Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV) DNA in plasma

BACKGROUND: Epstein-Barr virus (EBV) DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy) and an in-house assay (EBV RQ-PCR). RESULTS: The range of linearity and the degree of precision of the two assays were similar. The clinical sensitivity of Q-EBV PCR was higher for reference samples containing less than 1,000 EBV DNA copies/ml. The absolute quantitative results of the two methods were statistically correlated (R(2 )= 0.7789; p < 0.0001), with the systematic overestimation by EBV RQ-PCR possibly linked to different amplification efficiency in calibration standards. CONCLUSION: Both the commercial and the in-house assay may be appropriate for clinical use, but common standards are advisable for comparable absolute values, as these would improve the clinical utility of EBV DNA load measurement

Nosocomial outbreak of the pandemic Influenza A (H1N1) 2009 in critical hematologic patients during seasonal influenza 2010-2011: detection of oseltamivir resistant variant viruses

BACKGROUND: The pandemic influenza A (H1N1) 2009 (H1N1pdm09) virus infection caused illness and death among people worldwide, particularly in hematologic/oncologic patients because influenza infected individuals can shed virus for prolonged periods, thus increasing the chances for the development of drug-resistant strains such as oseltamivir-resistant (OST-r) variant. METHODS: The aim of our study was to retrospectively evaluate the clinical importance of OST-r variant in circulating strains of the pandemic H1N1pdm09 virus. By means of RT-PCR and Sanger sequencing we analysed the presence of OST-r variant in 76 H1N1pdm09 laboratory-confirmed cases, hospitalized at the hematologic/oncologic ward at Spedali Civili of Brescia –Italy. RESULTS: Out of 76 hospitalized hematologic/oncologic patients, 23 patients (30.2%) were infected by H1N1pdm09 virus. Further investigation revealed that 3 patients were positive for the OST-r variant carrying the H275Y mutation. All the 23 infected patients were immuno-compromised, and were under treatment or had been treated previously with oseltamivir. Three patients died (13%) after admission to intensive care unit and only one of them developed H275Y mutation. CONCLUSIONS: Our retrospective observational study shows that pandemic influenza A (H1N1) 2009 virus can cause significant morbidity and even mortality in hematologic/oncologic patients and confirms the high rate of nosocomial transmission of pandemic H1N1pdm09 virus in these critical subjects. Indeed, the reduction in host defences in these hospitalized patients favoured the prolonged use of antiviral therapy and permitted the development of OST-r strain. Strategies as diagnostic vigilance, early isolation of patients and seasonal influenza A(H1N1) vaccination may prevent transmission of influenza in high risk individuals

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

A diagnostic study comparing conventional and real-time PCR for Strongyloides stercoralis on urine and on faecal samples

Abstract Strongyloides stercoralis is a soil-transmitted helminth with a wide distribution in tropical and subtropical areas. The diagnosis of S. stercoralisinfection can be challenging, due to the low sensitivity of microscopic examination of stool samples and coproculture. In the last decade, different in-house molecular biology techniques for S. stercoralis have been implemented. They demonstrated good accuracy, although sensitivity does not seem sufficiently high yet. Recently, a novel PCR technique has been evaluated for the detection of S. stercoralis DNA in urine. Aim of this work was to compare the sensitivity of the real-time PCR (qPCR) on feces routinely used at the Centre for Tropical Disease (CTD) of Negrar, Verona, Italy, with that of the novel based PCR on urine. As secondary objective, we evaluated a Urine Conditioning Buffer ® (Zymoresearch) with the aim of improving nucleic acid stability in urine during sample storage/transport at ambient temperatures. Patients attending the CTD and resulting positive at routine screening with serology for S. stercoralis were invited, previous written consent, to supply stool and urine samples for molecular biology. A convenience sample of 30 patients was included. The sensitivity of qPCR on feces resulted 63%, and that of based PCR on urine was 17%. In all the samples treated with the Urine Conditioning Buffer ® there was no detectable DNA. In conclusion, the sensitivity of the novel technique resulted low, and needs further implementation before being considered as a valid alternative to the validated method

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

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper. Erratum for Statistical optimization of light intensity and CO2 concentration for lipid production derived from attached cultivation of green microalga Ettlia sp. [Sci Rep. 2018

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

Prevalence of Chagas disease and strongyloidiasis among HIV-infected Latin American immigrants in Italy – The CHILI study

INTRODUCTION: Screening HIV-positive migrants for neglected tropical diseases having potential for life-threatening reactivation, such as Chagas disease and strongyloidiasis is not widely implemented. We evaluated the prevalence of these infections among a large cohort of HIV-infected migrants from Latin America living in Italy. METHOD: Cross-sectional study evaluating the prevalence of Trypanosoma cruzi and Strongyloides stercoralis infections in HIV-infected migrants from Latin America enrolled in the Italian Cohort of Antiretroviral-Naïve patients (ICONA) between 1997 and 2018, based on serology performed on sera stored in the ICONA Foundation biobank. Screening for Chagas disease was performed using two commercial ELISA complemented by commercial Immunoblot and CLIA if discordant. Strongyloidiasis was evaluated using a commercial ELISA. RESULTS: 389 patients were analysed. Fifteen (3.86%) had at least one positive Chagas ELISA test. Prevalence of Chagas disease was 0.5% or 1.29% depending on the confirmatory technique. Serology for strongyloidiasis was positive in 16 (4.11%) patients. Only Nadir CD4+ T cell count was associated with discordant serology for Chagas disease (p = 0.046). CONCLUSIONS: The accuracy of seroassays for Chagas disease and strongyloidiasis in HIV-positive patients is unclear. To avoid missing potentially life-threatening infections, we suggest implementing additional diagnostic strategies in at-risk patients with inconclusive serology results

Relationship between pp65 antigenemia levels and real-time quantitative DNA PCR for Human Cytomegalovirus (HCMV) management in immunocompromised patients

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time PCR assays, which are more rapid and practical than pp65 antigenemia determination, are progressively becoming the preferred method for monitoring Human Cytomegalovirus (HCMV) reactivation. However, the relationship between HCMV DNA and antigenemia levels is still under investigation. The aim of this study was to analyse the relationship between HCMV DNA and pp65 antigenemia levels in order to identify clinically useful threshold values for the management of patients.</p> <p>Methods</p> <p>475 consecutive samples from 156 immunosuppressed patients were tested for HCMV by pp65 antigenemia and Real-time PCR assay.</p> <p>Results </p> <p>136 out of 475 consecutive samples derived from 48 patients showed evidence of HCMV infection. HCMV DNA was detected in 106 samples, pp65 antigen in 3, and both markers in 27. pp65 antigen detection was associated with higher HCMV DNA levels. The cut-off HCMV DNA level that best predicted pp65 antigenemia in this series of samples was 11,500 copies/ml, but different threshold levels could be observed for specific groups of patients. HCMV disease was observed in 5 out of 48 patients with active HCMV infection. The presence of clinical symptoms was associated with positive pp65 and with higher antigenemia levels. Higher HCMV DNA load at the onset of viral replication was correlated to the development of clinical symptoms.</p> <p>Conclusion</p> <p>Both pp65 antigenemia and HCMV DNA load can be useful for the prospective monitoring of immunocompromised subjects. Specific cut-off levels capable of triggering preemptive antiviral treatment should be determined in accordance to the type of test used and the characteristics of patients and prospectively validated.</p

Digital PCR: a new technology for diagnosis of parasitic infections

Parasitic infections are responsible for a significant burden of disease worldwide with international travel and immigration. More accurate diagnostic tools are necessary in support to parasite control and elimination programmes in endemic regions as well as for rapid case detection in non-endemic areas. Digital PCR (dPCR) is a powerful technology with recent applications in parasitology

Improved detection of DNA Schistosoma haematobium from eggs extracted by bead beating in urine

Diagnosis of Schistosoma haematobium relies primarily on microscopical analysis of urine. The method is time consuming and requires some expertise. Genus-specific real-time PCRs have been developed, but we still observed low sensitivity. In the present study, in order to achieve a more sensitive DNA detection of eggs of S. haematobium in urine samples, we wanted to develop a novel protocol of DNA extraction using mechanic disruption of eggs by bead beating as supplementary step. We tested Schistosoma spp. internal transcribed spacer 2 real-time PCR after both methods with and without bead beating. First, we preliminary assessed the DNA detection after bead beating using dilution of 2, 10, 50, and 90 eggs/10 mL, and the Ct value analysis showed significant improved DNA detection per each point of egg concentration using the novel supplementary step. Twenty microscopy positive and five microscopy negative urine samples were used to validate the procedure. All urines came from imported cases and admitted at center for tropical medicine, and were examined by microscopy. PCR results after novel method with bead beating showed 100% to be positive for S. haematobium, compared with 85% positive by our standard extraction procedure. Results confirmed mechanic disruption of eggs by bead beating before DNA extraction to be highly effective method for the detection of S. haematobium DNA in urine