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Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/μL for DNA samples diluted in water, 10 fg/μL for Fasciola/snail DNA scramble, and 100 fg/μL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories. [ABSTRACT FROM AUTHOR]

Chagas disease is a lingering Public Health problem in Latin America with ∼5.7 million people infected with Trypanosoma cruzi. Transmission is still taking place in most countries of the Americas, including the United States. Dogs are frequently infected with T. cruzi and its high infection prevalence is associated with increased risk of Chagas disease in humans. The city of Mérida in the Yucatan peninsula is endemic for Chagas disease and canines are frequently infected with T. cruzi. The objective of this study was to evaluate the performance of a qualitative point of care (POC) molecular test (RPA-LF, recombinase polymerase amplification-lateral flow) developed in our laboratory for identifying infected dogs. We used retrospective samples of dogs that came for consultation because of cardiac alterations and proved to be infected with T. cruzi as determined by enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative PCR (qPCR). The analytical sensitivity indicated that RPA-LF amplified T. cruzi DNA in samples containing almost equal to one to two parasites per reaction. Serial twofold dilutions of T. cruzi epimastigotes showed that the test had 95% (19/20) repeatability at concentrations of two parasites per reaction. The test showed no cross reactivity with human DNA or other protozoan parasites (Trypanosoma rangeli, Leishmania spp., and Plasmodium spp.). RPA-LF had the capacity to amplify all discrete typing units (DTUs I–VI) of T. cruzi that circulate in domestic or extradomestic environments. The RPA-LF had 93.2% (95% confidence interval 87.2–98.1) sensitivity and excellent agreement with qPCR used as gold standard (Cohen's Kappa test = 0.963). ELISA was positive in 96.6% (85/88) of dogs, which together with the molecular tests confirmed the frequent contact with infected triatomine bugs in the city of Mérida. These preliminary results on the diagnostic efficacy of the RPA-LF deserve further large-scale field testing of this POC test for T. cruzi infection in endemic areas. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis (CL) is highly prevalent in rural and sylvatic regions of Latin America, with an estimated 55,000 annual cases. Diagnosis in resource-limited areas still relies on microscopy of dermal scrapings, while more sensitive methods like PCR are not attainable due to costs and lack of adequate health infrastructure. Isothermal amplification of Leishmania DNA can be performed without sophisticated equipment and training and may become a point of care (POC) test for health care centers with scarce resources. We evaluated the efficacy of recombinase-polymerase-amplification (RPA-LF) to diagnose CL in 226 patients attending a clinic in Puerto Maldonado within the Peruvian Amazon basin. Conventional PCR targeting kinetoplast DNA (kDNA-PCR) was used as the gold standard. Eight of 226 patients were considered true negatives (microscopy, kDNA-PCR, and RPA-LF negative), while RPA-LF resulted positive in 186 of 204 kDNA-PCR positive patients, yielding 91.2% (confidence interval [CI] = 86.5-94.4%) sensitivity and 93% (CI 88.6-95.8%) positive predictive value. There were 14% (32/226) discrepant samples alternating positive and negative results in similar proportions between both tests. Quantitative PCR used to resolve the discrepancies suggested that they occurred in samples with scarce parasite numbers as determined by high cycle threshold (Ct) values (≥32; cutoff 35.5). Microscopy had the lowest sensitivity of all methods (45.4%). Nested real-time PCR performed in 71 samples determined that Leishmania ( Viannia ) braziliensis was highly prevalent (69/71), and Leishmania ( Viannia ) lainsoni was present in only two isolates. Results indicated that RPA-LF has POC potential for CL endemic areas, yet further simplification and optimization coupled with field validation will be necessary to confirm its broad applicability.

The parasites of the genus Cryptosporidium are important causes of diarrheal diseases, specifically cryptosporidiosis, worldwide. A major bottleneck for developing drugs and vaccines against cryptosporidiosis is the lack of methods to study gene function in this parasite. Silencing of genes by RNA interference (RNAi) is a powerful method to investigate gene function that has been widely used in the identification of targets for several pathogens. Unfortunately, as Cryptosporidium does not possess the enzymes of the RNAi pathway, its genes cannot be silenced by standard siRNA technology. To circumvent that problem, we have developed a novel strategy to knock down Cryptosporidium genes by reconstituting the effector arm of the siRNA pathway. We have induced silencing of several genes in Cryptosporidium by transfecting parasites with hybrid complexes formed between recombinant human Argonaute (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA). This novel methodology provides an effective strategy to study the role of selected genes in host-parasite interactions, and also can be used to identify potential targets for chemotherapy. The standardized methodology based on this strategy is described in this chapter.