Herpes simplex virus DNA testing by a loop-mediated isothermal amplification method for accurate clinical diagnosis and detection of mucosal viral shedding

Abstract

Herpes simplex virus (HSV) periodically forms characteristic blisters in the perioral and genital areas in a subset of people. Because of the lack of accurate tests for this common virus, various types of perioral/anogenital lesions are often misdiagnosed as herpes. Also, though asymptomatic HSV-positive people shed virus, the precise time course of symptoms and viral shedding is unclear. The loop-mediated isothermal amplification (LAMP) method amplifies target DNA sequences without thermal cycles, simpler and faster than polymerase chain reaction (PCR). To investigate clinico-laboratorial correlation and whether HSV can be detected in the oral cavity during symptom occurrence, we collected 445 specimens from 211 patients who visited our clinic with suspected herpetic lesions or non-symptomatic volunteers. DNA was extracted from swabs simultaneously taken from lesions (n = 219) and seemingly asymptomatic oral mucosa (n = 226). HSV-1 and HSV-2 DNA sequences were amplified by LAMP and validated by quantitative real-time PCR. The LAMP method detected HSV DNA almost as sensitively (97%) as PCR. Positivity for HSV DNA was found in 54% (40/74) of specimens from the perioral/oral area. Review of clinical images of recurrent herpes labialis revealed that HSV DNA was detected only from lesions located on the perioral skin and/or the dry, vermillion part of the lip; no HSV DNA was found in immunocompetent patients with lesions confined to the oral mucosa except primary infection. This observation may be an important principle for clinical diagnosis of recurrent herpes. HSV was detected in the oral mucosa in 2.7% (6/226) of samples; all of these patients had either primary infection or were immunosuppressed. Virus shedding in the mucosa was apparently tightly regulated by the immune system. Patients with suppressed or no immunity (naïve cases) did shed virus in the mucosa. LAMP is a simple method to reliably distinguish recurrent/primary herpes from other conditions.

Keywords: asymptomatic shedding; diagnostic test; herpes simplex; herpes simplex virus; loop-mediated isothermal amplification method.