MW Polyomavirus in diarrheal Italian infants.

BACKGROUND: MXPyV, like MWPyV, was identified in stool samples from children suffering diarrhea in Mexico. In this study, we used a home-made real time PCR to investigate the presence of this novel viruses in stool specimen collected from under-five-year-old children with gastroenteritis. METHODS: A total of 192 fecal specimens previously screened for RV, ADV, NoV, HPeV and SaV, were tested for MWPyV with Taqman real time PCR. RESULTS: The most detected virus was NoV GII (33.8%), followed by RV (21.3%), SaV (10.9%), HPeV (8%), NoV GI (6.7%) and Adv (1%). Real time PCR detected MWPyV in 1/192 (0.5%) patients. CONCLUSIONS: We detected MWPyV in 0.5% of fecal specimens collected from pediatric patients suffering gastroenteritis which is smaller than the previously reported in literature (4.4% in Australia and 12% Mexico).

Lack of detection of HPyV12 DNA using real-time PCR in Italian infants with diarrhea.

BACKGROUND: HPyV12 was found in organs of the digestive tract, in particular the liver but also in colon, rectum and feces. Until now, the prevalence of HPyV12 is not well characterized. METHODS: In this study, we investigate the presence of this novel polyomavirus DNA in stool specimens collected from under-five-year-old children with gastroenteritis compared to healthy infants. A total of 190 fecal specimens previously screened for rotavirus (RV) and adenovirus (ADV) and 80 fecal samples from healthy infants, were tested for HPyV12 DNA using a home-made real time PCR. All fecal specimens were tested for the presence of HPyV12 with specific primers and probes. RESULTS: None of 190 (0%) episodes of acute gastroenteritis was associated with HPyV12. We did not detect HPyV12 DNA in any of 80 control subjects, as well. CONCLUSIONS: Our study represents a pilot study aiming to clarify the current epidemiological pattern in pediatric Italian patients regarding the novel and rare HPyV12. Based on our negative data and the recent observations reported in literature, doubts remain on human tropism of the HPyV12 and epidemiology: these issues need further investigations.

Unexpected Listeria monocytogenes detection with a dithiothreitol-based device during an aseptic hip revision.

Prosthetic joint infection diagnosis is often difficult since biofilm-embedded microorganisms attach well to the prosthetic surfaces and resist their detection by conventional methods. DL-dithiothreitol has been described as a valid method for biofilm detachment on orthopedic devices. We report the case of an occasional detection of Listeria monocytogenes in a non immuno-compromised patient with a preoperative diagnosis of aseptic loosening. The infection diagnosis due to such rare bacteria was made postoperatively, thanks to a DL-dithiothreitol-based device. This may be considered a feasible approach for the microbiological analysis of prosthetic joint infection, considering that a prompt diagnosis of such biofilm-associated infections could bring some advantages, such as an early and appropriate antibiotic therapy administration and a reduction of undiagnosed infections.

Expression of the pol gene of human endogenous retroviruses HERV-K and -W in leukemia patients.

The human endogenous retroviruses (HERVs) are a family of endogenous retroviruses that integrated into the germ cell DNA of primates over 30 million years ago. HERV expression seems impaired in several diseases, ranging from autoimmune to neoplastic disorders. The purpose of this study was to evaluate the overall endogenous retroviral transcription profile in bone marrow (BM) samples. A total of 30 paediatric high-risk leukaemia patients (lymphoid and myeloid malignancies) were tested for HERVs virus gene expression. Our findings show that HERV-K expression was significantly higher in leukaemia patients when compared to healthy donors of a similar median age. We observed a significantly high expression of HERV-K in acute lymphoblastic leukemia (ALL) patients. In this study, we also found a relative overexpression of the endogenous retrovirus HERV-K in BM cells from the majority of leukemia samples analyzed, in particular in ALL. This overexpression might be related to lymphatic leukemogenesis and it warrants further investigations.

Development of a Low-Cost Stem-Loop Real-Time Quantification PCR Technique for EBV miRNA Expression Analysis.

MicroRNAs (miRNAs) are short, single stranded, non-coding RNA molecules. They are produced by many different species and are key regulators of several physiological processes. miRNAs are also encoded by the genomes of multiple virus families, such as herpesvirus family. In particular, miRNAs from Epstein Barr virus were found at high concentrations in different associated pathologies, such as Burkitt's lymphoma, Hodgkin disease, and nasopharyngeal carcinoma. Thanks to their stability, these molecules could possibly serve as biomarkers for EBV-associated diseases. In this study, a stem-loop real-time PCR for miR-BART2-5p, miR-BART15, and miR-BART22 EBV miRNAs detection and quantification has been developed. Evaluation of these miRNAs in 31 serum samples (12 from patients affected by primary immunodeficiency, 9 from X-linked agammaglobulinemia and 10 from healthy subjects) has been carried out. The amplification performance showed a wide dynamic range (10(8)-10(2) copies/reaction) and sensibility equal to 10(2) copies/reaction for all the target tested. Serum samples analysis, on the other hand, showed a statistical significant higher level of miR-BART22 in primary immunodeficiency patients (P = 0.0001) compared to other groups and targets. The results confirmed the potential use of this assay as a tool for monitoring EBV-associated disease and for miRNAs expression profile analysis.

Advances in Microbiology, Infectious Diseases and Public Health: Fungal Occurrence in the Hair and Skin of Symptomatic Pets in Turin, Italy.

Companion animals, often asymptomatic reservoir of fungi, can be important sources of infection in humans, due to the close contact with their owners. The present study was aimed to assess the occurrence of dermatophytes and other fungi isolated from pet dermatological lesions in Turin, Italy. Dermatological specimens were examined for fungal elements by direct microscopy and cultured to detect dermatophytes, other filamentous fungi and yeasts: 247 pets (118 cats, 111 dogs and 18 dwarf rabbits) were positive for fungal detection in culture. Microsporum canis was the most frequent dermatophyte in cats and dogs, whereas Trichophyton mentagrophytes was the most common in rabbits. Among the other fungi, for all examined pets, dematiaceous fungi were the most isolated, followed by Mucorales, penicilli, yeasts and yeast-like fungi, and aspergilli. No gender predisposition was detected for dermatophyte growth; on the contrary, for the other fungi male cats were more susceptible than female. The highest fungal occurrence was recorded in <1-year-old cats for dermatophytes, and in <5-year-old cats and dogs for the other fungi. Autumn was the period associated with a relevant incidence of fungal infection. Finally, fungi were more frequent in non pure-breed cats and in pure-breed dogs. These data underline the importance to timely inform pet owners about the potential health risk of infection caused not only by dermatophytes but also by non-dermatophyte fungi, routinely considered to be contaminants or harmless colonizers, since their role as source of zoonotic infections is not to be excluded.

Advances in Microbiology, Infectious Diseases and Public Health: Refractory Trichophyton rubrum Infections in Turin, Italy: A Problem Still Present.

Dermatophytosis caused by Trichophyton rubrum is the most common cutaneous fungal infection in industrialized countries and worldwide with high recurrence and lack of treatment response. In addition, patients with cutaneous and concurrent toenail lesions are often misdiagnosed and therefore treated with an inappropriate therapy. In this study, we evaluated five previously misdiagnosed cases of T.rubrum chronic dermatophytosis sustained by two variants at sites distant from the primary lesion. Our patients were successfully treated by systemic and topical therapy, and 1 year after the end of therapy follow-up did not show any recurrence of infection.Our data indicate that the localization of all lesions, the isolation and the identification of the causative fungus are essential to establish the diagnosis and the setting of a correct therapeutic treatment to avoid recurrences.

Caspofungin benefit on phagocytes from patients with renal dysfunction infected with multidrug-resistant Candida glabrata.

AIM: We evaluated the potential impact of caspofungin (CAS) on the functional activities of polymorphonuclear leukocytes (PMNs) from hemodialyzed patients (HDs) and renal transplant recipients (RTRs) against a multidrug-resistant clinical strain of Candida glabrata compared with those of PMNs from healthy subjects (HSs). MATERIALS & METHODS: Effects of CAS on PMN phagocytosis and intracellular killing towards multidrug-resistant C. glabrata were evaluated in 66 HDs, 54 RTRs and 30 HSs in the absence and presence of CAS at MIC and sub-MICs. RESULTS: When HD PMNs and RTR PMNs were exposed to both MICs and sub-MICs of CAS, their fungicidal activity against the multidrug-resistant C. glabrata strain was significantly higher than that of drug-free controls, with survival index values that overlapped with those achieved by HS PMNs. CONCLUSION: The obtained results underline the role of CAS in the restoration of the impaired PMN functions in HDs and RTRs. CAS might still constitute an effective therapeutic option for the treatment of invasive fungal infections caused by multidrug-resistant C. glabrata in patients with altered phagocyte-dependent innate immunity.

Positive interaction of thyme (red) essential oil with human polymorphonuclear granulocytes in eradicating intracellular Candida albicans.

The essential oils have started to be recognized for their potential antimicrobial role only in recent years. Clinical experience showed that the efficacy of antimicrobial agents depends not only on their direct effect on a given microorganism but also on the functional activity of the host immune system. Since data on the effects of essential oils on the innate immune system are scanty and fragmentary, the aim of this study was to evaluate the influence of thyme (red) essential oil (EO), at subinhibitory/inhibitory concentrations, on intracellular killing activity by human polymorphonuclear granulocytes (PMNs) against Candida albicans. In order to provide a frame of reference for the activity of this EO, its in vitro killing activity in the absence of PMNs was also evaluated.Results showed that EO at subminimal inhibitory (subMIC)/minimal inhibitory (MIC) concentrations significantly enhanced intracellular killing of C. albicans in comparison with EO-free controls and was comparable to the positive control (fluconazole). In in vitro killing assays without PMNs, we observed progressive growth of the yeast cells in the presence of EO subMIC/MIC concentrations. A positive antifungal interaction with phagocytes could explain why this EO, which appeared to be only fungistatic in time-kill assays, had efficacy in killing yeast cells once incubated with PMNs.

Functional and phenotypical impairment of polymorphonuclear cells in atopic dermatitis: an additional cause for the known susceptibility to infections?.

BACKGROUND: Atopic dermatitis (AD) patients present an high susceptibility to infections. The phagocytic activity of polymorphonuclear granulocytes (PMNs) is mediated by the interactions between Toll-like receptors (TLRs) and pathogen-associated molecular patterns. OBJECTIVE: To investigate functional activity and phenotype of PMNs in AD patients. METHODS: In vitro PMN phagocytosis and intracellular killing towards Klebsiella pneumoniae were evaluated in 24 AD patients; flow cytometry was applied to analyze PMN phenotype. RESULTS: PMNs from AD patients displayed both reduced phagocytic activity and intracellular killing against K. pneumoniae than healthy subjects (HS). CD11b, CD66b, TLR2, TLR4 and TLR5 median fluorescence intensity (MFI) on PMN membrane were significantly higher in AD patients than in HS. CONCLUSION: PMN functional impairment in AD patients could represent an additional cause of skin infections, coupled with other known defects in the innate immune system. The increased MFI of adhesion molecules and TLRs is rather a consequence of the increased skin barrier permeability to bacterial molecules capable of stimulating immunological reactions.

Role of caspofungin in restoring the impaired phagocyte-dependent innate immunity towards Candida albicans in chronic haemodialysis patients.

Phagocyte-dependent cellular immunity in chronic kidney disease patients undergoing haemodialysis treatment is frequently impaired owing to the uraemic state, resulting in an intrinsic susceptibility to developing invasive fungal infections with high mortality rates. Since synergism between phagocytic cells and antifungal drugs may be crucial for successful therapy, the aim of this study was to evaluate the effects exerted by caspofungin (CAS) on the functional activities of polymorphonuclear cells (PMNs) in haemodialysed patients (HDs) towards Candida albicans compared with those of PMNs from healthy subjects (HSs). PMNs were separated from venous blood samples of 66 HDs and 30 HSs (as controls), and measurement of phagocytic and intracellular fungicidal activities of HD-PMNs and HS-PMNs was performed in the presence of CAS at the minimum inhibitory concentration (MIC) and at sub-MICs. CAS-free controls were also included. In the drug-free test condition, no significant difference between the phagocytic activity of HD-PMNs and HS-PMNs was detected. In contrast, a progressive decline in the intracellular killing activity of HD-PMNs against proliferating yeasts was observed. CAS at MIC and sub-MIC levels was able to improve significantly the intracellular fungicidal activity of HD-PMNs against C. albicans, restoring their functionality. These findings provide evidence that CAS exerts a synergistic effect on HD-PMNs against C. albicans, being able to strength the depressed intracellular killing activity. These results corroborate the use of CAS as an effective therapeutic option for the treatment of invasive fungal infections in HDs, in whom even a marginal influence of antifungal drugs on host response may have a relevant effect.

Candida albicans infections in renal transplant recipients: effect of caspofungin on polymorphonuclear cells.

This study aimed to compare the caspofungin immunomodulating activities against Candida albicans on polymorphonuclear cells (PMNs) from renal transplant recipients (RTRs) and healthy subjects (HSs). RTR PMNs showed a significantly reduced fungicidal activity compared with that of HS PMNs. Addition of caspofungin to RTR PMNs significantly potentiated the yeast intracellular killing rate, achieving values similar to those observed for HS PMNs. These data show that caspofungin is suitable for invasive candidiasis treatment in patients with immune system-impaired components.

Functional and phenotypical alterations of polymorphonuclear cells in Sézary syndrome patients.

Sézary syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma (CTCL), has a poor prognosis and infections represent the most frequent cause of death. Polymorphonucleate granulocytes (PMNs) constitute an essential part of the innate immune system: their phagocytic and killing activity against pathogens is mediated by the interactions between Toll-like receptors (TLRs) and the Pathogen-associated molecular patterns (PAMPs). The aim of this study was to investigate PMN functional activity and phenotype in SS patients and their correlation with the onset of infectious complications. This prospective study enrolled 18 consecutive SS patients; PMN functional activity was evaluated by phagocytosis and intracellular killing tests towards Klebsiella pneumoniae. Flow-cytometry was applied to analyze PMN phenotype. PMNs from SS patients displayed a reduced phagocytic activity and intracellular killing against K. pneumoniae at 30 min and 60 min, more pronounced in SS patients with recurrent infections. CD11b and CD66b median fluorescence intensity (MFI) was significantly higher in SS than in healthy subjects, whereas CD62L MFI was decreased. No significant differences in TLR2, 4, 8 and 9 percentage expression or MFI were found. An increased TLR5 percentage expression was documented. The impairment in PMN functional activities in SS could favour the immune-suppression and raise infection risk.

Prevalence and significance of human parvovirus variants in skin from primary cutaneous T cell lymphomas, inflammatory dermatoses and healthy subjects.

Primary cutaneous T cell lymphomas (CTCL) represent a heterogeneous group of T lymphomas. Virus involvement in CTCL pathogenesis has been extensively investigated, but no data are available as to a causative role of parvovirus B19. The prevalence of parvovirus variants (B19, LaL1/K71, V9) was investigated by using two nested PCRs and a genotype-2 semiquantitative PCR (Q-PCR). Parvovirus DNA was detected in similar percentage in healthy skin controls (40%; n = 42), inflammatory dermatoses (ID) (41%; n = 80) and CTCL (34%; n = 76). Among variants, genotype-2 was more prevalent in ID (26%) and CTCL (22%) than in normal skin (14%; p < 0.05). genotype-3 was never found in normal skin and was rare in ID. The only four pathological skin samples with a quantifiable genome copies/mug DNA values in Q-PCR were ID. In conclusion, despite the skin represent a reservoir for genotype-1, parvovirus infection is not involved in the etiopathogenesis of CTCL.

Epstein-Barr virus in cutaneous T-cell lymphomas: evaluation of the viral presence and significance in skin and peripheral blood.

The importance of viral agents in the development of cutaneous T-cell lymphomas (CTCL) is still debated. For this purpose, we retrospectively evaluated the Epstein-Barr virus (EBV) presence in Sézary syndrome (SS), mycosis fungoides (MF), inflammatory dermatoses (ID), and healthy donors (HD) using different approaches: EBV-DNA was quantified in skin biopsies and peripheral blood using real-time PCR, EBV-encoded small RNA (EBER) transcripts were detected by in situ hybridization (ISH), and latent membrane protein1-2 antigens were detected by immunohistochemistry. Skin biopsies were EBV-DNA-positive in 8/30 (27%) SS, 7/71 (10%) MF, and 2/18 (11%) ID patients and in none of the 25 normal skin samples. Positive mRNA (EBER) signals, always confined to cerebriform T lymphocytes, were found in 5/30 SS patients (17%), whereas signals in all MF and ID patients were negative. The presence of EBV-DNA in skin and blood samples was associated with a significantly lower survival in MF/SS patients. In evaluating EBV serological status, most (>70%) SS, MF, and ID patients showed a serological reactivation demonstrated by the presence of anti-EA IgG. In conclusion, although the finding of EBV-DNA in CTCL does not prove its etiopathogenetic role and may be related instead to immunosuppression, our study demonstrates that it has prognostic relevance.

Non-organ-specific autoantibodies in renal transplant recipients: relation to BK virus infection.

Polyomavirus BK reactivation is common in renal transplant recipients and may cause nephropathy with significant graft dysfunction. The induction of anti-double stranded DNA (anti-dsDNA) antibodies by BKV has been described in experimental animals and during primary infection, and has been implicated in the pathogenesis of systemic lupus erythematosus. This study evaluated the occurrence of anti-dsDNA antibodies and non-organ-specific autoantibodies (NOSA) by indirect immunofluorescence before transplantation and at 3 and 6 months post-transplantation in 90 renal transplant recipients and the association with BKV reactivation, demographic and clinical features. Moreover, the relation to HCMV infection, as detected by pp65-antigenemia, was also evaluated. Post-transplantation NOSAs were present in 23/90 (25.6%) and anti-dsDNA antibodies in 17/90 (18.9%). BK viremia was detected in at least one serum sample in 22 patients: 9 anti-dsDNA antibody-positive vs 13 negative (p<0.01). No significant correlation between the occurrence of NOSAs and anti-dsDNA antibodies and demographic and clinical features was found. No significant association with pp65-antigenemia-positivity was found, although antigenemia was positive in 6/23 NOSA-positive patients (26.1%). Although a relation seems to exist between BKV and the occurrence of anti-dsDNA antibodies in renal transplant patients, the lack of correlation with other epidemiological and clinical features does not allow any conclusion. The role of autoimmune response in this context and the relation with other patient-related factors and infectious agents should be further investigated.

Variants of parvovirus B19: bioinformatical evaluation of nested PCR assays.

Variants of parvovirus B19 are currently grouped into three genotypes: 1 (reference B19 strains), 2 and 3. It has been evidenced that isolate K71 of genotype 2 is more prevalent in skin than the conventional B19 genotype 1. In this study we investigated the detection of parvovirus B19 genotypes by using two nested PCRs and evaluating the suitability of these assays by BLAST search of parvovirus isolates. Subsequently, we analyze the present genotypes in skin biopsies. The two nested PCRs employed in this study allow to amplify 41 isolates as confirmed by bioinformatical validation. The molecular epidemiological characterization of our casistics confirmed the presence of isolate K71 in human skin.

Multiplex-nested RT-PCR to evaluate latent and lytic Epstein Barr virus gene expression.

This paper describes the development of two multiplex-nested RT-PCR devised to evaluate latent/immortalizing (EBNA1, EBNA2, LMP1 and LMP2) and lytic [immediate early (Zebra), early, and late (VCA), respectively] Epstein Barr virus (EBV) transcripts. Subsequently, the assays have been validated evaluating the EBV latent/lytic gene expression in peripheral blood mononuclear cells (PBMC) from immunocompetent subjects (children with primary EBV infection, past EBV infection and no EBV infection) and from immunosuppressed patients (30 asymptomatic renal transplant recipients and 4 liver transplant patients with diagnosed post-transplant lymphoproliferative disorders [PTLD]). Our two multiplex-nested RT-PCR assays provide a reliable, rapid and sensitive system, enabling the simultaneous detection and identification of seven latent/immortalizing and lytic EBV transcripts. These assays could be employed in further investigations in order to evaluate the EBV transcriptional profile in EBV-related diseases both in immunocompetent and immunocompromised hosts.

Effect of dialysis membrane biocompatibility on polymorphonuclear granulocyte activity in dialysis patients.

BACKGROUND: Among patients with defects of the phagocytic component of the immune system, chronic haemodialysis patients are highly susceptible to microbial infections characterized by high morbidity/mortality, related to an impairment of the phagocytic response. Therefore the potential influence of dialysis membrane biocompatibility on the activity of polymorphonuclear (PMN) granulocytes from dialysis patients was investigated in this study. METHODS: Nineteen patients in haemodialysis were included in the protocol and divided into two groups: a control group (7 patients) and a study group (12 patients). The study group patients were treated for subsequent periods of 1 month with different dialysis membranes: low flux excebrane E membrane (CL-E), low flux polysulfone (PS). The control group patients were treated with a low flux modified cellulose membrane (SMC) for the entire observation period. The aetiology of end-stage renal disease included glomerulonephritis, nephroangiosclerosis and interstitial nephropathy. Following each period of treatment, clinical and haematological parameters were evaluated; phagocytosis and microbicidal activity of PMNs from uraemic patients against Klebsiella pneumoniae, the pathogen which can pose severe problems in immune depressed patients, were investigated in parallel. RESULTS: The data evidence that both clinical and haematological parameters remained unchanged during the study period and no differences were found among treatments. On the contrary, the PMN activity varied according to the type of the membrane. In fact, the use of both PS and CL-E, in contrast to SMC, resulted in a PMN functionality similar to that observed in healthy subjects. CONCLUSIONS: These results provide evidence that the depressed PMN activities in dialysis patients may be influenced by membrane biocompatibility in such a way to be totally restored.

Quantitative competitive-PCR assay to measure human parvovirus B19-DNA load in serum samples.

The B19 virus can persist in immunocompromised patients for several months and sometimes even years because of impaired immune response. Viremia in persistent and recurrent infection may range from very low to high titers and may be associated with chronic clinical manifestations, such as chronic anemia. Several recently developed techniques that quantify B19-DNA have improved laboratory diagnosis of the infection and can help guide the choice of treatment in persistent infections (i.e., intravenous immunoglobulin (IVIG) treatment vs immunosuppression reduction). Here we describe the development of a reliable internally controlled quantitative competitive (QC)- polymerase chain reaction (PCR) assay that measures B19-DNA load in serum samples by densitometric analysis of the amplification products for monitoring B19 infection in high-risk patients. A retrospective quantification of B19-DNA in the serum samples from 48 anemic transplanted patients by the QC-PCR assay we developed in our laboratory confirmed the presence of B19-DNA in 11 of 48 samples and showed a viral DNA load between 103 and 108 B19-DNA copies/mL depending on the patients' serostatus (the highest viral load was found in IgM-positive/IgG-negative patients, that is, in patients with active B19 infection at onset). The assay also confirmed B19-DNA negative patients. Our QC-PCR assay may be easily used in monitoring B19 prototype DNA load to follow persistent infections and to better understand the relation between active B19 infection and occurrence of anemia and to assess the efficacy of IVIG therapy or immunosuppression reduction in clearing the virus in high-risk patients.