The new millennium laboratory: molecular diagnostics goes clinical.

Applications of nucleic acid testing in most areas of the clinical laboratory have increased rapidly. The advantages of nucleic acid testing include enhanced specificity and sensitivity, ease of sample procurement, and more rapid turnaround time compared to conventional laboratory testing methods. However, the cost of testing is usually higher due to the need for additional laboratory space, specialized equipment, safety apparel, and the need for highly trained personnel. Most nucleic acid techniques currently used in a clinical setting can be categorized as either hybridization or amplification assays. Hybridization assays, including blotting techniques and microarrays, involve the complementary binding of an oligonucleotide probe of known DNA sequence with nucleic acid derived from the patient sample. To amplify small amounts of nucleic acid, assays such as the polymerase chain reaction and branched chain DNA employ either signal amplification or exponential amplification of target nucleic acid. Clinical applications of nucleic acid testing involve the detection of genetic diseases, e.g., sickle cell anemia and Huntington disease; and identification of infectious agents, e.g., HCV and HIV; or malignancies, e.g., chronic myelogenous leukemia and Burkitt lymphoma. Quantitative molecular assays also play important roles in predicting prognosis and monitoring responsiveness to therapy.

Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure.

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic-acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic-acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay.

Clinical utility of a quantitative polymerase chain reaction for diagnosis of cytomegalovirus disease in solid organ transplant patients.

BACKGROUND: Accurate and rapid diagnosis of human cytomegalovirus (HCMV) disease in solid organ transplant patients remains a challenge. We evaluated the clinical utility of a quantitative polymerase chain reaction (QPCR) method to diagnose transplant patients with HCMV disease. METHODS: A total of 429 plasma samples from 121 solid organ transplant patients were prospectively collected and evaluated for HCMV using a QPCR assay. To enhance the sensitivity of the QPCR assay, plasma samples were centrifuged in a manner designed to concentrate the virions before nucleic acid extraction. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) that contained the same primer sequences as for HCMV. Polymerase chain reaction products were detected by hybridization to 96-well microtiter plates coated with either a HCMV- or an IS-specific probe. RESULTS: A total of 103 patients had all samples negative by QPCR. None of the 103 patients developed HCMV disease during the study. In contrast, 18 patients showed at least 1 sample positive by the QPCR assay, but only 8 of these developed HCMV disease. The mean viral load value for patients without HCMV disease was 93 viral copies (vc) per ml of plasma (range: 35-325 vc/ml plasma) and for the 8 patients with HCMV disease was 67,686 vc/ml plasma (range: 167-1,325,000 vc/ml plasma) (P<0.001). Using a cut-off value of 100 vc/ml plasma and clinical diagnosis of HCMV disease, the QPCR assay showed a sensitivity of 100% and specificity of 99.1%. CONCLUSION: HCMV viral load may be useful in the diagnosis of HCMV disease in solid organ transplant patients.