Blood group genotyping in multi-transfused patients

Aim. To assess the possibility of using blood group genotyping in recipients who received transfusions for 3 months. Methods. The study included blood samples from 95 patients who received 3 or more erythrocyte transfusions within 3 months. The patients had the following diagnoses: multiple myeloma (n=7), beta thalassemia (n=4), non-Hodgkin's lymphomas (n=11), chronic myeloid leukemia (n=16), primary myelofibrosis (n=9), myelodysplastic syndrome (n=22), acute leukemia (n=21), aplastic anemia (n=5). Red blood cells phenotyping was performed in Diaclon Rh Subgroups+K Gel Cards. The Rh and Kell genotyping was performed by using RBC SSP-PCR kits FluoGene vERYfy (Inno-train Diagnostics, Germany). The standard RHD/RHCE alleles, as well as polymorphisms associated with KEL1/KEL2 [T698C (Met198Thr)] of the KEL gene were genotyped. Results. The concordance rate between serological and molecular genetic typing of RhCE and Kell blood groups for donors was 100%, while the patients results were discordant in 45.3% of cases. Discrepancies in antigens of the Rh system were registered in 41 patients: one antigen of the Rh system in 30 patients, two in 9 patients. Ten patients who had been previously phenotyped as RhCc were genotyped as RHCE*CC. 2 patients who had been previously phenotyped as Rhee were genotyped as RHCE*EE. In 2 patients, antigens D and C were not detected in the phenotype but were identified in the genotype. Discrepancies in antigen K were recorded in 2 patients, and the antigen was absent in the phenotype but was present in the genotype. The genotyping results were confirmed by serological typing at subsequent hospitalizations. Сonclusion. Blood group genotyping is a useful adjunct to traditional methods when serological typing is limited.

GENETIC TYPING OF LEUCONOSTOC MESENTEROIDES USING PCR

We studied the possibility of using RAPD-PCR with primers: ERIC1R-1, ERIC2-1, BOXA1R, BOXA2R and Rep-PCR with primers P15, P16, XD8, XD9, RAPD-mes, (GTG)5 to identify genetic heterogeneity of 9 strains and 8 isolates of Leuconostoc mesenteroides. Three clusters of cultures with a high level of bootstrap support were identified as a result of phylogenetic analysis obtained when typing Leuconostoc. The obtained results indicate the possibility of revealing genetic differences in the profile of the generated amplicons among Leuconostoc mesenteroides strains using the combined methods of Rep-PCR and RAPD-PCR.

Genetic analysis and pathogenicity of different sequence types of Streptococcus suis isolated from pigs in southern China

ABSTRACT In this study, 52 Streptococcus suis isolates from pigs in southern China were divided into four known sequence types (STs) and six new STs, using multilocus sequence typing. Ten representative isolates were selected from 10 STs for the analysis of whole genome sequences. Virulence was assessed in 10 isolates, which were classified into three pathogenic groups. The prevalence of virulence-associated factors in the moderately pathogenic group was higher than that in the highly pathogenic group. The isolates from ST1 complex and serotype 2 were allocated to the moderately pathogenic group, while the isolates from the highly pathogenic group belonged to the non-ST1 complex and non-serotype 2. Three clusters were obtained based on multilocus sequence typing sequences: cluster III isolates from the nasal cavity of healthy pigs were classified into the highly pathogenic group and showed many peculiarities compared with cluster I and II isolates in virulence genotypes, genetic typing and pathogenesis, indicating a potential independent evolutionary line. Our results suggest that S. suis infections in China are becoming more complicated with constantly mutating isolates, which makes it difficult to distinguish their virulence by recognized typing methods. Thus, increased investigation and monitoring of these infections should be a priority for the swine industry in China.

Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue

Human rabies post mortem diagnostic samples are often preserved in formalin. While immunohistochemistry (IHC) has been routinely used for rabies antigen detection in formalin-fixed tissue, the formalin fixation process causes nucleic acid fragmentation that may affect PCR amplification. This study reports the diagnosis of rabies in an individual from the Dominican Republic using both IHC and the LN34 pan-lyssavirus real-time RT-PCR assay on formalin-fixed brain tissue. The LN34 assay generates a 165 bp amplicon and demonstrated higher sensitivity than traditional PCR. Multiple efforts to amplify nucleic acid fragments larger than 300 bp using conventional PCR were unsuccessful, probably due to RNA fragmentation. Sequences generated from the LN34 amplicon linked the case to the rabies virus (RABV) strain circulating in the Ouest Department of Haiti to the border region between Haiti and the Dominican Republic. Direct sequencing of the LN34 amplicon allowed rapid and low-cost rabies genetic typing.