Detection of Herpesviridae in whole blood by multiplex PCR DNA-based microarray analysis after hematopoietic stem cell transplantation

Abstract

Viral infections are important causes of morbidity and mortality in patients after hematopoietic stem cell transplantation. The monitoring by PCR of Herpesviridae loads in blood samples has become a critical part of posttransplant follow-up, representing mounting costs for the laboratory. In this study, we assessed the clinical performance of the multiplex PCR DNA microarray Clart Entherpex kit for detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) as a screening test for virological follow-up. Two hundred fifty-five blood samples from 16 transplanted patients, prospectively tested by routine PCR assays, were analyzed by microarray. Routine PCR detected single or multiple viruses in 42% and 10% of the samples, respectively. Microarray detected single or multiple viruses in 34% and 18% of the samples, respectively. Microarray results correlated well with CMV and EBV detections by routine PCR (kappa tests = 0.79 and 0.78, respectively), whereas a weak correlation was observed with HHV-6 (0.43). HHV-7 was also detected in 48 samples by microarray. In conclusion, the microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV infections in blood. However, positive samples must be subsequently confirmed and viral loads must be quantified by PCR assays. Limitations were identified regarding HHV-6 detection. Although it is promising, is easy to use as a first-line test, and allows a reduction in the cost of analysis without undue delay in the reporting of the final quantitative result to the clinician, some characteristics of this microarray should be improved, particularly regarding quality control and the targeted virus panel, such that it could then be used as a routine test.

FIG 1

Comparison of qualitative PCR DNA microarray and quantitative real-time PCR results (CMV, n = 249; EBV, n = 244; HHV-6, n = 246). The viral loads (number of copies/ml of whole blood) are plotted on the y axis. The x axis indicates the positive (+) and negative (−) microarray results. The dashed lines represent the lower limits of quantification of PCR assays.

FIG 2

Microarray results for a sample with an HHV-6 viral load above 6 log₁₀ copies/ml of blood. (A) Undiluted sample. (B and C) Sample diluted 1:100 (B) and 1:1,000 (C) before the first multiplex PCR step of the microarray. (D and E) Sample diluted 1:100 (D) and 1:1,000 (E) before the hybridization step of the microarray.