Real-time automated PCR for early diagnosis and monitoring of cytomegalovirus infection after bone marrow transplantation

Abstract

The purpose of this study was to assess the usefulness of real-time automated PCR as a quantitative, highly reproducible, and sensitive method to detect cytomegalovirus (CMV) DNA in blood specimens. Intra- and interassay precision rates were 0.89% (small number of copies [L]), 1.43% (middle number of copies [M]), and 1.12% (high number of copies [H]), and 4.46% (L), 1.51% (M), and 2.28% (H), respectively. The linearity of this assay was obtained between 10 and 10(7) copies/well, with a minimum detection limit of 20 copies/well. Specimens from 55 of 70 healthy subjects were found to be positive for CMV antibody, but CMV DNA was not detected in any of them. In the qualitative assessment of each specimen, the results of the CMV antigenemia assay and those of the real-time PCR assay agreed in 80% (plasma specimens), 79% (all nucleated cells), and 86% (blood) of the cases examined. For eight patients diagnosed as having CMV infection or disease, no sample was positive in the antigenemia assay earlier than in the real-time PCR assay. Furthermore, the results of this assay could be obtained within 8 h. We concluded that the real-time PCR assay is useful for rapid diagnosis of CMV infection and monitoring of clinical courses.

FIG. 1

(A) Amplification profile of standards for CMV real-time PCR. Standard DNA at 10⁷ (solid circles), 10⁶ (open circles), 10⁵ (solid squares), 10⁴ (open squares), 10³ (solid triangles), 10² (open triangles), and 10 (solid diamonds) copies/well in duplicate was amplified for 50 cycles. (B) Standard curve for CMV real-time PCR. C_t values were plotted against various numbers of copies of standard DNA. The slope was −3.54 cycles/log decade. The correlation coefficient was 0.988.

FIG. 2

Association between maximum level of CMV antigenemia and maximum level of CMV DNA as determined by real-time PCR for each patient. There was a statistically significant correlation between the CMV antigenemia assay and ANC PCR (correlation coefficient, 0.873; P < 0.001) and between the CMV antigenemia assay and blood PCR.

FIG. 3

Clinical course of patients UPN85 and UPN93. (A) Patient UPN85 became positive in the shell-vial culture of peripheral blood. She also developed Aspergillus tracheobronchitis concomitantly under long-term steroid therapy for the treatment of acute GVHD and died of it on day 176. + and −, positive and negative in shell-vial culture, respectively. Open circles, white blood cells (WBC); solid circles, ANC; solid squares, plasma; solid triangles, whole blood. Bars and numbers show the number of CMV detected by the antigenemia assay. (B) Patient UPN93 died of grade III acute GVHD on day 60. Both patients responded well to ganciclovir. Results of real-time PCR for CMV accurately reflect their responses to the anti-CMV treatment.