Francesca Perandin

Comparison of S. stercoralis Serology Performed on Dried Blood Spots and on Conventional Serum Samples

Background: Dried blood spots (DBS) are used for epidemiological surveys on infectious diseases in settings where limited resources are available. In fact, DBS can help to overcome logistic difficulties for the collection, transport and storage of biological specimens. Objective: To evaluate the accuracy of Strongyloides stercoralis serology performed on DBS. Methods: A survey was proposed to children attending a school in the village of Borbon, Ecuador, and to their parents/guardians. Each participant gave consent to the collection of both serum and DBS specimens. DBS absorbed on filter papers were analyzed with a commercially available ELISA test for S. stercoralis antibodies, as well as with standard serology. The agreement between the two methods was assessed through the Cohen’s kappa coefficient. Results: The study sample was composed of 174 children and 61 adults, for a total of 235 serum and 235 DBS samples. The serology was positive in 31/235 (13%) serum samples, and in 27/235 (11%) DBS: 4 samples resulted discordant (positive at standard serology). Cohen’s kappa coefficient was 0.921 (95% CI 0.845 – 0.998), indicating a high rate of concordance. Conclusion: DBS are suitable for in field-surveys requiring serological testing for S. stercoralis.

A retrospective study comparing agar plate culture, indirect immunofluorescence and real-time PCR for the diagnosis of Strongyloides stercoralis infection

Strongyloides stercoralis is a parasite that can cause death in immunocompromised people. A proper diagnosis is hence essential. The real-time polymerase-chain reaction (RT-PCR) is a novel, promising diagnostic method, that detects the DNA of the parasite in stool samples. In this retrospective study, we compared the sensitivity of agar plate coproculture (APC), an in-house immunofluorescence test (IFAT) and an in-house RT-PCR for the diagnosis of S. stercoralis infection. The study sample was composed by 223 samples. Samples resulting positive to APC, IFAT and RT-PCR were 20, 140 and 25, respectively. When sensitivity was calculated against a composite reference standard, serology confirmed the best performance (sensitivity 95%), followed by RT-PCR (57%) and APC (45%). In conclusion, in a non-endemic setting, serology is the best screening method, while the combination of APC and RT-PCR does not seem a reasonable approach to increase sensitivity. Both methods can have a role as confirmatory tests for selected cases.

Accuracy of parasitological and immunological tests for the screening of human schistosomiasis in immigrants and refugees from African countries: An approach with Latent Class Analysis

Background Schistosomiasis is a neglected infection affecting millions of people, mostly living in sub-Saharan Africa. Morbidity and mortality due to chronic infection are relevant, although schistosomiasis is often clinically silent. Different diagnostic tests have been implemented in order to improve screening and diagnosis, that traditionally rely on parasitological tests with low sensitivity. Aim of this study was to evaluate the accuracy of different tests for the screening of schistosomiasis in African migrants, in a non endemic setting. Methodology/Principal findings A retrospective study was conducted on 373 patients screened at the Centre for Tropical Diseases (CTD) in Negrar, Verona, Italy. Biological samples were tested with: stool/urine microscopy, Circulating Cathodic Antigen (CCA) dipstick test, ELISA, Western blot, immune-chromatographic test (ICT). Test accuracy and predictive values of the immunological tests were assessed primarily on the basis of the results of microscopy (primary reference standard): ICT and WB resulted the test with highest sensitivity (94% and 92%, respectively), with a high NPV (98%). CCA showed the highest specificity (93%), but low sensitivity (48%). The analysis was conducted also using a composite reference standard, CRS (patients classified as infected in case of positive microscopy and/or at least 2 concordant positive immunological tests) and Latent Class Analysis (LCA). The latter two models demonstrated excellent agreement (Cohen’s kappa: 0.92) for the classification of the results. In fact, they both confirmed ICT as the test with the highest sensitivity (96%) and NPV (97%), moreover PPV was reasonably good (78% and 72% according to CRS and LCA, respectively). ELISA resulted the most specific immunological test (over 99%). The ICT appears to be a suitable screening test, even when used alone. Conclusions The rapid test ICT was the most sensitive test, with the potential of being used as a single screening test for African migrants.

Serological and molecular tests for the diagnosis of Strongyloides stercoralis infection in dogs

Strongyloides stercoralis can cause severe infection both in humans and dogs. Coproparasitological examination has low sensitivity for the diagnosis of this parasite; hence, different diagnostic techniques have been implemented. However, serology and molecular methods have been assessed almost exclusively in humans. In this study, two serologic assays and a real-time PCR (RT-PCR), routinely used for the diagnosis of strongyloidiasis in humans, have been tested for the diagnosis in dogs. Five dogs living in the same kennel in Bari, southern Italy, were diagnosed with S. stercoralis infection by detection of larvae in fecal samples processed by the Baermann method. Serum, fecal, and tissue (lungs, scraping of intestinal tract) samples from the same dogs were tested with two serologic assays (commercial ELISA, in-house IFAT) and with an in-house RT-PCR, routinely used for diagnosis in humans. IFAT was positive in all serum samples, ELISA in 3/7 (42.8%) samples. RT-PCR was positive in all pre-treatment fecal samples, in all fecal debris, and in intestinal scraping (three samples from the same deceased dog). The results suggest that IFAT and RT-PCR techniques routinely used for S. stercoralis diagnosis in humans could be useful for the diagnosis of the infection in dogs.

Schistosomiasis in immigrants, refugees and travellers in an Italian referral centre for tropical diseases

BackgroundSchistosomiasis is one of the most important neglected tropical diseases. If unrecognised and untreated, the chronic infection can lead to irreversible complications.MethodsRetrospective observational study aimed at describing clinical history, laboratory findings and imaging presentation of imported schistosomiasis diagnosed at the Centre for Tropical Diseases, Sacro Cuore Don Calabria Hospital of Negrar, Verona, Italy from 2010 to 2014. The aim of our study was to assess differences in demographic characteristics, clinical presentation, laboratory data and ultrasound findings between immigrants/visiting friends and relatives (VFR) from endemic countries (endemic group) and expatriates/travellers (non-endemic group).ResultsA total of 272 patients were retrieved: 234 in the endemic and 38 in the non-endemic group. Most of the patients acquired schistosomiasis in Africa (97.4%). Symptoms were reported by 52.9% of the patients; abdominal pain (36%), macroscopic hematuria (11.3%), and genito-urinary symptoms (7.4%) being the most frequently reported. Increased IgE and blood eosinophilia were observed in 169 (63.8%) and 130 (47.8%) patients, respectively. The proportion of positive serology was 250/272 (91.9%).The Circulating Cathodic Antigen CCA for Schistosoma mansoni was positive in 14/61 individuals (23%). At microscopy, infected subjects were 103/272 (37.9%). The species of Schistosoma found were S. haematobium (47.6%), S. mansoni (46.6%) or both (5.8%). Schistosomiasis was classified as confirmed in 103 (37.9%), probable in 165 (60.6%) and suspected in 4 (1.5%) cases using clinical presentation, laboratory data and ultrasound findings. The infection was further classified based on organ involvement: intestinal (17.9%), hepatosplenic (5.1%), urogenital (48.9%), and indeterminate (43.8%).The comparative analysis of endemic and non-endemic patients highlighted differences in sex and age. Endemic patients had more frequent ova identification (41.9% vs. 13.2%, P < 0.001) and increased IgE (70% vs. 26.3%, P < 0.001) when compared with non-endemic. Multivariate analyses showed that younger age, abnormal ultrasound findings and blood eosinophilia were significantly associated with positive microscopy (OR = 0.94, OR = 2.12, OR = 1.98, respectively).ConclusionsSymptoms, eosinophilia and abnormal ultrasound findings were present in about half of patients, without differences between groups. Many patients had positive serology but negative microscopy, indicating that schistosomiasis might be misdiagnosed. A combination of diagnostic tools may facilitate the diagnosis.

Tick-borne pathogens in removed ticks Veneto, northeastern Italy: A cross-sectional investigation

BACKGROUND In Italy, the incidence of tick-borne diseases in humans is underestimated, as they are not obligatorily notifiable. The aim of this study was to investigate the presence of tick-borne pathogens in ticks removed from human subjects in Veneto region (northeastern Italy), an area for which no published studies are yet available. METHOD Forty-five ticks prospectively removed from human subjects, between March and August 2016, were analysed for bacterial DNA. RESULTS Seven of 45 ticks were infected with bacteria, including human pathogens: 4 Rickettsia spp. (9%), including R. monacensis and R. helvetica; 3 Borrelia spp. and 1 Anaplasma phagocytophilum. Three subjects bitten by infected ticks reported symptoms. CONCLUSIONS Rickettsiosis and anaplasmosis, tick-borne diseases previously not considered in northeastern Italy, should not be neglected. A new survey for a longer period is required to obtain stronger epidemiological data.

A comprehensive analysis of the faecal microbiome and metabolome of Strongyloides stercoralis infected volunteers from a non-endemic area

Data from recent studies support the hypothesis that infections by human gastrointestinal (GI) helminths impact, directly and/or indirectly, on the composition of the host gut microbial flora. However, to the best of our knowledge, these studies have been conducted in helminth-endemic areas with multi-helminth infections and/or in volunteers with underlying gut disorders. Therefore, in this study, we explore the impact of natural mono-infections by the human parasite Strongyloides stercoralis on the faecal microbiota and metabolic profiles of a cohort of human volunteers from a non-endemic area of northern Italy (S+), pre- and post-anthelmintic treatment, and compare the findings with data obtained from a cohort of uninfected controls from the same geographical area (S−). Analyses of bacterial 16S rRNA high-throughput sequencing data revealed increased microbial alpha diversity and decreased beta diversity in the faecal microbial profiles of S+ subjects compared to S−. Furthermore, significant differences in the abundance of several bacterial taxa were observed between samples from S+ and S− subjects, and between S+ samples collected pre- and post-anthelmintic treatment. Faecal metabolite analysis detected marked increases in the abundance of selected amino acids in S+ subjects, and of short chain fatty acids in S− subjects. Overall, our work adds valuable knowledge to current understanding of parasite-microbiota associations and will assist future mechanistic studies aimed to unravel the causality of these relationships.

Preliminary comparison of an in-house real-time PCR with the automated BD Max Enteric Parasite Panel for the detection of Giardia intestinalis.

ABSTRACT Giardia intestinalis is a parasite that commonly causes diarrheal disease throughout the world. An accurate and rapid diagnosis is essential to reduce the infection. Classical diagnosis of giardiasis is performed by microscopic examination of stool samples, but in recent years many DNA-based methods have been developed. In this preliminary observational study, we compared the results of the commercial BD Max enteric parasite panel (EPP) with an in-house real-time (Rt) PCR for G. intestinalis. The study population was composed of 73 samples. Of these, 27 tested positive with both techniques and 39 tested negative. Seven samples were positive with the in-house Rt PCR and negative with the BD Max EPP. The Cohens kappa was 0.805 (95% CI 0.670–0.940). In conclusion, these preliminary results suggest that the Rt-PCR could possibly demonstrate higher sensitivity for the diagnosis of G. intestinalis than BD Max EPP, which tended to miss infection of low intensity.

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.