Abstract
Recent analysis of archived temporal arteries (TAs) acquired from 13 pathology laboratories in the US, Canada, Iceland, France, Germany and Israel from patients with pathologically-verified giant cell arteritis (GCA-positive) and TAs from patients with clinical features and laboratory abnormalities of GCA but whose TAs were pathologically negative (GCA-negative) revealed VZV antigen in most TAs from both groups. Despite formalin-fixation, VZV DNA was also found in many VZV-antigen positive sections that were scraped, subjected to DNA extraction, and examined by PCR with VZV-specific primers. Importantly, in past studies, the pathological diagnosis (GCA-positive or -negative) was known to the neurovirology laboratory. Herein, GCA-positive and GCA-negative TAs were provided by an outside institution and examined by 4 investigators blinded to the pathological diagnoses. VZV antigen was found in 3/3 GCA-positive TAs and in 4/6 GCA-negative TAs, and VZV DNA in 1/3 VZV antigen-positive, GCA-positive TAs and in 3/4 VZV antigen-positive, GCA-negative TAs. VZV DNA was also detected in one GCA-negative, VZV-antigen negative TA. Overall, the detection of VZV antigen in 78% of GCA-positive and GCA-negative TAs is consistent with previous reports on the prevalence of VZV antigen in patients with clinically suspect GCA.
Keywords: Varicella zoster virus, Giant cell arteritis
1. Introduction
Primary varicella zoster virus (VZV) infection causes chickenpox (varicella), after which virus becomes latent in ganglionic neurons along the entire neuraxis. In aging and immunocompromised individuals, a decline in cell-mediated immunity to VZV manifests clinically as zoster (shingles), dermatomal distribution pain and rash. Zoster may also be complicated by meningoencephalitis, myelitis and VZV vasculopathy, all of which are due to viral replication in infected tissue. Although intracerebral VZV vasculopathy has been recognized for decades, the discovery of VZV infection in extracerebral temporal arteries (TAs) of GCA-positive patients [Gilden, 2014; Gilden and Nagel, 2015] and in patients with clinical features and laboratory abnormalities of GCA but whose TAs were pathologically negative [Nagel et al., 2013] is recent. All virologic analyses from 2013–15 were conducted on GCA-positive and GCA-negative TAs in which the pathology report was known. In the present study, we searched for VZV antigen and VZV DNA in 10 archived TAs, but were masked to the pathological diagnosis of GCA-positive vs. GCA-negative.
2. Methods and methods
2.1 Human temporal arteries
One investigator (B.J.K.) provided 10 de-identified TAs from 9 subjects to the neurovirology laboratory at the University of Colorado for VZV analysis. Of the 10 TAs, 3 were GCA-positive and 7 were GCA-negative. A portion of all 10 TAs was formalin-fixed and paraffin-embedded (FFPE) for immunohistochemical analysis, and the remainder of 9 TAs was frozen in RNAlater® Stabilization Solution (ThermoFisher Scientific, Waltham, MA) at −80°C for nucleic acid analysis. All investigators in the neurovirology laboratory (D.G., M.A.N., T.W. and N.K.) were blinded to the diagnosis of GCA-positive or GCA-negative TA.
2.2 Immunohistochemical analysis of GCA-positive and GCA-negative TA biopsies
One-hundred 5-μm sections of all FFPE TAs were cut and alternate sections were analyzed immunohistochemicallyy using mouse monoclonal anti-VZV gE IgG1 antibody (Santa Cruz Biotechnology, Dallas, TX) and control mouse IgG1 antibody (Dako, Carpenteria, CA). Positive controls consisted of VZV-infected cadaveric cerebral arteries maintained for 14 days in vitro and immunostained with mouse anti-VZV gE IgG1 antibody, as previously described [Gilden et al., 2015]. Using light microscopy, 4 blinded readers (D.G., M.A.N., T.W. and NK) examined each section. A TA section was deemed positive or negative for VZV antigen only when all readers agreed.
2.3 PCR amplification of VZV DNA in FFPE TA sections containing VZV antigen
From each of the 7 TAs containing VZV antigen, all VZV antigen-positive sections were scraped with a scalpel and pooled, after which DNA was extracted and analyzed by PCR for the presence of VZV DNA as described [Gilden et. al., 2015]. Negative controls were provided by replacing template DNA with PCR-grade water.
2.4 PCR amplification of VZV DNA in frozen TA sections
Nine TAs frozen in RNAlater® Stabilization Solution were thawed at room temperature until the TA could readily be removed from the RNAlater® Stabilization Solution, after which the TA was immediately cut into 1-mm sections using a sterile scalpel and weighed in an RNase-free area. DNA was extracted and analyzed by PCR from every other 1-mm section for the presence of VZV DNA as described above. Remaining sections were frozen individually in RNAlater® Stabilization Solution at −20°C for future studies.
3. Results
Ten TAs from 9 subjects who underwent TA biopsy to diagnose GCA were examined for VZV by 4 investigators who were blinded to the pathological report of GCA-positive or GCA-negative. VZV antigen was found in 3/3 GCA-positive TAs and in 4/6 GCA-negative TAs (Fig. 1). VZV antigen was present mostly in the adventitia, but was seen in all arterial layers (Table 1). Despite formalin-fixation, VZV DNA was detected in 1/3 VZV antigen-positive GCA-positive TAs and in 3/4 VZV antigen-positive GCA-negative TAs. VZV DNA was also detected in one GCA-negative, VZV antigen-negative TA. VZV DNA was found in one frozen unfixed GCA-negative, VZV antigen-negative TA.
Fig. 1.
Varicella zoster virus (VZV) antigen in temporal arteries (TA) of patients with pathologically-verified giant cell arteritis (GCA) and patients with clinical features and laboratory abnormalities characteristic of GCA whose TAs were pathologically negative. Immunohistochemical analysis of TAs from 3 GCA-positive subjects revealed VZV antigen in the adventitia of all 3 TAs (A–C, pink color). VZV antigen was seen in the TAs of 4/6 GCA-negative subjects in the adventitia (G, thin arrow), media (G, dashed arrow) and thickened intima (G, thick arrow), in the adventitia alone (H and I) and in the media and thickened intima (J). No staining was seen in sections adjacent to those containing VZV antigen when mouse isotype IgG1 was substituted for mouse anti-VZV gE antibody (C–E, I–K). Magnification 600X.
Table 1.
Localization of VZV antigen in GCA-positive and GCA-negative TAs.
| Subject | Diagnosis | Localization of VZV antigen |
|---|---|---|
| 1 | GCA-positive | Adventitia, adjacent skeletal muscle |
| 2 | GCA-positive | Adventitia |
| 3 | GCA-positive | Adventitia |
| 4 | GCA-negative | Adventitia, intima |
| 5 | GCA-negative | Adventitia |
| 6 | GCA-negative | Adventitia, media, intima |
| 9 | GCA-negative | Adventitia, media, intima |
Blinded search for varicella zoster virus in giant cell arteritis (GCA)-positive and GCA-negative temporal arteries (TAs)
4. Discussion
For the first time in a research study, GCA-positive and GCA-negative TAs were analyzed for VZV by 4 investigators who were blinded to the pathologic report. The detection of VZV antigen in 3/3 GCA-positive TAs and in 4/6 GCA-negative TAs supports earlier larger studies that found VZV antigen in most GCA-positive and GCA-negative TAs [Gilden et al., 2015; Nagel et al., 2015]. Similarly, VZV DNA was detected by PCR in VZV antigen-positive sections from 1/3 GCA-positive TAs and from 3/4 GCA-negative TAs.
The methods employed here are not yet practical for routine diagnostic evaluation of TA biopsies. In the future, it may be reasonable to obtain fresh-frozen tissue at the time of biopsy for virological analysis. PCR analysis of DNA extracted from fresh-frozen unfixed tissue could eventually become part of the protocol. Future larger long-term studies will be required to determine if analysis of TA biopsies for VZV leads to changes in the management of GCA patients.
5. Conclusions
The work presented here further supports our laboratory’s findings that VZV plays a role in the pathogenesis of GCA. Future studies will be required to determine if routine testing of TA biopsy specimens for VZV is warranted and if antiviral treatment confers additional benefit to corticosteroid treatment.
HIGHLIGHTS.
VZV antigen is present in most GCA-positive and GCA-negative TAs
VZV DNA is present in many VZV antigen-positive TA sections
The diagnostic value of detecting VZV DNA in unfixed TAs remains to be determined
Acknowledgments
This work was supported in part by Public Health Service grants AG032958 (D.G., M.A.N.) and NS094758 (M.A.N.) from the National Institutes of Health, a Dennis W. Jahnigen Career Development Scholar Award from the American Geriatric Society (B.J.K), and by an unrestricted grant to the Department of Ophthalmology and Visual Sciences, University of Utah from Research to Prevent Blindness, Inc., New York, NY. The authors thank Drs. Bhupendra C.K. Patel and Alison V. Crum for acquiring surgical specimens used in this study, Marina Hoffman for editorial assistance and Cathy Allen for word processing and formatting of the manuscript.
Abbreviations
- TA
temporal artery
- GCA
giant cell arteritis
- VZV
varicella zoster virus
- FFPE
formalin-fixed and paraffin-embedded
Footnotes
Conflict of interest statement
All authors report no conflicts of interest.
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