Detection and genotyping of human papillomavirus by real-time PCR assay

BackgroundDiagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients.ObjectiveOur study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples.Study designValidation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity.ResultsData showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/10³ cells using in-house HPV (6, 11 and 16) viral load assays.ConclusionThe real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients.     

Donors for SARS-CoV-2 Convalescent Plasma for a Controlled Clinical Trial: Donor Characteristics,Content and Time Course of SARS-CoV-2 Neutralizing Antibodies

BackgroundConvalescent plasma is one of the treatment options for COVID-19 which is currently being investigated in many clinical trials. Understanding of donor and product characteristics is important for optimization of convalescent plasma.MethodsPatients who had recovered from CO­VID-19 were recruited as donors for COVID-19 convalescent plasma (CCP) for a randomized clinical trial of CCP for treatment of severe COVID-19 (CAPSID Trial). Titers of neutralizing antibodies were measured by a plaque-reduction neutralization test (PRNT). Correlation of antibody titers with host factors and evolution of neutralizing antibody titers over time in repeat donors were analysed.ResultsA series of 144 donors (41% females, 59% males; median age 40 years) underwent 319 plasmapheresis procedures providing a median collection volume of 850 mL and a mean number of 2.7 therapeutic units per plasmapheresis. The majority of donors had a mild or moderate course of COVID-19. The titers of neutralizing antibodies varied greatly between CCP donors (from <1:20 to >1:640). Donor factors (gender, age, ABO type, body weight) did not correlate significantly with the titer of neutralizing antibodies. We observed a significant positive correlation of neutralization titers with the number of reported COVID-19 symptoms and with the time from SARS-CoV-2 diagnosis to plasmapheresis. Neutralizing antibody levels were stable or increased over time in 58% of repeat CCP donors. Mean titers of neutralizing antibodies of first donation and last donation of repeat CCP donors did not differ significantly (1:86 at first compared to 1:87 at the last donation). There was a significant correlation of neutralizing antibodies measured by PRNT and anti-SARS-CoV-2 IgG and IgA antibodies which were measured by ELISA. CCP donations with an anti-SARS-CoV-2 IgG antibody content above the 25th percentile were substantially enriched for CCP donations with higher neutralizing antibody levels.ConclusionWe demonstrate the feasibility of collection of a large number of CCP products under a harmonized protocol for a randomized clinical trial. Titers of neutralizing antibodies were stable or increased over time in a subgroup of repeat donors. A history of higher number of COVID-19 symptoms and higher levels of anti-SARS-CoV-2 IgG and IgA antibodies in immunoassays can preselect donations with higher neutralizing capacity.     

Ethical Issues and Management of Fetal Hemolytic Anemia Caused by Anti-Rh17 in a Multipara with Rare −D– Phenotype

BackgroundThe development of allo-anti-Rh17 (anti-Hr0) in a −D– phenotype whose red blood cells (RBCs) lack CcEe antigens is most likely triggered by transfusion, transplantation, or pregnancy. Gene conversion is the predominating factor in generating RHD-CE-D and RHCE-D-CE hybrids like −D–.MethodsWe report here immunohematological and obstetrical data from 2 of the 5 pregnancies of a 24-year-old woman presenting with the −D– phenotype with anti-Rh17. Blood group typing, antibody screening, antibody differentiation, direct antiglobulin test (DAT), and antibody titers were performed by routine gel technology and tube testing. Additionally, molecular genetic analysis was performed. Fetal surveillance was done by sonographic evaluation of the fetal middle cerebral artery peak systolic velocity (MCA-PSV).ResultsBlood group typing showed O, C-c-D+E-e- and the DAT was negative. DNA sequencing revealed homozygosity for an RHCE-D(3–9)-CE null allele. Anti-Rh17 titers in the fourth pregnancy remained between 1:8 and 1:128, and no signs for a fetal anemia were observed. However, in the fifth pregnancy, the antibody titers increased up to 1:4,096. Signs of moderate fetal anemia were detected and cesarean section was performed at 34 + 6 weeks of gestation. The newborn presented with hemolytic anemia (cord blood hemoglobin [Hb] = 8.5 mg/dL). She received 2 compatible (small) packed RBC concentrates, phototherapy, and intravenous immunoglobulins.ConclusionOur case shows that the risk for hemolytic complications increases with the number of pregnancies of sensitized women. Only people who also lack CcEe antigens are compatible as donors. The role of such rare donors as lifesavers, their freedom, and voluntariness conflict with the urgent need for compatible blood.     

Comparative Real-Life Effectiveness and Safety of Dabigatran or Rivaroxaban vs. Vitamin K Antagonists: A High-Dimensional Propensity Score Matched New Users Cohort Study in the French National Healthcare Data System SNDS

Clinical trials have indicated that the direct-acting oral anticoagulants dabigatran and rivaroxaban have better risk/benefit profiles than do vitamin K antagonists (VKAs) for stroke prevention in non-valvular atrial fibrillation (NVAF). Our objective was to compare the 1-year real-life risk of major clinical events with dabigatran or rivaroxaban versus VKAs for NVAF. This was a high-dimensional propensity score (hdPS)-matched cohort study of new users of dabigatran, rivaroxaban or VKAs for NVAF in the French national healthcare systems database in 2013 followed-up for 1 year [22]. Hazard ratios (HRs) with 95% confidence intervals (CIs) for clinical events and death were determined during exposure. In 2013, a total of 103,101 new anticoagulant users had definite NVAF: 44,653 VKA, 27,060 dabigatran, and 31,388 rivaroxaban. In matched populations, HRs were as follows for dabigatran versus VKAs (20,489 per group): stroke and systemic embolism (SSE) 0.75 (95% CI 0.63–0.88), clinically relevant bleeding (CRB) 0.58 (95% CI 0.51–0.66), hemorrhagic stroke (HS) 0.22 (95% CI 0.14–0.36), gastrointestinal bleeding (GIB) 0.98 (95% CI 0.80–1.19), acute coronary syndrome (ACS) 0.79 (95% CI 0.65–0.95), death 0.74 (95% CI 0.67–0.82), composite (any of the above) 0.71 (95% CI 0.66–0.76). For matched rivaroxaban versus VKA (23,053 per group) HRs were as follows: SSE 0.98 (95% CI 0.85–1.14), CRB 0.83 (95% CI 0.75–0.92), HS 0.65 (95% CI 0.49–0.87), GIB 1.08 (95% CI 0.90–1.30), ACS 0.84 (95% CI 0.71–1.00), death 0.77 (95% CI 0.71–0.84), composite 0.84 (95% CI 0.79–0.89). Numbers needed to treat to observe one fewer death were 49 ± 0.05 with dabigatran or rivaroxaban versus VKAs. Consistent with results from clinical trials and other observational studies, dabigatran and rivaroxaban were at least as effective and safer than VKAs for the prevention of thromboembolic events in NVAF over 1 year in the French population. European Medicines Agency EUPAS 13017 (www.encepp.eu) Clinicaltrials.gov id NCT02785354.