Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients

Abstract

A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.

FIG. 1.

Standard curves of the RQ-PCR virus assays described. Serial logarithmic dilutions were analyzed by using standard amplification conditions. The C_t (x axis) of each of the dilutions is plotted against the cycle number (y axis). The slope, y-axis intercept (Y-Inter.), and correlation coefficient are displayed in each graph. HSV 1, herpes simplex virus type 1; B19, PVB19; JCV, JC virus; EV, enterovirus; RSV, respiratory syncytial virus; Inf A, influenza A virus; PIV 1, parainfluenza virus type 1.

FIG. 1.

Standard curves of the RQ-PCR virus assays described. Serial logarithmic dilutions were analyzed by using standard amplification conditions. The C_t (x axis) of each of the dilutions is plotted against the cycle number (y axis). The slope, y-axis intercept (Y-Inter.), and correlation coefficient are displayed in each graph. HSV 1, herpes simplex virus type 1; B19, PVB19; JCV, JC virus; EV, enterovirus; RSV, respiratory syncytial virus; Inf A, influenza A virus; PIV 1, parainfluenza virus type 1.

FIG. 1.

Standard curves of the RQ-PCR virus assays described. Serial logarithmic dilutions were analyzed by using standard amplification conditions. The C_t (x axis) of each of the dilutions is plotted against the cycle number (y axis). The slope, y-axis intercept (Y-Inter.), and correlation coefficient are displayed in each graph. HSV 1, herpes simplex virus type 1; B19, PVB19; JCV, JC virus; EV, enterovirus; RSV, respiratory syncytial virus; Inf A, influenza A virus; PIV 1, parainfluenza virus type 1.

FIG. 1.

Standard curves of the RQ-PCR virus assays described. Serial logarithmic dilutions were analyzed by using standard amplification conditions. The C_t (x axis) of each of the dilutions is plotted against the cycle number (y axis). The slope, y-axis intercept (Y-Inter.), and correlation coefficient are displayed in each graph. HSV 1, herpes simplex virus type 1; B19, PVB19; JCV, JC virus; EV, enterovirus; RSV, respiratory syncytial virus; Inf A, influenza A virus; PIV 1, parainfluenza virus type 1.

FIG. 1.

Standard curves of the RQ-PCR virus assays described. Serial logarithmic dilutions were analyzed by using standard amplification conditions. The C_t (x axis) of each of the dilutions is plotted against the cycle number (y axis). The slope, y-axis intercept (Y-Inter.), and correlation coefficient are displayed in each graph. HSV 1, herpes simplex virus type 1; B19, PVB19; JCV, JC virus; EV, enterovirus; RSV, respiratory syncytial virus; Inf A, influenza A virus; PIV 1, parainfluenza virus type 1.

FIG. 2.

Kinetics of BKV load during hemorrhagic cystitis. Documentation of BKV infection of the urinary bladder and clearance of the virus by serial RQ-PCR analysis of urine samples during the posttransplant period. The virus load (y axis) is plotted against the time after bone marrow transplantation (BMT) (x axis).

FIG. 3.

Kinetics of EBV load in PB in a posttransplant lymphoproliferative disease. Serial RQ-PCR analysis documents the reactivation of a latent EBV infection by revealing constantly increasing virus copy numbers. This retrospective analysis of virus proliferation kinetics, which heralded the development of EBV-associated malignant lymphoma, underlines the potential of molecular detection and monitoring of EBV load to provide a basis for early initiation of preemptive antiviral treatment. BMT, bone marrow transplantation.

FIG. 4.

Kinetics of AdV load in PB. The curve shows the appearance and expansion of AdV in PB. Observation of the first 10-fold increase in virus load (arrow) preceded the onset of clinical symptoms (star) by more than 3 weeks. The monitoring of AdV in PB may therefore serve as a basis for early initiation of preemptive antiviral treatment. BMT, bone marrow transplantation.

FIG. 5.

Kinetics of CMV load in PB in response to antiviral treatment. Monitoring of CMV by RQ-PCR in serial plasma (solid line) and PB leukocyte (dashed line) samples during the posttransplant period reveals viral reactivation by rising levels of CMV DNAemia and subsequent clearance of the virus following antiviral therapy. BMT, bone marrow transplantation.