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Background: The role that the genetic diversity of natural Trypanosoma cruzi populations plays in response to trypanocidal treatment of chronic Chagas disease (CD) patients remains to be understood. We analysed the genetic polymorphisms of parasite bloodstream populations infecting chronic CD patients enrolled in the E1224 clinical trial., Methods: A total of 506 baseline and post-treatment follow-up samples from 188 patients were analysed. T. cruzi satellite DNA (satDNA) was amplified and sequenced using cruzi1/cruzi2 primers, and samples with TcI/III, TcII, TcIV or hybrid satDNA sequences were identified. Minicircle signatures were obtained after kinetoplast DNA amplification using 121/122 primers and restriction enzyme digestion. Genetic distances between baseline and post-treatment minicircle signatures were estimated using the Jaccard coefficient., Results: At baseline, 74.3% TcII, 17.9% hybrid and 7.8% TcI/III satDNA sequences were found, whereas at the end of follow-up the distribution was 55.2% TcII, 35.2% hybrid and 9.5% TcI/III. The placebo arm was the treatment group with the highest variation of satDNA sequences between baseline and post-treatment follow-up. Genetic distances between baseline and post-treatment minicircle signatures were similar among all treatment arms. No association between minicircle signature variability and satDNA type distribution was found., Conclusions: Genetic variability of T. cruzi bloodstream populations during post-treatment follow-up did not differ from that observed during chronic infection in the absence of treatment, suggesting that there were no selection events of E1224-resistant parasite populations. This is the first report documenting the genetic polymorphism of natural T. cruzi populations in chronic patients in the context of clinical trials with trypanocidal drugs., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Data on the distribution and abundance of Lutzomyia spp. (Diptera: Psychodidae) in Bolivia is scarce. Sand flies from an area of Leishmania (Viannia) braziliensis endemicity in the Isiboro-Secure National Park in the Department of Cochabamba were captured and identified to species. In total, 945 sand flies (789 females and 156 males) belonging to 15 species were collected from the four collection points in two study villages in 2007. With 549 (58.1%) specimens, Lutzomyia shawi was the most abundant species, followed by Lutzomyia (Trichophoromyia) sp. (22.2%), Lutzomyia llanosmartinsi (8.3%), Lutzomyia antunesi (4.3%), and Lutzomyia olmeca (2.1%). Abundance and species composition varied between rainy and dry seasons, with 99.3% of all sand flies being collected outdoors. Because of species abundance and confirmed Leishmania infection in previous entomological collections, we believe Lu. shawi is the vector of L. (Viannia) braziliensis in Isiboro-Secure National Park.

Human-made and environmental changes constitute a major risk factor for the (re-)emergence and spread of leishmaniasis; surveillance of the transmission cycle is essential in this context. This study integrated entomological and molecular parasitological techniques to document the transmission pattern of a peridomestic focus of Leishmania in the Isiboro Secure area of Bolivia. First the spatial distribution and relative density of phlebotomine sand flies, genus Lutzomyia, were established. Lutzomyia shawi was the predominant species in domestic and peridomestic environments (90% from all collections). Second, direct application of the hsp70 PCR to sand fly extracts detected Leishmania infections in Lu. shawi only, and gave an estimated infection rate of 0.21 to 0.38%. The cleavage of the hsp70 amplicon with restriction enzymes (hsp70 PCR-RFLP) allowed identification of Le. (V.) braziliensis and Le. (V.) guyanensis in Lu. shawi captured in the same village. These two parasite species were also found in humans from the study region, supporting the co-existence of two transmission cycles involving the same sand fly species. This study demonstrated the use of PCR-RFLP in the identification of Leishmania in sand fly pools which could lead to the development of methods for screening large sand fly populations in Latin America.

Species identification is highly relevant for improved prognosis and adequate treatment of American tegumentary leishmaniasis (ATL). PCR-based methods are available for this purpose but should be simplified to improve accessibility. As a first step in this process, this paper describes a simplified protocol for collection of clinical samples. Using samples from 44 Bolivian patients with confirmed ATL, we demonstrated that hsp70 PCR-RFLP on skin scrapings collected with a tooth pick allowed identification of the parasite species with a sensitivity of 95% and specificity of 100%. Our method should greatly facilitate individual patient management and epidemiological surveillance of ATL.