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Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.

Background microRNAs play a critical role in many biological processes such as cell proliferation and maturation, apoptosis, regulation of chronic inflammation and development of cancer. Methods In this study is described a protocol for the isolation of RNA from serum and subsequent determination of miRNA expression levels using TaqMan-based MGB Real-Time PCR detection. RNA was extracted using two different isolation methods including available kits RNAzol and a modified RNAzol protocol. In all cases, RNA was eluted in RNase free H2O, kept frozen until analysis and the presence of contaminants assessed by NanoDrop spectrophotometry. Results Higher RNA quantity was observed in RNAzol (378.8 ng/μl) vs RNAzol modified protocol (226.5 ng/μl) and a better performance in terms of RNA extraction yield and purity. Subsequently, measurements of endogenous miRNAs (RNU43), cellular miRNAs (mir155 and mir146a) and EBV miRNAs (mirBART2-5p, mirBART15 and mirBART22) were performed by RT-qPCR. Conclusion In contrast to the findings in terms of purity and quantity, the amplifiable RNA was more abundant using RNAzol modified protocol compared to not modified protocol.

Polyomavirus BK latently persist in different sites, including the renourinary tract, and may reactivate causing nephropathy in renal transplant recipients or hemorrhagic cystitis in bone marrow recipients. Based on the sequence of the VP1 gene, four genotypes have been described, corresponding to the four serologically differentiated subtypes I-IV, with different prevalence and geographic distribution. In this study, the development and clinical validation of four different Real-Time PCR assays for the detection and discrimination of BKV genotypes as a substitute of DNA sequencing are described. 379 BK VP1 sequences, belonging to the main four genotypes, were aligned and "hot spots" of mutation specific for all the strains or isolates were identified. Specific primers and probes for the detection and discrimination of each genotype by four Real-Time PCR assays were designed and technically validated. Subsequently, the four Real-Time PCR assays were used to test 20 BK-positive urine specimens from renal transplant patients, and evidenced a prevalence of BK genotype I, as previously reported in Europe. Results were confirmed by sequencing. The availability of a rapid and simple genotyping method could be useful for the evaluation of BK genotypes prevalence and studies on the impact of the infecting genotype on viral biological behavior, pathogenic role, and immune evasion strategies.

Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP)+874∗T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donor's blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p

Human cytomegalovirus (HCMV) is a virus belonging to the Beta Herpes virus family. Its genome contains many different genes clustered in immediate early, early and late genes. This last cluster includes UL99, a late gene that encodes for a tegument protein called pp28. In immunocompetent patients, HCMV infection occurs asymptomatically, while its reactivation in immunocompromised patients can be a cause of pneumonia, retinitis and gastrointestinal diseases. To prevent or to contrast HCMV infection, several drugs (such as Ganciclovir, Acyclovir, Foscarnet) are available, and their efficiency is evaluated by HCMV DNA load monitoring, as also for antiviral resistance onset that may occur after the therapy. In this study is described the development of a Real Time PCR for the detection and quantification of UL99 transcript and the clearance of this target compared to HCMV DNA, both in vitro and in vivo on bronchoalveolar lavage samples.

Immunofluorescenza diretta (DFA) su cellule HEP2 e R-Mix Too per la rilevazione degli Adenovirus

The occurrence of human rhinoviruses (HRV) and its relationship to clinical and histopathological findings were investigated in 127 bronchoalveolar lavage specimens from 36 lung transplant recipients by real-time RT-PCR. In addition, 286 samples from 235 other immunocompromised and immunocompetent patients were also studied. HRV was detected in 41.7% of lung transplant recipients vs 14.5% of other patients (p0.0001), and no differences in viral load were observed. Acute respiratory insufficiency was found in 15 cases, three of which were HRV positive (viral load, 6.3 x 10(6) RNA copies/ml in one patient with chronic graft dysfunction). A diagnosis of pneumonia was made in 10 out of 127 cases, two of which were HRV positive (viral load, 10(3)-10(4) in cases of co-infection). Acute rejection was diagnosed in 12 cases, three of which were HRV positive (viral load, 10(3) in two cases of co-infection and 10(5) in a single infection). HRV infection may involve the lower respiratory tract, particularly in the presence of an impaired pulmonary background, such as a transplanted lung. Clinical evaluation should take into account the viral load, with a load of10(5) possibly being associated with clinical symptoms, although lower loads can be detected in both symptomatic and asymptomatic patients.

Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study, a cRNA-standard-based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us. Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intra- and inter-test variability, sensitivity, and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra- and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07 log was found, showing a significant underrate using plasmid standard-based quantification. The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.

Introduction. Human Bocavirus (HBov) is a ubiquitous parvovirus predominantly associated with respiratory tract infections in pediatric patients.The results of the few studies conducted in adult patients are conflicting, with data of prevalence around 0.8%, thus making difficult to evaluate the HBOV clinical role. In this study the prevalence and clinical role of HBoV in adult hospitalized patients were evaluated. Methods. The presence of HBoV was evaluated in 514 bronchoalveolar lavages (BAL), obtained over a period of 24 months from 341 adult patients (mean age, 56.5 ± 16.2 years, range 19-85), by real-time TaqMan PCR.The BAL was performed for the presence of symptoms/signs of suspected infection of the lower airways or for surveillance in the transplanted lung patients (month 1 and every 3 months). Results. 12/341 patients (3.5%) were positive for HBoV; in particular: 1/45 (2.2%) lung transplanted, 1/20 (5%) liver transplanted and 2/26 (7.7%) bone marrow transplanted, while no kidney (n=19) or heart (n=13) transplant patients resulted positive. 8/218 (3.6%) not transplantpatient were positive, in particular 1.8% patients with haematological cancers and 1.8% with other disease.The prevalence of HBoV did not differ between transplanted and not transplanted patients, depending on the type of transplant, and between immunocompetent and immunocompromised individuals. Conclusions. HBoV can be detected at low frequency in BAL of adult patients with acute lower respiratory tract, further studies would show whether the virus plays a role as a single pathogen or whether the altered lung background could help the viral infection.

Introduction: The Cytomegalovirus (CMV) is the major cause of morbidity and mortality in solid organ (SOT) and bone marrow (BMT) transplantation. An early reconstitution of immune response is crucial in limiting CMV replication; on the other hand, a late reconstitution may determine CMV reactivation with possible evolution to symptomatic phase.The Enzyme-Linked Immunosorbent Spot (EliSPOT) assay is a useful tool for monitoring the CMV-specific immune response recovery in transplant patients.This study propose the development and optimization of a interferon-gamma-(IFN-γ)-based EliSPOT assay for the detection of CMV immune response. Methods: CD3 + lymphocytes were separated using Robosep (negative selection, HLA ® EasySep WB Human T Cell Enrichment Kit, Stemcell Technology,Vancouver, Canada).The EliSPOT assay was optimized using the Human IFN gamma ELISPOT Kit and a modified protocol provided by Nanogen Advanced Diagnostics (Buttigliera Alta, Italy). Different parameters were analyzed: number of cells (200,000 - 300,000), antigens (CMV peptide mix and whole CMV antigen), incubation time (18 - 20 - 22 - 24h), number of washes, incubation conditions with secondary antibody and conditions of substrate development. Results: Herein we report the optimal conditions identified. 200,000 CD3+ cells per well were stimulated with CMV peptide mix antigens. Cells were stimulated for 18h at 37 °C in humidified atmosphere; wells were washed and secondary antibody was added and incubated at room temperature for 2h.After incubation a second series of washes were performed. Substrate was added and incubation for 15 minutes was carried out.The reaction was detected by plate reader AID ELISPOT (Strassberg, Germany). Conclusions:We developed an EliSPOT assay for the detection of CMV-immune response and reconstitution.All variables were compared to previous published protocols.The improvement of the EliSPOT assay for the detection of CMV-specific immune response represents the first step for result evaluation in clinical practice.

The respiratory syncytial virus (RSV) is the major cause of respiratory infection in children, with bronchopneumonia and bronchiolitis. In the adult the clinical manifestation is lighter than in infant, however, in the case of immunocompromised patients (transplant recipients or patients in immunosuppressive treatment) infection can lead to death. Immunofluorescence is a diagnostic procedures which can detect RSV infection and the presence of viable virus in the biological sample of the patient. In this study, indirect and direct immunofluorescence techniques using monoclonal antibodies directed to RSV have been compared. For the comparison HEP2 shell vials, that are susceptible to RSV, were used.These cells were infected with different virus titres, ranging from 102 to 10-3 TCID50. Subsequently, different primary or Fluorescein isothiocyanate antibody dilutions were tested (1:40, 1:80, 1:160), for indirect and direct immunofluorescence evaluation, respectively, at three incubation periods after infection (48h, 72h, 96h). Indirect immunofluorescence showed a sensitivity of 101 TCID50, with a cells positive detection even at lower dilutions up to 10-1 TCID50 at 72 hours. For direct immunofluorescence, the same sensitivity was observed with 1:40 antibody dilution at 72h, with a detection limit of 10-1 TCID50. Indirect immunofluorescence showed optimal RSV detection at 72h after infection with 1:80 primary antibody dilution, with a sensitivity of 101 TCID50. An equivalent sensitivity has been observed with direct immunofluorescence at 72h, 1:40 antibody dilution, thus representing an advantage in terms of time.

Objectives: Human herpesvirus-7 (HHV-7) is a highly seroprevalent virus that, following primary infection, establishes latency or persistence in some tissues, including lung. The aim of this study was to investigate the prevalence of HHV-7 in the lower respiratory tract of hospitalized adult patients. Methods: The prevalence of HHV-7 DNA was determined by quantitative real-time PCR in 212 bronchoalveolar lavage (BAL) samples obtained from 153 patients. The molecular epidemiology and clinical role of HHV-7 were evaluated. Results: HHV-7 DNA was positive in 44 of 212 specimens (20.7%), obtained from 40 of 153 patients (26.1%), in particular 22/68 (32.35%) and 18/86 (20.9%) in transplant and non-transplant patients, respectively (1 patient evaluated both before and after transplantation). No significant difference according to transplant condition or discharge diagnosis was found. Viral load was >100,000 genome equivalents/ml BAL in 6/22 (27.3%) transplant recipients and 4/18 (22.2%) non-transplant patients (p = n.s.). Conclusions: The evaluation of HHV-7 DNA in BAL may be useful to investigate its potential role in lower respiratory tract infection, alone or in association with other viral and/or non-viral pathogens and to distinguish latency from reactivation.

Human Rhinoviruses (HRV) are the most common viral agents, being responsible for upper as well as lower respiratory tract infections. Evidence demonstrating that HRV disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of HRV infections and the increasing number of immunocompromised patients underline the need for including HRV in virological diagnostics of acute lower respiratory tract illness. This article describes the development and optimization of a reverse transcription (RT) real-time PCR assay for quantification of HRV RNA in clinical samples. Efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated on bronchoalveolar lavage (BAL) specimens obtained from immunocompetent and immunocompromised patients.