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Strongyloidiasis is a neglected, soil-transmitted helminth infection prevalent worldwide. The true burden of strongyloidiasis is unclear due to the lack of sensitive, field-friendly diagnostic tests. PCR tests to detect Strongyloides DNA in stool are sensitive and specific, but the need for expensive equipment limits their use in endemic regions. Isothermal PCR amplification tests are easier to deploy while maintaining sensitivity and specificity. We developed and evaluated a recombinase polymerase amplification lateral flow assay (RPA-LFA) to detect Strongyloides stercoralis in human stool samples. Three hundred stool samples were collected in three communities in the jungle of Cusco, Peru. Samples were tested for S. stercoralis larvae using microscopy (Baermann’s, agar plate culture (APC), and rapid sedimentation), real-time PCR, and RPA-LF for Strongyloides DNA. The RPA-LFA showed an analytical limit of detection of 20 pg/µL. The prevalence of S. stercoralis was 27%, 38%, 46.3%, and 46% using microscopy, PCR, microscopy/PCR, and RPA-LFA, respectively. RPA-LFA had a sensitivity and specificity of 59.3% and 58.9%, 66.2% and 71.4%, and 77.2% and 73.1% when microscopy, microscopy/PCR, and real-time PCR were used as the gold standards, respectively. The Strongyloides RPA-LFA is a novel, fast, highly sensitive, and specific molecular method with the potential for deployment in endemic regions.

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.

Triclabendazole (TCBZ) resistance is an emerging problem in fascioliasis that is not well understood. Studies including small numbers of parasites fail to capture the complexity of susceptibility variations between and within Fasciolahepatica populations. As the first step to studying the complex resistant phenotype–genotype associations, we characterized a large sample of adult F. hepatica with diverging TCBZ susceptibility. We collected parasites from naturally infected livestock slaughtered in the Cusco and Cajamarca regions of Peru. These parasites were exposed to TCBZ sulfoxide (TCBZ.SO) in vitro to determine their susceptibility. We used a motility score to determine the parasite’s viability. We titrated drug concentrations and times to detect 20% non-viable (susceptible conditions) or 80% non-viable (resistant conditions) parasites. We exposed 3348 fully motile parasites to susceptible (n = 1565) or resistant (n = 1783) conditions. Three hundred and forty-one (21.8%) were classified as susceptible and 462 (25.9%) were classified as resistant. More resistant parasites were found in Cusco than in Cajamarca (p < 0.001). Resistant parasites varied by slaughterhouse (p < 0.001), month of the year (p = 0.008), fluke length (p = 0.016), and year of collection (p < 0.001). The in vitro susceptibility to TCBZ.SO in wildtype F. hepatica was associated with geography, season, and morphometry.