Michael A. Miles

Background: Chagas disease remains a significant public health problem in Latin America. There are only two chemotherapy drugs, nifurtimox and benznidazole, and both may have severe side effects. After complete chemotherapy of acute cases, seropositive diagnosis may revert to negative. However, there are no definitive parasitological or serological biomarkers of cure. Methods: Following a pilot study with seven Bolivian migrants to Spain, we tested 71 serum samples from chronic patients (mean age 12.6 years) inhabiting the Argentine Chaco region. Benznidazole chemotherapy (5–8 mg/kg day, twice daily for 60 days) was administered during 2011–2016. Subsequently, pre-and post-chemotherapy serum samples were analysed in pairs by IgG1 and IgG ELISA using two different antigens and Chagas Sero K-SeT rapid diagnostic tests (RDT). Molecular diagnosis by kDNA-PCR was applied to post-treatment samples. Results: Pilot data demonstrated IgG1 antibody decline in three of seven patients from Bolivia 1 year post-treatment. All Argentine patients in 2017 (averaging 5 years post-treatment), except one, were positive by conventional serology. All were kDNA-PCR-negative. Most (91.5%) pre-treatment samples were positive by the Chagas Sero K-SeT RDT, confirming the predominance of TcII/V/VI. IgG1 and IgG of Argentine patients showed significant decline in antibody titres post-chemotherapy, with either lysate (IgG, P = 0.0001, IgG1, P = 0.0001) or TcII/V/VI peptide antigen (IgG, P = 0.0001, IgG1, P = 0.0001). IgG1 decline was more discriminative than IgG. Antibody decline after treatment was also detected by the RDT. Incomplete treatment was associated with high IgG1 post-treatment titres against lysate (P = 0.013), as were IgG post-treatment titres to TcII/V/VI peptide (P = 0.0001). High pre-treatment IgG1 with lysate was associated with Qom ethnicity (P = 0.045). No associations were found between gender, age, body mass index and pre- or post-treatment antibody titres. Conclusions: We show that following chemotherapy of early chronic Chagas disease, significant decline in IgG1 antibody suggests cure, whereas sustained or increased IgG1 is a potential indicator of treatment failure. Due to restricted sensitivity, IgG1 should not be used as a diagnostic marker but has promise, with further development, as a biomarker of cure. Graphical We show that following chemotherapy of early chronic Chagas disease, a significant decline in IgG1 antibody suggests cure, whereas sustained or increased IgG1 is a potential indicator of treatment failure. Due to restricted sensitivity, IgG1 should not be used as a diagnostic marker but has promise, with further development, as a biomarker of cure.

Background: Visceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL. Methodology/Principal findings: Sampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis. Conclusions/Significance: Seroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance.

Significance: Parasites are interesting models for studying speciation processes because they have a high potential for specialization, thanks to the intimate ecological association with their hosts and vectors. Yet little is known about the circumstances under which new parasite lineages emerge. Here we studied the genome diversity of parasites of the Leishmania braziliensis species complex that inhabit both Amazonian and Andean biotas in Peru. We identify three major parasite lineages that occupy particular ecological niches and show that these emerged during forestation changes over the past 150,000 y. We furthermore discovered that meiotic recombination between Amazonian and Andean lineages resulted in full-genome hybrids presenting mixed mitochondrial genomes, providing insights into the genetic consequences of hybridization in parasitic protozoa.

The clinical manifestation of leishmaniases depends on parasite species, host genetic background, and immune response. Manifestations of human leishmaniases are highly variable, ranging from self-healing skin lesions to fatal visceral disease. The scope of standard model hosts is insufficient to mimic well the wide disease spectrum, which compels the introduction of new model animals for leishmaniasis research. In this article, we study the susceptibility of three Asian rodent species (Cricetulus griseus, Lagurus lagurus, and Phodopus sungorus) to Leishmania major and L. donovani. The external manifestation of the disease, distribution, as well as load of parasites and infectiousness to natural sand fly vectors, were compared with standard models, BALB/c mice and Mesocricetus auratus. No significant differences were found in disease outcomes in animals inoculated with sand fly- or culture-derived parasites. All Asian rodent species were highly susceptible to L. major. Phodopus sungorus showed the non-healing phenotype with the progressive growth of ulcerative lesions and massive parasite loads. Lagurus lagurus and C. griseus represented the healing phenotype, the latter with high infectiousness to vectors, mimicking best the character of natural reservoir hosts. Both, L. lagurus and C. griseus were also highly susceptible to L. donovani, having wider parasite distribution and higher parasite loads and infectiousness than standard model animals.

Background: Triatomine bugs, the vectors of Chagas disease, associate with vertebrate hosts in highly diverse ecotopes. When these blood-sucking bugs adapt to new microhabitats, their phenotypes may change. Although understanding phenotypic variation is key to the study of adaptive evolution and central to phenotype-based taxonomy, the drivers of phenotypic change and diversity in triatomines remain poorly understood. Methods/Findings: We combined a detailed phenotypic appraisal (including morphology and morphometrics) with mitochondrial cytb and nuclear ITS2 DNA-sequence analyses to study Rhodnius ecuadoriensis populations from across the species’ range. We found three major, naked-eye phenotypic variants. Southern-Andean bugs (SW Ecuador/NW Peru) from house and vertebrate-nest microhabitats are typical, light-colored, small bugs with short heads/wings. Northern-Andean bugs (W Ecuador wet-forest palms) are dark, large bugs with long heads/wings. Finally, northern-lowland bugs (coastal Ecuador dry-forest palms) are light-colored and medium-sized. Wing and (size-free) head shapes are similar across Ecuadorian populations, regardless of habitat or naked-eye phenotype, but distinct in Peruvian bugs. Bayesian phylogenetic and multispecies-coalescent DNA-sequence analyses strongly suggest that Ecuadorian and Peruvian populations are two independently-evolving lineages, with little within-lineage structuring/differentiation. Conclusions: We report sharp naked-eye phenotypic divergence of genetically similar Ecuadorian R. ecuadoriensis (house/nest southern-Andean vs . palm-dwelling northern bugs; and palm-dwelling Andean vs . lowland); and sharp naked-eye phenotypic similarity of typical, yet genetically distinct, southern-Andean bugs from house and nest (but not palm) microhabitats (SW Ecuador vs . NW Peru). This remarkable phenotypic diversity within a single nominal species likely stems from microhabitat adaptations possibly involving predator-driven selective pressure (yielding substrate-matching camouflage coloration) and a shift from palm-crown to vertebrate-nest microhabitats (yielding smaller bodies and shorter heads and wings). These findings shed new light on the origins of phenotypic diversity in triatomines, warn against excess reliance on phenotype-based triatomine-bug taxonomy, and confirm the Triatominae as an informative model-system for the study of phenotypic change under ecological pressure. Author summary: Triatomine bugs feed on the blood of vertebrates including humans and transmit the parasite that causes Chagas disease. The bugs, of which 150+ species are known, are highly diverse in size, shape, and color. Some species look so similar that they are commonly confused, whereas a few same-species populations look so different that they were thought to be separate species. Despite the crucial role of naked-eye phenotypes in triatomine-bug identification and classification (which are essential for vector control-surveillance), the origins of this variation remain unclear. Here, we describe a striking case of phenotypic divergence, with genetically similar bugs looking very different from one another, and phenotypic convergence, with bugs from two genetically distinct populations (likely on their way to speciation) looking very similar – and all within a single nominal species, Rhodnius ecuadoriensis . Phenotypically divergent populations occupy different ecological regions (wet vs . dry) and microhabitats (palm-crowns vs . vertebrate nests), whereas convergent populations occupy man-made and nest (but not palm) microhabitats. These findings suggest that triatomines can ‘respond’ to ecological novelty by changing their external, naked-eye phenotypes as they adapt to new microhabitats. We therefore warn that phenotypic traits such as overall size or color may confound triatomine-bug species identification and classification.

Background: Trypanosoma cruzi, the protozoan agent of Chagas disease, is comprised of at least 6 genetic lineages (TcI-TcVI). Their geographical distribution, clinical associations and reservoir hosts are not fully elucidated, as genotyping is hampered due to the difficulty in isolating representative populations of organisms. Lineage-specific serological techniques may address these issues. Methods: Trypanosoma cruzi lineage-specific serological assays were performed on human, canine, feline and armadillo sera from the Gran Chaco in northern Argentina, a region of ongoing transmission. Synthetic peptides representing lineage-specific epitopes of the trypomastigote small surface antigen (TSSA) were used in ELISA, and the TcII/V/VI shared epitope peptide (TSSApep-II/V/VI) was used in the Chagas Sero K-SeT rapid diagnostic test (RDT). Results: Chagas Sero K-SeT RDT, using Protein G to detect human and canine IgG, was at least as sensitive as TSSApep-II/V/VI ELISA using specific secondary antibodies. For sera from humans TSSApep-II/V/VI seroprevalence by Chagas Sero K-SeT was 273/393 (69.5%), for dogs 48/73 (65.8%) and for armadillos 1/7 (14.3%); by ELISA for cats 5/19 (26.3%). The seroprevalence for humans was similar to that for Bolivian patients, amongst whom we previously observed an association of TSSApep-II/V/VI seropositivity with severity of cardiomyopathy. In humans, prevalence of TSSApep-II/V/VI recognition was associated with locality, and with increasing and decreasing age within the Qom and Creole populations, respectively. For dogs TSSApep-II/V/VI recognition was associated with being born before community-wide insecticide spraying (P = 0.05) and with Qom household (P < 0.001). Conclusions: We show here that Chagas Sero K-SeT RDT can replace ELISA for TSSApep-II/V/VI serology of humans and dogs; for humans there were statistically significant associations between a positive Chagas Sero K-SeT RDT and being resident in Area IV, and for dogs association with Qom household or with being born before the mass spraying campaign; we also show that with cats the TcII/V/VI epitope can be detected by ELISA. We assessed the lineage distribution in an unprecedented 83% of the human T. cruzi-seropositive population. These results form the basis for more detailed studies, enabling rapid in-the-field surveillance of the distribution and clustering of these lineages among humans and mammalian reservoirs of T. cruzi infection.

This chapter focuses on trypanosomiasis and leishmaniasis and demonstrates how comparative genetic and genomic analyses have informed and will continue to empower diagnostics discovery, and have led to the development of low-cost, point-of-care, rapid diagnostic tests, with both clinical and research applications. Also highlighted is the still underexploited potential of a systematic combined ‘omics’ approach to both the identification of candidate vaccines and production of prototype diagnostics.

Parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases, ranging from self-healing but disfiguring cutaneous lesions to destructive mucocutaneous presentations or usually fatal visceral disease. Visceral leishmaniasis due to Leishmania donovani is endemic in Ethiopia where it has also been responsible for major epidemics. The presence of hybrid genotypes has been widely reported in surveys of natural populations, genetic variation reported in a number of Leishmania species, and the extant capacity for genetic exchange demonstrated in laboratory experiments. However, patterns of recombination and evolutionary history of admixture that produced these hybrid populations remain unclear, as most of the relevant literature examines only a limited number (typically fewer than 10) genetic loci. Here, we use whole-genome sequence data to investigate Ethiopian L. donovani isolates previously characterised as hybrids by microsatellite and multi-locus sequencing. To date there is only one previous study on a natural population of Leishmania hybrids, based on whole-genome sequence. The current findings demonstrate important differences. We propose hybrids originate from recombination between two different lineages of Ethiopian L. donovani occurring in the same region. Patterns of inheritance are more complex than previously reported with multiple, apparently independent, origins from similar parents that include backcrossing with parental types. Analysis indicates that hybrids are representative of at least three different histories. Furthermore, isolates were highly polysomic at the level of chromosomes with startling differences between parasites recovered from a recrudescent infection from a previously treated individual. The results demonstrate that recombination is a significant feature of natural populations and contributes to the growing body of evidence describing how recombination, and gene flow, shape natural populations of Leishmania . Author Summary: Leishmaniasis is a spectrum of diseases caused by the protozoan parasite Leishmania . It is transmitted by sandfly insect vectors and is responsible for an enormous burden of human suffering. In this manuscript we examine Leishmania isolates from Ethiopia that cause the most serious form of the disease, namely visceral leishmaniasis, which is usually fatal without treatment. Historically the general view was that such parasites reproduce clonally, so that their progeny are genetically identical to the founding cells. This view has changed over time and it is increasingly clear that recombination between genetically different Leishmania parasites occurs. The implication is that new biological traits such as virulence, resistance to drug treatments or the ability to infect new species of sandfly could emerge. The frequency and underlying mechanism of such recombination in natural isolates is poorly understood. Here we perform a detailed whole genome analysis on a cohort of hybrid isolates from Ethiopia together with their potential parents to assess the genetic nature of hybrids in more detail. Results reveal a complex pattern of mating and inbreeding indicative of multiple mating events that has likely shaped the epidemiology of the disease agent. We also show that some hybrids have very different relative amounts of DNA (polysomy) the implications of which are discussed. Together the results contribute to a fuller understanding of the nature of genetic recombination in natural populations of Leishmania .