Population genomics and geographic dispersal in Chagas disease vectors: Landscape drivers and evidence of possible adaptation to the domestic setting.

Accurate prediction of vectors dispersal, as well as identification of adaptations that allow blood-feeding vectors to thrive in built environments, are a basis for effective disease control. Here we adopted a landscape genomics approach to assay gene flow, possible local adaptation, and drivers of population structure in Rhodnius ecuadoriensis, an important vector of Chagas disease. We used a reduced-representation sequencing technique (2b-RADseq) to obtain 2,552 SNP markers across 272 R. ecuadoriensis samples from 25 collection sites in southern Ecuador. Evidence of high and directional gene flow between seven wild and domestic population pairs across our study site indicates insecticide-based control will be hindered by repeated re-infestation of houses from the forest. Preliminary genome scans across multiple population pairs revealed shared outlier loci potentially consistent with local adaptation to the domestic setting, which we mapped to genes involved with embryogenesis and saliva production. Landscape genomic models showed elevation is a key barrier to R. ecuadoriensis dispersal. Together our results shed early light on the genomic adaptation in triatomine vectors and facilitate vector control by predicting that spatially-targeted, proactive interventions would be more efficacious than current, reactive approaches.

Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity.

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/µl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

The potential role of the gut microbiota in shaping host energetics and metabolic rate.

It is increasingly recognized that symbiotic microbiota (especially those present in the gut) have important influences on the functioning of their host. Here, we review the interplay between this microbial community and the growth, metabolic rate and nutritional energy harvest of the host. We show how recent developments in experimental and analytical methods have allowed much easier characterization of the nature, and increasingly the functioning, of the gut microbiota. Manipulation studies that remove or augment gut microorganisms or transfer them between hosts have allowed unprecedented insights into their impact. Whilst much of the information to date has come from studies of laboratory model organisms, recent studies have used a more diverse range of host species, including those living in natural conditions, revealing their ecological relevance. The gut microbiota can provide the host with dietary nutrients that would be otherwise unobtainable, as well as allow the host flexibility in its capacity to cope with changing environments. The composition of the gut microbial community of a species can vary seasonally or when the host moves between environments (e.g. fresh and sea water in the case of migratory fish). It can also change with host diet choice, metabolic rate (or demands) and life stage. These changes in gut microbial community composition enable the host to live within different environments, adapt to seasonal changes in diet and maintain performance throughout its entire life history, highlighting the ecological relevance of the gut microbiota. Whilst it is evident that gut microbes can underpin host metabolic plasticity, the causal nature of associations between particular microorganisms and host performance is not always clear unless a manipulative approach has been used. Many studies have focussed on a correlative approach by characterizing microbial community composition, but there is now a need for more experimental studies in both wild and laboratory-based environments, to reveal the true role of gut microbiota in influencing the functioning of their hosts, including its capacity to tolerate environmental change. We highlight areas where these would be particularly fruitful in the context of ecological energetics.

Hosts and vectors of Trypanosoma cruzi discrete typing units in the Chagas disease endemic region of the Paraguayan Chaco.

Active Trypanosoma cruzi transmission persists in the Gran Chaco region, which is considered hyperendemic for Chagas disease. Understanding domestic and sylvatic transmission cycles and therefore the relationship between vectors and mammalian hosts is crucial to designing and implementing improved effective control strategies. Here we describe the species of triatomine vectors and the sylvatic mammal reservoirs of T. cruzi, in different localities of the Paraguayan and Bolivian Chaco. We identify the T. cruzi genotypes discrete typing units (DTUs) and provide a map of their geographical distribution. A total of 1044 triatomines and 138 sylvatic mammals were captured. Five per cent of the triatomines were microscopically positive for T. cruzi (55 Triatoma infestans from Paraguay and one sylvatic Triatoma guasayana from Bolivia) and 17 animals (12·3%) comprising eight of 28 (28·5%) Dasypus novemcinctus, four of 27 (14·8%) Euphractus sexcinctus, three of 64 (4·7%) Chaetophractus spp. and two of 14 (14·3%) Didelphis albiventris. The most common DTU infecting domestic triatomine bugs was TcV (64%), followed by TcVI (28%), TcII (6·5%) and TcIII (1·5%). TcIII was overwhelmingly associated with armadillo species. We confirm the primary role of T. infestans in domestic transmission, armadillo species as the principal sylvatic hosts of TcIII, and consider the potential risk of TcIII as an agent of Chagas disease in the Chaco.

Importation of Hybrid Human-Associated Trypanosoma cruzi Strains of Southern South American Origin, Colombia.

We report the characterization of Trypanosoma cruzi of southern South American origin among humans, domestic vectors, and peridomestic hosts in Colombia using high-resolution nuclear and mitochondrial genotyping. Expanding our understanding of the geographic range of lineage TcVI, which is associated with severe Chagas disease, will help clarify risk of human infection for improved disease control.

Ecological host fitting of Trypanosoma cruzi TcI in Bolivia: mosaic population structure, hybridization and a role for humans in Andean parasite dispersal.

An improved understanding of how a parasite species exploits its genetic repertoire to colonize novel hosts and environmental niches is crucial to establish the epidemiological risk associated with emergent pathogenic genotypes. Trypanosoma cruzi, a genetically heterogeneous, multi-host zoonosis, provides an ideal system to examine the sylvatic diversification of parasitic protozoa. In Bolivia, T. cruzi I, the oldest and most widespread genetic lineage, is pervasive across a range of ecological clines. High-resolution nuclear (26 loci) and mitochondrial (10 loci) genotyping of 199 contemporaneous sylvatic TcI clones was undertaken to provide insights into the biogeographical basis of T. cruzi evolution. Three distinct sylvatic parasite transmission cycles were identified: one highland population among terrestrial rodent and triatomine species, composed of genetically homogenous strains (Ar = 2.95; PA/L = 0.61; DAS = 0.151), and two highly diverse, parasite assemblages circulating among predominantly arboreal mammals and vectors in the lowlands (Ar = 3.40 and 3.93; PA/L = 1.12 and 0.60; DAS = 0.425 and 0.311, respectively). Very limited gene flow between neighbouring terrestrial highland and arboreal lowland areas (distance ~220 km; FST = 0.42 and 0.35) but strong connectivity between ecologically similar but geographically disparate terrestrial highland ecotopes (distance >465 km; FST = 0.016-0.084) strongly supports ecological host fitting as the predominant mechanism of parasite diversification. Dissimilar heterozygosity estimates (excess in highlands, deficit in lowlands) and mitochondrial introgression among lowland strains may indicate fundamental differences in mating strategies between populations. Finally, accelerated parasite dissemination between densely populated, highland areas, compared to uninhabited lowland foci, likely reflects passive, long-range anthroponotic dispersal. The impact of humans on the risk of epizootic Chagas disease transmission in Bolivia is discussed.

Reproductive clonality in protozoan pathogens--truth or artefact?

The debate around the frequency and importance of genetic exchange in parasitic protozoa is now several decades old. Recently, fresh assertions have been made that predominant clonal evolution explains the population structures of several key protozoan pathogens. Here, we present an alternative perspective. On the assumption that much apparent clonality may be an artefact of inadequate sampling and study design, we review current research to define why sex might be so difficult to detect in protozoan parasite populations. In doing so, we contrast laboratory models of genetic exchange in parasitic protozoa with natural patterns of genetic diversity and consider the fitness advantage of sex at different evolutionary scales. We discuss approaches to improve the accuracy of efforts to characterize genetic exchange in the field. We also examine the implications of the first population genomic studies for the debate around sex and clonality in parasitic protozoa and discuss caveats for the future.

Molecular epidemiologic source tracking of orally transmitted Chagas disease, Venezuela.

Oral outbreaks of Chagas disease are increasingly reported in Latin America. The transitory presence of Trypanosoma cruzi parasites within contaminated foods, and the rapid consumption of those foods, precludes precise identification of outbreak origin. We report source attribution for 2 peri-urban oral outbreaks of Chagas disease in Venezuela via high resolution microsatellite typing.

Comparative genomic analysis of human infective Trypanosoma cruzi lineages with the bat-restricted subspecies T. cruzi marinkellei.

BACKGROUND: Trypanosoma cruzi marinkellei is a bat-associated parasite of the subgenus Schizotrypanum and it is regarded as a T. cruzi subspecies. Here we report a draft genome sequence of T. c. marinkellei and comparison with T. c. cruzi. Our aims were to identify unique sequences and genomic features, which may relate to their distinct niches. RESULTS: The T. c. marinkellei genome was found to be ~11% smaller than that of the human-derived parasite T. c. cruzi Sylvio X10. The genome size difference was attributed to copy number variation of coding and non-coding sequences. The sequence divergence in coding regions was ~7.5% between T. c. marinkellei and T. c. cruzi Sylvio X10. A unique acetyltransferase gene was identified in T. c. marinkellei, representing an example of a horizontal gene transfer from eukaryote to eukaryote. Six of eight examined gene families were expanded in T. c. cruzi Sylvio X10. The DGF gene family was expanded in T. c. marinkellei. T. c. cruzi Sylvio X10 contained ~1.5 fold more sequences related to VIPER and L1Tc elements. Experimental infections of mammalian cell lines indicated that T. c. marinkellei has the capacity to invade non-bat cells and undergo intracellular replication. CONCLUSIONS: Several unique sequences were identified in the comparison, including a potential subspecies-specific gene acquisition in T. c. marinkellei. The identified differences reflect the distinct evolutionary trajectories of these parasites and represent targets for functional investigation.

Contemporary cryptic sexuality in Trypanosoma cruzi.

Clonal propagation is considered to be the predominant mode of reproduction among many parasitic protozoa. However, this assumption may overlook unorthodox, infrequent or cryptic sexuality. Trypanosoma cruzi, which causes Chagas disease, is known to undergo non-Mendelian genetic exchange in the laboratory. In the field, evidence of extant genetic exchange is limited. In this study, we undertook intensive sampling of T. cruzi Discrete Typing Unit I in endemic eastern Colombia. Using Fluorescence-activated cell sorting, we generated 269 biological clones from 67 strains. Each clone was genotyped across 24 microsatellite loci. Subsequently, 100 representative clones were typed using 10 mitochondrial sequence targets (3.76 Kbp total). Clonal diversity among humans, reservoir hosts and vectors suggested complex patterns of superinfection and/or coinfection in oral and vector-borne Chagas disease cases. Clonal diversity between mother and foetus in a congenital case demonstrates that domestic TcI genotypes are infective in utero. Importantly, gross incongruence between nuclear and mitochondrial markers is strong evidence for widespread genetic exchange throughout the data set. Furthermore, a confirmed mosaic maxicircle sequence suggests intermolecular recombination between individuals as a further mechanism of genetic reassortment. Finally, robust dating based on mitochondrial DNA indicates that the emergence of a widespread domestic TcI clade that we now name TcI(DOM) (formerly TcIa/VEN(Dom)) occurred 23 000 ± 12 000 years ago and was followed by population expansion, broadly corresponding with the earliest human migration into the Americas.

Genome plasticity driven by aneuploidy and loss of heterozygosity in Trypanosoma cruzi.

Trypanosoma cruzi the causative agent of Chagas disease shows a marked genetic diversity and divided into at least six Discrete Typing Units (DTUs). High intra genetic variability has been observed in the TcI DTU, the most widely distributed DTU, where patterns of genomic diversity can provide information on ecological and evolutionary processes driving parasite population structure and genome organization. Chromosomal aneuploidies and rearrangements across multigene families represent an evidence of T. cruzi genome plasticity. We explored genomic diversity among 18 Colombian T. cruzi I clones and 15 T. cruzi I South American strains. Our results confirm high genomic variability, heterozygosity and presence of a clade compatible with the TcIdom genotype, described for strains from humans in Colombia and Venezuela. TcI showed high structural plasticity across the geographical region studied. Differential events of whole and segmental aneuploidy (SA) along chromosomes even between clones from the same strain were found and corroborated by the depth and allelic frequency. We detected loss of heterozygosity (LOH) events in different chromosomes, however, the size and location of segments under LOH varied between clones. Genes adjacent to breakpoints were evaluated, and retrotransposon hot spot genes flanked the beginning of segmental aneuploidies. Our results suggest that T. cruzi genomes, like those of Leishmania, may have a highly unstable structure and there is now an urgent need to design experiments to explore any potential adaptive role for the plasticity observed.

Deploying an In Vitro Gut Model to Assay the Impact of the Mannan-Oligosaccharide Prebiotic Bio-Mos on the Atlantic Salmon (Salmo salar) Gut Microbiome.

Alpha mannose-oligosaccharide (MOS) prebiotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and their associated metabolism. Bio-Mos is a commercially available prebiotic currently used in the agri-feed industry, but studies show contrasting results of its effect on fish performance and feed efficiency. Thus, detailed studies are needed to investigate the effect of MOS supplements on the fish microbiome to enhance our understanding of the link between MOS and gut health. To assess Bio-Mos for potential use as a prebiotic growth promoter in salmonid aquaculture, we have modified an established Atlantic salmon in vitro gut model, SalmoSim, to evaluate its impact on the host microbial communities. The microbial communities obtained from ceca compartments from four adult farmed salmon were inoculated in biological triplicate reactors in SalmoSim. Prebiotic treatment was supplemented for 20 days, followed by a 6-day washout period. Inclusion of Bio-Mos in the media resulted in a significant increase in formate (P = 0.001), propionate (P = 0.037) and 3-methyl butanoic acid (P = 0.024) levels, correlated with increased abundances of several, principally, anaerobic microbial genera (Fusobacterium, Agarivorans, Pseudoalteromonas). DNA metabarcoding with the 16S rDNA marker confirmed a significant shift in microbial community composition in response to Bio-Mos supplementation with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to complementin vivo trials of microbiome modulators. IMPORTANCE In this paper we report the results of the impact of a prebiotic (alpha-MOS supplementation) on microbial communities, using an in vitro simulator of the gut microbial environment of the Atlantic salmon. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production by the microbiota from salmon pyloric ceca and correlates with a significant shift in microbial community composition with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to augment in vivo trials of microbiome modulators.

Tele-entomology and tele-parasitology: A citizen science-based approach for surveillance and control of Chagas disease in Venezuela.

Chagas Disease (CD), a chronic infection caused by the Trypanosoma cruzi parasite, is a Neglected Tropical Disease endemic to Latin America. With a re-emergence in Venezuela during the past two decades, the spread of CD has proved susceptible to, and inhibitable by a digital, real-time surveillance system effectuated by Citizen Scientists in communities throughout the country. The #TraeTuChipo (#BringYourKissingBug) campaign implemented in January 2020, has served as such a strategy counting on community engagement to define the current ecological distribution of CD vectors despite the absence of a functional national surveillance program. This pilot campaign collected data through online surveys, social media platforms, and/or telephone text messages. A total of 79 triatomine bugs were reported from eighteen Venezuelan states; 67 bugs were identified as Panstrongylus geniculatus, 1 as Rhodnius pictipes, 1 as Triatoma dimidiata, and 10 as Triatoma maculata. We analyzed 8 triatomine feces samples spotted from 4 Panstrongylus geniculatus which were confirmed positive by qPCR for T. cruzi . Further molecular characterization of discrete typing units (DTUs), revealed that all samples contained TcI, the most highly diverse and broadly distributed strain of T. cruzi. Moreover, analysis of the mitochondrial 12S gene revealed Myotis keaysi, Homo sapiens, and Gallus gallus as the main triatomine feeding sources. This study highlights a novel Citizen Science approach which may help improve the surveillance systems for CD in endemic countries.

Genome-scale multilocus microsatellite typing of Trypanosoma cruzi discrete typing unit I reveals phylogeographic structure and specific genotypes linked to human infection.

Trypanosoma cruzi is the most important parasitic infection in Latin America and is also genetically highly diverse, with at least six discrete typing units (DTUs) reported: Tc I, IIa, IIb, IIc, IId, and IIe. However, the current six-genotype classification is likely to be a poor reflection of the total genetic diversity present in this undeniably ancient parasite. To determine whether epidemiologically important information is "hidden" at the sub-DTU level, we developed a 48-marker panel of polymorphic microsatellite loci to investigate population structure among 135 samples from across the geographic distribution of TcI. This DTU is the major cause of resurgent human disease in northern South America but also occurs in silvatic triatomine vectors and mammalian reservoir hosts throughout the continent. Based on a total dataset of 12,329 alleles, we demonstrate that silvatic TcI populations are extraordinarily genetically diverse, show spatial structuring on a continental scale, and have undergone recent biogeographic expansion into the southern United States of America. Conversely, the majority of human strains sampled are restricted to two distinct groups characterised by a considerable reduction in genetic diversity with respect to isolates from silvatic sources. In Venezuela, most human isolates showed little identity with known local silvatic strains, despite frequent invasion of the domestic setting by infected adult vectors. Multilocus linkage indices indicate predominantly clonal parasite propagation among all populations. However, excess homozygosity among silvatic strains and raised heterozygosity among domestic populations suggest that some level of genetic recombination cannot be ruled out. The epidemiological significance of these findings is discussed.

Remarkable genetic diversity of Trypanosoma cruzi and Trypanosoma rangeli in two localities of southern Ecuador identified via deep sequencing of mini-exon gene amplicons.

BACKGROUND: Trypanosoma cruzi, the causative agent of Chagas disease, and T. rangeli are kinetoplastid parasites endemic to Latin America. Although closely related to T. cruzi and capable of infecting humans, T. rangeli is non-pathogenic. Both parasite species are transmitted by triatomine bugs, and the presence of T. rangeli constitutes a confounding factor in the study of Chagas disease prevalence and transmission dynamics. Trypanosoma cruzi possesses high molecular heterogeneity: seven discrete typing units (DTUs) are currently recognized. In Ecuador, T. cruzi TcI and T. rangeli KP1(-) predominate, while other genetic lineages are seldom reported. METHODS: Infection by T. cruzi and/or T. rangeli in different developmental stages of triatomine bugs from two communities of southern Ecuador was evaluated via polymerase chain reaction product size polymorphism of kinetoplast minicircle sequences and the non-transcribed spacer region of the mini-exon gene (n = 48). Forty-three mini-exon amplicons were also deep sequenced to analyze single-nucleotide polymorphisms within single and mixed infections. Mini-exon products from ten monoclonal reference strains were included as controls. RESULTS: Trypanosoma cruzi genetic richness and diversity was not significantly greater in adult vectors than in nymphal stages III and V. In contrast, instar V individuals showed significantly higher T. rangeli richness when compared with other developmental stages. Among infected triatomines, deep sequencing revealed one T. rangeli infection (3%), 8 T. cruzi infections (23.5%) and 25 T. cruzi + T. rangeli co-infections (73.5%), suggesting that T. rangeli prevalence has been largely underestimated in the region. Furthermore, deep sequencing detected TcIV sequences in nine samples; this DTU had not previously been reported in Loja Province. CONCLUSIONS: Our data indicate that deep sequencing allows for better parasite identification/typing than amplicon size analysis alone for mixed infections containing both T. cruzi and T. rangeli, or when multiple T. cruzi DTUs are present. Additionally, our analysis showed extensive overlap among the parasite populations present in the two studied localities (c.28 km apart), suggesting active parasite dispersal over the study area. Our results highlight the value of amplicon sequencing methodologies to clarify the population dynamics of kinetoplastid parasites in endemic regions and inform control campaigns in southern Ecuador.

SARS-CoV-2 spread across the Colombian-Venezuelan border.

INTRODUCTION: Venezuela and Colombia both adopted measures of containment early in response to the COVID-19 pandemic. However, Venezuela's ongoing humanitarian crisis has decimated its health care system, and forced millions of Venezuelans to flee through its porous border with Colombia. The extensive shared border, and illegal cross-border transit through improvised trails between the two countries are major challenges for public health authorities. We report the first SARS-CoV-2 genomes from Venezuela, and present a snapshot of the SARS-CoV-2 epidemiologic landscape in the Colombian-Venezuelan border region. METHODS: We sequenced and assembled viral genomes from total RNA extracted from nasopharyngeal (NP) clinical specimens using a custom reference-based analysis pipeline. Three assemblies obtained were subjected to typing using the Phylogenetic Assignment of Named Global Outbreak LINeages 'Pangolin' tool. A total of 376 publicly available SARS-CoV-2 genomes from South America were obtained from the GISAID database to perform comparative genomic analyses. Additionally, the Wuhan-1 strain was used as reference. RESULTS: We found that two of the SARS-CoV-2 genomes from Venezuela belonged to the B1 lineage, and the third to the B.1.13 lineage. We observed a point mutation in the Spike protein gene (D614G substitution), previously reported to be associated with increased infectivity, in all three Venezuelan genomes. Additionally, three mutations (R203K/G204R substitution) were present in the nucleocapsid (N) gene of one Venezuelan genome. CONCLUSIONS: Genomic sequencing demonstrates similarity between SARS-CoV-2 lineages from Venezuela and viruses collected from patients in bordering areas in Colombia and from Brazil, consistent with cross-border transit despite administrative measures including lockdowns. The presence of mutations associated with increased infectivity in the 3 Venezuelan genomes we report and Colombian SARS-CoV-2 genomes from neighboring borders areas may pose additional challenges for control of SARS-CoV-2 spread in the complex epidemiological landscape in Latin American countries. Public health authorities should carefully follow the progress of the pandemic and its impact on displaced populations within the region.

Meiotic sex in Chagas disease parasite Trypanosoma cruzi.

Genetic exchange enables parasites to rapidly transform disease phenotypes and exploit new host populations. Trypanosoma cruzi, the parasitic agent of Chagas disease and a public health concern throughout Latin America, has for decades been presumed to exchange genetic material rarely and without classic meiotic sex. We present compelling evidence from 45 genomes sequenced from southern Ecuador that T. cruzi in fact maintains truly sexual, panmictic groups that can occur alongside others that remain highly clonal after past hybridization events. These groups with divergent reproductive strategies appear genetically isolated despite possible co-occurrence in vectors and hosts. We propose biological explanations for the fine-scale disconnectivity we observe and discuss the epidemiological consequences of flexible reproductive modes. Our study reinvigorates the hunt for the site of genetic exchange in the T. cruzi life cycle, provides tools to define the genetic determinants of parasite virulence, and reforms longstanding theory on clonality in trypanosomatid parasites.

Venezuela's humanitarian crisis, resurgence of vector-borne diseases, and implications for spillover in the region.

In the past 5-10 years, Venezuela has faced a severe economic crisis, precipitated by political instability and declining oil revenue. Public health provision has been affected particularly. In this Review, we assess the impact of Venezuela's health-care crisis on vector-borne diseases, and the spillover into neighbouring countries. Between 2000 and 2015, Venezuela witnessed a 359% increase in malaria cases, followed by a 71% increase in 2017 (411 586 cases) compared with 2016 (240 613). Neighbouring countries, such as Brazil, have reported an escalating trend of imported malaria cases from Venezuela, from 1538 in 2014 to 3129 in 2017. In Venezuela, active Chagas disease transmission has been reported, with seroprevalence in children (<10 years), estimated to be as high as 12·5% in one community tested (n=64). Dengue incidence increased by more than four times between 1990 and 2016. The estimated incidence of chikungunya during its epidemic peak is 6975 cases per 100 000 people and that of Zika virus is 2057 cases per 100 000 people. The re-emergence of many vector-borne diseases represents a public health crisis in Venezuela and has the possibility of severely undermining regional disease elimination efforts. National, regional, and global authorities must take action to address these worsening epidemics and prevent their expansion beyond Venezuelan borders.

Environmental DNA: A New Low-Cost Monitoring Tool for Pathogens in Salmonid Aquaculture.

Environmental DNA (eDNA) metabarcoding is a relatively new monitoring tool featuring in an increasing number of applications such as the facilitation of the accurate and cost effective detection of species in environmental samples. eDNA monitoring is likely to have a major impact on the ability of salmonid aquaculture industry producers and their regulators to detect the presence and abundance of pathogens and other biological threats in the surrounding environment. However, for eDNA metabarcoding to develop into a useful bio-monitoring tool it is necessary to (a) validate that sequence datasets derived from amplification of metabarcoding markers reflect the true species' identity, (b) test the sensitivity under different abundance levels and environmental noise and (c) establish a low-cost sequencing method to enable the bulk processing of field samples. In this study, we employed an elaborate experimental design whereby different combinations of five biological agents were crossed at three abundance levels and exposed to sterile pre-filtered and unfiltered seawater, prior to coarse filtering and then eDNA ultrafiltration of the resultant material. We then benchmarked the low-cost, scalable, Ion Torrent sequencing method against the current gold-standard Illumina platform for eDNA surveys in aquaculture. Based on amplicon-seq of the 18S SSU rDNA v9 region, we were able to identify two parasites (Lepeophtheirus salmonis and Paramoeba perurans) to species level, whereas the microalgae species Prymnesium parvum, Pseudo-nitzschia seriata, and P. delicatissima could be assigned correctly only to the genus level. Illumina and Ion Torrent provided near identical results in terms of community composition in our samples, whereas Ion Torrent was more sensitive in detecting species richness when the medium was unfiltered seawater. Both methods were able to reflect the difference in relative abundance between treatments in 4 out of 5 species when samples were exposed to the unfiltered seawater, despite the significant amount of background noise from both bacteria and eukaryotes. Our findings indicate that eDNA metabarcoding offers significant potential in the monitoring of species harmful to aquaculture and for this purpose, the low-cost Ion Torrent sequencing is as accurate as Illumina in determining differences in their relative abundance between samples.

Variability of Cutaneous Leishmaniasis Lesions Is Not Associated with Genetic Diversity of Leishmania tropica in Khyber Pakhtunkhwa Province of Pakistan.

Leishmania tropica is the causative agent of cutaneous leishmaniasis in Pakistan. Here, intraspecific diversity of L. tropica from northern Pakistan was investigated using multilocus microsatellite typing. Fourteen polymorphic microsatellite markers were typed in 34 recently collected L. tropica isolates from Pakistan along with 158 archival strains of diverse Afro-Eurasian origins. Previously published profiles for 145 strains of L. tropica originating from different regions of Africa, Central Asia, Iran, and Middle East were included for comparison. Six consistently well-supported genetic groups were resolved: 1) Asia, 2) Morroco A, 3) Namibia and Kenya A, 4) Kenya B/Tunisia and Galilee, 5) Morocco B, and 6) Middle East. Strains from northern Pakistan were assigned to Asian cluster except for three that were placed in a geographically distant genetic group; Morocco A. Lesion variability among these Pakistani strains was not associated with specific L. tropica genetic profile. Pakistani strains showed little genetic differentiation from strains of Iraq, Afghanistan, and Syria (FST = 0.00-0.06); displayed evidence of modest genetic flow with India (FST = 0.14). Furthermore, genetic structuring within these isolates was not geographically defined. Pak-Afghan cluster was in significant linkage disequilibrium (IA = 1.43), had low genetic diversity, and displayed comparatively higher heterozygosity (FIS = -0.62). Patterns of genetic diversity observed suggest dominance of a minimally diverse clonal lineage within northern Pakistan. This is surprising as a wide clinical spectrum was observed in patients, suggesting the importance of host and other factors. Further genotyping studies of L. tropica isolates displaying different clinical phenotypes are required to validate this potentially important observation.