Evaluation of IFN-γ polymorphism+874 T/A in patients with recurrent tonsillitis by PCR real time mismatch amplification mutation assay (MAMA real time PCR).

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<0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject (p=0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard.

Detection of Mimivirus in bronchoalveolar lavage of ventilated and nonventilated patients.

BACKGROUND/AIMS: We investigated the prevalence of Mimivirus in bronchoalveolar lavage (BAL) specimens from ventilated versus nonventilated patients. METHODS: The occurrence of Mimivirus DNA was evaluated by two previously developed real-time PCR assays in 69 BAL specimens: 30 from patients on mechanical ventilation for at least 48 h and 39 from nonventilated patients from different clinical settings, including lung transplant recipients. RESULTS: None of the BAL specimens from ventilated and nonventilated patients resulted positive for Mimivirus. CONCLUSION: This study, similarly to other studies that used molecular assays to detect Mimivirus, found no occurrence of the virus in the lower respiratory tract, thus being in contrast to serological investigations which reported a significant association between Mimivirus and the development of pneumonia. Gene polymorphism could explain these results or, alternatively, it could be hypothesized that Mimivirus does not represent a common cause of lower respiratory tract infection in either ventilated or nonventilated patients. Further studies on a larger population of patients from a different clinical setting evaluating both serology and DNA detection in lower respiratory tract specimens, including BAL and possibly tissue samples, could allow a better definition of the epidemiological and pathological role of Mimivirus in the development of pneumonia.

In vitro CMV-infection model in fresh and glycerolized skin graft.

Viral infections, especially cytomegalovirus (CMV), are a cause of death in burned patients. Aim of this study was to perform an in vitro CMV-infection model comparing fresh and glycerol-treated fibroblasts and keratinocytes. Cells were plated in plates for the two conditions. Each plate was set up with CMV dilutions. Immunofluorescence and real time PCR assays were performed. The assays were negative in both fresh and glycerolized keratinocytes. For fibroblasts, CMV-DNA was positive in both conditions and immunofluorescence test only in fresh cells. Glycerol at 85% confirms its strong virucidal effect as reported also for other viruses.

Detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients.

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 (p < 0.0001), 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 of >10(5) possibly being associated with clinical symptoms, although lower loads can be detected in both symptomatic and asymptomatic patients.

Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease and may contribute to the onset and development of Crohn's disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species.

Human cytomegalovirus glycoprotein B genotyping from bronchoalveolar lavage specimens.

The genes encoding glycoprotein complexes of human cytomegalovirus are often polymorphic; in particular, glycoprotein B (gB), which is essential for both in vivo and in vitro replication, is encoded by the highly polymorphic gene UL55. In this study, the distribution of gB genotypes was investigated in 44 bronchoalveolar lavage specimens from adult patients positive for human cytomegalovirus DNA by a multiplex nested fast PCR able to amplify 5 gB genotypes (gB1-gB5). The distribution of gB genotypes was as follows: 12 (27.3%) gB1, 11 (25%) gB2, 9 (20.4%) gB3, 4 (9.1%) gB4, 0 gB5, and 8 (18.2%) mixed genotypes. No difference in prevalence was found in relation to clinical features, including immunological status, non-transplant or transplant condition, and type of transplanted organ, or in follow-up specimens; while gB4 and gB3 were shown to be significantly more prevalent in patients with respiratory insufficiency, and gB4 and gB2 in those with pneumonia. The prevalence of gB genotypes in the lower respiratory tract was similar to that previously reported using other specimen types and patients, with gB1 found to be the most prevalent. The association of gB genotypes with specific clinical features should be further investigated.

Non-organ-specific and anti-endothelial antibodies in relation to CMV infection and acute rejection in renal transplant recipients.

The presence of non-organ-specific (NOSA) and anti-endothelial antibodies (AECAs) and the onset of rejection in relation to cytomegalovirus (CMV) infection was investigated in 96 renal transplant recipients: 48 CMV pp65-antigenemia-negative (group 1) and 48 positive (group 2). The presence of autoantibodies (autoAbs) was evaluated before and following renal transplantation (first three months) by indirect immunofluorescence. Before transplantation, none of the patients was positive to AECAs, while eight (8.3%) were positive to NOSAs. Post-transplantation, AECA were found in none of patients from group 1 vs. 15/48 (31.2%) from group 2 (p<0.05); NOSAs were detected in 9/48 (18.8%) and 9/48 patients from group 1 and 2, respectively. An acute rejection was diagnosed in ten cases: six of interstitial type (antigenemia-, and AECA-negative; two NOSA-positive); four of vascular type (all of them NOSA-negative, 3/4 antigenemia-, and AECA-positive). CMV infection did not seem to be significantly associated with the appearance of NOSAs, while there was a significant correlation with the occurrence of AECAs. No significant correlation was found between acute rejection and the occurrence of NOSAs, while 75% of the cases of vascular rejection was associated to CMV infection and AECA-positivity, suggesting the pathogenic role of CMV-mediated endothelial damage.

Detection of human herpesvirus-7 DNA in bronchoalveolar lavage.

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 cytomegalovirus load in fresh and glycerolized skin grafts.

This study evaluated the detection of Human Cytomegalovirus (HCMV)-DNA in donors' skin samples. HCMV-DNA was quantified in 100 skin specimens, including 50 fresh samples and as many corresponding glycerol-preserved specimens by a home-made Real Time PCR. HCMV-DNA was detected in 19/50 (38%) fresh specimens and 23/50 (46%) glycerol-preserved (p = n.s.). Nevertheless, the mere detection of HCMV-DNA does not imply the presence of infectious virions and therefore does not imply a risk of HCMV transmission, as treatment with glycerol is particularly efficacious in inactivating viral particles. Therefore, HCMV serology confirms its pivotal role in the setting of skin grafting.

Polyomaviruses BK- And JC-DNA quantitation in kidney allograft biopsies.

BACKGROUND: Polyomavirus-associated nephropathy (PVAN) is one of the most common viral disease affecting renal allograft, with BK being the most frequent causal agent and JCV being considered responsible in <3% of the cases. OBJECTIVES: To quantify polyomaviruses BK and JC load by real-time TaqMan PCR in tissue specimens (renal and ureteral) from kidney transplant recipients. STUDY DESIGN AND METHODS: One-hundred-thirty-eight specimens (125 kidneys, 13 ureters) obtained from 109 patients were evaluated by quantitative real-time PCR for the detection of BKV- and JCV-DNA. Demographic, virological, and histopathological data were collected. RESULTS: BKV-DNA was positive in 32 of 109 patients (29.6%) and JCV-DNA in 20 of 109 patients (18.3%). The highest BK viral loads (>10(4) genome equivalents/cell) were found in two renal samples with histopathologically confirmed PVAN; while JC viral load was >10(4) genome equivalents/cell in one ureteral sample. CONCLUSIONS: Although quantitation of viral DNA on renal allograft biopsies could be complementary to histopathological evaluation and the highest viral load are detectable in renal specimens with PVAN, the identification of a diagnostic cut-off should require further studies.

Polyomavirus-associated nephropathy: critical issues in virological monitoring.

Polyomavirus-associated nephropathy is one of the most common viral complications in renal transplant recipients. Viral replication is the single common feature of all patients at risk of nephropathy. Therefore, screening for virus replication allows for the identification of patients at risk of developing nephropathy and permits earlier intervention, in particular a pre-emptive reduction of immunosuppression, with improvement of outcome. This paper describes the modes of virological monitoring and its role in the clinical management of renal transplant patients.

What role for human rhinoviruses in the lower respiratory tract?

Human rhinoviruses (HRV) usually cause upper airway infections. However, viral replication in the tracheobronchial tree has been disclosed, although its clinical role is poorly known. We evaluated the prevalence of HRV in 159 bronchoalveolar lavages from 88 patients and describe a lung transplant recipient with a high HRV load in association with acute rejection. HRV was detected in 22/88 patients (25.0%): 7/18 lung transplant recipients, 11/41 immunocompetent, and 4/29 immunocompromised (p = n.s.). No lung disease was significantly associated with HRV positivity. It should be recommended to include HRV in the virological diagnostic work-up of lower respiratory specimens to elucidate their role.

Detection of PARV4, genotypes 1 and 2, in healthy and pathological clinical specimens.

The molecular epidemiology and tissue distribution of Human Parvovirus 4 (PARV4) and its variant PARV5 (Parvoviridae family) are poorly known. The aim of this study was to investigate the epidemiological role and prevalence of PARV4/5 by a nested-PCR on different clinical specimens, including blood samples from healthy donors, healthy and pathological skin samples, and bronchoalveolar lavages (BAL). Among blood specimens, 2/53 were positive; 3/37 and 23/105 of healthy and pathological skin specimens resulted positive, respectively, whereas no BAL was positive. PARV4/5 may be present in different healthy and pathological samples, suggesting the need for further investigating its tissue distribution.

Reverse transcriptase-polymerase chain reaction to evaluate human cytomegalovirus lytic gene expression.

This paper describes the development of four Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assays devised to evaluate lytic (UL123, immediate-early; UL54, early; UL65, late; and UL99, true late) human cytomegalovirus (HCMV) transcripts. Subsequently, the assays have been validated evaluating the HCMV lytic gene expression in 28 samples (peripheral blood leukocytes, PBLs) from 14 renal transplant recipients. Although the assessment of HCMV transcriptional profile could be useful to evaluate viral reactivation state, further studies on dynamics of viral transcripts in different clinical settings are needed to determine the role of transcriptional profiling in virological monitoring and therapeutic management in immunocompromised patients.

Monitoring of BK virus replication in the first year following renal transplantation.

BACKGROUND: BK virus-associated nephropathy (BKVAN) is one of the most common viral diseases affecting renal allografts. Screening for viral replication may allow for earlier intervention with reduced allograft loss. A plasma viral load >10(4) copies/mL of BKV DNA is recommended for a presumed diagnosis of BKVAN. METHODS: We monitored BKV load on serum and urine samples by Real-Time TaqMan PCR in 229 renal transplant recipients in the first year post-transplantation. Overall, 2025 serum and 2025 urine samples were evaluated. A graft biopsy was performed in 47/229 patients to investigate the declining renal function. Operating characteristics [sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV)] and receiver operating characteristic (ROC) curve analysis at different viral load values were calculated. RESULTS: Serum BKV viral load was >10(4) in 5/229 patients (2.2%). A histological diagnosis of BKVAN was made in 3/229 patients (1.3%): 3/5 (60.0%) among those with serum viral load >10(4) and 3/4 (75.0%) in those with >1.6 x 10(4). Operating characteristics of a serum BK load of 10(4) for the diagnosis of BKVAN were as follows: sensitivity, 100%; specificity, 99.1%; NPV, 100%; PPV, 59.4%. Specificity and PPV rose to 99.6% and 75.0% when using a cut-off level of 1.6 x 10(4) copies/mL. CONCLUSIONS: The recommended level of BK viraemia of 10(4) copies/mL is useful to identify patients at risk of BKVAN, although specificity and PPV increase by using a cut-off level of 1.6 x 10(4) copies/mL. BK replication may occur in the first 3 months post-transplantation and subsequently recede. Therefore, the temporal profile of BKV replication has to be accurately evaluated and occasionally elevated values should prompt a closer monitoring.

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.

Egg and Milk Proteins as Hidden Allergens in Food: 5-Year (2010 to 2014) Results of Food Allergen Monitoring in Piedmont, Italy.

Cow's milk and egg allergies are two of the most common food allergies. Manufacturers of food products containing milk or eggs or their derivatives as an ingredient are required by European Union regulations to list their presence on the ingredient label. Under European Union legislation, member states are mandated to carry out food safety monitoring programs to verify compliance with food labeling requirements. Through the Regional Integrated Plan for Food Safety, the Piedmont (Italy) regional authority carries out an annual program to determine the presence of undeclared allergens in foods. In the 5-year period from 2010 to 2014, a total of 1,566 food samples were analyzed for the presence of hidden egg and milk proteins. The average positive percentage was 2.8% (3.6% egg and 2% milk proteins). Comparison between the allergen concentration and the published eliciting dose (ED) for egg proteins (0.03 mg) and for total milk proteins (0.1 mg) indicated a high risk of allergen exposure for sensitized consumers. The calculated exposure was up to 135× (for milk) the ED01 reported in the literature. Food manufacturers will need to improve their allergen control programs to reduce allergen exposure and risk.

Wound botulism after traumatic open fracture in Italy.

Seventeen days after a traumatic open fracture, a Clostridium botulinum wound infection was diagnosed, with self-limiting symptoms. This is the first report of wound botulism in Italy and the authors discuss the possible role of aerosolized contamination of the wound prior to hospital admission.

Consumers' Perception and Knowledge of Food Safety: Results of Questionnaires Accessible on IZSalimenTO Website.

The present survey was undertaken to investigate consumers' knowledge of the main foodborne agents and dietary regimen during pregnancy. Data were collected using monthly questionnaires available on IZSalimenTO website between March 2013 and January 2014. Hepatitis A virus questionnaire: 20 respondents (77%) recognized berries as foodstuff linked to the outbreak of hepatitis A. The majority correctly indicated as precautionary advice to boil berries before consumption. Botulism questionnaire: 29 respondents (62%) indicated pesto as food involved in botulism alert in July 2013. The risk of infant botulism in infant less than 1 year old due to honey consumption is known by 24 respondents (51%). Main foodborne disease questionnaire: the risk of infection by Salmonella after the consumption of foods made with raw eggs is known by the majority (94%; N=17) as well as the treatments to be applied in order to make fresh fish safe from parasites (76%). Pregnancy questionnaire: 20 respondents (74%) believed that washing vegetables and fruits with sodium bicarbonate or chlorate solution is able to inactivate Toxoplasma; only 4 (15%) reported both raw meat and vegetables washed with sodium bicarbonate as food at risk. Results indicate that all consumers should be trained on behaviour and dietary regimen to be adopted in pregnancy and in infant <1 year old. The website may be considered as a useful tool to assess consumers' knowledge: both the news section and the contents published may be a source of information and education for consumers on food safety.

Foodborne botulism associated with home-preserved turnip tops in Italy.

In Italy, foodborne botulism is a rare disease mainly due to home-preserved food. In the case reported here, clinical diagnosis was performed on the basis of clinical signs and referred consumption of home-preserved turnip tops in oil. Definitive diagnosis was performed by detection of botulinum toxin in sera and neuro-toxigenic organisms in stools and leftover food. This case report highlights the need of a high medical awareness, prompt clinical diagnosis, and synergic collaboration among the health authorities for a correct management of botulism as well as disease containment.