Lyophilized Matrix Containing Ready-to-Use Primers and Probe Solution for Standardization of Real-Time PCR and RT-qPCR Diagnostics in Virology.

Real-time molecular techniques have become the reference methods for direct diagnosis of pathogens. The reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. We describe here a process of preparation of oligonucleotide primers and hydrolysis probe in a single tube at predefined optimized concentrations that are stabilized via lyophilization (Lyoph-P&P). Lyoph-P&P was compared versus the classic protocol using extemporaneously prepared liquid reagents using (i) sensitivity study, (ii) long-term stability at 4 °C, and (iii) long-term stability at 37 °C mimicking transportation without cold chain. Two previously published molecular assays were selected for this study. They target two emerging viruses that are listed on the blueprint of the WHO as to be considered for preparedness and response actions: chikungunya virus (CHIKV) and Rift Valley fever phlebovirus (RVFV). Results of our study demonstrate that (i) Lyoph-P&P is stable for at least 4 days at 37 °C supporting shipping without the need of cold chain, (ii) Lyoph-P&P rehydrated solution is stable at +4 °C for at least two weeks, (iii) sensitivity observed with Lyoph-P&P is at least equal to, often better than, that observed with liquid formulation, (iv) validation of results observed with low-copy specimens is rendered easier by higher fluorescence level. In conclusion, Lyoph-P&P holds several advantages over extemporaneously preparer liquid formulation that merit to be considered when a novel real-time molecular assay is implemented in a laboratory in charge of routine diagnostic activity.

Molecular epidemiology of KI and WU polyomaviruses in healthy blood donors, south-eastern France.

The epidemiology of human polyomaviruses KI (KIPyV) and WU (WUPyV) in healthy populations is described poorly in the literature. The frequency of KIPyV and WUPyV viraemia was evaluated in a cohort of blood donors from south-eastern France. Plasma samples (n=640) were investigated for the presence of KIPyV/WUPyV DNA using a conserved real-time PCR detection system (VP2 gene). Three plasma samples (3/640; ∼0.5%) exhibited a positive fluorescence signal, with a low viral load (<500 copies/ml plasma); no additional amplicons were identifiable by agarose gel analysis. Sequencing highlighted the KIPyV origin of the three amplified sequences and the occurrence of point mutations. The sustained detection of KIPyV DNA in two serial samples (9 months) was in favor of a possible persistence of the virus in blood of healthy individuals. Further studies will be needed in order to explore both the prevalence and potential clinical impact of KIPyV/WUPyV on infected hosts.

Parvovirus 4 in French in-patients: a study of hemodialysis and lung transplant cohorts.

The epidemiology and the clinical implication of human parvovirus 4 (PARV4) in human populations is still under evaluation. The distribution of PARV4 DNA was determined in cohorts of French hemodialysis and lung transplant patients. Plasma samples (n=289) were tested for PARV4 by real-time PCR assay (ORF2), and amplification products selected at random were sequenced. Analysis of available serological and biological markers was also undertaken. Fifty-seven samples out of 185 (30.8%) were positive for PARV4 DNA in the cohort of hemodialysis patients. A higher prevalence of the virus was identified in patients with markers of HBV infection. PARV4 was also identified in 14 out of 104 samples (13.5%) from lung transplant recipients, with no clear-cut association with available clinical markers. Point mutations located on the zone of real-time detection were identified for some amplification products. This study describes the detection of PARV4 in the blood of hemodialysis and lung transplanted patients with significant difference in prevalence in these two cohorts. Further studies will be needed in order to understand better both the potential implication in host health and the natural history of this virus.

Molecular analysis of inactive and active RHD alleles in native Congolese cohorts.

BACKGROUND: In Africa, RHD alleles have not been fully characterized. The purpose of this study was to identify inactive and active RHD alleles at the molecular level in Congolese cohorts. STUDY DESIGN AND METHODS: Blood samples were collected from people living in central Congo populated by Teke ethnic group. A total of 110 D- and 40 D+ samples from Congo-Brazzaville and Teke groups, respectively, were selected for RHD genotyping using allele-specific primer polymerase chain reaction and sequencing. RESULTS: In the 110 D- samples, RHD exon amplifications were observed in 7 samples that were subsequently identified by sequencing as weak D type 4 variants. In the remaining 103 D- samples, the frequencies of RHD gene deletion, RHDpsi pseudogene, and RHD-CE-D(s) hybrid gene were 0.75685, 0.20560, and 0.04468, respectively. In the D+ samples, 26 individuals carried at least a regular RHD gene; 9 carried aberrant RHD alleles belonging to the African D clusters, that is, DAU, DIVa, and weak D type 4; 3 carried RHDpsi in trans with a DAU allele including one novel RHD allele (V279M, S333N, T379M) named DAU-7; and 2 others were partially determined. CONCLUSION: This study revealed a high frequency of weak D type 4 alleles that confirmed the need to use indirect antiglobulin test to improve transfusion safety in the Congo and in countries hosting Congolese people. Findings also indicated that there is a geographic variation in RHD allele distribution and showed that RHD gene deletion is the most prevalent cause of the D- phenotype in the Congolese population.

DNA-based typing of Kell, Kidd, MNS, Dombrock, Colton, and Yt blood group systems in the French Basques.

The Basques demonstrate peculiar characteristics regarding blood group systems. Although ABO, Rhesus, and Duffy have been extensively studied in this population, the distribution of other groups remains largely unknown. Therefore, we evaluated the frequency of less-explored- or still noninvestigated blood groups using DNA-based assays and interpreted these data in the view of population genetics. Polymorphisms of KEL (Kell), SLCA14A1 (Kidd), GYPA/GYPB (MNS), ART4 (Dombrock), AQP1 (Colton), and ACHE (Yt) blood group genes were determined from a sample of more than 100 autochthonous French Basques using allele-specific primer PCR (PCR-ASP) methods. Our results were compared with those previously obtained by the use of serology from both Basque and non-Basque European populations. MNS*1 and JK*1 allele frequencies were comparable with those reported from Basque samples. Conversely, the KEL*1 allele frequency differed significantly. To our knowledge, this is the first time that the other three systems are studied in the Basque population. DO*1 and CO*1 allele frequencies, being respectively 0.35 and 0.96, were significantly inferior to those published from various European populations. There were some discrepancies regarding these six blood systems when comparing molecular typing with serology. These findings may be explained by differences in either criteria for individual selection or technical assays. Nevertheless, these results constitute additional data to be included in the chapter of Basque biological anthropology.

HLA-DRB1 polymorphism is associated with Kell immunisation.

K immunisation is observed in some polytransfused patients and pregnant women but does not occur in all cases of K incompatibility. This study analysed the role of genetic background in this selective response to K antigen by investigating HLA-DRB1 alleles associated with K immunisation in a southern European population. HLA-DRB1 genotyping was performed by polymerase chain reaction sequence-specific oligonucleotide/sequence-specific primer procedures in 54 K immunised patients and 200 healthy controls. The frequency of HLA-DRB1*11 was significantly higher in K immunised patients than healthy controls: 31 of 54 (57%) vs. 56 of 200 (28%) (P(c) < 0.001). In the remaining K immunised HLA-DRB1*11-negative patients, the frequency of HLA-DRB1*13 was increased: 14 of 23 (61%) vs. 49 of 144 in healthy controls (34%) (P < 0.02). The combined frequency of the two HLA-DRB1 alleles (HLA-DRB1*11 and HLA-DRB1*13) was 83% in K immunised patients when compared with 52% in healthy controls (P(c) < 0.001). K and k differ by a single amino acid T193 (M). The DRB1*11 and DRB1*13 alleles share a HLA-DRB1 gene sequence containing S in position 13, D in 70 and A in 74, and coding for the P4 pocket within the HLA-DR binding groove. This feature of the HLA-DRB1 gene could be involved in the K peptide presentation through a polymorphism ligand specific for the T193 (M) of K. In conclusion, this study demonstrated a high frequency of HLA-DRB1*11 or HLA-DRB1*13 alleles in K immunised patients, which could be due to specific K peptide presentation by HLA-DR molecules.

HLA-DRB1 alleles and Jk(a) immunization.

BACKGROUND: In transfusion medicine, anti-Jk(a) has been implicated in hemolytic transfusion reactions. Development of anti-Jk(a) after transfusion does not always occur after Jk(a-) patients receive at least 1 unit of Jk(a+) blood unit. This study was designed to identify HLA-DRB1 alleles associated with predisposition to Jk(a) immunization after blood transfusion or pregnancy. STUDY DESIGN AND METHODS: Genotyping by polymerase chain reaction and sequence-specific oligonucleotide probe nonradioactive hybridization/sequence-specific primers was performed in 20 Jk(a)-immunized patients and 200 controls from the same southern European population. RESULTS: Genotyping showed that HLA-DRB1*01 was significantly more frequent in Jk(a)-immunized patients than controls: 55 percent versus 17 percent (odds ratio [OR], 5.9; confidence interval [CI], 2.3-15.5; corrected p value<0.05). Because HLA-DRB1*0101, DRB1*0102, and DRB1*1001 share a common sequence in their B1 chain, that is, F in 13, R in 71, and A in 74, HLA genetic predisposition was analyzed by comparing patients and controls with respect to the distribution of F13/R71/A74-positive and -negative alleles. Results demonstrated greater positivity of the F13/R71/A74 sequence (DRB1*0101, *0102, or *1001) in patients than in controls: 65 percent versus 19.5 percent (OR, 7.7; CI, 2.9-20.5; p<0.001). CONCLUSION: In conclusion, HLA-DRB1*0101, DRB1*0102, and DRB1*1001, which share a common DRB1 sequence, appeared to be overrepresented in Jk(a)-immunized patients.

Comparative analysis of NK cell subset distribution in normal and lymphoproliferative disease of granular lymphocyte conditions.

We have characterized the heterogeneity of human blood NK cell subsets defined by expression of KIR, lectin like receptors and NK cell differentiation markers within a cohort of 51 healthy Caucasian individuals. High inter-individual variability in cell surface expression of most NK cell markers is observed. Range values defining NK cell subsets in healthy donors were further used as references to characterize 14 patients with NK-type lymphoproliferative disease of granular lymphocytes (NK-LDGL). Alterations of the KIR repertoire were noted in all NK-LDGL patients. NK cell expansions were classified as oligoclonal KIR(+) or as non-detectable KIR ((nd)KIR) using anti-KIR2DL1/2DS1, anti-KIR2DL2/2DL3/2DS2, anti-KIR3DL1 and anti-KIR2DS4 monoclonal antibodies. A major reduction in the size of the CD56(bright) NK cell subset was a constant feature of NK-LDGL. Altered distribution of CD94(+), CD161(+), and CD162R(+) NK cell subsets was also observed in NK-LDGL patients. Considering the potential role of NK cells in eliminating tumors or virus-infected cells, the reference values defined in this study should be valuable to characterize both quantitative and qualitative alterations of the NK cell repertoire in pathological conditions and to monitor NK cell reconstitution following hematopoietic transplantation.

Duffy blood group genotyping in French Basques using polymerase chain reaction with allele-specific primers (PCR-ASP).

Blood group antigens such as Duffy represent interesting models for population genetics studies. The distribution of the Duffy blood group was determined using PCR in a sample of Basque (n = 126) and non-Basque (n = 110) patients from the general hospital of the French Basque Country. The frequency of FY*A allele was significantly lower among autochthonous French Basques (P < 0.001). This result, obtained for the first time by PCR analysis in this population, was within the range of previous historical studies in various Basque subpopulations using traditional hemagglutination methods. When compared with European data, our Basque sample demonstrated the lowest FY*A/FY*B ratio, this fact confirming hemotypology data published before the molecular biology era. Our results with FY*X were quite similar to those reported in European populations.

Genetic characterization of the population of Grande Comore Island (Njazidja) according to major blood groups.

The Comorian population is historically considered a blend of influences from African Bantus, Arabs, and possibly Austronesians. In this study we present the first genetic data on the current Comorian population. Serologic analysis of the six major blood group systems (ABO, RH, KEL, FY, JK, and MNS) was performed on 164 individuals from Grande Comore Island (Njazidja). In addition, Duffy genotypes were determined by polymerase chain reaction using allele-specific primers. Our findings establish a high frequency of the Fy(a- b-) phenotype (86%), presenting the same genetic background as in sub-Saharan Africa. Analysis of genetic frequencies, distances, and admixture with other populations indicates that African Bantus made the main contribution to the gene pool (73.2%+/-15.5%). The Arab contribution from the Arabian peninsula was smaller (24.2%+/-7%) and the Indonesian contribution was minor (2.6%+/-9%). The major Bantu contribution was commensurate with the Bantu cultural influence. The contribution from the Arabian peninsula seemed in relation to its permeating religious and linguistic influence. As with the language, the Indonesian contribution to the Comorian gene pool was small. These results are in agreement with historical, sociological, and linguistic data.

Distribution of rhesus blood group system in the French basques: a reappraisal using the allele-specific primers PCR method.

OBJECTIVES: To determine for the first time using PCR the distribution of Rhesus (Rh) blood group in French Basques and compare these results with those obtained by serology in the same sample and in the historical series from various Basque subgroups. METHODS: Rh polymorphism was determined in a sample of 127 autochthonous French Basques using allele-specific primers (ASP) PCR and traditional serological technique. Statistical comparisons were performed between both techniques and with the data published from various Basque subpopulations. RESULTS: No intra-sample discrepancies were detected between ASP-PCR and serology. A high frequency of RH Ddel exceeding 0.50, as typically described from several decades, was found here (0.511), as the peculiar frequency of cde (0.456) and cDE (0.073) haplotypes. This profile, obtained by molecular analysis, was within the range of previous historical studies in various Basque subpopulations using serological approach. The Rh polymorphism among the reviewed autochthonous Basque samples indicates a heterogeneous pattern of distribution, with individuals from the Alava province demonstrating the most atypical profile. CONCLUSIONS: Molecular biology approach using PCR confirms the peculiar pattern of Rh polymorphism which was previously defined by serology among Basques. Nevertheless, this distribution profile is not homogeneous within the Basque area.

Functional categorization of HLA-DRB1 alleles in rheumatoid arthritis: the protective effect.

Because of past recombination event, human leukocyte antigen (HLA) alleles that are not closely related in overall sequence may come to resemble each other in areas coding for peptide binding regions (PBR) of HLA molecules. Peptide binding is likely to be important for the role of HLA molecules in autoimmune disease. As a result, it has been suggested that a strategy of searching for HLA disease associations that groups alleles in functional categories based on PBR motifs may be more successful than conventional strategies based on studying different alleles. Using such functional categorization, we examined the possibility of discriminating subcategories of HLA-DRB1 alleles associated with rheumatoid arthritis (RA) in a Southern French population. HLA-DRB1 genotyping was performed by polymerase chain reaction with sequence-specific oligonucleotide hybridization or sequence-specific primers. HLA-DRB1 alleles were classified according to a functional categorization that defined seven similar subregion structures or restrictive supertype patterns (RSPs) within pocket 4 of HLA-DR peptide binding groove as the molecular basis for grouping these alleles. HLA-DRB1* RSPs "A," "De," "Q," "Dr," "E," " R," and "a" association with susceptibility or resistance to disease was then studied in 200 RA patients versus 200 controls. DRB1* RSP "A" containing the shared epitope alleles (DRB1*0101, *0102, *0401, *0404, *0405, *0408, *1001, *1402; odds ratio [OR] = 4.35; pc < 0.001) had a predisposing effect, with double-dose effect as expected, OR 6.68 (pc < 0.001). Among the six remaining RSPs, two had significantly protective effect: DRB1* RSP "De" (DRB1*0103, *0402, *1102, *1103, *1301, *1302, *1304; OR = 0.33; p(c) < 0.001), and DRB1* RSP "Q" (DRB1*0701; OR = 0.40; pc < 0.001). One had non-significantly protective effect: DRB1* RSP "Dr" (DRB1*08, *1101, *1104, *1106, *12, *1303, *16; OR = 0.68; p < 0.05, pc = not significant [NS]). Three had neutral effect: HLA-DRB1* RSPs "E" (DRB1*0403, *0407, *0901, *1401; OR = 0.71; p = NS), " R" (DRB1*0301, *0302; OR = 0.76; p = NS), and "a" (DRB1*1501, *1502; OR = 0.94; p = NS). The functional categorization allowed us to discriminate among the HLA-DRB1 alleles those that confer a predisposing effect, a neutral effect, and a protective effect in RA.

Genetic analysis of full-length genomes and subgenomic sequences of TT virus-like mini virus human isolates.

The phylogenetic relationship between the complete genomic sequences of ten Japanese and one French isolate of TT virus-like mini virus (TLMV) was investigated. Analysis of the variability of the nucleotide sequences and the detection of signature patterns for overlapping genes suggested that ORFs 1 and 2 are probably functional. However, this was not the case for a putative third ORF, ORF3. Throughout the viral genome, several nucleotide or amino acid motifs that are conserved in circoviruses such as TT virus (TTV) and chicken anaemia virus were identified. Phylogenetic analysis distinguished three main groups of TLMV and allowed the identification of putative recombination breakpoints in the untranslated region. TLMV genomes were detected by PCR in the plasma of 38/50 French blood donors tested and were also identified in peripheral blood mononuclear cells, faeces and saliva. A phylogenetic study of 37 TLMV strains originating from France, Japan and Brazil showed that groupings were not related to geographical origin.