Evaluation of the siemens HIV antigen-antibody immunoassay.

Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibodies are available on the international market and are currently used for blood donor screening and for HIV diagnosis. In this study we evaluated the performance of the novel automated fourth-generation ADVIA Centaur® HIV Ag/Ab Combo assay. The assay detected seroconversion at the same bleed or at least one bleed earlier in panels with respect to other assays and showed a detection efficacy equal to those of other assays in a low-titer panel. Samples obtained from blood donors (n = 2,778) or from HIV-positive patients (HIV-1 B subtype, n = 82; non-B subtype, n = 71) were also tested, showing a good correlation with other fourth-generation assays. We assessed the performance of 3 fourth-generation assays for detecting in utero transmitted anti-HIV antibodies and found a more specific detection efficiency with the ADVIA Centaur HIV Ag/Ab Combo assay compared to the other fourth-generation assays.

Performance of genotypic tropism testing on proviral DNA in clinical practice: results from the DIVA study group.

OBJECTIVE: The DIVA study is aimed at setting up a standardized genotypic tropism-testing on proviral-DNA for the routine clinical diagnostic-laboratory. METHODS: Twelve local centres and 5 reference centres (previously cross-validated) were identified. For inter-center validation-procedure, 60 peripheral-blood mononuclear cells (PBMCs) aliquots from 45 HAART-treated patients were randomly chosen for population V3 sequencing on proviral-DNA at local HIV centre and at reference-laboratory. Viral tropism was predicted by Geno2Pheno algorithm (False Positive Rate [FPR] = 20%) as proposed by the European-Guidelines. Quantification of total HIV-1 DNA was based on a method described by Viard (2004). RESULTS: Quantification of HIV-1 DNA was available for 35/45 (77.8%) samples, and gave a median value of 598 (IQR:252- 1,203) copies/10 PBMCs. A total of 56/60 (93.3%) samples were successfully amplified by both the reference and the local virological centers. The overall concordance of tropism prediction between local and reference centers was 54/56 (96.4%). Results of tropism prediction by local centers were: 33/54 (61.1%) R5 and 21/54 (38.9%) X4/DM. CONCLUSION: There was high concordance in the genotypic tropism prediction based on proviral DNA among different virological centers throughout Italy. Our results are in line with other European studies, and support the use of genotypic tropism testing on proviral DNA in patients with suppressed plasma HIV-1 RNA candidate to CCR5-antagonist treatment.

Tracking the 2009 H1N1 influenza virus in the Italian region Campania.

A novel swine-origin influenza A (H1N1) virus affecting humans was detected in April 2009 in Mexico, Canada, and USA. The S-OIV infection caused a mild to severe febrile respiratory disease throughout the world. Here, we briefly review the main features of influenza A viruses, which caused also other pandemics in the past, and focus in particular on the epidemiology data of the H1N1 influenza in the Italian region Campania, which resulted the most affected by the S-OIV and the one with more lethal cases. In Campania, the peak of influenza preceded of about 2 weeks the incidence peak at the national level. Moreover, the percentage of H1N1-positive patients was much higher in the main town Naples, compared to the other Campania provinces. The age group from 7 months to 17 years was the most affected by the H1N1 infection (43.45%), similarly to what reported at the national level. Here, we discuss the possible reasons of the high H1N1 incidence in Campania and the implications that these findings could have on the future prevention campaigns.

Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver.

BACKGROUND: Clinical use of porcine cell-based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow-up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. METHODS: Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center-BAL (AMC-BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real-time polymerase chain reaction assay has been used. RESULTS: Eight patients who received a liver transplant after AMC-BAL treatment are still alive under long-term pharmacological immunosuppression. The current clinical follow-up ranges from 5.6 to 8.7 yr after BAL treatment. A new q-real-time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥ 5 copies per 1 × 10(5) PBMCs. The linear dynamic range was from 5 × 10(0) to 5 × 10(6) copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. CONCLUSION: Up to 8.7 yr after exposure to treatment with porcine liver cell-based BAL, no PERV infection has been found in long-term immunosuppressed patients and in HCWs by a new highly sensitive and specific q-real-time PCR assay.

Difficulties in classifying a/g recombinants: methodological problems or genetic variability?

Thirty pol gene plasma-derived sequences clustering with the circulating recombinant form (CRF) 02_AG (IbNG) (bootstrap 100%) were evaluated to analyze the genomic composition. Subtype assignment was also phylogenetically confirmed by C2-V3 region analysis for 18/21 sequences evaluated. Thereafter, we compared the genomic recombination of the CRF02_AG/IbNG prototype as predicted by bootscanning and Jumping HMMER software (jpHMM) to that of our strains. With these methods, 27% and 50%, respectively, of our clinical sequences demonstrated the same pol structure as the prototype CRF02_A/G-IbNG. However, in subtrees built for each segment predicted by jpHMM (with a bootstrap value of more than 75%), all fragments clustered with IbNG and were distinct from A and G clades. Overall, our sequences resulted in true members of CRF02_AG-IbNG, which, however, appeared to be a subtype phylogenetically separate from A or G, at least with regard to the pol gene.

Toscana virus central nervous system infections in southern Italy.

Toscana virus was detected by reverse transcription-nested PCR in 5.6% of cerebrospinal fluid (CSF) samples from patients with meningitis and encephalitis during the summer in southern Italy. The central nervous system infections were associated with young adults and with a substantially benign clinical course. Presenting features and CSF findings are also discussed in the present report.

No evidence of in vitro and in vivo porcine endogenous retrovirus infection after plasmapheresis through the AMC-bioartificial liver.

BACKGROUND: Currently a number of bioartificial livers (BAL) based on porcine liver cells have been developed as a treatment to bridge acute liver failure patients to orthotopic liver transplantation or liver regeneration. These xenotransplantation related treatments hold the risk of infection of treated patients by porcine endogenous retrovirus (PERV) released from the porcine cells, as in vitro infection experiments and transplantations in immunocompromised mice have shown that PERV is able to infect human cells. The Academic Medical Center (AMC)-BAL, unlike other BALs, is characterized by direct contact between porcine liver cells and human plasma, and might therefore be permissive for PERV transfer. METHODS: Prior to a clinical phase I trial, human plasma perfused through the AMC-BAL was investigated for PERV DNA and RNA. Moreover productive infectivity was analyzed by exposing the plasma to HEK-293 cells that were subsequently tested for PERV DNA, PERV RNA and reverse transcriptase activity. RESULTS: Although PERV DNA was detected in the perfused plasma, no productive infectivity was detected. Consequently fourteen patients were treated with the AMC-BAL and monitored for PERV transmission. Immediately after treatment the plasma of the patients was positive for PERV DNA, most probably due to porcine liver cell lysis. The PERV DNA was cleared within 2 weeks post-treatment and no PERV RNA was detected. No productive infectivity in human embryonic kidney (HEK)-293 cells exposed to plasma of treated patients was detectable. CONCLUSION: To conclude, no release of infective PERV particles from the AMC-BAL was observed. Therefore we consider the AMC-BAL as safe, however careful surveillance of patients will be continued.

Bridging a patient with acute liver failure to liver transplantation by the AMC-bioartificial liver.

Recently a phase I clinical trial has been started in Italy to bridge patients with acute liver failure (ALF) to orthotopic liver transplantation (OLT) by the AMC-bioartificial liver (AMC-BAL). The AMC-BAL is charged with 10 x 10(9) viable primary porcine hepatocytes isolated from a specified pathogen-free (SPF) pig. Here we report a patient with ALF due to acute HBV infection. This patient was treated for 35 h by two AMC-BAL treatments and was bridged to OLT. There was improvement of biochemical and clinical parameters during the treatment. No severe adverse events were observed during treatment and follow-up of 15 months after hospital discharge. Possible porcine endogenous retrovirus (PERV) activity could not be detected in the patient's blood or blood cells up to 12 months after treatment.

Bridging a Patient with Acute Liver Failure to Liver Transplantation by the AMC-Bioartificial Liver.

Recently a phase I clinical trial has been started in Italy to bridge patients with acute liver failure (ALF) to orthotopic liver transplantation (OLT) by the AMC-bioartificial liver (AMC-BAL). The AMC-BAL is charged with 10 × 109 viable primary porcine hepatocytes isolated from a specified pathogen-free (SPF) pig. Here we report a patient with ALF due to acute HBV infection. This patient was treated for 35 h by two AMC-BAL treatments and was bridged to OLT. There was improvement of biochemical and clinical parameters during the treatment. No severe adverse events were observed during treatment and follow-up of 15 months after hospital discharge. Possible porcine endogenous retrovirus (PERV) activity could not be detected in the patient's blood or blood cells up to 12 months after treatment.