As the Adenovirus-Induced Hepatitis Mystery Continues, an Understanding of Testing is Vital

From October 2021 through May 2022, approximately 109 cases of acute hepatitis of unknown origin in children were publicly reported.¹ Since May 3, 2023, an additional 282 cases (391 total pediatric patients) have been reported in 46 states and jurisdictions and in at least 11 countries in Europe and the Americas.¹

The recent increase in cases under investigation were identified retrospectively since October 1, 2021, and may not ultimately be linked to this investigation. No additional deaths have been reported since February 2023, and the proportion of patients requiring liver transplants has decreased from 15% to 9% from May 5, 2022, through May 3, 2023. Additional testing details were updated by the CDC on May 11, 2022. The CDC continues to investigate the cause and any contributing factors (e.g., immune response to adenovirus, coinfections, environmental exposures, medications) related to these pediatric hepatitis cases.¹

What Are Adenoviruses?
Adenoviruses are a group of viruses that can cause mild to severe infection almost anywhere in the human body. They usually infect the respiratory system and can cause cold- or flu-like symptoms. Most adenovirus infections are mild and require only symptom relief for treatment. For these reasons, an adenovirus infection is often difficult to diagnose based on clinical appearance and symptoms and could be associated with any number of respiratory virus agents (coronaviruses, rhinoviruses, etc.) or other viruses (Herpesviridae, Picornaviridae family, etc.).

This digitally-colorized transmission electron microscopic (TEM) image revealed some of the ultrastructural details exhibited by a small cluster of adenovirus virions.

Image courtesy: CDC / Dr. G. William Gary, Jr.

Adenoviruses typically cause respiratory illnesses, such as a common cold, conjunctivitis (an infection in the eye sometimes called pink eye), croup, bronchitis, or pneumonia. In the pediatric population, adenoviruses usually cause infections in the respiratory tract and intestinal tract.² Approximately 100 or more serologically distinct types of adenoviruses have been identified, including approximately 49 types that infect humans.² The Adenoviridae family is divided into 2 genera: the mammalian adenoviruses (mastadenoviruses) and the avian adenoviruses (aviadenoviruses).²

It’s important to consider that many human adenoviruses may cause mild to subclinical infections and may not be a typical test request by physicians or even part of a serious differential diagnosis consideration. However, health care providers may request testing for adenoviruses in certain situations—for example, if an individual has severe illness or pneumonia, is immunocompromised, or if an outbreak is being investigated.

Adenovirus Diagnostics
Many laboratories can test for the presence of human adenoviruses, but the extent of viral testing capability can vary. For example, the gold standard for adenovirus laboratory testing is cell culture, but this can be time consuming and subjective. Collected samples (usually respiratory, ocular, or gastrointestinal) are layered on a specific type of monolayer of cells in test tubes. If present, adenovirus will infect the cells and multiply in the cells. As the virus amplifies, the cells will present with the cytopathic effect (CPE) of rounded cells.

Under a magnification of 125X, this photomicrograph revealed the cytopathic effects (CPE) exhibited by a Hep-2 cell culture, after the addition of adenovirus type-2 virus. Normally, you would see a monolayer of uniformly sized, polygonal-shaped cells, dispersed across the field. However, after being inoculated with the adenovirus type-2 virus, there were now many ovoid-shaped, granulated cells, some arranged in clusters of multinucleated giant cells with interconnecting fibrils that bridge areas devoid of cells. These acellular regions represented cells that were destroyed, due to the cytopathic effect (CPE).

Image courtesy: CDC / Dr. Kirsh

The CPE can be unique to a specific virus or virus family and be utilized as a screening test. Cell culture and CPE screening take specialized observation and can create a time lapse for identification. Due to this issue, one can seek specialized and rapid testing via a state health department to discuss available testing options. Additional information from the CDC on adenovirus testing is available.³

Laboratories can detect and type human adenoviruses via the following³:

• Molecular detection (eg, polymerase chain reaction)
• Partial or full genome sequencing
• Antigen detection (eg, immunoassay)
• Virus isolation (cell culture and CPE)
• Virus neutralization with type-specific antisera
  

It All Starts with a Specimen
Like any good medical laboratory professional will tell a health care provider or others, the accuracy of any strong laboratory medicine test begins with the accurate collection of the correct specimen.⁴ The types of specimens you should collect for human adenovirus detection depend on the patient’s clinical presentation and type of infection. Before collecting any specimens, talk with a laboratory professional and a clinician familiar with adenoviruses. The timing of collection can be critical, and generally one should collect specimens within a week of symptom onset for a higher likelihood of success at adenovirus viability.

Consider the following guidance for collecting specimens based on the infection site³:

• Respiratory infections: Typically, a health care provider should collect upper respiratory specimens via a nasopharyngeal swab and/or oropharyngeal (throat) swab and should also collect a lower respiratory specimen such as sputum when evidence of a lower respiratory infection exists. A serum specimen also may be helpful in some cases.
• Eye infections: Collect a conjunctival swab when there is clinical evidence of a conjunctival or eye infection.
• Gastroenteritis infections: The CDC advises that providers refer to recommendations for collection of laboratory specimens associated with outbreaks of gastroenteritis. (Go to cdc.gov for more information).

The Ongoing Outbreak
The CDC and World Health Organization (WHO) continue to work with other public health officials globally to learn more about these cases of acute hepatitis in children to determine the cause, implement disease management strategies, and further refine control and prevention actions. Of the previously reported 109 cases, in the US the American Academy of Pediatrics writes in its Red Book¹:

• Cases were reported mostly in young children (median age, 2 years).
• More than half were a confirmed adenovirus infection, most commonly Human adenovirus F type 41.
• Hospitalization was 90%
• Liver transplants were required in 14% of patients
• 5 patients died and the rest recovered
• Within the past 7 months, pediatric hepatitis cases of unknown cause have been reported in a number of states including the following: Alabama, Arizona, California, Colorado, Delaware, Florida, Georgia, Idaho, Illinois, Indiana, Louisiana, Michigan, Minnesota, Missouri, North Carolina, North Dakota, Nebraska, New York, Ohio, Pennsylvania, Puerto Rico, Tennessee, Texas, Washington, and Wisconsin.

Guidance
Although the adenovirus is one culprit to consider, the clinical picture is not clearly fitting the severity of infection presentation. Human adenovirus F type 41, the implicated adenovirus type, has not historically been linked to such a clinical presentation. Adenoviruses are common pathogens that usually cause self-limited infections. Human adenovirus F type 41 usually induces diarrhea, vomiting, and fever, with possible respiratory symptoms. Although cases of hepatitis in immunocompromised children with adenovirus infection have been reported, Human adenovirus F type 41 is not known to be a cause of hepatitis in otherwise healthy children.⁵

The WHO noted that “factors such as increased susceptibility [among] young children following a lower level of circulation of adenovirus during the COVID-19 pandemic, the potential emergence of a novel adenovirus, as well as SARS-CoV-2 coinfection need to be further investigated. Hypotheses related to [adverse] effects from the COVID-19 vaccines are currently not supported, as most affected children did not receive COVID-19 vaccination. Other infectious and noninfectious explanations need to be excluded to fully assess and manage the risk.”⁵

The world can expect to see more cases being detected. And with these ongoing case detections, it may take additional time, testing, and global patience before we can confirm the underlying cause.

References

1. Red Book Online outbreaks: hepatitis cases possibly associated with adenoviral infection. American Academy of Pediatrics. May 4, 2023. Accessed May 25, 2023. https://publications.aap.org/redbook/resources/20171?autologincheck=redirected

2. Doerfler W. Chapter 67: adenoviruses. In: Baron S, ed. Medical Microbiology. 4th ed. University of Texas Medical Branch at Galveston; 1996. Accessed May 25, 2023. https://www.ncbi.nlm.nih.gov/books/NBK8503/#:~:text=More%20than%20100%20serologically%20distinct,49%20types%20that%20infect%20humans

3. Adenoviruses: laboratory testing and specimen collection. CDC. Updated March 7, 2023. Accessed May 25, 2023. https://www.cdc.gov/adenovirus/specimen-collection.html

4. How do blood tests work? Medical laboratory scientists explain the pathway from blood draw to diagnosis and treatment. Rohde R, Moore N. The Conversation. February 25, 2023. Accessed May 25, 2023
https://theconversation.com/how-do-blood-tests-work-medical-laboratory-scientists-explain-the-pathway-from-blood-draw-to-diagnosis-and-treatment-196874

5. Multi-country—acute, severe hepatitis of unknown origin in children. World Health Organization. April 23, 2022. Accessed May 25, 2023. https://www.who.int/emergencies/disease-outbreak-news/item/2022-DON376