Hepatitis E virus (HEV) infection is a public health concern and recognized as the most common cause of acute viral hepatitis worldwide. It is estimated that 20 million HEV infections occur annually, with more than 3 million symptomatic cases and &#;60,000 deaths (1). It can cause not only endemic hepatitis in developing countries with poor sanitation and limited medical resources but also sporadic hepatitis in developed countries due to contact with HEV-contaminated meat (2) or blood transfusion (3). The prognosis of HEV infection is generally good. However, it is a concern in patients with underlying chronic liver disease (4) and women in their second or third trimester of pregnancy (5). More recently, it was reported that chronic HEV infections have become a significant problem in immunocompromised patients (6).
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HEV is a positive-stranded RNA virus (7). It has extensive genetic diversity. Genotypes 1 and 2 can infect only humans, while genotypes 3 and 4 can infect both humans and swine, which makes it hard to eliminate due to the existence of the animal reservoir (8).
A large proportion of hepatitis E cases go unrecognized or are frequently misdiagnosed as drug-induced liver injury (9, 10). Lack of knowledge and awareness of the disease among clinicians is part of the reason. Meanwhile, the accurate diagnosis of hepatitis E is still challenging.
After HEV infection, anti-HEV IgM antibody is induced after an incubation period ranging from 15 to 60&#;days, and it disappears early in the convalescent period. Anti-HEV IgG may remain detectable for many years (11). Acute hepatitis E is commonly diagnosed by detecting anti-HEV IgM and/or rising IgG levels in the serum (12). In some immunosuppressed patients with chronic HEV infection, anti-HEV antibodies remained negative (13). Therefore, molecular diagnosis of HEV RNA is highly recommended (14). Viral RNA can be detected in the serum and/or feces during the incubation period or early acute phase of disease by reverse transcription-PCR (RT-PCR). It provides a specific and highly sensitive approach to the diagnosis of HEV infection. Because RT-PCR is relatively expensive and technically challenging, the assay is not easily accessible, especially in developing countries where HEV is hyperendemic. Detection of HEV antigen (Ag) has been suggested as a convenient and cost-efficient alternative to RT-PCR, since HEV Ag production may parallel that of HEV RNA. It was reported that the method of HEV Ag detection had good concordance with HEV RNA detection and could serve as a useful tool in early diagnosis of infection (15&#;18).
The current study is dedicated to comparing the diagnostic performance of the Wantai anti-HEV IgM assay and the HEV Ag assay with the HEV RNA detection assay as a reference. We first evaluated the anti-HEV IgM assay and found it had high sensitivity (99.4%) but poor specificity (74.3%). By retrospective follow-up of samples collected serially from HEV-infected patients, we found the positivity of anti-HEV IgM may last for a long period, which contributed to the low positive predictive value (PPV) of this assay (48.6%). Meanwhile, the diagnostic accuracy of the HEV Ag assay was evaluated, and factors affecting its detection efficiency were explored. Finally, we used regression modeling techniques and receiver operating characteristics (ROC) curve analysis to propose a multifactorial model, which was shown to outperform the anti-HEV IgM or the HEV Ag assay in the diagnosis of current HEV infection when HEV RNA detection is not available.
This noninterventional study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki and approved by the Human Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. Informed consent was waived, since the research involved no more than minimal risk to the subjects. All medical information was coded and kept confidential as required.
Serum samples from subjects who presented with symptoms of hepatitis or came for disease screening purposes were sent to the laboratory for routine testing of anti-HEV antibodies. From August to December , &#;8,700 samples were tested for anti-HEV antibodies and 565 samples were positive for anti-HEV IgM. A total of 809 samples were subsequently tested for HEV RNA based on sample availability, including 325 anti-HEV IgM-positive samples and 484 anti-HEV IgM-negative samples. Most (479/484) of the anti-HEV IgM-negative samples were included because their alanine aminotransferase (ALT) levels were above the upper limit of normal (ULN; 64&#;IU/ml). The other 5 anti-HEV IgM-negative samples with ALT levels within the normal range were included because they were the follow-up samples of anti-HEV IgM-positive subjects. Available leftover specimens were collected and stored at &#;20°C until analyzed. Subjects with HEV RNA detected in serum were defined as current HEV infection. Diagnostic performance evaluation of different assays was based on comparison with HEV RNA.
To explore the duration of the anti-HEV antibody response, we retrospectively identified patients with sequential specimens from the entire set of 809 samples. A total of 143 samples, collected from 58 patients with multiple testing, were available. The kinetics of anti-HEV antibodies and the diagnosis performance of the HEV Ag assay were studied with these serial samples. When we noticed the flaws of the anti-HEV IgM and the HEV Ag assay in the diagnosis of current HEV infection, a model combining anti-HEV IgM, HEV Ag, and ALT was developed. Another set of 67 IgM-positive single-visit samples with available record of ALT level and enough volume (>0.5&#;ml) for HEV testing then were randomly selected from the entire set, and the performance characteristics of the model were tested. Since the results of these 143 sequential samples are the basis of the proposed model, they are defined as the training set. The other 67 single-visit samples are defined as the test set.
HEV antibodies were detected in 50 μl of serum samples using the chemiluminescence microparticle immunoassay (CMIA), developed by Wantai BioPharm (Beijing, China). It uses a recombinant antigen corresponding to 394 to 606 amino acid residues of the HEV capsid protein (19). The test was performed and interpreted in accordance with the manufacturer's instructions. The results were expressed as cutoff index (COI). A COI of &#;1 and a COI of <1 were defined as a positive result and a negative result, respectively. The CMIA was approved for the diagnostic testing of anti-HEV antibodies by the National Medical Products Administration (NMPA) of China.
HEV Ag was measured with the Wantai HEV Ag enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. In this system, goat polyclonal anticapsid antibodies are used for antigen capture, and enzyme-linked monoclonal antibodies against the capsid protein are used for detection. Briefly, 100&#;μl of serum sample was added to each well of the microplate, which was precoated with anti-HEV capsid polyclonal antibodies. The microplate was sealed and incubated at 37°C for 60&#;min. After washing the wells with washing buffer five times, a second horseradish peroxidase-conjugated monoclonal anti-HEV capsid antibody was added, followed by further incubation at 37°C for 30&#;min and washing five times with the washing solution again. Subsequently, 100 μl of tetramethylbenzidine substrate solution was added and incubated for 15&#;min at 37°C. The reaction was stopped by addition of the stop solution, and the absorbance was immediately measured using dual-wavelength detection (450 and 630&#;nm) with a microplate reader (Multiskan FC microplate reader; Thermo Fisher Scientific). The cutoff value was 0.12 plus the mean absorbance of three negative controls, as recommended by the manufacturer. The absorbance ratio of each specimen to the cutoff value (S/CO) was used to indicate HEV Ag status, with positivity defined as a ratio of &#;1.0. The HEV Ag ELISA kit is intended for research use only.
The level of HEV RNA in clinical samples was determined by a laboratory-developed test (LDT) of RT-PCR. HEV RNA was extracted from 140 μl of serum samples using a TIANamp virus RNA kit (Tiangen, Beijing, China) according to the manufacturer's instructions, and 50 μl of RNA solution was eluted for each sample. The one-step RT-PCR was performed with a HiScript II one-step quantitative RT-PCR probe kit (Vazyme, Nanjing, China). Each reaction mixture included 8&#;μl of extracted RNA, 10&#;μl of 2×&#;one-step Q probe mix, 1&#;μl of one-step probe enzyme mix, 0.1 μM fluorescence probe, and 0.2 μM each primer. RT-PCR conditions included an RT step of 50°C for 15 min, a predenature step of 95°C for 30&#;s, followed by 45 cycles of 95°C for 10&#;s and 60°C for 30&#;s. The sequences of the primers and the probe were described and validated previously (20&#;22). The forward and reverse primers were 5&#;-GGT GGT TTC TGG GGT GAC-3&#; and 5&#;-AGG GGT TGG TTG GAT GAA-3&#;, respectively, and the probe was 5&#;-FAM-TGA TTC TCA GCC CTT CGC-TAMRA-3&#;. The amplification was carried out on a ViiA 7 real-time PCR system (Thermo Fisher Scientific) according to the manufacturer&#;s instructions. HEV RNA was in vitro transcribed from pGEM-7Zf(-)-TWE (23) by T7 RNA polymerase to obtain a positive strand as the RNA standard for quantitative RT-PCR (23). The RNA standard was titrated by measuring the optical density at 260&#;nm with a spectrophotometer. A standard curve was generated from serial dilutions of the standard. The threshold cycle values were plotted as a function of the input HEV RNA viral copy numbers. The LDT was validated against the commercial promoter HEV RNA detection kit (ACON, Hangzhou, China), which was approved for the diagnostic testing of HEV RNA by the NMPA of China to provide a qualitative dichotomous (positive/negative) result. The linearity (see Table S1 in the supplemental material), limit of detection (Table S2), and agreement across specimens (Table S3) were compared between the LDT and the promoter assay.
Samples with positive anti-HEV IgM were tested for HEV RNA individually, and samples with negative anti-HEV IgM were pooled (4 to 5 specimens were mixed into 1 specimen) and tested for HEV RNA. When positive HEV RNA was detected from a pooled sample testing, the subsequent testing of the individual samples from the pool was required.
To test the effect of the anti-HEV IgG on the detection of HEV Ag, serum samples that were either positive or negative for anti-HEV IgG but negative for both anti-HEV IgM and HEV RNA were collected and pooled separately as dilution sera to a final volume of &#;5&#;ml each. Two serum samples collected from HEV RNA-positive patients (A and B) were 10-fold serially diluted with these pooled anti-HEV IgG-positive or IgG-negative sera to generate 1/10, 1/100, and 1/1,000 dilutions. The original samples and the serially diluted samples were then subjected to HEV Ag detection by ELISA or HEV RNA detection by RT-PCR in duplicates.
Statistical analyses were performed using SAS version 9.4 (SAS Institute Inc, Cary, NC) and MedCalc statistical software version 19.3 (MedCalc Software, Ostend, Belgium). Graphs were generated using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA).
Categorical variables were described as proportion (percent), and continuous variables were described as means ± standard errors of the means (SEM) or median (range). Comparisons between two groups were performed using t test, Mann&#;Whitney U test for continuous variables, and chi-square test for categorical variables as appropriate. Correlation analyses were performed with the Spearman's method. A two-tailed P value of&#;<0.05 was considered statistically significant.
Diagnostic performance was determined with ROC curve analyses, followed by calculation of 95% confidence intervals (CI) using the binomial exact method. Area under the ROC curve (AUROC) of different diagnostic methods was compared using the method of DeLong et al. (24). Detailed diagnostic values were evaluated by calculating the sensitivity, specificity, PPV, negative predictive value (NPV), and accuracy. The optimum cutoff value for diagnosis is selected by maximizing the sum of sensitivity and specificity.
Model estimation was done with the LOGISTIC procedure of SAS. Data of the 143 sequential samples were used to build a model, whereas data of the 67 single-visit patients were used to test the model. Variables of age, gender, ALT/ULN, anti-HEV IgM, anti-HEV IgG, and HEV Ag were included in the logistic regression analysis. Logarithmic transformation of anti-HEV IgM, IgG, and HEV Ag levels and square root transformation of ALT/ULN levels were applied to improve the normality of the distribution. Variables that were significantly different between subjects with and without HEV RNA detected by univariate analysis (P&#;&#;&#;0.05) were included in the stepwise multiple logistic regression analysis to identify independent variables associated with current HEV infection. Variables with a P value&#;of <0.05 by multivariate analysis were kept, and the estimate coefficient output by SAS was used to construct a model. The goodness of fit of the model was assessed using the Akaike information criterion (AIC), and the discrimination ability was assessed by AUROC.
A total of 809 samples were tested for HEV RNA. Among them, 158 out of the 325 anti-HEV IgM-positive samples and 1 out of the 484 anti-HEV IgM-negative samples had HEV RNA detected. The performance of the anti-HEV IgM assay in diagnosing current HEV infection was summarized in Table 1. The sensitivity and the NPV was quite good (&#;99%), followed by accuracy (&#;79%) and specificity (&#;74%). The main flaw of the anti-HEV IgM assay was its low PPV, only &#;49%, suggesting that half of the cases with positive anti-HEV IgM are not current HEV infection. The only case who was negative for anti-HEV IgM but positive for HEV RNA had another sequential sample collected 1 day later. He was found to have anti-HEV IgM seroconversion and a rising level of anti-HEV IgG at the later time point. HEV RNA was detected in both samples, and acute HEV infection was confirmed.
To understand why the anti-HEV IgM assay gives low PPV, we performed a retrospective follow-up study: 58 subjects who had serial sera collected and positive anti-HEV IgM specimen(s) at least once were identified from the entire set of 809 samples. Among them, 14 subjects (1 to 14) remained negative for HEV RNA throughout the observation period, while the other 44 patients (15 to 58) were confirmed as acute HEV infection by detecting positive HEV RNA in the first or following samples collected initially. A total of 143 samples were collected from these 58 subjects, with, on average, 2.5 samples per subject and a median follow-up of 3&#;months. Twenty-five of the 44 patients with acute HEV infection cleared the virus (19, 21, 23, 24, 26, 27, 33, 34, 36, 39, and 44 to 58), while the other 19 patients had follow-up duration of less than 3&#;months and remained positive for HEV RNA at the last visit. Patients 15 to 44 had a midterm follow-up period within 3&#;months, while patients 45 to 58 had long-term follow-up lasting from 90&#;days to 414&#;days. The anti-HEV IgM and IgG levels were plotted against the follow-up days (Fig. 1). For the patients who remained negative for HEV RNA (1 to 14), the levels of their anti-HEV IgM were relatively low, and most of them showed neither large increases nor large decreases (Fig. 1A and B). These 14 patients were probably in the convalescent period from recent infection or had nonspecific cross-reactive antibodies to HEV due to unknown reasons. In contrast, for the patients with acute HEV infection (15 to 58), the levels of anti-HEV IgM increased rapidly at the beginning but decreased slowly during the convalescent period, which remained positive for as long as 0.5 to 1&#;year (Fig. 1C and E). For example, patients 51 and 58 remained positive for 6&#;months, patients 54 and 55 were positive for 8&#;months, and 57 remained positive for more than 1&#;year. The levels of anti-HEV IgG of these 44 patients increased gradually and remained positive throughout the period of observation (Fig. 1D and F). The unexpected long period of anti-HEV IgM positivity interfered with the diagnosis of current HEV infection and explained the low PPV of this assay.
HEV Ag has been reported as a useful serological marker for diagnosing HEV infection. Next, we used the 143 sequential samples from the antibody kinetics study to study the diagnostic efficacy of the HEV Ag assay. Of the 143 samples, 72 samples were negative for both HEV RNA and HEV Ag. Only 46 out of the 71 HEV RNA-positive samples were positive for HEV Ag. The sensitivity of the HEV Ag assay was 64.8%. Both the specificity and the PPV were 100%. The accuracy was &#;83%, and the NPV was &#;74% (Table 1).
To understand why some samples showed discordant HEV RNA and HEV Ag results, we categorized the samples according to their HEV RNA levels. HEV Ag levels increased with HEV RNA levels (Fig. 2A). The discordance was mainly found in the HEV RNA low viral load groups (Fig. 2B). Among the 71 HEV RNA-positive samples, HEV Ag was detected in 3/16 (18.75%) samples in the HEV RNA range below 105 copies/ml, 12/22 (54.5%) samples in the range of 105 to 106 copies/ml, 21/23 (91.3%) samples in the range of 106 to 107 copies/ml, and 10/10 (100%) samples in the range above 107 copies/ml.
To explore other factors that may affect the detection efficiency of HEV Ag, we determined the correlation between the levels of HEV Ag and HEV RNA, anti-HEV IgG, IgM, or ALT/ULN in the 71 HEV RNA-positive samples (Fig. 3). The Spearman&#;s correlation coefficient (r) was 0. between HEV Ag and HEV RNA (P&#;<&#;0.), 0. between HEV Ag and ALT/ULN (P&#;<&#;0.), &#;0. between HEV Ag and anti-HEV IgG (P&#;<&#;0.), and &#;0. between HEV Ag and anti-HEV IgM (P&#;=&#;0.), respectively. Thus, both HEV RNA level and ALT/ULN had strong positive linear association with HEV Ag level, while anti-HEV IgG level showed a moderate to strong negative linear relationship to HEV Ag level and anti-HEV IgM level had no obvious linear relationship to HEV Ag level.
With that said, we hypothesized that the presence of anti-HEV IgG interferes with the detection of HEV Ag. To test this idea, we serially diluted two HEV RNA-positive serum samples (A and B) with either anti-HEV IgG-positive or IgG-negative serum (Table S3). The original samples and the diluted samples were then subjected to HEV Ag detection by ELISA or HEV RNA detection by RT-PCR (Fig. 4). The HEV RNA levels in serial dilutions by either positive or negative anti-HEV IgG serum were comparable (Fig. 4A and B). However, the HEV Ag level was much higher in dilutions with negative anti-HEV IgG serum (Fig. 4C and D), suggesting that the presence of anti-HEV IgG interfered with the detection of HEV Ag.
Considering the high seropositivity of anti-HEV IgG in areas where HEV is endemic, we reason that the HEV Ag assay alone is not good enough to diagnose current HEV infection, since it will cause misdiagnosis when the HEV RNA level is low or the anti-HEV IgG level is high. Thus, we decided to establish a model combining the key factors that differentiate current HEV infection from past infection or other liver diseases. Multivariate analyses showed that the levels of HEV Ag, anti-HEV IgM, and ALT/ULN were significantly independent variables associated with current HEV infection (Table S4). By stepwise multiple logistic regression analysis, a model for predicting current HEV infection was proposed. The model combining the 3&#;key factors in parameter estimates of 3.08 (lgAg), 5.14 (lgIgM), and 2.64(ALTULN), respectively, was identified with the highest AUROC (referred to as [IgM+Ag+ALT]). The model was shown as
logit&#;(p)=2.64×ALTULN+3.08×lg&#;(Ag)+5.14×lg&#;(IgM)In the above-mentioned 143 sequential samples, 3 samples in the HEV RNA-positive group and 10 samples in the HEV RNA-negative group did not have records of ALT levels, so they were not included. A weighted sum score of 7.741 of the parameter estimates was associated with the highest sum of sensitivity and specificity and determined as the cutoff value. Sixty-two out of the 68 HEV RNA-positive samples had values above the cutoff, and 59 out of the 62 HEV RNA-negative samples had values below the cutoff. The diagnostic performance of the model was visualized by ROC curve (Fig. 5A) and summarized in Table 1. It achieved a sensitivity of 91.2% and a specificity of 95.2% with AUROC of 0.98. Both the PPV and the NPV were above 90%.
The diagnostic performance of the HEV Ag assay and the model was repeatedly evaluated in the 67 randomly selected anti-HEV IgM-positive samples. Results confirmed that the diagnostic performance of the model (AUROC and 95% CI, 0.97, 0.94 to 1.00) was better than that of HEV Ag (AUROC and 95% CI, 0.87, 0.78 to 0.95) (Table 1 and Fig. 5B).
If the HEV Ag assay is not available, a simplified model combining only anti-HEV IgM and ALT/ULN level was proposed and shown as follows. Parameters were scaled down and rounded for the ease of clinical application.
logit&#;(p)=ALTULN+2×lg&#;(IgM)A weighted sum score of 3.921 of the parameter estimates was determined as the cutoff value. It achieved a sensitivity of 94.1% and a specificity of 85.5% with an AUROC of 0.96 in the training set. The sensitivity, specificity, and AUROC was 84.8%, 85.7%, and 0.95 in the test set, respectively (Fig. 5B and Table 1).
In this study, we evaluated the diagnostic performance of HEV serological markers for current HEV infection. Our findings show that anti-HEV IgM and HEV Ag are on the opposite ends in the diagnosis of HEV infection. A positive anti-HEV IgM result does not necessarily mean there is current HEV infection, while a negative anti-HEV IgM result basically excludes the possibility of HEV infection, except for very rare cases when patients seek medical care during the window period before anti-HEV IgM appears or patients are immunosuppressed and unable to produce anti-HEV IgM. In contrast, a positive result of HEV Ag confirms current HEV infection, while a negative result of HEV Ag does not guarantee there is no HEV infection, especially when the patient has a low viral RNA level or high anti-HEV IgG level.
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The concordance between HEV Ag and HEV RNA is 118/143 (82.5%) in the training set and 48/67 (71.6%) in the test set. The HEV Ag assay gave correct diagnosis on all HEV RNA-negative samples but only correctly diagnosed 46/71 (64.5%) HEV RNA-positive samples in the training set and 27/46 (58.7%) in the test set.
It is understandable to have less HEV Ag detected at low levels of HEV RNA, since the HEV Ag assay was reported to be less sensitive than RT-PCR (17, 25, 26). The low efficiency of HEV Ag detection at the presence of anti-HEV IgG is somewhat unexpected. By revisiting the literature, we found the phenomenon was described previously. Zhao et al. used an in-house-developed HEV Ag detection assay and found that the sensitivity of the HEV Ag assay was inversely proportional to the concentration of anti-HEV antibodies in serum (17). In our speculation, epitope competition might lead to the reduced detection efficiency of HEV Ag in the presence of anti-HEV IgG. The anti-HEV IgG antibodies in serum may compete with the anti-capsid polyclonal antibodies used in the ELISA kit to bind with the capsid protein. An analysis of serial samples from HEV-infected monkeys showed that HEV Ag could be detected prior to the appearance of anti-HEV antibodies, almost simultaneously with the fecal RNA, but HEV Ag disappeared 2 to 3&#;weeks earlier than the RNA, when anti-HEV IgG levels increased (15). Trémeaux et al. showed the HEV Ag assay gave more frequently positive results in immunocompromised patients at the acute phase (26). Behrendt et al. found significantly higher levels of HEV Ag in chronically infected individuals than acutely infected patients (25). These results have several things in common. The easier detection of HEV Ag in either immunocompromised patients at the acute phase or chronic patients probably benefits from the lower level of anti-HEV IgG in these subjects. Another interesting result was shown by Geng et al. (27). They found that HEV Ag was detected more frequently in urine than HEV RNA, and the ratio of HEV Ag to HEV RNA in the urine was significantly higher than in sera and feces. It is possible that since less anti-HEV IgG is secreted into urine than sera (since IgGs are normally too large to pass through the tubules of kidney), it is easier to obtain HEV Ag detection results. These findings indicate that the accuracy of the HEV Ag assay will be affected especially in regions where HEV is endemic and where the anti-HEV IgG seroprevalence is high (28, 29). It is necessary to improve the HEV Ag detection assay in the future to make it less susceptible to concomitant substances in the serum.
The exact duration of anti-HEV antibody response remains uncertain. Dawson et al. reported IgM antibodies disappeared in specimen collected 3.5&#;years later, and IgG antibodies continued to be detected 4.5&#;years after the acute-phase infection (11). Myint et al. showed that in acute hepatitis E patients, anti-HEV IgM can persist for an average of 5&#;months (30). Riveiro-Barciela et al. reported an unexpectedly long persistence of anti-HEV IgM: 4 out of 5 (80%) subjects were positive for anti-HEV IgM with the Wantai assay during the second year after acute hepatitis E, and 2 out of 12 (17%) patients were still positive by the Wantai assay after 3&#;years (31). In our retrospective follow-up analysis, most patients with confirmed HEV infection experienced a rapid increase of anti-HEV IgM shortly after the acute phase and a slow decrease of anti-HEV IgM thereafter. Four patients were positive for anti-HEV IgM during the 3- to 6-month follow-up (45 to 48), 6 were positive during the 6- to 12-month follow-up (50, 51, 53, 54, 55, and 58), and 1 was positive after a year (57). One patient turned negative after &#;5&#;months, one turned negative after &#;8&#;months, and another one turned negative after 13&#;months. Generally, patients with higher levels of anti-HEV IgM had longer durations of being positive. We currently do not know why the levels of anti-HEV IgM varied among the patients. It may be related to the immune status of the patient per se but not to the previous exposure to HEV, since the patients who had relatively low levels of anti-HEV IgM at presentation were all positive for anti-HEV IgG (22, 23, 32, and 34).
The study has several limitations. First, HEV RNA and HEV Ag were analyzed retrospectively. RNA may degrade in stored sera over time, and the detection accuracy may be impaired. Second, the follow-up duration was not evenly distributed. For some patients, the intervals between sequential samples were too short to describe the long-term trend. For other patients, their samples were collected from 2 time points, and we can only make a rough estimation of their anti-HEV IgM seroconversion. Third, further modification or validation may be necessary before the multifactorial model can be applied to testing results from other products, considering the sensitivity and specificity difference between them (32). It also may not be readily applied to immunosuppressed patients. Fourth, the model is still complicated. To ease the application in clinical settings, an algorithm is proposed. When an immunocompetent person has elevated levels of liver enzymes and is suspected of HEV infection, anti-HEV IgM should be tested first, since it has very high sensitivity. When the anti-HEV IgM testing gives a positive result, HEV Ag testing is recommended. Positive results of HEV Ag can confirm HEV infection. If the HEV Ag assay gives a negative result, diagnosis based on the [IgM+Ag+ALT] model is needed. If the HEV Ag assay is not available, the simplified [IgM+ALT] model could correctly diagnose &#;88% of the cases at a cutoff value greater than 3.921.
In conclusion, we explored the merits and demerits of the anti-HEV IgM and HEV Ag assay and developed a model with satisfactory performance in the diagnosis of current HEV infection when HEV RNA detection is not available. It is a useful tool in clinical decision making, especially in developing countries where HEV is endemic.
Acute hepatitis E (AHE) is caused by hepatitis E virus (HEV) in developing countries where sanitation is suboptimal, however, epidemiological investigation indicated HEV infection also occurs among individuals in industrialized countries with no history of travel to epidemic regions [1&#;3]. Like hepatitis A, it is transmitted by the fecal&#;oral route and contaminated water or food supplies have been implicated in major outbreaks. However, mortality rate of AHE is 0.5&#;4% in the general population and up to 20% among pregnant women [4].
The HEV genome is a single-stranded, positive-sense RNA, approximately 7.2 kb in length. It contains a short 5&#; untranslated region (UTR), three open reading frames (ORFs: ORF1, ORF2 and ORF3), and a short 3&#; UTR that is terminated by a poly(A) tract [5]. Although HEV sequences have been classified into four genotypes according to either the complete genome sequence or the nucleotide 80&#;450 of ORF1 [1, 6], only the single serotype was identified until now [4]. Genotype 1 is distributed in various developing countries in Asia and Africa; genotype 2 has been found in Mexico and Africa; genotype 3 is widely distributed and has been isolated from sporadic cases of an acute HEV infection and/or domestic pigs in the United States, several European countries, and Japan; genotype 4 is found mainly in Asian countries and contains strains from human and domestic pigs in China [7].
Clinical diagnosis of AHE is mainly done by a blood test that detects specific antibodies to HEV [8]. The immunoglobulin M (IgM) class of antibody against HEV (Anti-HEV IgM) is a diagnostic for recent or ongoing HEV infection for its short duration [9]. But the anti-HEV IgM alone should not be seen as evidence for infection because false-positive results are often caused by rheumatoid factors and immunoglobulin G (IgG.). Although the IgG class of antibody against HEV (Anti-HEV IgG) is generally only used as the post-infectious index for its long duration, detection of newly elicited anti-HEV IgG also can be the proof to diagnose AHE [10]. Therefore, the diagnosis of AHE should be based on both the serological evidence and clinical manifestation.
Commercially available assays were produced with synthetic or recombinant peptides specified by open reading frames 2 and 3 of different strains of the HEV genome. P239 antigen is a bacterially expressed recombinant peptide corresponding to aa368&#;aa606 of HEV ORF2 of a genotype 1 strain of HEV. The outstanding features of this peptide are that it naturally interacts with one another to form homodimers under physiological conditions and that such is strongly recognizable by HEV reactive human sera [11].
In our previous study, we found that the duration of anti-HEV IgA is longer than anti-HEV IgM, but the clinical and epidemiological implications of anti-HEV IgA in finding HEV infection remain to be clarified. In this study, we constructed the indirect ELISA assay with p239 antigen to detect anti-HEV IgA and further demonstrated the value of anti-HEV IgA in diagnosis of AHE.
About 245 patients with AHE in this study were recruited from the Department of Infectious Disease of Tongji Hospital in Wuhan, China, during the period of July to July . All of the 245 patients showed clinical manifestation (jaundice or elevated serum aminotransferase levels) of the acute hepatitis. They were positive for HEV RNA and (or) anti-HEV (verified by monoclonal antibody and antigen blocking test) but negative for IgM antibody to hepatitis A virus (anti-HAV), hepatitis B virus surface antigen (HBsAg), IgM antibody to hepatitis B virus core antigen (anti-HBc), and antibody to hepatitis C virus (anti-HCV). Drug-induced hepatitis and autoimmune hepatitis were also excluded. Among these 245 patients, 84 serum samples from 84 patients were positive for HEV RNA. The patients were 14&#;92 years of age (mean = 51 ± 13 years), male/female = 5:1, alanine aminotransferase (ALT) = 2,327 ± 1,520 U/l. In addition, 2,210 control serum samples were collected from Tongji Hospital and Xiamen Blood Center. All 2,210 control samples were negative for HEV RNA and normal for serum ALT, aspartate aminotransferase (AST) and total serum bilirubin (T-Bil). Samples testing false-positive for anti-HEV IgM or anti-HEV IgA were verified by antigen and monoclonal antibody blocking test, respectively. Among them, 1,313 serum samples were negative for both anti-HEV IgM and anti-HEV IgG. Of the remaining 897 serum samples, 15 were positive for anti-HEV IgM and 868 were positive for anti-HEV IgG. All serum samples were stored at &#;80°C before detection.
Viral RNA was extracted from 200 μl of serum samples with Trizol LS reagent (Invitrogen). Reverse transcription of the extracted RNA was carried out in a 20-μl reaction mixture containing 20 U of RNAsin (Promega), 10× RT buffer (Promega), 1 mM each dNTP (Takara), 5 U of AMV reverse transcriptase (Promega), and 2.5 uM of reverse transcription primer E5:5&#;-ctacacgaaaccgaragw-3&#; (r = a OR g, w = a OR c). The mixture was incubated at room temperature for 5 min, then at 42°C for 60 min, and at 95°C for 5 min. About 2 μl of the obtained cDNA was added to a 20-μl reaction mixture containing 0.5 mM each of the primers E5 and E1:5&#;-ctgtttaaycttgctgacac-3&#; (y = c OR t), 1 U of Taq DNA polymerase (Takara), and 10× PCR buffer (Takara), overlaid with 20 μl of mineral oil, and subjected to 35 cycles of PCR in a thermo-cycler (94°C, 40 s; 53°C, 40 s; 72°C, 40 s). About 2 μl of the first-round PCR product was amplified for a further 25 cycles (94°C, 40 s; 53°C, 40 s; 72°C, 40 s) using the internal primers E2:5&#;-gacagaattgatttcgtcg-3&#;) and E4:5&#;-gtcctaatactrttggttgt-3&#; (r = a OR g). The length of product corresponding to ORF2 sequence is 189 bp (6,298 nt&#;6,486 nt).
Anti-HEV IgM (capture method) and anti-HEV IgG were detected by using commercially available kits (BeiJing Wantai Biological Pharmacy Enterprise Co., LTD.) based on a recombinant HEV antigen, pE2. This is a structural peptide coded by an ORF2 sequence derived from a Chinese genotype 1 strain of HEV. For anti-HEV IgM, 100 μl of each samples was added to each well at a dilution of 1:10 in sample diluent. The microplates were incubated at 37°C for 30 min and then were washed five times with wash buffer. About 100 μl of horseradish peroxidase (HRP) conjugated pE2 antigen was added to each well. The microplates were incubated at 37°C for 30 min and then washed five times with washing buffer. Then, 100 μl tetramethyl benzidine buffer was added to each well. The microplates were incubated at 37°C for 15 min in the dark and then 50 μl of stop buffer was added to each well. For anti-HEV IgG, the procedures were the same as that of anti-HEV IgM except that HRP-labeled pE2 antigen against human IgG replaced by the HRP conjugated monoclonal antibody.
Absorbance (A) value was measured at 450 nm. According to the protocols provided by the manufacturer, anti-HEV IgM cut-off value = 0.26+ negative control mean A value and anti-HEV IgG cut-off value = 0.16+ negative control mean A value. Samples with A values higher than the cut-off value were considered positive, and samples with other A values were considered negative.
The well of the microplates was coated with 100 μl p239 antigen (2.66 μg/ml in carbonate buffer PH 9.6) and incubated at 4°C overnight after 37°C for 2 h. After removal of the coating buffer, 200 μl of the 3%PBS-BSA was added to every well and incubated at 37°C for 2 h. Then the blocking buffer was discarded and the microplates were dehumidified for 2 h. To test the anti-HEV IgA, 100 μl of each sample was added to each well at a dilution of 1:10 in 1% PBST-BSA. The microplates were incubated at 37°C for 30 min and then were washed five times with 1% PBST. A total of 100 μl of horseradish peroxidase conjugated goat monoclonal anti-human IgA (Wuhan Boster Biological Technology, LTD.) at a dilution of 1: was added to each well. The microplates were then incubated at 37°C for 30 min and then washed five times with washing buffer. Then, 100 μl of tetramethyl benzidine buffer was added to each well. The microplates were incubated at 37°C for 15 min in the dark and then 50 μl stop buffer (2 mol/l sulphuric acid) was added to each well. The OD value of each well was read at 450 nm.
The specificity of anti-HEV IgA and anti-HEV IgM assay was verified by monoclonal antibody against p239 antigen (8H3 and 8C11) [12] and unlabeled pE2 antigen. The serum samples were diluted to adjust its OD value to below 1.5. For the anti-HEV IgA, the solid surface was incubated with saturating levels (1:100) of 8H3 and 8C11 at 37°C for 30 min prior to the addition of serum samples. Then the microplates were washed five times with washing buffer and added to the serum samples. The follow-up procedures were the same as above in ELISA for anti-HEV IgA. For the anti-HEV IgM, the microplates were incubated at saturating levels (100 μg/ml) of unlabeled pE2 antigen at 37°C for 30 min after the addition of serum samples. Then the microplates were washed five times with washing buffer and added to the HRP-labeled pE2 antigen. The follow-up procedures were the same as above in ELISA for anti-HEV IgM. The OD value of the tested sample was reduced by no less than 70%. All samples positive for anti-HEV IgA and (or) anti-HEV IgM were verified by blocking test.
Chi-square test was performed using SPSS version 13.0. All tests were two-tailed and P-values of P < 0.05 were considered significant.
To determine the cut-off value of anti-HEV IgA assay, 1,313 serum samples with normal ALT and negative for both anti-HEV IgM and anti-HEV IgG were used as a panel in the present study. The OD values of anti-HEV IgA ranged from 0.001 to 0.33. The OD value of 0.211 (mean + 7SD) was used as the cut-off value for anti-HEV IgA assay. Using the cut-off value, the remaining 897 serum samples considered not to have been infected recently with HEV were tested anti-HEV IgA, anti-HEV IgM, and anti-HEV IgG. Among the total 2,210 serum samples, anti-HEV IgA, anti-HEV IgM, and anti-HEV IgG were detected in nine (Table 1), 15, and 868 samples, respectively. Among the nine serum samples testing positive for anti-HEV IgA, no sample was positive for anti-HEV IgM and seven samples were positive for anti-HEV IgG. All nine samples positive for anti-HEV IgA and 15 samples positive for anti-HEV IgM were false-positive, confirmed by monoclonal antibody blocking test and antigen blocking test, respectively. The specificity of anti-HEV IgA assay was 99.6% (/) (95% CI: 99.2&#;99.8%).
About 84 serum samples positive for HEV RNA from 84 patients with AHE were collected in 40 days after disease onset (Table 2). All the samples were genotype 4 HEV infection verified by sequencing. The positive rate of anti-HEV IgA, anti-HEV IgM, and anti-HEV IgG in serum samples positive for HEV RNA was 96.3% (81/84) (95% CI: 89.8&#;98.8%), 97.6% (82/84) (95% CI: 91.7&#;99.3%) and 88.1% (74/84) (95% CI: 79.5&#;93.4%), respectively. OD value of three samples negative for anti-HEV IgA was 0.007, 0.009, and 0.048, respectively. The difference between anti-HEV IgA and anti-HEV IgM was not statistically significant (P = 0.65). However, the difference between total positive rate of anti-HEV IgA and anti-HEV IgM compared to anti-HEV IgG were statistically significant (P = 0.043 and 0.017, respectively). Not only anti-HEV IgA but also anti-HEV IgM may be negative in serum sample positive for HEV RNA, but no samples were negative for anti-HEV IgA and anti-HEV IgM simultaneously.
Among 245 patients diagnosed with AHE, nine were negative for anti-HEV IgM in acute period (within 20 days after disease onset) but positive for anti-HEV IgA and two were positive for HEV RNA (Table 3). The two patients were infected with genotype 4 HEV verified by sequencing. For patient No. 46, anti-HEV IgG were detected at 10 days after disease onset, but the anti-HEV IgM was still negative. Anti-HEV IgM was positive at 3 days after disease onset and could not be detected at 9 days after disease onset in patient No. 97. For patient No. 156, anti-HEV IgM, anti-HEV IgG, and HEV RNA all were positive at 3 days after disease onset, but anti-HEV IgM was negative at 15 days after disease onset. For patient No. 219, anti-HEV IgM switched from positive to negative at 13 days after disease onset. Both anti-HEV IgM and anti-HEV IgG of patient No. 391 and patient No. 402 switched from negative to positive at 15 and 20 days after disease onset, respectively.
We also found four serum samples from above 245 patients with AHE were negative for anti-HEV IgA but positive for anti-HEV IgM in acute period. Among the four samples, HEV RNA was detected in two samples (Table 4). Anti-HEV IgA of patients No. 11, 21, and 78 switched from negative to positive at 15, 20, and 20 days after disease onset, respectively. But anti-HEV IgA of patient No. 249 was still negative at 40 days after disease onset. For patient No. 21, anti-HEV IgG was detected at 20 days after disease onset.
Among the 245 AHE patients, 13 samples collected at acute period with anti-HEV IgM or anti-HEV IgA alone were found and 4 samples of them were positive for HEV RNA. No sample was detected only positive for anti-HEV IgG.
Although HEV RNA is the gold standard for the diagnosis of AHE, it has not been widely available for its complex procedure. In addition, not only is the duration of HEV viremia very short (about 2 weeks on average) in most of the AHE patients but also the low HEV viral loads, so that the patients negative for HEV RNA can not be ruled out for the diagnosis of AHE [13, 14]. Currently, the diagnosis of AHE is mainly based on the serological detection. The presence of anti-HEV IgM is the marker of recent HEV infection, but the sandwich and indirect ELISA methods for detecting anti-HEV IgM have two disadvantages. One is the reduced sensitivity due to competition among virus-specific IgM, IgA, and IgG for antigen-binding sites. Another is that IgM-rheumatoid factor in sera from patients with rheumatoid arthritis presumably induced false-positive results. Yu et al. [15] constructed the class-capture enzyme immunoassay that eliminates the competing IgG antibodies, IgA antibodies at the beginning of the assay so as to enhance the reaction between anti-HEV IgM and HEV antigen. But sensitivity of the capture system depends on the capacity of solidified antibodies against the total human IgM antibodies containing the IgM specific against the HEV antigen. In addition, there is no agreement about the duration of anti-HEV IgG and that the duration from 6 months to 14 years was reported by different investigators [16&#;18]. Anti-HEV IgG is generally used in epidemiological investigation and as the post-infectious index for its long duration, but anti-HEV IgG does not exist throughout the lifetime, thus the positive rate of anti-HEV IgG in a population cannot completely reflect the post-infection rate.
Chau, Tokita and Takahashi et al. [19&#;24] reported that anti-HEV IgA can be a useful supplementary marker for recent HEV infection. In a previous study [13], we found that the duration of anti-HEV IgA was longer than that of anti-HEV IgM. The positive rate of anti-HEV IgA in AHE patients was 100, 100, 97, 93, 63, and 30% in the second week and the 1&#;5 month after the disease onset, respectively. The positive rate of anti-HEV IgM was 100, 100, 77, 57, 20, and 3% in the second week and the first 1&#;5 month, respectively. In the present study, we constructed the anti-HEV IgA indirect ELISA assay with p239 antigen and determined the cut-off value. The specificity of anti-HEV IgA assay was 99.6% (/) (95% CI: 99.2&#;99.8%) and no samples were found to be false-positive for either anti-HEV IgA or anti-HEV IgM. Takahashi et al. [22] had the same findings that anti-HEV IgM and anti-HEV IgA may be false-positive but specificity of combined detection of anti-HEV IgM and anti-HEV IgA was 100%. Single anti-HEV IgM, anti-HEV IgA, and anti-HEV IgG could not confirm whether the diagnosis of AHE for either anti-HEV IgM or anti-HEV IgA may be false-positive, and anti-HEV IgG can be positive at both ongoing infection and post-infection. In 84 serum samples positive for HEV RNA, whether anti-HEV IgA or anti-HEV IgM may be negative, but no samples negative for both anti-HEV IgA and anti-HEV IgM. Using the combined detection of anti-HEV IgA and anti-HEV IgM, the diagnosis sensitivity of AHE was improved to 100% in the PCR positive group. However, 84 serum samples positive for HEV RNA in this study all were of genotype 4 HEV infection. The significance of anti-HEV IgA in other genotype HEV infection remains to be clarified. Herremans et al. [25] reported IgA responses were more prominent in the patients with genotype 1 HEV infection compared to those with the genotype 3 infection, but the differences between genotype 1 and genotype 3-infected patients could be explained by the use of the homologous genotype 1 antigens in the assay. Although only one serotype of HEV was reported, the immunoreactivities of polypeptides from various HEV genotype isolates were different [26]. Therefore, it is necessary to synthesize various polypeptides from various HEV genotype isolates to clarify the significance of anti-HEV antibodies.
In clinical practice, the clinical manifestation of many patients was similar with acute hepatitis, but the indexes of HAV, HBV, HCV, HEV, EBV, CMV and autoimmune hepatitis and drug-induced hepatitis were negative. In this study, all nine serum samples collected from nine patients diagnosed as AHE in acute period (within 20 days after disease onset) were negative for anti-HEV IgM and two samples were infected verified by HEV genotype 4 by sequencing. All nine serum samples positive for anti-HEV IgA showed that some patients were negative for anti-HEV IgM in acute period and anti-HEV IgA assay can be a supplementary for diagnosis of AHE especially in patients negative for anti-HEV IgM in acute period.
IgA is the major secretory immunoglobulin and is widely distributed in all mucosal secretions as a dimeric molecule linked by a joining chain and a third molecule, the secretory piece. In the circulation, IgA occurs in both monomeric and polymeric forms. There are two serologically and structurally distinct IgA subclasses. IgAl makes up 90% of circulating IgA while the IgA found in secretions comprises 50% IgA1 and 50% IgA2. IgA deficiency is the most common reason for the primary immunodeficiency. There is a marked variability in the prevalence in different ethnic groups, with a frequency of 1/ among Chinese [27, 28]. Therefore, anti-HEV IgA of AHE patients may be a false-negative caused by IgA deficiency in the circulation.
Usually, mean plus 2(or 3) SD is used as the cut-off value for normal distribution data providing a specificity of 95% (or 99%). Using a higher cut-off value would increase the specificity at the expense of sensitivity. In this study, an OD value of three samples negative for anti-HEV IgA in HEV RNA positive group was 0.007, 0.009, and 0.048, respectively. Therefore, the sensitivity of anti-HEV IgA in HEV RNA-positive group was 96.3% using the cut-off value of both mean plus 2SD (0.101) and mean plus 7SD (0.211). However, the cut-off value of mean plus 7SD (0.211) can improve the specificity from 94.5% (122/) to 99.6% (9/) compared to mean plus 2SD (0.101). However, an OD value above 0.211 has little improvement to specificity of anti-HEV IgA. Moreover, an OD value of 0.211 (mean + 7SD) made both the sensitivity and specificity of combined detection of anti-HEV IgA and anti-HEV IgM were 100% so that 0.211 (mean + 7SD) was used as the cut-off value in this study. Certainly, more control samples and patient samples need to be detected to further optimize the cut-off value of anti-HEV IgA assay.
In conclusion, based on the results obtained in this study, anti-HEV IgA assay can be a useful supplement for diagnosis of acute HEV infection especially in patients negative for anti-HEV IgM in acute period.
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