First autochthonous dengue outbreak in Italy, August 2020.

In August 2020, during the coronavirus disease (COVID-19) pandemic, five locally acquired cases of dengue virus type 1 were detected in a family cluster in Vicenza Province, North-East Italy where Aedes albopictus mosquitoes are endemic. The primary case was an importation from West Sumatra, Indonesia. This is the first outbreak of autochthonous dengue reported in Italy. During the COVID-19 pandemic, screening of febrile travelers from endemic countries is crucial in areas where competent vectors are present.

The production method affects the efficacy of platelet derivatives to expand mesenchymal stromal cells in vitro.

BACKGROUND: The use of fetal bovine serum (FBS) as a media supplement for the ex vivo expansion of bone-marrow derived mesenchymal stromal cells (BM-MSC) has been discouraged by regulatory agencies, due to the risk of transmitting zoonoses and to elicit immune reactions in the host once transplanted. Platelet derivatives are valid FBS substitutes due to their content of growth factors that can be released disrupting the platelets by physical methods or physiological stimuli. We compared platelet derivatives produced by freezing/thawing (platelet lysates, PL) or after CaCl2 activation (platelet releasate surnatant rich in growth factors, PR-SRGF) for their content in growth factors and their ability to support the ex vivo expansion of BM-MSC. METHODS: The cytokine content in the two platelet derivatives was evaluated. BM-MSC were expanded in complete medium containing 10, 7.5 and 5% PL or PR-SRGF and the cell phenotype, clonogenic capacity, immunomodulation properties and tri-lineage differentiation potential of the expanded cells in both media were investigated. RESULTS: The concentration of PDGF-AB, PDGF-AA, PDGF-BB in PR-SRGF resulted to be respectively 5.7×, 1.7× and 2.3× higher compared to PL. PR-SRGF promoted a higher BM-MSC proliferation rate compared to PL not altering BM-MSC phenotype. Colony forming efficiency of BM-MSC expanded in PR-SRGF showed a frequency of colonies significantly higher than cells expanded in PL. BM-MSC expanded in PL or PR-SRGF maintained their immunomodulatory properties against activated lymphocytes even if BM-MSC expanded in FBS performed significantly better. CONCLUSIONS: The method used to release platelet factors significantly affects the enrichment in growth factors and overall product performance. The standardization of the production process of platelet derivatives and the definition of their release criteria requires further investigation.

Extracorporeal therapy in the treatment of sepsis: In vitro assessment of the effect of an absorbent cartridge on the circulating bacterial concentration and its interaction with the antibiotic therapy.

Sepsis is one of the major causes of death worldwide. In its physiopathological process, a broad spectrum of pro and antiinflammatory mediators plays a strategic role, leading to a sepsis induced state of immunoparalysis. The rationale behind the employment of extracorporeal purification techniques as a complement to therapy for sepsis is based on their ability to remove the mediators involved. Until now, attention was focused on the immunomodulation allowed by purification therapies. However, the focus of studies on the application possibilities that these techniques offer as a supplement to antimicrobial therapy and resuscitation of critically ill patients must be extended. In this study, the possible removal by adsorption that the Jafron® HA330 cartridge operates against bacteria (S. aureus) was evaluated in vitro. Subsequently, it was evaluated whether the adsorptive capabilities toward bacteria were maintained by using a cartridge functionalized with Vancomycin and whether the latter maintains its bactericidal activity. This study showed that HA330 reduces the circulating bacterial load, even in the presence of pre-adsorbed Vancomycin. Vancomycin, once adsorbed by the cartridge, does not guarantee its bactericidal activity during the 2-h of hemoperfusion treatment.

Co-infection by two linezolid-resistant coagulase-negative staphylococci with two different resistance determinants.

Linezolid resistance among Gram-positive pathogens is being reported with increasing frequency. We examined 14 linezolid-resistant coagulase-negative staphylococci (CoNS) isolated from blood cultures obtained from patients admitted to the Intensive Care Unit of Vicenza General Hospital, Italy. The species identification yielded 10 Staphylococcus epidermidis, 3 Staphylococcus hominis, and 1 Staphylococcus capitis. Minimal inhibitory concentrations of linezolid ranged between 16 and 32 mg/l. By sequencing domain V of the 23S rRNA gene, 4 isolates were found to harbour a G2576T mutation and 10 isolates a G2447T mutation. None of the strains under study presented either the cfr gene or cardinal mutations in the L3, L4, or L22 riboproteins. In this clinical collection of linezolid-resistant CoNS the G2447T mutation was dominantly associated with S. epidermidis, while the G2576T mutation was found in other CoNS species. Two different CoNS species endowed with either mutation were isolated from 2 patients.

Evaluating the role of a fully automated SARS-CoV-2 antigen ECLIA immunoassay in the management of the SARS COV 2 pandemic on general population.

OBJECTIVES: Chemiluminescence immunoassay (CLIA) automated assays (fourth-generation antigen test) for SARS-CoV-2 detection are promising because of their analytical productivity, but have lower sensitivity and specificity than rt-PCR assays. The authors of this paper evaluated a recent immunoassay implemented on Siemens Atellica IM, investigating how much this could affect the actual feasibility of this diagnostic during the pandemic. METHODS: From the three-day routine 134 positive and 241 negative swab samples by rt-PCR test were evaluated, selected as 1/3 positive - 2/3 negative. RESULTS: Using rt-PCR as gold standard, the specificity of immunoassay was 96.7%, while sensitivity was 68.0%. Sensitivity is inversely proportional to the viral load: 100% for cycles threshold (CT) values from 14 to 29, 95% until 30 CT, then 85, 74, 72, 68%, for 31-35 CT respectively. CONCLUSIONS: Our study confirms the reliability of the fourth-generation antigen assay in recognizing negative samples. Conversely, sensitivity appears to be less reliable (68.0%) than reported in the literature. This could be due to a non-randomized study group: many swab samples were taken from patients with expected low viral load (hospitalized for COVID for more than 10-12 days or asymptomatic patients for epidemiological surveillance). The strong correlation of sensitivity and viral load could prove significant to track the infectiousness of infected people, as previous studies reported that a viral load of at least 10E6 copies of RNA/mL, corresponding to 25 CT, is the threshold of transmission of the disease.

Electrospun Chitosan Functionalized with C12, C14 or C16 Tails for Blood-Contacting Medical Devices.

Medical applications stimulate the need for materials with broad potential. Chitosan, the partially deacetylated derivative of chitin, offers many interesting characteristics, such as biocompatibility and chemical derivatization possibility. In the present study, porous scaffolds composed of electrospun interwoven nanometric fibers are produced using chitosan or chitosan functionalized with aliphatic chains of twelve, fourteen or sixteen methylene groups. The scaffolds were thoroughly characterized by SEM and XPS. The length of the aliphatic tail influenced the physico-chemical and dynamic mechanical properties of the functionalized chitosan. The electrospun membranes revealed no interaction of Gram+ or Gram- bacteria, resulting in neither antibacterial nor bactericidal, but constitutively sterile. The electrospun scaffolds demonstrated the absence of cytotoxicity, inflammation response, and eryptosis. These results open the door to their application for blood purification devices, hemodialysis membranes, and vascular grafts.

A flow cytometric assay for the quantification of MSC lysis by peripheral blood mononucleated cells.

Mesenchymal stromal cells (MSC) are attractive candidates for the treatment of acute graft versus host disease (aGvHD) or autoimmune disorders. However, mechanisms of MSC recognition remain unclear and there are evidences that MSC are not totally immunoprivileged. Data suggest that MSC undergo apoptosis after infusion in presence of cytotoxic cells and their death could drive immunosuppression. In GvHD patients, that activity was associated with clinical response. It is mandatory to develop an in vitro potency testing predictor of the "in vivo" response to the therapy. We describe a flow cytometric assay based on differential immunostaining of target and effector cells where BM MSC are enumerated with fluorospheres to determine the loss of target cells after co-culture with PB MNC. 6/13 (46%) of BM MSC lots were lysed by PB MNC and the lysis was proportional to the E/T cell ratio. The method overcomes the problems linked to the use of dyes or radioactive, evidencing the limitations linked to the use of a single vital dye and proposing a precise gating strategy based on absolute cell counts where cells are left untouched. The assay is easy and could be used to predict the response of the patients to the therapy.

Autochthonous dengue outbreak in Italy 2020: clinical, virological and entomological findings.

BACKGROUND: In August 2020, in the context of COVID-19 pandemics, an autochthonous dengue outbreak was identified for the first time in Italy. METHODS: Following the reporting of the index case of autochthonous dengue, epidemiological investigation, vector control and substances of human origin safety measures were immediately activated, according to the national arbovirus surveillance plan. Dengue cases were followed-up with weekly visits and laboratory tests until recovery and clearance of viral RNA from blood. RESULTS: The primary dengue case was identified in a young woman, who developed fever after returning from Indonesia to northern Italy, on 27 July 2020. She spent the mandatory quarantine for COVID-19 at home with relatives, six of whom developed dengue within two weeks. Epidemiological investigation identified further five autochthonous dengue cases among people who lived or stayed near the residence of the primary case. The last case of the outbreak developed fever on 29 September 2020. Dengue cases had a mild febrile illness, except one with persistent asthenia and myalgia. DENV-1 RNA was detected in blood and/or urine in all autochthonous cases, up to 35 days after fever onset. All cases developed IgM and IgG antibodies which cross-reacted with West Nile virus (WNV) and other flaviviruses. Sequencing of the full viral genome from blood samples showed over 99% nucleotide identity with DENV-1 strains isolated in China in 2014-2015; phylogenetic analysis classified the virus within Genotype I. Entomological site inspection identified a high density of Aedes albopictus mosquitoes, which conceivably sustained local DENV-1 transmission. Aedes koreicus mosquitoes were also collected in the site. CONCLUSIONS: Areas in Europe with high density of Aedes mosquitoes should be considered at risk for dengue transmission. The presence of endemic flaviviruses, such as WNV, might pose problems in the laboratory diagnosis.

Compliance of clinical microbiology laboratories with recommendations for the diagnosis of bloodstream infections: Data from a nationwide survey in Italy.

In 2014, the Italian Working Group for Infections in Critically Ill Patient of the Italian Association of Clinical Microbiologists updated the recommendations for the diagnostic workflow for bloodstream infections (BSI). Two years after publication, a nationwide survey was conducted to assess the compliance with the updated recommendations by clinical microbiology laboratories. A total of 168 microbiologists from 168 laboratories, serving 204 acute care hospitals and postacute care facilities, were interviewed during the period January-October 2016 using a questionnaire consisting of nineteen questions which assessed the level of adherence to various recommendations. The most critical issues were as follows: (a) The number of sets of blood cultures (BC) per 1,000 hospitalization days was acceptable in only 11% of laboratories; (b) the minority of laboratories (42%) was able to monitor whether BCs were over or under-inoculated; (c) among the laboratories monitoring BC contamination (80%), the rate of contaminated samples was acceptable in only 12% of cases;(d) the Gram-staining results were reported within 1 hr since BC positivity in less than 50% of laboratories. By contrast, most laboratories received vials within 2-4 hr from withdrawal (65%) and incubated vials as soon as they were received in the laboratory (95%). The study revealed that compliance with the recommendations is still partial. Further surveys will be needed to monitor the situation in the future.

Anti SARS-CoV-2 antibodies monitoring in a group of residents in a long term care facility during COVID-19 pandemic peak.

Objectives Clinical laboratories plays a key role in screening, diagnosis and containment of the Coronavirus 2019 infection epidemic. The etiological diagnosis presupposes the isolation of virus genetic material in the patient's biological sample but laboratory diagnostics also make use of searching possibility for immunoglobulin (Ig)G, IgM classes antibodies. The characteristics of the antibody response are not yet completely clear. Methods This study describes a serological monitoring of subjects, elderly nursing care residence guests, interested by a very large infection outbreak. After first nasopharyngeal swab, all the positive subjects (43) were monitored for the persistence of the virus infection through nasopharyngeal swab after 20 days (16-24), 32 days (28-36) and after 49 days (47-50). At the same time, during the second (day 32) and third (day 49) follow up, all the guests were investigated for IgM and IgG anti SARS-CoV-2 antibodies, by using a quantitative chemiluminescence method. Results Thirty two days after performing the first diagnostic swab, 39 of 43 patients (90%) had IgG higher than the cut off value. After 49 days the four patients with negative IgG were still negative. The comparison of the levels of IgG-Ab between the controls shows a significant decrease in concentrations (-10%). Conclusions Our study confirms that in most patients affected by COVID-19 there is a typical antibody response with IgG-Ab present in 90% of nursing care COVID-19 positive residence guests. For IgM-Ab only 23% of tested subjects were positive on the 32nd and 49th day of illness, always in parallel with the IgG-Ab positivity.

Genomic analysis of cardiac surgery-associated Mycobacterium chimaera infections in Italy.

One hundred and twenty-two Mycobacterium chimaera strains isolated in Italy from cardiac surgery-related patients, cardiac surgery-unrelated patients and from heater-cooler units, were submitted to whole-genome sequencing and to subsequent SNP analysis. All but one strains isolated from cardiac surgery-related patients belonged to Subgroup 1.1 (19/23) or Subgroup 1.8 (3/23). Only 28 out of 79 strains isolated from heater-cooler units belonged to groupings other than 1.1 and 1.8. The strains isolated from cardiac surgery-unrelated patients were instead distributed across the phylogenetic tree. Our data, the first on isolates from Italy, are in agreement with a recent large genomic study suggesting a common source, represented by strains belonging to Subgroups 1.1 and 1.8, of cardiac surgery-related Mycobacterium chimaera infections. The strains belonging to groupings other than 1.1 and 1.8 isolated from heather-cooler units evidently resulted from contaminations at hospital level and had no share in the Mycobacterium chimaera outbreak. One Mycobacterium chimaera strain investigated in this study proved distant from every previously known Mycobacterium chimaera Groups (1, 2, 3 and 4) and we propose to assign to a novel group, named "Group 5".

In vitro effects of glycyrrhetinic acid on the growth of clinical isolates of Candida albicans.

Compounds derived from Glycyrrhiza glabra L. root have been used widely for centuries for their numerous therapeutic properties. The present study aimed to test the in vitro activity against Candida albicans strains of the compound 18-beta glycyrrhetinic acid (18-beta GA), derived from the root of Glycyrrhiza species. This antimicrobial activity was assessed using the National Committee for Clinical Laboratory Standards (NCCLS) method on C. albicans strains that were isolated from patients with recurrent vulvovaginal candidiasis (RVVC). The in vitro growth of the C. albicans strains was markedly reduced, in a pH-dependent manner, by relatively low doses (6.2 microg/mL) of 18-beta GA. The results demonstrate that 18-beta GA is a promising biological alternative for the topical treatment of recurrent vulvovaginal candidiasis (RVVC).

Over Expressed TKTL1, CIP-2A, and B-MYB Proteins in Uterine Cervix Epithelium Scrapings as Potential Risk Predictive Biomarkers in HR-HPV-Infected LSIL/ASCUS Patients.

High oncogenic risk human papillomaviruses (HR-HPVs) promote cervical carcinoma development, the fourth most common feminine cancer. A slow oncodevelopmental phase-defined histopathologically as Cervical Intraepithelial Neoplasia (CIN) grades 1-3, or cytologically as Low- or High-grade Squamous Intraepithelial Lesions (LSIL or HSIL)-precedes the malignancy. Cervical carcinoma screenings through HR-HPV genotyping and Pap smears are regularly performed in Western countries. Faulty cytology screening or genotyping or patients' non-compliance with follow-ups can let slip an oncoprogression diagnosis. Novel biomarker tests flanking HR-HPV genotyping and cytology could objectively predict the risk of disease progression thus helping triage LSIL/ASCUS patients. Here, anonymized leftovers of fresh cervical epithelium scrapings from twice (LSIL/ASCUS and HR-HPV DNA)-positive and twice (Pap smear- and HR-HPV DNA)-negative (control) patients in a proteome-preserving solution served to assess the biomarker worth of three cervical carcinoma-related proteins, i.e., B-MYB (or MYBL2), Cancerous Inhibitor of PP2A (CIP-2a), and transketolase-like1 (TKTL1). Leftovers anonymity was strictly kept and storage at -80°C, protein extraction, immunoblotting, and band densitometry were blindly performed. Only after tests completion, the anonymous yet code-corresponding HR-HPV-genotyping and cytology data allowed to assign each sample to the twice-positive or twice-negative group. Descriptive statistics showed that the three proteins levels significantly increased in the twice-positive vs. twice-negative scrapings. Diagnostic ROC curve analysis identified each protein's Optimal Decision Threshold (OTD) showing that TKTL1 and CIP-2a are stronger risk predictive biomarkers (Sensitivity, 0.91-0.93; Specificity, 0.77-0.83) than B-MYB. Logistic Regression coupled with Likelihood-Ratio Tests confirmed that a highly significant relation links increasing TKTL1/CIP-2a/B-MYB protein levels in twice-positive cervical scrapings to the risk of HR-HPV-driven oncoprogression. Finally, a 3 year clinical follow-up showed that 13 patients (50% of total) of the twice-positive group with biomarker values over OTDs compliantly underwent scheduled colposcopy and biopsy. Of these, 11 (i.e., 84.7%) received a positive histological diagnosis, i.e., CIN1 (n = 5; 38.5%) or CIN2/CIN2+ (n = 6; 46,2%). Therefore, TKTL1/CIP-2a/B-MYB protein levels could objectively predict oncoprogression risk in twice (HR-HPV- and Pap smear)-positive women. Further studies will assess the translatability of these findings into clinical settings.

Staphylococcus caprae meningitis following intraspinal device infection.

A case is reported of Staphylococcus caprae meningitis due to infection of an intraspinal analgesia pump. The subclinical and pauci-symptomatic clinical course of the infection strongly suggested a chronic device contamination.

Prevalence of cervical colonization by Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium in childbearing age women by a commercially available multiplex real-time PCR: An Italian observational multicentre study.

BACKGROUND: Mycoplasmas are frequently isolated from the genital tract. New molecular PCR-based methods for the detection of mycoplasmas can better define the real epidemiology of these microorganisms. The aim of this study was to evaluate the prevalence of mycoplasmas in a population of childbearing age women by means of PCR. METHODS: This 21-month multicentre observational study was conducted at four Italian clinical microbiology laboratories. Women reporting symptoms of vaginitis/cervicitis, or with history of infertility, pregnancy, miscarriage or preterm birth were included. Detection of Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium was performed from cervical swabs by means of a commercially available multiplex real-time PCR. RESULTS: a total of 1761 women fulfilled the inclusion criteria and were included in the study. The overall prevalence was: U. parvum 38.3%, U. urealyticum 9%, M. hominis 8.6% and M. genitalium 0.6%. The proportion of foreign patients positive for U. parvum was significantly higher compared to Italian patients (37% vs 30.1%, p = 0.007) and also for overall mycoplasma colonization (53.4% vs 45.8%, p = 0.011). The number of symptomatic patients positive for M. hominis was significantly higher than that of negative controls (2.9% vs 1%, p = 0.036). A significant positive trend in mycoplasma colonization was found in relation to the pregnancy week for U. urealyticum (p = 0.015), M. hominis (p = 0.044) and for overall mycoplasma colonization (p = 0.002). CONCLUSION: multiplex RT-PCR can be a valuable tool to evaluate the real epidemiology of cervical mycoplasma colonization.

Protective effect of urate oxidase on uric acid induced-monocyte apoptosis.

Uremic patients have a higher risk of infection and malignancy than normal subjects. Previous studies have deomonstrated that monocytes isolated from uremic patients display an increased apoptosis rate compared to normal subjects; furthermore uremic plasma can increase apoptosis rates on U937, a human monocytic cell line. In several pathological conditions, precipitation of uric acid crystals can lead to renal insufficiency or acute renal failure by different mechanisms. In recent studies uric acid has been shown to induce inflammatory response from monocytes and it has been suggested to be involved in cell dysfunction. Rasburicase is a new recombinant urate oxidase developed to prevent and treat hyperuricaemia in patients with cancer or renal failure; it degrades uric acid to allantoin, a less toxic and more soluble product. In the present study, we aimed at determining whether uric acid may be a factor affecting U937 apoptosis, and whether urate oxidase may reduces or even prevent uric acid induced cell apoptosis. Hoechst staining and internucleosome ledder fragmentation of DNA showed that uric acid increased the percentage of apoptotic cells comparing to the control and that when the U937 cells were incubated with uric acid and urate oxidase the percentage of apoptosis significantly decreased (from 43+/-7% to 19+/- 3%, p<0.05). Also, the activity of caspase-8 and caspase-3 showed the same trend (caspase 3: from 2.7+/-0.53 to 1.6+/-0.42; caspase-8: from 2.2+/-0.43 to 1.3+/-0.57). A reduction of intracellular reduced glutathione (GSH) concentration was found in uric acid treated cells while the addition of urate oxidase in the uric acid incubated cells decreased the GSH extrusion. The concentration of TNF-alpha was increased in the sample incubated with uric acid comparing to the control. Uric acid is an inducer of apoptosis on U937 cell line, and therefore it may be a component of the mosaic of uremic toxins both in acute and chronic renal disease. We can hypothesize that uric acid might be directly involved in the apoptotic process trough the activation of both death receptor and mitochondrial-mediated pathways. We have, also, demonstrated that urate oxidase is able to prevent at least in part, the effect of uric acid on U937 apoptosis. This effect might be a result of different mechanisms of action.

Epstein-Barr virus DNA load in chronic lymphocytic leukemia is an independent predictor of clinical course and survival.

The relation between Epstein-Barr virus (EBV) DNA load and clinical course of patients with chronic lymphocytic leukemia (CLL) is unknown. We assessed EBV DNA load by quantitative PCR at CLL presentation in mononuclear cells (MNC) of 220 prospective patients that were enrolled and followed-up in two major Institutions. In 20 patients EBV DNA load was also assessed on plasma samples. Forty-one age-matched healthy subjects were tested for EBV DNA load on MNC. Findings were validated in an independent retrospective cohort of 112 patients with CLL. EBV DNA load was detectable in 59%, and high (≥2000 copies/µg DNA) in 19% of patients, but it was negative in plasma samples. EBV DNA load was significantly higher in CLL patients than in healthy subjects (P < .0001). No relation was found between high EBV load and clinical stage or biological variables, except for 11q deletion (P = .004), CD38 expression (P = .003), and NOTCH1 mutations (P = .05). High EBV load led to a 3.14-fold increase in the hazard ratio of death and to a shorter overall survival (OS; P = .001). Poor OS was attributable, at least in part, to shorter time-to-first-treatment (P = .0008), with no higher risk of Richter's transformation or second cancer. Multivariate analysis selected high levels of EBV load as independent predictor of OS after controlling for confounding clinical and biological variables. EBV DNA load at presentation is an independent predictor of OS in patients with CLL.

Rapid detection of Paenibacillus larvae from honey and hive samples with a novel nested PCR protocol.

The bacterial pathogen Paenibacillus larvae is the causative agent of American foulbrood disease in honeybees (Apis mellifera). A touchdown nested PCR protocol was developed to detect the presence of P. larvae spores directly in honey and hive samples. This approach allows early discovery of the bacteria even at concentrations below pathogenic levels, opening the door to new prophylactic approaches against American foulbrood and real-time epidemiological studies.

Four year longitudinal study of Mycobacterium tuberculosis complex isolates in a region of North-Eastern Italy.

Recent reports have suggested a change of Mycobacterium tuberculosis complex genetic diversity in Western Europe due to an increasing proportion of imported cases of tuberculosis (TB). This study analyzed a total of 705 M. tuberculosis strains isolated from 2006 to 2009 in Veneto, a North-Eastern Italian region, to see the impact of foreign-born cases vs. Italian patients on prevailing TB epidemiology. Strains were genotyped using spoligotyping followed by comparison with international genotyping database SITVIT2. Six spoligotyping clusters with suspected phylogeographical specificity for imported cases, were typed by 15-loci MIRUs for a finer characterization. Overall, 410 (58.16%) strains were isolated from foreign-born patients, while 295 (41.84%) were isolated from Italian patients. Older patients (>70 years, i.e., 46.4% of cases) predominated among Italians while younger age groups prevailed among foreign-born patients. Our results suggest that despite a high proportion of reactivation of latent TB infection in elderly Italian-born patients, active TB transmission between foreign-born and Italian patients may be ongoing, and argue in favor of an increased TB surveillance among immigrants to combat TB epidemic in Italy.