Role of real-time polymerase chain reaction in diagnosing Hepatitis E, the commonest cause of acute hepatitis in adult patients seeking institutional care.

Background: This cross-sectional study was performed with the aim of determining the prevalence of hepatitis E virus (HEV) infection among acute hepatitis patients attending a tertiary care teaching hospital in a developing country and to determine the relative performance of prevalent diagnostic assays in establishing its diagnosis. Materials and Methods: A total of 46 adult patients were included in this study, all of whom presented with jaundice of <4 weeks' duration and elevation of AST and ALT above 500 U/L. The prevalence of HEV among patients with acute hepatitis was calculated on the basis of the proportion of recruited patients reacting positively in serum anti-HEV immunoglobulin M (IgM) and real-time polymerase chain reaction (RT-PCR) assays. Results: Among the recruited patients, 11 (23.91%) and 15 (32.6%) patients were positive for anti-HEV IgM and RT-PCR, respectively. The two tests demonstrated poor inter-test agreement, thereby implying the necessity of performing both tests for reliable diagnosis of acute HEV virus infection. We also observed a significant difference in the duration of illness between RT-PCR positive and negative patients (P = 0.008). The mean (±SD) duration of illness in the two groups was 8.6 (±3.50) and 11.66 (± 5.15) days, respectively. Combining the results of IgM ELISA and RT-PCR, we observed that 23 out of 46 patients (50%) had evidence of acute HEV virus infection among our patients. Conclusion: Our study suggests that HEV is the commonest cause of acute hepatitis in adult patients attending a tertiary care teaching hospital and that the diagnostic algorithm for its confirmation should include both IgM ELISA and RT-PCR assays.

Laboratory Diagnosis of HEV Infection.

Serological and nucleic acid tests for detecting hepatitis E virus (HEV) have been developed for both epidemiologic and diagnostic purposes. The laboratory diagnosis of HEV infection depends on the detection of HEV antigen or HEV RNA in the blood, stool, and other body fluids, and serum antibodies against HEV (immunoglobulin [Ig]A, IgM, and IgG). Anti-HEV IgM antibodies and low avidity IgG can be detected during the acute phase of the illness and can last approximately 12 months, representing primary infection, whereas anti-HEV IgG antibodies can last more than several years, representing remote exposure. Thus, the diagnosis of acute infection is based on the presence of anti-HEV IgM, low avidity IgG, HEV antigen, and HEV RNA, while epidemiological investigations are mainly based on anti-HEV IgG. Although significant progress has been made in developing and optimizing different formats of HEV assays, improving their sensitivity and specificity, there are many shortcomings and challenges in inter-assay concordance, validation, and standardization. This article reviews the current knowledge on the diagnosis of HEV infection, including the most common available laboratory diagnostic techniques.

Epidemiology of Suspected and Confirmed Acute Hepatitis E Cases Reported Among Los Angeles County Residents, 2017-2019.

In a 3-year period, 38 of 48 persons testing positive for hepatitis E virus (HEV) immunoglobulin M in Los Angeles County did not meet the acute HEV case definition. Healthcare providers should restrict HEV serologic testing for persons with clinically compatible symptoms or epidemiologic risk factors.

Hepatitis E Virus Capsid Antigen (HEV-Ag) - A practical diagnostic biomarker in the HEV outbreak scenario.

BACKGROUND: The increased global incidence of hepatitis E virus (HEV) infections, warrants accurate and affordable diagnostics across different geographical regions. The soluble and highly conserved HEV open reading frame 2 (ORF2) capsid antigen (HEV-Ag) is detectable in self-limited acute enteric hepatitis by HEV-Ag ELISA which is a promising serological assay in settings where HEV-RNA testing is not feasible. Our aim was to assess the HEV-Ag biomarker in an HEV outbreak in a low income country. METHODS: A prospective single center longitudinal study during HEV outbreaks in the Chittagong, Bangladesh region between October 2018 and October 2019 was conducted based on recruitment of acute jaundice cases with clinical signs and symptoms of suspect HEV infections. Acute HEV infection was defined as a positive test result for anti-HEV IgM antibodies. RESULTS: Forty four of the 51 enrolled enteric hepatitis cases (86 %) were confirmed HEV by anti-HEV IgM ELISA at day 0 hospital entry. The anti-HEV-IgM and IgG were positive in all patients and did not reveal significant differences; neither between the time points day 0 and follow-up hospitalization on day 2-6 or day 7-10 nor between RNA-positive (n = 36) versus RNAnegative (n = 8) HEV groups. The HEV-Ag positivity was higher in viral RNA-positive (29/36, 81 %) than the viral RNA-negative (1/8, 12 %) group, p < 0.001 and the HEV-Ag levels positively correlated with viremia, r = 0.77, p < 0.0001. All non-HEV cases; n = 7 tested negative anti-HEV IgM and HEV-Ag and 5 of 7 (71 %) tested anti-HAV IgM positive. CONCLUSIONS: The HEV-Ag ELISA is a reliable and practical diagnostic tool in this acute HEV outbreak.

Unexpected long-lasting anti-HEV IgM positivity: Is HEV antigen a better serological marker for hepatitis E infection diagnosis?

Hepatitis E virus (HEV) is the leading cause of acute hepatitis worldwide. The minimum criterion for diagnosis of acute infection is detection of anti-HEV antibodies, although there are scant data on IgM duration. Our aim was to assess the persistence of HEV markers after acute self-limited hepatitis E. HEV serological tests (IgM by Mikrogen and Wantai and HEV-Ag) and HEV RNA were carried out in two cohorts: (a) patients with prior acute hepatitis E (ALT >10 x ULN plus positive IgM ± HEV RNA) currently self-limited and (b) 50 blood donors with positive HEV RNA. Among 25 cases of prior acute hepatitis E, after a median follow-up of 34 months, all presented undetectable HEV RNA. However, anti-HEV IgM remained detectable in 14 (56%) by Mikrogen, 6 (24%) by Wantai and none for HEV-Ag. Anti-HEV IgM tested positive in 80%-100% within the second year and 17%-42% over 3 years later, by Wantai and Mikrogen, respectively. Among HEV RNA-positive donors, 12 (25%) tested positive for either IgM by Mikrogen or Wantai, 9 (18%) for both and 18 (36%) for HEV-Ag. HEV-Ag positivity was more likely as HEV RNA was higher (14% if <2.2 log IU/mL; 64% if RNA ≥ 3.7). Overall, HEV-Ag performed best, with a positive predictive value of 100% and diagnostic accuracy of 57%. Anti-HEV IgM exhibited unexpectedly long persistence after a self-limited acute hepatitis E. HEV-Ag had the best performance and could be especially useful in settings where HEV RNA is not available.

[The estimation of the hepatitis E proportion in the etiological structure of acute viral hepatitis in certain regions of of Kyrgyzstan.]

Despite the fact that the Kyrgyz Republic (KR) belongs to the highly endemic regions of the world for hepatitis E, the true extent of the spread of this infection in the country remains poorly understood. It was estimated the prevalence of serological markers of hepatitis E virus (HEV) infection among patients with acute viral hepatitis (AVH) from the regions of the Kyrgyz Republic with a high level of seroprevalence previously established by us. Blood sera samples of hepatitis patients who were admitted to hospitals of Kyrgyzstan in the period 2018-2019 were examined by the enzyme immunoassay method using the kits «DS-ELISA-Anti-HEVIgG¼ and «DS-ELISA-ANTI-HEV-IgM¼ (RPC Diagnostic Systems, Russia). IgG and IgM antibodies to HEV were detected in 103 of 344 studied samples (29.9%). Most often, seropositive specimens were detected among people of age groups under 20 and over 40 years old. Hepatitis with the fecal-oral mode of transmission was dominated in the structure of AVH: the specific gravity of hepatitis E was 47.9%, hepatitis A - 35.32%. Markers of mixed infections with other hepatitis viruses have been detected in 40.4% IgM-positive individuals. Thus, high prevalence of serological markers of HEV infection in the territory of Kyrgyzstan during the interepidemic period had been shown. The necessity of including the determination of serological markers of hepatitis E into the algorithm for the comprehensive diagnosis of AVH in patients of all age groups with liver pathology had been confirmed.

Hepatitis E virus infection as a promoting factor for hepatocellular carcinoma in Cameroon: Preliminary Observations.

OBJECTIVES: To determine the seroprevalence of hepatitis E virus (HEV) infection in patients with chronic hepatitis and/or hepatocellular carcinoma (HCC) and to assess its potential consequences for disease progression. METHODS: We conducted a prospective case-control study on patients with HCC hepatitis B or C related and non-HCC patients including patients with CLD and patients without clinical evidence of liver disease. Anti-HEV IgG and IgM were tested by ELISA using commercially available kits. Liver damage was assessed by alanine aminotransferase, aspartate aminotransferase, platelets and prothrombin measurements. RESULTS: We observed a significant anti-HEV IgG carriage in HCC patients compared to non-HCC subjects with CLD (41.8% vs 12.6%; P=9.1 E-6; OR=4.8, 95%CI: 2.3-10.6). HCC patients with HEV infection display more profound alterations of circulating liver enzymes, platelets count and prothrombin time than HCC patients without sero-reactivity to HEV. CONCLUSION: Overall, this study indicates a high prevalence of HEV infection in Cameroonian patients with CLD and HCC. These data suggest either that patients with liver tumors are more susceptible to hepeviral infection or that, in a tropical context, HEV might promote the progression of liver diseases towards tumor.

Retrospective Study Evaluating Seroprevalence of Hepatitis E Virus in Blood Donors and in Swine Veterinarians in Italy (2004).

Hepatitis E is an emerging viral disease in developed countries, with sporadic cases occasionally linked to the consumption of raw or undercooked pork, wild boar or deer meat. Cases due to transfusion or transplantation have also been reported. In developed countries, hepatitis E is considered a zoonosis and pig is the main reservoir. In the last few years, several studies conducted in Europe reported variable seroprevalence rates among the general population, ranging between 0.26% and 52.5%. A higher seroprevalence was described among workers who come in contact with pigs. The aim of this retrospective study was to evaluate the seroprevalence of anti-HEV IgG and IgM antibodies in blood donors (170) and in pig veterinarians (83). Archival sera were collected in Italy in 2004. The observed seroprevalence was 9.64% and 8.82% in veterinarians and blood donors, respectively. Overall, only three sera from blood donors were positive for IgM, but no HEV-RNA was detected.

Laboratory Diagnosis of HEV Infection.

Serological and nucleic acid tests for detecting hepatitis E virus (HEV) have been developed for both epidemiologic and diagnostic purposes. The laboratory diagnosis of HEV infection depends on the detection of HEV antigen, HEV RNA, and serum antibodies against HEV (immunoglobulin [Ig]A, IgM, and IgG). Anti-HEV IgM antibodies can be detected during the acute phase of the illness and can last approximately 4 or 5 months, representing recent exposure, whereas anti-HEV IgG antibodies can last more than 10 years, representing remote exposure. Thus, the diagnosis of acute infection is based on the presence of anti-HEV IgM, HEV antigen, and HEV RNA, while epidemiological investigations are mainly based on anti-HEV IgG. Although significant progress has been made in developing and optimizing different formats of HEV assays, improving their sensitivity and specificity, there are many shortcomings and challenges in inter-assay concordance, validation, and standardization. This article reviews the current knowledge on the diagnosis of HEV infection, including the most common available laboratory diagnostic techniques.

Hepatitis E in blood donors: investigation of the natural course of asymptomatic infection, Germany, 2011.

Asymptomatic hepatitis E virus (HEV) infections have been found in blood donors from various European countries, but the natural course is rarely specified. Here, we compared the progression of HEV viraemia, serostatus and liver-specific enzymes in 10 blood donors with clinically asymptomatic genotype 3 HEV infection, measuring HEV RNA concentrations, plasma concentrations of alanine/aspartate aminotransferase, glutamate dehydrogenase and bilirubin and anti-HEV IgA, IgM and IgG antibodies. RNA concentrations ranged from 77.2 to 2.19×10(5) IU/mL, with viraemia lasting from less than 10 to 52 days. Donors showed a typical progression of a recent HEV infection but differed in the first detection of anti-HEV IgA, IgM and IgG and seropositivity of the antibody classes. The diagnostic window between HEV RNA detection and first occurrence of anti-HEV antibodies ranged from eight to 48 days, depending on the serological assay used. The progression of laboratory parameters of asymptomatic HEV infection was largely comparable to the progression of symptomatic HEV infection, but only four of 10 donors showed elevated liver-specific parameters. Our results help elucidate the risk of transfusion-associated HEV infection and provide a basis for development of screening strategies. The diagnostic window illustrates that infectious blood donors can be efficiently identified only by RNA screening.

High hepatitis E virus seroprevalence with absence of chronic infection in HIV-infected patients.

BACKGROUND & AIMS: The seroprevalence of the hepatitis E virus (HEV) and its chronicity rate in the HIV-infected population has not been well established. As a result, the magnitude of this emerging disease in this population cannot be established. METHODS: Prospective study that included HIV-infected patients followed up between September 2012 and May 2013. All included patients were tested for anti-HEV IgG/IgM. In patients with confirmed anti-HEV IgG/IgM positivity, RT-PCR was performed. In patients where HEV RNA was amplified, a second RT-PCR assay was performed 6 months later to identify transient or chronic HEV infections. RESULTS: Eight hundred and ninety-four HIV-infected patients were enrolled in the study. Of these patients, 399 (44.6%) were monoinfected with HIV; 462 (51.6%) were co-infected with HIV/HCV; 12 (1.3%) were co-infected with HIV/HBV; and 21 (2.3%) were co-infected with HIV/HCV/HBV. In 88 patients, anti-HEV IgG/IgM was detected (seroprevalence: 9.8% [95% CI: 8.02%-11.9%]). In five patients (0.5%; 95% CI: 0.2%-1.2%), HEV RNA was detected; 5.7% (95% CI: 2.1%-12.1%) of the patients were anti-HEV IgG/IgM positive. None of these patients showed detectable HEV RNA six months later. CONCLUSION: HEV infection is frequent in HIV-infected patients but developing a chronic HEV infection may be considered an uncommon liver disease in this population.

Occurrence of hepatitis E virus infection in acute hepatitis in Thailand.

Hepatitis E virus (HEV) infection is one of the enterically transmitted types of hepatitis. The present study was undertaken to estimate the occurrence of HEV infection in sporadic acute hepatitis in Thailand. Serum samples were obtained from 614 suspected acute hepatitis patients at two large hospitals in Bangkok during 2008, 2009, and 2011. Acute hepatitis E was identified by the presence of anti-HEV IgM (4.8%) using indirect ELISA kits and/or HEV RNA (4.5%) by a semi-nested reverse transcription-polymerase chain reaction assay. HEV IgM was the most common marker for detection (77%) at diagnosis, either by positive HEV IgM alone or together with HEV RNA, whereas HEV RNA alone was detected in 23% of patients. Overall, 4.2% of cases (26 out of 614) were acute HEV infection with the highest attack rate in the elderly age group. In addition, nucleotide sequence analysis of five HEV samples revealed 92.8-99.8% homology. All viruses were clustered into HEV genotype 3 and were similar genetically to swine HEV strains previously detected in the same area. Therefore, the occurrence of HEV infection with closely related to swine genotype 3 was approximately 4-5% of acute hepatitis cases in Thailand. Anti-HEV IgM was the most common marker at diagnosis.

Assessment of dried blood samples as an alternative less invasive method for detection of Hepatitis E virus marker in an outbreak setting.

Hepatitis E virus (HEV) is the causative agent of hepatitis E. It can be asymptomatic, associated with acute self-limiting hepatitis or acute liver failure. The conventional diagnosis of HEV infection relies on anti-HEV IgM serology. The collection of blood samples by venepunture for laboratory confirmation is often difficult during an outbreak. Thus, testing the specimens of dried blood spots (DBS) on filter papers can prove to be a feasible alternative. The present study aimed to evaluate the applicability of anti-HEV IgM detection from DBS samples and the stability of anti-HEV IgM detection at varied time interval, at various storage temperatures. Paired blood and DBS sample were collected from 44 jaundiced patients and eight healthy controls during HEV outbreaks. The DBS were tested for anti-HEV IgM by available ELISA kit with in-house modifications. Three cut offs were determined, that is, the CO1: kit cut-off, CO2: mean of negative controls above 3SD and CO3: area under Receiver operating Curve. The sensitivity of anti-HEV IgM detection ranged from 86-91%. The maximum sensitivity (91%) and specificity (100%) was obtained using CO3. Maximum stability of anti-HEV IgM antibodies (100%) was observed till 65 days at 4°C. Storage at 37°C significantly reduced anti-HEV IgM positivity, wherein 42.85% sample became negative by 45 days. DBS showed good sensitivity and specificity for detecting anti-HEV IgM and can be considered an alternate to serum sample. Moreover, anti-HEV IgM was stable at 4°C, which makes DBS a preferred method for storage and transportation of the sample to reference laboratory.

Role of hepatitis E virus antigen in confirming active viral replication in patients with acute viral hepatitis E infection.

BACKGROUND: Demonstration of active viral replication is important in serologically confirmed cases of hepatitis E virus (HEV) infection to assess prognosis. Detection of HEV RNA by reverse transcriptase polymerase chain reaction (rtPCR) is the gold standard for demonstration of active viremia. OBJECTIVES: The present study was designed to compare the diagnostic utility of HEV antigen (Ag) with HEV IgM and HEV rtPCR in detecting the active viral replication. STUDY DESIGN: Blood samples from 156 probable cases of acute viral hepatitis (AVH) were collected. Screening for hepatitis B surface antigen (HBsAg), antibody to hepatitis C virus (anti HCV), IgM antibody to hepatitis A virus (HAV IgM) was done on the Architect platform (Abbott Laboratories, IL, USA). HEV IgM ELISA (Wantai Biological, Beijing, China), HEV-Ag ELISA (Wantai Biological, Beijing, China) and HEV rtPCR were done on all the samples. RESULTS: Out of 156 AVH cases in 56 (35.8%) a diagnosis of HEV was confirmed. Positivity being; anti-HEV IgM 44/56, HEV RNA 20/56, and HEV antigen 17/56 in established cases. Male to female ratio was 3:1. The median age was 40 (range 14-71) years. HEV Ag showed a good concordance with HEV RNA (k=0.635, p<0.001). However HEV IgM did not show any concordance with HEV RNA (k=0.14). CONCLUSION: HEV antigen assay can be used as an additional diagnostic marker to confirm active viral replication in serologically positive samples.