Comparison of In-House Real-Time Quantitative PCR to the Adenovirus R-Gene Kit for Determination of Adenovirus Load in Clinical Samples

Abstract

In the context of hematopoietic stem cell transplantation, adenovirus infections are associated with relevant mortality and morbidity. Detection of adenovirus DNA by quantitative PCR is the "gold standard" for these patients. A total of 150 samples, namely, 78 whole-blood, 22 cerebrospinal fluid, 24 digestive biopsy, and 26 stool samples, from 29 patients, including 24 hematopoietic stem cell transplant recipients, were tested for the detection of adenovirus using an in-house real-time quantitative PCR assay (A. Heim, C. Ebnet, G. Harste, and P. Pring-Akerblom, J. Med. Virol. 70:228-239, 2003) and the commercially available Adenovirus R-Gene kit. Adenovirus DNA was automatically isolated from whole-blood samples (Magna Pure LC system; Roche) or was manually extracted from other specimens (QIAamp; Qiagen) using the appropriate kit. The intra-and interassay reproducibilities and sensitivities were evaluated with cell culture supernatant dilutions. Of the 150 samples tested, 86 were found to be positive and 55 were found to be negative using both techniques. Nine (6%) discordant results were obtained. In most cases, discrepant results concerned samples with low viral loads. Quantitative results for all concordant positive samples were analyzed using the Spearman correlation test. A good correlation between the results of the in-house assay and those of the kit assay was obtained (r ‫؍‬ 0.95; P < 0.001). Regarding the threshold cycle value for internal control spiked samples, none of the 150 samples tested contained a PCR inhibitor. In conclusion, a relevant correlation of results between the in-house assay and the kit assay, as well as the high-quality reproducibility and sensitivity of the kit assay, warranted its use for follow-up of hematopoietic stem cell transplantation recipients.

Adenovirus Infections in Immunocompetent and Immunocompromised Patients

“…The monitoring of patients in the immunocompromised setting requires tests permitting reliable detection of all potentially relevant HAdV types in different clinical specimens, as well as accurate quantitative assessment of viral loads. Among the currently available commercial kits, this requirement appears to be met by the Adenovirus R-gene kit (199), and possibly the ELITe MGB kit, although the latter seems to be approved for wholeblood analysis only.…”

“…Complete HAdV genome sequences are now available in public databases, thereby permitting at least in silico control of the primers and probes used to ensure coverage of any newly identified HAdV type by the assay used. However, in view of the clinical importance of adenoviral infections, the availability of commercial, regularly updated diagnostic tests (199,267) is highly desirable to facilitate standardized diagnostics under stringent quality control conditions. Commercial assays should permit pan-HAdV screening, quantification of viral loads, and, ideally, typing at least to the species level in order to provide a basis for more specifically targeted treatment approaches (251,268).…”

How I treat adenovirus in hematopoietic stem cell transplant recipients

“…The quality of these tests is similar and can be assured by joining the quality control for molecular diagnostics panel testing. 18,19 Panels with 3 concentrations of DNA are sent to different laboratories; results are submitted, analyzed, and compared; and the quality is reported to the originating laboratories. 18 In some cases, AdV can be detected significantly earlier at local sites, such as in stool or nasopharyngeal aspirate (NPA).…”

“…9 (81) 5 (46) CDV 1 mg/kg intravenously 3 times a week, for 3 weeks 3 (19) 12 (75) 2 (13) Listed are all clinical studies that used cidofovir in patients who received HSCT either for preemptive treatment when symptoms are not yet present or for therapeutic treatment when disease symptoms are present. Only studies with n Ͼ 5 were presented.…”

Adenovirus infections in Bordeaux University Hospital 2008–2010: Clinical and virological features

“…32,33 Using this method, our laboratory passed the quality control for molecular diagnostics (QCMD) HAdV 2011 panel. Results were expressed as either DNA copies/ml (in whole blood, bronchoalveolar lavage fluids and urine), DNA copies/g stools or DNA copies g −1 DNA (respiratory swabs, upper respiratory secretions and biopsies).…”