Performance Evaluation of Different Commercial Serological Kits for Diagnosis of Acute Hepatitis E Viral Infection

Hepatitis E is the infection of the liver caused by a virus known as the hepatitis E virus (HEV) and has posed severe public health hazards around the world. HEV has four major genotypes (1–4) that are globally distributed into different epidemiological patterns based on socioeconomic factors and ecology (Lu et al. 2006). HEV genotypes 1 and 2 infect humans solely (Ahmad et al. 2011). Generally, genotype 1 accounts for the epidemics in some parts of Asia, while genotype 2 is more prevalent in Africa, Mexico, and other developing countries (Colson et al. 2012). Genotypes 3 and 4 are zoonotic with an expanded host range (Okamoto 2007), while there have been noted chronic HEV infections in immunosuppressed patients (Honer zu Siederdissen et al. 2014). Genotype 3 is prevalent worldwide, while genotype 4 is mainly present in Asia. Besides, genotypes 5 and 6, which primarily infect wild boar, have been found in Japan (Sato et al. 2011; Takahashi et al. 2011). Recently, new genotypes, known as HEV-7 and HEV-8, were also found to infect camels and humans (Al-Sadeq et al. 2017).

Currently, HEV’s diagnosis depends on specific serological and nucleic acid tests, as the clinical manifestations and routine laboratory measures of HEV are similar to those of other acute hepatitis (Zhang et al. 2019). There are four major methods for diagnosing hepatitis E, including the detection of anti-HEV IgM and IgG antibodies, the antigen (Ag), and HEV RNA. Presently, the clinical diagnosis of acute hepatitis E cases mainly depends on the serological detection of anti-HEV antibodies (Dreier and Juhl 2014). However, equivalence, sensitivity, and specificity in the results of the HEV Enzyme-linked Immunosorbent Assay (ELISA) kits tend to differ between manufacturers, leading to discrepancies in the rates of anti-HEV antibodies among different populations (Herremans et al. 2007; Drobeniuc et al. 2010), together with the HEV genome heterogeneity, and the different antigenic structure of HEV proteins. Moreover, cross-reactions of anti-HEV IgM with the Epstein-Barr virus (EBV) and cytomegalovirus (CMV) antibodies have been reported, which cause false-positive results (Hyams et al. 2014). Currently, the development of the HEV RNA assay kits is in the early stages in China and has not yet been widespread. Thus, the clinical diagnosis of HEV infection still mainly relies on serological assays with a few reports of hepatitis E misdiagnoses occurring in China.

In the present study, the performance of four commercial serological assays and PCR assay for the detection of HEV infection was evaluated, and the possibility of misdiagnosing of this infection using serological detection alone was determined.

Performance of anti-HEV IgM assays. The diagnostic performance characteristic of each anti-HEV IgM kit was determined by the ROC curve. As shown in Table I, the area under the curve (AUC) value of the ELISA kits ranged from 0.909 to 0.93. The sensitivity among the 86 acute HEV infections ranged from 82.6% to 86% (Table I), suggesting an excellent agreement (Kappa: 0.819–1) in anti-HEV IgM antibody detection between the ELISA kits (Table II). All the anti-HEV IgM assays evaluated demonstrated good specificities (97.8–100%), except for two false-positive results obtained from the RD cases detected by the Genelabs ELISA kit. No positive results were shown in CMV/EBV infected patients by all the assays (Table I), which demonstrated that there were no cross-reactions of IgM against CMV and EBV.

Performance of anti-HEV IgG assays. The AUC value (0.827–0.929) of the four distinct anti-HEV IgG kits are shown in Table III. The sensitivity among the acute HEV infection patients ranged from 87.2% to 91.9% and showed a substantial agreement with Kappa coefficients from 0.752 to 0.927 between each different ELISA kit (Table III and IV). However, the specificities of anti-HEV IgG among the non-HEV population showed significant inconsistency between each ELISA kit (Table IV). The specificity of the Kehua (96.9–98.9%) and Genelab (96.7–98.9%) tests were significantly higher when compared with the Wantai (64.6–81.3%) and Lizhu assays (69.8–83.5%) (Table III).

The HEV Ag and HEV RNA assays. A total of 36 acute HEV patients were positive for the HEV Ag using the Wantai ELISA kit, with no positive cases found among the non-HEV infection patients. A total of 44 acute HEV infection patients were positive for HEV RNA. The consistency rate between the HEV Ag and RNA was 90.7% (78/86), as shown in Table V. The two methods had a substantial agreement with a Kappa coefficient of 0.787 (0.656–0.918). Furthermore, six of the 86 samples were positive for HEV RNA but negative for anti-HEV IgM and IgG antibodies, and the HEV Ag by all ELISA kits (Fig. 1).

Fig. 1.

To date, the identification of serological markers in HEV infections using accurate diagnostic assays remain a challenge. There are a plethora of issues regarding the specificity and sensitivity of HEV serological assays in epidemiological and clinical settings that require urgent attention. In this present study, we evaluated four dominant, commercially available anti-HEV IgM and IgG assays, as well as the HEV Ag and HEV RNA to investigate the misdiagnosis’s current status rely on the current measurements.

Anti-HEV IgM appears in the early phase of acute hepatitis E. The antibodies can be detected as early as four days after the onset of jaundice and last up to five months (Kuniholm et al. 2009). There are two main methods in anti-HEV IgM serological assays: the capture method with anti-human IgM μ chain (Wantai, Kehua and Lizhu), and the indirect method (Genelabs). The sensitivity and specificity of different methods present variations in anti-HEV IgM assays with a recent study demonstrating a high cross-reactivity of HEV IgM compared to EBV and CMV (Drobeniuc et al. 2010). However, in the present study, no false-positive results due to cross-reaction with EBV- or CMV-infected patients were observed, indicating the highly specific nature of the anti-HEV IgM assays. Moreover, these findings were found to be consistent with those of the other groups, including immunocompromised patients (Abravanel et al. 2013) and infections with HEV genotype 3 (Legrand-Abravanel et al. 2009). The Genelabs ELISA kit detected two false-positive results obtained from patients with RD. This finding supports the opinion that the capture method using the anti-human IgM μ chain is more specific than the indirect method using the anti-HEV IgM assay.

In general, the detection of anti-HEV IgG is usually used as an indicator of past infection. However, the appearance of the anti-HEV IgG antibody is early, which could be used in the clinical diagnosis of acute HEV infection (Aggarwal and Jameel 2011). The Qatar research group found that Wantai HEV-IgG assays revealed high sensitivity and specificity with excellent Kappa concordance using different enzyme immunoassays in assessing seroprevalence of HEV antibodies (Al-Absi et al. 2018). However, a significant discrepancy in anti-HEV IgG results between different assay kits in the non-HEV population was found in our study. The positive rates were significantly higher by the Wantai and Lizhu kits than those of Kehua and Genelabs. A Korean research compared anti-HEV IgG antibody results using the Genelabs and Wantai ELISA kits to estimate HEV serum prevalence in the Korean population (Park et al. 2012). They found a significant inconsistency in the results between the two assays, which was also observed in our study. Therefore, epidemiological investigations of HEV in the population may lead to significant inconsistencies when different kits are used. The Kehua and Genelabs IgG assays had high specificities in the non-HEV population and could be used in the clinical diagnosis of HEV. On the other hand, the Wantai and Lizhu IgG assays were more suitable for epidemiological investigations because the positive rates in the non-HEV population were too high to distinguish the acute HEV infection from the previous disease.

In this study, all four anti-HEV IgG serological assays used the indirect method. There are two major types of antigens coated on the plates for binding of anti-HEV IgG antibodies, including synthetic peptides and recombinant proteins (Innis et al. 2002; Ulanova et al. 2008). The use of the recombinant ORF2-encoded protein in numerous serological studies has revealed its significant efficacy in the identification of antibodies against various HEV strains (Christensen et al. 2008; Kuniholm et al. 2009). Since recombinant proteins can replicate the HEV neutralizing epitope better than the synthetic peptides, the results in the Wantai anti-HEV IgG assay were more sensitive. A French research group also substantiated that the Wantai IgG assay was the most sensitive amongst all other eight commercial ELISA kits used to detect HEV of genotypes 1 and 3 (Abravanel et al. 2013). The results suggested that the anti-HEV IgM assay was superior in the diagnosis of acute HEV infection due to its good specificity when paired with the Wantai anti-HEV IgG assay, which could improve the accuracy of diagnosis.

The latest reports have indicated that this novel HEV Ag is a resourceful serum marker to detect the acute HEV infection and has a good consistency with HEV RNA (Zhang et al. 2006; Zhao et al. 2015; Fraga et al. 2018; Zhang et al. 2019). Our findings in this study also supported this view. However, all the HEV Ag positive samples showed positive anti-HEV IgM results (Fig. 1), which provided no direct evidence to support that Ag detection could improve diagnostic efficiency. Furthermore, six samples were only positive for HEV RNA but negative for anti-HEV IgM and IgG, as well as Ag in all ELISA kits employed, which showed that 6.98% of acute HEV infection patients have a chance to be misdiagnosed if reliant on serological assays detection alone. It indirectly indicates that HEV RNA detection can improve diagnostic efficiency. However, despite the highly specific and sensitive capability of some PCR assays for the detection of HEV RNA, their utility has been restricted due to the short period of HEV viremia detection. Therefore, the incidence of acute HEV infection cannot be completely ruled out by a negative HEV PCR result.

In conclusion, for the successful diagnosis of acute viral hepatitis E, a combination of nucleic acid and serological tests is imperative to provide excellent specificity and sensitivity to the diagnosis. However, we also observed significant inconsistencies between the serological and HEV RNA assays; thereby, caution is warranted while interpreting the results of both serological and molecular tests in HEV diagnosis.