We created a multiplex, quantitative, real-time PCR assay that amplifies cytomegalovirus (CMV) and human DNA in the same reaction tube, allowing for a viral load determination that is normalized to measured human DNA. The assay targets a conserved region of the CMV DNA polymerase gene that is not affected by known drug resistance mutations. All 36 strains of CMV detected by culture or qualitative PCR in a population of lung transplant recipients were detected. The assay detected 1 to 10 copies of CMV plasmid DNA. The analytic sensitivity was not affected by the presence of DNA from 10 6 human cells but was reduced approximately 10-fold by alkaline lysates of leukocyte preparations. CMV quantitation was linear over a range of 10 1 to 10 6 copies. The intraassay and interassay coefficients of variation were 29 and 40%. Human DNA was regularly detected in patient plasma samples, and the amount was increased by storage of blood at room temperature before plasma separation and by plasma separation techniques that allowed leukocyte contamination. Applied to whole blood, the assay provides a measurement of CMV DNA in relation to cellular content without a need for cell counting procedures. Applied to plasma, the assay can reveal artifactual increases in plasma CMV levels resulting from leukocyte contamination. Further study of the utility of this assay to monitor patient populations at risk for CMV disease is warranted.Cytomegalovirus (CMV) continues to be an important cause of morbidity and occasional mortality in immunocompromised patients. While the impact of CMV has been lessened by the use of ganciclovir, concerns including drug toxicity, cost, and drug resistance have stimulated efforts to find the most efficient strategies for using the drug, some of which involve using a diagnostic test to monitor for infection (15). The ideal diagnostic test should be sufficiently sensitive to detect infection at an early stage before clinically significant disease has occurred, but not so sensitive that only a small proportion of patients with a positive test would actually develop disease. Quantitative nucleic acid detection assays are the most likely to be useful in this way because they combine inherent analytic sensitivity with the ability to define threshold levels that could be used to initiate treatment. A number of studies have shown relationships between the level of CMV DNA in blood and the likelihood of disease (2, 5, 7, 10-13, 17, 20, 26). The stability of DNA is an additional advantage that is important because specimens may have to be transported before testing (18, 21).The development of real-time PCR technology has simplified nucleic acid quantification and is coming into more widespread use. Real-time, quantitative PCR has several advantages over older forms of quantitative PCR assays. Experience to date has reported comparable sensitivity but superior reproducibility and precision compared to previous methods, with a wide dynamic range (14, 19). The fluorescence-based real-time assays also allow for multiple...
We developed a multiplex, quantitative, real-time, polymerase chain reaction assay for cytomegalovirus (CMV) and used it to measure the CMV viral load in weekly blood specimens from 43 lung transplant recipients. The median viral load in blood samples immediately preceding bronchoscopy was 1150 copies/microg human DNA for 12 subjects with pneumonitis compared to 91 copies for 31 subjects without (P=0.02, Mann-Whitney U test). Each log10 increase in CMV viral load resulted in an increase of 1.92 in the odds ratio for CMV pneumonitis (95% confidence interval 1.03-3.56). CMV viral load was elevated (>100 copies/microg human DNA) for a median of 21 days before bronchoscopy in those subjects with pneumonitis versus 0 days in those without (P=0.004). We conclude that the risk of CMV pneumonitis after lung transplantation is related to the level of CMV DNA in blood. Quantitative PCR should be evaluated prospectively for the preemptive management of CMV in lung transplant recipients.
Correspondence 625cultures have ∼85%-95% yield, the latter procedure appears to be superior and the best choice for effective therapy. We neglected to mention the amylase level of our patient in our report; it was 22 U (normal range, 15-125 U), which suggested that our patient's ascites was not the result of pancreatitis, pancreatic pseudocyst, mesenteric vein thrombosis, pancreatic and some other nonpancreatic neoplasms.We look forward to the use of PCR technology and newer biomolecular probes, which should provide us with more sensitive and specific tests to diagnose tuberculous peritonitis, which is the 6th most common manifestation of extrapulmonary tuberculosis. References 1. Jang SS, Hansen LM, Breher JE, et al. Antimicrobial susceptibilities of equine isolates of Clostridium difficile and molecular characterization of metronidazole-resistant strains.
The incidence of Clostridium difficile and its cytotoxic activity were determined in the feces of 122 children under 1 year of age. Samples were obtained from children receiving antibiotics and with (52 cases) or without (26 cases) diarrhea, from children with diarrhea who did not receive antibiotics (22 cases), and from healthy children (22 cases). Isolation of C. difficile in feces from children in all groups was similar (mean 23.4%) except for the group with non-antibiotic-associated diarrhea (4.5%). In both groups of children receiving antibiotics, with or without diarrhea, the cytotoxin was detected in 7.6% of the cases. In the group with non-antibioticassociated diarrhea, none of the samples was positive for cytotoxicity. In healthy children, cytotoxin was positive in 4.5% of the cases.
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