A mystery may have been solved

Long-time followers of my blog may remember the perplexing occurrence and significant number of cases of hepatitis (liver infection/inflammation) in 2022, first noted in the U.K. occurring in COVID-19-unvaccinated children under the age of 16 years that we have since referred to as acute hepatitis of unknown origin (AHUO) at a time when fewer mitigation measures were being taken to protect children in schools against airborne transmission of viruses. Clinically significant hepatitis is not a predominant feature of most cases of COVID-19, yet, many of these children were quite ill with the liver disease, some critically ill and still some requiring liver transplantation. Tests for the usual suspects (e.g., hepatitis A, B, C, D and E viruses) were all negative. After all the testing and review of cases, the major remaining differential diagnoses were narrowed down to adeno-associated virus co-infection with adenovirus or autoimmune hepatitis. We may now have a different answer.

We have known since the early days of the pandemic that SARS-Co-V-2 RNA can be detected in the stool of patients with acute COVID-19. Subsequent investigation and a publication by Zuo et al demonstrated that SARS-CoV-2 RNA can continue to be detected in some COVID-19 patients in the convalescent phase of the infection (when symptoms are resolving or have resolved).

Given mounting evidence of viral persistence in some people following SARS-CoV-2 infection and the fact that detection of viral RNA after the infection has supposedly resolved can be an indication of viral persistence, a group of investigators began to search for evidence of the virus in other organs of the gastrointestinal tract Residual SARS-CoV-2 viral antigens detected in GI and hepatic tissues from five recovered patients with COVID-19 | Gut. Using conventional immunohistochemistry (this is a process by which investigators create antibodies that are specific to the protein they wish to search for, tag these antibodies with a substance that will show up under the microscope, inject the antibodies into the tissues, then cut up the tissue into sections that can be stained and examined under a microscope), these investigators examined the tissues obtained from five patients who seemed to have recovered from COVID-19.

The protein they targeted was the nucleocapsid protein (NP) of the virus, as this protein is not present in any of the vaccines used in the U.S. or most other countries, only being an issue in the few countries that utilized whole inactivated virus in their vaccines, such as China. Thus, in those who have not received inactivated whole virus vaccine, the presence of NP (and by extension antibodies to NP) is a consequence and indicator of prior infection regardless of vaccine status.

These researchers detected SARS-CoV-2 NP in the colon, appendix (this is a finger-like projection extending from the colon very close to the location where the small intestine connects to the large intestine), ileum (this is the last part of the small intestine just before it connects to the large intestine (or colon), hemorrhoid tissue, liver, gallbladder and lymph nodes from these five patients who recovered from COVID-19, ranging from 9 to 180 days after testing negative for SARS-CoV-2 by nasal swabs. In fact, two of the five had NP detected in each of these locations, an indicator of how far and extensively the virus spreads within the body even in “mild” cases. The findings were validated by confirmatory identification of SARS-CoV-2 spike (S) protein and in some cases the presence of SARS-CoV-2 RNA.

Building on these two studies, with good evidence for viral persistence that can involve the liver as long as six months after even what appeared to be a “mild” infection, in an environment of a rapidly evolving virus with increasing immune escape capabilities, and especially at the time (2022) when we were seeing new Omicron variants as often as every several months, the case for autoimmune hepatitis has become strengthened.

Given the persistence of nucleocapsid protein embedded within liver cells following a prior infection, the potential for hepatitis due to cell-mediated killing of the liver cells as a consequence of efforts to clear the virus or autoimmune hepatitis upon subsequent reinfection with a similar, but distinct antigenic variant of SARS-CoV-2 are reasonable hypotheses.

This leads us to a new study published this year, Characteristic immune cell interactions in livers of children with acute hepatitis revealed by spatial single-cell analysis identify a possible postacute sequel of COVID-19.

These researchers examined the liver biopsies obtained from 12 patients (mean age 9.5 years) who were hospitalized between February 2022 and December 2022 in European countries who tested negative for acute adenoviral or SARS-CoV-2 infection and who met the WHO criteria for AHUO. Three of the 12 showed serological evidence of past adenoviral infection and 10 of the 12 had a history or serological evidence of past COVID-19. Eight of the 12 were known to have had a recent bout of COVID-19 with a median onset of 2 months prior to the hospitalization for AHUO. Unlike the original cohort noted in the U.K., a third of these children had a history of past COVID-19 vaccination.

Imaging mass cytometry (my apologies in advance to laboratory scientists who might be reading this as I have no expertise in this area – this is a technique that utilizes metal tags of antibodies designed for up to 40 antigens found in different types of cells that is applied to a tissue specimen. A machine then scans the tissue with a laser that generates signals from the metal tags that create detailed pictures of the cells in the tissue. The pictures reveal the location of the antigens targeted within the cells to allow identification of their intracellular locations as well as to help us understand how they are interacting with the cell) identified significant infiltration of the liver tissue with CD8+T- cells, which are cytotoxic T-cells (the cellular part of the immune response that identifies infected cells and kills them in order to rid the body of the virus within). The degree of infiltration and immune activation correlated with the severity of the hepatitis.

Further, the investigators detected SARS-CoV-2 antigens in ACE2- expressing cells in the areas of the liver with significant pathology in 11/12 samples using several different detection methods (we have known for some time that not everyone who has had COVID-19 will test positive on serological testing (measurement of antibodies in the blood).

The identification of immune-mediated liver injury associated with the detection of SARS-CoV- 2 antigens suggests a possible association of AHUO with prior SARS-CoV- 2 infection, further suggesting that the hepatic disease could have manifested as a part of Long COVID or post-acute sequelae of COVID-19.

PCR testing for adenovirus in liver specimens was negative in all of the subjects. Conversely, detection of SARS-CoV-2 proteins occurred in 11 of the 12 subjects. Notable was the extent of immune-cell infiltration in all subjects, and the degree of infiltration correlated with the clinical severity of each child’s course of illness. Further, the colocation of infiltrating cytotoxic T-cells in areas where SARS-CoV-2 proteins were detected suggests an antigen-mediated pathology.

The fact that the SARS-CoV-2 virus itself was not detected in the liver tissues and cells raises the concern that has already been raised as a possible underlying explanation for Long COVID, that virus is persisting and residing in areas of the body that are more difficult for the immune system to surveil and remove virus from (one candidate in these patients was already identified in the earlier studies I referenced above – the small and large intestines). In this instance, the thought would be that the persisting virus is replicating, although likely far less than in an acute infection, and shedding viral proteins that are entering the venous system that returns blood from the bowels that passes through the liver where the proteins can be deposited prior to the blood returning to the right side of the heart. Support for this theory is the fact that a biomarker for a disturbance of the gut integrity with subsequent leak into the vascular system (zonulin) has been detected in plasma along with spike protein. As a result, in at least some cases, immunopathology results and hepatitis may be the presenting illness as a result of liver cell injury due to the infiltration of immune cell infiltration targeting cells for destruction due to the presence of viral protein.

Of interest, this same situation of persistent SARS-CoV-2 virus in the GI tract with SARS-CoV-2 RNA detected in stool weeks after initial infection at a time when nasal swab testing for SARS-CoV-2 had long since turned negative has been postulated as the mechanism behind MIS-C (multi-system inflammatory syndrome in children) that we often saw present weeks to months following acute infection.

Of course, nothing from this study rules out the possibility that adeno-associated virus 2 (AAV2) contributes to the development or increases the risk for AHUO, but clearly, prior or co-infection with AAV2 does not appear to be necessary for the development of AHUO.

In conclusion, it appears that AHUO may be a post-COVID-19 immune-mediated hepatitis, another potential part of the spectrum of illnesses that may occur as part of the post-acute sequelae of COVID-19.