Persing, D. H.

A total of 60 cerebrospinal fluid (CSF) specimens from patients manifesting symptoms resembling viral central nervous system (CNS) disease were examined for the presence of herpes simplex virus (HSV), human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), cytomegalovirus, varicella-zoster virus, Borrelia burgdorferi, and Tropheryma whippelii DNA by PCR. Of 30 specimens which were selected on the basis of HSV DNA positivity, 2 were concomitantly positive for HHV-6 DNA and 1 was positive for EBV DNA. In the three specimens positive for more than one herpesvirus, amplicons generated with virus-specific primer sets hybridized specifically to the corresponding virus-specific probe. Sequence analysis of the two amplified DNA fragments demonstrated that they were derived from distinct herpesviruses. Of 22 patients with clinically diagnosed encephalitis, 2 of 3 patients coinfected with HSV and HHV-6 died, compared to 1 of 19 (5%) patients infected with only HSV. Of 30 CSF specimens that were negative for HSV DNA, EBV DNA was detected in one sample. These data indicated the presence of DNA specific for two distinct herpesviruses in the same CSF specimen, providing molecular evidence that coinfection with this group of viruses may occur in the CNS.

False-positive results because of carryover contamination by previously amplified nucleic acids are currently the greatest impediment to routine implementation of nucleic acid amplification protocols. We evaluated three methods for inactivation of a 156-bp Borrelia burgdorferi polymerase chain reaction (PCR) product: (i) post-PCR cross-linking with isopsoralen (IP), (ii) pre-PCR treatment of a dU-containing PCR product with uracil N-glycosylase (UNG), and (iii) post-PCR alkaline hydrolysis (primer hydrolysis) of PCR products synthesized by using primers containing 3' ribose residues. The sensitivities of the PCR performed under the conditions of each protocol were comparable. Inactivation of amplified DNA was highly efficient for all three protocols; the IP and UNG protocols eliminated at least to 3 x 10(9) copies of the product. The primer hydrolysis protocol varied in efficiency depending on the number and position of the 3' ribose residues, but inactivation ranged from 10(4) to 10(9) copies. We conclude that with some modifications, all three systems are effective for eliminating amplified DNA products. Routine implementation of at least one method should help to avoid false-positive results because of carryover contamination.

We describe a novel procedure for direct covalent coupling of horseradish peroxidase to rRNA and ribotyping by using enhanced chemiluminescence. Compared with their 32P-end-labeled counterparts, chemiluminescent rRNA probes are stable and easy to synthesize and provide results as good as or superior to those obtained with isotopic labeling. Direct chemiluminescent labeling of Escherichia coli rRNA produces a sensitive, universal probe suitable for clinical laboratory use in the investigation of nosocomial outbreaks.

ABSTRACT A branched-DNA (bDNA) signal amplification method was used to detect the mecA gene directly from blood culture broth growing staphylococci. BACTEC blood culture bottles with positive growth indices and containing staphylococcus-like organisms as shown by Gram stain were tested for the presence of the mecA gene. Comparison of test results was done among 225 patients (one blood culture from each patient). Compared with PCR, the sensitivity and specificity of the bDNA method are 100 and 99%, respectively. The bDNA test is carried out in a 96-well format and requires approximately 6 h to perform. Our preliminary results suggest that direct detection of the mecA gene by bDNA signal amplification is (i) sensitive enough to detect mecA directly from blood culture bottles without the requirement for subculture and (ii) as sensitive and specific as the PCR-based method.