Rapid diagnosis of herpes simplex encephalitis by nested polymerase chain reaction assay of cerebrospinal fluid.

With the aim of improving early diagnosis of herpes simplex encephalitis a polymerase chain reaction (PCR) assay with two "nested" primer pairs was developed for the amplification of herpes simplex virus DNA in cerebrospinal fluid (CSF). Southern blotting was used to confirm the specificity of the amplification. The assay was applied to 151 CSF samples from 43 consecutive patients with herpes simplex encephalitis verified by the finding of herpes simplex virus/viral antigen in a brain biopsy sample or at necropsy (13) and/or intrathecal production of IgG antibody to the virus (40). As controls, 87 CSF samples from 60 patients with acute febrile focal encephalopathy (initially suspected to be herpes simplex encephalitis but excluded by the absence of intrathecal antibody synthesis) were tested. PCR detected herpes simplex virus DNA in 42 of the 43 patients with proven herpes simplex encephalitis; all but 1 were positive in the first CSF sample taken. The 1 PCR-negative patient had been treated with acyclovir from 20 h after the onset of symptoms. All the control subjects were PCR negative, as were 270 internal contamination controls. The PCR result remained positive in samples drawn up to 27 days after the onset of neurological symptoms. This method is a rapid and non-invasive means to diagnose herpes simplex encephalitis; it is highly sensitive and specific.

CMV PCR monitoring in leucocytes of transplant patients.

BACKGROUND: The presumed latency of cytomegalovirus (CMV) in leucocytes and the sensitivity of the polymerase chain reaction (PCR) raise a question of its clinical value. OBJECTIVES: To develop and standardize a CMV PCR as a diagnostic tool for CMV infection in solid organ and bone marrow transplant patients by comparing it to a likewise standardized isolation, rapid isolation and to clinical symptoms. STUDY DESIGN: The material comprised 822 EDTA peripheral blood samples from 96 solid organ and 119 bone marrow transplant patients. One sample from each of 21 healthy bone marrow donors and 25 blood donors were used as controls. Two million leucocytes were lysed and one-tenth of a volume was used in a nested PCR employing immediate early gene primers. RESULTS: The limit of detection was approximately 10 gene copies of a CMV DNA clone and 1 TCID(50) of extracted DNA from a cell suspension. The specificity was >/=0.99 when tested in CMV seronegative individuals. The positive and negative predictive values were 0.62 and 1.00, respectively. When PCR was compared to virus isolation/rapid culture in individual patients, PCR was positive more frequently in solid organ transplant patients than was CMV isolation/rapid culture, but the difference was not significant in bone marrow transplant patients. In isolation-positive patients, PCR became positive in samples taken 1-2 weeks earlier. In 54 solid organ transplant patients with PCR-positive samples, CMV-associated symptoms were present in 29/31 patients with CMV isolated from blood but in only 5/23 patients without viraemia. In 17 bone marrow transplant patients treated with ganciclovir, PCR became negative during or immediately after treatment in 14/20 (70%) episodes. This was true of 5/12 (42%) solid organ transplant patients. CONCLUSION: Screening of transplant patients with CMV PCR can be standardized at a clinically relevant level so that antiviral therapy can be instituted early.