Strongyloides stercoralis infection: an underlying cause of invasive bacterial infections of enteric origin. Results from a prospective cross-sectional study of a northern Italian tertiary hospital.

PURPOSE OF THE STUDY: We assessed the prevalence of S. stercoralis in a cohort of inpatients with invasive bacterial infections of enteric origin to investigate whether the parasite may facilitate these bacterial infections even in the absence of larval hyperproliferation. METHODS: We performed a prospective cross-sectional study in a hospital in northern Italy. Subjects admitted due to invasive bacterial infection of enteric origin and potential previous exposure to S. stercoralis were systematically enrolled over a period of 10 months. S. stercoralis infection was investigated with an in-house PCR on a single stool sample and with at least one serological method (in-house IFAT and/or ELISA Bordier). Univariate, bi-variate and logistic regression analyses were performed. RESULTS: Strongyloidiasis was diagnosed in 14/57 patients (24.6%; 95% confidence interval 14.1-37.8%) of which 10 were Italians (10/49, 20.4%) and 4 were migrants (4/8, 50.0%). Stool PCR was performed in 43/57 patients (75.4%) and no positive results were obtained. Strongyloidiasis was found to be significantly associated (p ≤ 0.05) with male gender, long international travels to areas at higher endemicity, deep extra-intestinal infectious localization and solid tumors. In the logistic regression model, increased risk remained for the variables deep extra-intestinal infectious localization and oncologic malignancy. CONCLUSIONS: Our findings suggest a new role of chronic strongyloidiasis in favoring invasive bacterial infections of enteric origin even in the absence of evident larval dissemination outside the intestinal lumen. Further well-designed studies should be conducted to confirm our results, and possibly establish the underlying mechanisms.

Humoral Effect of SARS-CoV-2 mRNA vaccination with booster dose in solid tumor patients with different anticancer treatments.

Introduction: Cancer patients are at risk for serious complications in case of SARS-CoV-2 infection. In these patients SARS-CoV-2 vaccination is strongly recommended, with the preferential use of mRNA vaccines. The antibody response in cancer patients is variable, depending on the type of cancer and antitumoral treatment. In solid tumor patients an antibody response similar to healthy subjects has been confirmed after the second dose. Only few studies explored the duration of immunization after the two doses and the effect of the third dose. Methods: In our study we explored a cohort of 273 solid tumor patients at different stages and treated with different anticancer therapies. Results and Discussion: Our analysis demonstrated that the persistence of the neutralizing antibody and the humoral response after the booster dose of vaccine was not dependent on either the tumor type, the stage or type of anticancer treatment.

Evaluation of Nine Commercial Serological Tests for the Diagnosis of Human Hepatic Cyst Echinococcosis and the Differential Diagnosis with Other Focal Liver Lesions: A Diagnostic Accuracy Study.

The differential diagnosis of hepatic cystic echinococcosis (CE) may be challenging. When imaging is insufficient, serology can be applied, but no consensus diagnostic algorithm exists. We evaluated the performances of nine serological tests commercialized in Europe for the diagnosis of "echinococcosis". We performed a diagnostic accuracy study using a panel of sera from patients with hepatic CE (n = 45 "liquid" content stages, n = 25 "solid" content stages) and non-CE focal liver lesions (n = 54 with "liquid" content, n = 11 with "solid" content). The diagnosis and staging of CE were based on ultrasound (gold standard). Nine commercial seroassays (5 ELISA, 2 WB, 1 Chemiluminescence Immunoassay [CLIA] and 1 Immunochromatographic test [ICT]) were the index tests. Sensitivity (Se) ranged from 43 to 94% and from 31 to 87%, and specificity (Sp) from 68 to 100% and from 94 to 100%, when borderline results were considered positive or negative, respectively. Three seroassays (2 ELISA, 1 WB) were excluded from further analyses due to poor performances. When tests were combined, Sp was 98-100%. The best results were obtained using the WB-LDBIO alone (Se 83%) or as a third test after two non-WB tests (Se 67-86%). A validated WB or two non-WB tests, read with stringent criteria (borderline = negative and considered positive only if concordant positive), possibly confirmed by the WB, appear sensible approaches.

Risk of transfusion-transmitted malaria: evaluation of commercial ELISA kits for the detection of anti-Plasmodium antibodies in candidate blood donors.

BACKGROUND: Transfusion with Plasmodium-infected blood represents a risk for malaria transmission, a rare but severe event. Several non-endemic countries implement a strategy for the screening of candidate blood donors including questionnaire for the identification of at-risk subjects and laboratory testing of blood samples, often serology-based, with temporary deferral from donation for individuals with a positive result. In Italy, the most recent legislation, issued in November 2015, introduced the use of serological tests for the detection of anti-Plasmodium antibodies. METHODS: In the absence of a gold standard for malaria serology, the aim of this work was to evaluate five commercial ELISA kits, and to determine their accuracy (sensitivity and specificity) in comparison to immuno-fluorescence antibody test (IFAT), and their agreement (concordance of results). Serum samples from malaria patients or from subjects with malaria history (N = 64), malaria naïve patients with other parasitic infections (N = 15), malaria naïve blood donors (N = 8) and malaria exposed candidate blood donors (N = 36) were tested. RESULTS: The specificity of all ELISA kits was 100%, while sensitivity ranged between 53 and 64% when compared to IFAT on malaria patients samples. When tested on candidate blood donors' samples, ELISA kits showed highly variable agreement (42-94%) raising the possibility that the same individual could be included or excluded from donation depending on the test in use by the transfusion centre. CONCLUSIONS: These preliminary results indicate how the lack of a gold standard for malaria serology must be taken into account in the application and future revision of current legislation. There is need of developing more sensitive serological assays. Moreover, the adoption of a unique serological test at national level is recommended, as well as the development of screening algorithms based on multiple laboratory tests, including molecular assays.

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.

Case Report: Molecular Confirmation of Lobomycosis in an Italian Traveler Acquired in the Amazon Region of Venezuela.

Lobomycosis is a chronic skin mycosis endemic in Amazon regions characterized by chronic nodular or keloidal lesions caused by Lacazia loboi, an uncultivable fungus. Imported cases in nonendemic countries are rare and diagnosed after years. We describe a case of lobomycosis in a healthy 55-year-old Italian traveler who had acquired the infection during 5-day-honeymoon in the Amazon region of Venezuela in 1999. Several weeks after return, he recalled pruritus and papular skin lesions on the left lower limb, subsequently evolving to a plaque-like lesion. Blastomycosis and cryptococcosis were hypothesized based on microscopic morphology of yeast-like bodies found in three consecutive biopsies, although fungal cultures were always negative. In 2016, exfoliative cytology and a biopsy specimen examination showed round yeast-like organisms (6-12 µm), isolated or in a chain, connected by short tubular projections fulfilling the morphologic diagnostic criteria of Lacazia spp. The microscopic diagnosis was confirmed by molecular identification.

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.

Performance evaluation of the automated NucliSens easyMAG nucleic acid extraction platform in comparison with QIAamp Mini kit from clinical specimens.

The performance of the NucliSens easyMAG platform for the extraction of nucleic acid from different clinical specimens was compared with a manual procedure. A total of 308 specimens were analyzed: 209 plasma samples collected for virus detection and quantification of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (n = 70), and 29 for HIV genotyping for drug resistance. Linearity of extraction was tested on dilution series of CMV and EBV; the correlation coefficient (R(2)) for standard curves based on repeated extraction runs was 0.99 for CMV and EBV. Inter- and intrarun variability was in accordance with previous studies, and the correlation between automated and manual extraction was very high. The concordant results were 95.7% for CMV and 100% for EBV. The results of sequence analysis for HIV drug resistance showed a concordance in 24 of 29 specimens. The NucliSens easyMAG is extremely easy to perform, is automated, and resulted in a strong reduction of hands-on time compared with manual protocol.

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 (R2 = 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.

Mycobacterium szulgai identification by hsp65 gene sequencing in an HIV-positive patient with non-Hodgkin's lymphoma.

Mycobacterium szulgai, described for the first time in 1972, is a rare human pathogen that mainly causes pulmonary non-tubercular mycobacteriosis. We report its isolation and identification from a bronchoalveolar lavage specimen by hsp65 gene sequencing analysis in an HIV-positive patient with non-Hodgkin's lymphoma.

Rapid detection and identification of Brachyspira aalborgi from rectal biopsies and faeces of a patient.

This study reports for the first time the detection of Brachyspira aalborgi in faeces and rectal biopsies of a female suffering for 3-4 months of abdominal pain with long-standing mucosal diarrhoea, rectal bleeding and suspected carcinoma of the rectum. After pre-treatment of samples (faeces and biopsies) with a liquid medium (trypticase soy broth-TSB) containing foetal calf serum (FCS, 10%) and spectinomycin and rifampicin (TSB-SR) the first detection of B. aalborgi isolate HBS1 was observed after 48 h in the primary plates of selective blood agar modified medium (BAM) containing spectinomycin and rifampicin (BAM-SR), where growth zones were signalled by a small weakly beta-haemolytic halo. Attempts to subculture spirochaetes in agar media failed. The new HBS1 isolate was only propagated in TSB broth and at electron microscopy it showed 4 endoflagella inserted at each tapered end. The phenotypic characterization of HBS1 demonstrated absence of hippurate hydrolysis, indole production, alpha-galactosidase, alpha- and beta-glucosidase activities in accordance with the B. aalborgi type strain. Rapid identification of B. aalborgi isolate HBS1 was performed directly from faeces and rectal biopsies and subsequently from pure cultures by a genetic method based on 16S DNA restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR). The sequence of 16S DNA amplicon of the isolate HBS1 was found 99.2% corresponding to that of the B. aalborgi type strain. Our results encourage further investigations for the development of a suitable selective agar medium for the isolating and cultivating B. aalborgi from human specimens.

Detection of HTLV-I tax-rex and pol gene sequences of thymus gland in a large group of patients with myasthenia gravis.

We report in this study the use of polymerase chain reaction (PCR) for the amplification of the genomic DNA, isolated from thymic tissue, using the primers flanking HTLV-I/II tax-rex genes and the sequence method to analyze the HTLV-I pol sequence of 27 Italian patients with myasthenia gravis. These molecular methods showed that 92.5% of patients tested positive for tax gene and 55% for pol genes; 55.5% samples were positive for both the tax gene of HTLV-I/II, and the pol gene of HTLV-I. Histologic investigation of the thymus showed that 15 samples had thymic hyperplasia, 93% tested positive for the tax gene, and 40% tested positive for both the tax and pol genes of HTLV-I. In contrast, 91.6% of thymoma-positive samples were positive for tax gene I/II and 75% positive for both genes, tax and pol type I. The sequence analysis of PCR product for tax and pol genes confirmed that these amplified products were HTLV-I, with minimal variations. Our date suggested that either HTLV-I or part of the virus genome is involved in the etiopathogenesis of myasthenia gravis.