Reliable Determination of Fetal RhD Status by RHD Genotyping from Maternal Plasma.

BACKGROUND: Immunoprophylaxis with IgG anti-D is a standard prevention of hemolytic disease of the fetus and newborn. Fetal Rhesus D (RhD) blood group genotyping from maternal plasma of RhD-negative pregnant women allows targeted prophylaxis with IgG anti-D in RhD-positive pregnancies only. We set up a reliable protocol for prenatal RHD genotyping. METHODS: 153 pregnant Caucasian RhD-negative women were tested in the 27th week (range 7-38th week) of pregnancy. 18 of them were alloimmunized to the RhD antigen. The fetal RHD genotype was determined based on an automated DNA extraction and real-time polymerase chain reaction method. Intron 4 and exons 5, 7 and 10 of the RHD gene and the SRY gene were targeted. RESULTS: The fetal RhD status and gender was 100% correctly predicted in all 153 pregnancies (55 RhD-positive males, 45 RhD-positive females; 23 RhD-negative males, 30 RhD-negative females). CONCLUSION: The accuracy and applicability of our protocol for non-invasive fetal RhD determination allows the correct management of RhD-incompatible pregnancies. Our protocol could prevent unnecessary immunoprophylaxis in 53 of 153 cases. We therefore recommend that non-invasive fetal RHD genotyping is introduced as an obligatory part of prenatal screening.

Intrahost Norovirus Evolution in Chronic Infection Over 5 Years of Shedding in a Kidney Transplant Recipient.

Noroviruses are the leading cause of acute gastroenteritis, and they can affect humans of all age groups. In immunocompromised patients, norovirus infections can develop into chronic diarrhea or show prolonged asymptomatic virus shedding. Chronic norovirus infections are frequently reported for solid organ transplant recipients, with rapid intrahost norovirus evolution seen. In this report, we describe a case of chronic norovirus infection in an immunocompromised patient who was followed up for over 5 years. The purpose of the study was to specify the norovirus evolution in a chronically infected immunocompromised host and identify possible selection sites in norovirus capsid protein. During the follow-up period, 25 sequential stool samples were collected and nine of them were selected to generate amplicons covering viral RNA-dependent RNA polymerase (RdRp) and viral capsid protein (VP1) genes. Amplicons were sequenced using next-generation sequencing. Single nucleotide polymorphisms were defined, which demonstrated a nearly 3-fold greater mutation rate in the VP1 genome region compared to the RdRp genome region (7.9 vs. 2.8 variable sites/100 nucleotides, respectively). This indicates that mutations in the virus genome were not accumulated randomly, but are rather the result of mutant selection during the infection cycle. Using ShoRAH software we were able to reconstruct haplotypes occurring in each of the nine selected samples. The deduced amino-acid haplotype sequences were aligned and the positions were analyzed for selective pressure using the Datamonkey program. Only 12 out of 25 positive selection sites were within the commonly described epitopes A, B, C, and D of the VP1 protein. New positive selection sites were determined that have not been described before and might reflect adaptation of the norovirus toward optimal histo-blood-group antigen binding, or modification of the norovirus antigenic properties. These data provide new insights into norovirus evolutionary dynamics and indicate new putative epitope "hot-spots" of modified and optimized norovirus-host interactions.

Optimization of purification of human cell-free mRNA from plasma.

Cell-free RNA has a potential for diagnosis and prognosis of many diseases. Our aim was to optimize a commercially available QIAamp UltraSens Virus Kit (Qiagen, Hilden, Germany) for isolation of mRNA from human plasma. The amount of carrier RNA to bind plasma mRNA, the centrifugal force to pellet the mRNA-carrier RNA complex, the incubation time for proteolysis, and the centrifugal force to bind mRNA to the silica gel membrane were modified in order to maximize the yield of isolated mRNA. The isolated cell-free mRNA was detected for cycA using TaqMan real-time quantitative RT-PCR. The lowest threshold cycle (Ct) was obtained with the use of 4.0 microL of carrier RNA. Pelleting with a centrifugal force of 1500 xg yielded the lowest Ct, but caused difficulties in resuspending the pellet. Therefore, we suggest using a lower centrifugal force of 1300 xg. The duration of proteolysis had no effect on Ct. To bind mRNA to the silica gel membrane, a centrifugal force of 5000 xg is recommended. Our results show that the UltraSens Virus Kit is an appropriate choice for isolating mRNA from human plasma, with imprecision expressed as coefficient of variation below 2%.