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Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children.
Pediatr Infect Dis J. 2003 Aug; 22(8):736-9.PI

Abstract

BACKGROUND

Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples.

METHODS

A serum sample was available from patients classified serologically as primary EBV infection (n = 28), EBV-seronegative (n = 25) and EBV-seropositive (n = 26). Patients were classified as having EBV infectious mononucleosis if they had specified clinical findings and > or =10% atypical lymphocytes in peripheral blood or had a positive Monospot test result. DNA was purified by a spin column method and tested in PCR reactions with primers for EBV DNA polymerase gene and internal control targets. Amplification of the two PCR products was measured in real time with separate TaqMan DNA probes labeled with various fluorescent reporters.

RESULTS

The mean age of study patients was 9 years, 4 months. Twenty-one (75%) of the patients in the primary EBV infection group, one (4%) of the seronegatives and none of the seropositives had detectable EBV DNA. Within the primary infection group, those with detectable virus were more likely than those without detectable virus to have evidence of lymphadenopathy (14 of 16 vs.1 of 5; P = 0.011), higher mean atypical (11.7 vs.0.9%; P = 0.002) and absolute atypical (1.5 vs.0.1 x 109/l; P = 0.004) lymphocyte count, higher mean absolute lymphocyte count (4.7 vs.2.3 x 109/l; P = 0.026) and higher mean aspartate aminotransferase value (119.8 vs.37.3 IU/l; P = 0.036). Ten patients, all in the primary infection group, had EBV infectious mononucleosis, and all had positive PCR results. No sample contained PCR inhibitors.

CONCLUSIONS

A real time TaqMan PCR assay allows rapid identification of patients with primary EBV infection and those with EBV infectious mononucleosis.

Authors+Show Affiliations

Division of Pediatric Emergency Medicine, Department of Pediatrics, Children's Hospital of Pittsburgh/University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Evaluation Study
Journal Article

Language

eng

PubMed ID

12913777

Citation

Pitetti, Raymond D., et al. "Clinical Evaluation of a Quantitative Real Time Polymerase Chain Reaction Assay for Diagnosis of Primary Epstein-Barr Virus Infection in Children." The Pediatric Infectious Disease Journal, vol. 22, no. 8, 2003, pp. 736-9.
Pitetti RD, Laus S, Wadowsky RM. Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children. Pediatr Infect Dis J. 2003;22(8):736-9.
Pitetti, R. D., Laus, S., & Wadowsky, R. M. (2003). Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children. The Pediatric Infectious Disease Journal, 22(8), 736-9.
Pitetti RD, Laus S, Wadowsky RM. Clinical Evaluation of a Quantitative Real Time Polymerase Chain Reaction Assay for Diagnosis of Primary Epstein-Barr Virus Infection in Children. Pediatr Infect Dis J. 2003;22(8):736-9. PubMed PMID: 12913777.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children. AU - Pitetti,Raymond D, AU - Laus,Stella, AU - Wadowsky,Robert M, PY - 2003/8/13/pubmed PY - 2003/9/27/medline PY - 2003/8/13/entrez SP - 736 EP - 9 JF - The Pediatric infectious disease journal JO - Pediatr Infect Dis J VL - 22 IS - 8 N2 - BACKGROUND: Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples. METHODS: A serum sample was available from patients classified serologically as primary EBV infection (n = 28), EBV-seronegative (n = 25) and EBV-seropositive (n = 26). Patients were classified as having EBV infectious mononucleosis if they had specified clinical findings and > or =10% atypical lymphocytes in peripheral blood or had a positive Monospot test result. DNA was purified by a spin column method and tested in PCR reactions with primers for EBV DNA polymerase gene and internal control targets. Amplification of the two PCR products was measured in real time with separate TaqMan DNA probes labeled with various fluorescent reporters. RESULTS: The mean age of study patients was 9 years, 4 months. Twenty-one (75%) of the patients in the primary EBV infection group, one (4%) of the seronegatives and none of the seropositives had detectable EBV DNA. Within the primary infection group, those with detectable virus were more likely than those without detectable virus to have evidence of lymphadenopathy (14 of 16 vs.1 of 5; P = 0.011), higher mean atypical (11.7 vs.0.9%; P = 0.002) and absolute atypical (1.5 vs.0.1 x 109/l; P = 0.004) lymphocyte count, higher mean absolute lymphocyte count (4.7 vs.2.3 x 109/l; P = 0.026) and higher mean aspartate aminotransferase value (119.8 vs.37.3 IU/l; P = 0.036). Ten patients, all in the primary infection group, had EBV infectious mononucleosis, and all had positive PCR results. No sample contained PCR inhibitors. CONCLUSIONS: A real time TaqMan PCR assay allows rapid identification of patients with primary EBV infection and those with EBV infectious mononucleosis. SN - 0891-3668 UR - https://www.unboundmedicine.com/medline/citation/12913777/Clinical_evaluation_of_a_quantitative_real_time_polymerase_chain_reaction_assay_for_diagnosis_of_primary_Epstein_Barr_virus_infection_in_children_ L2 - https://doi.org/10.1097/01.inf.0000078157.90639.96 DB - PRIME DP - Unbound Medicine ER -