汤一苇教授：中枢神经系统病毒感染的分子诊断和监测
注：文字内容来自会议光盘
Departments of Pathology and Medicine, Vanderbilt University Medical Center, Nashville,Tennessee 37232-5310, USA
Yi-Wei Tang
Emergence of PCR-led molecular techniques has initiated a revolution in the field of diagnosticmicrobiology. With its speed and high molecular sensitivity, PCR, along with its derivatives such asreverse transcriptase (RT)-PCR, multiplex PCR, broad-range PCR, and real-time PCR, has great potentialfor the diagnosis and monitoring of central nervous system (CNS) infections caused by viral pathogens. Inaddition to PCR, nucleic acid sequence-based amplification method has been reported for enterovirusdetection with good sensitivity and specificity.
Viral loads in CNS are usually low, any techniques that can enhance the test sensitivity while keepingthe specificity is desirable. We have been using a colorimetric microtiter plate PCR system (PCR-EIA) todetect a panel of pathogens in cerebrospinal fluid (CSF). This system combines a target amplification in thePCR step and an antigen-antibody signal amplification in the EIA step, which significantly improves theanalytical sensitivity to 10-20 copies/reaction for herpesvirus and enterovirus detection in CSF. Anotherrecently reported system is an Invader Plus method (Third Wave Technologies, Madison, WI) for thequalitative detection and differentiation of HSV-1 and HSV-2 in CSF. The method combines PCR targetamplification and Invader technique signal amplification in a single, closed-tube, continuous-reactionformat, giving an analytical sensitivity of approximately 10 copies per reaction. The GeneXpert Dx system(Cepheid, Sunnyvale, CA) is a fully integrated and automated nucleic acid sample preparation,amplification, and real-time detection system. The system-based enterovirus assay, which is used toprovide rapid and on-demand diagnosis of aseptic meningitis, has recently received the US FDA approval.The complete automation and rapid-result capability of the enterovirus assay make it uniquely suited forurgent and stat testing. A duplex real-time TaqMan PCR device has been developed by TrimGen (Sparks,MD) to simultaneously detect and differentiate HSV and enteroviruses in one tube. The system provides arapid and sensitive procedure by enhancing the 5’-exonuclease activity owned by Taq polymerase. Thisassay is predicted very useful in summer season as an urgent testing when both HSV and enterovirusinfections are on the differential diagnostic list.
Detection of several viral pathogens in CSF by PCR-based assays has become the first line diagnostictool, especially for herpesviruses and enteroviruses. Epstein-Barr virus (EBV) DNA can be used as a tumormarker of lymphoma of the CNS with excellent sensitivity and specificity. PCR of CSF is more reliablethan clinical features in the diagnosis of CMV-related neurological disease in patients with AIDS. PCRanalysis allows recognition of both overt VZV disease of the CNS and subclinical reactivation of VZVinfection in HIV-infected patients. Molecular assay allows rapid detection of enterovirus RNA in CSF withexcellent sensitivity and specificity and significant shortness of hospital stay. Detection of JC virus DNA inCSF has been proven to be both sensitive and specific for the diagnosis of progressive multifocalleukoencephalopathy, a demyelinating disease of the CNS seen primarily in AIDS patients. RT-PCR hasbeen used for the diagnosis of mumps virus CNS disease, and may soon become the “gold standard” testfor the diagnosis of this condition. On the other hand, PCR methods have been approved complementary toother laboratory methods. While IgM serology is accepted as the test of choice for West Nile encephalitis, areal-time PCR method used to analyze specimens obtained during the 1999 New York outbreak detectedthe presence of West Nile virus sequences in CSF of all 4 individuals with fatal outcomes, and in one offour who survived. PCR assays have been useful to diagnose HHV-8 related lymphomas in HIV ornon-HIV patients in CNS.
Both qualitative and quantitative molecular methods have been used in therapy efficacy evaluation andprognosis prediction. PCR analysis for HSV DNA in CSF represents the method of choice for the diagnosisand follow-up of treatment of herpes encephalitis in immunocompetent patients. Quantitative PCR can beused to monitor the response to treatment or progression of disease. The level of both measles viral RNAand antigen in the brain of subacute sclerosing panencephalittis (SSPE) correlate with disease progressionand measles viral loads may be used for disease monitoring. Quantitation of human herpesvirus-6 genomein CSF may help evaluate the viral replication level and the implication of disease. Molecular assays havebeen useful to detect and confirm viral coinfections in CNS. Tropical spastic paraparesis/human T leukemiavirus type 1-associated myelopathy has been reported in HIV-1 coinfected patients.
olecular methods have emerged to detect antimicrobial-drug resistance in clinical settings and havesubstantially contributed to our understanding of the spread and genetics of resistance. These assays mayoffer advantages over phenotypic assays. PCR followed by direct sequencing has been used to detect themutations existed in the thymidine kinase gene of HSV and phosphotransferase gene of CMV, which arerelated to acyclovir- and ganciclovir-resistances respectively. A similar technique has been used to detectthe mutations in the thymidine kinase gene of HSV and phosphotransferase gene of CMV, which arerelated to acyclovir- and ganciclovir-resistances respectively. There is considerable interest in theapplication of molecular methods directly to clinical specimens to provide a more rapid means ofidentification of resistance, resulting in improved patient outcome and reductions in overall costs.Three CNS disease cases, caused by enteroviruses, HIV-1 and cytomegalovirus respectively, will bepresented to demonstrate molecular diagnosis and monitoring of viral diseases in laboratories.
编辑: yang 作者:汤一苇
以下网友留言只代表网友个人观点，不代表网站观点
