In-house RT-PCR detection of hepatitis A virus in human plasma pools used for the manufacture of medicinal products.

Hepatitis A virus (HAV) is the most frequent cause of viral hepatitis worldwide and is transmitted through the fecal-oral route. However, HAV can also be transmitted by blood-derived products. This is due to the fact that viremia occurs during the asymptomatic phase of HAV infection, enabling infected blood or plasma donations to occur. Viral inactivation/removal steps are included during manufacturing of plasmaderived products. However, HAV is a small non-enveloped virus very difficult to remove with traditional viral inactivation procedures. To accomplish European guidelines for pooled human plasma (treated for virus inactivation), plasma manufacturers have been implementing HAV nucleic acid test (NAT) screening on plasma pools. In this study, we validate an in-house multiplex reverse-transcription real-time PCR (RT-PCR) assay targeting HAV RNA and an internal control with hydrolysis probes for amplicon detection. The HAV RNA test was validated by assessing limit of detection, robustness, sensitivity and specificity according to European Pharmacopoeia (Ph. Eur.) guidelines. Our assay is able to detect 100 IU/mL of all human HAV genotypes that have been described so far. The multiplex assay shows remarkable sensitivity with a 95% lower limit of detection of 5.2 IU/mL. Also, our HAV test shows good robustness, precision, and specificity. We conclude that our assay broadly meets the requirements for its purpose. The implementation of this test in the production process of plasma-derived products will increase their safety.

Krüppel-like factor 6 (KLF6) requires its amino terminal domain to promote villous trophoblast cell fusion.

INTRODUCTION: Villous cytotrophoblast (vCTB) cells fuse to generate and maintain the syncytiotrophoblast layer required for placental development and function. Krüppel-like factor 6 (KLF6) is a ubiquitous transcription factor with an N-terminal acidic transactivation domain and a C-terminal zinc finger DNA-binding domain. KLF6 is highly expressed in placenta, and it is required for proper placental development. We have demonstrated that KLF6 is necessary for cell fusion in human primary vCTBs, and in the BeWo cell line. MATERIALS AND METHODS: Full length KLF6 or a mutant lacking its N-terminal domain were expressed in BeWo cells or in primary vCTB cells isolated from human term placentas. Cell fusion, gene and protein expression, and cell proliferation were analyzed. Moreover, Raman spectroscopy and atomic force microscopy (AFM) were used to identify biochemical, topography, and elasticity cellular modifications. RESULTS: The increase in KLF6, but not the expression of its deleted mutant, is sufficient to trigger cell fusion and to raise the expression of ß-hCG, syncytin-1, the chaperone protein 78 regulated by glucose (GRP78), the ATP Binding Cassette Subfamily G Member 2 (ABCG2), and Galectin-1 (Gal-1), all molecules involved in vCTB differentiation. Raman and AFM analysis revealed that KLF6 reduces NADH level and increases cell Young's modulus. KLF6-induced differentiation correlates with p21 upregulation and decreased cell proliferation. Remarkable, p21 silencing reduces cell fusion triggered by KLF6 and the KLF6 mutant impairs syncytialization and decreases syncytin-1 and ß-hCG expression. DISCUSSION: KLF6 induces syncytialization through a mechanism that involves its regulatory transcriptional domain in a p21-dependent manner.

Human parvovirus B19 frequency among blood donors after an epidemic outbreak: relevance of the epidemiological scenario for transfusion medicine.

A retrospective, cross-sectional study was conducted to determine the frequency of human parvovirus B19 (B19V) infected individuals, viral loads and immunity among blood donors from Argentina, in a post-epidemic outbreak period. B19V DNA and specific IgG were tested in minimum study samples of donors attending a blood bank at Córdoba, Argentina, in 2014. Anti-B19V IgM and viral loads were determined in B19V-positive plasma samples. Seven of 731 samples (0.96%) resulted positive, corresponding to individuals aged 32-53 years, four of them repeat donnors and three first-time donors. Viral loads were <103 IU/mL. None had IgM and 6/7 had IgG, one of them at a high level (in the range of 100-200 IU/ml, and the remaining 5 at low to medium level, 5-50 IU/ml). Thus one case was classified as acute infection (DNA+/IgM-/IgG-) and six as potentially persistent infections (DNA+/IgM-/IgG+). No coinfections with other pathogens of mandatory control in the pre-transfusion screening were detected. Prevalence of IgG was 77.9% (279/358). This study provides the first data of B19V prevalence in blood donors in Argentina, demonstrating high rates of acute and persistent B19V infections and high prevalence of anti-B19V IgG in a post-epidemic period. Further research is needed to elucidate mechanisms/factors for B19V persistence as well as follow-up of recipients in the context of haemo-surveillance programs, contributing to the knowledge of B19V and blood transfusion safety.

Parvovirus B19 in HIV+ adult patients with different CD4+ lymphocyte counts.

PURPOSE: Human parvovirus B19 (B19V) can cause anemia in immunocompromised patients. We aimed to investigate the presence of B19V in HIV+ adults with different CD4+ T cell counts, to recognise the frequency of B19V in these different conditions and its possible association with anemia. METHODOLOGY: We studied B19V specific IgM, IgG and DNA in 98 HIV+ patients and in 52 healthy individuals. HIV load, CD4+ counts and haemoglobin level were also determined in the patients. RESULTS: No individual in the control group had detectable IgM, 41/52 (78.8 %) had IgG and 5/52 (9.6 %) had B19V DNA. Among HIV+ patients, we found 5/98 (5.1 %) IgM+, 66/98 (67.3 %) IgG+ and 15/98 (15.3 %) had B19V DNA (no significant differences between the two groups compared). Considering the CD4+ cell range in HIV patients, 37 had <200 CD4+ cells ml-1, 31 had 200-500, and 30 had >500. Anti-B19V IgG prevalence in patients with >500 CD4+ cells ml-1 was significantly higher than in the rest (P=0.004) and compared to the control (P=0.046). B19V DNA concentration was always <103 IU ml-1, including 5 healthy individuals and 15 HIV+ patients. There was no significant association between B19V IgM or DNA and anemia nor between B19V DNA and HIV load. CONCLUSIONS: The results indicate that B19V is not a high-risk factor for anemia in adult HIV+ patients under HAART treatment. Further studies will contribute to elucidate the mechanisms and significance of B19V DNA prevalence/persistence in adults, independently of the CD4+ cell status.