Improved access to better HLA-matched hematopoietic cells for allogeneic transplant: analysis of donors and cord blood units selected for Canadian patients in 2018.

BACKGROUND: Patients undergoing hematopoietic cell transplantation (HCT) often require use of an unrelated donor or cord blood unit (CBU). An understanding of evolving practices in graft selection is needed for optimization of donor recruitment and cord blood collection. STUDY DESIGN AND METHODS: Each donor workup (WU) requested in 2018 involving a Canadian (CDN) patient and unique donor product or CBU was reviewed (n = 598). Degree of HLA match; product origin (domestic or international [INT]); and non-HLA factors including donor age, sex, cytomegalovirus (CMV), and ABO compatibility were analyzed for WUs that proceeded to transplant (n = 414). We also analyzed changes compared to a similar analysis performed in 2013. RESULTS: The majority of transplants used matched unrelated donors (MUDs; n = 323; 78%) and were most often young (≤35 years), male, INT donors (n = 136). The proportion of transplants involving MUDs, as opposed to mismatched unrelated donors or CBUs, increased by 12.4% compared with 2013. When young, male, CDN MUDs were identified in patient search reports but not selected, CMV mismatching and ABO incompatibility were most likely to have influenced the decision to use an INT MUD. Consistent with global trends, CBU transplants decreased compared to 2013; however, the degree of HLA matching improved significantly, and 27% of transplanted CBUs were procured from the Canadian Blood Services Cord Blood Bank. CONCLUSIONS: Access to MUDs and better HLA-matched CBUs by CDN patients has increased since 2013. Ongoing recruitment of young registrants and cord blood donors with diverse HLA haplotypes will support selection of donors with optimal non-HLA characteristics.

Unrelated donor choices for allogeneic hematopoietic cell transplantation in Canada: an evaluation of factors influencing donor selection.

BACKGROUND: Utilization of unrelated donors and cord blood units (CBUs) for allogeneic hematopoietic cell transplantation continues to increase. Understanding the practices of donor selection by transplant centers is critical for unrelated donor registries and cord blood banks to optimize registry composition and inventory to meet patient need. STUDY DESIGN AND METHODS: Unrelated donor and CBU selection practices of Canadian transplant centers served by Canadian Blood Services' OneMatch Stem Cell & Marrow Network (OM) were reviewed, including HLA match level, locus of disparity, age, sex, and product choice (donor vs. CBU). RESULTS: HLA-matched donors within OM and/or international (INT) registries were preferentially investigated, underscoring the primary importance of HLA matching. In the case of HLA-mismatched donors, HLA-A disparities were most common while DRB1 mismatches were least common. Advanced age, sex, and lack of donor availability were the most frequent reasons that high-probability OM donors were overlooked in favor of INT donors. High-probability 10 of 10 HLA-matched female donors from OM were often avoided in favor of INT male donors. Use of female donors, however, increased in cases restricted to more HLA-disparate donor options. Caucasian patients were more likely to find 10 of 10 matched donors, whereas use of mismatched donors and CBUs were more prevalent among non-Caucasian patients. CONCLUSIONS: Recruitment and retention of young, male donors from diverse ethnic backgrounds may increase the usage of histocompatible OM donors for patients in need.

Biofilm-forming skin microflora bacteria are resistant to the bactericidal action of disinfectants used during blood donation.

BACKGROUND: A one-step skin disinfection method containing 2% chlorhexidine-gluconate (CHG) and 70% isopropyl alcohol (IPA) is currently used by blood suppliers worldwide. Reports of bacterially contaminated platelet concentrates (PCs) indicate that skin disinfection is not fully effective. Approximately 20% of skin microflora exist as surface-attached aggregates (biofilms), known for displaying increased resistance to disinfectants. This study was aimed at determining whether skin microflora biofilm-positive Staphylococcus epidermidis and Staphylococcus capitis are resistant to CHG and/or IPA. STUDY DESIGN AND METHODS: Free-floating cells and mono or dual (1 : 1 ratio) biofilms of S. epidermidis and S. capitis were exposed to CHG, IPA, or CHG/IPA for 30 seconds, simulating skin disinfection practices. Residual viable cells were quantified by colony counting. Morphology of disinfectant-treated S. epidermidis biofilms was examined by scanning electron microscopy. Treated S. epidermidis and S. capitis biofilms were inoculated into PCs and bacterial concentrations were determined on Days 0 and 5 of storage. RESULTS: Treatment of staphylococcal biofilm cells with all disinfectants caused cell damage and significant reduction in viability, with CHG/IPA being the most effective. However, biofilms were significantly more resistant to treatment than free-floating cells. Disinfectant-treated S. epidermidis proliferated better in PCs than S. capitis, especially when grown as monospecies biofilms. CONCLUSION: Although CHG/IPA is effective in reducing the viability of S. epidermidis and S. capitis biofilms, these organisms are not completely eliminated. Furthermore, disinfectant-treated staphylococcal biofilms multiply well in PCs. These results demonstrate that the biofilm-forming capability of the skin microflora reduces the bactericidal efficiency of blood donor skin disinfectants.

Characterization of the growth dynamics and biofilm formation of Staphylococcus epidermidis strains isolated from contaminated platelet units.

Bacterial contamination of platelet concentrates (PCs) poses the highest transfusion-associated infectious risk, with Staphylococcus epidermidis being a predominant contaminant. Herein, the growth dynamics of 20 S. epidermidis strains in PCs and regular media were characterized. Strains were categorized as fast (short lag phase) or slow (long lag phase) growers in PCs. All strains were evaluated for the presence of the biofilm-associated icaAD genes by PCR, their capability to produce extracellular polysaccharide (slime) on Congo red agar plates and their ability to form surface-attached aggregates (biofilms) in glucose-supplemented trypticase soy broth (TSBg) using a crystal violet staining assay. A subset of four strains (two slow growers and two fast growers) was further examined for the ability for biofilm formation in PCs. Two of these strains carried the icAD genes, formed slime and produced biofilms in TSBg and PCs, while the other two strains, which did not carry icaAD, did not produce slime or form biofilms in TSBg. Although the two ica-negative slime-negative strains did not form biofilms in media, they displayed a biofilm-positive phenotype in PCs. Although all four strains formed biofilms in PCs, the two slow growers formed significantly more biofilms than the fast growers. Furthermore, growth experiments of the two ica-positive strains in plasma-conditioned platelet bags containing TSBg revealed that a slow grower isolate was more likely to escape culture-based screening than a fast grower strain. Therefore, this study provides novel evidence that links S. epidermidis biofilm formation with slow growth in PCs and suggests that slow-growing biofilm-positive S. epidermidis would be more likely to be missed with automate culture.

Enhanced pathogenicity of biofilm-negative Staphylococcus epidermidis isolated from platelet preparations.

BACKGROUND: The platelet (PLT) storage environment triggers the formation of surface-attached aggregates known as biofilms by the common PLT contaminant Staphylococcus epidermidis. The biofilm matrix is largely composed of polysaccharide intercellular adhesin (PIA) mediated by the icaADBC operon. However, PIA-negative S. epidermidis has been reported to form biofilms in PLT concentrates (PCs). Since biofilm formation is associated with increased virulence, this study was aimed at determining if PIA-negative S. epidermidis grown in PCs presents enhanced virulence using the nematode Caenorhabditis elegans as a host model for bacterial pathogenesis. STUDY DESIGN AND METHODS: Biofilm-positive S. epidermidis ATCC 35984 and 9142, which carry the icaADBC operon, and biofilm-negative S. epidermidis ATCC 12228 and 9142 ΔicaA were grown in regular media and in PCs and biofilm formation was quantified using a crystal violet assay. The virulence of these strains after passage through PCs was tested using nematode killing assays. Nematode survival was calculated using the Kaplan-Meier method and statistical differences were determined by log-rank analysis. RESULTS: All S. epidermidis strains were able to form biofilms in PCs. Although persistence of a biofilm-positive phenotype in the biofilm-negative strains grown in PCs was not observed after passage in regular medium, the virulence of all strains was significantly increased as demonstrated by shortened life spans of the nematodes in C. elegans killing assays. CONCLUSION: Our findings highlight the potential of an increased risk of nosocomial infections caused by S. epidermidis in transfusion recipients since PC storage conditions promote biofilm formation, and possibly pathogenicity, of strains traditionally known to be attenuated for virulence.

Biofilm formation by Staphylococcus capitis strains isolated from contaminated platelet concentrates.

Bacterial contamination of platelet concentrates (PCs) poses the greatest infectious risk in modern transfusion medicine despite the implementation of measures such as improved skin disinfection and first aliquot diversion. The majority of PC contaminants are commensal skin flora introduced by venipuncture at the time of blood collection. The predominant organisms are Gram-positive coagulase-negative staphylococci such as Staphylococcus capitis. This bacterium has been implicated in numerous instances of infection and sepsis, likely for its ability to form surface-associated communities of micro-organisms encased in extracellular materials, known as biofilms. In the present study, five strains of S. capitis isolated from contaminated PCs were assessed for their ability to produce extracellular polysaccharide (slime), a canonical indicator of biofilm-formation ability, on Congo red agar plates. Biofilm formation was evaluated in both glucose-enriched trypticase soy broth (TSBg) and in PCs by using a crystal violet staining assay. The chemical nature of the biofilms was evaluated by disruption assays using sodium metaperiodate and proteinase K. In addition, biofilm architecture was observed by scanning electron microscopy. The presence of the biofilm-associated icaR and icaADBC genes was also examined by PCR. While only two out of the five S. capitis strains formed biofilms in TSBg, all strains formed biofilms in PCs. The ability of strains to produce extracellular polysaccharide and their possession of wild-type ica genes were not exclusive predictors of biofilm formation in TSBg or PCs; different profiles of biofilm markers were observed among isolates. This is likely due to the proteinaceous composition of the S. capitis biofilm matrix. Interestingly, an ica-negative, non-slime-producing isolate was capable of biofilm formation in PCs. Together, these data indicate that the platelet storage environment stimulates biofilm formation in S. capitis in the absence of extracellular polysaccharide production and that multiple bacterial factors and regulatory elements are likely involved in biofilm formation in this milieu.

Interaction of host cellular proteins with components of the hepatitis delta virus.

The hepatitis delta virus (HDV) is the smallest known RNA pathogen capable of propagation in the human host and causes substantial global morbidity and mortality. Due to its small size and limited protein coding capacity, HDV is exquisitely reliant upon host cellular proteins to facilitate its transcription and replication. Remarkably, HDV does not encode an RNA-dependent RNA polymerase which is traditionally required to catalyze RNA-templated RNA synthesis. Furthermore, HDV lacks enzymes responsible for post-transcriptional and -translational modification, processes which are integral to the HDV life cycle. This review summarizes the known HDV-interacting proteins and discusses their significance in HDV biology.

The hepatitis delta virus RNA genome interacts with eEF1A1, p54(nrb), hnRNP-L, GAPDH and ASF/SF2.

Because of its extremely limited coding capacity, the hepatitis delta virus (HDV) takes over cellular machineries for its replication and propagation. Despite the functional importance of host factors in both HDV biology and pathogenicity, little is known about proteins that associate with its RNA genome. Here, we report the identification of several host proteins interacting with an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA, using mass spectrometry on a UV crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation was used to confirm the interactions of eEF1A1, p54(nrb), hnRNP-L, GAPDH and ASF/SF2 with the right terminal stem-loop domain of HDV genomic RNA in vitro, and with both polarities of HDV RNA within HeLa cells. Our discovery that HDV RNA associates with RNA-processing pathways and translation machinery during its replication provides new insights into HDV biology and its pathogenicity.

The hepatitis delta virus RNA genome interacts with the human RNA polymerases I and III.

The hepatitis delta virus (HDV) relies on human transcriptional machinery for its replication and transcription. Although the involvement of RNA polymerase II in HDV RNA biosynthesis is established, the contribution of additional polymerases remains uncertain. Here, we demonstrate the interaction of both RNA polymerase I and III with HDV RNA, both in vitro and in human cells. Binding of these polymerases occurs near the terminal stem-loop domains of both polarities of the HDV RNA genome. Based on interactions of HDV RNA with numerous host polymerases, our results suggest a higher level of complexity of HDV biology than previously envisioned.

The human RNA polymerase II interacts with the terminal stem-loop regions of the hepatitis delta virus RNA genome.

The hepatitis delta virus (HDV) is an RNA virus that depends on DNA-dependent RNA polymerase (RNAP) for its transcription and replication. While it is generally accepted that RNAP II is involved in HDV replication, its interaction with HDV RNA requires confirmation. A monoclonal antibody specific to the carboxy terminal domain of the largest subunit of RNAP II was used to establish the association of RNAP II with both polarities of HDV RNA in HeLa cells. Co-immunoprecipitations using HeLa nuclear extract revealed that RNAP II interacts with HDV-derived RNAs at sites located within the terminal stem-loop domains of both polarities of HDV RNA. Analysis of these regions revealed a strong selection to maintain a rod-like conformation and demonstrated several conserved features. These results provide the first direct evidence of an association between human RNAP II and HDV RNA and suggest two transcription start sites on both polarities of HDV RNA.

Binding of the polypyrimidine tract-binding protein-associated splicing factor (PSF) to the hepatitis delta virus RNA.

The hepatitis delta virus (HDV) has a very limited protein coding capacity and must rely on host proteins for its replication. A ribonucleoprotein complex was detected following UV cross-linking between HeLa nuclear proteins and an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA. Mass spectrometric analysis of the complex revealed the polypyrimidine tract-binding protein-associated splicing factor (PSF) as a novel HDV RNA-interacting protein. Co-immunoprecipitation demonstrated the interaction between HDV RNA and PSF both in vitro in HeLa nuclear extract and in vivo within HeLa cells containing both polarities of the HDV genome. Analysis of the binding of various HDV-derived RNAs to purified, recombinant PSF further confirmed the specificity of the interaction and revealed that PSF directly binds to the terminal stem-loop domains of both polarities of HDV RNA. Our findings provide evidence of the involvement of a host mRNA processing protein in the HDV life cycle.