Seroreversion of IgG anti‐HEV in HIV cirrhotic patients: A long‐term multi‐sampling longitudinal study

Abstract The aim of our study was to evaluate HEV antibody kinetics in HIV/HCV‐coinfected patients with cirrhosis. A longitudinal retrospective study was designed. Patients were followed up every 6 months; anti‐HEV IgG and IgM antibodies levels and HEV‐RNA by qPCR were analysed. The prevalence and incidence of every HEV infection marker were calculated. The kinetics of anti‐HEV IgG and IgM during the follow‐up were evaluated. Seventy‐five patients comprised the study population. The seroprevalence observed was 17.3%. None showed IgM antibodies or HEV‐RNA at baseline. None showed detectable HEV viral load during the study period. After a median follow‐up of 5.1 years, two of 62 seronegative patients (3.2%) seroconverted to IgG antibody. The incidence for IgM was 2.7%. Of the 13 patients with IgG seropositivity at baseline, five (38.5%) seroreverted. Meanwhile, of the two patients who exhibited IgM positivity during the study, one (50%) showed intermittent positivity. We found that HEV seropositivity is common in HIV/HCV‐coinfected cirrhotic patients. A remarkable rate of IgG seroreversions and IgM intermittence was found, limiting the use of antibodies for the diagnosis of HEV infection in this population.


INTRODUCTION
Hepatitis E virus (HEV) is the most common cause of acute hepatitis around the world and, consequently, is a major global health issue . In most cases, HEV infections are usually subclinical and self-limiting (Hoofnagle et al., 2012). However, HEV infection could have a worse prognosis in several groups of patients, such as immunosuppressed patients (Rivero-Juarez et al., 2019), pregnant women (Karna et al., 2020) or patients with underlying chronic liver disease (Kamar et al., 2012).
For the diagnosis of HEV infection, both serological and molecular markers are employed (Lu et al., 2021). The most sensitive method is the detection of HEV RNA, which allows the diagnosis of both acute and chronic infections. Nevertheless, its use requires more complex and expensive methods than HEV antibody detection. For this reason, clinical guidelines recommend the detection of anti-HEV IgM as a screening approach for the diagnosis of acute HEV infection (European Association for the Study of the Liver, 2018; Rivero-Juárez et al., 2020). In contrast, the presence of IgG antibodies is indicative of past infection, thus is not of diagnostic value but is useful for epidemiological studies. Therefore, anti-HEV IgG antibody levels could be used to identify individuals who could be protected against HEV in the long term (Su et al., 2017;Zhang et al., 2014). However, several studies highlight that the clinical value of antibody determination (IgG and IgM) could limit the diagnostic value and, consequently, jeopardize the management of patients with HEV. First, it has been reported that the loss of IgG antibodies (seroreversions) in immunosuppressed patients might imply the risk of reinfection in these patients . Second, it has been communicated that IgM could persist for a long time; thus, its use for the diagnosis of acute infection could be limited. Consequently, studies evaluating the kinetics of HEV antibodies over time are needed to optimize the management of HEV infection in high-sensitivity populations. For these reasons, we designed a study to evaluate the anti-HEV antibody kinetics in a cohort of HIV-infected patients with cirrhosis.

Study design and patients
This was a longitudinal retrospective study including HIV cirrhotic patients in follow-up at the Hospital Universitario Reina Sofia de Cordoba (Spain) between January 2012 and October 2020. Inclusion criteria in the cohort were as follows: (i) diagnosis of liver cirrhosis by transient liver elastography (Vergara et al., 2007) or F4 METAVIR fibrosis score by histological examination, and (ii) no history of liver decompensation prior to inclusion in the cohort. Patients were followed up every 6 months collecting a blood sample for the analysis.

Variable collection and definition of HEV infection
In all the samples, anti-HEV IgG and IgM antibodies and HEV RNA were evaluated. HEV infection was defined as detectable HEV-RNA and/or positivity to IgM, independent of the IgG result. Regarding HEV antibodies, four categories were defined to classify the serostatus of the patients: (i) HEV seropositivity, defined as positivity to IgG and/or IgM antibodies at baseline, (ii) HEV seroconversion, defined as positivity to anti-HEV IgG and/or IgM antibodies during the follow-up in patients with negative markers in the previous visit, (iii) IgG seroreversion, defined as undetectable anti-HEV IgG antibody in patients with a previous positivity, and (iv) persistence of anti-HEV IgM antibody, defined as the positivity of anti-HEV IgM antibodies for more than 6 months (Goel & Aggarwal, 2020).

Molecular and serological determination for HEV
Serum was obtained by centrifugation for 10 min at 400 × g and stored at −80 • C until required for analysis. Samples were tested for anti-HEV IgG and anti-HEV IgM antibodies by commercial ELISA (recomWell HEV IgG/IgM®; Mikrogen Diagnostik, Neuried, Germany) using an automated procedure (Automatic ELISA workstation DS2®, Dynex Technologies). The analyses were carried out in accordance with the instructions provided by the manufacturer using a cut-off value ≥ 24 U/ml for positive samples. Antibodies quantitative value are presented in U/ml, not related with the WHO international standard. The specimens with a value-to-cut-off ratio between 20 and 24 U/ml were considered borderline. Confirmatory testing was performed using immunoblotting (recomLine HEV IgG/IgM®; Mikrogen Diagnostik, Neuried, Germany), following a manual procedure according to the manufacturer's instructions for all positive samples. RNA was extracted from 400 μl of serum using the commercial QIAamp Mini Elute Virus Spin Kit (QIAgen, Hilden, Germany) by an automated procedure (QIAcube. QIAgen, Hilden, Germany). The purified RNA was eluted in a volume of 50 μl. RT q-PCR for HEV was performed using the QIAgen One-Step PCR Kit (QIAgen, Hilden, Germany), following an inhouse protocol described previously by our group (Frías et al., 2021).

Ethics statement
This study was designed and conducted in accordance with the Declaration of Helsinki. The Ethics and Clinical Trials Committee (CEIC) of Córdoba approved the study protocol, obtaining the informed consent of each patient. The SSPA Biobank has coordinated the collection, processing, handling and assignment of the biological samples used in this study in accordance with the standard procedures established for this purpose.

Study population
Seventy-five patients were included in the study. Of these, 67 (89.3%) were males and eight (10.6%) were females, with a median age of 53 years (IQR: 49-56 years). In Table 1, we show the baseline characteristics of the population. Seventy-four patients (98.6%) were treated for HCV during the study, and 67 (90.5%) of them achieved sustained virological response (SVR). All patients were under antiretroviral therapy, all with undetectable HIV viral load. No patient received any blood transfusion or intravenous immunoglobulin therapy during the study.

Prevalence and incidence ratio of HEV seropositivity
The baseline prevalence of anti-HEV IgG antibodies was 17.3% (13 out of 75 [95% CI: 10.3%-27.6%]). IgM antibodies or HEV-RNA were detected in any of the patients. In Figure 1

Seroreversion of anti-HEV IgG
Seroreversions for anti-HEV IgG antibody were evaluated by moni-   Table S1.

Persistence of anti-HEV IgM antibodies
Two patients (2.7%) presented seroconversion to IgM antibodies during the study (Figure 1). One of them seroconverted to IgG and IgM antibody in the last visit of the study (

DISCUSSION
In the natural course of HEV infection, after an approximate incubation period of 2 weeks, HEV-RNA can be detected in blood up to approximately 3 weeks after the onset of symptoms . Thereafter, an initial short-lived IgM antibody response, persisting for 6 months, is followed by IgG antibodies . Consequently, the detection of IgM indicates a recent infection (Goel & Aggarwal, 2020). However, several studies have found that anti-HEV IgM antibodies may persist for more than 6 months (Myint et al., 2006;Norder et al., 2016;Riveiro-Barciela et al., 2020).
In a cohort of 25 patients with self-limited acute HEV infection, it was found that 24%-56% (depending on the assay employed) of individuals showed positivity for anti-HEV IgM antibodies at baseline and that IgM remained detectable after a median follow-up of 34 months (Riveiro-Barciela et al., 2020). Another study that retrospectively evaluated samples from 62 adults diagnosed with acute HEV during two outbreaks reported that 25% of individuals with positive IgM antibodies remained positive for IgM for at least 14 months (Myint et al., 2006). In addition, a study carried out in Sweden found one individual out of 27 who presented persistence of IgM antibodies but only for 7 months (Norder et al., 2016). Nevertheless, these studies present methodological differences from ours; we longitudinally analysed multiple samples during a long follow-up, whereas the other studies analysed only baseline and final samples (Norder et al., 2016;Riveiro-Barciela et al., 2020) or different nearby points during a short follow-up (Myint et al., 2006).

TA B L E 2 Timeline of the dynamics of anti-HEV
In our study, we do not find IgM persistence. Nevertheless, we found After the acute phase of the infection, anti-HEV IgG antibodies are produced to persist for at least several years and confer immunity against HEV for a long time (Su et al., 2017;Zhang, 2014). Thus, IgG detection indicates previous exposure to HEV (Goel & Aggarwal, 2020).
However, studies have shown that IgG seroreversions may occur in a proportion of patients (Faber et al., 2018), associated with immunosuppression (Kaba et al., 2011;Pineda et al., 2014) and with low baseline antibody titres (Servant-Delmas et al., 2016). In our study, we found seroreversion for anti-HEV IgG antibody in five out of 13 (38.5%) patients with a mean follow-up of 3.36 years. This fact has important implications because the loss of acquired immunity could confer susceptibility to reinfection with HEV. However, we did not find recent infections in these patients during the study period, so we were unable to evaluate this point. Several factors associated with seroreversion have been identified. In one study, it was observed that a low CD4+ count (<200 cells/μl) was associated with seroreversions of the IgG antibody in HIV patients (Pineda et al., 2014). Another study conducted in a Swiss cohort including 735 HIV-infected patients found that the prevalence of IgG antibody was lower in patients with low CD4+ counts (Kenfak-Foguena et al., 2011). In our study, we did not find a relationship between CD4+ cell count and IgG antibody seroreversions, showing that all seroreverted patients had a high CD4+ cell count. However, it has been also shown that HIV-infected individuals had a lower humoral response, independently of the CD4+ cells count (Abravanel et al., 2017). Thus, the seroreversion for IgG antibodies could be related with this, but could not evaluate this point in our study. Other factors affecting seroreversion should be evaluated.
On the other hand, we did not find HEV infection to be the main trigger of liver decompensation in cirrhotic HCV/HIV-coinfected patients in our study. Of the 15 patients who decompensated in our study, none had liver injury associated with HEV infection. Similarly, studies carried out in cirrhotic patients in France or the United Kingdom found low liver decompensation due to HEV infection, 3.5% and 3.2%, respectively (Blasco-Perrin et al., 2015;Haim-Boukobza et al., 2015).
In addition, a study conducted in the United States found an incidence of 4.5% anti-HEV IgG antibodies in decompensated patients (Samala et al., 2016). These differences observed between Asia and America and Europe are not related to the prevalence and incidence of HEV infection in this population, because studies carried out in patients with chronic hepatitis C in the United States or with chronic liver diseases in Spain showed prevalences comparable to our study (Samala et al., 2016;Vázquez-Morón et al., 2019). Likewise, the incidence rates reported in the United Kingdom (2/1000 patient-years) and the United States (7/1000 patient-years) were similar to those observed in our cohort (Khuroo et al., 2016). Therefore, in Europe and America, exposure to HEV among patients with underlying chronic liver disease seems to be common. For this reason, although HEV infection is frequent in patients with underlying chronic liver disease in countries where genotype 3 is the main cause of infection, this seems not to be a leading cause of liver decompensation or death in this population.
Several limitations should be noted. The main limitation of our study is the relatively low number of patients, which could be insufficient to identify any HEV infection as a risk factor for liver decompensation and death. However, the longitudinal nature of our study together with the multisample analysis allows us to observe the dynamics of anti-HEV IgG/IgM antibodies of each individual at different points as well as assess their association with the possible outcomes. Furthermore, although the association of HEV with decompensation in cirrhosis has been found to be independent of the aetiology (Wang et al., 2020), we only included patients with cirrhosis caused by HCV, not patients with cirrhosis from other causes (chronic hepatitis B, alcohol intake, moderate to severe fatty liver, autoimmune liver disease or any other aetiology).
In conclusion, seroreversion of anti-HEV IgG antibody is frequent in cirrhotic population.
HEV infection does not seem to be associated with death or liver decompensation in this population of our setting. People at high risk of developing a severe course of HEV infection or the chronification of it (such as cirrhotic) must be specifically informed of the risk involved in eating undercooked pork products and game animals, and to avoid the consumption of these products, even in those carrying anti-HEV IgG antibodies because of the high rate of seroreversion.