Plasmodium vivax is the most prevalent human malaria parasite outside of Africa. Yet, studies aimed to identify genes with signatures consistent with natural selection are rare. Here, we present a comparative analysis of the pattern of genetic variation of five sequenced isolates of P. vivax and its divergence with two closely related species, Plasmodium cynomolgi and Plasmodium knowlesi, using a set of orthologous genes. In contrast to Plasmodium falciparum, the parasite that causes the most lethal form of human malaria, we did not find significant constraints on the evolution of synonymous sites genome wide in P. vivax. The comparative analysis of polymorphism and divergence across loci allowed us to identify 87 genes with patterns consistent with positive selection, including genes involved in the "exportome" of P. vivax, which are potentially involved in evasion of the host immune system. Nevertheless, we have found a pattern of polymorphism genome wide that is consistent with a significant amount of constraint on the replacement changes and prevalent negative selection. Our analyses also show that silent polymorphism tends to be larger toward the ends of the chromosomes, where many genes involved in antigenicity are located, suggesting that natural selection acts not only by shaping the patterns of variation within the genes but it also affects genome organization.

Our genetic analyses of uncharacterized bunyaviruses isolated in Peru identified a possible reassortant virus containing small and large gene segment sequences closely related to the Caraparu virus and a medium gene segment sequence potentially derived from an unidentified group C orthobunyavirus. Neutralization tests confirmed serologic distinction among the newly identified virus and the prototype and Caraparu strains. This virus, named Itaya, was isolated in 1999 and 2006 from febrile patients in the cities of Iquitos and Yurimaguas in Peru. The geographic distance between the 2 cases suggests that the Itaya virus could be widely distributed throughout the Amazon basin in northeastern Peru. Identification of a new Orthobunyavirus species that causes febrile disease in humans reinforces the need to expand viral disease surveillance in tropical regions of South America.

Detection of histidine-rich protein 2 (HRP2) from the malaria parasite Plasmodium falciparum provides evidence for active or recent infection, and is utilized for both diagnostic and surveillance purposes, but current laboratory immunoassays for HRP2 are hindered by low sensitivities and high costs. Here we present a new HRP2 immunoassay based on antigen capture through a bead-based system capable of detecting HRP2 at sub-picogram levels. The assay is highly specific and cost-effective, allowing fast processing and screening of large numbers of samples. We utilized the assay to assess results of HRP2-based rapid diagnostic tests (RDTs) in different P. falciparum transmission settings, generating estimates for true performance in the field. Through this method of external validation, HRP2 RDTs were found to perform well in the high-endemic areas of Mozambique and Angola with 86.4% and 73.9% of persons with HRP2 in their blood testing positive by RDTs, respectively, and false-positive rates of 4.3% and 0.5%. However, in the low-endemic setting of Haiti, only 14.5% of persons found to be HRP2 positive by the bead assay were RDT positive. Additionally, 62.5% of Haitians showing a positive RDT test had no detectable HRP2 by the bead assay, likely indicating that these were false positive tests. In addition to RDT validation, HRP2 biomass was assessed for the populations in these different settings, and may provide an additional metric by which to estimate P. falciparum transmission intensity and measure the impact of interventions.

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

Recent anti-malarial resistance monitoring in Angola has shown efficacy of artemether-lumefantrine (AL) in certain sites approaching the key 90% lower limit of efficacy recommended for artemisinin-based combination therapy. In addition, a controversial case of malaria unresponsive to artemisinins was reported in a patient infected in Lunda Sul Province in 2013.

Mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of Plasmodium falciparum are associated with resistance to anti-folate drugs, most notably sulphadoxine-pyrimethamine (SP). Molecular studies document the prevalence of these mutations in parasite populations across the African continent. However, there is no systematic review examining the collective epidemiological significance of these studies. This meta-analysis attempts to: 1) summarize genotype frequency data that are critical for molecular surveillance of anti-folate resistance and 2) identify the specific challenges facing the development of future molecular databases.

Plasmodium vivax (Pv) is the second most important human malaria parasite. Recent data indicate that the impact of Pv malaria on the health and economies of the developing world has been dramatically underestimated. Pv has a unique feature in its life cycle. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain as dormant forms called hypnozoites, which cause relapses of malaria months to several years after the primary infection. Elimination of malaria caused by Pv will be facilitated by developing a safe, highly effective drug that eliminates Pv liver stages, including hypnozoites. Identification and development of such a drug would be facilitated by the development of a medium to high throughput assay for screening drugs against Pv liver stages. We undertook the present pilot study to (1) assess the feasibility of producing large quantities of purified, vialed, cryopreserved Pv sporozoites and (2) establish a system for culturing the liver stages of Pv in order to assess the effects of drugs on the liver stages of Pv. We used primaquine (PQ) to establish this assay model, because PQ is the only licensed drug known to clear all Pv hepatocyte stages, including hypnozoites, and the effect of PQ on Pv hepatocyte stage development in vitro has not previously been reported. We report that we have established the capacity to reproducibly infect hepatoma cells with purified, cyropreserved Pv spz from the same lot, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. These data provide the foundation for finalizing a medium throughput, high content assay to identify new drugs for the elimination of all Pv liver stages.

RTS,S is the most advanced malaria vaccine candidate, currently under phase-III clinical trials in Africa. This Plasmodium falciparum vaccine contains part of the central repeat region and the complete C-terminal T cell epitope region (Th2R and Th3R) of the circumsporozoite protein (CSP). Since naturally occurring polymorphisms at the vaccine candidate loci are critical determinants of the protective efficacy of the vaccines, it is imperative to investigate these polymorphisms in field isolates. In this study we have investigated the genetic diversity at the central repeat, C-terminal T cell epitope (Th2R and Th3R) and N-terminal T cell epitope regions of the CSP, in P. falciparum isolates from Madhya Pradesh state of India. These isolates were collected through a 5-year prospective study aimed to develop a well-characterized field-site for the future evaluation of malaria vaccine in India. Our results revealed that the central repeat (63 haplotypes, n = 161) and C-terminal Th2R/Th3R epitope (24 haplotypes, n = 179) regions were highly polymorphic, whereas N-terminal non-repeat region was less polymorphic (5 haplotypes, n = 161) in this population. We did not find any evidence of the role of positive natural selection in maintaining the genetic diversity at the Th2R/Th3R regions of CSP. Comparative analysis of the Th2R/Th3R sequences from this study to the global isolates (n = 1160) retrieved from the GenBank database revealed two important points. First, the majority of the sequences (~61%, n = 179) from this study were identical to the Dd2/Indochina type, which is also the predominant Th2R/Th3R haplotype in Asia (~59%, n = 974). Second, the Th2R/Th3R sequences in Asia, South America and Africa are geographically distinct with little allele sharing between continents. In conclusion, this study provides an insight on the existing polymorphisms in the CSP in a parasite population from India that could potentially influence the efficacy of RTS,S vaccine in this region.

Tropical countries are thought to play an important role in the global behavior of respiratory infections such as influenza. The tropical country of Ecuador has almost no documentation of the causes of acute respiratory infections. The objectives of this study were to identify the viral agents associated with influenza like illness (ILI) in Ecuador, describe what strains of influenza were circulating in the region along with their epidemiologic characteristics, and perform molecular characterization of those strains.

Molecular methods elucidate evolutionary and ecological processes in parasites, where interaction between hosts and parasites enlighten the evolution of parasite lifestyles and host defenses. Population genetics of Plasmodium vivax parasites accurately describe transmission dynamics of the parasites and evaluation of malaria control measures. As a first generation vaccine candidate against malaria, the Circumsporozoite Protein (CSP) has demonstrated significant potential in P. falciparum. Extensive polymorphism hinders the development of a potent malaria vaccine. Hence, the genetic diversity of Pvcsp was investigated for the first time in 60 Sri Lankan clinical isolates by obtaining the nucleotide sequence of the central repeat (CR) domain and examining the polymorphism of the peptide repeat motifs (PRMs), the genetic diversity indices and phylogenetic relationships. PCR amplicons determined size polymorphism of 610, 700 and 710 bp in Pvcsp of Sri Lanka where all amino acid sequences obtained were of the VK210 variant, consisting variable repeats of 4 different PRMs. The two most abundant PRMs of the CR domain, GDRADGQPA and GDRAAGQPA consisted ~2-4 repeats, while GNRAAGQPA was unique to the island. Though, different nucleotide sequences termed repeat allotypes (RATs) were observed for each PRM, these were synonymous contributing to a less polymorphic CR domain. The genetic diversity of Pvcsp in Sri Lanka was due to the number of repetitive peptide repeat motifs, point mutations, and intragenic recombination. The 19 amino acid haplotypes defined were exclusive to Sri Lanka, whereas the 194 Pvcsp sequences of global isolates generated 57 more distinct a.a. haplotypes of the VK210 variant. Strikingly, the CR domain of both VK210 and VK247 variants was under purifying selection interpreting the scarcity of CSP non-synonymous polymorphisms. Insights to the distribution of RATs in the CR region with geographic clustering of the P. vivax VK210 variant were revealed. The cladogram reiterated this unique geographic clustering of local (VK210) and global isolates (VK210 and VK247), which was further validated by the elevated fixation index values of the VK210 variant.

Understanding the spatial clustering of Plasmodium falciparum populations can assist efforts to contain drug-resistant parasites and maintain the efficacy of future drugs. We sequenced single nucleotide polymorphisms (SNPs) in the dihydropteroate synthase gene (dhps) associated with sulfadoxine resistance and 5 microsatellite loci flanking dhps in order to investigate the genetic backgrounds, genetic relatedness, and geographic clustering of falciparum parasites in the Democratic Republic of the Congo (DRC). Resistant haplotypes were clustered into subpopulations: one in the northeast DRC, and the other in the balance of the DRC. Network and clonal lineage analyses of the flanking microsatellites indicate that geographically-distinct mutant dhps haplotypes derive from separate lineages. The DRC is therefore a watershed for haplotypes associated with sulfadoxine resistance. Given the importance of central Africa as a corridor for the spread of antimalarial resistance, the identification of the mechanisms of this transit can inform future policies to contain drug-resistant parasite strains.

Determining the source of malaria outbreaks in Ecuador and identifying remaining transmission foci will help in malaria elimination efforts. In this study, the genetic signatures of Plasmodium falciparum isolates, obtained from an outbreak that occurred in northwest Ecuador from 2012 to 2013, were characterized.

Human parainfluenza viruses (HPIVs) are common viral causes of community-acquired pneumonia, particularly in children. The four types of HPIV have world-wide distribution; however, limited information exists about the epidemiological profile of HPIV in Latin-America.

In vivo efficacy assessments of antimalarials are essential for ensuring effective case management. In Ethiopia, chloroquine (CQ) without primaquine is the first-line treatment for Plasmodium vivax in malarious areas, but artemether-lumefantrine (AL) is also commonly used.

Oropouche (ORO) virus, a member of the Simbu serogroup, is one of the few human pathogens in the Orthobunyavirus genus in the family Bunyaviridae. Genetic analyses of ORO-like strains from Iquitos, Peru, identified a novel reassortant containing the S and L segments of ORO virus and the M segment of a novel Simbu serogroup virus. This new pathogen, which we named Iquitos (IQT) virus, was first isolated during 1999 from a febrile patient in Iquitos, an Amazonian city in Peru. Subsequently, the virus was identified as the cause of outbreaks of "Oropouche fever" during 2005 and 2006 in Iquitos. In addition to the identification of 17 isolates of IQT virus between 1999 and 2006, surveys for neutralizing antibody among Iquitos residents revealed prevalence rates of 14.9% for ORO virus and 15.4% for IQT virus. Limited studies indicate that prior infection with ORO virus does not seem to protect against disease caused with the IQT virus infection. Identification of a new Orthobunyavirus human pathogen in the Amazon region of Peru highlights the need for strengthening surveillance activities and laboratory capabilities, and investigating the emergence of new pathogens in tropical regions of South America.

False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2-based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies.

Haemosporidians are a diverse group of vector-borne parasitic protozoa that includes the agents of human malaria; however, most of the described species are found in birds and reptiles. Although our understanding of these parasites' diversity has expanded by analyses of their mitochondrial genes, there is limited information on these genes' evolutionary rates. Here, 114 mitochondrial genomes (mtDNA) were studied from species belonging to four genera: Leucocytozoon, Haemoproteus, Hepatocystis, and Plasmodium. Contrary to previous assertions, the mtDNA is phylogenetically informative. The inferred phylogeny showed that, like the genus Plasmodium, the Leucocytozoon and Haemoproteus genera are not monophyletic groups. Although sensitive to the assumptions of the molecular dating method used, the estimated times indicate that the diversification of the avian haemosporidian subgenera/genera took place after the Cretaceous-Paleogene boundary following the radiation of modern birds. Furthermore, parasite clade differences in mtDNA substitution rates and strength of negative selection were detected. These differences may affect the biological interpretation of mtDNA gene lineages used as a proxy to species in ecological and parasitological investigations. Given that the mitochondria are critically important in the parasite life cycle stages that take place in the vector and that the transmission of parasites belonging to particular clades has been linked to specific insect families/subfamilies, this study suggests that differences in vectors have affected the mode of evolution of haemosporidian mtDNA genes. The observed patterns also suggest that the radiation of haemosporidian parasites may be the result of community-level evolutionary processes between their vertebrate and invertebrate hosts.

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.

There is an increased interest in potential zoonotic malarias. To date, Plasmodium malariae that infects humans remains indistinguishable from Plasmodium brasilianum, which is widespread among New World primates. Distributed throughout tropical Central and South America, the Callitrichidae are small arboreal primates in which detection of natural Plasmodium infection has been extremely rare. Most prior screening efforts have been limited to small samples, the use of low-probability detection methods, or both. Rarely have screening efforts implemented a longitudinal sampling design. Through an annual mark-recapture program of two sympatric callitrichids, the emperor (Saguinus imperator) and saddleback (Saguinus fuscicollis) tamarins, whole blood samples were screened for Plasmodium by microscopy and nested PCR of the cytochrome b gene across four consecutive years (2012-2015). Following the first field season, approximately 50% of the samples collected each subsequent year were from recaptured individuals. In particular, out of 245 samples from 129 individuals, 11 samples from 6 individuals were positive for Plasmodium, and all but one of these infections was found in S. imperator. Importantly, the cytochrome b sequences were 100% identical to former isolates of P. malariae from humans and P. brasilianum from Saimiri sp. Chronic infections were detected as evidenced by repeated infections (7) from two individuals across the 4-year study period. Furthermore, 4 of the 5 infected emperor tamarins were part of a single group spanning the entire study period. Overall, the low prevalence reported here is consistent with previous findings. This study identifies two new natural hosts for P. brasilianum and provides evidence in support of chronic infections in wildlife populations. Given that callitrichids are often found in mixed-species associations with other primates and can be resilient to human-disturbed environments, they could contribute to the maintenance of P. malariae populations if future work provides entomological and epidemiological evidence indicating human zoonotic infections.

The ability to identify mixed-species infections and track the origin of Plasmodium parasites can further enhance the development of treatment and prevention recommendations as well as outbreak investigations. Here, we explore the utility of using the full Plasmodium mitochondrial genome to classify Plasmodium species, detect mixed infections, and infer the geographical origin of imported P. falciparum parasites to the United States (U.S.). Using the recently developed standardized, high-throughput Malaria Resistance Surveillance (MaRS) protocol, the full Plasmodium mitochondrial genomes of 265 malaria cases imported to the U.S. from 2014-2017 were sequenced and analyzed. P. falciparum infections were found in 94.7% (251/265) of samples. Five percent (14/265) of samples were identified as mixed- Plasmodium species or non-P. falciparum, including P. vivax, P. malariae, P. ovale curtisi, and P. ovale wallikeri. P. falciparum mitochondrial haplotypes analysis revealed greater than eighteen percent of samples to have at least two P. falciparum mitochondrial genome haplotypes, indicating either heteroplasmy or multi-clonal infections. Maximum-likelihood phylogenies of 912 P. falciparum mitochondrial genomes with known country origin were used to infer the geographical origin of thirteen samples from persons with unknown travel histories as: Africa (country unspecified) (n = 10), Ghana (n = 1), Southeast Asia (n = 1), and the Philippines (n = 1). We demonstrate the utility and current limitations of using the Plasmodium mitochondrial genome to classify samples with mixed-infections and infer the geographical origin of imported P. falciparum malaria cases to the U.S. with unknown travel history.