TRYPOMASTIGOTE SMALL SURFACE ANTIGEN (TSSA) AS IMMUNOLOGICAL MARKER FOR T. cruzi LINEAGES ANALYSIS IN DIFFERENT REGIONS OF LATIN AMERICA.

SARTOR P.; RISSO M.,; BURGOS J ,; BRICEÑO L.,; GUHL F.; ESPINOZA B.; MONTEÓN V.; RUSSOMANDO G.; TRIANA O.; SCHIJMAN A.; BOTTASSO O; LEGUIZAMÓN, S

Armacao de Buzios, RJ

Congreso; XIII International Congress of Protistology. XXV Annual Meeting of the Brazilian Society of Protozoology. XXXVI Annual Meeting on Basic Research in Chagas Disease.; 2009

Background. Based on genetic studies Trypanosoma cruzi population was grouped into the two major lineages T. cruzi I (TCI) and T. cruzi II (TCII). Genotyping studies have demonstrated a polarized geographic distribution. TCI was found in both the sylvatic and domestic transmission cycles from the North of Brazil northwards, while was mainly related to the sylvatic cycle in southern South America. In the southern cone of Latin America TCII shows a high prevalence in the domestic cycle and is the causative agent of most human infections. TSSA (trypomastigote small surface antigen) I and TSSA II are coded by two alleles (tssa I and tssa II) which are present in TCI or TCII genomes, respectively. Since the detection of anti-TSSA antibodies has been proposed as an immunological marker able to identify T. cruzi lineages directly on human samples, a study employing this method was carried out in countries from different parts from the Latin American continent. Material and Methods. Human serum samples: Mexico (n=182), Venezuela (n=156), Colombia (n=199), Paraguay (n=85) and Argentina (n=83). Negative controls: sera from individuals with leishmaniasis or other infectious or non-infectious diseases (n=85) and healthy individuals without epidemiological risk (n=16). T. cruzi diagnosis was performed by using conventional serology tests (CS). Also trans-sialidase inhibition assay (TIA, an improved accuracy assay for T. cruzi detection) was used to confirm diagnosis in samples from Paraguayan patients, a population at high risk of infection. Anti-TSSA I and anti-TSSA II antibodies were detected by western-blotting. Statistical comparisons were performed by the test for proportions, the Chi square and Fisher’s exact test if applicable. Results. Negative controls were not reactive to TSSA except for 1 patient suffering from malaria. CS and TSSA correlation is presented in the Table. When TIA was assayed in Paraguayan samples a large number of CS negative sera was identified as T. cruzi infected thus improving negative correlation from 47.7% to 72.7% (Table). TIA sensitivity allows the identification of infections that were previously diagnosed as negative by CS. Reactivity to TSSA of samples from Argentina, Paraguay, Mexico, Colombia and Venezuela is shown in the Table.   Region Sample Origin TSSA   CS/TSSA copositive    CS/TSSA         conegative * TSSA/TIA correlation I II I/II 1 Paraguay 2 (3%) 58 (92%) 3 (5%)   95.8%  (89.28%)* 47.7% (72.7%)* Argentina 0 (0 %) 57 (100 %) 0 (0 %) 90.5% 100.0% 2 Venezuela 20 (40%) 21 (42%) 9 (18 %) 42.7% 82.0% Colombia  20 (19%) 51 (47 %) 37 (34 %) 62.0% 63.4% 3 Mexico 13 (26%) 24 (47 %) 14 (27 %) 36.14% 78.0% Significant differences on regional lineage distribution were found when comparing Region 1 (Argentina and Paraguay) with Region 2 (Venezuela and Colombia, p<0.00001) or Region 3 (Mexico, p<0.00001). In contrast, neither Region 2 vs. Region 3 analysis nor the comparisons between countries from the same region rendered significant differences in parasite lineage distribution. The relative prevalence of TSSA I (TCI) was superior in Mexico, Colombia and Venezuela compared to each of the countries of Region 1 (p < 0.005 for all cases). The same result was obtained when TSSA I+II reactivity (TCI +TCII) was assessed but lower statistical significance were found for differences between Venezuela vs. Paraguay (p=0.05) and Venezuela vs. Argentina (p < 0.01). The proportion of TSSA II (TCII) reactivity in sera from Argentina and Paraguay was significantly superior than the one detected for samples from the other three studied countries (p < 0.0001 for all cases). Conclusions. By testing TSSA we have confirmed the presence of TCII in southern South America even in specific areas of Paraguay where TCI may be expected given the sylvatic environment. In patients from Venezuela, samples reactive to TSSA I and TSSA II were detected in similar proportions. Also mixed infections (samples reactive to both TSSA I and TSSA II) were found in Venezuela for the first time in this work. In Colombia we have found a high prevalence of TCII. Our results with Mexican samples allowed the novel description of TCII in human infections in this country. The comparative region analysis confirmed TCII as preferentially associated to human infections in the southern cone of the continent but TCI and TCII in the north.