Stefano Gambarino

Development of a RT Real-time PCR for the detection and quantification of human rhinoviruses

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.

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 donors 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.

Comparison of Two Available RNA Extraction Protocols for microRNA Amplification in Serum Samples

microRNAs play a critical role in many biological processes such as cell proliferation and maturation, apoptosis, regulation of chronic inflammation and development of cancer.

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.

Genotyping of Polyomavirus BK by Real Time PCR for VP1 Gene

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.

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.

Evaluation of UL99 transcript as a target for antiviral treatment efficacy.

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.