Diversity and selection analyses identify transmission-blocking antigens as the optimal vaccine candidates in Plasmodium falciparum.

Background: A highly effective vaccine for malaria remains an elusive target, at least in part due to the under-appreciated natural parasite variation. This study aimed to investigate genetic and structural variation, and immune selection of leading malaria vaccine candidates across the Plasmodium falciparum 's life cycle. Methods: We analyzed 325 P. falciparum whole genome sequences from Zambia, in addition to 791 genomes from five other African countries available in the MalariaGEN Pf3k Rdatabase. Ten vaccine antigens spanning three life-history stages were examined for genetic and structural variations, using population genetics measures, haplotype network analysis, and 3D structure selection analysis. Findings: Among the ten antigens analyzed, only three in the transmission-blocking vaccine category display P . falciparum 3D7 as the dominant haplotype. The antigens AMA1, CSP, MSP1 19 and CelTOS, are much more diverse than the other antigens, and their epitope regions are under moderate to strong balancing selection. In contrast, Rh5 , a blood stage antigen, displays low diversity yet slightly stronger immune selection in the merozoite-blocking epitope region. Except for CelTOS , the transmission-blocking antigens Pfs25 , Pfs48/45 , Pfs230 , Pfs47 , and Pfs28 exhibit minimal diversity and no immune selection in epitopes that induce strain-transcending antibodies, suggesting potential effectiveness of 3D7-based vaccines in blocking transmission. Interpretations: These findings offer valuable insights into the selection of optimal vaccine candidates against P. falciparum . Based on our results, we recommend prioritizing conserved merozoite antigens and transmission-blocking antigens. Combining these antigens in multi-stage approaches may be particularly promising for malaria vaccine development initiatives. Funding: Purdue Department of Biological Sciences; Puskas Memorial Fellowship; National Institute of Allergy and Infectious Diseases (U19AI089680). Research in context: Evidence before this study: Decades of research on the most virulent malaria parasite, Plasmodium falciparum , have yielded multiple antigen candidates of pre-erythrocytic, blood-stage, and transmission-blocking vaccines in varying stages of development from preclinical development to more advanced clinical trials. The malaria vaccine, RTS,S/AS01, which was constructed using the C-terminal and NANP repeat region of the Circumsporozoite Protein ( CSP ) from the African reference strain 3D7, was approved and recommended for use in 2021. However, the vaccine's lower efficacy is likely a result of the genetic polymorphism of the target antigen shown by studies on natural variation in CSP . Similarly, another more recent pre-erythrocytic vaccine, R21/Matrix-M, showed great promise in clinical trials and was recommended in late 2023 by the WHO for use for prevention of malaria in children, but is also multi-dose and CSP -based. To maximize vaccine efficacy, it would be more strategic to first understand diversity and variation of antigens across the three types of vaccine classes, targeting various stages of the P. falciparum life cycle. Previous studies have reported analyses of vaccine candidate antigens but were mostly limited to pre-erythrocytic and blood-stage antigens, with less focus on transmission-blocking antigens. These studies revealed that most of the pre-erythrocytic and blood-stage antigens are of high diversity due to balancing selection, posing challenges for vaccine design to encompass the antigenic variation. A search conducted on PubMed on April 1, 2024, for relevant published research which used the terms "malaria vaccine", " Plasmodium falciparum " [not " vivax "], "selection" and "diversity" yielded 48 studies between 1996 and the present day, with only 14 published studies in the past 3 years. This emphasizes the need for more studies assessing genetic diversity and selection of potential P. falciparum vaccine candidates to aid in more effective vaccine development efforts. A similar search with the terms "transmission-blocking vaccine", "malaria", " Plasmodium falciparum ", not " vivax ", "selection" and "diversity" without any date or language restrictions revealed three relevant studies. This warrants future studies to explore transmission-blocking vaccines in this context. Added value of this study: By comparing the genetic and structural analyses of transmission-blocking antigens with pre-erythrocytic and blood-stage antigens, we identify promising P. falciparum vaccine antigens characterized by their conservation with low balancing selection and the presence of infection/transmission-blocking epitopes, which are essential for informing the development of new malaria vaccines. This comprehensive workflow can be adopted for studying the genetic and structural variation of other P. falciparum vaccine targets before developing the next generation of malaria vaccines for effectiveness against natural parasite populations. Implications of this study: Our suggested strategies for designing malaria vaccines include two possible approaches. We emphasize the development of a multi-stage vaccine that combines critical components such as anti-merozoite ( Rh5 ) and transmission-blocking antigens ( Pfs25 , Pfs28 , Pfs48/45 , Pfs230 ). Alternatively, we suggest the creation of transmission-blocking vaccines specifically targeting Pfs25 , Pfs28 and Pfs48/45 . These innovative approaches show great potential in advancing the development of more potent and effective malaria vaccines for the future.

Genomics reveals heterogeneous Plasmodium falciparum transmission and selection signals in Zambia.

BACKGROUND: Genomic surveillance is crucial for monitoring malaria transmission and understanding parasite adaptation to interventions. Zambia lacks prior nationwide efforts in malaria genomic surveillance among African countries. METHODS: We conducted genomic surveillance of Plasmodium falciparum parasites from the 2018 Malaria Indicator Survey in Zambia, a nationally representative household survey of children under five years of age. We whole-genome sequenced and analyzed 241 P. falciparum genomes from regions with varying levels of malaria transmission across Zambia and estimated genetic metrics that are informative about transmission intensity, genetic relatedness between parasites, and selection. RESULTS: We provide genomic evidence of widespread within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in Zambia. Our analysis reveals country-level clustering of parasites from Zambia and neighboring regions, with distinct separation in West Africa. Within Zambia, identity by descent (IBD) relatedness analysis uncovers local spatial clustering and rare cases of long-distance sharing of closely related parasite pairs. Genomic regions with large shared IBD segments and strong positive selection signatures implicate genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Furthermore, differences in selection signatures, including drug resistance loci, are observed between eastern and western Zambian parasite populations, suggesting variable transmission intensity and ongoing drug pressure. CONCLUSIONS: Our findings enhance our understanding of nationwide P. falciparum transmission in Zambia, establishing a baseline for analyzing parasite genetic metrics as they vary over time and space. These insights highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

Malaria is caused by a parasite that is spread to humans via mosquito bites. It is a leading cause of death in children under five years old in sub-Saharan Africa. Analysis of the malaria parasite's complete set of DNA (its genome) can help us to understand transmission of the disease and how this changes in response to different strategies to control the disease. We analyzed the genomes of malaria parasites from children across Zambia. Our study revealed that 77% of children harbored multiple parasite strains, which suggests that local transmission (transmission between people within the same local area) is high. Genetic evidence for long-distance transmission was rarer. Furthermore, our findings suggest parasites are evolving in response to antimalarial drugs. Our study enhances our understanding of malaria dynamics in Zambia and may help to inform strategies for improved surveillance and control.

Investigating the rise of Omicron variant through genomic surveillance of SARS-CoV-2 infections in a highly vaccinated university population.

Novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge as the coronavirus disease 2019 (COVID-19) pandemic extends into its fourth year. Understanding SARS-CoV-2 circulation in university populations is vital for effective interventions in higher education settings and will inform public health policy during pandemics. In this study, we generated 793 whole-genome sequences collected over an entire academic year in a university population in Indiana, USA. We clearly captured the rapidity with which Delta variant was wholly replaced by Omicron variant across the West Lafayette campus over the length of two academic semesters in a community with high vaccination rates. This mirrored the emergence of Omicron throughout the state of Indiana and the USA. Further, phylogenetic analyses demonstrated that there was a more diverse set of potential geographic origins for Omicron viruses introduction into campus when compared to Delta. Lastly, statistics indicated that there was a more significant role for international and out-of-state migration in the establishment of Omicron variants at Purdue. This surveillance workflow, coupled with viral genomic sequencing and phylogeographic analyses, provided critical insights into SARS-CoV-2 transmission dynamics and variant arrival.

Genomics reveals heterogeneous Plasmodium falciparum transmission and population differentiation in Zambia and bordering countries.

Genomic surveillance plays a critical role in monitoring malaria transmission and understanding how the parasite adapts in response to interventions. We conducted genomic surveillance of malaria by sequencing 241 Plasmodium falciparum genomes from regions with varying levels of malaria transmission across Zambia. We found genomic evidence of high levels of within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in the country. We identified country-level clustering of parasites from Zambia and neighboring countries, and distinct clustering of parasites from West Africa. Within Zambia, our identity by descent (IBD) relatedness analysis uncovered spatial clustering of closely related parasite pairs at the local level and rare cases of long-distance sharing. Genomic regions with large shared IBD segments and strong positive selection signatures identified genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Together, our findings enhance our understanding of P. falciparum transmission nationwide in Zambia and highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

A GPU-accelerated compute framework for pathogen genomic variant identification to aid genomic epidemiology of infectious disease: a malaria case study.

As recently demonstrated by the COVID-19 pandemic, large-scale pathogen genomic data are crucial to characterize transmission patterns of human infectious diseases. Yet, current methods to process raw sequence data into analysis-ready variants remain slow to scale, hampering rapid surveillance efforts and epidemiological investigations for disease control. Here, we introduce an accelerated, scalable, reproducible, and cost-effective framework for pathogen genomic variant identification and present an evaluation of its performance and accuracy across benchmark datasets of Plasmodium falciparum malaria genomes. We demonstrate superior performance of the GPU framework relative to standard pipelines with mean execution time and computational costs reduced by 27× and 4.6×, respectively, while delivering 99.9% accuracy at enhanced reproducibility.

Genomic Surveillance of SARS-CoV-2 in a University Community: Insights Into Tracking Variants, Transmission, and Spread of Gamma (P.1) Variant.

Background: Using a combination of data from routine surveillance, genomic sequencing, and phylogeographic analysis, we tracked the spread and introduction events of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants focusing on a large university community. Methods: Here, we sequenced and analyzed 677 high-quality SARS-CoV-2 genomes from positive RNA samples collected from Purdue University students, faculty, and staff who tested positive for the virus between January 2021 and May 2021, comprising an average of 32% of weekly cases across the time frame. Results: Our analysis of circulating SARS-CoV-2 variants over time revealed periods when variants of concern (VOC) Alpha (B.1.1.7) and Iota (B.1.526) reached rapid dominance and documented that VOC Gamma (P.1) was increasing in frequency as campus surveillance was ending. Phylodynamic analysis of Gamma genomes from campus alongside a subsampling of >20 000 previously published P.1 genomes revealed 10 independent introductions of this variant into the Purdue community, predominantly from elsewhere in the United States, with introductions from within the state of Indiana and from Illinois, and possibly Washington and New York, suggesting a degree of domestic spread. Conclusions: We conclude that a robust and sustained active and passive surveillance program coupled with genomic sequencing during a pandemic offers important insights into the dynamics of pathogen arrival and spread in a campus community and can help guide mitigation measures.

A genotyping assay to determine geographic origin and transmission potential of Plasmodium falciparum malaria cases.

As countries work towards malaria elimination, it is important to monitor imported cases to prevent reestablishment of local transmission. The Plasmodium falciparum Pfs47 gene has strong geographic population structure, because only those parasites with Pfs47 haplotypes compatible with the mosquito vector species in a given continent are efficiently transmitted. Analysis of 4,971 world-wide Pfs47 sequences identified two SNPs (at 707 and 725 bp) as sufficient to establish the likely continent of origin of P. falciparum isolates. Pfs47 sequences from Africa, Asia, and the New World presented more that 99% frequency of distinct combinations of the SNPs 707 and 725 genotypes. Interestingly, Papua New Guinea Pfs47 sequences have the highest diversity in SNPs 707 and 725. Accurate and reproducible High-Resolution Melting (HRM) assays were developed to genotype Pfs47 SNPs 707 and 725 in laboratory and field samples, to assess the geographic origin and risk of local transmission of imported P. falciparum malaria cases.

Performance evaluation of RDT, light microscopy, and PET-PCR for detecting Plasmodium falciparum malaria infections in the 2018 Zambia National Malaria Indicator Survey.

BACKGROUND: Zambia continues to advance on the path to elimination with significant reductions in malaria morbidity and mortality. Crucial components that have contributed to progress thus far and are necessary for achieving the national malaria elimination goals include properly identifying and treating all malaria cases through accurate diagnosis. This study sought to compare and assess the diagnostic performance of Rapid Diagnostic Tests (RDT) and Light Microscopy (LM) with photo-induced electron transfer polymerase chain reaction (PET-PCR) as the gold standard using 2018 Malaria Indicator Survey (MIS) data across Zambia to better understand diagnostic accuracy metrics and how these vary across a transmission gradient. METHODS: Cross-sectional samples collected in a nationally representative survey from 7 provinces in Zambia were tested for the presence of malaria parasites by light microscopy (LM), rapid diagnostic test (RDT) and the gold standard PET-PCR. Diagnostic performance was assessed including sensitivity, specificity, negative- and positive-predictive values across a wide malaria transmission spectrum. Diagnostic accuracy metrics were measured, and statistically significant differences were calculated between test methods for different outcome variables. RESULTS: From the individuals included in the MIS, the overall prevalence of Plasmodium falciparum malaria was 32.9% by RDT, 19.4% by LM, and 23.2% by PET-PCR. Herein, RDT and LM diagnostic performance was compared against gold standard PET-PCR with LM displaying a higher diagnostic accuracy than RDTs (91.3% vs. 84.6% respectively) across the transmission spectrum in Zambia. However, the performance of both diagnostics was significantly reduced in low parasitaemia samples. Consistent with previous studies, RDT diagnostic accuracy was predominantly affected by a high rate of false positives. CONCLUSIONS: RDTs and LM both perform well across a range of transmission intensities within their respective target applications, i.e., in the community, for the former, where ease of use and speed of result is critical, and at the health facility, for the latter, where accuracy is prioritized. However, the performance of both diagnostic methods is adversely affected by low parasitaemia infections. As Zambia moves towards elimination more sensitive tools may be required to identify the last cases.

Phylogenetic Complexity of Morphologically Identified Anopheles squamosus in Southern Zambia.

Despite dramatic reductions in malaria cases in the catchment area of Macha Hospital, Choma District, Southern Province in Zambia, prevalence has remained near 1-2% by RDT for the past several years. To investigate residual malaria transmission in the area, this study focuses on the relative abundance, foraging behavior, and phylogenetic relationships of Anopheles squamosus specimens. In 2011, higher than expected rates of anthropophily were observed among "zoophilic" An. squamosus, a species that had sporadically been found to contain Plasmodium falciparum sporozoites. The importance of An. squamosus in the region was reaffirmed in 2016 when P. falciparum sporozoites were detected in numerous An. squamosus specimens. This study analyzed Centers for Disease Control (CDC) light trap collections of adult mosquitoes from two collection schemes: one performed as part of a reactive-test-and-treat program and the second performed along a geographical transect. Morphological identification, molecular verification of anopheline species, and blood meal source were determined on individual samples. Data from these collections supported earlier studies demonstrating An. squamosus to be primarily exophagic and zoophilic, allowing them to evade current control measures. The phylogenetic relationships generated from the specimens in this study illustrate the existence of well supported clade structure among An. squamosus specimens, which further emphasizes the importance of molecular identification of vectors. The primarily exophagic behavior of An. squamosus in these collections also highlights that indoor vector control strategies will not be sufficient for elimination of malaria in southern Zambia.

Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern Zambia.

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI's nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An. sp. 6. Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases.

Genetic Diversity of Anopheles coustani (Diptera: Culicidae) in Malaria Transmission Foci in Southern and Central Africa.

Despite ongoing malaria control efforts implemented throughout sub-Saharan Africa, malaria remains an enormous public health concern. Current interventions such as indoor residual spraying with insecticides and use of insecticide-treated bed nets are aimed at targeting the key malaria vectors that are primarily endophagic and endophilic. Anopheles coustani s.l., an understudied vector of malaria, is a species previously thought to exhibit mostly zoophilic behavior. Like many of these understudied species, An. coustani has greater anthropophilic tendencies than previously appreciated, is often both endophagic and exophagic, and carries Plasmodium falciparum sporozoites. The aim of this study was to explore genetic variation of An. coustani mosquitoes and the potential of this species to contribute to malaria parasite transmission in high transmission settings in Zambia and the Democratic Republic of the Congo (DRC). Morphologically identified An. coustani specimens that were trapped outdoors in these study sites were analyzed by PCR and sequencing for species identification and bloodmeal sources, and malaria parasite infection was determined by ELISA and qPCR. Fifty An. coustani s.s. specimens were confirmed by analysis of mitochondrial DNA cytochrome c oxidase subunit I (COI) and ribosomal internal transcribed spacer region 2 (ITS2). Maximum likelihood phylogenetic analysis of COI and ITS2 sequences revealed two distinct phylogenetic groups within this relatively small regional collection. Our findings indicate that both An. coustani groups have anthropophilic and exophagic habits and come into frequent contact with P. falciparum, suggesting that this potential alternative malaria vector might elude current vector control measures in northern Zambia and southern DRC.

Stability and detection of nucleic acid from viruses and hosts in controlled mosquito blood feeds.

Monitoring the presence and spread of pathogens in the environment is of critical importance. Rapid detection of infectious disease outbreaks and prediction of their spread can facilitate early responses of health agencies and reduce the severity of outbreaks. Current sampling methods are sorely limited by available personnel and throughput. For instance, xenosurveillance utilizes captured arthropod vectors, such as mosquitoes, as sampling tools to access blood from a wide variety of vertebrate hosts. Next generation sequencing (NGS) of nucleic acid from individual blooded mosquitoes can be used to identify mosquito and host species, and microorganisms including pathogens circulating within either host. However, there are practical challenges to collecting and processing mosquitoes for xenosurveillance, such as the rapid metabolization or decay of microorganisms within the mosquito midgut. This particularly affects pathogens that do not replicate in mosquitoes, preventing their detection by NGS or other methods. Accordingly, we performed a series of experiments to establish the windows of detection for DNA or RNA from human blood and/or viruses present in mosquito blood meals. Our results will contribute to the development of xenosurveillance techniques with respect to optimal timing of sample collection and NGS processing and will also aid trap design by demonstrating the stabilizing effect of temperature control on viral genome recovery from blood-fed mosquitoes.

High Plasmodium falciparum genetic diversity and temporal stability despite control efforts in high transmission settings along the international border between Zambia and the Democratic Republic of the Congo.

BACKGROUND: While the utility of parasite genotyping for malaria elimination has been extensively documented in low to moderate transmission settings, it has been less well-characterized in holoendemic regions. High malaria burden settings have received renewed attention acknowledging their critical role in malaria elimination. Defining the role for parasite genomics in driving these high burden settings towards elimination will enhance future control programme planning. METHODS: Amplicon deep sequencing was used to characterize parasite population genetic diversity at polymorphic Plasmodium falciparum loci, Pfama1 and Pfcsp, at two timepoints in June-July 2016 and January-March 2017 in a high transmission region along the international border between Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC). RESULTS: High genetic diversity was observed across both seasons and in both countries. No evidence of population structure was observed between parasite populations on either side of the border, suggesting that this region may be one contiguous transmission zone. Despite a decline in parasite prevalence at the sampling locations in Haut-Katanga Province, no genetic signatures of a population bottleneck were detected, suggesting that larger declines in transmission may be required to reduce parasite genetic diversity. Analysing rare variants may be a suitable alternative approach for detecting epidemiologically important genetic signatures in highly diverse populations; however, the challenge is distinguishing true signals from potential artifacts introduced by small sample sizes. CONCLUSIONS: Continuing to explore and document the utility of various parasite genotyping approaches for understanding malaria transmission in holoendemic settings will be valuable to future control and elimination programmes, empowering evidence-based selection of tools and methods to address pertinent questions, thus enabling more efficient resource allocation.

The Babesia observational antibody (BAOBAB) study: A cross-sectional evaluation of Babesia in two communities in Kilosa district, Tanzania.

BACKGROUND: Babesia, a tick-borne genus of intraerythrocytic parasites, is understudied in humans outside of established high-endemic areas. There is a paucity of data on Babesia in Africa, despite evidence that it is regionally present. A pilot study suggested that Babesia was present in a rural district of Tanzania. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was conducted July-August 2017: residents in a case hamlet that had clustering of subjects with high signal-to-cut off (S/CO) ratios for antibodies against B. microti in the pilot study, and a control hamlet that had lacked significant signal, were evaluated for B. microti. Subjects aged ≥15yrs (n = 299) underwent clinical evaluation and household inspections; 10ml whole blood was drawn for Babesia transcription mediated amplification (TMA), B. microti indirect fluorescent antibody testing (IFA) and rapid diagnostic testing (RDT) for Plasmodium spp. Subjects aged <15yrs (n = 266) underwent a RDT for Plasmodium and assessment by ELISA for B. microti antibodies. A total of 570 subjects participated (mean age 22 [<1 to 90yrs]) of whom 50.7% were female and 145 (25.5%) subjects were Plasmodium RDT positive (+). In those <15yrs, the median ELISA S/CO was 1.11 (IQR 0.80-1.48); the median S/CO in the case (n = 120) and control (n = 146) hamlets was 1.19 (IQR 0.81-1.48) and 1.06 (IQR 0.80-1.50) respectively (p = 0.4). Children ≥5yrs old were more likely to have a higher S/CO ratio than those <5yrs old (p<0.001). One hundred (38%) subjects <15yrs were Plasmodium RDT+. The median S/CO ratio (children <15yrs) did not differ by RDT status (p = 0.15). In subjects ≥15yrs, no molecular test was positive for Babesia, but four subjects (1.4%) were IFA reactive (two each at titers of 128 and 256). CONCLUSIONS/SIGNIFICANCE: The findings offer further support for Babesia in rural Tanzania. However, low prevalence of seroreactivity questions its clinical significance.

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.

Complete Anopheles funestus mitogenomes reveal an ancient history of mitochondrial lineages and their distribution in southern and central Africa.

Anopheles funestus s.s. is a primary vector of malaria in sub-Saharan Africa. Despite its important role in human Plasmodium transmission, evolutionary history, genetic diversity, and population structure of An. funestus in southern and central Africa remains understudied. We deep sequenced, assembled, and annotated the complete mitochondrial genome of An. funestus s.s. for the first time, providing a foundation for further genetic research of this important malaria vector species. We further analyzed the complete mitochondrial genomes of 43 An. funestus s.s. from three sites in Zambia, Democratic Republic of the Congo, and Tanzania. From these 43 mitogenomes we identified 41 unique haplotypes that comprised 567 polymorphic sites. Bayesian phylogenetic reconstruction confirmed the co-existence of two highly divergent An. funestus maternal lineages, herein defined as lineages I and II, in Zambia and Tanzania. The estimated coalescence time of these two mitochondrial lineages is ~500,000 years ago (95% HPD 426,000-594,000 years ago) with subsequent independent diversification. Haplotype network and phylogenetic analysis revealed two major clusters within lineage I, and genetic relatedness of samples with deep branching in lineage II. At this time, data suggest that the lineages are partially sympatric. This study illustrates that accurate retrieval of full mitogenomes of Anopheles vectors enables fine-resolution studies of intraspecies genetic relationships, population differentiation, and demographic history. Further investigations on whether An. funestus mitochondrial lineages represent biologically meaningful populations and their potential implications for malaria vector control are warranted.

RTS,S/AS01 malaria vaccine mismatch observed among Plasmodium falciparum isolates from southern and central Africa and globally.

The RTS,S/AS01 malaria vaccine encompasses the central repeats and C-terminal of Plasmodium falciparum circumsporozoite protein (PfCSP). Although no Phase II clinical trial studies observed evidence of strain-specific immunity, recent studies show a decrease in vaccine efficacy against non-vaccine strain parasites. In light of goals to reduce malaria morbidity, anticipating the effectiveness of RTS,S/AS01 is critical to planning widespread vaccine introduction. We deep sequenced C-terminal Pfcsp from 77 individuals living along the international border in Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC) and compared translated amino acid haplotypes to the 3D7 vaccine strain. Only 5.2% of the 193 PfCSP sequences from the Zambia-DRC border region matched 3D7 at all 84 amino acids. To further contextualize the genetic diversity sampled in this study with global PfCSP diversity, we analyzed an additional 3,809 Pfcsp sequences from the Pf3k database and constructed a haplotype network representing 15 countries from Africa and Asia. The diversity observed in our samples was similar to the diversity observed in the global haplotype network. These observations underscore the need for additional research assessing genetic diversity in P. falciparum and the impact of PfCSP diversity on RTS,S/AS01 efficacy.

Identification of Novel Viruses in Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis Ticks.

Ticks carry a wide range of known human and animal pathogens and are postulated to carry others with the potential to cause disease. Here we report a discovery effort wherein unbiased high-throughput sequencing was used to characterize the virome of 2,021 ticks, including Ixodes scapularis (n = 1,138), Amblyomma americanum (n = 720), and Dermacentor variabilis (n = 163), collected in New York, Connecticut, and Virginia in 2015 and 2016. We identified 33 viruses, including 24 putative novel viral species. The most frequently detected viruses were phylogenetically related to members of the Bunyaviridae and Rhabdoviridae families, as well as the recently proposed Chuviridae. Our work expands our understanding of tick viromes and underscores the high viral diversity that is present in ticks. IMPORTANCE The incidence of tick-borne disease is increasing, driven by rapid geographical expansion of ticks and the discovery of new tick-associated pathogens. The examination of the tick microbiome is essential in order to understand the relationship between microbes and their tick hosts and to facilitate the identification of new tick-borne pathogens. Genomic analyses using unbiased high-throughput sequencing platforms have proven valuable for investigations of tick bacterial diversity, but the examination of tick viromes has historically not been well explored. By performing a comprehensive virome analysis of the three primary tick species associated with human disease in the United States, we gained substantial insight into tick virome diversity and can begin to assess a potential role of these viruses in the tick life cycle.

Genomic insights into the ancient spread of Lyme disease across North America.

Lyme disease is the most prevalent vector-borne disease in North America and continues to spread. The disease was first clinically described in the 1970s in Lyme, Connecticut, but the origins and history of spread of the Lyme disease bacteria, Borrelia burgdorferi sensu stricto (s.s.), are unknown. To explore the evolutionary history of B. burgdorferi in North America, we collected ticks from across the USA and southern Canada from 1984 to 2013 and sequenced the, to our knowledge, largest collection of 146 B. burgdorferi s.s. genomes. Here, we show that B. burgdorferi s.s. has a complex evolutionary history with previously undocumented levels of migration. Diversity is ancient and geographically widespread, well pre-dating the Lyme disease epidemic of the past ~40 years, as well as the Last Glacial Maximum ~20,000 years ago. This means the recent emergence of human Lyme disease probably reflects ecological change-climate change and land use changes over the past century-rather than evolutionary change of the bacterium.

Distinct parasite populations infect individuals identified through passive and active case detection in a region of declining malaria transmission in southern Zambia.

BACKGROUND: Substantial reductions in the burden of malaria have been documented in parts of sub-Saharan Africa, with elimination strategies and goals being formulated in some regions. Within this context, understanding the epidemiology of low-level malaria transmission is crucial to achieving and sustaining elimination. A 24 single-nucleotide-polymorphism Plasmodium falciparum molecular barcode was used to characterize parasite populations from infected individuals identified through passive and active case detection in an area approaching malaria elimination in southern Zambia. METHODS: The study was conducted in the catchment area of Macha Hospital in Choma District, Southern Province, Zambia, where the parasite prevalence declined over the past decade, from 9.2% in 2008 to less than 1% in 2013. Parasite haplotypes from actively detected, P. falciparum-infected participants enrolled in a serial cross-sectional, community-based cohort study from 2008 to 2013 and from passively detected, P. falciparum-infected individuals enrolled at five rural health centres from 2012 to 2015 were compared. Changes in P. falciparum genetic relatedness, diversity and complexity were analysed as malaria transmission declined. RESULTS: Actively detected cases identified in the community were most commonly rapid diagnostic test negative, asymptomatic and had submicroscopic parasitaemia. Phylogenetic reconstruction using concatenated 24 SNP barcode revealed a separation of parasite haplotypes from passively and actively detected infections, consistent with two genetically distinct parasite populations. For passively detected infections identified at health centres, the proportion of detectable polyclonal infections was consistently low in all seasons, in contrast with actively detected infections in which the proportion of polyclonal infections was high. The mean genetic divergence for passively detected infections was 34.5% for the 2012-2013 transmission season, 37.8% for the 2013-2014 season, and 30.8% for the 2014-2015 season. The mean genetic divergence for actively detected infections was 22.3% in the 2008 season and 29.0% in the 2008-2009 season and 9.9% across the 2012-2014 seasons. CONCLUSIONS: Distinct parasite populations were identified among infected individuals identified through active and passive surveillance, suggesting that infected individuals detected through active surveillance may not have contributed substantially to ongoing transmission. As parasite prevalence and diversity within these individuals declined, resource-intensive efforts to identify the chronically infected reservoir may not be necessary to eliminate malaria in this setting.