Identification of miRNAs of Strongyloides stercoralis L1 and iL3 larvae isolated from human stool.

Strongyloidiasis is a neglected tropical disease caused by the soil-transmitted nematode by Strongyloides stercoralis, that affects approximately 600 million people worldwide. In immunosuppressed individuals disseminated strongyloidiasis can rapidly lead to fatal outcomes. There is no gold standard for diagnosing strongyloidiasis, and infections are frequently misdiagnosed. A better understanding of the molecular biology of this parasite can be useful for example for the discovery of potential new biomarkers. Interestingly, recent evidence showed the presence of small RNAs in Strongyloididae, but no data was provided for S. stercoralis. In this study, we present the first identification of miRNAs of both L1 and iL3 larval stages of S. stercoralis. For our purpose, the aims were: (i) to analyse the miRNome of L1 and iL3 S. stercoralis and to identify potential miRNAs of this nematode, (ii) to obtain the mRNAs profiles in these two larval stages and (iii) to predict potential miRNA target sites in mRNA sequences. Total RNA was isolated from L1 and iL3 collected from the stool of 5 infected individuals. For the miRNAs analysis, we used miRDeep2 software and a pipeline of bio-informatic tools to construct a catalog of a total of 385 sequences. Among these, 53% were common to S. ratti, 19% to S. papillosus, 1% to Caenorhabditis elegans and 44% were novel. Using a differential analysis between the larval stages, we observed 6 suggestive modulated miRNAs (STR-MIR-34A-3P, STR-MIR-8397-3P, STR-MIR-34B-3P and STR-MIR-34C-3P expressed more in iL3, and STR-MIR-7880H-5P and STR-MIR-7880M-5P expressed more in L1). Along with this analysis, we obtained also the mRNAs profiles in the same samples of larvae. Multiple testing found 81 statistically significant mRNAs of the total 1553 obtained (FDR < 0.05; 32 genes expressed more in L1 than iL3; 49 genes expressed more in L3 than iL1). Finally, we found 33 predicted mRNA targets of the modulated miRNAs, providing relevant data for a further validation to better understand the role of these small molecules in the larval stages and their valuein clinical diagnostics.

SARS-CoV-2 rapid antigen test in comparison to RT-PCR targeting different genes: A real-life evaluation among unselected patients in a regional hospital of Italy.

We assessed the performance of the Panbio rapid antigen detection (RAD) test for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and we compared it with the routine reverse transcriptase-polymerase chain reaction (RT-PCR)-based molecular test in a population of 4167 unselected patients admitted to IRCCS Sacro Cuore Don Calabria Hospital. Analysis stratified by cycling threshold (Ct ) value of SARS-CoV-2 gene targets indicated that antigen (Ag)-positive Ct values were significantly lower compared to Ag-negative values (p < 0.0001). Overall, we found discordance in 140, tested negative by RAD and positive by RT-PCR, and in 4 resulted positive by RAD and negative by RT-PCR. RAD test achieved a sensitivity and specificity of 66.82% and 99.89%, respectively. The positive predictive value was shown to be 97.87% while the negative predictive value was shown to be 97.62%. In our context, the RAD test showed a reliable diagnostic response in subjects that displayed high Ct values, corresponding to high viral load, while low ability was displayed to identify positive cases with medium-low Ct values, thus presenting low viral load and where confirmatory RT-PCR was needed. Our finding supports the use of the RAD test in real-life settings where a high volume of swabs is being processed but with caution when interpreting a positive test result in a low prevalence setting.

Molecular techniques for the genomic viral RNA detection of West Nile, Dengue, Zika and Chikungunya arboviruses: a narrative review.

Introduction: Molecular technology has played an important role in arboviruses diagnostics. PCR-based methods stand out in terms of sensitivity, specificity, cost, robustness, and accessibility, and especially the isothermal amplification (IA) method is ideal for field-adaptable diagnostics in resource-limited settings (RLS).Areas covered: In this review, we provide an overview of the various molecular methods for West Nile, Zika, Dengue and Chikungunya. We summarize literature works reporting the assessment and use of in house and commercial assays. We describe limitations and challenges in the usage of methods and opportunities for novel approaches such as NNext-GenerationSequencing (NGS).Expert opinion: The rapidity and accuracy of differential diagnosis is essential for a successful clinical management, particularly in co-circulation area of arboviruses. Several commercial diagnostic molecular assays are available, but many are not affordable by RLS and not usable as Point-of-care/Point-of-need (POC/PON) such as RReal-TimeRT-PCR, Array-based methods and NGS. In contrast, the IA-based system fits better for POC/PON but it is still not ideal for the multiplexing detection system. Improvement in the characterization and validation of current molecular assays is needed to optimize their translation to the point of care.

Prevalence of Strongyloidiasis in a Cohort of Migrants in Italy and Accuracy of a Novel ELISA Assay for S. stercoralis Infection, a Cross-Sectional Study.

Strongyloides stercoralis infection is a life-threatening neglected tropical disease. Diagnostic issues have caused an underestimation of its global burden. The choice of appropriate diagnostic tests for the screening of populations at risk of the infection, such as migrants from endemic countries, is of paramount importance. From November 2017 to July 2018, all migrants presenting to the National Institute for Health Migration and Poverty (INMP) in Rome, Italy were offered screening tests for S. stercoralis infection. The study objective was to estimate the prevalence of strongyloidiasis in the study population and the accuracy of a novel ELISA assay. The following tests were carried out at the IRCCS Sacro Cuore Don Calabria hospital in Negrar, Verona: stool microscopy, real-time PCR for S. stercoralis, in-house immunofluorescence test (IFAT), a commercial ELISA assay (Bordier ELISA), and a novel ELISA assay (Euroimmun ELISA). A latent class analysis (LCA) model set up with test results, clinical variables, and eosinophilia indicated a prevalence around 7.5%, in line with previous findings. The sensitivity and the specificity of Euroimmun ELISA were 90.6% (95% CI 80.5-100) and 87.7% (95CI 84.5-91.0); these results indicate that the novel ELISA assay would be suitable for screening of migrants from endemic countries.

Assessment of Real-Time Polymerase Chain Reaction for the Detection of Trichostrongylus spp. DNA from Human Fecal Samples.

Sporadic cases of Tricostrongylosis are reported in humans. Diagnosis of enteric Trichostrongylus relies primarily on coproscopic analysis but morphological identification is difficult because of similarity among nematode species. The method is time consuming and requires some expertise. To overcome these limitations, we developed a molecular approach by real-time polymerase chain reaction (PCR) to provide a rapid, specific, and sensitive tool to detect Trichostrongylus spp. in human feces. We designed primers and probe specific for Trichostrongylus rDNA region 5.8S and internal transcribed spacer 2. Three Italian family clusters were analyzed and DNA sequencing was performed to confirm real-time PCR results comparing with known GenBank sequence data. Sequence analysis showed ≥ 99% identity to Trichostrongylus colubriformis and Trichostrongylus axei. This study provides a molecular methodology suitable for fast and specific detection of Trichostrongylus in fecal specimens and to distinguish the zoonotic species.

Molecular Biology Can Change the Classic Laboratory Approach for Intestinal Protozoan Infections.

For many years microscopy has been considered the mainstay of the diagnosis of parasitic infections. In our laboratory, before the advent of molecular biology, the approach for the identification of parasitic infections in stools was the microscopic exam of three samples. Once we adopted molecular biology, a real-time PCR on one single sample was added to the classical coproparasitological exam of three samples. Given the high sensitivity of real-time PCR (Rt-PCR), we then decided to evaluate if a change of our routine was justified. In detail, we intended to assess if a much more practical routine, based on the analysis of a single fecal sample, was sufficiently sensitive to replace the routine described above. The new approach to be evaluated included, on the same and unique fecal sample, a classical coproparasitological exam plus Rt-PCR. The data obtained showed that the sensitivity of the new proposed approach remains very high, despite the reduction of coproparasitological exams from three to one, with the advantage of reducing costs and saving time, both for patients and for the laboratory.

Four clusters of Trichostrongylus infection diagnosed in a single center, in Italy.

Trichostrongylus spp. are parasites that are seldom recognized as a cause of eosinophilia and gastroenteric symptoms in industrialized countries. The index of suspicion raises when several members of a same household present eosinophilia. We report four clusters of Trichostrongylus infection diagnosed in a single center, in northern Italy. Patients came from four different provinces of three Italian Regions. Some patients presented symptoms (abdominal pain and diarrhea were the most frequent ones, reported by 67 and 42% of our patients, respectively), while other were asymptomatic. All of them presented eosinophilia, that was severe (>5000 eosinophils/mmc) in 58% cases. Obtaining an accurate history from patients, investigating possible ingestion of vegetables contaminated by organic manure or sheep dejections, is particularly important to achieve diagnosis, also in light of the low sensitivity of parasitological tests.

Acute strongyloidiasis in Italian tourists returning from Southeast Asia.

Strongyloidiasis is a soil-transmitted helmithiasis with worldwide distribution. Contrary to chronic form, hyperinfestation and life-threatening dissemination, first (invasive) stages of the disease are not well characterized. This paper describes two cases of acute strongyloidiasis in travelers returning from Southeast Asia and highlights the need to take strongyloidiasis into account also among acute travel-related illnesses.

Evaluation of an indirect immunofluorescence assay for strongyloidiasis as a tool for diagnosis and follow-up.

The diagnostic accuracy of an indirect immunofluorescence antibody test (IFAT) for Strongyloides stercoralis at different serum antibody titers was evaluated. To assess diagnostic sensitivity, sera from 156 patients with known strongyloidiasis were collected. Negative control sera were obtained from a composite group of 427 subjects (blood donors and hospitalized patients). With an area under the receiver-operating characteristic plot of 0.98, the IFAT showed a high level of diagnostic accuracy for strongyloidiasis. An antibody titer of > or = 1:20, with 97% sensitivity and 98% specificity, was identified as the diagnostic threshold with the best overall performance. Cross-reactions were evaluated with 41 additional samples from patients with other known helminth infections, and the IFAT detected low-titer positivity in only one subject with filariasis. A positive IFAT result at an antibody dilution of > or = 1:80 was virtually 100% specific, with 71% sensitivity. To test the usefulness of the IFAT as a monitoring tool, the changes in specific-antibody titers after treatment in a group of 155 patients were evaluated. Seroreversion or a decrease in antibody titer of twofold or more was observed in 60% of the patients. Response to treatment was directly correlated to the initial antibody titer, and a baseline titer of > or = 1:80 was identified as the best predictor of response. In conclusion, a positive IFAT result at an antibody dilution of >/=1:20 is the optimal cutoff for screening. A titer of > or = 1:80, with virtually no false-positive result, is a reliable cutoff for a serological assessment of treatment efficacy and for inclusion in clinical trials.