Molecular typing of Strongyloides stercoralis in Latin America, the clinical connection.

This study analysed Strongyloides stercoralis genetic variability based on a 404 bp region of the cox1 gene from Latin-American samples in a clinical context including epidemiological, diagnosis and follow-up variables. A prospective, descriptive, observational study was conducted to evaluate clinical and parasitological evolution after ivermectin treatment of 41 patients infected with S. stercoralis. Reactivation of the disease was defined both by clinical symptoms appearance and/or direct larvae detection 30 days after treatment or later. We described 10 haplotypes organized in two clusters. Most frequent variants were also described in the Asian continent in human (HP24 and HP93) and canine (HP24) samples. Clinical presentation (intestinal, severe, cutaneous and asymptomatic), immunological status and eosinophil count were not associated with specific haplotypes or clusters. Nevertheless, presence of cluster 1 haplotypes during diagnosis increased the risk of reactivation with an odds ratio (OR) of 7.51 [confidence interval (CI) 95% 1.38­44.29, P = 0.026]. In contrast, reactivation probability was 83 times lower if cluster 2 (I152V mutation) was detected (OR = 0.17, CI 95% 0.02­0.80, P = 0.02). This is the first analysis of S. stercoralis cox1 diversity in the clinical context. Determination of clusters during the diagnosis could facilitate and improve the design of follow-up strategies to prevent severe reactivations of this chronic disease.

Development of a New LAMP Assay for the Detection of Ancylostoma caninum DNA (Copro-LAMPAc) in Dog Fecal Samples.

Ancylostoma caninum is a zoonotic nematode which is able to affect animals and humans. Diagnosis in the definitive host and environmental detection are key to prevent its dissemination and achieve control. Herein, a new coprological LAMP method for the detection of A. caninum (Copro-LAMPAc) DNA was developed. DNA extraction was performed using a low-cost method and a fragment of the cox-1 gene was used for primer design. The analytical sensitivity, evaluated with serial dilutions of genomic DNA from A. caninum adult worms, was 100 fg. A specificity of 100% was obtained using genomic DNA from the host and other pathogens. The Copro-LAMPAc was evaluated using environmental canine fecal samples. When compared with gold standard optical microscopy in epidemiological studies, it proved to be more sensitive. This new LAMP assay can provide an alternative protocol for screening and identification of A. caninum for epidemiological studies in endemic areas.

Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati.

Toxocariasis is a zoonotic disease caused mainly by Toxocara canis and Toxocara cati and diagnosis in dogs and cats is an important tool for its control. For this reason, a new coprological loop-mediated isothermal amplification (LAMP) assay was developed for the simultaneous detection of these species. The primer set was designed on a region of the mitochondrial cox-1 gene. Amplification conditions were evaluated using a temperature gradient (52°C to 68°C), different incubation times (15­120 min), and different concentrations of malachite green dye (0.004­0.4% w/v). The analytical sensitivity was evaluated with serial dilutions of genomic DNA from T. canis and T. cati adult worms, and with serial dilutions of DNA extracted from feces using a low-cost in-house method. The specificity was evaluated using genomic DNA from Canis lupus familiaris, Felis catus, Escherichia coli, Toxascaris leonina, Ancylostoma caninum, Echinococcus granulosus sensu stricto and Taenia hydatigena. The LAMP assay applied to environmental fecal samples from an endemic area showed an analytical sensitivity of 10­100 fg of genomic DNA and 10−5 serial dilutions of DNA extracted from feces using the low-cost in-house method; with a specificity of 100%. Additionally, the total development of the assay was carried out in a basic laboratory and per-reaction reagent cost decreased by ~80%. This new, low-cost tool can help identify the most common agents of toxocariasis in endemic areas in order to manage prevention strategies without having to rely on a laboratory with sophisticated equipment.