Identification of highly conserved Trypanosoma cruzi antigens for the development of a universal serological diagnostic assay.

Chagas Disease is an important neglected tropical disease caused by Trypanosoma cruzi. There is no gold standard for diagnosis and commercial serological tests perform poorly in certain locations. By aligning T. cruzi genomes covering parasite genetic and geographic diversity, we identified highly conserved proteins that could serve as universal antigens for improved diagnosis. Their antigenicity was tested in high-density peptide microarrays using well-characterized plasma samples, including samples presenting true infections but discordant serology. Individual and combination of epitopes were also evaluated in peptide-ELISAs. We identified >1400 highly conserved T. cruzi proteins evaluated in microarrays. Remarkably, T. cruzi positive controls had a different epitope recognition profile compared to serologically discordant samples. In particular, multiple T. cruzi antigens used in current tests and their strain-variants, and novel epitopes thought to be broadly antigenic failed to be recognized by discordant samples. Nonetheless, >2000 epitopes specifically recognized by IgGs from both positive controls and discordant samples were identified. Evaluation of selected peptides in ELISA further illustrated the extensive variation in antibody profiles among subjects and a peptide combination could outperform a commercial ELISA, increasing assay sensitivity from 52.3% to 72.7%. Individual variation in antibody profiles rather than T. cruzi diversity appears to be the main factor driving differences in serological diagnostic performance according to geography, which will be important to further elucidate. ELISA with a combination of peptides recognized by a greater number of individuals could better capture infections, and further development may lead to an optimal antigen mixture for a universal diagnostic assay.

Genetic diversity of Trypanosoma cruzi infecting raccoons (Procyon lotor) in 2 metropolitan areas of southern Louisiana: implications for parasite transmission networks.

Trypanosoma cruzi, the aetiological agent of Chagas disease, exists as an anthropozoonosis in Louisiana. Raccoons are an important reservoir, as they demonstrate high prevalence and maintain high parasitaemia longer than other mammals. Given the complex nature of parasite transmission networks and importance of raccoons as reservoirs that move between sylvatic and domestic environments, detailing the genetic diversity of T. cruzi in raccoons is crucial to assess risk to human health. Using a next-generation sequencing approach targeting the mini-exon, parasite diversity was assessed in 2 metropolitan areas of Louisiana. Sequences were analysed along with those previously identified in other mammals and vectors to determine if any association exists between ecoregion and parasite diversity. Parasites were identified from discrete typing units (DTUs) TcI, TcII, TcIV, TcV and TcVI. DTUs TcII, TcV and TcVI are previously unreported in raccoons in the United States (US). TcI was the most abundant DTU, comprising nearly 80% of all sequences. All but 1 raccoon harboured multiple haplotypes, some demonstrating mixed infections of different DTUs. Furthermore, there is significant association between DTU distribution and level III ecoregion in Louisiana. Finally, while certain sequences were distributed across multiple tissues, others appeared to have tissue-specific tropism. Taken together, these findings indicate that ongoing surveillance of T. cruzi in the US should be undertaken across ecoregions to fully assess risk to human health. Given potential connections between parasite diversity and clinical outcomes, deep sequencing technologies are crucial and interventions targeting raccoons may prove useful in mitigating human health risk.

Locally Transmitted Trypanosoma cruzi in a Domestic Llama (Lama glama) in a Rural Area of Greater New Orleans, Louisiana, USA.

Trypanosoma cruzi-associated megaesophagus was diagnosed in a domestic Louisiana-born llama with no significant travel history. The llama resided in the same rural area of greater New Orleans, Louisiana, where the first human autochthonous case of Chagas disease was identified in the state. Venous blood from the llama tested positive for T. cruzi kinetoplastid DNA by conventional PCR. The cardiac evaluation was unremarkable, while thoracic radiographs revealed generalized megaesophagus. The llama received supportive care, but was ultimately humanely euthanized. The esophagus was severely distended throughout its length on necropsy, and histologic evaluation showed no microscopic changes in esophageal tissue and minimal to mild lymphoplasmacytic inflammation in cardiac tissue. T. cruzi DNA was detected by conventional PCR in the esophagus, small intestine, and blood despite no protozoan organisms being observed in multiple tissue sections examined. This report contributes to the growing body of evidence of local transmission of T. cruzi in the southern United States, and Chagas disease should be considered a differential diagnosis when evaluating llamas and other large animal species for esophageal dysfunction. There is little research describing megaesophagus or Chagas disease in llamas, and this report aims to increase awareness about this zoonotic disease that is becoming more frequently reported in the southern United States.

Assessing Trypanosoma cruzi Parasite Diversity through Comparative Genomics: Implications for Disease Epidemiology and Diagnostics.

Chagas disease is an important vector-borne neglected tropical disease that causes great health and economic losses. The etiological agent, Trypanosoma cruzi, is a protozoan parasite endemic to the Americas, comprised by important diversity, which has been suggested to contribute to poor serological diagnostic performance. Current nomenclature describes seven discrete typing units (DTUs), or lineages. We performed the first large scale analysis of T. cruzi diversity among 52 previously published genomes from strains covering multiple countries and parasite DTUs and assessed how different markers summarize this genetic diversity. We also examined how seven antigens currently used in commercial serologic tests are conserved across this diversity of strains. DTU structuration was confirmed at the whole-genome level, with evidence of sub-DTU diversity, associated in part to geographic structuring. We observed very comparable phylogenetic tree topographies for most of the 32 markers investigated, with clear clustering of sequences by DTU, and a few of these markers suggested some degree of intra-lineage diversity. At least three of the currently used antigens represent poorly conserved sequences, with sequences used in tests quite divergent from sequences in many strains. Most markers are well suited for estimating parasite diversity to DTU level, and a few are particularly well-suited to assess intra-DTU diversity. Analysis of antigen sequences across all strains indicates that antigenic diversity is a likely explanation for limited diagnostic performance in Central and North America.

Genetic diversity of Trypanosoma cruzi parasites infecting dogs in southern Louisiana sheds light on parasite transmission cycles and serological diagnostic performance.

BACKGROUND: Chagas disease is a neglected zoonosis of growing concern in the southern US, caused by the parasite Trypanosoma cruzi. We genotyped parasites in a large cohort of PCR positive dogs to shed light on parasite transmission cycles and assess potential relationships between parasite diversity and serological test performance. METHODOLOGY/PRINCIPAL FINDINGS: We used a metabarcoding approach based on deep sequencing of T. cruzi mini-exon marker to assess parasite diversity. Phylogenetic analysis of 178 sequences from 40 dogs confirmed the presence of T. cruzi discrete typing unit (DTU) TcI and TcIV, as well as TcII, TcV and TcVI for the first time in US dogs. Infections with multiple DTUs occurred in 38% of the dogs. These data indicate a greater genetic diversity of T. cruzi than previously detected in the US. Comparison of T. cruzi sequence diversity indicated that highly similar T. cruzi strains from these DTUs circulate in hosts and vectors in Louisiana, indicating that they are involved in a shared T. cruzi parasite transmission cycle. However, TcIV and TcV were sampled more frequently in vectors, while TcII and TcVI were sampled more frequently in dogs. CONCLUSIONS/SIGNIFICANCE: These observations point to ecological host-fitting being a dominant mechanism involved in the diversification of T. cruzi-host associations. Dogs with negative, discordant or confirmed positive T. cruzi serology harbored TcI parasites with different mini-exon sequences, which strongly supports the hypothesis that parasite genetic diversity is a key factor affecting serological test performance. Thus, the identification of conserved parasite antigens should be a high priority for the improvement of current serological tests.

Interactions among Triatoma sanguisuga blood feeding sources, gut microbiota and Trypanosoma cruzi diversity in southern Louisiana.

Integrating how biodiversity and infectious disease dynamics are linked at multiple levels and scales is highly challenging. Chagas disease is a vector-borne disease, with specificities of the triatomine vectors and Trypanosoma cruzi parasite life histories resulting in a complex multihost and multistrain life cycle. Here, we tested the hypothesis that T. cruzi transmission cycles are shaped by triatomine host communities and gut microbiota composition by comparing the integrated interactions of Triatoma sanguisuga in southern Louisiana with feeding hosts, T. cruzi parasite and bacterial microbiota in two habitats. Bugs were collected from resident's houses and animal shelters and analysed for genetic structure, blood feeding sources, T. cruzi parasites, and bacterial diversity by PCR amplification of specific DNA markers followed by next-generation sequencing, in an integrative metabarcoding approach. T. sanguisuga feeding host communities appeared opportunistic and defined by host abundance in each habitat, yielding distinct parasite transmission networks among hosts. The circulation of a large diversity of T. cruzi DTUs was also detected, with TcII and TcV detected for the first time in triatomines in the US. The bacterial microbiota was highly diverse and varied significantly according to the DTU infecting the bugs, indicating specific interactions among them in the gut. Expanding such studies to multiple habitats and additional triatomine species would be key to further refine our understanding of the complex life cycles of multihost, multistrain parasites such as T. cruzi, and may lead to improved disease control strategies.

Raccoons As an Important Reservoir for Trypanosoma cruzi: A Prevalence Study from Two Metropolitan Areas in Louisiana.

Raccoons are an important reservoir for Trypanosoma cruzi infection, having been reported to maintain a high and lengthy parasitemia. Although raccoon populations have historically been abundant in Louisiana, the prevalence rate of T. cruzi infection in raccoons in this state is unknown. Here, we tested raccoon tissues from two urban areas in Louisiana, namely Orleans Parish (OP) and East Baton Rouge Parish (EBRP), to investigate prevalence in these areas using direct detection through polymerase chain reaction. Overall, 33.6% of raccoons tested were positive. The prevalence in OP (42.9%) was significantly higher than the prevalence in EBRP (23.2%). There was no significant difference in prevalence between sexes or based on age, but there was a significant difference in infection prevalence based on season of trapping. These results suggest the importance of raccoons as a reservoir host, maintaining T. cruzi infection and potentially posing a risk to human health.

An Improved Approach to Trypanosoma cruzi Molecular Genotyping by Next-Generation Sequencing of the Mini-exon Gene.

Trypanosoma cruzi, the etiological agent of Chagas disease, is a protozoan parasite usually transmitted by triatomines. As the parasite can infect all mammals and the vectors can be found across a broad range of ecologies, transmission cycles are quite complex, and extensive genetic diversity exists within the parasite population. Seven main evolutionary lineages, named "discrete typing units," have been described, but a large amount of intra-lineage heterogeneity is also observed. To date, typing methods used to elucidate both inter-lineage and intra-lineage diversity have faced limitations, with some approaches unable to determine all levels of diversity and others requiring investigation of numerous markers and often the selective process of isolation of live parasites. Here, we present a method for parasite genotyping using next-generation sequencing of the mini-exon gene marker, to assign lineage and describe intra-lineage diversity directly from biological samples. This approach is sensitive enough to detect the presence of multiclonal infections and low-frequency parasite genotypes within this context, providing an unprecedented description of T. cruzi assemblages in hosts and vectors.

Molecular Genotyping of Trypanosoma cruzi by Next-Generation Sequencing of the Mini-Exon Gene Reveals Infections With Multiple Parasite Discrete Typing Units in Chagasic Patients From Yucatan, Mexico.

The diversity of Trypanosoma cruzi parasites infecting humans is still poorly understood. We used deep sequencing to analyze this diversity in chagasic patients from Mexico. Such information is crucial to understand transmission cycles and to identify determinants of epidemiological and clinical characteristics of the infection. We analyzed parasite mini-exon spliced-leader sequences following amplification of blood DNA by polymerase chain reaction and deep sequencing. Chagasic patients presented a diverse assemblage of parasite haplotypes covering TcI, TcII, TcV, and TcVI discrete typing units, with a mean (±SEM) of 3.9 ± 0.7 haplotypes/patient, and 47% harbored infections with multiple discrete typing units. Most parasite haplotypes from patients were identical or similar to those for Triatoma dimidiata from the same region, confirming their local circulation. Infection with multiple T. cruzi strains may influence serological diagnostic test results and disease progression in patients and should be taken into account to evaluate associations between parasite diversity and clinical aspects of T. cruzi infections.

Trypanosoma cruzi diversity in naturally infected nonhuman primates in Louisiana assessed by deep sequencing of the mini-exon gene.

BACKGROUND: Trypanosoma cruzi is a zoonotic pathogen of increasing relevance in the USA, with a growing number of autochthonous cases identified in recent years. The identification of parasite genotypes is key to understanding transmission cycles and their dynamics and consequently human infection. Natural T. cruzi infection is present in captive nonhuman primate colonies in the southern USA. METHODS: We investigated T. cruzi genetic diversity through a metabarcoding and next-generation sequencing approach of the mini-exon gene to characterize the parasite genotypes circulating in nonhuman primates in southern Louisiana. RESULTS: We confirmed the presence of T. cruzi in multiple tissues of 12 seropositive animals, including heart, liver, spleen and gut. The TcI discrete typing unit (DTU) predominated in these hosts, and specifically TcIa, but we also detected two cases of coinfections with TcVI and TcIV parasites, unambiguously confirming the circulation of TcVI in the USA. Multiple mini-exon haplotypes were identified in each host, ranging from 6 to 11. CONCLUSIONS: The observation of multiple T. cruzi sequence haplotypes in each nonhuman primate indicates possible multiclonal infections. These data suggest the participation of these nonhuman primates in local parasite transmission cycles and highlight the value of these naturally infected animals for the study of human Chagas disease.