International review of blood donation nucleic acid amplification testing.

BACKGROUND AND OBJECTIVES: Nucleic acid amplification testing (NAT), in blood services context, is used for the detection of viral and parasite nucleic acids to reduce transfusion-transmitted infections. This project reviewed NAT for screening blood donations globally. MATERIALS AND METHODS: A survey on NAT usage, developed by the International Society of Blood Transfusion Working Party on Transfusion-transmitted Infectious Diseases (ISBT WP-TTID), was distributed through ISBT WP-TTID members. Data were analysed using descriptive statistics. RESULTS: Forty-three responses were received from 32 countries. Increased adoption of blood donation viral screening by NAT was observed over the past decade. NAT-positive donations were detected for all viruses tested in 2019 (proportion of donations positive by NAT were 0.0099% for human immunodeficiency virus [HIV], 0.0063% for hepatitis C virus [HCV], 0.0247% for hepatitis B virus [HBV], 0.0323% for hepatitis E virus [HEV], 0.0014% for West Nile virus [WNV] and 0.00005% for Zika virus [ZIKV]). Globally, over 3100 NAT-positive donations were identified as NAT yield or solely by NAT in 2019 and over 22,000 since the introduction of NAT, with HBV accounting for over half. NAT-positivity rate was higher in first-time donors for all viruses tested except WNV. During 2019, a small number of participants performed NAT for parasites (Trypanosoma cruzi, Babesia spp., Plasmodium spp.). CONCLUSION: This survey captures current use of blood donation NAT globally. There has been increased NAT usage over the last decade. It is clear that NAT contributes to improving blood transfusion safety globally; however, there is a need to overcome economic barriers for regions/countries not performing NAT.

An international review of the characteristics of viral nucleic acid-amplification testing (NAT) reveals a trend towards the use of smaller pool sizes and individual donation NAT.

BACKGROUND AND OBJECTIVES: Nucleic acid-amplification testing (NAT) is used for screening blood donations/donors for blood-borne viruses. We reviewed global viral NAT characteristics and NAT-yield confirmatory testing used by blood operators. MATERIALS AND METHODS: NAT characteristics and NAT-yield confirmatory testing used during 2019 was surveyed internationally by the International Society of Blood Transfusion Working Party Transfusion-Transmitted Infectious Diseases. Reported characteristics are presented herein. RESULTS: NAT was mainly performed under government mandate. Human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV) NAT was performed on all donors and donation types, while selective testing was reported for West Nile virus, hepatitis E virus (HEV), and Zika virus. Individual donation NAT was used for HIV, HCV and HBV by ~50% of responders, while HEV was screened in mini-pools by 83% of responders performing HEV NAT. Confirmatory testing for NAT-yield samples was generally performed by NAT on a sample from the same donation or by NAT and serology on samples from the same donation and a follow-up sample. CONCLUSION: In the last decade, there has been a trend towards use of smaller pool sizes or individual donation NAT. We captured characteristics of NAT internationally in 2019 and provide insights into confirmatory testing approaches used for NAT-yields, potentially benefitting blood operators seeking to implement NAT.

A novel competition ELISA for the rapid quantification of SARS-CoV-2 neutralizing antibodies in convalescent plasma.

BACKGROUND: COVID-19 convalescent plasma (CCP) ideally contains high titers of (neutralizing) anti-SARS-CoV-2 antibodies. Several scalable immunoassays for CCP selection have been developed. We designed an enzyme-linked immunosorbent assay (ELISA) that measures neutralizing antibodies (of all isotypes) in plasma by determining the level of competition between CCP and a mouse neutralizing antibody for binding to the receptor binding domain (RBD) of SARS-CoV-2. METHODS: Plasma was collected from 72 convalescent individuals and inhibition of viral infection was determined by plaque reduction neutralization (PRNT50). The level of neutralizing antibodies was measured in the novel competition ELISA and in a commercially available ELISA that measures inhibition of recombinant ACE2 binding to immobilized RBD. These results were compared with a high throughput chemiluminescent microparticle immunoassay (CMIA). RESULTS: The results from both ELISAs were correlating, in particular for high titer CCP (PRNT50 ≥ 1:160) (Spearman r = .73, p < .001). Moderate correlation was found between the competition ELISA and CMIA (r = .57 for high titer and r = .62 for low titer CCP, p < .001). Receiver operator characteristic analysis showed that the competition ELISA selected CCP with a sensitivity and specificity of 61% and 100%, respectively. However, discrimination between low and high titer CCP had a lower resolution (sensitivity: 34% and specificity: 89%). CONCLUSION: The competition ELISA screens for neutralizing antibodies in CCP by competition for just a single epitope. It exerts a sensitivity of 61% with no false identifications. These ELISA designs can be used for epitope mapping or for selection of CCP.

Hepatitis B virus prevalence in first-time blood donors in Flanders, Belgium: Impact of universal vaccination and migration.

BACKGROUND: Transfusion-transmissible infections such as hepatitis B virus (HBV) remain a major concern for the safety of blood transfusion. This cross-sectional study aimed to assess the trend of HBV prevalence and associated risk factors among a first-time donor population in a low endemic country. STUDY DESIGN AND METHODS: Between 2010 and 2018, blood samples were collected from first-time donors presented at donor collection sites of Belgian Red Cross-Flanders. They were tested for hepatitis B surface antigen (HBsAg), hepatitis B core antibodies (anti-HBc), and HBV DNA, HIV and hepatitis virus C (HCV) antibodies and RNA, and syphilis antibodies. RESULTS: A total of 211,331 first-time blood donors (43.7% males, median age 25 years) were analyzed. HBsAg prevalence decreased from 0.06% in 2010 to 0.05% in 2018 (p = .004) and this declining trend was accompanied by an increased number of donors in the HBV vaccinated birth cohort (p < .001). HBsAg prevalence was 0.33% in foreign-born donors and 0.02% in Belgian natives (p < .001). Multivariate risk profiling showed that anti-HBc positivity was significantly associated with mainly foreign-born donors (odds ratio [OR] = 9.24) but also with older age (OR = 1.06), male gender (OR = 1.32), year of blood donation (OR = 0.94), and co-infections with HCV (OR = 4.31) or syphilis (OR = 4.91). DISCUSSION: The decreasing trend in HBV prevalence could mainly be explained by the introduction of the universal HBV vaccination. Being born in endemic areas was the most important predictor for HBV infection while the co-infections with syphilis suggest unreported sexual risk contacts.

Phylogenetic analysis reveals three distinct epidemiological profiles in Dutch and Flemish blood donors with hepatitis B virus infection.

During 2006-2016, hepatitis B virus (HBV) was detected in nearly 400 blood donors in the Netherlands and Flanders. Donor demographics and self-reported risk factors as disclosed during the donor exit interview were compared to HBV phylogenies of donor and reference sequences. First-time donors with chronic HBV-infection were often immigrants (67%) infected with genetically highly diverse strains of genotypes A (32%), B (8%), C (6%), D (53%) and E to H (1%). Each subtype was strongly associated with donor ethnicity. In contrast, 57/62 (93%) of acute/recent HBV infections occurred among indigenous donors, of whom 67% was infected with one specific widely circulating epidemic HBV-A2 lineage. HBV typing identified three distinct epidemiological profiles: the import of chronic HBV infections through migration, longstanding transmission of non-epidemic HBV-A2 strains within western-Europe, and the active transmission of one epidemic HBV-A2 strain most likely fueled by sexual risk behavior.

Extended-Spectrum beta-lactamase (ESBL) producing Enterobacter aerogenes phenotypically misidentified as Klebsiella pneumoniae or K. terrigena.

BACKGROUND: Enterobacter aerogenes and Klebsiella pneumoniae are common isolates in clinical microbiology and important as producers of extended spectrum beta-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of E. aerogenes can be misidentified as K. pneumoniae by routine laboratories using standard biochemical identification and using identification automates. RESULTS: Ten clinical isolates, identified as K. pneumoniae or K. terrigena with the routinely used biochemical tests and with API-20E, were identified as E. aerogenes by tDNA-PCR - an identification that was confirmed by 16S rRNA gene sequencing for five of these isolates. Misidentification also occurred when using the automated identification systems Vitek 2 and Phoenix, and was due to delayed positivity for ornithine decarboxylase and motility. Subculture and prolonged incubation resulted in positive results for ornithine decarboxylase and for motility. It could be shown by RAPD-analysis that the E. aerogenes strains belonged to different genotypes. CONCLUSIONS: Clinical E. aerogenes isolates can be easily misidentified as Klebsiella due to delayed positivity for ornithine decarboxylase and motility. The phenomenon may be widespread, since it was shown to occur among genotypically unrelated strains from different hospitals and different isolation dates. A useful clue for correct identification is the presence of an inducible beta-lactamase, which is highly unusual for K. pneumoniae. In several instances, the use of genotypic techniques like tDNA-PCR may circumvent problems of phenotypic identification.

Identification of cultured isolates of clinically important yeast species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region (ITS2).

BACKGROUND: The number of patients candidate to yeast infection has increased during the last years. Also the variety of species of clinical importance has increased. Correct species identification is often important for efficient therapy, but is based on phenotypic features and is sometimes time-consuming and depends largely on the expertise of technicians. Therefore, we evaluated the feasability of PCR-based amplification of the Internally Transcribed Spacer region 2, followed by fragment size analysis on the ABI Prism 310 for the identification of clinically important yeasts. RESULTS: A rapid DNA-extraction method, based on simple boiling freezing was introduced. Of the 25 species tested, 22 could be identified unambiguously by scoring the length of the ITS2-region. No distinction could be made between the species T. asteroides and T. inkin and between T. mucoides and T. ovoides. The two varieties of Cryptococcus neoformans (var. neoformans and var. gattii) could be differentiated from each other due to a one bp length difference of the ITS2 fragment. The three C. laurentii isolates were split in two groups according to their ITS2-fragment lengths, in correspondence with the phylogenetic groups described previously. Since the obtained fragment lenghts compared well to those described previously, an internationally usable library of ITS2 fragment lengths can be constructed. CONCLUSIONS: The existing ITS2 size based library enables identification of most of the clinically important yeast species, within 6 hours starting from a single colony, can be easily updated when new species are described. Data can be exchanged between laboratories.

Bacteremia due to Moraxella atlantae in a cancer patient.

A gram-negative alkaline phosphatase- and pyrrolidone peptidase-positive rod-shaped bacterium (CCUG 45702) was isolated from two aerobic blood cultures from a female cancer patient. No identification could be reached using phenotypic techniques. Amplification of the tRNA intergenic spacers revealed fragments with lengths of 116, 133, and 270 bp, but no such pattern was present in our reference library. Sequencing of the 16S rRNA gene revealed its identity as Moraxella atlantae, a species isolated only rarely and published only once as causing infection. In retrospect, the phenotypic characteristics fit the identification as M. atlantae (formerly known as CDC group M-3). Comparative 16S rRNA sequence analysis indicates that M. atlantae, M. lincolnii, and M. osloensis might constitute three separate genera within the MORAXELLACEAE: After treatment with amoxicillin-clavulanic acid for 2 days, fever subsided and the patient was dismissed.