Trypanosoma cruzi IV Causing Outbreaks of Acute Chagas Disease and Infections by Different Haplotypes in the Western Brazilian Amazonia

Background

Chagas disease is an emergent tropical disease in the Brazilian Amazon Region, with an increasing number of cases in recent decades. In this region, the sylvatic cycle of Trypanosoma cruzi transmission, which constitutes a reservoir of parasites that might be associated with specific molecular, epidemiological and clinical traits, has been little explored. The objective of this work is to genetically characterize stocks of T. cruzi from human cases, triatomines and reservoir mammals in the State of Amazonas, in the Western Brazilian Amazon.

Methodology/Principal Findings

We analyzed 96 T. cruzi samples from four municipalities in distant locations of the State of Amazonas. Molecular characterization of isolated parasites from cultures in LIT medium or directly from vectors or whole human blood was performed by PCR of the non-transcribed spacer of the mini-exon and of the 24 S alfa ribosomal RNA gene, RFLP and sequencing of the mitochondrial cytochrome c oxidase subunit II (COII) gene, and by sequencing of the glucose-phosphate isomerase gene. The T. cruzi parasites from two outbreaks of acute disease were all typed as TcIV. One of the outbreaks was triggered by several haplotypes of the same DTU. TcIV also occurred in isolated cases and in Rhodnius robustus. Incongruence between mitochondrial and nuclear phylogenies is likely to be indicative of historical genetic exchange events resulting in mitochondrial introgression between TcIII and TcIV DTUs from Western Brazilian Amazon. TcI predominated among triatomines and was the unique DTU infecting marsupials.

Conclusion/Significance

DTU TcIV, rarely associated with human Chagas disease in other areas of the Amazon basin, is the major strain responsible for the human infections in the Western Brazilian Amazon, occurring in outbreaks as single or mixed infections by different haplotypes.     

Multilocus sequence typing (MLST) for lineage assignment and high resolution diversity studies in Trypanosoma cruzi

Background

Multilocus sequence typing (MLST) is a powerful and highly discriminatory method for analysing pathogen population structure and epidemiology. Trypanosoma cruzi, the protozoan agent of American trypanosomiasis (Chagas disease), has remarkable genetic and ecological diversity. A standardised MLST protocol that is suitable for assignment of T. cruzi isolates to genetic lineage and for higher resolution diversity studies has not been developed.

Methodology/Principal Findings

We have sequenced and diplotyped nine single copy housekeeping genes and assessed their value as part of a systematic MLST scheme for T. cruzi. A minimum panel of four MLST targets (Met-III, RB19, TcGPXII, and DHFR-TS) was shown to provide unambiguous assignment of isolates to the six known T. cruzi lineages (Discrete Typing Units, DTUs TcI-TcVI). In addition, we recommend six MLST targets (Met-II, Met-III, RB19, TcMPX, DHFR-TS, and TR) for more in depth diversity studies on the basis that diploid sequence typing (DST) with this expanded panel distinguished 38 out of 39 reference isolates. Phylogenetic analysis implies a subdivision between North and South American TcIV isolates. Single Nucleotide Polymorphism (SNP) data revealed high levels of heterozygosity among DTUs TcI, TcIII, TcIV and, for three targets, putative corresponding homozygous and heterozygous loci within DTUs TcI and TcIII. Furthermore, individual gene trees gave incongruent topologies at inter- and intra-DTU levels, inconsistent with a model of strict clonality.

Conclusions/Significance

We demonstrate the value of systematic MLST diplotyping for describing inter-DTU relationships and for higher resolution diversity studies of T. cruzi, including presence of recombination events. The high levels of heterozygosity will facilitate future population genetics analysis based on MLST haplotypes.     

Trypanosoma cruzi I and IV Stocks from Brazilian Amazon Are Divergent in Terms of Biological and Medical Properties in Mice

Background

In the Brazilian Amazon, clinical and epidemiological frameworks of Chagas disease are very dissimilar in relation to the endemic classical areas of transmission, possibly due to genetic and biological characteristics of the circulating Trypanosoma cruzi stocks. Twenty six T. cruzi stocks from Western Amazon Region attributed to the TcI and TcIV DTUs were comparatively studied in Swiss mice to test the hypothesis that T. cruzi clonal structure has a major impact on its biological and medical properties.

Methodology/Principal Findings

Seventeen parameters were assayed in mice infected with 14 T. cruzi strains belonging to DTU TcI and 11 strains typed as TcIV. In comparison with TcI, TcIV stocks promoted a significantly shorter pre-patent period (p<0.001), a longer patent period (p<0.001), higher values of mean daily parasitemia (p = 0.009) and maximum of parasitemia (p = 0.015), earlier days of maximum parasitemia (p<0.001) and mortality (p = 0.018), higher mortality rates in the acute phase (p = 0.047), higher infectivity rates (p = 0.002), higher positivity in the fresh blood examination (p<0.001), higher positivity in the ELISA at the early chronic phase (p = 0.022), and a higher positivity in the ELISA at the late chronic phase (p = 0.003). On the other hand TcI showed higher values of mortality rates in the early chronic phase (p = 0.014), higher frequency of mice with inflammatory process in any organ (p = 0.005), higher frequency of mice with tissue parasitism in any organ (p = 0.027) and a higher susceptibility to benznidazole (p = 0.002) than TcIV. Survival analysis showing the time elapsed from the day of inoculation to the beginning of the patent period was significantly shorter for TcIV strains and the death episodes triggered following the infection with TcI occurred significantly later in relation to TcIV. The notable exceptions come from positivity in the hemocultures and PCR, for which the results were similar.

Conclusion/Significance

T. cruzi stocks belonging to TcI and TcIV DTUs from Brazilian Amazon are divergent in terms of biological and medical properties in mice.     

A Genome-Wide Analysis of Genetic Diversity in Trypanosoma cruzi Intergenic Regions

Background

Trypanosoma cruzi is the causal agent of Chagas Disease. Recently, the genomes of representative strains from two major evolutionary lineages were sequenced, allowing the construction of a detailed genetic diversity map for this important parasite. However this map is focused on coding regions of the genome, leaving a vast space of regulatory regions uncharacterized in terms of their evolutionary conservation and/or divergence.

Methodology

Using data from the hybrid CL Brener and Sylvio X10 genomes (from the TcVI and TcI Discrete Typing Units, respectively), we identified intergenic regions that share a common evolutionary ancestry, and are present in both CL Brener haplotypes (TcII-like and TcIII-like) and in the TcI genome; as well as intergenic regions that were conserved in only two of the three genomes/haplotypes analyzed. The genetic diversity in these regions was characterized in terms of the accumulation of indels and nucleotide changes.

Principal Findings

Based on this analysis we have identified i) a core of highly conserved intergenic regions, which remained essentially unchanged in independently evolving lineages; ii) intergenic regions that show high diversity in spite of still retaining their corresponding upstream and downstream coding sequences; iii) a number of defined sequence motifs that are shared by a number of unrelated intergenic regions. A fraction of indels explains the diversification of some intergenic regions by the expansion/contraction of microsatellite-like repeats.     

Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients

Chagas disease caused by Trypanosoma cruzi is a complex disease that is endemic and an important problem in public health in Latin America. The T. cruzi parasite is classified into six discrete taxonomic units (DTUs) based on the recently proposed nomenclature (TcI, TcII, TcIII, TcIV, TcV and TcVI). The discovery of genetic variability within TcI showed the presence of five genotypes (Ia, Ib, Ic, Id and Ie) related to the transmission cycle of Chagas disease. In Colombia, TcI is more prevalent but TcII has also been reported, as has mixed infection by both TcI and TcII in the same Chagasic patient. The objectives of this study were to determine the T. cruzi DTUs that are circulating in Colombian chronic Chagasic patients and to obtain more information about the molecular epidemiology of Chagas disease in Colombia. We also assessed the presence of electrocardiographic, radiologic and echocardiographic abnormalities with the purpose of correlating T. cruzi genetic variability and cardiac disease. Molecular characterization was performed in Colombian adult chronic Chagasic patients based on the intergenic region of the mini-exon gene, the 24Sα and 18S regions of rDNA and the variable region of satellite DNA, whereby the presence of T.cruzi I, II, III and IV was detected. In our population, mixed infections also occurred, with TcI-TcII, TcI-TcIII and TcI-TcIV, as well as the existence of the TcI genotypes showing the presence of genotypes Ia and Id. Patients infected with TcI demonstrated a higher prevalence of cardiac alterations than those infected with TcII. These results corroborate the predominance of TcI in Colombia and show the first report of TcIII and TcIV in Colombian Chagasic patients. Findings also indicate that Chagas cardiomyopathy manifestations are more correlated with TcI than with TcII in Colombia.     

Genetic Diversity of Giardia duodenalis: Multilocus Genotyping Reveals Zoonotic Potential between Clinical and Environmental Sources in a Metropolitan Region of Brazil

Background

Giardia duodenalis is a flagellate protozoan that parasitizes humans and several other mammals. Protozoan contamination has been regularly documented at important environmental sites, although most of these studies were performed at the species level. There is a lack of studies that correlate environmental contamination and clinical infections in the same region. The aim of this study is to evaluate the genetic diversity of a set of clinical and environmental samples and to use the obtained data to characterize the genetic profile of the distribution of G. duodenalis and the potential for zoonotic transmission in a metropolitan region of Brazil.

Methodology/Principal Findings

The genetic assemblages and subtypes of G. duodenalis isolates obtained from hospitals, a veterinary clinic, a day-care center and important environmental sites were determined via multilocus sequence-based genotyping using three unlinked gene loci. Cysts of Giardia were detected at all of the environmental sites. Mixed assemblages were detected in 25% of the total samples, and an elevated number of haplotypes was identified. The main haplotypes were shared among the groups, and new subtypes were identified at all loci. Ten multilocus genotypes were identified: 7 for assemblage A and 3 for assemblage B.

Conclusions/Significance

There is persistent G. duodenalis contamination at important environmental sites in the city. The identified mixed assemblages likely represent mixed infections, suggesting high endemicity of Giardia in these hosts. Most Giardia isolates obtained in this study displayed zoonotic potential. The high degree of genetic diversity in the isolates obtained from both clinical and environmental samples suggests that multiple sources of infection are likely responsible for the detected contamination events. The finding that many multilocus genotypes (MLGs) and haplotypes are shared by different groups suggests that these sources of infection may be related and indicates that there is a notable risk of human infection caused by Giardia in this region.     

Retrospective distribution of Trypanosoma cruzi I genotypes in Colombia

Trypanosoma cruzi is the aetiological agent of Chagas disease, which affects approximately eight million people in the Americas. This parasite exhibits genetic variability, with at least six discrete typing units broadly distributed in the American continent. T. cruzi I (TcI) shows remarkable genetic diversity; a genotype linked to human infections and a domestic cycle of transmission have recently been identified, hence, this strain was named TcIDom. The aim of this work was to describe the spatiotemporal distribution of TcI subpopulations across humans, insect vectors and mammalian reservoirs in Colombia by means of molecular typing targeting the spliced leader intergenic region of mini-exon gene. We analysed 101 TcI isolates and observed a distribution of sylvatic TcI in 70% and TcIDom in 30%. In humans, the ratio was sylvatic TcI in 60% and TcIDom in 40%. In mammal reservoirs, the distribution corresponded to sylvatic TcI in 96% and TcIDom in 4%. Among insect vectors, sylvatic TcI was observed in 48% and TcIDom in 52%. In conclusion, the circulation of TcIDom is emerging in Colombia and this genotype is still adapting to the domestic cycle of transmission. The epidemiological and clinical implications of these findings are discussed herein.     

Sex, subdivision, and domestic dispersal of Trypanosoma cruzi lineage I in southern Ecuador

Background

Molecular epidemiology at the community level has an important guiding role in zoonotic disease control programmes where genetic markers are suitably variable to unravel the dynamics of local transmission. We evaluated the molecular diversity of Trypanosoma cruzi, the etiological agent of Chagas disease, in southern Ecuador (Loja Province). This kinetoplastid parasite has traditionally been a paradigm for clonal population structure in pathogenic organisms. However, the presence of naturally occurring hybrids, mitochondrial introgression, and evidence of genetic exchange in the laboratory question this dogma.

Methodology/Principal Findings

Eighty-one parasite isolates from domiciliary, peridomiciliary, and sylvatic triatomines and mammals were genotyped across 10 variable microsatellite loci. Two discrete parasite populations were defined: one predominantly composed of isolates from domestic and peridomestic foci, and another predominantly composed of isolates from sylvatic foci. Spatial genetic variation was absent from the former, suggesting rapid parasite dispersal across our study area. Furthermore, linkage equilibrium between loci, Hardy-Weinberg allele frequencies at individual loci, and a lack of repeated genotypes are indicative of frequent genetic exchange among individuals in the domestic/peridomestic population.

Conclusions/Significance

These data represent novel population-level evidence of an extant capacity for sex among natural cycles of T. cruzi transmission. As such they have dramatic implications for our understanding of the fundamental genetics of this parasite. Our data also elucidate local disease transmission, whereby passive anthropogenic domestic mammal and triatomine dispersal across our study area is likely to account for the rapid domestic/peridomestic spread of the parasite. Finally we discuss how this, and the observed subdivision between sympatric sylvatic and domestic/peridomestic foci, can inform efforts at Chagas disease control in Ecuador.     

Hidden Sylvatic Foci of the Main Vector of Chagas Disease Triatoma infestans: Threats to the Vector Elimination Campaign?

Background

Establishing the sources of reinfestation after residual insecticide spraying is crucial for vector elimination programs. Triatoma infestans, traditionally considered to be limited to domestic or peridomestic (abbreviated as D/PD) habitats throughout most of its range, is the target of an elimination program that has achieved limited success in the Gran Chaco region in South America.

Methodology/Principal Findings

During a two-year period we conducted semi-annual searches for triatomine bugs in every D/PD site and surrounding sylvatic habitats after full-coverage spraying of pyrethroid insecticides of all houses in a well-defined rural area in northwestern Argentina. We found six low-density sylvatic foci with 24 T. infestans in fallen or standing trees located 110–2,300 m from the nearest house or infested D/PD site detected after insecticide spraying, when house infestations were rare. Analysis of two mitochondrial gene fragments of 20 sylvatic specimens confirmed their species identity as T. infestans and showed that their composite haplotypes were the same as or closely related to D/PD haplotypes. Population studies with 10 polymorphic microsatellite loci and wing geometric morphometry consistently indicated the occurrence of unrestricted gene flow between local D/PD and sylvatic populations. Mitochondrial DNA and microsatellite sibship analyses in the most abundant sylvatic colony revealed descendents from five different females. Spatial analysis showed a significant association between two sylvatic foci and the nearest D/PD bug population found before insecticide spraying.

Conclusions

Our study shows that, despite of its high degree of domesticity, T. infestans has sylvatic colonies with normal chromatic characters (not melanic morphs) highly connected to D/PD conspecifics in the Argentinean Chaco. Sylvatic habitats may provide a transient or permanent refuge after control interventions, and function as sources for D/PD reinfestation. The occurrence of sylvatic foci of T. infestans in the Gran Chaco may pose additional threats to ongoing vector elimination efforts.     

Helicobacter pylori from Gastric Cancer and Duodenal Ulcer Show Same Phylogeographic Origin in the Andean Region in Colombia

Background

A recent report has shown that the phylogenetic origin of Helicobacter pylori based on multi-locus sequence typing (MLST) was significantly associated with the severity of gastritis in Colombia. However, the potential relationship between phylogenetic origin and clinical outcomes was not examined in that study. If the phylogenetic origin rather than virulence factors were truly associated with clinical outcomes, identifying a population at high risk for gastric cancer in Colombia would be relatively straightforward. In this study, we examined the phylogenetic origins of strains from gastric cancer and duodenal ulcer patients living in Bogota, Colombia.

Methods

We included 35 gastric cancer patients and 31 duodenal ulcer patients, which are considered the variant outcomes. The genotypes of cagA and vacA were determined by polymerase chain reaction. The genealogy of these Colombian strains was analyzed by MLST. Bacterial population structure was analyzed using STRUCTURE software.

Results

H. pylori strains from gastric cancer and duodenal ulcer patients were scattered in the phylogenetic tree; thus, we did not detect any difference in phylogenetic distribution between gastric cancer and duodenal ulcer strains in the hpEurope group in Colombia. Sixty-six strains, with one exception, were classified as hpEurope irrespective of the cagA and vacA genotypes, and type of disease. STRUCTURE analysis revealed that Colombian hpEurope strains have a phylogenetic connection to Spanish strains.

Conclusions

Our study showed that a phylogeographic origin determined by MLST was insufficient for distinguishing between gastric cancer and duodenal ulcer risk among hpEurope strains in the Andean region in Colombia. Our analysis also suggests that hpEurope strains in Colombia were primarily introduced by Spanish immigrants.     

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).

Methods/Principal Findings

The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.

Conclusions/Significance

Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.     

Spatial genetic structure reflects extensive clonality,low genotypic diversity and habitat fragmentation in Grevillea renwickiana (Proteaceae), a rare,sterile shrub from south-eastern Australia

Background and Aims

The association of clonality, polyploidy and reduced fecundity has been identified as an extinction risk for clonal plants. Compromised sexual reproduction limits both their ability to adapt to new conditions and their capacity to disperse to more favourable environments. Grevillea renwickiana is a prostrate, putatively sterile shrub reliant on asexual reproduction. Dispersal is most likely limited by the rate of clonal expansion via rhizomes. The nine localized populations constituting this species provide an opportunity to examine the extent of clonality and spatial genotypic diversity to evaluate its evolutionary prospects.

Methods

Ten microsatellite loci were used to compare genetic and genotypic diversity across all sites with more intensive sampling at four locations (n = 185). The spatial distribution of genotypes and chloroplast DNA haplotypes based on the trnQ–rps16 intergenic spacer region were compared. Chromosome counts provided a basis for examining genetic profiles inconsistent with diploidy.

Key Results

Microsatellite analysis identified 46 multilocus genotypes (MLGs) in eight multilocus clonal lineages (MLLs). MLLs are not shared among sites, with two exceptions. Spatial autocorrelation was significant to 1·6 km. Genotypic richness ranged from 0 to 0·33. Somatic mutation is likely to contribute to minor variation between MLGs within clonal lineages. The eight chloroplast haplotypes identified were correlated with eight MLLs defined by ordination and generally restricted to single populations. Triploidy is the most likely reason for tri-allelic patterns.

Conclusions

Grevillea renwickiana comprises few genetic individuals. Sterility has most likely been induced by triploidy. Extensive lateral suckering in long-lived sterile clones facilitates the accumulation of somatic mutations, which contribute to the measured genetic diversity. Genetic conservation value may not be a function of population size. Despite facing evolutionary stagnation, sterile clonal species can play a vital role in mitigating ecological instability as floras respond to rapid environmental change.     

Estimating the extent of vaccine-derived poliovirus infection

Background

Eight outbreaks of paralytic polio attributable to circulating vaccine-derived poliovirus (cVDPV) have highlighted the risks associated with oral poliovirus vaccine (OPV) use in areas of low vaccination coverage and poor hygiene. As the Polio Eradication Initiative enters its final stages, it is important to consider the extent to which these viruses spread under different conditions, so that appropriate strategies can be devised to prevent or respond to future cVDPV outbreaks.

Methods and Findings

This paper examines epidemiological (temporal, geographic, age, vaccine history, social group, ascertainment), and virological (type, genetic diversity, virulence) parameters in order to infer the numbers of individuals likely to have been infected in each of these cVDPV outbreaks, and in association with single acute flaccid paralysis (AFP) cases attributable to VDPVs. Although only 114 virologically-confirmed paralytic cases were identified in the eight cVDPV outbreaks, it is likely that a minimum of hundreds of thousands, and more likely several million individuals were infected during these events, and that many thousands more have been infected by VDPV lineages within outbreaks which have escaped detection.

Conclusions

Our estimates of the extent of cVDPV circulation suggest widespread transmission in some countries, as might be expected from endemic wild poliovirus transmission in these same settings. These methods for inferring extent of infection will be useful in the context of identifying future surveillance needs, planning for OPV cessation and preparing outbreak response plans.     

Multilocus genotyping of human Giardia isolates suggests limited zoonotic transmission and association between assemblage B and flatulence in children

Background

Giardia intestinalis is one of the most common diarrhea-related parasites in humans, where infection ranges from asymptomatic to acute or chronic disease. G. intestinalis consists of eight genetically distinct genotypes or assemblages, designated A–H, and assemblages A and B can infect humans. Giardiasis has been classified as a possible zoonotic disease but the role of animals in human disease transmission still needs to be proven. We tried to link different assemblages and sub-assemblages of G. intestinalis isolates from Swedish human patients to clinical symptoms and zoonotic transmission.

Methodology/Principal Findings

Multilocus sequence-based genotyping of 207 human Giardia isolates using three gene loci: ß-giardin, glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) was combined with assemblage-specific tpi PCRs. This analysis identified 73 patients infected with assemblage A, 128 with assemblage B, and six with mixed assemblages A+B. Multilocus genotypes (MLGs) were easily determined for the assemblage A isolates, and most patients with this genotype had apparently been infected through anthroponotic transmission. However, we also found evidence of limited zoonotic transmission of Giardia in Sweden, since a few domestic human infections involved the same assemblage A MLGs previously reported in Swedish cats and ruminants. Assemblage B was detected more frequently than assemblage A and it was also more common in patients with suspected treatment failure. However, a large genetic variability made determination of assemblage B MLGs problematic. Correlation between symptoms and assemblages was found only for flatulence, which was significantly more common in children less than six years of age infected with assemblage B.

Conclusions/Significance

This study shows that certain assemblage A subtypes are potentially zoonotic and that flatulence is connected to assemblage B infections in young children. Determination of MLGs from assemblages A and B can be a valuable tool in outbreak situations and to help identify possible zoonotic transmission.     

Dynamics of Sylvatic Chagas Disease Vectors in Coastal Ecuador Is Driven by Changes in Land Cover

Background

Chagas disease is a serious public health problem in Latin America where about ten million individuals show Trypanosoma cruzi infection. Despite significant success in controlling domiciliated triatomines, sylvatic populations frequently infest houses after insecticide treatment which hampers long term control prospects in vast geographical areas where vectorial transmission is endemic. As a key issue, the spatio-temporal dynamics of sylvatic populations is likely influenced by landscape yet evidence showing this effect is rare. The aim of this work is to examine the role of land cover changes in sylvatic triatomine ecology, based on an exhaustive field survey of pathogens, vectors, hosts, and microhabitat characteristics'' dynamics.

Methodology and Principal Findings

The study was performed in agricultural landscapes of coastal Ecuador as a study model. Over one year, a spatially-randomized sampling design (490 collection points) allowed quantifying triatomine densities in natural, cultivated and domestic habitats. We also assessed infection of the bugs with trypanosomes, documented their microhabitats and potential hosts, and recorded changes in landscape characteristics. In total we collected 886 individuals, mainly represented by nymphal stages of one triatomine species Rhodnius ecuadoriensis. As main results, we found that 1) sylvatic triatomines had very high T. cruzi infection rates (71%) and 2) densities of T. cruzi-infected sylvatic triatomines varied predictably over time due to changes in land cover and occurrence of associated rodent hosts.

Conclusion

We propose a framework for identifying the factors affecting the yearly distribution of sylvatic T. cruzi vectors. Beyond providing key basic information for the control of human habitat colonization by sylvatic vector populations, our framework highlights the importance of both environmental and sociological factors in shaping the spatio-temporal population dynamics of triatomines. A better understanding of the dynamics of such socio-ecological systems is a crucial, yet poorly considered, issue for the long-term control of Chagas disease.     

Phylodynamics and Dispersal of HRSV Entails Its Permanence in the General Population in between Yearly Outbreaks in Children

Background

Human respiratory syncytial virus (HRSV) is one of the major etiologic agents of respiratory tract infections among children worldwide.

Methodology/Principal Findings

Here through a comprehensive analysis of the two major HRSV groups A and B (n = 1983) which comprise of several genotypes, we present a complex pattern of population dynamics of HRSV over a time period of 50 years (1956–2006). Circulation pattern of HRSV revealed a series of expansions and fluctuations of co-circulating lineages with a predominance of HRSVA. Positively selected amino acid substitutions of the G glycoprotein occurred upon population growth of GB3 with a 60-nucleotide insertion (GB3 Insert), while other genotypes acquired substitutions upon both population growth and decrease, thus possibly reflecting a role for immune selected epitopes in linkage to the traced substitution sites that may have important relevance for vaccine design. Analysis evidenced the co-circulation and predominance of distinct HRSV genotypes in Brazil and suggested a year-round presence of the virus. In Brazil, GA2 and GA5 were the main culprits of HRSV outbreaks until recently, when the GB3 Insert became highly prevalent. Using Bayesian methods, we determined the dispersal patterns of genotypes through several inferred migratory routes.

Conclusions/Significance

Genotypes spread across continents and between neighboring areas. Crucially, genotypes also remained at any given region for extended periods, independent of seasonal outbreaks possibly maintained by re-infecting the general population.     

Epidemiology,Risk Factors and Genotypes of HBV in HIV-Infected Patients in the Northeast Region of Colombia: High Prevalence of Occult Hepatitis B and F3 Subgenotype Dominance

Introduction

Chronic hepatitis B virus (HBV) infection is an increasing cause of morbidity and mortality in human immunodeficiency virus (HIV)-infected individuals. HIV-positive patients are commonly co-infected with HBV due to shared routes of transmission.

Objectives

Our aim was to determine the risk factors, prevalence, genotypes, and mutations of the Surface S gene of HBV, and occult hepatitis B infection (OBI) among patients infected with HIV in a northeastern Colombian city.

Methods

A cross-sectional study was conducted with 275 HIV-positive patients attending an outpatient clinic in Bucaramanga, Colombia during 2009–2010. Blood samples were collected and screened for serological markers of HBV (anti-HBs, anti-HBc and HBsAg) through ELISA assay. Regardless of their serological profile, all samples were tested for the HBV S gene by nested-PCR and HBV genotypes were determined by phylogenetic inference. Clinical records were used to examine demographic, clinical, virological, immunological and antiretroviral therapy (ART) variables of HIV infection.

Results

Participants were on average 37±11 years old and 65.1% male. The prevalence of HIV-HBV coinfection was 12% (95%CI 8.4–16.4) of which 3.3% had active HBV infection and 8.7% OBI. The prevalence of HIV-HBV coinfection was associated with AIDS stage and ART treatment. Sequence analysis identified genotype F, subgenotype F3 in 93.8% of patients and genotype A in 6.2% of patients. A C149R mutation, which may have resulted from failure in HBsAg detection, was found in one patient with OBI.

Conclusions

The present study found a high prevalence of HIV-HBV coinfection with an incidence of OBI 2.6-fold higher compared to active HBV infection. These findings suggest including HBV DNA testing to detect OBI in addition to screening for HBV serological markers in HIV patients.     

Detection of recombination events,haplotype reconstruction and imputation of sires using half-sib SNP genotypes

Background

Identifying recombination events and the chromosomal segments that constitute a gamete is useful for a number of applications in genomic analyses. In livestock, genotypic data are commonly available for half-sib families. We propose a straightforward but computationally efficient method to use single nucleotide polymorphism marker genotypes on half-sibs to reconstruct the recombination and segregation events that occurred during meiosis in a sire to form the haplotypes observed in its offspring. These meiosis events determine a block structure in paternal haplotypes of the progeny and this can be used to phase the genotypes of individuals in single half-sib families, to impute haplotypes of the sire if they are not genotyped or to impute the paternal strand of the offspring’s sequence based on sequence data of the sire.

Methods

The hsphase algorithm exploits information from opposing homozygotes among half-sibs to identify recombination events, and the chromosomal regions from the paternal and maternal strands of the sire (blocks) that were inherited by its progeny. This information is then used to impute the sire’s genotype, which, in turn, is used to phase the half-sib family. Accuracy (defined as R²) and performance of this approach were evaluated by using simulated and real datasets. Phasing results for the half-sibs were benchmarked to other commonly used phasing programs – AlphaPhase, BEAGLE and PedPhase 3.

Results

Using a simulated dataset with 20 markers per cM, and for a half-sib family size of 4 and 40, the accuracy of block detection, was 0.58 and 0.96, respectively. The accuracy of inferring sire genotypes was 0.75 and 1.00 and the accuracy of phasing was around 0.97, respectively. hsphase was more robust to genotyping errors than PedPhase 3, AlphaPhase and BEAGLE. Computationally, hsphase was much faster than AlphaPhase and BEAGLE.

Conclusions

In half-sib families of size 8 and above, hsphase can accurately detect block structure of paternal haplotypes, impute genotypes of ungenotyped sires and reconstruct haplotypes in progeny. The method is much faster and more accurate than other widely used population-based phasing programs. A program implementing the method is freely available as an R package (hsphase).     

Multiple mitochondrial introgression events and heteroplasmy in trypanosoma cruzi revealed by maxicircle MLST and next generation sequencing

Background

Mitochondrial DNA is a valuable taxonomic marker due to its relatively fast rate of evolution. In Trypanosoma cruzi, the causative agent of Chagas disease, the mitochondrial genome has a unique structural organization consisting of 20–50 maxicircles (∼20 kb) and thousands of minicircles (0.5–10 kb). T. cruzi is an early diverging protist displaying remarkable genetic heterogeneity and is recognized as a complex of six discrete typing units (DTUs). The majority of infected humans are asymptomatic for life while 30–35% develop potentially fatal cardiac and/or digestive syndromes. However, the relationship between specific clinical outcomes and T. cruzi genotype remains elusive. The availability of whole genome sequences has driven advances in high resolution genotyping techniques and re-invigorated interest in exploring the diversity present within the various DTUs.

Methodology/Principal Findings

To describe intra-DTU diversity, we developed a highly resolutive maxicircle multilocus sequence typing (mtMLST) scheme based on ten gene fragments. A panel of 32 TcI isolates was genotyped using the mtMLST scheme, GPI, mini-exon and 25 microsatellite loci. Comparison of nuclear and mitochondrial data revealed clearly incongruent phylogenetic histories among different geographical populations as well as major DTUs. In parallel, we exploited read depth data, generated by Illumina sequencing of the maxicircle genome from the TcI reference strain Sylvio X10/1, to provide the first evidence of mitochondrial heteroplasmy (heterogeneous mitochondrial genomes in an individual cell) in T. cruzi.

Conclusions/Significance

mtMLST provides a powerful approach to genotyping at the sub-DTU level. This strategy will facilitate attempts to resolve phenotypic variation in T. cruzi and to address epidemiologically important hypotheses in conjunction with intensive spatio-temporal sampling. The observations of both general and specific incidences of nuclear-mitochondrial phylogenetic incongruence indicate that genetic recombination is geographically widespread and continues to influence the natural population structure of TcI, a conclusion which challenges the traditional paradigm of clonality in T. cruzi.     

<Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> Transmission Among Captive Nonhuman Primates,Wildlife, and Vectors

Natural infection of captive nonhuman primates (NHPs) with Trypanosoma cruzi (agent of Chagas disease) is an increasingly recognized problem in facilities across the southern USA, with negative consequences for NHP health and biomedical research. We explored a central Texas NHP facility as a nidus of transmission by characterizing parasite discrete typing units (DTU) in seropositive rhesus macaques (Macaca mulatta), identifying the wildlife reservoirs, and characterizing vector infection. In seropositive NHPs, we documented low and intermittent concentrations of circulating T. cruzi DNA, with two DTUs in equal proportions, TcI and TcIV. In contrast, consistently high concentrations of T. cruzi DNA were found in wild mesomammals at the facility, yet rodents were PCR-negative. Strong wildlife host associations were found in which raccoons (Procyon lotor) harbored TcIV and opossums (Didelphis virginiana) harbored TcI, while skunks (Mephitis mephitis) were infected with both DTUs. Active and passive vector surveillance yielded three species of triatomines from the facility and in proximity to the NHP enclosures, with 17% T. cruzi infection prevalence. Interventions to protect NHP and human health must focus on interrupting spillover from the robust sylvatic transmission in the surrounding environment.