Cytotaxonomy of Trypanosoma cruzi (Chagas, 1909): Differentiation of T. cruzi I (TcI) and T. cruzi II (TcII) Genotypes Using Cytogenetic Markers

Abstract.

Chagas disease is a public health problem caused by the Trypanosoma cruzi, and the T. cruzi I (TcI) and T. cruzi II (TcII) groups are considered important genotypes from the clinical point of view. Currently, the groups need to be molecularly analyzed for their identification; thus, we cytogenetically analyzed these groups with the objective of developing more accessible techniques for the characterization of these parasites. TcI and TcII groups were differentiated by nucleus characterization with lacto-acetic orcein (TcI—nucleus with positive heteropycnosis and TcII—nucleus with negative heteropycnosis), emphasizing the importance of the application of this technique for epidemiological and clinical studies of Chagas disease.

Chagas disease is caused by the protozoan Trypanosoma cruzi (Chagas, 1909) and represents a serious public health problem.¹ It is estimated that eight million people worldwide are infected by T. cruzi² and about 70 million people are living in areas with contamination risk.³

Transmission of T. cruzi can occur, for example, through organ transplantation of an infected donor, laboratory accidents, or ingestion of contaminated food or liquids (sugarcane, acai, and raw meat). In addition, it is important to note that the transmission can be also congenital (vertically between mother and child) or can happen through blood transfusion. The latter two forms lead to infections in urban and non-endemic areas and are targeted by control measures in an attempt to reduce disease.^4,5 However, despite nonvector forms, the main mechanism of transmission of T. cruzi is through triatomines.⁶

There are seven discrete typing units (DTUs) for T. cruzi, classified based on different molecular markers,⁷ namely, T. cruzi I (TcI), T. cruzi II (TcII), T. cruzi III (Tc III), T. cruzi IV (Tc IV), T. cruzi V (Tc V), T. cruzi VI (Tc VI), and T. cruzi VII (Tc VII).^8,9 TcI and TcII groups are important DTUs from the clinical point of view because TcI is associated with chronic cases of cardiomyopathy and severe meningoencephalitis and TcII is related to chronic forms of Chagas disease that cause cardiac and digestive manifestations.^7,10–12

TcI has a wide geographic distribution in the American continent (South, Central, and North America)⁷ and has been reported as the most frequently isolated of all mammalian taxa throughout the geographic range of the parasite, in a wide variety of biomes and habitats.^7,13 TcII has a more restricted distribution [Southern Cone and North (sporadic)]⁷ that, although initially was mainly associated with human infection,¹⁴ more recently has been isolated from a wide range of mammalian species in various biomes.^7,13,15,16 However, when compared with the number of TcI reservoirs, it has been proposed that this DTU occurs in more focal cycles.¹⁷

Methods that are currently used to determine T. cruzi DTUs require isolation and molecular analysis for their identification⁷; therefore, it is important to develop more accessible techniques for the identification of DTUs. Based on this priority, the present work aimed to cytogenetically analyze the TcI and TcII groups of T. cruzi.

As a representative of the TcI group, we used the Bolivia strain, isolated from feces from specimens of Triatoma infestans (Klug, 1834) captured in Vitichi, Bolivia.¹⁸ As a representative of TcII, we used the Y strain, isolated from a human case.¹⁴ The maintenance of populations of T. cruzi may be related to intrinsic characteristics of the parasite, such as its infection ability.¹⁹ To minimize this potential confounding factor, the strains used in the study were cryopreserved at −80° and then grown in LIT medium.

TcI and TcII strains were cultured in LIT medium in the epimastigote form of the parasite, and slides were prepared with 100 μL of culture spread. After drying, the material was fixed with methanol. Subsequently, the slides were stained by lacto-acetic orcein,²⁰ with modifications described by Alevi et al.²¹ and analyzed using the Jenaval light microscope (Zeiss), coupled with a digital camera and an image analyzer system AxioVision LE 4.8 (Copyright ©2006-2009 Carl Zeiss Imaging Solutions Gmb H). The images were magnified by a factor of 1,000.

Cytogenetic analyses allowed us to differentiate TcI and TcII groups of T. cruzi by characterization of the protozoan nucleus because TcI presented as a nucleus with positive heteropycnosis (Figure 1A, arrow), whereas TcII showed the nucleus with negative heteropycnosis (Figure 1B, arrow).

Figure 1.

TcI (A) and TcII (B) groups of Trypanosoma cruzi in epimastigote form stained with orcein. Note the difference in the protozoa nuclei: TcI presented the nucleus with positive heteropycnosis (A, arrow) and TcII presented the nucleus with negative heteropycnosis (B, arrow). Bar: 10 μm. This figure appears in color at www.ajtmh.org.

Currently, T. cruzi DTUs cannot be differentiated by classic analyses with Giemsa, which only allow evaluation of the presence/absence of this protozoan in biological material samples,²² and molecular analyses are necessary for the classification of DTUs.⁷ Based on current findings, the cytogenetic characterization by orcein may help in entoepidemiological studies, allowing investigators to evaluate if the triatomines are infected by T. cruzi and, above all, the type of DTU that is present in these vectors. Furthermore, it may also help in the clinical diagnosis of Chagas disease.

Although T. cruzi DTUs share a common ancestry, TcI and TcII are distant from the phylogenetic point of view.^8,9,23 The dates of divergence date between TcI and TcII have been estimated between 88 and 37 million years ago (based on small subunit rDNA)²⁴ or between 16 and 3 million years ago (based on dihydrofolate reductase-thymidylate synthase and trypanothione reductase genes).²⁵ These estimates support the nuclear divergences observed for the analyzed strains.

By means of the cytogenetic characterization of the parasite nucleus, the lacto-acetic orcein stain was shown to be an efficient technique for the differentiation of T. cruzi belonging to the TcI and TcII DTUs. These results may complement epidemiological studies and help in the clinical diagnosis of Chagas disease.