Current Laboratory Detection Methods of Herpes Simplex Virus

Magdalena Lewicka

48980

Presentation Plan
1. Introduction 2. HSV Clinical Features and Presentation 3. Laboratory Diagnosis 4. Conclusion

1. Introduction

Localisation of infection: - orofacial skin - genital skin - eye - meninges - brain - visceral organs of nevborn Symproms: - vesicles, ulcers, lesion changes on the skin - aroudn half of cases is asymptomatic

2. HSV Clinical Features and Presentation

Family:

Herpesviridae

Subfamily: Alphaherpesvirinae Genus :

Simplexvirus

HSV consists of enveloped double stranded DNA After primary infection by person-to-person contact HSV is capable to establish letancy in the host withsubsequent reactivation and recurrent disease.

2. HSV Clinical Features and Presentation

Two serotypes: HSV-1 and HSV-2: both: infect squamous epithelium are transmissed by direct contact with Infected epidermal or mucosal surface have unique antigenic profiles and distinct epidemiology and clinical disease patterns

2. HSV Clinical Features and Presentation

HSV - 1 age of primary infection transmission sites of infection latency site CNS infections neonatal infections children oral secretions mouth, lips, eyes, fingers trigeminal ganglion cause of most encephalitis rare, limited to skin

HSV - 2 teens, adults intimate sexual contact Genital sites, mouth, lips, fingers sacral autonomic ganglion cause of Mollaret's recurent lymphocytic meningitis cause of majority of infections

3. Laboratory Diagnosis

3.1. Direct Smear 3.2. Virus Culture 3.3. Nucleic Acid Amplification Assay 3.4. Serologic Diagnosis

3. Laboratory Diagnosis 3.1. Direct Smear

Examination of vesicle fluid in search of Tznack cells. Wright Giemsa staining of intracellular inclusions and fused, multinucleated squamous epithelial cells.

rapid, easy, inexpensive

requires intact infected cells with distinct inclusions or fused giant cells Wright-Giemsa stained Tznack cells ~60% sensitivity with vesicle lesions very poor sensitivity with ulcer lesions positive result also in case of other infections (varicella-zoster virus, chickenpox, and shingles)

3. Laboratory Diagnosis 3.1. Direct Smear

Diresc Fluorescent Antibody (DFA) stain
sensitivity varies with stage of lesions: 85% vesicles 60% pustules 20% ulcers <10% crusted lesions monoclonal antibody can be used to differentiate HVP-1 and HVP-2

Immunohistochemistry - immunoperoxidase stain

performed on formaline fixed, paraffine embadded biopsy tissue sections to detect HSV antigen (antibodies are visualized via a peroxidase-catalyzed reaction) in situ hybridization can also identify HSV DNA in tissue biopsy specimens

3. Laboratory Diagnosis 3.2. virus culture

Swab samples of vesicles or ulcers substance are collected to viral transport medium with antibiotics and can be refrigerated overnight. Cell lines for virus proliferation: human fibroblast, rabbit kidney, mink lung etc.

HSV grows rapidly, cytophatic effect (CPE) is usually apparent after 1-3 days of incubation and includes balooning degradation, swelling and rounding changes Daily, microscopic examination of samples practical gold standard CPE suspisious for HSV should be confirmed with DFA stain of cells Cell lines may support growth of other viruses

3. Laboratory Diagnosis 3.2. virus culture

Shell vial culture
The specimen inoculated by centrifugation onto the cell surface, and after a 24-h incubation, virus in the monolayer cells is identified by DFA staining. more rapid than tube culture positive and negative results in 1-2 days

3. Laboratory Diagnosis 3.2. virus culture

Enzyme Linkec Virus Incubation System (ELVIS) Culture
Genetically modified baby hamster kidney cells produces galactosidase only in cells with HSV growth. Enzymes activity is detected by colorimetric step.

easy to perform and interpret reliable result in 24 h commercially available cell line mix viable viruses required

3. Laboratory Diagnosis
3.3. Nucleic Acid Amplification Assay PCR
identifies HSV in any kind of specimen HSV DNA extraction, amplification and detection steps can be accomplished in a few hours HSV-1 and HSV-2 easily differentiated by melting-curve (Tm) analysis

3. Laboratory Diagnosis
3.3. Nucleic Acid Amplification Assay PCR
single, closed reactional vessel lower risk of contamination is not dependent on viability automated user-friendly methods under development cost competitive with culture

new gold standard for detection of HSV

3. Laboratory Diagnosis
3.4. Serologic Diagnosis

In primary infections antibodies appear within 4-7 days after infection and reach a peak at 2-4 weeks.

Antibody to common antigens Enzyme immunoassay (EIA) Immunofluorescence assay (IFA) Easy to perform Cannot separate HSV-1 and HSV-2 antibodies Heterotypic antibody response to other herpesviruses common (HCV-1, HSV2, varicella-zoster virus)

3. Laboratory Diagnosis
3.4. Serologic Diagnosis

HSV glycoprotein gG antibody EIA with unique gG HSV-1 and HSV-2 antigens Specific HSV-1 and HSV-2 antibody detection Easy to perform Expensive Detection of IgG antibody in partner of pregnant seronegative women predicts risk of neonatal HSV Immunoblot assay Detects antibodies to specific HSV-1 and HSV-2 Viral antigens separated by electrophoresis Serologic gold standard for differentiating HSV-1 from HSV-2 infection

4. Conclusion

Tradition methods: Direct smear Virus culture easy, cheap, time consuming

New methods: PCR immunoassay improved specificity and accuracy especially useful in asymptomatic cases

Thank you for your atention :)

Bibliography

Harpes simplex virus infections and current methods for laboratory detection - Michael Costello M.T.
(ASCP), Ph.D.,1 Linda Sabatini, Ph.D., HCLD,1 and Peggy Yungbluth, M.D.,1,2 1ACL Laboratories, Rosemont, Illinois, 2Department of Pathology, St. Francis Hospital, Evanston, Illinois

Comparison of Western blot (immunoblot) and glycoproteinG-specific immunodot assay for detecting antibodies to herpes simplex virus types 1 and 2 in human sera. Ashley, R.L. et al. 1988. J.
Clin. Microbiol. 26:662-667