Lab Diagnosis of Epstein–Barr Virus

Nivedhana Subburaju¹

¹Department of Microbiology, Rainbow Children’s Hospital, Chennai, Tamil Nadu, India

Corresponding Author: Nivedhana Subburaju, Department of Microbiology, Rainbow Children’s Hospital, Chennai, Tamil Nadu, India, Phone: +91 9941938029, e-mail: nivedhana24@gmail.com

Received on: 04 September 2023; Accepted on: 08 October 2023; Published on: 14 December 2023

ABSTRACT

This article aims to summarize the hematological and biochemical findings in acute Epstein–Barr virus (EBV) infection and serological diagnosis of infectious mononucleosis (IM).

How to cite this article: Subburaju N. Lab Diagnosis of Epstein–Barr Virus. Pediatr Inf Dis 2023;5(4):138–140.

Source of support: Nil

Conflict of interest: None

Keywords: Atypical lymphocytes, Epstein–Barr virus, Heterophile antibodies, Infectious mononucleosis, Viral capsid antigen

An accurate diagnosis of infectious mononucleosis (IM), typically resulting from the Epstein–Barr virus (EBV), is often achievable through an assessment of clinical, hematologic, and serologic features. It is prudent to consider the possibility of EBV-induced IM in adolescents who manifest a triad of symptoms, including fever, tonsillar pharyngitis, and lymphadenopathy. The latter is commonly observed in posterior cervical, posterior auricular, and inguinal regions. Additionally, fatigue is a prevalent symptom, and approximately 50–60% of cases may present with splenomegaly. Furthermore, a maculopapular rash may develop in some individuals, enhancing the clinical profile for diagnosis.¹

Hematologic findings²^,³:

Leukocytosis is common, with a white blood cell (WBC) count typically ranging from 10,000 to 20,000 cells/mm³. The WBC count usually returns to normal within 3 weeks.
Marked lymphocytosis is a key feature, where the term “mononucleosis” reflects an increase in lymphocytes. This is defined as an absolute lymphocyte count greater than 4,500 cells/mm³ or a differential count exceeding 50% on a blood smear. It indicates the activation of a cluster of differentiation 8 (CD8⁺) cytotoxic T cells in response to virus-induced B cell proliferation, reaching its peak at 7–21 days.
Atypical lymphocytosis (Fig. 1), with >10% of total lymphocytes, is observed. These are activated CD8⁺ cytotoxic T cells and CD16⁺ natural killer cells. Atypical lymphocytes show nuclear alterations and an increase in basophilic cytoplasm. They may include “monocytoid” and “plasmacytoid” lymphocytes.
Atypical lymphocytosis resolves as the patient recovers, but the virus is not eliminated from the host.
Atypical lymphocytes are also seen in other conditions like toxoplasmosis, viral hepatitis, mumps, cytomegalovirus (CMV), acute human immunodeficiency virus (HIV) infection, rubella, roseola, and some drug reactions.
Transient monocytosis may be present.
Absolute neutropenia is accompanied by an increase in band cells and metamyelocytes.
Mild thrombocytopenia may occur.
Uncommon hematologic manifestations include hemolytic or aplastic anemia, thrombocytopenia, hemolytic-uremic syndrome, and disseminated intravascular coagulation.
Hemolytic anemia, occurring in 1–3% of cases, may be associated with an anti-I cold agglutinin.
Bone marrow findings often show increased cellularity with collections of mononuclear cells forming loose granulomas.

Fig. 1: Atypical lymphocytes in infectious mononucleosis (with abundant basophilic cytoplasm)

BIOCHEMICAL–LIVER FUNCTION ABNORMALITIES

Elevated transaminases are a common finding in 85–100% of cases, often accompanied by mild bilirubin elevation. Clinical jaundice is rare. In rare instances, primary EBV infection can lead to hemophagocytic lymphohistiocytosis, characterized by abnormal liver function tests, and elevated serum ferritin levels.²

Serological Tests

Plasma B cells infected by the EBV generate heterophile antibodies, specialized polyclonal immunoglobulin M (IgM) antibodies with the unique capability to agglutinate red blood cells from phylogenetically unrelated species.¹^,⁴^,⁵

The Paul–Bunnell test assesses the agglutination of sheep red blood cells by patient sera, while the monospot test gauges the agglutination of horse red blood cells following serum preabsorption with guinea pig cells.

Heterophile antibodies typically exhibit 40–60% positivity within the 1st week of illness, escalating to 80–90% positivity 1 month post onset.

The sensitivity of these tests ranges from 81 to 95%, and they demonstrate negative, and positive predictive values of 78–88 and 95%, respectively.

Advantages

Technical ease, rapid turnaround time, and low cost.
Point-of-care tests, including immunochromatography, latex agglutination, and immunofiltration methods.
Reactive heterophile antibodies in symptomatic patients are diagnostic of EBV infection, and although nonspecific, eliminate the need for additional testing for specific antibodies to EBV.

Disadvantages

False Negatives

Early infection rates—25% in the 1st week, 5–10% in the 2nd week, and 5% in the 3rd week.
Young children, particularly those under 4 years old, prefer EBV-specific serology for accurate testing.

False Positives

Reported in various infections such as malaria, HIV, CMV, rubella, viral hepatitis, and tularemia.
Also observed in conditions like leukemia, lymphoma, pancreatic cancer, and systemic lupus erythematosus.
Heterophile antibodies may persist at low levels for up to 1 year after IM.

Epstein–Barr Virus (EBV)-specific Antibodies

The diagnostic gold standard for IM involves EBV-specific antibodies, specifically IgM and IgG directed against viral capsid antigens (VCA). These antibodies provide high sensitivity (97%) and specificity (94%), proving particularly valuable in cases where patients suspected of IM show negative heterophile test results or do not align with classic diagnostic criteria.

Viral Capsid Antigen

The IgM and IgG antibodies targeting VCA are typically detectable at the onset of illness due to the virus’s extended incubation period (4–8 weeks).

The IgM levels, serving as a reliable marker for acute infection, decrease after 3 months, while IgG VCA antibodies persist for life.

It is important to acknowledge that VCA antibodies may yield positive results in the presence of other herpesviruses (e.g., CMV, herpes simplex virus (HSV) 1 and 2). Additionally, during intense immune activation in other illnesses, EBV, IgM, and VCA antibodies may be spuriously elevated.

Nuclear Antigen

Immunoglobulin G (IgG) antibodies to epstein-barr virus nuclear antigen (EBNA), a protein expressed during latency, become detectable 6–12 weeks after symptom onset and persist for life. Early positivity for EBNA IgG effectively excludes acute EBV infection and should prompt consideration of EBV-negative causes of mononucleosis. Heterophile-negative mononucleosis most commonly stems from CMV infection, with other potential causes including HIV, infectious hepatitis, human herpesvirus 6, HIV-1 and -2, toxoplasmosis, and certain drug ingestion (e.g., p-aminosalicylic acid, phenytoin).

In summary, while IgM and VCA antibody positivity indicates a probable acute EBV infection, the diagnosis is reinforced by the presence of IgM and VCA and the absence of IgG and EBNA antibodies (Fig. 2).

Fig. 2: Response time in diagnosis of EBV infection

EBV VIRAL LOAD [DNA-POLYMERASE CHAIN REACTION (PCR)]

Here are key points regarding the use of EBV DNA-PCR in diagnosing and monitoring EBV infections¹:

Epstein–Barr virus (EBV) DNA-PCR is crucial for investigating primary or reactivated EBV infections in immunocompromised patients, given that serological tests may yield unreliable results in this population.
It can serve as a confirmatory assay when antibody test results are inconclusive.
“Chronic active EBV infection,” a rare and life-threatening lymphoproliferative disorder, is associated with EBV viremia.
Viral genomes are typically detectable in the blood of about 90% of patients approximately 2 weeks after the onset of symptoms. PCR-positive patients often exhibit lymphadenopathy, a higher presence of atypical lymphocytes, and elevated aminotransferase levels.
For immunocompetent patients with suspected EBV infection, measuring EBV viral load is generally not recommended, as it does not offer therapeutic guidance.
Epstein–Barr virus (EBV) can cause posttransplant lymphoproliferative disease, and monitoring EBV viral load in transplant recipients can assist in assessing the risk and developing strategies for preemptive treatment.

DIFFERENTIAL DIAGNOSIS

Patients should also have a diagnostic evaluation for group I streptococcal infection by culture or antigen testing, the most common cause of acute bacterial pharyngitis.¹

REFERENCES

1. Infectious mononucleosis - UpToDate.

2. Leukocytic disorders. Henry’s Clinical Diagnosis and Management by Laboratory Methods, 24th edition.

3. Paediatric Infectious Disease. Cummings Otolaryngology: Head and Neck surgery, 7th edition.

4. Infectious mononucleosis. Oxford Handbook of Infectious Diseases and Microbiology, 2nd edition.

5. UK Standards for Microbiology Investigations – EBV serology.

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