Real-time polymerase chain reaction based algorithm for differential diagnosis of Kinetoplastidean species of zoonotic relevance.

Kinetoplastids are a group of flagellated protozoa that infect a vast repertoire of mammals and insect vectors. From a zoonotic point of view, domestic animals are critical reservoirs for transmission of Kinetoplastidean parasites. Due to their proximity to humans, they assume substantial epidemiological importance in the context of these zoonoses and consequently in public health. Their reliable identification is relevant to understand their eco-epidemiological involvement in transmission cycles. This work aimed to develop an algorithm based on sequential Real-Time PCR (qPCR) assays targeted to different loci (24S alpha rDNA, ITS1 and Hsp70) allowing distinction among Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma evansi and Leishmania species in biological samples collected from mammalian reservoirs and triatomine vectors. The algorithm includes a first qPCR test targeted to endogenous genes conserved within mammals and within triatomine vectors as internal controls of DNA sample integrity and/or qPCR inhibition. This algorithm was evaluated in biological samples from domestic cattle (N = 14), dogs (N = 19) and triatomines (N = 19). Analytical sensitivity of 24S alpha rDNA for detection of T. rangeli was 10 fg of DNA, with a linear range between 10 fg and 10 ng. For T. cruzi it varied depending on the Discrete typing unit. The ITS1 qPCR showed an analytical sensitivity of 100 pg/reaction and 100 fg/reaction of Leishmania spp. and T. evansi DNAs. In mammal field samples, four T. cruzi 24S alpha rDNA sequences and fourteen ITS1 amplicons specific for T. evansi were detected. qPCR-HRM analysis directed to the Hsp70 gene diagnosed two dogs with Leishmania infantum infection. Among 19 triatomine field samples, T. cruzi was detected in five; T. rangeli in eight and one specimen showed a mixed infection. This diagnostic algorithm can provide more accurate records of kinetoplastidean infection burden in vectors and reservoirs, relevant to update current eco-epidemiological maps in co-endemic regions.

Development and evaluation of a duplex TaqMan qPCR assay for detection and quantification of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts.

BACKGROUND: A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. RESULTS: The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq's) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq's between 24 and 33. CONCLUSIONS: This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.

High effectiveness of an adulticide-larvicide formulation for field control of sandflies (Diptera: Psychodidae) in the city of Clorinda, Argentina.

In Argentina, Leishmania infantum (syn. L. chagasi) is the etiologic agent of human visceral leishmaniosis (HVL), and Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) is the main vector. The objective of this study was to evaluate the effectiveness and residual effect of two commercial insecticide formulations, one with permethrin and pyriproxyfen as active ingredients (Dragon Max®) and the other with only permethrin (Flop®) for the control of sandflies. Both formulations were applied in chicken coops and other surroundings structures of the peridomicile of urban houses in Clorinda, Formosa (Argentina). Entomological monitoring was carried out weekly for 44 weeks after the intervention. The results showed great effectiveness and residual effect up to 21 weeks post-intervention for Dragon Max®. This result could be explained by the excellent larvicidal activity of the Insect Growth Regulator (IGR) pyriproxyfen against the immature forms of phlebotomines and by the delay on the restoration of the natural threshold of the vector population in treated sites.

Spatial population dynamics and temporal analysis of the distribution of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in the city of Clorinda, Formosa, Argentina.

BACKGROUND: Lutzomyia longipalpis, the vector for the causal agent of visceral leishmaniasis (VL), has extended its distribution in the southern cone in the Americas. The first urban record of Lu. longipalpis in Argentina was from the City of Clorinda in 2004. The aim of this study was to analyse the monthly distribution and abundance of Lu. longipalpis and to evaluate its association with environmental and climatic variables in Clorinda City, Province of Formosa. METHODS: Phlebotominae sampling was performed using CDC light mini-traps that were placed in different sites of the city between January 2012 and December 2013. Environmental variables including the normalised difference vegetation index, normalized difference water index, land surface temperature and precipitation were evaluated using a spatiotemporal model. RESULTS: A total of 4996 phlebotomine sandflies were captured during the study period, and eight species were reported: Lu. longipalpis, Migonemyia migonei, Nyssomyia whitmani, Ny. neivai, Brumptomyia guimaraesi, Evandromyia cortelezzii/sallesi, Psathyromyia bigeniculata and Expapillata firmatoi. This is the first urban record of Ex. firmatoi in Argentina. Lutzomyia longipalpis was the most abundant species between 2012 and 2013, and it appeared in all the sampled sites. Moreover, the model applied showed that ground humidity and temperature were significantly associated with the abundance of Lu. longipalpis. CONCLUSIONS: This longitudinal approach at city scale allows for modelling that explains more than 60% of the temporal variability of the abundance of Lu. longipalpis based exclusively on satellite obtained data. The results support the hypothesis of steady 'hot spots' of abundance with time, while other sites could change its abundance due to eventual microenvironment changes. The Lu. longipalpis abundance driving factors are breeding site-related variables, highlighting the importance both for modelling and surveillance to use lag data.