Gabriella Gregori

Polyomavirus BK DNA quantification assay to evaluate viral load in renal transplant recipients

BACKGROUND Several studies have disclosed a correlation between polyomavirus BK (BKV) and interstitial nephritis in renal transplant recipients and its quantification in urine and serum is therefore required to assess the role of BKV infection in nephropathy. OBJECTIVE This paper describes a urine and serum BKV-DNA quantification protocol devised to evaluate the viral load. STUDY DESIGN Screening of samples containing > or =10(3)/ml viral genome copies by a semi-quantitative polymerase chain reaction (PCR) assay is followed by precise quantification of the samples containing a high number of viral genomes in a quantitative-competitive (QC)-PCR assay. Generation of the competitor construct relied on the different sizes of wild-type and competitor amplicons. RESULTS AND CONCLUSIONS Screening by semi-quantitative PCR selects samples with a high number of viral genomes for use in the more labor-intensive and -expensive QC-PCR assay and thus provides a handy means for quantitative DNA analysis of large numbers of samples. The results obtained in BKV-DNA quantification in urine and serum samples from 51 renal transplant recipients (22 on treatment with tacrolimus (FK506) and 29 on cyclosporine A (Cy A)) are interesting: BKV-DNA findings (43.1%) in urine samples are in agreement with the BKV urinary shedding reported in literature (5-45%). With regard to immunosuppressive treatment, the percentage of activation of the infection (revealed by BKV-DNA detection in urine samples) in the two groups of therapy is similar (40.9% vs 44.8%). The observation that the viral load in urine is dissociated with that of serum suggests that both parameters should be investigated in evaluation of the pathogenetic role of BKV reactivation in renal transplant recipients. Moreover, our BKV-DNA quantification protocol could be used to monitor viral load in urine and serum samples from renal transplant recipients so as to detect those at risk of nephropathy and monitor their response to immunosuppression reduction therapy if it occurs.

Kinetics and prediction of HBsAg loss during therapy with analogues in patients affected by chronic hepatitis B HBeAg negative and genotype D

In patients affected by chronic hepatitis because of HBV infection, long‐term suppressive therapy with nucleos(t)ides analogues in the HBeAg− patients has shown low effects on HBsAg titre (qHBsAg) decrease, and HBsAg loss is difficult to achieve. Thus, in this type of patients the main goals of antiviral therapy is the suppression of HBV‐DNA and ALT normalization.

Recent outbreak of aseptic meningitis in Italy due to Echovirus 30 and phylogenetic relationship with other European circulating strains

BACKGROUND Enteroviruses (EVs) are common human viral pathogens, causing a variety of diseases, including aseptic meningitis. Recently, EV aseptic meningitis outbreaks have been reported across Europe, but, in Italy, knowledge of recent EV molecular epidemiology is very limited. OBJECTIVES We report an outbreak of EV aseptic meningitis in 10 adults in North-Western Italy, from October to November 2012. Patients were parents or close relatives of children <5 years old attending the same class of a nursery school, suffering from a mild febrile upper respiratory disease. Phylogenetic relationship with other European circulating strains was analyzed updating E30 circulation in Italy in recent years. STUDY DESIGN EVs were detected from cerebrospinal fluid (CSF) specimens with a real-time reverse transcription polymerase chain reaction and virus isolation was achieved from rectal and pharyngeal swabs. For cluster definition and phylogenetic studies, viral VP1 region was directly amplified and sequenced from CSF. RESULTS EVs were identified in CSF from all patients and from rectal and pharyngeal swabs in 7 of them. Direct sequencing of CSF revealed the presence of the same Echovirus 30 (E30) in all patients and phylogenetic analysis identified it as a diverging clade within E30 genotype VII, the most recent strain circulating in UK, Finland and Denmark since 2006. CONCLUSION Molecular techniques allowed the rapid identification and typing of E30 from CSF. Phylogenetic analysis revealed that the cluster might be due to a new E30 variant within the genotype VII currently circulating in Europe, thus updating the epidemiology of EV circulation in Italy.

Combination of conventional blood cultures and the SeptiFast molecular test in patients with suspected sepsis for the identification of bloodstream pathogens

We evaluated performances of the molecular test SeptiFast (SF) for the detection of agents of bloodstream infection (BSI) in patients with suspected sepsis, the majority of them under antibiotic treatment and at high prevalence of HIV-1 infection (10.5%). Matched SF and blood culture (BC) samples (n=1186) from 1024 patients were studied. Two hundred fifty-one episodes of BSI out of 1144 were identified with the combined methods (22%). SF identified more episodes of BSI than BC: 206 versus 176 (χ(2)=7.008, P=0.0081) and a significantly higher number of Gram-negative bacteria than BC (77 versus 53, χ(2)=9.12; P=0.0025), as well as of polymicrobial infections (χ(2)=4.50, P=0.0339). In conclusion, SF combined with BC improved the diagnosis of sepsis, especially in immunocompromised patients.

Diagnosis of dengue fever in North West Italy in travelers from endemic areas: A retrospective study

BACKGROUND Domestic outbreaks of Dengue (DENV) fever from imported cases have to be considered a possible risk in non-endemic countries where Dengue vectors are present, such as in Italy. OBJECTIVE To review imported acute/recent DENV infections in a one-year survey in a North West Italy region where the presence of Aedes albopictus is documented. STUDY DESIGN We retrospectively reviewed laboratory and clinical records of Italian febrile travelers from Dengue endemic areas referring to the local reference Centre for Infectious Disease, covering a population of about 4 million people. RESULTS Acute/recent DENV infection was identified in 15 out of 91 travelers from endemic areas (16.5%) including 12 primary and 3 secondary infections; in 6 patients the virus was detectable in blood according to molecular real-time Polymerase Chain Reaction-based assays: in 9 patients the diagnosis of DENV infection was accomplished by the combination of specific IgM reactivity, high IgG titers, IgG seroconversion from negative to positive and increasing (four-fold) IgG titers in paired serum samples. Two cases of DENV infections were imported from South Egypt in patients travelling together, confirming the importance of returning travelers as sentinels of a rapidly changing epidemiology in specific geographic areas. CONCLUSIONS Our findings outline the high rate of imported Dengue infection in North West Italy and emphasize the need for a continued Dengue surveillance in non-endemic countries as well as a careful evaluation and follow-up of febrile patients returning from Dengue endemic countries.

A(H1N1)pdm09 hemagglutinin D222G and D222N variants are frequently harbored by patients requiring extracorporeal membrane oxygenation and advanced respiratory assistance for severe A(H1N1)pdm09 infection

In patients with A(H1N1)pdm09 infection, severe lung involvement requiring admission to intensive care units (ICU) has been reported. Mutations at the hemagglutinin (HA) receptor binding site (RBS) have been associated with increased virulence and disease severity, representing a potential marker of critical illness.

Rectal lymphogranuloma venereum

A 42-year-old man was referred by his gastroenterologist after endoscopy with a suspected rectal neoplasm. For 2 months he had complained of anorectal pain, bleeding, mucous discharge, tenesmus and constipation. Digital rectal examination showed rectal stenosis and endoscopy revealed an ulcerating bleeding mass between 7 and 10 cm from the anal verge (Fig. 1). Multiple biopsies showed granulation tissue. On further questioning, the patient reported unprotected receptive anal intercourses with multiple male partners, also while abroad in the UK and Spain. He was on antiretroviral therapy (raltegravir and atazanavir) for human immunodeficiency virus (HIV) stage A1 infection. The CD4 cell count was 550 ⁄ mm and the HIV viral load was undetectable. Gram staining of a rectal smear showed more than 30 polymorphonuclear leucocytes per highpower field. A rectal swab processed with a COBAS TaqMan CT Test version 2.0 (Roche, Branchburg, New Jersey, USA) for qualitative detection of Chlamydia trachomatis DNA, tested positive for C. trachomatis. An ‘in-house’ genovar-specific nucleic acid amplification test demonstrated lymphogranuloma venereum (LGV) C. trachomatis infection [1]. Treatment with doxycycline (100 mg twice daily for 21 days) resolved the symptoms within 15 days. Follow-up proctoscopy confirmed complete healing. At 12 months the patient was disease free.

Epstein-Barr Virus May Contribute to Central Nervous System Involvement in HIV-positive Individuals

Epstein-Barr virus (EBV) often accesses the central nervous system (CNS) where it may lead to blood brain barrier (BBB) integrity disruption, facilitating the migration of immune cells into brain parenchyma. Our aim was to study the association between cerebrospinal fluid (CSF) EBV DNA and HIV-1 compartmental replication. 281 HIV-positive adults undergoing lumbar punctures for clinical reasons (excluding those with lymphoproliferative disorders) and CSF samples were examined. CSF virological, neurodamage (tau, p-tau, 1-42 beta amyloid) and immune activation (neopterin and S100beta) markers were measured by immune-enzymatic, ELISA and PCR validated methods. Two hundred eighty one patients were included; 111 (40.5 %) were naïve for antiretroviral treatment. CSF EBV DNA was detectable in 25 (21.9%) naïve and 26 (16%) treated patients at low levels (<100 and 146 copies/mL). Naïve EBV+ subjects presented higher CSF HIV RNA, biomarkers (t-tau, p-tau, neopterin) and higher rates of pleocytosis. Treated EBV+ individuals showed pleocytosis, higher CSF HIV RNA, CSF to serum albumin ratio, IgG index and neopterin. No association was observed between detectable CSF EBV DNA and the rate of CSF escape. In patients with plasma HIV RNA <20 copies/mL (n=97) CSF EBV DNA was detectable in 13 subjects (13.4%) and it was associated with pleocytosis, higher CSF HIV RNA and neopterin levels. EBV DNA was detectable in a considerable proportion of HIV-positive patients and it was associated with higher levels of CSF HIV RNA and neuronal damage/inflammation biomarkers. The role of EBV reactivation in HIV-associated CNS disorders warrant further studies. Importance EBV is a human gamma-herpesvirus with a seroprevalence in adults approaches 95% and the pattern of clinical manifestations is very heterogeneous and varies from asymptomatic or mild viral infection to a tightly linked with several malignancies as nasopharyngeal carcinoma, Hodgkin’s lymphoma and Burkitt’s lymphoma. HIV-infected and immunocompetent patients were both at risk of primary infection and complications linked to EBV. Primary tropism of EBV is for lymphocytes (type B, T and NK), epithelial, endothelial and smooth muscle cells and establishes lifelong latent infection. Central nervous system could be affected by this herpesvirus in primary infection and reactivation and EBV-DNA is not an uncommon finding in CSF in HIV-infected population. The significance of our research is in identifying the presence of a link between HIV and EBV CNS replication.