Necrotizing pneumonia in children: Report of 25 cases between 2008 and 2018 at a French tertiary care center.

BACKGROUND: Necrotizing pneumonia (NP) is a serious and rare disease in children. Pediatric data on NP are limited and the impact of the 13-valent pneumococcal conjugate vaccine has been very poorly evaluated. PATIENTS AND METHODS: We conducted a retrospective study at Toulouse University Hospital between 2008 and 2018. Children who presented with thin-walled cavities in the areas of parenchymal consolidation on imaging were included in the study. RESULTS: The incidence of NP did not decrease during this period. Bacterial identification occurred in 56% of cases (14/25) and included six cases of Streptococcus pneumoniae, five of Staphylococcus aureus, two of Streptococcus pyogenes, and one of Streptococcus viridans. Streptococcus pneumoniae NP are more frequently associated with empyema/parapneumonic effusion compared to S. aureus NP (p = 0.02). Patients with S. pyogenes NP more often required volume expansion than did S. pneumoniae cases (p = 0.03). When comparing children born before and after implementation of the 13-valent pneumococcal conjugate vaccine, we identified a relative modification of the bacterial epidemiology, with an increase in the proportion of S. pyogenes NP and S. aureus NP and a decrease in the proportion of NP caused by S. pneumoniae. CONCLUSION: Future studies are needed to assess the epidemiology of NP in children. Continued surveillance of identified pneumococcal serotypes is essential to document epidemiological changes in the coming years.

Effect of the COVID-19 Pandemic on Stillbirths in Canada and the United States.

OBJECTIVE: There is uncertainty regarding the effect of the COVID-19 pandemic on population rates of stillbirth. We quantified pandemic-associated changes in stillbirth rates in Canada and the United States. METHODS: We carried out a retrospective study that included all live births and stillbirths in Canada and the United States from 2015 to 2020. The primary analysis was based on all stillbirths and live births at ≥20 weeks gestation. Stillbirth rates were analyzed by month, with March 2020 considered to be the month of pandemic onset. Interrupted time series analyses were used to determine pandemic effects. RESULTS: The study population included 18 475 stillbirths and 2 244 240 live births in Canada and 134 883 stillbirths and 22 963 356 live births in the United States (8.2 and 5.8 stillbirths per 1000 total births, respectively). In Canada, pandemic onset was associated with an increase in stillbirths at ≥20 weeks gestation of 1.01 (95% confidence interval [CI] 0.56-1.46) per 1000 total births and an increase in stillbirths at ≥28 weeks gestation of 0.35 (95% CI 0.16-0.54) per 1000 total births. In the United States, pandemic onset was associated with an increase in stillbirths at ≥20 weeks gestation of 0.48 (95% CI 0.22-0.75) per 1000 total births and an increase in stillbirths at ≥28 weeks gestation of 0.22 (95% CI 0.12-0.32) per 1000 total births. The increase in stillbirths at pandemic onset returned to pre-pandemic levels in subsequent months. CONCLUSION: The COVID-19 pandemic's onset was associated with a transitory increase in stillbirth rates in Canada and the United States.

Dancing the Nanopore limbo - Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction.

BACKGROUND: While genome-resolved metagenomics has revolutionized our understanding of microbial and genetic diversity in environmental samples, assemblies of short-reads often result in incomplete and/or highly fragmented metagenome-assembled genomes (MAGs), hampering in-depth genomics. Although Nanopore sequencing has increasingly been used in microbial metagenomics as long reads greatly improve the assembly quality of MAGs, the recommended DNA quantity usually exceeds the recoverable amount of DNA of environmental samples. Here, we evaluated lower-than-recommended DNA quantities for Nanopore library preparation by determining sequencing quality, community composition, assembly quality and recovery of MAGs. RESULTS: We generated 27 Nanopore metagenomes using the commercially available ZYMO mock community and varied the amount of input DNA from 1000 ng (the recommended minimum) down to 1 ng in eight steps. The quality of the generated reads remained stable across all input levels. The read mapping accuracy, which reflects how well the reads match a known reference genome, was consistently high across all libraries. The relative abundance of the species in the metagenomes was stable down to input levels of 50 ng. High-quality MAGs (> 95% completeness, ≤ 5% contamination) could be recovered from metagenomes down to 35 ng of input material. When combined with publicly available Illumina reads for the mock community, Nanopore reads from input quantities as low as 1 ng improved the quality of hybrid assemblies. CONCLUSION: Our results show that the recommended DNA amount for Nanopore library preparation can be substantially reduced without any adverse effects to genome recovery and still bolster hybrid assemblies when combined with short-read data. We posit that the results presented herein will enable studies to improve genome recovery from low-biomass environments, enhancing microbiome understanding.

Obstetric Intervention and Perinatal Outcomes During the Coronavirus Disease 2019 (COVID-19) Pandemic.

OBJECTIVE: To quantify pandemic-related changes in obstetric intervention and perinatal outcomes in the United States. METHODS: We carried out a retrospective study of all live births and fetal deaths in the United States, 2015-2021, with data obtained from the natality, fetal death, and linked live birth-infant death files of the National Center for Health Statistics. Analyses were carried out among all singletons; singletons of patients with prepregnancy diabetes, prepregnancy hypertension, and hypertensive disorders of pregnancy; and twins. Outcomes of interest included preterm birth, preterm labor induction or preterm cesarean delivery, macrosomia, postterm birth, and perinatal death. Interrupted time series analyses were used to estimate changes in the prepandemic period (January 2015-February 2020), at pandemic onset (March 2020), and in the pandemic period (March 2020-December 2021). RESULTS: The study population included 26,604,392 live births and 155,214 stillbirths. The prepandemic period was characterized by temporal increases in preterm birth and preterm labor induction or cesarean delivery rates and temporal reductions in macrosomia, postterm birth, and perinatal mortality. Pandemic onset was associated with absolute decreases in preterm birth (decrease of 0.322/100 live births, 95% CI 0.506-0.139) and preterm labor induction or cesarean delivery (decrease of 0.190/100 live births, 95% CI 0.334-0.047) and absolute increases in macrosomia (increase of 0.046/100 live births), postterm birth (increase of 0.015/100 live births), and perinatal death (increase of 0.501/1,000 total births, 95% CI 0.220-0.783). These changes were larger in subpopulations at high risk (eg, among singletons of patients with prepregnancy diabetes). Among singletons of patients with prepregnancy diabetes, pandemic onset was associated with a decrease in preterm birth (decrease of 1.634/100 live births) and preterm labor induction or cesarean delivery (decrease of 1.521/100 live births) and increases in macrosomia (increase of 0.328/100 live births) and perinatal death (increase of 9.840/1,000 total births, 95% CI 3.933-15.75). Most changes were reversed in the months after pandemic onset. CONCLUSION: The onset of the coronavirus disease 2019 (COVID-19) pandemic was associated with a transient decrease in obstetric intervention (especially preterm labor induction or cesarean delivery) and a transient increase in perinatal mortality.

Temporal disturbance of a model stream ecosystem by high microbial diversity from treated wastewater.

Microbial communities in freshwater streams play an essential role in ecosystem functioning via biogeochemical cycling. Yet, the impacts of treated wastewater influx into stream ecosystems on microbial strain diversity remain mostly unexplored. Here, we coupled full-length 16S ribosomal RNA gene Nanopore sequencing and strain-resolved metagenomics to investigate the impact of treated wastewater on a mesocosm system (AquaFlow) run with restored river water. Over 10 days, community Bray-Curtis dissimilarities between treated and control mesocosm decreased (0.57 ± 0.058 to 0.26 ± 0.046) based on ribosomal protein S3 gene clustering, finally converging to nearly identical communities. Similarly, strain-resolved metagenomics revealed a high diversity of bacteria and viruses after the introduction of treated wastewater; these microbes also decreased over time resulting in the same strain clusters in control and treatment at the end of the experiment. Specifically, 39.2% of viral strains detected in all samples were present after the introduction of treated wastewater only. Although bacteria present at low abundance in the treated wastewater introduced additional antibiotic resistance genes, signals of naturally occurring ARG-encoding organisms resembled the resistome at the endpoint. Our results suggest that the previously stressed freshwater stream and its microbial community are resilient to a substantial introduction of treated wastewater.

Clinical relevance of cell-free DNA quantification and qualification during the first month after lung transplantation.

Background: Many studies have reported the relevance of donor-derived cfDNA (dd-cfDNA) after lung transplantation (LTx) to diagnose and monitor acute rejection (AR) or chronic rejection or infection (INF). However, the analysis of cfDNA fragment size has not been studied. The aim of this study was to determine the clinical relevance of dd-cfDNA and cfDNA size profiles in events (AR and INF) during the first month after LTx. Methods: This prospective, single-center study includes 62 LTx recipients at the Marseille Nord Hospital, France. Total cfDNA quantification was performed by fluorimetry and digital PCR, dd-cfDNA by NGS (AlloSeq cfDNA-CareDX®), and the size profile by BIABooster (Adelis®). A bronchoalveolar lavage and transbronchial biopsies at D30 established the following groups: not-injured and injured graft (AR, INF, or AR+INF). Results: Quantification of total cfDNA was not correlated with the patient's status at D30. The percentage of dd-cfDNA was significantly higher for injured graft patients at D30 (p=0.0004). A threshold of 1.72% of dd-cfDNA correctly classified the not-injured graft patients (negative predictive value of 91.4%). Among recipients with dd-cfDNA >1.72%, the quantification of small sizes (80-120bp) >3.70% identified the INF with high performance (specificity and positive predictive value of 100%). Conclusion: With the aim of considering cfDNA as a polyvalent non-invasive biomarker in transplantation, an algorithm combining the quantification of dd-cfDNA and small sizes of DNA may significantly classify the different types of allograft injuries.

Virus-Host Dynamics in Archaeal Groundwater Biofilms and the Associated Bacterial Community Composition.

Spatial and temporal distribution of lytic viruses in deep groundwater remains unexplored so far. Here, we tackle this gap of knowledge by studying viral infections of Altivir_1_MSI in biofilms dominated by the uncultivated host Candidatus Altiarchaeum hamiconexum sampled from deep anoxic groundwater over a period of four years. Using virus-targeted direct-geneFISH (virusFISH) whose detection efficiency for individual viral particles was 15%, we show a significant and steady increase of virus infections from 2019 to 2022. Based on fluorescence micrographs of individual biofilm flocks, we determined different stages of viral infections in biofilms for single sampling events, demonstrating the progression of infection of biofilms in deep groundwater. Biofilms associated with many host cells undergoing lysis showed a substantial accumulation of filamentous microbes around infected cells probably feeding off host cell debris. Using 16S rRNA gene sequencing across ten individual biofilm flocks from one sampling event, we determined that the associated bacterial community remains relatively constant and was dominated by sulfate-reducing members affiliated with Desulfobacterota. Given the stability of the virus-host interaction in these deep groundwater samples, we postulate that the uncultivated virus-host system described herein represents a suitable model system for studying deep biosphere virus-host interactions in future research endeavors.

Imaging Follow-up of Pediatric Transphyseal Anterior Cruciate Ligament Reconstruction: A Case Report.

CASE: We are reporting on the x-ray and magnetic resonance imaging (MRI) follow-up of an 8-year-old boy who underwent transphyseal anterior cruciate ligament reconstruction with a 4-strand semitendinosus tendon graft. His graft was followed for 5 years by MRI, demonstrating a satisfactory but slow and gradual ligamentization process. CONCLUSION: The long-term MRI follow-up demonstrated the satisfactory outcome of the transphyseal reconstruction, although with a slower and more progressive ligamentization process than in adult patients.

Evaluation of Next-Generation Sequencing and Crystal Digital PCR for Chimerism Monitoring of Post-Allogeneic Hematopoietic Stem Cell Transplantation.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for most hematologic diseases. To evaluate the level of donor engraftment, chimerism must be carefully monitored after HSCT. Short tandem repeats, quantitative PCR (qPCR), and, more recently, digital PCR (dPCR) are widely used to determine the proportions of donor and recipient cells after HSCT. The screening and quantification of chimerism have been evaluated by 2 new methods: a ready-to-use next-generation sequencing (NGS)-based method using the Devyser ChimerismNGS kit and an original combination of the Stilla crystal digital PCR (cdPCR) platform with 3-color multiplexing capacity using GenDX KMRtrack reagents. The genotyping of 4 HSCT pairs by cdPCR using 11 triplex mixes of the GenDX KMRtype kit was consistent at 98.8% with qPCR. Informative samples (n = 20) from 6 donor-recipient pairs and 1 external proficiency test demonstrated the reliability of the results (0.1% to 50%) for the 2 methods. The methods are also highly sensitive (0.1%) and accurate. The chimerism values of the 2 methods are correlated and concordant with those of the reference methods. In addition, the ADVYSER software (Devyser) is user-friendly and well adapted to chimerism monitoring. In conclusion, these 2 innovative methods are easy to perform and user-friendly in all molecular, hematology, and immunogenetic laboratories and allow the genotyping and monitoring of chimerism with high performance and sensitivity.

Pulmonary Alveolar Microlithiasis in Children Less than 5 Years of Age.

OBJECTIVE: To collect all published cases up to January 2019 of pulmonary alveolar microlithiasis (PAM) in patients age 5 years and under and to compare their characteristics with those of the 1022 cases in the most recent all-age cohort published in 2015. STUDY DESIGN: We identified 28 cases of PAM worldwide in children age 5 years and under, accounting for only 2%-3% of all cases. RESULTS: Children seem more frequently symptomatic, notably with more cough and severe acute respiratory failure, but had no reported extrapulmonary manifestation. Children with PAM evidenced less typical radiologic findings, with frequent ground glass opacities not reported in adult cases and milder calcifications as less frequent, smaller, and mainly restricted to the lower lobes. CONCLUSIONS: PAM remains an uncommon diagnosis in young children, as symptoms and radiologic findings are less specific. Physicians should be aware to look for calcifications in chest computed tomography at mediastinal window and avoid elution of the bronchoalveolar lavage to find microliths. Collecting longitudinal data through an international registry would help in characterizing PAM to predict disease progression and plan lung transplantation.

A cross-sectional study to screen community health volunteers for hip/knee-osteoarthritis and osteoporosis.

CONTEXT: Osteoarthritis (OA) is a degenerative disease mainly affecting hip and knee joints, and osteoporosis is characterized by diminution of bone mass. Both these diseases have a substantial economic impact on society. Community health volunteers (CHVs) being peripheral health workers are prone to such diseases owing to their sociodemographic and occupational profile. AIM: This study was conducted to estimate the proportion of hip/knee OA and osteoporosis among CHVs and understand determinants of their current bone health status. MATERIALS AND METHODS: Screening for OA was done using a tool adopted from a previous study after obtaining due permissions. Weight, height, blood pressure, and bone mineral density of all participants were recorded. Statistical tests such as Chi-square and multiple logistic regression were used for analysis of data. RESULTS: Out of 80 participants, 50 (62.5%) had increased body mass index (overweight + obese), 10 (12.5%) were hypertensive, 14 (17.5%) CHVs screened positive for hip OA, and 29 (36.3%) were positive for knee OA. Hip OA was associated with advancing age, parity, and obesity. Knee OA was associated with age and exercise. In total, 16.3% subjects were found to have osteoporosis and 61.2% had osteopenia. CONCLUSIONS: This study showed that a remarkable proportion of CHVs had bone and joint problems. CHVs must, therefore, receive preventive measures such as health education and screening for these diseases.

The CD31 molecule: a possible neuroprotective agent in acute ischemic stroke?

BACKGROUND: The transmembrane receptor molecule CD31 is known to have immunomodulatory functions, suggesting a possible neuroprotective effect in the context of acute ischemic stroke by restricting an over-activation of secondary immunological processes. This study examines the density of CD31+ cells in mechanically extracted thrombi of stroke patients with the aim to test whether the occurrence of CD31+ cells was associated with a beneficial clinical outcome in those patients. METHODS: Thrombi of 122 consecutive patients with large anterior circulation stroke were collected during intracranial mechanical recanalization. Out of these, 86 immunostained specimens of adequate quality could be analysed. The density of CD31+ cells was quantified and compared with clinical outcome data of the affected patients. RESULTS: The density of CD31+ cells was positively related to early patient improvement (ΔNIHSS, r = 0.283, p = 0,012) with an even clearer relationship after exclusion of patients who died in the early hospital phase (r = 0.371, p = 0.001). This finding stayed stable also in the multivariate analysis after corrrection for other outcome-influencing factors (p = 0.049). CONCLUSION: This study shows a stable relation between CD31+ cells and early clinical improvement of patients with acute ischemic stroke. This finding is in line with recent reports showing immunomodulatory and potential neuroprotective effects of CD31, suggesting that CD31 may be a promising neuroprotective agent in stroke patients.

[Haploidentical hematopoietic stem cell transplantation: Guidelines from the Francophone society of marrow transplantation and cellular therapy (SFGM-TC)]. / Greffe de cellules-souches hématopoïétiques haplo-identiques : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

Haploidentical hematopoietic stem cell transplantation (HSCT) is being increasingly used due to improvement of the transplantation procedures allowing a reduction of graft-versus-host-disease (GVHD) and of transplant-related mortality (TRM). Such improvements have been particularly observed after administration of T-replete HSCT graft associated to an in vivo T cell depletion by the administration of high-doses of cyclophosphamide (HD-Cy) after transplantation. Here, we have analyzed the results of haplo-identical T replete HSC transplants, in particular, when performed with post-transplant HD-Cy in order to provide recommendations for the clinical practice. Criteria of choice for a haploidentical donor by priority order are absence of donor-specific antibodies (DSA) and to prioritize: CMV seronegative recipient/donor couples, ABO matching in case of deserythrocytation, male donor for a male recipient, the youngest donor. There is no clear argument in favor of the use of bone marrow versus peripheral blood stem cells (PBSC) after non myeloablative conditioning regimen, while after ablative conditioning PBSC seem to be associated with higher risks of GVHD without obvious impact on survival. Results of haploidentical HSCT, confirmed by several groups, are interesting in lymphomas (in particular Hodgkin disease) and for acute leukemia. Outcomes of patients rely on age, disease status at transplant and conditioning intensity. At equivalent disease risk, results of haploidentical HSCT seem comparable to those of HLA matched HSCT, raising the question of the classification of such transplants as alternatives. In all cases, we recommend to include patients in prospective clinical trials.

[Violence against women: an involvement of all players]. / Les violences faites aux femmes: une implication de tous.

Every year in France, 201,000 women are victims of physical and/or sexual violence perpetrated by their partner or ex-partner. At the same time, 83,000 are victims of rape or attempted rape, by a known or unknown aggressor. The impact of this violence on the health and overall quality of life of female victims is considerable. Faced with the extent of these violences, initiatives are being set up, instigated by the government and numerous players in the field.

Assessing the HLA diversity of cord blood units collected from a birth clinic caring for pregnant women in an ethnically diverse metropolitan area.

BACKGROUND: New strategies are emerging in cord blood banking where focusing on birth clinics caring for a high number of mothers belonging to ethnic minorities could offer new possibilities for allotransplantation both for patients of European origin and for patients from ethnic minorities or mixed ancestries. STUDY DESIGN AND METHODS: Marseilles Cord Blood Bank works with one university birth clinic caring for a culturally and sociologically diverse population. Stringent French legal restrictions apply to recording the geographic origin of parents. To circumvent this limitation and evaluate the contribution of newly banked cord blood units (CBUs) to increasing HLA diversity, we applied an algorithm that allows for the determination of parents' putative haplotypes and thus grossly deduce information on their ancestry. Generic resolution HLA-A, HLA-B, and allelic resolution HLA-DRB1 genotyping for 328 CBUs and 2691 unrelated donors (UDs) between January 2009 and May 2012 were performed. Enrichment from international CBU registry with nonreferenced generic HLA-A, HLA-B, and allelic HLA-DRB1 phenotypes was compared between CBUs identified with one or two non-European haplotypes and CBUs identified with two European haplotypes. RESULTS: Marseilles CBUs display an increased proportion of HLA antigens frequently expressed in African populations compared to UDs. Whereas 93% of 199 CBUs identified with one or two non-European haplotypes enrich international CBU registry, this result is reduced to 42% for the 129 CBUs identified with two European haplotypes. CONCLUSION: This study supports a new method to assess HLA diversity. However, such an increased of HLA diversity raises questions about frequencies of CBUs released and clinical relevance from their uses.

HLA-G UTR haplotype conservation in the Malian population: association with soluble HLA-G.

The HLA-G molecule plays an important role in immunomodulation. In a previous study carried out on a southern French population our team showed that HLA-G haplotypes, defined by SNPs in the coding region and specific SNPs located in 5'URR and 3'UTR regulatory regions, are associated with differential soluble HLA-G expression (sHLA-G). Furthermore, the structure of these HLA-G haplotypes appears to be conserved in geographically distant populations. The aim of our study is to confirm these expectations in a sub-Saharan African population and to explore additional factors, such as HLA-A alleles, that might influence sHLA-G expression. DNA and plasma samples were collected from 229 Malians; HLA-G and HLA-A genotyping were respectively performed by the Snap Shot® method and by Luminex™ technology. sHLA-G dosage was performed using an ELISA kit. HLA-G and HLA-A allelic and haplotypic frequencies were estimated using an EM algorithm from the Gene[Rate] program. Associations between genetic and non genetic parameters with sHLA-G were performed using a non-parametric test with GRAPH PAD Prism 5. Our results reveal a good conservation of the HLA-G UTR haplotype structure in populations with different origins and demographic histories. These UTR haplotypes appear to be involved in different sHLA-G expression patterns. Specifically, the UTR-2 haplotype was associated with low sHLA-G levels, displaying a dominant negative effect. Furthermore, an allelic effect of both HLA-G and HLA-A, as well as non genetic parameters, such as age and gender possibly linked to osteogenesis and sexual hormones, also seem to be involved in the modulation of sHLA-G. These data suggest that further investigation in larger cohorts and in populations from various ethnical backgrounds is necessary not only to detect new functional polymorphism in HLA-G regulatory regions, but also to reveal the extent of biological phenomena that influence sHLA-G secretion and this might therefore have an impact on transplantation practice.

RHCE*cE734C allele encodes an altered c antigen and a suppressed E antigen not detected with standard reagents.

BACKGROUND: The RH blood group system has many RHCE variant alleles that have arisen through gene conversion or nucleotide changes. Two probands, with red blood cells (RBCs) that were D+C+E-c+(w) e+ were sent to our laboratories to resolve the weak c expression. STUDY DESIGN AND METHODS: Hemagglutination tests were performed by automated and manual procedures. Genomic DNA analysis was performed by sequencing of Exons 1 to 10 of RHCE and RHD. RESULTS: The probands' RBCs did not react with standard monoclonal anti-E reagents from Bio-Rad, Diagast, DiaMed, Immucor, Ortho, and Quotient. The RBCs reacted variably with anti-c reagents from Diagast, DiaMed, Immucor, or Ortho and did not react with the Quotient anti-c reagent. Surprisingly, sequencing results of RHCE showed the presence of C/G at Position 676 (E/e polymorphism) and the association of the E polymorphism with a 734T>C transition in Exon 5 of the RHCE, encoding a Leu245Pro amino acid substitution in the mature RhcE polypeptide. Replacement of leucine 245 by proline in the eighth transmembrane domain of the RhcE protein may have a steric effect on the protein such that most anti-E reagents do not bind and the interaction between anti-c and c antigen is also affected. CONCLUSION: We report a novel RHCE*cE allele, RHCE*cE734C, which was assigned the provisional ISBT allele name RHCE*cE.14 or RHCE*03.14. It was found in two probands whose RBCs had weakened c expression and typed E- with conventional anti-E reagents. These data, once again, highlight the fact that the genotype does not always reflect the phenotype.

Characterization of novel RHD alleles: relationship between phenotype, genotype, and trimeric architecture.

BACKGROUND: RH1 is one of the most clinically important blood group antigens in the field of transfusion and prevention of fetomaternal incompatibilities. New variant RHD alleles are regularly identified and their characterization is essential to ensuring patient safety. STUDY DESIGN AND METHODS: Blood samples with uncertain RhD phenotypes not resolved by our first-line SNaPshot assay were sequenced for all 10 RHD exons. RHD zygosity was investigated. Flow cytometry was performed to determine RhD antigen density and epitope pattern. RESULTS: Seven novel RHD alleles were identified. Six, that is, RHD(T55P), RHD(A85G), RHD(G132R), RHD(G132E), RHD(D403V), and DAR(T203A), resulted from nucleotide polymorphisms. The seventh, that is, RHD(S182WfsX46), resulted from a 4-bp deletion that led to a reading frame shift and the appearance of a premature stop codon. Study of RhD expression of the first five alleles at hemizygous state showed greatly reduced antigen densities ranging from 50 to 618 antigens per red blood cell (RBC). DAR(T203A) was classified as a partial D antigen with a weakened reactivity profile similar to that of DAR. As expected, no D antigen was detected on RBCs carrying the RHD(S182WfsX46) allele. In parallel, RhD expression of RHD(G336R)/weak D type 58, RHD(F410V), and suspected RHD(1-9)-CE was determined to be less than or equal to 50 antigens per RBC. RhAG/RhD(2) trimer model supports the observed phenotypes. CONCLUSION: Although the frequency of the new RHD alleles presented herein is low, their phenotypic and genotypic description adds to the repertoire of reported RHD alleles. These data can be useful for optimization of molecular screening tools.

Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles.

BACKGROUND: Molecular RHD blood group typing is very efficient for managing donors and patients carrying any of the various molecular types of weak D and DEL. The purpose of the work was to develop a multiplex polymerase chain reaction (PCR) SNaPshot assay for simultaneous detection of weak D and DEL alleles that are prevalent in Europeans, Africans, and Asians. STUDY DESIGN AND METHODS: Preliminary profiling was carried out on single-nucleotide polymorphisms (SNPs) associated with 13 prevalent RHD alleles, that is, weak D Types 1, 2, 3, 4.0, 4.0.1, 4.1, 4.2, 5, 11, 15, and 17; RHD(IVS3+1g>a); and RHD(K409K). Multiplex PCR was used to amplify six RHD regions encompassing 14 SNPs. Identification was obtained by incorporation of the complementary dye single base at the 3'-end of each probe-primer. A prospective analysis was then carried out on 152 blood samples from patients (n = 53) and donors (n = 88) with equivocal RhD serology and pregnant women (n = 11). RESULTS: After validation, our SNaPshot assay allowed direct genotyping of 82.9% of samples overall and 100% of samples harboring weak D Types 1, 2, 3, and 4.1 alleles. In the remaining 17.1% of samples overall, sequence investigation allowed accurate genotyping. In addition, four novel RHD alleles were identified, that is, RHD(S256P), RHD(L390L), RHD(F410V), and RHD(IVS4-2a>g). CONCLUSION: The SNaPshot assay described herein is a helpful supplementary tool for resolving doubtful RhD serology. By allowing accurate identification of weak D and DEL alleles this assay should allow better management of the donors and the patients genotyped weak D Types 1, 2, 3, and 4.1 who can receive D+ blood units.

Prevalence of IgG antibodies against West Nile virus in blood donors during the 2003 outbreak in Tunisia.

This study aimed to evaluate the prevalence of anti-West Nile virus (WNV) IgG among two populations of Tunisian blood donors living in areas where human outbreaks of WNV have occurred. Cohorts A (Monastir) and B (Mahdia) included 742 and 102 blood donors respectively. Sera were tested by IgG ELISA test and results were confirmed by PRNT test. WNV neutralizing antibodies were detected in 32 (4.3%) and in 14 (13.7%) sera in cohorts A and B respectively. The prevalence of anti-WNV IgG was significantly higher in cohort B than in cohort A (P<0.001) and was significantly lower in females than in males (P<0.001).