One of the most commonly used molecular test for malaria diagnosis is the polymerase chain reaction (PCR)-based amplification of the 18S ribosomal DNA (rDNA) gene. Published diagnostic assays based on the 18S gene include the "gold standard" nested assay, semi-nested multiplex assay, and one tube multiplex assay. To our knowledge, no one has reported whether the two multiplex methods are better at detecting mixed Plasmodium infections compared to the nested assay using known quantities of DNA in experimentally mixed cocktails.

Molluscum contagiosum virus (MCV) causes an innocuous yet persistent skin infection in immunocompetent individuals and is spread by contact with lesions. Studies point to atopic dermatitis (AD) as a risk factor for MCV infection; however, there are no longitudinal studies that have evaluated this hypothesis.

We sequenced and annotated the genomes of four P. vivax strains collected from disparate geographic locations, tripling the number of genome sequences available for this understudied parasite and providing the first genome-wide perspective of global variability in this species. We observe approximately twice as much SNP diversity among these isolates as we do among a comparable collection of isolates of P. falciparum, a malaria-causing parasite that results in higher mortality. This indicates a distinct history of global colonization and/or a more stable demographic history for P. vivax relative to P. falciparum, which is thought to have undergone a recent population bottleneck. The SNP diversity, as well as additional microsatellite and gene family variability, suggests a capacity for greater functional variation in the global population of P. vivax. These findings warrant a deeper survey of variation in P. vivax to equip disease interventions targeting the distinctive biology of this neglected but major pathogen.

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.

Plasmodium vivax is the most prevalent malarial species in South America and exerts a substantial burden on the populations it affects. The control and eventual elimination of P. vivax are global health priorities. Genomic research contributes to this objective by improving our understanding of the biology of P. vivax and through the development of new genetic markers that can be used to monitor efforts to reduce malaria transmission. Here we analyze whole-genome data from eight field samples from a region in Cordóba, Colombia where malaria is endemic. We find considerable genetic diversity within this population, a result that contrasts with earlier studies suggesting that P. vivax had limited diversity in the Americas. We also identify a selective sweep around a substitution known to confer resistance to sulphadoxine-pyrimethamine (SP). This is the first observation of a selective sweep for SP resistance in this species. These results indicate that P. vivax has been exposed to SP pressure even when the drug is not in use as a first line treatment for patients afflicted by this parasite. We identify multiple non-synonymous substitutions in three other genes known to be involved with drug resistance in Plasmodium species. Finally, we found extensive microsatellite polymorphisms. Using this information we developed 18 polymorphic and easy to score microsatellite loci that can be used in epidemiological investigations in South America.

Multiplicity of infection (MOI) refers to the average number of distinct parasite genotypes concurrently infecting a patient. Although several studies have reported on MOI and the frequency of multiclonal infections in Plasmodium falciparum, there is limited data on Plasmodium vivax. Here, MOI and the frequency of multiclonal infections were studied in areas from South America where P. vivax and P. falciparum can be compared.

The study of the three protagonists in malaria-the Plasmodium parasite, the Anopheles mosquito, and the human host-is key to developing methods to control and eventually eliminate the disease. Genomic technologies, including the recent development of next-generation sequencing, enable interrogation of this triangle to an unprecedented level of scrutiny, and promise exciting progress toward real-time epidemiology studies and the study of evolutionary adaptation. We discuss the use of genomics by the International Centers of Excellence for Malaria Research, a network of field sites and laboratories in malaria-endemic countries that undertake cutting-edge research, training, and technology transfer in malarious countries of the world.

Several of the intended Plasmodium falciparum vaccine candidate antigens are highly polymorphic and could render a vaccine ineffective if their antigenic sites were not represented in the vaccine. In this study, characterization of genetic variability was performed in major B and T-cell epitopes within vaccine candidate antigens in isolates of P. falciparum from Peru.

Malaria incidence has reached staggering numbers in Venezuela. Commonly, Bolívar State accounted for approximately 70% of the country cases every year. Most cases cluster in the Sifontes municipality, a region characterized by an extractive economy, including gold mining. An increase in migration to Sifontes, driven by gold mining, fueled a malaria spillover to the rest of the country and the region. Here samples collected in 2018 were compared with a previous study of 2003/2004 to describe changes in the parasites population structures and the frequency of point mutations linked to anti-malarial drugs.

To clarify the role of bats in the ecology of Ebola viruses, we assessed the prevalence of Ebola virus antibodies in a large-scale sample of bats collected during 2015-2017 from countries in Africa that have had previous Ebola outbreaks (Guinea, the Democratic Republic of the Congo) or are at high risk for outbreaks (Cameroon). We analyzed 4,022 blood samples of bats from >12 frugivorous and 27 insectivorous species; 2-37 (0.05%-0.92%) bats were seropositive for Zaire and 0-30 (0%-0.75%) bats for Sudan Ebola viruses. We observed Ebola virus antibodies in 1 insectivorous bat genus and 6 frugivorous bat species. Certain bat species widespread across Africa had serologic evidence of Zaire and Sudan Ebola viruses. No viral RNA was detected in the subset of samples tested (n = 665). Ongoing surveillance of bats and other potential animal reservoirs are required to predict and prepare for future outbreaks.

Reported urban malaria cases are increasing in Latin America, however, evidence of such trend remains insufficient. Here, we propose an integrated approach that allows characterizing malaria transmission at the rural-to-urban interface by combining epidemiological, entomological, and parasite genotyping methods.

Emperor Geese (Anser canagicus) are iconic waterfowl endemic to Alaska and adjacent areas of northeastern Russia that are considered to be near threatened by the International Union for Conservation. This species has been identified as harboring diverse viruses and parasites which have, at times, been associated with disease in other avian taxa. To better assess if disease represents a vulnerability for Emperor Geese breeding on the Yukon-Kuskokwim Delta, Alaska, we evaluated if haemosporidian parasites were associated with decreased mass or survival among adult female nesting birds captured during 2006-2016. Through molecular analyses, we detected genetically diverse Leucocytozoon, Haemoproteus, and Plasmodium parasites in 28%, 1%, and 1% of 607 blood samples screened in triplicate, respectively. Using regression analysis, we found evidence for a small effect of Leucocytozoon infection on the mass of incubating adult female Emperor Geese. The estimated mass of infected individuals was approximately 43 g (95% CI: 20-67 g), or approximately 2%, less than uninfected birds when captured during the second half of incubation (days 11-25). We did not, however, find support for an effect of Leucocytozoon infection on survival of adult female nesting Emperor Geese using a multi-state hidden Markov framework to analyze mark-resight and recapture data. Using parasite mitochondrial DNA cytochrome b sequences, we identified 23 haplotypes among infected Emperor Geese. Leucocytozoon haplotypes clustered into three phylogenetically supported clades designated as 'L. simondi clade A', 'L. simondi clade B', and 'other Leucocytozoon'. We did not find evidence that parasites assigned to any of these clades were associated with differential mass measures among nesting adult female Emperor Geese. Collectively, our results provide negligible evidence for Leucocytozoon parasites as causing detrimental effects to adult female Emperor Geese breeding on the Yukon-Kuskokwim Delta.

Monkeypox virus (MPXV), a member of the Orthopoxvirus (OPXV) genus, is a zoonotic virus, endemic to central and western Africa that can cause smallpox-like symptoms in humans with fatal outcomes in up to 15% of patients. The incidence of MPXV infections in the Democratic Republic of the Congo, where the majority of cases have occurred historically, has been estimated to have increased as much as 20-fold since the end of smallpox vaccination in 1980. Considering the risk global travel carries for future disease outbreaks, accurate epidemiological surveillance of MPXV is warranted as demonstrated by the recent Mpox outbreak, where the majority of cases were occurring in non-endemic areas. Serological differentiation between childhood vaccination and recent infection with MPXV or other OPXVs is difficult due to the high level of conservation within OPXV proteins. Here, a peptide-based serological assay was developed to specifically detect exposure to MPXV. A comparative analysis of immunogenic proteins across human OPXVs identified a large subset of proteins that could potentially be specifically recognized in response to a MPXV infection. Peptides were chosen based upon MPXV sequence specificity and predicted immunogenicity. Peptides individually and combined were screened in an ELISA against serum from well-characterized Mpox outbreaks, vaccinee sera, and smallpox sera collected prior to eradication. One peptide combination was successful with ~86% sensitivity and ~90% specificity. The performance of the assay was assessed against the OPXV IgG ELISA in the context of a serosurvey by retrospectively screening a set of serum specimens from the region in Ghana believed to have harbored the MPXV-infected rodents involved in the 2003 United States outbreak.

Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine-pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum.

Since the eradication of smallpox, there have been increases in poxvirus infections and the emergence of several novel poxviruses that can infect humans and domestic animals. In 2015, a novel poxvirus was isolated from a resident of Alaska. Diagnostic testing and limited sequence analysis suggested this isolate was a member of the Orthopoxvirus (OPXV) genus but was highly diverged from currently known species, including Akhmeta virus. Here, we present the complete 210,797 bp genome sequence of the Alaska poxvirus isolate, containing 206 predicted open reading frames. Phylogenetic analysis of the conserved central region of the genome suggested the Alaska isolate shares a common ancestor with Old World OPXVs and is diverged from New World OPXVs. We propose this isolate as a member of a new OPXV species, Alaskapox virus (AKPV). The AKPV genome contained host range and virulence genes typical of OPXVs but lacked homologs of C4L and B7R, and the hemagglutinin gene contained a unique 120 amino acid insertion. Seven predicted AKPV proteins were most similar to proteins in non-OPXV Murmansk or NY_014 poxviruses. Genomic analysis revealed evidence suggestive of recombination with Ectromelia virus in two putative regions that contain seven predicted coding sequences, including the A-type inclusion protein.

P. cynomolgi, a malaria-causing parasite of Asian Old World monkeys, is the sister taxon of P. vivax, the most prevalent malaria-causing species in humans outside of Africa. Because P. cynomolgi shares many phenotypic, biological and genetic characteristics with P. vivax, we generated draft genome sequences for three P. cynomolgi strains and performed genomic analysis comparing them with the P. vivax genome, as well as with the genome of a third previously sequenced simian parasite, Plasmodium knowlesi. Here, we show that genomes of the monkey malaria clade can be characterized by copy-number variants (CNVs) in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites and CNVs in the P. cynomolgi genome, providing a map of genetic variation that can be used to map parasite traits and study parasite populations. The sequencing of the P. cynomolgi genome is a critical step in developing a model system for P. vivax research and in counteracting the neglect of P. vivax.

Plasmodium falciparum is distributed throughout the tropics and is responsible for an estimated 230 million cases of malaria every year, with a further 1.4 billion people at risk of infection. Little is known about the genetic makeup of P. falciparum populations, despite variation in genetic diversity being a key factor in morbidity, mortality, and the success of malaria control initiatives. Here we analyze a worldwide sample of 519 P. falciparum isolates sequenced for two housekeeping genes (63 single nucleotide polymorphisms from around 5000 nucleotides per isolate). We observe a strong negative correlation between within-population genetic diversity and geographic distance from sub-Saharan Africa (R(2) = 0.95) over Africa, Asia, and Oceania. In contrast, regional variation in transmission intensity seems to have had a negligible impact on the distribution of genetic diversity. The striking geographic patterns of isolation by distance observed in P. falciparum mirror the ones previously documented in humans and point to a joint sub-Saharan African origin between the parasite and its host. Age estimates for the expansion of P. falciparum further support that anatomically modern humans were infected prior to their exit out of Africa and carried the parasite along during their colonization of the world.

The Hoatzin (Opisthocomus hoazin) is the only extant member of the order Opisthocomiformes. This unique South American bird lives in the riparian lowland vegetation characteristic of the Amazon and Orinoco basins. Hoatzins nest in communal social units close to water bodies; they are strictly folivores being the only bird with pregastric fermentation in the crop. Because of the complex logistics involved in capturing this bird, there is a knowledge gap on its parasites. This study documents two distant lineages of haemosporidian parasites (Plasmodium spp.) in a juvenile and two adults sampled in the Cojedes state, Venezuela. Although negative by microscopy, the parasite identification was possible by using molecular methods. We estimated the phylogenetic relationships on the parasite cytochrome b (cytb, 480 bp) gene and the mitochondrial DNA. We found one of the parasites lineages in two individuals (nestling and adult), and the corresponding fragment of cytb was identical to a one found in Wood Stork (Mycteria americana) from Brazil. The other lineage, found in an adult, has an identity of 469 out of 478 bp (98%) with Plasmodium sp. GAL-2012 (isolate THAMB08) from Brazil. Although a morphological description of these parasites was not possible, this is the first molecular study focusing on Hoatzin haemosporidian parasites and the first documentation of Plasmodium infections in the Hoatzin from Venezuela. Furthermore, we reported microfilaria in two adults as well as hematological parameters for six individuals. Information on hematological parameters could contribute to establishing the necessary baseline to detect underlying conditions, such as infections, in this bird species.

Foamy viruses are complex retroviruses that have been shown to be transmitted from nonhuman primates to humans. In Bangladesh, infection with simian foamy virus (SFV) is ubiquitous among rhesus macaques, which come into contact with humans in diverse locations and contexts throughout the country. We analyzed microsatellite DNA from 126 macaques at six sites in Bangladesh in order to characterize geographic patterns of macaque population structure. We also included in this study 38 macaques owned by nomadic people who train them to perform for audiences. PCR was used to analyze a portion of the proviral gag gene from all SFV-positive macaques, and multiple clones were sequenced. Phylogenetic analysis was used to infer long-term patterns of viral transmission. Analyses of SFV gag gene sequences indicated that macaque populations from different areas harbor genetically distinct strains of SFV, suggesting that geographic features such as forest cover play a role in determining the dispersal of macaques and SFV. We also found evidence suggesting that humans traveling the region with performing macaques likely play a role in the translocation of macaques and SFV. Our studies found that individual animals can harbor more than one strain of SFV and that presence of more than one SFV strain is more common among older animals. Some macaques are infected with SFV that appears to be recombinant. These findings paint a more detailed picture of how geographic and sociocultural factors influence the spectrum of simian-borne retroviruses.

During 2012, 2013 and 2015, we collected small mammals within 25 km of the town of Boende in Tshuapa Province, the Democratic Republic of the Congo. The prevalence of monkeypox virus (MPXV) in this area is unknown; however, cases of human infection were previously confirmed near these collection sites. Samples were collected from 353 mammals (rodents, shrews, pangolins, elephant shrews, a potamogale, and a hyrax). Some rodents and shrews were captured from houses where human monkeypox cases have recently been identified, but most were trapped in forests and agricultural areas near villages. Real-time PCR and ELISA were used to assess evidence of MPXV infection and other Orthopoxvirus (OPXV) infections in these small mammals. Seven (2.0%) of these animal samples were found to be anti-orthopoxvirus immunoglobulin G (IgG) antibody positive (six rodents: two Funisciurus spp.; one Graphiurus lorraineus; one Cricetomys emini; one Heliosciurus sp.; one Oenomys hypoxanthus, and one elephant shrew Petrodromus tetradactylus); no individuals were found positive in PCR-based assays. These results suggest that a variety of animals can be infected with OPXVs, and that epidemiology studies and educational campaigns should focus on animals that people are regularly contacting, including larger rodents used as protein sources.