Chieko Matsumoto

Analysis of HBV infection after blood transfusion in Japan through investigation of a comprehensive donor specimen repository.

BACKGROUND: To understand the risk of transfusion‐transmitted viral infection, it is important to precisely assess cases of infection that follow transfusion.

Detection and quantitation of HBV DNA by semi-nested PCR in donated blood: comparison with HBV serological markers

To detect and quantitate hepatitis B virus (HBV) DNA, semi-nested polymerase chain reaction (PCR) method was designed for amplifying the HBV core region DNA. Cloned HBV core region DNA was used as a quantitation control, and upon electrophoresis of the semi-nested PCR product, one, two, or three bands of amplified DNA were observed using a small (< 50 mol), moderate (around 200 mol), or large (> or = 1250 mol) quantity of the template DNA, respectively. Using this semi-nested PCR method, HBV DNA was quantitated in donated blood and tested for HBV serological markers. Most of the HBV surface antigen (HBsAg) high titer samples showed three bands on the electrophoresis, indicating a high level of HBV DNA, while most of the HBsAg low titer samples showed one band, indicating a low level of HBV DNA. HBV DNA was detected in 7 out of 36 HBsAg-undetectable and anti-HBc-positive samples (19.4%) but all 7 showed one band, indicating a low level of HBV DNA. In almost all of the HBV e antigen-positive samples the HBsAg titer was high, and three bands were observed indicating a high level of HBV DNA.

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.

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.

High-throughput HBV DNA and HCV RNA detection system using a nucleic acid purification robot and real-time detection PCR: its application to analysis of posttransfusion hepatitis.

BACKGROUND: A high‐throughput detection system was developed for HBV DNA and HCV RNA.

Immunoglobulin E oligomers identified in blood components activate mast cells: relevance to anaphylactic transfusion reaction

BACKGROUND: In most cases of anaphylactic transfusion reaction, the mechanisms underlying its development are unclear. We found a donor whose transfused blood components were implicated in two cases of anaphylactic transfusion reaction, and we found that the donor plasma showed mast cell degranulation activity.

Standardization of Quantitative PCR for Human T-cell Leukemia Virus Type 1 in Japan: A Collaborative Study

ABSTRACT 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.

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.

First report of human immunodeficiency virus transmission via a blood donation that tested negative by 20-minipool nucleic acid amplification in Japan

The Japanese Red Cross (JRC) blood centers screen donated blood for infectious agents using serologic assays and nucleic acid amplification testing (NAT). A multiplex NAT for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus Type 1 (HIV-1) with a minipool (MP) format comprising 50 seronegative samples was started in 2000. During the implementation of the 50-MPNAT in 2003, HIV-1 was transmitted through fresh-frozen plasma (FFP) from one blood donor during the window period. To reinforce NAT screening, the pool size was decreased to 20 in 2004. Since 20-MP-NAT implementation in 2004, we have found 19 donations that were seronegative but positive for HIV in the 20-MP-NAT. The rate of HIV-infected donations that were positive only in the NAT was approximately 1 in 2.7 million. No transfusiontransmitted HIV infection (TT-HIV) has been reported in Japan since the 20-MP-NAT was introduced. In November 2013, anti-HIV was detected in a blood sample from a repeat male blood donor aged in his 40s. Western blotting (New LAV Blot 1, Bio-Rad, Hercules, CA), real-time reverse transcription–polymerase chain reaction assay (Cobas TaqScreen HIV, Roche, Basel, Switzerland), and transcription-mediated amplification assay using a kit (Procleix Ultrio ABD, Novartis Diagnostics, Emeryville, CA) confirmed HIV-1 infection. A qualitative NAT for HIV-1 (Cobas TaqMan, Roche) detected a plasma HIV-1 viral load of 4.7 × 10 copies/mL. A cryopreserved sample of plasma from his previous donation in February 2013 was retested in accordance with the Japanese guidelines for lookback studies on blood products. Using individual donation (ID-) NAT, the Cobas TaqScreen HIV (plasma input volume, 850 μL; 95% limit of detection [LOD], 24.3 IU/mL) detected HIV-1 RNA in an archived blood sample from his previous donation, whereas the Procleix (plasma input volume, 500 μL; 95% LOD, 19.6 IU/mL) did not. Each of these NAT assays was performed as a single test. The low plasma volume in the archival sample did not allow for repeat analysis. Red blood cell (RBC) and FFP components were prepared from the previous donation and transfused into two recipients. The RBCs were transfused to a female patient in her 80s. A pretransfusion sample and a posttransfusion sample collected 9 months after transfusion were HIV seronegative. The latter sample was also negative for HIV RNA. The FFP was transfused 8 months after donation to a male patient in his 60s, from whom a pretransfusion sample was seronegative for HIV. Serologic tests and NAT assay identified HIV-1 infection in this recipient at 34 days after transfusion, and the plasma HIV-1 viral load was 1.1 × 10 copies/mL (Fig. 1). The viral sequences determined in blood samples from both the donor (postseroconversion donation) and the FFP recipient differed by only one among 341 nucleotides in the env region (99.7% identity) and by four of 2800 nucleotides in the pol region (99.9% identity). Such high genetic similarity among the sequences supported the notion that HIV had been transferred from the donor to the FFP recipient. Isolates of HIV-1 from the donor and recipient were Subtype B, which is the most common among individuals infected with HIV-1 in Japan. Major antiretroviral drug-resistant mutations were not detected in either the donor or the recipient. Sequencing the HIV-1 5′-long terminal repeat, which was the target region of our NAT screen, did not detect HIV-1 mutations that caused false-negative NAT results. To estimate the HIV-1 viral load in the implicated blood, the sensitivity of both the Cobas TaqScreen HIV and the Procleix was reassessed by probit analysis using serial threefold dilutions (four replicates per dilution) of postseroconverted plasma (4.7 × 10 copies/mL) from the donor, which revealed that the 95% LOD of both NAT screens was 10 copies/mL. The archived blood sample from the implicated donation was reactive in the Cobas, but not in the Procleix screen; therefore, we speculated that the viral load in the donor plasma was approximately at the detection limit of the two NATs. Thus, the estimated total amount of HIV-1 in the FFP (containing

Risk for transmission of pandemic (H1N1) 2009 virus by blood transfusion.

To the Editor: Influenza A pandemic (H1N1) 2009 virus emerged in early 2009 in Mexico and has since spread worldwide. In Japan, the first outbreak of the novel influenza was reported in May 2009 (1) and became pandemic in November. Although no cases of transfusion-transmitted influenza have been published, evidence exists of brief viremia before onset of symptoms (2,3). The possibility of transmission of this virus through transfusion of donated blood is of concern. The Japanese Red Cross Blood Centers have intercepted blood products with accompanying postdonation information indicating possible pandemic (H1N1) 2009 infection and attempted to identify the viral genome in those products by using nucleic acid amplification technology (NAT). During June–November 2009, blood samples were collected from plasma and erythrocyte products that had been processed from donations; postdonation information indicated diagnosis of pandemic (H1N1) 2009 infection soon after donation. Viral RNA was extracted from plasma samples and erythrocyte fractions by using a QIAamp Virus Biorobot MDx kit (QIAGEN, Valencia, CA, USA) and a High Pure Viral Nucleic Acid Large Volume kit (Roche Diagnostics, Indianapolis, IN, USA), respectively. RNA samples were subjected to real-time reverse transcription–PCR (RT-PCR) of hemagglutinin (HA) and matrix (M) genes of influenza A by using PRISM 7900 (Applied Biosystems, Foster City, CA, USA). The RT-PCR of HA was specific for pandemic (H1N1) 2009 virus, whereas the RT-PCR of M was designed to detect both pandemic (H1N1) 2009 and seasonal influenza A viruses. The sequences of probes and primers were synthesized according to the protocols developed by the Japanese National Institute of Infectious Diseases (4). Either 200 μL of a plasma sample or 100 µL of packed erythrocytes was used for each test, and the test was performed 2× for each gene in each sample. Before the investigation using donated blood samples, the sensitivity of the NAT system was checked by spiking experiments. Viral particles of pandemic (H1N1) 2009 virus (A/California/04/2009 [H1N1]), donated by the National Institute of Infectious Diseases, were spiked into plasma and erythrocyte samples from healthy volunteers. Viral RNA was detected in the plasma samples spiked with viral particles corresponding to 300 genome equivalents/mL and in the packed erythrocyte samples spiked with viral particles corresponding to 3,000 genome equivalents/mL. NAT was conducted by using 96 plasma and 67 erythrocyte samples obtained from 96 blood donors who had symptoms of influenza within 7 days postdonation. For 20 donors, pandemic (H1N1) 2009 was diagnosed within 1 day postdonation and, for another 20, within 2 days postdonation (Figure). Pandemic (H1N1) 2009 virus was not found in any of the samples tested, but it was consistently detected in the external positive control. These results suggest that the viremia with pandemic (H1N1) 2009 virus, if any, is very low and can be missed by current NAT or that the viremic period is too brief to identify viremia. Although the risk for transmission of pandemic influenza by transfusion seems to be low, further investigation is needed to elucidate this risk. Figure Number of blood donations from persons for whom pandemic (H1N1) 2009 infection was diagnosed postdonation and time between donation and diagnosis, by donor age, Japan.

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.