Molecular detection of viral causes of encephalitis and meningitis in New York State

Abstract

The etiology of encephalitis and meningitis, serious diseases of the central nervous system (CNS), in most cases remains unknown. The importance of establishing a diagnosis however, becomes even more important as advances are made in effective therapy. Molecular methods of detection, in particular, PCR, are being used routinely and have established a place in the arsenal of tools for diagnosis of CNS infections. In this study a viral etiological agent was detected by PCR in 340 of the total 2,357 specimens from patients who exhibited symptoms of encephalitis or meningitis. The detection rate increased from 8.9% during the first year of the study to 14.8% during the second year of the study with improved methodology and an expanded panel of viral agents. Methods were enhanced by developing real-time PCR assays (some multiplexed), using increased automation, superior nucleic acid extraction, and reverse transcription (RT) methods, and incorporation of an internal extraction control. Additionally, adenovirus and human herpes virus 6 (HHV-6) were added to the original panel of 10 viruses that included enteroviruses, herpesviruses, and arboviruses. The most common viruses detected were enteroviruses (129; 5.5%), Epstein-Barr virus (EBV) (85; 3.6%), herpes simplex viruses (HSVs) 1 and 2 (67; 2.8%), and varicella zoster virus (VZV) (44; 1.9%).

Detection and Molecular Typing of Human Adenoviruses Associated with Respiratory Illnesses in Kerala

“…The viral DNA was eluted in 50 mL elution buffer. The extracted DNA was tested for adenovirus by real-time PCR using the published hexon gene based primers and probe (16) with the 2 × Platinum Quantitative PCR Supermix -UDG (Invitrogen, Carlsbad, CA, USA) in 20 mL reaction volume. Amplification was carried out at 959 C for 10 min of initial denaturation, followed by 40 cycles of 959 C for 15 sec and 609 C for 1 min in an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA).…”

Molecular Method Development and Establishment of a Database for Clinical and Epidemiological Herpes Simplex Virus 1 Strain Comparisons

“…Subsequently, a blinded clinical study for verification of the assay and analysis algorithm included the testing of residual portions of 147 samples (lesion, skin, oral, or genital swabs and cultured isolates) collected and received for viral diagnosis between 2004 and 2011. All samples were previously found to be positive for HSV-1 by real-time PCR (16) or conventional culture with confirmation and typing by immunofluorescent staining (MicroTrak HSV1/HSV2 Culture Identification/Typing Test; Trinity Biotech, Wicklow, Ireland). All samples were stored at Ϫ70°C after initial testing.…”

“…A nested PCR was performed with 1 l of the first-round product as the sample. If the nested PCR also failed to produce an amplicon, the presence of HSV-1 was checked by real-time PCR for HSV-1 (16). This occurred with five specimens in the study, and they were shown to be negative or to contain very low concentrations of HSV-1 DNA (data not shown).…”

Presence of Cross-Reactions with Other Viral Encephalitides in the Indirect Fluorescent-Antibody Test for Diagnosis of Rabies

“…A total of 135 CSF samples from viral encephalitis patients were tested with the rabies IFA assay. The sample set included 10 cases of Epstein-Barr virus (EBV), one of eastern equine encephalitis virus (EEEV), one of human herpesvirus 6 (HHV-6), one of HHV-6 plus enterovirus, four of enterovirus, six of herpes simplex virus 2 (HSV-2), and one of EBV plus varicella-zoster virus (VZV) infection, all confirmed by real-time PCR assays with CSF samples (6). Thirty CSF samples were from patients with serologically diagnosed West Nile virus infections and five CSF samples were from patients with serologically diagnosed Powassan virus encephalitis.…”