Universal nucleic acid donor screening revealed epidemiological features of hepatitis E and prevented transfusion-transmitted infection in Japan.

BACKGROUND: More than 45 cases of transfusion-transmitted hepatitis E virus infection (TT-HEV) have been reported in Japan. Therefore, in 2020, universal individual donation nucleic acid amplification testing (ID-NAT) was implemented for HEV. STUDY DESIGN AND METHODS: We characterized HEV NAT-positive blood donors. The number of new HEV infections and the asymptomatic infection rate were estimated using the HEV NAT-positive rate. HEV RNA quantitation, phylogenetic analysis, and antibody tests were performed, and the residual risk of TT-HEV was assessed based on the lookback study results. RESULTS: A total of 5,075,100 blood donations were screened with ID-NAT during the first year of implementation, among which 2804 (0.055%; males: 0.060%, females: 0.043%) were NAT-positive with regional differences. Approximately 270,000 new HEV infection cases were estimated to occur annually in Japan, with an asymptomatic infection rate of 99.9%. The median HEV RNA concentration, excluding cases below the limit of quantification, was 205 IU/mL. Among the 1113 cases where the genotype could be determined, HEV-3 and HEV-4 accounted for 98.8% (1100) and 1.2% (13), respectively. The maximum duration of HEV viremia, including the pre- and post-ID-NAT window periods, was estimated to be 88.2 days. Within the 3 years since ID-NAT implementation, no confirmed cases of breakthrough TT-HEV were observed. DISCUSSION: Multiple indigenous HEV strains are prevalent in Japan, infecting a significant number of individuals. However, since the implementation of ID-NAT, TT-HEV has been prevented due to the test's high sensitivity.

Calibrating Hepatitis E Virus Serological Assays Using Asymptomatic Specimens Obtained in Japan.

This study aimed to calibrate hepatitis E virus (HEV) serological assays. We optimized the previously developed in-house HEV antibody enzyme-linked immunosorbent assay (ELISA) by setting the cutoff with an in-house serological performance panel consisting of broad HEV antibody titers and subtracting nonspecific background values for anti-HEV IgM, IgA, and IgG. We also compared the assay's performance with that of commercial serological assay kits (four kits for IgM, one for IgA, and two for IgG). Although all serological assays readily detected HEV antibodies at high titers in the symptomatic hepatitis E population, considerable variations between assays were observed in the asymptomatic population. The in-house ELISA showed a higher sensitivity for HEV IgM, IgA, and IgG than the commercial kits and detected the seroconversion of HEV IgM and IgG earlier when testing a commercially available HEV seroconversion panel. The low sensitivity of the commercial kits was due to the high setting of the original cutoff, which was demonstrated by receiver operating characteristic analysis. However, the corrected cutoff value reduced assay specificity. Background subtraction is essential to achieve high specificity because the in-house ELISA without background subtraction reduced its specificity. These results indicate that asymptomatic specimens and background subtraction contribute to the optimization of HEV serological assays. IMPORTANCE Accurate diagnosis of hepatitis E virus (HEV) infection is essential for public health surveillance and for preventing HEV-contaminated blood transfusion. Anti-HEV IgM or IgA is used as a reliable marker of recent HEV infection. However, considerable variability in the sensitivity and specificity of HEV antibody detection is observed among several commercially available assay kits. In addition, none of the HEV antibody detection methods have been approved by the U.S. Food and Drug Administration (FDA). Here, we show that the in-house enzyme-linked immunosorbent assay (ELISA) could detect HEV IgM and IgA more sensitively than commercial kits in the asymptomatic population. We also suggest that the assay performance of commercial kits might be improved by optimizing the cutoff and reducing nonspecific background noise. A sensitive serological (IgM or IgA) assay in addition to HEV RNA testing will contribute to accurate diagnosis of acute HEV infection because HEV RNA-positive duration is relatively short.

Broadly Reactive Real-Time RT-PCR Assay for the Detection of Hepatitis E Virus and Simultaneous Genotyping by Single Nucleotide Polymorphism Analysis.

Hepatitis E virus (HEV) infection is a global public health concern. Although HEV infection is usually asymptomatic and self-limiting, extrahepatic manifestations and chronic infections in immunocompromised patients have been described. HEV strains infecting humans have been classified into four main genotypes. In this study we have developed and validated a novel sensitive real-time RT-PCR assay for the detection of all four HEV genotypes. Simultaneous discrimination of genotypes 1, 2, and 4 from genotype 3 by single nucleotide polymorphism (SNP) analysis was possible. In all, 201 serum samples from cases and carriers previously tested for HEV by nested RT-PCR were analyzed. Twenty-seven HEV-positive samples could not be typed by the nested RT-PCR and nucleotide sequencing, but were newly typed by SNP analysis. As polymorphisms were present at the primer or probe binding site, we adopted a degenerate primer and mixed probes. When a mixed probe was added, the fluorescence intensity increased, facilitating genotype determination. IMPORTANCE The distribution of HEV-3 and HEV-4 has been changing. HEV-4, which had been predominantly found in Asia, is now being detected in other parts of the world, and there are now reports of chronic infections. Additionally, neurological disorders have frequently been reported in patients with acute or chronic HEV infections. HEV-4 has also been shown to lead to a higher severity in terms of acute hepatitis than does HEV-3. Early typing can provide useful information regarding the route of infection and for tailoring treatment to the expected course of the disease. The present method afforded a good detection rate even when polymorphisms were present within the target region for viral gene detection. We believe that this method can be applied to the analysis of mutation-prone viral genes in the future.

Trends in hepatitis E virus infection: Analyses of the long-term screening of blood donors in Hokkaido, Japan, 2005-2019.

BACKGROUND: After experiencing several cases of transfusion-transmitted hepatitis E (TT-HE) in Hokkaido, Northern Japan, hepatitis E virus (HEV) screening in blood donors, using a nucleic acid amplification test (NAT), was introduced in 2005. STUDY DESIGN AND METHODS: The frequency of HEV RNA-positive donations (2005-2019) was investigated, and the HEV RNA-positive specimens were phylogenetically analyzed. In August 2014, the 20-pooled NAT (20P-NAT) was replaced with an individual-NAT (ID-NAT) system. RESULTS: Until 2019, the frequency of HEV RNA-positive donors was 0.011% (289/2,638,685) with 20P-NAT and 0.043% (597/1,379,750) with ID-NAT, and no TT-HE cases were observed in Hokkaido. The prevalence among male, but not female donors, increased significantly between 2015 and 2019. Eighty-nine percent of HEV isolates from donors were genotype 3 and the remainder were genotype 4, and many clusters existed in each genotype. ALT levels at the time of donation were significantly higher in donors with genotype 4. Four subgenotypes, namely 3a (37%), 3b (41%), 3e (6%), and 4c (10%), comprised 94% of the total. During this period, the most identified subgenotype, 3a, transitioned to 3b. Majority of the HEV strains within the same clusters were detected in the same geographical region around the same period. Many of the human HEV isolates were shown to coexist closely with animal HEV isolates phylogenetically. CONCLUSION: In Hokkaido, multiple divergent HEV strains have been circulating, and small outbreaks of hepatitis E have occurred in the last 15 years. The results suggested that HEV NAT can contribute significantly in ensuring safety during blood transfusions.

IFN-λ3 as a host immune response in acute hepatitis E virus infection.

BACKGROUND AND AIM: Hepatitis E virus (HEV) is mainly transmitted orally, either waterborne or zoonotic foodborne. Intestinal viruses such as rotavirus are known to induce type III interferon (IFN) in the gastrointestinal (GI) tract where type III IFN dominantly functions in comparison with type I IFN. Therefore, the aim of this study is to investigate the significance of type III IFN (IFN-λ3) in acute hepatitis E. METHODS: IFN-λ3 and HEV RNA levels in the sera of patients with acute HEV infection and in the supernatant of HEV-inoculated cells were measured, using an in-house high-sensitivity method and reverse transcription-polymerase chain reaction, respectively. RESULTS: High serum IFN-λ3 levels were found in the early phase of acute HEV infection, which normalized after resolution. Interestingly, serum IFN-λ3 levels correlated well with serum HEV RNA titers in the same sera, both of which showed the peak before the robust increase of transaminases. In vitro experiments demonstrated that HEV replicated well in the cells with little IFN-λ3 induction (Caco-2, A549) and recombinant IFN-λ3 inhibited HEV replication in a dose-dependent manner. In contrast, in HT-29 cells, a colon cancer cell line, HEV poorly replicated and induced IFN-λ3 in a titer-dependent manner. CONCLUSIONS: These clinical and experimental observations suggest that HEV induced IFN-λ3 as a host innate immune response, which may play a protective role against HEV.

Development of a World Health Organization International Reference Panel for different genotypes of hepatitis E virus for nucleic acid amplification testing.

BACKGROUND: Globally, hepatitis E virus (HEV) is a major cause of acute viral hepatitis. Epidemiology and clinical presentation of hepatitis E vary greatly by location and are affected by the HEV genotype. Nucleic acid amplification technique (NAT)-based assays are important for the detection of acute HEV infection as well for monitoring chronic cases of hepatitis E. OBJECTIVES: The aim of the study was to evaluate a panel of samples containing different genotypes of HEV for use in nucleic NAT-based assays. STUDY DESIGN: The panel of samples comprises eleven different members including HEV genotype 1a (2 strains), 1e, 2a, 3b, 3c, 3e, 3f, 4c, 4g as well as a human isolate related to rabbit HEV. Each laboratory assayed the panel members directly against the 1st World Health Organization (WHO) International Standard (IS) for HEV RNA (6329/10) which is based upon a genotype 3 a strain. RESULTS: The samples for evaluation were distributed to 24 laboratories from 14 different countries and assayed on three separate days. Of these, 23 participating laboratories returned a total of 32 sets of data; 17 from quantitative assays and 15 from qualitative assays. The assays used consisted of a mixture of in-house developed and commercially available assays. The results showed that all samples were detected consistently by the majority of participants, although in some cases, some samples were detected less efficiently. CONCLUSIONS: Based on the results of the collaborative study the panel (code number 8578/13) was established as the "1st International Reference Panel (IRP) for all HEV genotypes for NAT-based assays" by the WHO Expert Committee on Biological Standardization. This IRP will be important for assay validation and ensuring adequate detection of different genotypes and clinically important sub-genotypes of HEV.

Rare case of transfusion-transmitted hepatitis E from the blood of a donor infected with the hepatitis E virus genotype 3 indigenous to Japan: Viral dynamics from onset to recovery.

AIM: The transfusion transmission of hepatitis E can occur even in non-endemic areas in the world as autochthonous hepatitis E has been increasingly reported in developed countries where the hepatitis E virus (HEV) is not prevalent. We investigated the post-transfusion transmission of hepatitis E in a patient by molecularly confirming its presence, and characterized the viral kinetics of HEV in this case. METHODS: A Japanese man underwent re-thoracotomy for hemostasis followed by platelet transfusion. After the transfusion, the blood donor was found to be HEV positive. The donated blood was re-examined and was found to contain HEV. Throughout the prospective follow up of the patient, we analyzed the viral kinetics, chronological anti-HEV antibody level changes and disease progression during the entire course of HEV infection from transfusion until the end of viremia. RESULTS: Sequence analysis of the strains isolated from both the donor and the patient who contracted acute hepatitis E showed an identical match for 326 nucleotides in open reading frame 1. Two strains belonged to HEV genotype 3 indigenous to Japan. CONCLUSION: To the best of our knowledge, this is the first detailed report on the entire natural course of hepatitis E from viral transmission, then clearance, to replication preceding liver injury caused by HEV genotype 3, which is responsible for autochthonous infection in developed countries. The findings provide valuable insights into the mechanism of the transfusion transmission of HEV and subsequent viral dynamics.

World Health Organization International Standard to harmonize assays for detection of hepatitis E virus RNA.

Nucleic acid amplification technique-based assays are a primary method for the detection of acute hepatitis E virus (HEV) infection, but assay sensitivity can vary widely. To improve interlaboratory results for the detection and quantification of HEV RNA, a candidate World Health Organization (WHO) International Standard (IS) strain was evaluated in a collaborative study involving 23 laboratories from 10 countries. The IS, code number 6329/10, was formulated by using a genotype 3a HEV strain from a blood donation, diluted in pooled human plasma and lyophilized. A Japanese national standard, representing a genotype 3b HEV strain, was prepared and evaluated in parallel. The potencies of the standards were determined by qualitative and quantitative assays. Assay variability was substantially reduced when HEV RNA concentrations were expressed relative to the IS. Thus, WHO has established 6329/10 as the IS for HEV RNA, with a unitage of 250,000 International Units per milliliter.

Impact of chemiluminescent enzyme immunoassay screening for human parvovirus B19 antigen in Japanese blood donors.

BACKGROUND: To reduce the risk of human parvovirus B19 (B19V) transmission through contaminated blood for transfusion and plasma-derived products, the Japanese Red Cross (JRC) Blood Centers introduced B19V antigen screening by chemiluminescent enzyme immunoassay (CLEIA-B19V) in 2008. STUDY DESIGN AND METHODS: Donor samples that were positive by CLEIA-B19V screening were tested for B19V DNA. The sensitivity of CLEIA-B19V was tested using samples of all three genotypes and B19V DNA-positive donations. B19V DNA-positive donations and pooled plasma were quantitatively assayed for B19V DNA. B19V DNA-positive donations were phylogenetically analyzed by polymerase chain reaction direct sequencing. RESULTS: The sensitivity of CLEIA-B19V was inferred to be approximately 6.3 log IU/mL with the genotype samples and 6.4 log IU/mL with B19V DNA-positive donor samples. Of 417 CLEIA-B19V-positive samples from 1,035,560 donations in Hokkaido, Japan, 101 were positive for B19V DNA. The 198 strains of B19V DNA-positive donations in Hokkaido over the past 15 years clustered exclusively with Genotype 1. After introduction of CLEIA-B19V, the viral load for B19V DNA in all 772 pooled plasma for fractionation from donors in nationwide Japan did not exceed 4 log IU/mL. CONCLUSION: CLEIA-B19V can detect all three genotypes of B19V (viral load >6.3 log IU/mL) and limit the viral load (<4 log IU/mL) in pooled plasma, and thus such screening has further reduced the risk of transfusion-transmitted B19V infection. These results show that CLEIA-B19V screening at the JRC Blood Centers can be an alternative approach to comply with recommendations regarding B19V in the United States and Europe.

A nationwide survey for hepatitis E virus prevalence in Japanese blood donors with elevated alanine aminotransferase.

BACKGROUND: Although we reported two cases of transfusion-transmitted hepatitis E in Japan, the prevalence of hepatitis E virus (HEV) in Japanese blood donors is not very clear. STUDY DESIGN AND METHODS: Blood samples of donors who were deferred from donation because of elevated alanine aminotransferase (ALT) levels were collected from all Japanese Red Cross Blood Centers and subjected to HEV tests. RESULTS: Among the 41 donors with elevated ALT levels higher than 500 IU per L in Hokkaido, HEV RNA was detected in 8 (19.5%) samples. In 1389 donor samples with ALT levels of higher than 200 IU per L in nationwide Japan, the numbers of positive HEV RNA, immunoglobulin M (IgM) anti-HEV, and immunoglobulin G (IgG) anti-HEV samples were 15 (1.1%), 14 (1.0%), and 45 (3.2%), respectively. Although RNA-positive donors were predominantly male and found in any geographic area of Japan, they tended to be higher in number in eastern Japan including Hokkaido and lower in number in western Japan. Of the 23 HEV-positive samples, 19 were Genotype 3 and 4 were Genotype 4. DNA sequences of the 9 isolates showed more than 98.5 percent homology with the known swine HEV isolates. In 1062 donor samples with ALT levels of 61 to 199 IU per L, the percentages of IgM and IgG anti-HEV-positive samples were 0.1 and 2.7 percent, respectively, although there was no HEV RNA-positive sample. CONCLUSION: HEV markers (HEV RNA and anti-HEV) were detected in donors with elevated ALT levels who were widely distributed over Japan. The prevalence and incidence were higher in eastern Japan than in western Japan.

A case of transfusion-transmitted hepatitis E caused by blood from a donor infected with hepatitis E virus via zoonotic food-borne route.

BACKGROUND: Five cases of transfusion transmission of hepatitis E virus (HEV) have been reported so far. The infection routes of the causative donors remain unclear, however. Also, the progress of virus markers in the entire course of HEV infection has not been well documented. STUDY DESIGN AND METHODS: Nucleic acid testing was performed by real-time reverse transcription-polymerase chain reaction targeting the open reading frame 2 region of HEV. Full-length nucleotide sequences of HEV RNA were detected by direct sequencing. RESULTS: Lookback study of a HEV-positive donor revealed that the platelets (PLTs) donated from him 2 weeks previously contained HEV RNA and were transfused to a patient. Thirteen relatives including the donor were ascertained to enjoy grilled pork meats together in a barbecue restaurant 23 days before the donation. Thereafter, his father died of fulminant hepatitis E and the other 6 members showed serum markers of HEV infection. In the recipient, HEV was detected in serum on Day 22 and reached the peak of 7.2 log copies per mL on Day 44 followed by the steep increase of alanine aminotransferase. Immunoglobulin G anti-HEV emerged on Day 67; subsequently, hepatitis was resolved. HEV RNA sequences from the donor and recipient were an identical, Japan-indigenous strain of genotype 4. HEV RNA was detectable up to Day 97 in serum, Day 85 in feces, and Day 71 in saliva. CONCLUSION: A transfusion-transmitted hepatitis E case by blood from a donor infected via the zoonotic food-borne route and the progress of HEV markers in the entire course are demonstrated. Further studies are needed to clarify the epidemiology and the transfusion-related risks for HEV even in industrialized countries.

Transfusion-transmitted hepatitis E caused by apparently indigenous hepatitis E virus strain in Hokkaido, Japan.

BACKGROUND: In industrialized countries, sporadic cases of hepatitis E have been reported in individuals who have never been in an endemic area. Hepatitis E virus (HEV) infection commonly occurs via the fecal-oral route but a potential risk of transfusion transmission route has been suggested. STUDY DESIGN AND METHODS: A 67-year-old Japanese male patient who had never been abroad received a transfusion of blood from 23 voluntary donors and developed acute hepatitis with unknown etiology after transfusion. His blood samples were tested for viral markers of hepatitis viruses. RESULTS: HAV, HBV, HCV, CMV, and EBV were ruled out as causative agents in this case. The patient's blood sample in the acute phase contained HEV RNA as well as IgM and IgG anti-HEV. HEV RNA was also detected in one of the FFP units transfused. The donor had no history of traveling abroad and had a normal ALT level at the time of donation. The PCR products from the patient and the donor showed complete identity for two distinct regions of HEV within open reading frame 1. CONCLUSION: The patient was infected with HEV via transfused blood from a volunteer donor. A potential risk of posttransfusion hepatitis E should be considered even in nonendemic countries.