are Biomarkers of Protective Immunity to Malaria in a Cohort of Young Children from Papua New Guinea.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates parasite sequestration to the cerebral microvasculature via binding of DBLß domains to intercellular adhesion molecule 1 (ICAM1) and is associated with severe cerebral malaria. In a cohort of 187 young children from Papua New Guinea (PNG), we examined baseline levels of antibody to the ICAM1-binding PfEMP1 domain, DBLß3PF11_0521, in comparison to four control antigens, including NTS-DBLα and CIDR1 domains from another group A variant and a group B/C variant. Antibody levels for the group A antigens were strongly associated with age and exposure. Antibody responses to DBLß3PF11_0521 were associated with a 37% reduced risk of high-density clinical malaria in the follow-up period (adjusted incidence risk ratio [aIRR] = 0.63 [95% confidence interval {CI}, 0.45 to 0.88; P = 0.007]) and a 25% reduction in risk of low-density clinical malaria (aIRR = 0.75 [95% CI, 0.55 to 1.01; P = 0.06]), while there was no such association for other variants. Children who experienced severe malaria also had significantly lower levels of antibody to DBLß3PF11_0521 and the other group A domains than those that experienced nonsevere malaria. Furthermore, a subset of PNG DBLß sequences had ICAM1-binding motifs, formed a distinct phylogenetic cluster, and were similar to sequences from other areas of endemicity. PfEMP1 variants associated with these DBLß domains were enriched for DC4 and DC13 head structures implicated in endothelial protein C receptor (EPCR) binding and severe malaria, suggesting conservation of dual binding specificities. These results provide further support for the development of specific classes of PfEMP1 as vaccine candidates and as biomarkers for protective immunity against clinical P. falciparum malaria.

Correction to: Mapping malaria by combining parasite genomic and epidemiologic data.

The original article [1] contained an error in the presentation of Figure 1; this error has now been rectified and Figure 1 is now presented correctly.