Risk of transfusion-transmitted infection with severe acute respiratory syndrome coronavirus 2 from blood donors in Japan.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) essentially affects respiratory organs and tissues. SARS-CoV-2 RNAemia is often associated with more severe cases of coronavirus disease 2019 (COVID-19) compared to cases without RNAemia. To determine the impact of the pandemic on transfusion medicine, particularly transfusion-related infection, we examined the frequency of blood donation with RNAemia, the viral RNA (vRNA) concentration, and any possibility of transfusion-transmitted infection (TTI) among transfusion recipients. STUDY DESIGN AND METHODS: vRNA was examined in plasma/serum samples from 496 of 513 blood donors who reported having been infected with SARS-CoV-2 within 2 weeks of donation among a total of ca. 9.9 million blood donations in Japan between January 15, 2020, and December 31, 2021. The clinical course of patients transfused with the blood component containing vRNA was also examined. RESULTS: vRNA was detected in 23 of 496 samples. The median period from blood donation to COVID-19 onset was 1 day in 16 RNAemia-positive donors. Most samples had vRNA concentrations below the limit of quantification. Three patients were transfused with either a packed red blood cell or platelet concentrate that tested positive for vRNA, showing no COVID-19 symptoms and testing negative for vRNA in post-transfusion blood. CONCLUSION: The rate of RNAemia was 4.6% among blood donors who were found to be infected with SARS-CoV-2 shortly after donation, and vRNA concentrations in their donated blood were extremely low. There was no evidence of TTI in the recipients transfused with RNAemia-positive blood components. TTI risk in SARS-CoV-2 is negligible.

Evaluation of human T-cell leukemia virus in vitro diagnostics using plasma specimens collected in Japan.

BACKGROUND: In vitro diagnostics (IVDs) for primary detection test/screening of human T-cell leukemia virus (HTLV) have recently been updated to new-generation products in Japan. In this study, the performance of these products was evaluated and discussed in terms of the usability of HTLV diagnosis in Japan. METHODS: The performance of 10 HTLV IVDs for primary detection test and confirmatory/discriminatory test was evaluated. Plasma specimens that had been declared ineligible for transfusion were provided by the Japanese Red Cross Blood Center. RESULTS: The diagnostic specificity of the IVDs was 100% (160/160). Six sandwich assays resulted in all HTLV-1/HTLV-positive specimens being positive (46/46). On the other hand, one sandwich assay, IVD under development 2 (UD2), resulted in one HTLV-1-positive and one HTLV-positive specimen being negative (44/46, 95.7%). One indirect assay, HISCL HTLV-1, could not detect one HTLV-positive specimen (45/46, 97.8%), but the updated product, UD1, correctly detected it (46/46, 100%). Serodia HTLV-I, based on a particle agglutination assay, resulted in 44 of the 46 positive specimens, but could not detect two specimens (44/46, 95.7%). ESPLINE HTLV-I/II, based on an immunochromatography assay (ICA), was able to diagnose all specimens as positive (46/46, 100%). CONCLUSIONS: Six sandwich assays and an ICA demonstrated high diagnostic sensitivity and specificity and are recommended for use in HTLV diagnosis in conjunction with confirmatory/discriminatory test using the INNO-LIA HTLV-I/II Score.

Establishment of a novel diagnostic test algorithm for human T-cell leukemia virus type 1 infection with line immunoassay replacement of western blotting: a collaborative study for performance evaluation of diagnostic assays in Japan.

BACKGROUND: The reliable diagnosis of human T-cell leukemia virus type 1 (HTLV-1) infection is important, particularly as it can be vertically transmitted by breast feeding mothers to their infants. However, current diagnosis in Japan requires a confirmatory western blot (WB) test after screening/primary testing for HTLV-1 antibodies, but this test often gives indeterminate results. Thus, this collaborative study evaluated the reliability of diagnostic assays for HTLV-1 infection, including a WB-based one, along with line immunoassay (LIA) as an alternative to WB for confirmatory testing. RESULTS: Using peripheral blood samples from blood donors and pregnant women previously serologically screened and subjected to WB analysis, we analyzed the performances of 10 HTLV-1 antibody assay kits commercially available in Japan. No marked differences in the performances of eight of the screening kits were apparent. However, LIA determined most of the WB-indeterminate samples to be conclusively positive or negative (an 88.0% detection rate). When we also compared the sensitivity to HTLV-1 envelope gp21 with that of other antigens by LIA, the sensitivity to gp21 was the strongest. When we also compared the sensitivity to envelope gp46 by LIA with that of WB, LIA showed stronger sensitivity to gp46 than WB did. These findings indicate that LIA is an alternative confirmatory test to WB analysis without gp21. Therefore, we established a novel diagnostic test algorithm for HTLV-1 infection in Japan, including both the performance of a confirmatory test where LIA replaced WB on primary test-reactive samples and an additional decision based on a standardized nucleic acid detection step (polymerase chain reaction, PCR) on the confirmatory test-indeterminate samples. The final assessment of the clinical usefulness of this algorithm involved performing WB analysis, LIA, and/or PCR in parallel for confirmatory testing of known reactive samples serologically screened at clinical laboratories. Consequently, LIA followed by PCR (LIA/PCR), but neither WB/PCR nor PCR/LIA, was found to be the most reliable diagnostic algorithm. CONCLUSIONS: Because the above results show that our novel algorithm is clinically useful, we propose that it is recommended for solving the aforementioned WB-associated reliability issues and for providing a more rapid and precise diagnosis of HTLV-1 infection.

Development of reference material with assigned value for human T-cell leukemia virus type 1 quantitative PCR in Japan.

Quantitative PCR (qPCR) of human T-cell leukemia virus type 1 (HTLV-1) provirus is used for HTLV-1 testing and for assessment of risk of HTLV-1-related diseases. In this study, a reference material was developed for standardizing HTLV-1 qPCR. Freeze-dried TL-Om1 cells diluted with Jurkat cells were prepared and an assigned value for proviral load (PVL) of 2.71 copies/100 cells was determined by digital PCR. Nine Japanese laboratories using their own methods evaluated the PVLs of this reference material as 1.08-3.49 copies/100 cells. The maximum difference between laboratories was 3.2-fold. Correcting measured PVLs by using a formula incorporating the assigned value of this reference material should minimize such discrepancies.

Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens.

Analysis of HTLV-1 proviral load (PVL) and antibody detected with various kinds of tests in Japanese blood donors to understand the relationship between PVL and antibody level and to gain insights toward better antibody testing.

Adult T-cell leukemia/lymphoma (ATL) occurs in approximately 5% of individuals infected with human T-cell leukemia virus type 1 (HTLV-1). A high proviral load (PVL; more than four copies per 100 peripheral blood mononuclear cells (PBMCs) or 1.6 copies per 100 blood leukocytes) and being male are risk factors for ATL development. Whether anti-HTLV-1 antibody level is related to such risk is unknown. Here, PVL and antibody levels were examined using real-time PCR and other tests in 600 HTLV-1 positive screened Japanese blood donors to understand the relationship between PVL and antibody level in asymptomatic carriers and to gain insights toward better antibody testing for HTLV-1 infection. The 430 donors in whom proviral DNA was detected were considered as true positives for HTLV-1 infection. Among donors aged 40 years or older, more males than females had a PVL corresponding to more than 1.6% infected leukocytes, and an antibody titer below the median (P = 0.0018). In antibody tests using an HTLV-1 positive cell line or Env antigens there was a large discrepancy in antibody titer among 13 provirus-positive samples, probably suggesting that antibody-based screening tests should incorporate multiple HTLV-1 antigens, such as Gag and Env antigens.

Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study.

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.

Establishment of culture systems for Genotypes 3 and 4 hepatitis E virus (HEV) obtained from human blood and application of HEV inactivation using a pathogen reduction technology system.

BACKGROUND: It has been demonstrated that the hepatitis E virus (HEV) can be transmitted via blood transfusion, and the risk of HEV transmission via transfusion has become a major global concern. An HEV culture system for blood-derived HEV has been sought to obtain valuable knowledge of the virus and the risk of HEV infection through blood products. STUDY DESIGN AND METHODS: We endeavored to establish an HEV culture system using RNA-positive blood specimens for Genotypes (G) 3 and 4 and applied this system to evaluate tissue culture infectious dose (TCID). We applied this method to investigate the potential of the Mirasol pathogen reduction technology (PRT) system (Terumo BCT) to inactivate live HEV in contaminated platelet samples (PLTs). PLTs were spiked with cultured HEV G3 or G4 and then treated with the Mirasol PRT system. PLTs were examined before and after the treatment for HEV load using TCID titration. RESULTS: We successfully established two strains for HEV production: the JRC-HE3 strain for G3 and the UA1 strain for G4. The Mirasol PRT system expressed more than 3 log inactivation for JRC-HE3 and more than 2 log inactivation for UA1. CONCLUSION: The Mirasol PRT system inactivated greater than 2 to 3 logs of live HEV in PLTs and can potentially be used to lower the possibility of blood-borne HEV transmission. The G3 and G4 HEV inocula identified in this study and the hepatoma cell culture system provide a new means to assess HEV infectious titer and to evaluate other pathogen reduction strategies.

Performance evaluation of Espline HTLV-I/II, a newly developed rapid immunochromatographic antibody test for different diagnostic situations.

IMPORTANCE: The World Health Organization estimated that 5-10 million people are infected with human T-cell leukemia virus type 1 (HTLV-1). This number is likely to be underestimated because reliable endemic data are available for only approximately 1.5 billion people worldwide. The point-of-care test is a powerful tool for the easy and quick detection of infections without the requirement for expensive instruments and laboratory equipment. Espline HTLV-I/II, a newly developed rapid immunochromatographic antibody test that was evaluated in this study, might significantly advance our understanding of the global epidemiology of HTLV-1 infection.

No viremia of pandemic (H1N1) 2009 was demonstrated in blood donors who had donated blood during the probable incubation period.

BACKGROUND: In the spring of 2009, the novel swine-origin influenza A (pandemic [H1N1] 2009) virus emerged and spread globally. Although no established cases of transfusion-transmitted influenza have been reported, the widespread outbreak of pandemic (H1N1) 2009 caused serious concern regarding the safety of blood products. The Japanese Red Cross Blood Centers have intercepted blood products with accompanying postdonation information indicating possible pandemic (H1N1) 2009 infection. To study the risk of transmission of pandemic (H1N1) 2009 by blood transfusion, we searched for the viral genome in such products using nucleic acid amplification technology. STUDY DESIGN AND METHODS: Between June and December 2009, blood components were collected from 579 blood donors who were diagnosed as or strongly suspected of having pandemic (H1N1) 2009 within 7 days after donation. Viral RNA was extracted from plasma and red blood cell (RBC) products, and RNA samples were subjected to real-time reverse transcription-polymerase chain reaction of the hemagglutinin and matrix genes of the pandemic (H1N1) 2009 virus. RESULTS: A total of 565 plasma and 413 RBC products from the 579 blood donors were available. No viral RNA of the pandemic (H1N1) 2009 was detected in any of the blood samples from the 579 blood donors. CONCLUSION: No viremia of pandemic (H1N1) 2009 was demonstrated in any of the 579 blood donors who had most likely donated blood during the incubation period. It is considered that the risk of transmitting pandemic (H1N1) 2009 by blood transfusion is extremely low.

Genetic Analysis of HIV-1 in Japan: a Comprehensive Analysis of Donated Blood.

In Japan, the number of human immunodeficiency virus (HIV)-1 infections remains relatively low; nevertheless, the annual incidence of HIV-1 infection has not decreased. New infections remain a great concern, and an improved understanding of epidemiological trends is critical for public health. The env C2V3 and pol sequences of HIV-1 RNA from 240 early (1996-2001) and 223 more recent (2010-2012) blood donations were used to compare the distribution of virus subtypes and to generate phylogenetic trees. Subtype B was clearly predominant in both early and more recent donations (both were 88.3%), and CRF01_AE was the second most common subtype. Phylogenetic analysis revealed a peculiar epidemiological transition. Compared to early subtype B isolates from 2 major endemic areas (Tokyo and Osaka), the more recent subtype B isolates formed fewer tight clusters in phylogenetic trees (from 8 to 2 clusters in Tokyo and 5 to zero clusters in Osaka). Furthermore, mixing of HIV-1 infections between these 2 endemic areas appear to increase. Analysis of phylogenetic trees suggested that local outbreaks have become smaller in Japan; however, intermixing of viral types between these 2 areas was more evident in the more recent samples.