Claudia Herrera

Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans.

Genetic variability in the Trypanosoma cruzi I group has recently been revealed in Colombian isolates from humans, reservoirs and vectors. Genomic rearrangements and the polymorphic regions in taxonomic markers, such as the miniexon gene, have led to the development of molecular tools to identify phylogenetic haplotypes in T. cruzi isolates. From genetic polymorphisms found in T. cruzi I isolates, they have been classified into four haplotypes according to their epidemiologic transmission cycles. Haplotype Ia is associated with domestic isolates, from Rhodnius prolixus; haplotype Ib, with the domestic and peridomestic cycle, mainly associated with Triatoma dimidiata; haplotype Ic is a poorly characterized group, which has been associated with the peridomestic cycle; and haplotype Id has been related to the sylvatic cycle. In order to demonstrate that the circulating T. cruzi I isolates in Colombia can be classified in the four proposed haplotypes, specific primers were designed on polymorphic regions of the miniexon genes intergenic sequences. This set of primers allowed the molecular characterization of 33 Colombian isolates, classifying them into three of the four proposed haplotypes (Ia, Ib and Id). Results obtained from maximum parsimony and maximum-likelihood-based phylogenetic analyses correlated with the molecular classification of the isolates and their transmission cycles. This study brings insights into the Chagas disease epidemiology and the parasites transmission dynamics.

Genetic Variability and Phylogenetic Relationships within Trypanosoma cruzi I Isolated in Colombia Based on Miniexon Gene Sequences

Phylogenetic studies of Trypanosoma cruzi have identified the existence of two groups: T. cruzi I and T. cruzi II. There are aspects that still remain unknown about the genetic variability within the T. cruzi I group. Given its epidemiological importance, it is necessary to have a better understanding of T. cruzi transmission cycles. Our purpose was to corroborate the existence of haplotypes within the T. cruzi I group and to describe the genetic variability and phylogenetic relationships, based on single nucleotide polymorphisms (SNPs) found in the miniexon gene intergenic region, for the isolates from different hosts and epidemiological transmission cycles in Colombian regions. 31 T. cruzi isolates were molecularly characterized. Phylogenetic relationships within T. cruzi I isolates showed four haplotype groups (Ia–Id), associated with their transmission cycle. In previous studies, we reported that haplotype Ia is mainly associated with the domestic cycle and domiciliated Rhodnius prolixus. Haplotype Ib is associated with the domestic cycle and peridomestic cycle, haplotype Ic is closely related with the peridomestic cycle, and haplotype Id is strongly associated with the sylvatic cycle. The phylogenetic methodologies applied in this study are tools that bolster the associations among isolates and thus shed light on Chagas disease epidemiology.

Genotype diversity of Trypanosoma cruzi in small rodents and Triatoma sanguisuga from a rural area in New Orleans, Louisiana

BackgroundChagas disease is an anthropozoonosis caused by the protozoan parasite Trypanosoma cruzi that represents a major public health problem in Latin America. Although the United States is defined as non-endemic for Chagas disease due to the rarity of human cases, the presence of T. cruzi has now been amply demonstrated as enzootic in different regions of the south of the country from Georgia to California. In southeastern Louisiana, a high T. cruzi infection rate has been demonstrated in Triatoma sanguisuga, the local vector in this area. However, little is known about the role of small mammals in the wild and peridomestic transmission cycles.MethodsThis study focused on the molecular identification and genotyping of T. cruzi in both small rodents and T. sanguisuga from a rural area of New Orleans, Louisiana. DNA extractions were prepared from rodent heart, liver, spleen and skeletal muscle tissues and from cultures established from vector feces. T. cruzi infection was determined by standard PCR using primers specific for the minicircle variable region of the kinetoplastid DNA (kDNA) and the highly repetitive genomic satellite DNA (satDNA). Genotyping of discrete typing units (DTUs) was performed by amplification of mini-exon and 18S and 24Sα rRNA genes and subsequent sequence analysis.ResultsThe DTUs TcI, TcIV and, for the first time, TcII, were identified in tissues of mice and rats naturally infected with T. cruzi captured in an area of New Orleans, close to the house where the first human case of Chagas disease was reported in Louisiana. The T. cruzi infection rate in 59 captured rodents was 76%. The frequencies of the detected DTUs in such mammals were TcI 82%, TcII 22% and TcIV 9%; 13% of all infections contained more than one DTU.ConclusionsOur results indicate a probable presence of a considerably greater diversity in T. cruzi DTUs circulating in the southeastern United States than previously reported. Understanding T. cruzi transmission dynamics in sylvatic and peridomestic cycles in mammals and insect vectors will be crucial to estimating the risk of local, vector-borne transmission of T. cruzi to humans in the United States.

Interest and limitations of Spliced Leader Intergenic Region sequences for analyzing Trypanosoma cruzi I phylogenetic diversity in the Argentinean Chaco.

Internal and geographical clustering within Trypanosoma cruzi I (TcI) has been recently revealed by using Multilocus Microsatellite Typing and sequencing of the Spliced-Leader Intergenic Region (SL-IR). In the present work, 14 isolates and 11 laboratory-cloned stocks obtained from a geographically restricted area in Chaco Province, Argentina, were analyzed by PCR and sequencing of SL-IR. We were able to differentiate 8 different genotypes that clustered into 4 groups. One of these groups was classified within the formerly described haplotype A and another one within the recently described SL-IR group E. Both were phylogenetically well-supported. In contrast, none of the stocks from the Chaco province were grouped within the cluster previously named haplotype D despite the fact that they shared a similar microsatellite motif in the SL-IR. No evidence of recombination or gene conversion within these stocks was found. On the other hand, multiple ambiguous alignments in the microsatellite region of SL-IR, affecting the tree topology and relationships among groups were detected. Finally, since there are multiple copies of the SL-IR, and they are arranged in tandem, we discuss how molecular processes affecting this kind of sequences could mislead phylogenetic inference.

Toxoplasmosis in military personnel involved in jungle operations.

Tropical diseases, mainly leishmaniasis and malaria, increased among Colombian military personnel due to intensive operations in the jungle in the last ten years; as a result the Colombian army developed important preventive strategies for malaria and leishmaniasis. However, no knowledge exists about toxoplasmosis, an emergent disease in military personnel. We compared the prevalence of IgG anti-Toxoplasma antibodies by ELISA and of parasitaemia by a real time PCR assay, in 500 professional soldiers that operated in the jungle with a group of 501 soldiers working in an urban zone (Bogotá). We found that the prevalence was significantly different between both groups of soldiers (80% in soldiers operating in jungle vs. 45% in urban soldiers, adjusted OR 11.4; CI 95%: 3.8-34; p<0.0001). All soldiers operating in the jungle drink unboiled and chlorine untreated lake or river water. In urban soldiers, these risk factors along with eating wild animal meat or eating tigrillo (little spotted cat) were significantly associated with a higher prevalence. Characteristic toxoplasmic choriorretinal lesions were found in 4 soldiers that operated in the jungle (0.8%) and in one urban soldier (0.19%). All soldiers before being deployed in jungle operations should be tested for Toxoplasma antibodies and to receive adequate health information about the routine use of personnel filters to purify their water for consumption.

Complex evolutionary pathways of the intergenic region of the mini-exon gene in Trypanosoma cruzi TcI: A possible ancient origin in the Gran Chaco and lack of strict genetic structuration

The TcI discrete typing unit (DTU) of Trypanosoma cruzi is the most abundant and widely spread in the Americas. It is found in a wide range of triatomine and mammal species, which are distributed throughout the Americas in sylvatic and domestic environments. Previous studies based on intergenic sequences of the mini-exon gene (SL-IR) have identified five genotype groups within TcI. Based in the large number of sequences available in GenBank, the present study conducted an exhaustive revision of the sequence variability of the SL-IR within TcI using 244 sequences from isolates, cellular or molecular clones, from 11 Latin American countries. First, the evolutionary branching between strains was examined by analyzing only the single nucleotide polymorphism (SNP) deleting the microsatellite region and the gaps from the total alignment. Then the variability of the microsatellite region was re-analyzed alone using principal component analysis (PCA). After haplotype reconstruction using the PHASE algorithm, because of the presence of several ambiguous nucleotides in the SNP region, a total of 131 different haplotypes were obtained. The topology reveals how difficult it is to identify an obvious structure in TcI for most of the parameters examined. Somewhat genetic and geographical structures exist, but no structure was depicted with cycle and host origins. Indeed, the long-lasting evolution with possible recombination events, the occurrence of several waves of geographical dispersions (old and recent), and the high flow of strains between sylvatic and domestic cycles partially hide the major evolutionary trends within TcI. Moreover, we identified several problems in previous analyses, and concluded that in absence of supplementary studies of TcI phylogeny with other genetic markers, it is hazardous to use only the mini-exon intergenic region as a relevant marker of the substructure within TcI.

Molecular identification and genotyping of Trypanosoma cruzi DNA in autochthonous Chagas disease patients from Texas, USA

The parasitic protozoan Trypanosoma cruzi, the causative agent of Chagas disease, is widely distributed throughout the Americas, from the southern United States (US) to northern Argentina, and infects at least 6 million people in endemic areas. Much remains unknown about the dynamics of T. cruzi transmission among mammals and triatomine vectors in sylvatic and peridomestic eco-epidemiological cycles, as well as of the risk of transmission to humans in the US. Identification of T. cruzi DTUs among locally-acquired cases is necessary for enhancing our diagnostic and clinical prognostic capacities, as well as to understand parasite transmission cycles. Blood samples from a cohort of 15 confirmed locally-acquired Chagas disease patients from Texas were used for genotyping T. cruzi. Conventional PCR using primers specific for the minicircle variable region of the kinetoplastid DNA (kDNA) and the highly repetitive genomic satellite DNA (satDNA) confirmed the presence of T. cruzi in 12/15 patients. Genotyping was based on the amplification of the intergenic region of the miniexon gene of T. cruzi and sequencing. Sequences were analyzed by BLAST and phylogenetic analysis by Maximum Likelihood method allowed the identification of non-TcI DTUs infection in six patients, which corresponded to DTUs TcII, TcV or TcVI, but not to TcIII or TcIV. Two of these six patients were also infected with a TcI DTU, indicating mixed infections in those individuals. Electrocardiographic abnormalities were seen among patients with single non-TcI and mixed infections of non-TcI and TcI DTUs. Our results indicate a greater diversity of T. cruzi DTUs circulating among autochthonous human Chagas disease cases in the southern US, including for the first time DTUs from the TcII-TcV-TcVI group. Furthermore, the DTUs infecting human patients in the US are capable of causing Chagasic cardiac disease, highlighting the importance of parasite detection in the population.

Chagas Disease Has Not Been Controlled in Ecuador.

A recent study by Cartelle Gestal et al. reported an analysis of data from the Ministry of Public Health on the epidemiological situation of neglected tropical diseases in Ecuador [1]. Based on a misleading definition of Chagas disease cases not corresponding to that of the Ministry of Public Health [2], the authors concluded that the government had mounted successful control campaigns, and as a result Chagas disease (among others) had been effectively controlled as no cases in children under age five had been reported since 2009. Ecuador is thus identified as one of the first countries to control Chagas disease. While we certainly agree that efforts have been made in terms of Chagas disease surveillance and control campaigns in Ecuador, a more comprehensive analysis of available data, from both the Ministry of Public Health and the literature, provides a very different picture, and the claim that Chagas disease is controlled made by Cartelle Gestal et al. seems largely inadequate and sends an equivocal message which can undermine current control efforts. As mentioned in this study, the Chagas disease control program in the country was formally established in 2003–2004, in response to recommendations from a technical consultation through PAHO/WHO [3] and field studies [4,5]. This consultation and data provided a baseline to prioritize activities. It reported a national seroprevalence of Trypanosoma cruzi infection of 1.38%, corresponding to 165–170,000 seropositive patients in the country. Three regions were prioritized: the coastal region (seroprevalence of 1.99%), the Amazon region (1.75%) and the southern highlands (0.65%). The incidence was estimated at 36 cases/100,000 inhabitants/year, resulting in 4,400 new cases each year [3]. Today, the most recent estimates from the WHO suggest the presence of nearly 200,000 seropositive patients and a current incidence of 14 cases/100,000 inhabitants/year [6]. An in depth analysis of the complete records from the Ministry of Public Health from 2004–2014, indicates a total of 915 reported human cases in the country, with a major increase over the years followed by a decrease in the past two years [7]. This increase reflects the efforts at improving the epidemiologic surveillance program, but it is clear that there is still significant underreporting of cases in the country. Indeed, several independent and recent seroprevalence studies in different regions and communities point out relatively high levels of seroprevalence of T. cruzi infection (ranging from 0.6 to 13.3%), and persistent active parasite transmission, as evidenced by the detection of seropositive children [8–12]. Additionally, there are reports of Chagas disease cases in regions where the Ministry of Public Health has no records of patients, further highlighting current underreporting [5,11,12]. Furthermore, while during the last decade Ecuador has achieved near 100% blood screening coverage for T. cruzi infection, the 15 participating blood banks regularly report seropositive blood donors to the External Performance Evaluation of Serological Screening Program administered by the Pontifical Catholic University of Ecuador. The vector control program was effectively started in 2004. However, due to limited human and financial resources, there have been important variations in the geographic coverage of the surveillance and control activities from year to year [7]. Importantly, a total of 12 provinces have not been included in these activities, representing an area larger than the covered provinces. Therefore, the available data do not correspond to a systematic national coverage, and thus still present an incomplete picture of the current transmission of Chagas disease in Ecuador. In the 11 provinces in which surveillance and control activities have been performed, house infestation by triatomines is still observed in many regions [7,13]. While vector control activities have had a significant effect and allowed reducing the infestation level, particularly in coastal Ecuador, these need to be sustained to avoid reinfestation and provide long-term effects. Also, while insecticide spraying may be effective against Triatoma dimidiata, a possibly domiciliated species which is poised for elimination in Ecuador, alternative control strategies may be needed against intrusive triatomine species such as Rhodnius ecuadoriensis or Panstrongylus howardi or for occasional exposure outside of homes [14–19]. Moreover, no formal vector control intervention has been implemented in the Amazon region, where nearly half of the cases of the country seem to originate [7], and active transmission still occurs through triatomine species including Rhodnius robustus and R. pictipes [8,9]. Especially in the Amazon, human activities (deforestation, urbanization) disturb the natural balance between the vectors, their wild hosts and the parasite, favoring the emergence of new transmission cycles in which humans may be included [8,9,11,20]. An accurate description of the situation of Chagas disease in Ecuador should mention that access to diagnosis throughout the country is limited and case detection during the last two decades has been sporadic and geographically restricted. Indeed, only one laboratory in the whole country, at the Instituto Nacional de Investigacion en Salud Publica (INSPI), performs official confirmation of anti-T. cruzi seropositivity and releases Nifurtimox for the treament of patients. In fact, we believe that lack of awareness by health care personnel in areas with active vectorial transmission, combined with lack of diagnostic capacity elsewhere in the country, have resulted in a gross under reporting of cases in Ecuador. Taken together, these data and studies highlight that Chagas disease is all but controlled in Ecuador, contrary to what is stated by Cartelle Gestal et al. While it is clear that disease surveillance and vector control activities from the Ministry of Public Health have improved over the years, these need to (i) reach national coverage to ensure the inclusion of all endemic provinces, and (ii) be sustained to ensure that what has been achieved can result in long-term control of the disease. These represent a clear challenge at a time when the Ministry of Public Health is undergoing major structural reorganization and many of its activities are being decentralized or interrupted. Indeed, there is a decrease in reported human cases and in vector controls activities observed in the past two years in Ecuador [7], which may reflect the interruption of the National Chagas Program and the Servicio Nacional de Control y Vigilancia de Enfermedades Transmitidas por Vectores Artropodos (SNEM) in late 2015. Their actions have not been replaced yet, so that there is currently no Chagas vector control program in the country. This can strongly jeopardize the results achieved so far and may be a lost opportunity to eliminate vectorial transmission with domiciliated vectors in some regions of Ecuador. Finally, as in many other countries in Latin America, current activities for Chagas disease control in Ecuador still need to improve treatment access and care for Chagas disease patients [21–23] as well as to better understand the importance of congenital transmission in the epidemiology of the disease [9,24]. Thus, control of Chagas disease in the country will only be reached if the programs from the Ministry of Public Health are strengthened and expanded. The National Chagas disease control programs in other Latin America countries such as Brazil, Argentina, or Colombia (among others) can provide key examples of successful strategies for Chagas disease surveillance and control, as well as of the challenges encountered for their implementation. Additionally, research needs to be performed to further expand our understanding of triatomine infestation and T. cruzi transmission cycles in the different specific endemic areas, to help further tailor surveillance and interventions. More than claiming that Chagas disease is controlled, we need to promote further political commitment to sustain current achievements in Chagas disease surveillance and control in Ecuador and to ensure that the goals of the London declaration on neglected tropical diseases [25] are met in the near future.

Striking Divergence in Toxoplasma ROP16 Nucleotide Sequences From Human and Meat Samples

BACKGROUND ROP16 is a protein kinase of Toxoplasma gondii identified in the mouse model as a virulent marker, but it is unknown whether this finding is relevant in human toxoplasmosis. METHODS We obtained the Toxoplasma ROP16 locus DNA sequence in samples from 12 patients with ocular toxoplasmosis, 1 sample from a patient with congenital toxoplasmosis, 22 samples from soldiers operating in the jungle, 2 samples from urban soldiers, and 10 samples from meat for human consumption. An enzyme-linked immunosorbent assay specific for antibodies against the ROP16 mouse-virulent peptide was performed in 46 serum specimens from patients with ocular toxoplasmosis and in 28 serum specimens from patients with chronic asymptomatic infection, of whom 19 had congenital infection and 11 had toxoplasmic lymphadenitis. RESULTS We found a striking divergence of the ROP16 nucleotide sequences. Ten of 12 sequences (83.3%) from patients with ocular toxoplasmosis clustered with those of mouse-virulent strains, whereas 7 of 7 ROP16 sequences (100%) from meat were clustered with those of mouse-avirulent strains. Only 11 of 104 serum specimens (10.5%) had specific antibodies against the mouse-virulent peptide, and there was no association between clinical forms and positive results of serological assays. CONCLUSIONS The majority of ROP16 nucleotide sequences from Colombian patients with ocular toxoplasmosis belonged to the group of mouse-virulent strains.

Validation of a Poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations.

Trypanosoma cruzi is the causative agent of American trypanosomiasis, a complex zoonotic disease that affects more than 10million people in the Americas. Strains of this parasite possess a significant amount of genetic variability and hence can be divided into at least six discrete typing units (DTUs). The life cycle of this protist suggests that multiclonal infections may emerge due to the likelihood of contact of triatomine insects with more than 100 mammal species. To date, there have been a few studies on but no consensus regarding standardised methodologies to identify multiclonal infections caused by this parasite. Hence, the aim of this study was to develop and validate a limiting dilution assay (LDA) to identify multiclonal infections in T. cruzi populations by comparing the feasibility and reliability of this method with the widely applied solid phase blood agar (SPBA) methodology. We cloned reference strains belonging to three independent genotypes (TcI, TcII, and TcIV) and mixed infections (TcI+TcII) using LDA and SPBA; the comparison was conducted by calculating the feasibility and reliability of the methods employed. Additionally, we implemented LDA in strains recently isolated from Homo sapiens, Rhodnius prolixus, Triatoma venosa, Panstrongylus geniculatus, Tamandua tetradactyla, Rattus rattus, Didelphis marsupialis and Dasypus novemcinctus, with the aim of resolving multiclonal infections using molecular characterization employing SL-IR (spliced leader intergenic region of mini-exon gene), the 24Sα rDNA gene and microsatellite loci. The results reported herein demonstrate that LDA is an optimal methodology to distinguish T. cruzi subpopulations based on microsatellite markers by showing the absence of multiple peaks within a single locus. Conversely, SPBA showed patterns of multiple peaks within a single locus suggesting multiclonal events. The biological consequences of these results and the debate between multiclonality and aneuploidy are discussed.