Improved quantitative PCR protocols for adenovirus and CMV with an internal inhibition control system and automated nucleic acid isolation

• Published in: Journal of medical virology Vol. 84; no. 6; pp. 890 - 896
• Main Authors: Henke-Gendo, C., Ganzenmueller, T., Kluba, J., Harste, G., Raggub, L., Heim, A.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.06.2012; Wiley; Wiley Subscription Services, Inc
• Subjects: Adenovirus; Adenoviruses; Adenoviruses, Human - genetics; Adenoviruses, Human - isolation & purification; Automation - methods; Biological and medical sciences; Blood; Blood - virology; broncho-alveolar lavage fluid; Bronchoalveolar Lavage Fluid - virology; Cytomegalovirus; Cytomegalovirus - genetics; Cytomegalovirus - isolation & purification; DNA, Viral - isolation & purification; Feces - virology; Fundamental and applied biological sciences. Psychology; Human viral diseases; Humans; Infectious diseases; Medical sciences; Microbiology; Miscellaneous; Polymerase chain reaction; quantitation; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Reference Standards; Sensitivity and Specificity; Specimen Handling - methods; stool; TaqMan; Viral diseases; viral load; Viral Load - methods; Viral Load - standards; Virology; Human; stool; broncho-alveolar lavage fluid; Adenoviridae; quantitation; Taq-Man; Virus; Polymerase chain reaction; Isolation; Feces; Viral load; Nucleic acid; Quantitative analysis
• ISSN: 0146-6615; 1096-9071
• DOI: 10.1002/jmv.23285

With the establishment of routine virus load (DNAemia) screening for Human adenovirus (HAdV) and Cytomegalovirus (CMV) in post‐transplant care quality standards for quantitative PCR‐assays are increasing. Established real‐time PCR assays were improved with a fully automated DNA‐extraction and with a competitive internal control DNA packaged into a lambda phage which serves as an extraction and amplification control in each sample. HAdV and CMV DNA were detected and quantified simultaneously in various types of diagnostic samples like blood, feces or respiratory tract materials. Inhibition was observed in 0.33–0.66% of over 14,000 diagnostic samples, an infrequent but nevertheless not negligible event, which is observed mainly in stool samples. CMV viral load in broncho‐alveolar lavage fluid (BALF) ranged between positive but below the quantitation limit of 1,000 copies/ml up to 1.8 × 107 copies/ml with a median of 6.0 × 103 copies/ml. Forty‐one (4.7%) BALF samples had a viral load above 5.0 × 105 copies/ml, which was proposed as a threshold for the diagnosis of pneumonia. HAdV viral loads ranged between positive but below the quantitation limit of 1,000 copies/ml to a very high concentration of 1.3 × 1011 copies/ml in stool and BALF samples. A HAdV‐DNAemia of >104 copies/ml was found only in patients with stool viral load of above 105 copies/ml. These data support the hypothesis that quantitation in diagnostic materials other than blood may give valuable diagnostic information and that further evaluation of this approach is reasonable. J. Med. Virol. 84:890–896, 2012. © 2012 Wiley Periodicals, Inc.