Michael A. Miles

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

The revised Trypanosoma cruzi subspecific nomenclature: Rationale, epidemiological relevance and research applications

The protozoan Trypanosoma cruzi, its mammalian reservoirs, and vectors have existed in nature for millions of years. The human infection, named Chagas disease, is a major public health problem for Latin America. T. cruzi is genetically highly diverse and the understanding of the population structure of this parasite is critical because of the links to transmission cycles and disease. At present, T. cruzi is partitioned into six discrete typing units (DTUs), TcI-TcVI. Here we focus on the current status of taxonomy-related areas such as population structure, phylogeographical and eco-epidemiological features, and the correlation of DTU with natural and experimental infection. We also summarize methods for DTU genotyping, available for widespread use in endemic areas. For the immediate future multilocus sequence typing is likely to be the gold standard for population studies. We conclude that greater advances in our knowledge on pathogenic and epidemiological features of these parasites are expected in the coming decade through the comparative analysis of the genomes from isolates of various DTUs.

Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy

Leishmaniasis is a geographically widespread severe disease, with an increasing incidence of two million cases per year and 350 million people from 88 countries at risk. The causative agents are species of Leishmania, a protozoan flagellate. Visceral leishmaniasis, the most severe form of the disease, lethal if untreated, is caused by species of the Leishmania donovani complex. These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. We have conducted a multifactorial genetic analysis that includes DNA sequences of protein-coding genes as well as noncoding segments, microsatellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of ≈18,000 characters for each of 25 geographically representative strains. Genotype is strongly correlated with geographical (continental) origin, but not with current taxonomy or clinical outcome. We propose a new taxonomy, in which Leishmania infantum and L. donovani are the only recognized species of the L. donovani complex, and we present an evolutionary hypothesis for the origin and dispersal of the species. The genus Leishmania may have originated in South America, but diversified after migration into Asia. L. donovani and L. infantum diverged ≈1 Mya, with further divergence of infraspecific genetic groups between 0.4 and 0.8 Mya. The prevailing mode of reproduction is clonal, but there is evidence of genetic exchange between strains, particularly in Africa.

The identification by isoenzyme patterns of two distinct strain-groups of Trypanosoma cruzi, circulating independently in a rural area of Brazil

Culture forms of 17 Trypanosoma cruzi stocks, primarily isolated from a rural area of endemic Chagas disease at São Felipe, Bahia, Brazil, were compared by the electrophoretic patterns of six enzymes: aspartate aminotransferase, alanine aminotransferase, glucose-6-phosphate dehydrogenase, malate dehydrogenase (decarb-oxylating) (NADP+), glucosephosphate isomerase and phosphoglucomutase. Two markedly distinct combinations of isoenzyme patterns were seen, justifying the arrangement of the 17 stocks into two strain-groups, each of which was enzymically homogeneous. One combination was characteristic of the 11 domestic stocks of T. cruzi derived from both human infections and domiciliated animals; the second was characteristic of the six sylvatic stocks derived from opossums and a sylvatic triatomine species. The enzyme patterns were independent of the original host and the type of culture medium used. Distinction of the two strain-groups accords with epidemiological evidence that the domestic and sylvatic transmission cycles in São Felipe do not overlap. It is suggested that the diverse enzyme characters of the two strain-groups circulating in São Felipe reflect diverse origins; the domestic form of T. cruzi probably invaded the area from the south of Brazil with the domestic triatomine vector, Panstrongylus megistus.

Emerging Chagas disease in Amazonian Brazil

In the Amazon Basin, Trypanosoma cruzi infection is enzootic, involving a variety of wild mammals and at least 10 of the 16 reported silvatic triatomine bug species. Human cases of Chagas disease are increasing, indicating that the disease may be emerging as a wider public health problem in the region: 38 cases from 1969 to 1992, and 167 in the past eight years. This article reviews the status of Chagas disease in Amazonian Brazil, including known reservoirs and vectors, and the genetic diversity of T. cruzi. At least three subspecific groups of T. cruzi-T. cruzilZ1, T. cruziZ3 and T. cruziZ3/Z1 ASAT--are present. It appears that T. cruzil has an extant capacity for genetic exchange. Attention is also drawn to the risk of domestic endemicity, in addition to the tasks facing the disease control authorities.

Further enzymic characters of Trypanosoma cruzi and their evaluation for strain identification

Abstract Starch-gel electrophoresis of 38 enzymes was attempted with extracts of Trypanosoma cruzi culture forms. 18 of the enzymes that gave discrete electrophoretic bands were selected for routine characterization of T. cruzi stocks; the enzymes were: aspartate aminotransferase (E.C. 2.6.1.1, ASAT); alanine aminotransferase (E.C.2.6.1.2, ALAT); phosphoglucomutase (E.C.2.7.5.1, PGM); glucosephosphate isomerase (E.C.5.3.1.9, GPI); malate dehydrogenase (oxaloacetate decarboxylating) (NADP+) (E.C.1.1.1.40, ME); glucose 6-phosphate dehydrogenase (E.C.1. 1.1.49, G6PD); malate dehydrogenase (E.C.1.1.1.37, MDH); aconitate hydratase (E.C.4.2.1.3, ACON); isocitrate dehydrogenase (NADP+) (E.C.1.1.1.42, ICD); alcohol dehydrogenase (NADP+) (E.C.1.1.1.2, ADH); lactate dehydrogenase (E.C.1.1.1.27, LDH); aminopeptidase (cytosol) (E.C.3.4.11.1, PEP); pyruvate kinase (E.C.2.7.1.40, PK); phosphoglycerate kinase (E.C.2.7.2.3, PGK); enolase (E.C.4.2.1.11, ENO); hexokinase (E.C.2.7.1.1, HK); mannosephosphate isomerase (E.C.5.3.1.8, MPI); and glutamate dehydrogenase (E.C. 1.4.1.2, GD). ADH (NADP+) in the genus Trypanosoma, and PGK, MPI and ENO, in T. cruzi, were apparently demonstrated for the first time. Between six and 18 enzymes were used to characterize more than 250 T. cruzi stocks, newly isolated from a wide range of sources in northern and central Brazil. All stocks were identified as belonging to T. cruzi zymodemes 1, 2 or 3, as originally defined—that is, by combination of electrophoretic patterns of ASAT, ALAT, PGM, GPI, ME and G6PD. The composite range of results with all enzymes confirmed the presence of three principal T. cruzi zymodemes, but some enzymic characters overlapped between zymodemes and others suggested subgroups within individual zymodemes. Seven (MDH, ACON, LDH, PK, PGK, ENO, HK) of the 18 enzymes did not distinguish the three zymodemes; five (ASAT, PGM, GPI, ICD, PEP) distinguished all three zymodemes; 10 (ASAT, ALAT, PGM, GPI, ME, G6PD, ICD, ADH, PEP, GD) distinguished zymodemes 1 and 2, of which seven plus MPI and eight plus MPI separated zymodemes 1 from 3 and 2 from 3 respectively. T. cruzi stocks were taken from a small area of the natural species distribution; the full range of enzymic characters within the species T. cruzi is expected to be far more complex. The epidemiological distribution of the zymodemes continued to accord with local transmission cycles and supported the hypothesis that distinct T. cruzi strains might be responsible for the enigmatic distribution of chronic Chagass disease. Some of the difficulties in the empirical selection of new electrophoretic methods and the interpretation of results were presented, and the present and prospective significance of T. cruzi enzymic characters was discussed. Until the stability and genetic basis of T. cruzi enzymic characters are better understood it is recommended that isoenzymic profiles be confirmed routinely, both before and after stocks are used experimentally, as representative of a given zymodeme. A multiple biochemical approach to T. cruzi strain identification is recommended, using characters suitable for a numerical taxonomy.

The ecotopes and evolution of triatomine bugs (triatominae) and their associated trypanosomes

Triatomine bug species such as Microtriatoma trinidadensis, Eratyrus mucronatus, Belminus herreri, Panstrongylus lignarius, and Triatoma tibiamaculata are exquisitely adapted to specialist niches. This suggests a long evolutionary history, as well as the recent dramatic spread a few eclectic, domiciliated triatomine species. Virtually all species of the genus Rhodnius are primarily associated with palms. The genus Panstrongylus is predominantly associated with burrows and tree cavities and the genus Triatoma with terrestrial rocky habitats or rodent burrows. Two major sub-divisions have been defined within the species Trypanosoma cruzi, as T. cruzi 1 (Z1) and T. cruzi 2 (Z2). The affinities of a third group (Z3) are uncertain. Host and habitat associations lead us to propose that T. cruzi 1 (Z1) has evolved in an arboreal, palm tree habitat with the triatomine tribe Rhodniini, in association with the opossum Didelphis. Similarly we propose that T. cruzi (Z2) and Z3 evolved in a terrestrial habitat in burrows and in rocky locations with the triatomine tribe Triatomini, in association with edentates, and/or possibly ground dwelling marsupials. Both sub-divisions of T. cruzi may have been contemporary in South America up to 65 million years ago. Alternatively, T. cruzi 2 (Z2) may have evolved more recently from T. cruzi 1 (Z1) by host transfers into rodents, edentates, and primates. We have constructed a molecular phylogeny of haematophagous vectors, including triatomine bugs, which suggests that faecal transmission of trypanosomes may be the ancestral route. A molecular clock phylogeny suggests that Rhodnius and Triatoma diverged before the arrival, about 40 million years ago, of bats and rodents into South America.

The molecular epidemiology and phylogeography of Trypanosoma cruzi and parallel research on Leishmania: looking back and to the future

Trypanosoma cruzi is the protozoan agent of Chagas disease, and the most important parasitic disease in Latin America. Protozoa of the genus Leishmania are global agents of visceral and cutaneous leishmaniasis, fatal and disfiguring diseases. In the 1970s multilocus enzyme electrophoresis demonstrated that T. cruzi is a heterogeneous complex. Six zymodemes were described, corresponding with currently recognized lineages, TcI and TcIIa-e--now defined by multiple genetic markers. Molecular epidemiology has substantially resolved the phylogeography and ecological niches of the T. cruzi lineages. Genetic hybridization has fundamentally influenced T. cruzi evolution and epidemiology of Chagas disease. Genetic exchange of T. cruzi in vitro involves fusion of diploids and genome erosion, producing aneuploid hybrids. Transgenic fluorescent clones are new tools to elucidate molecular genetics and phenotypic variation. We speculate that pericardial sequestration plays a role in pathogenesis. Multilocus sequence typing, microsatellites and, ultimately, comparative genomics are improving understanding of T. cruzi population genetics. Similarly, in Leishmania, genetic groups have been defined, including epidemiologically important hybrids; genetic exchange can occur in the sand fly vector. We describe the profound impact of this parallel research on genetic diversity of T. cruzi and Leishmania, in the context of epidemiology, taxonomy and disease control.

Genomic diversity in the Leishmania donovani complex

The Leishmania donovani complex is considered to be composed of 3 species; L. donovani , L. infantum and L. chagasi , although this classification has been challenged. Genotypic relationships within the complex were evaluated at different levels by: binding of the probe Lmet9, specific for L. chagasi and Old World Leishmania spp.; partial sequencing of a constitutive major surface protease single gene ( mspC ) and random amplification of polymorphic DNA (RAPD). The Old World Leishmania spp. and the L. donovani complex have a monophyletic origin. Leishmania chagasi clearly belongs to the L. donovani complex but it is indistinguishable from L. infantum , which suggests introduction of L. chagasi into the New World in recent history. Leishmania infantum / L. chagasi was identified as a monophyletic group within the L. donovani complex but L. donovani may be paraphyletic. Diversity within L. donovani is substantial and phylogeographical patterns of association were found.

Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I Reveals Phylogeographic Structure and Specific Genotypes Linked to Human Infection

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