Varicella Zoster Virus lLaboratory Case Definition (LCD)

The Public Health Laboratory Network have developed a standard case definition for the diagnosis of diseases which are notifiable in Australia. This page contains the laboratory case definition for Varicella zoster virus

Page last updated: 12 April 2010

Authorisation: PHLN

Consensus Date: 24 December 2009

1 PHLN summary laboratory definition

1.1 Condition:

Chicken pox (Varicella); Shingles (Herpes Zoster)

1.1.1 Definitive Criteria

Varicella zoster infections (chickenpox, herpes zoster) are usually diagnosed clinically, but laboratory diagnosis is recommended when the clinical picture is atypical or complicated.
  • Detection of varicella zoster virus (VZV) by direct immunofluorescence assay (DFA); OR
  • Detection of (VZV) by Nucleic Acid Amplification (NAA); OR
  • Isolation of VZV by cell culture; OR
  • Seroconversion with VZV-specific IgG antibody

1.1.2 Suggestive Criteria

Detection of VZV IgM by enzyme immunoassay or other validated serological assay.

1.1.3 Special Considerations / Guide for Use

  • Laboratory confirmation is required usually for atypical presentations, particularly in the immunocompromised patient and for distinguishing between Herpes Simplex Virus (HSV) infection and herpes zoster. Direct methods of diagnosis are made when there is clinical manifestation of the disease where vesicular fluid or scrapings from the base of the fresh lesion are used.
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2 Introduction

Varicella-zoster virus (VZV), also known as human herpes virus 3 (HHV-3), is a highly communicable alpha-herpesvirus, which causes varicella (chicken-pox) and herpes-zoster (shingles). It is a DNA virus with a lipid envelope surrounding a nucleocapsid.

Varicella is a common childhood disease causing fever and a generalized pruritic vesicular rash after which it becomes latent in the dorsal root ganglia. Children usually acquire varicella between 5 to 10 years of age with the highest rates of hospitalisation in children under the age of 4. VZV has a worldwide geographic distribution, but infection is more prevalent in temperate climates with a strong seasonality occurring most often during late winter and spring (18). The virus is spread by inhalation of respiratory aerosols, and to a much lesser extent aerosolised virus particles from vesicular skin lesions. After an incubation period of approximately 2 weeks a characteristic cropping vesicular rash moves from the scalp and trunk to the periphery accompanied by mild fever. Lesions evolve from vesicles to crust over 8-12 hours.

Complications include skin infection, benign cerebral ataxia in children and post infections encephalitis in adults, and pneumonitis. The immunocompromised, neonates, adults and perhaps pregnant women may be at increased risk of severe disease. Neonates are at increased risk if maternal infection is 4 days before to 48 hours after birth.

Herpes-zoster is the reactivation of latent VZV causing a localised, painful, unilateral vesicular rash involving one or adjacent dermatomes usually the ophthalmic division of the trigeminal nerve and T3-L2. It is typically preceded by a 1-4 day prodrome of pain and parasthesia. Zoster is uncommon before the age of 12 years, and most cases occur over the age of 40. The incidence of herpes zoster increases in the aged and immunocompromised patients. A significant proportion of patients with ophthalmic zoster will develop ocular complications: up to 50% of patients with zoster over 50 years of age will develop postherpetic neuralgia, characterised by persistent severe pain in the affected dermatome. Immunocompromised patients may develop disseminated zoster with visceral, central nervous system and pulmonary involvement. In both adults and children with AIDS, VZV may cause cutaneous, ophthalmic and neural complications.

Congenital varicella syndrome characterised by cicatricial skin lesions, limb deformities, a variety of ocular abnormalities, and CNS disease appears to occur in less than 5% of infants contracting varicella in pregnancy.

Live attenuated varicella vaccine is currently available as a monovalent vaccine. The vaccine is derived from the Oka VZV strain but has some genetic differences (9). Vaccine failure is known as ‘breakthrough varicella’ and is defined as a case of wild-type varicella post vaccination. Breakthrough varicella infections are generally mild with fewer lesions than natural infection and can be contagious (15). Cases of chickenpox or shingles occurring in vaccinated persons can be caused by either wild-type or vaccine virus. Monitoring of these and other potential vaccine-related complications, as well as transmission events, requires that the vaccine strain be differentiated from wild-type viruses. Characterisation of varicella-zoster virus strains and differentiation from the vaccine strain (vOka) is achieved only by molecular genotyping methods. These methods have classified the wild-types into clades separated by geographic regions. Detection and quantitation of viremia during VZV infections are potentially useful for diagnostic, prognostic and therapeutic monitoring purposes (6).

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3 Tests

3.1 Clinical specimens

Swabs taken from the base of fresh vesicular lesions are suitable for nucleic acid testing, genotyping, direct immunofluorescent assay (DFA) and culture. Viral transport swabs are used for sampling. Alternatively, rayon or Dacron-tipped swabs with plastic-coated shafts can be used. After sampling from vesicular lesions, the swab should be immediately placed in viral transport medium (VTM) and stored at 4ºC until processed. Cerebrospinal fluid can also be used for testing in the appropriate clinical situation. Acceptable specimens for quantitative real-time PCR analysis are whole blood or plasma, serum or peripheral blood mononuclear cells (PBMCs). A significant proportion of patients with uncomplicated varicella have DNA detectable in blood and CSF during the acute phase of illness. A smaller proportion of zoster patients have detectable viraemia.

3.2 Direct Immunofluorescent Assay

Specific fluorescent-antibody detection of VZV is a rapid, simple and practical method for early diagnosis. Swabs or scrapings from lesions are spread over a 10mm diameter area in 50 µl of saline on a clean microscope slide and air dried. The slides are fixed in cold acetone for 10 minutes and stained with a Fluorescein–labelled conjugate (monoclonal antibody - VZV glycoprotein complex gp98-gp62) containing Evans Blue counterstain (e.g., MeriFluor VZV, Meridian Bioscience, Inc., Cincinnati, USA). Slides are read using fluorescent microscopy.

3.2.1 Test Sensitivity

The sensitivity and negative predictive value of the assay is reported in the range 97-98% (2).

3.2.2 Test Specificity

When clinical diagnosis is used as the gold standard, DFA is 100% specific for VZV.

3.2.3 Suitable Test Acceptance Criteria

Staining of a second cell smear with a bivalent fluorescein isothiocyanate-conjugated mouse monoclonal antibody specific for both herpes simplex virus types 1 and 2 (HSV-1, HSV-2).

3.2.4 Suitable Reference Controls

Incorporation of appropriate positive VZV and HSV antigen control slides.

3.3 Cell Culture

Viral isolation in conventional cell culture is considered to be a definitive diagnostic test for the detection of VZV. The disadvantage is that growth of VZV in culture can be difficult and too slow to be of clinical use. Tubes of human diploid lung fibroblasts (e.g., MRC-5; ATCC, Rockville, USA) are inoculated with 0.2 ml of clinical specimen and incubated at 37ºC and examined daily for cytopathic effects (CPE) for the first 7 days and then every other day for 21 days. Direct immunofluorescence is used to confirm suspected VZV isolates.

3.3.1 Test Sensitivity

Viral culture has a reported sensitivity ranging from 49.4% (2) to 65% (3). The sensitivity of VZV culture is also dependant on the speed of processing after specimen collection. The virus is labile and difficult to culture from lesions that are over 5 days old (14). Suggestion has been made that the administration of antiviral therapy prior to sampling has an influence on the propagation of VZV in culture.

3.3.2 Test Specificity

VZV culture is a presumptive test based on observation of focal cytopathic effects (CPE) in MRC-5 cells. CPE appears as small groups of swollen, granular, refractile cells progressing linearly along the long axis of the fibroblasts. VZV CPE can then be confirmed with DFA or PCR.

3.4 Nucleic Acid Detection

3.4.1 Qualitative PCR

PCR permits the rapid and highly sensitive detection of VZV from clinical specimens in the routine clinical laboratory. Various platforms are currently used for validated laboratory testing.

3.4.2 Quantitative PCR

Quantitative real-time polymerase chain reaction may be used for the detection and quantitation of VZV nucleic acids. Monitoring of VZV viral load in blood and CSF can be done in complicated cases which fail to respond to treatment. Two chemistries are currently used for the amplification and detection of VZV gene targets; sequence-specific probes and intercalating dsDNA fluorescent dyes.

3.4.3 Test Details

Gene specific primers and FRET (fluorescent resonance energy transfer) labelled probes or alternatively, a dsDNA intercalating fluorescent dye followed by melt curve analysis can be used for both the detection and quantitation of VZV. Suitable gene targets used in PCR detection include ORF 29, 62 (IE63) and ORF 63 (IE63) (4, 5, 7, 13).

For quantitation, a standard curve comprising of plasmid constructs containing the VZV gene target of interest is used as a standard and incorporated into each PCR test assay. Hence, the VZV genome equivalents (geq) of the test samples are interpolated from the standard curve.

3.4.4 Test Sensitivity

Sensitivity of PCR for diagnosis of varicella and zoster significantly exceed that of other methods and approaches 100%. Gene specific probe based assays have shown higher sensitivity to detecting VZV DNA than fluorescent dye based methods (5, 13). However, the use of fewer than ten copies of target may produce inconsistent results due to the random distribution inherent in dilutions of samples with low copy numbers.

3.4.5 Test Specificity

Specificity of the assay is dependant on the primer/probe design..

3.4.6 Predictive Values and Relevant Populations

The predictive value of a positive result is high in symptomatic individuals. In asymptomatic and immunocompromised individuals, predictability is compromised by no or low VZV viral loads.

3.4.7 Suitable Test Acceptance Criteria

Varicella-zoster positive control, a negative control and a non-template control should be included in each assay.

3.4.8 Suitable Internal Controls

An amplification control sequence is incorporated in the same tube to ensure that any VZV-negative samples do not contain PCR inhibitors and as a check on DNA sample integrity.

3.5 VZV-Specific Antibody Detection

Serology diagnosis is more important for determining the immune status before prophylactic therapy. Paired acute and convalescent sera are used for the diagnosis of primary Varicella infection, but this is less reliable for herpes zoster where specific antibodies are already present. For this reason, collection of the first sample as soon as possible after the onset of the rash is important to show a rising titre. However, cross reactivity between HSV and VZV can make the interpretation of results very difficult.

Recent infection is suggested by the detection of serum VZV-specific IgM antibodies. The antibodies occur within days of onset of infection and to a lesser extent after further exposure.
Immunity to varicella is relatively rarely an indication for a laboratory test, in part because of the high predictive value of a history of varicella, and the relative high prevalence of immunity in those without such a history. This may change as varicella vaccine displaces naturally acquired immunity. Post-immunization serological testing for immunity is not generally indicated because of the high level of immunity conferred by the vaccine – 95 % of adults seroconvert after receiving two doses of Oka vaccine. Post-vaccination antibody status may be checked in health-care workers who provide care to high risk patients. In vaccinated children, an assumption of immunity is made unless a vaccine associated rash or breakthrough infection occurs.

A positive VZV antibody titre obtained using a high quality test correlates well with immunity. FAMA (fluorescent antibody-to-membrane antigen) is the most sensitive test for immunity but rarely available. Latex agglutination is a highly sensitive alternative that is more widely available. Commercially available enzyme-labeled antigens (ELA) may fail to detect a proportion of immune individuals.

Two test methods used to measure VZV immune status are:
  1. Enzyme-linked Immunosorbent Assays (ELISA) - Enzygnost® Anti-VZV/IgG and IgM (Dade Behring)
  2. Enzyme-linked fluorescent immunoassay (ELFA) – VIDAS VZV immunoglobulin G automated immuno assay (bioMérieux,Vitek Inc.)
Enzyme-linked Immunosorbent assays are the preferred diagnostic test for determining serum antibodies. However, taking into consideration the number of clinical samples and the capacity of the diagnostic laboratory, patient sera may be initially screened by ELFA with equivocal sera re-tested by ELISA. Generally, the VIDAS VZV screen for IgG is not indicated.

The VZV VIDAS IgG system is an enzyme-linked fluorescent immunoassay utilising a virus coated Solid-Phase-Receptacle (SPR) to which antibody in serum binds. A fluorescent product is formed when Anti-Human IgG conjugated with Alkaline Phosphatase reacts with substrate 4-Methylumbelliferyl Phosphate. All reagents and test serum are contained within the reagent strips and the assay is accomplished automatically within the VIDAS instrument.

Another technique for identifying VZV antibodies is the indirect-immunofluorescence assay (IIFA) in which bound antibody is detected by a fluorescent-labelled anti-immunoglobulin. The stained VZV-infected cells are examined using fluorescent microscopy and the antibody titre determined by the highest dilution of a serum sample that allows the clear detection of fluorescence.

3.5.1 ELISA Test Details

A number of sensitive assays are available to measure antibody to VZV including enzyme immunoassay (EIA), latex agglutination and fluorescent antibody to membrane antigen (FAMA) which is a reference test of limited availability.

Suitable specimens are serum or plasma samples (EDTA/heparinised/citrated plasma). The samples should be stored for not more than 3 days at 2-8ºC. Various high quality commercial ELISA kits are available and are the most commonly used diagnostic tests.,

Another technique for identifying VZV antibodies is the indirect-immunofluorescence assay (IIFA) in which bound antibody is detected by a fluorescent-labelled anti-immunoglobulin. The stained VZV-infected cells are examined using fluorescent microscopy and the antibody titre determined by the highest dilution of a serum sample that allows the clear detection of fluorescence.

3.5.2 ELISA Test Sensitivity

Enzygnost® Anti-VZV/IgG was shown to have a sensitivity of 99.3% in a test comprising of 145 test samples (1).

3.5.3 ELISA Test Specificity

The test is specific for VZV IgG or IgM only. A specificity of 100% was found in a 54 test sample study (1).

3.5.4 Predictive value and Relevant Populations

Relevant populations for testing, apart from clinical indications, include possible screening for prenatal care programmes and determining immunity status in health-care workers.

3.5.5 Suitable Reference Controls

Human serum containing IgG and IgM antibodies to VZV antigens is provided as a reference within the kit.

3.5.6 Suitable External (QAP) Programme(s)

A QAP for Varicella zoster-specific antibody detection is available through the Royal College of Pathologists Association of Australia.

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4 Agreed typing & subtyping methods

4.1 Genotyping Methods

Varicella-zoster infection caused by wild-type or vaccine strains of the virus can be differentiated by genotyping. Three methods are in (1)current use to geographically distinguish between the varicella vaccine strain (vOka) and the following VZV wild-type stains: African/Asian (A), European (B), European (C) and Japanese [pOka, European] (J). The varicella vaccine strain vOka (Varivax® and Varilrix ®) and VZV wild-type strain ‘Dumas’ (Genbank Accession No. X04370) are used as laboratory reference control strains.

4.1.1 Multi-SNP (Single Nucleotide Polymorphism) genotyping of strains

SNP genotyping of VZV strains is achieved by the detection of gene mutations by allelic discrimination using gene specific probes (dual labelled) (12) or the High Resolution Melt analysis (HRMA) of a temperature shift in a melt curve detected by a dsDNA saturating fluorescent dye (17). The five VZV gene targets, ORF 1, 21 37, 60 and 62 can be used to discriminate vOka from and between wild-type strains.

4.1.2 DNA sequence variation in ORF 22

This genotyping method is based on DNA sequencing of a short region in VZV ORF 22. Four polymorphic positions in a 447-bp fragment of ORF 22 (10) distinguish European strains from Japanese (J) and mosaic (M1-M4) strains.

4.1.3 PCR-RFLP

PCR followed by restriction fragment length (PCR-RFLP) analysis of two polymorphic loci – a PstI restriction site in ORF 38, and a BglI restriction site in ORF 54 (8, 16). This method separates VZV wild-type isolates from the Oka vaccine strain. The vaccine virus strain is PstI ¯ BglII+ at these loci.

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5 References

1Enzygnost Anti VZV/IgG. Enzyme immunoassay for the qualitative detection and quantitative determination of IgG antibodies to Varicella-Zoster virus (VZV) in human serum and plasma. Dade Behring. July edition 2003, p8, Marburg, GmbH.

2 Coffin, S. E., and R. L. Hodinka. 1995. Utility of direct immunofluorescence and virus culture for detection of varicella-zoster virus in skin lesions. J Clin Microbiol 33:2792-5.

3 Dahl, H., J. Marcoccia, and A. Linde. 1997. Antigen detection: the method of choice in comparison with virus isolation and serology for laboratory diagnosis of herpes zoster in human immunodeficiency virus-infected patients. J Clin Microbiol 35:347-9.

4 de Jong, M. D., J. F. Weel, T. Schuurman, P. M. Wertheim-van Dillen, and R. Boom. 2000. Quantitation of varicella-zoster virus DNA in whole blood, plasma, and serum by PCR and electrochemiluminescence. J Clin Microbiol 38:2568-73.

5 Engelmann, I., D. R. Petzold, A. Kosinska, B. G. Hepkema, T. F. Schulz, and A. Heim. 2008. Rapid quantitative PCR assays for the simultaneous detection of herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6 DNA in blood and other clinical specimens. J Med Virol 80:467-77.

6 Hawrami, K., and J. Breuer. 1999. Development of a fluorogenic polymerase chain reaction assay (TaqMan) for the detection and quantitation of varicella zoster virus. J Virol Methods 79:33-40.

7 Kimura, H., S. Kido, T. Ozaki, N. Tanaka, Y. Ito, R. K. Williams, and T. Morishima. 2000. Comparison of quantitations of viral load in varicella and zoster. J Clin Microbiol 38:2447-9.

8 LaRussa, P., O. Lungu, I. Hardy, A. Gershon, S. P. Steinberg, and S. Silverstein. 1992. Restriction fragment length polymorphism of polymerase chain reaction products from vaccine and wild-type varicella-zoster virus isolates. J Virol 66:1016-20.

9 Lau, Y. L., S. J. Vessey, I. S. Chan, T. L. Lee, L. M. Huang, C. Y. Lee, T. Y. Lin, B. W. Lee, K. Kwan, S. M. Kasim, C. Y. Chan, K. M. Kaplan, D. J. Distefano, A. L. Harmon, A. Golie, J. Hartzel, J. Xu, S. Li, H. Matthews, J. C. Sadoff, and A. Shaw. 2002. A comparison of safety, tolerability and immunogenicity of Oka/Merck varicella vaccine and VARILRIX in healthy children. Vaccine 20:2942-9.

10 Loparev, V. N., E. N. Rubtcova, V. Bostik, D. Govil, C. J. Birch, J. D. Druce, D. S. Schmid, and M. C. Croxson. 2007. Identification of five major and two minor genotypes of varicella-zoster virus strains: a practical two-amplicon approach used to genotype clinical isolates in Australia and New Zealand. J Virol 81:12758-65.

11 (delete if sole reference in text is deleted) Macartney, K. K., P. Beutels, P. McIntyre, and M. A. Burgess. 2005. Varicella vaccination in Australia. J Paediatr Child Health 41:544-52.

12 Parker, S. P., M. Quinlivan, Y. Taha, and J. Breuer. 2006. Genotyping of varicella-zoster virus and the discrimination of Oka vaccine strains by TaqMan real-time PCR. J Clin Microbiol 44:3911-4.

13 Quinlivan, M. L., K. Ayres, H. Ran, S. McElwaine, M. Leedham-Green, F. T. Scott, R. W. Johnson, and J. Breuer. 2007. Effect of viral load on the outcome of herpes zoster. J Clin Microbiol 45:3909-14.

14 Schmidt, N. J., D. Gallo, V. Devlin, J. D. Woodie, and R. W. Emmons. 1980. Direct immunofluorescence staining for detection of herpes simplex and varicella-zoster virus antigens in vesicular lesions and certain tissue specimens. J Clin Microbiol 12:651-5.

15 Seward, J. F., J. X. Zhang, T. J. Maupin, L. Mascola, and A. O. Jumaan. 2004. Contagiousness of varicella in vaccinated cases: a household contact study. JAMA 292:704-8.

16 Takada, M., T. Suzutani, I. Yoshida, M. Matoba, and M. Azuma. 1995. Identification of varicella-zoster virus strains by PCR analysis of three repeat elements and a PstI-site-less region. J Clin Microbiol 33:658-60.

17 Toi, C. S., and D. E. Dwyer. 2008. Differentiation between vaccine and wild-type varicella-zoster virus genotypes by high-resolution melt analysis of single nucleotide polymorphisms. J Clin Virol 43:18-24.

18 Whitley, R. J. 1990. Varicella-Zoster virus infections, p. 235-263. In R. W. GJ Galasso, TC Merigan (ed.), Antiviral agents and viral diseases of man. Raven Press, New York.

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