Bryson

Journal of the
American Academy of
Dermatology
movinyl deoxyuridine and human immunoglobulin in
herpes simplex virus-infected mice. Antiviral Res 1987;
7:227-35.
54. Gangemi JD, Nachtigal M, Barnhart D, Krech L,
Jani P. Therapeutic efficacy of lyposome-encapsulated
ribavirin and muramyl tripeptide in experimental infec-
tion with influenza or herpes simplex virus. J Infect Dis
1987;155:510-7.
New diagnostic tests for herpes
varicella zoster infections
simplex and
Al vi n R. Sol omon, M. D. Galveston, TX
Laboratory tests for herpetic infections can be divided into (1) morphologic,
(2) immunomorphologic, (3) serologic, and (4) virologic. Tzanck smears
are easy to do, inexpensive, and compare favorably with cultures and
immunofluorescence tests for specificity and sensitivity, but they require
considerable experience to interpret accurately and they cannot differentiate
between herpes type 1, herpes type 2, and varicella zoster infections.
Biopsies are useful when clues to a diagnosis are being sought.
Peroxidase-antiperoxidase and avidin-biotin tests present technical difficulties
but interpretational difficulties are low and the results are available in a
few hours. They can distinguish between herpes type 1, herpes tyi~e 2, and
varicella zoster virus, as can immunofluorescence using monoclonal antibodies.
Serologic tests are used primarily to distinguish between primary and recurrent
herpes simplex infections. Virus isolation in tissue cultures is the gold standard
for identifying herpes simplex virus but it is not 100% specific or 100%
sensitive. Restriction endonuctease analysis identifies types and strains of virus
by their deoxyribonucleic acid composition and it is very useful in
epidemiologic studies. Ability to find virus by whatever method is influenced
by the stage of the lesion. As lesions age, less infectivity and antigen result
in less sensitivity of the tests. (J AM ACAD DERMATOL 1988;18:218-21.)
I have sorted through t he ma ny different labo-
rat ory tests used for diagnosing herpet i c infections
to distill t hem down to t he ones that woul d be
useful to t he practicing dermatologist. The tests
can be divided into four basic types: (1) morpho-
logic, (2) i mmunom0rphol ogi c, (3) serologic, and
(4) virologic. 1,2
From the Department of Dermatology, University of Texas Medical
Branch.
Reprint requests to: Alvin R. Solomon, M.D., Department of Der-
matology, University of Texas Medical Branch, Galveston, TX
77550.
218
I started with t he morphol ogi c t ype of di agnost i c
methodologies t hat is typified by the Tzanck
smear, a t echni que used by many physi ci ans, es-
pecially der mat ol ogi st s) Some of the charact er-
istics and special features of t he Tzanck smear are
(1) the Tzanck smear is a t echni que, not a stain.
Several t ypes of stains may be used, and (2) the
speci men is obt ai ned by scraping t he l esi ons, an
easy technique wi t h l ow morbidity; (3) t he tech-
nical difficulty of doing the Tzanck preparat i on is
low; (4) t he interpretative difficulty with t he tech-
ni que is hi ghl y vari abl e and depends on t he ex-
peri ence of the i nt er pr et er - - di f f i cul t wi t h inac-
Volume 18
Number 1, Part 2
January 1988
Tests f or herpes simplex and varicella zoster 219
c ur a t e i nt er pr et at i ons i f t he i nt er pr et or is not ex-
p e r i e n c e d ; (5) t he t est is near l y uni ver sal l y
avai l abl e. The onl y e qui pme nt r equi r ed is a mi -
c r o s c o p e and a way t o do t he st ai ns ei t her manual l y
or a u t o ma t e d ( bl ood f i l m st ai ners); (6) t he t i me f or
d o i n g t he t est is ver y s h o r t - - a f ew mi nut es; (7)
t he t es t is i nexpens i ve; (8) t he sensi t i vi t y of t he
t es t i s ver y good but t he per cent posi t i vi t y o f t he
t est var i es wi t h t he st age o f t he l esi on. Posi t i vi t y
i s 7 0 % or mor e i n ves i cl es , about 55% i n pust ul es,
a n d v e r y l ow i n cr ust s, a l t hough s ome crust ed l e-
s i ons are posi t i ve i n her pes s i mpl e x i nfect i ons; (9)
t he speci f i ci t y i s hi gh, t her e be i ng about 3% f al se
p o s i t i v e test r esul t s i n e xpe r i e nc e d hands; (10) it
is a cos t - ef f ect i ve wa y o f det er mi ni ng t hat a her pes
s i mp l e x vi rus or var i cel l a zos t er vi rus i nf ect i on
is p r e s e n t but it c a nnot di f f er ent i at e bet ween
h e r p e s 1 and her pes 2 or vari cel l a zost er vi rus
i nf e c t i ons . 4-6
T h e Tzanck t est has a var yi ng degr ee of user
f r i endl i nes s . Th e novi c e ma y wi sh to r el y on find-
i ng mul t i nuc l e a t e d gi ant v i mc y t e s wi t h mol de d nu-
cl ei be c a us e t hes e are t he mo s t pat hognomoni c.
Th e mo r e e xpe r i e nc e d per s on r ecogni zes a sort o f
h o mo g e n i z a t i o n of nucl ear c hr oma t i n or gr ound
gl ass appear ance t hat occur s i n i nf ect ed epi der mal
cel l s a n d i n mo n o n u c l e a r cel l s as wel l . I nt r anucl ear
i n c l u s i o n s ( Cowdr y t ype A) are n o t seen wi t h any
d e g r e e of r egul ar i t y wh e n monoc hr oma t i c st ai ns
are u s e d . The t ype of st ai n to us e i s l argel y a mat t er
o f p e r s o n a l pr ef er ence. I t end t o use me t hyl e ne
bl ue . He ma t oxyl i n- e os i n st ai n can. be us ed but i t
is mo r e di f f i cul t to do. The Ro ma n o v s k y st ai ns,
s uc h as t he Gi e ms a a nd Wr i ght st ai ns, gi ve good
r es ul t s and are easy t o us e i f you have an aut omat i c
b l o o d f i l m stainer. Th e best st ai n t o use f or nucl ear
mo r p h o l o g y and det ai l is t he Papani col aou st ai n,
wh i c h makes t he s me a r easi er t o i nt erpret . Thi s
s t ai n can be done i n t he offi ce but it is mor e dif-
f i cul t t o do. One mu s t fix t he s mear i mmedi at el y
i n a l c ohol . It i s a mul t i s t e p st ai n, it takes l onger
t o d o , and t he cost is hi gher , but specificity and
and s ens i t i vi t y are about t he s a me as for t he ot hers.
T h e next pr oc e dur e i n t he mor phol ogi c di ag-
nos i s o f her pes is t he bi opsy. Bi ops y is not of t en
d o n e (or hel pf ul ) wh e n cl assi c l esi ons of her pes
s i mp l e x , her pes zost er, or var i cel l a are pr esent . It
i s o f gr eat est va l ue wh e n t he gr oss mor phol ogy of
t he l es i ons is n o t di agnos t i c or char act er i st i c a nd
one i s s ear chi ng for cl ues t hat mi g h t l e a d to t he
di agnosi s. Thi s i s mos t l i kel y t o b e the c a s e i n t he
ever - i ncr easi ng n u mb e r of i mmu n o c o mp r o mi s e d
pat i ent s whose he r pe t i c l es i ons ar e bi zarre and f ar
f r om t he cl assi c cl i ni cal a ppe a r a nc e . Th e bi ops y
can be done b y s have, p u n c h , or we dge exci s i on
t echni ques . I nt er pr et at i on o f t he bi ops y s p e c i me n
ma y b e easi er t h a n i nt er pr et at i on o f Tz a nc k s mear s
becaus e one i s abl e t o eval uat e hi s t ol ogi c as we l l
as cyt ol ogi c f i ndi ngs. I t ma y t ake 24 hour s to ge t
t he s peci mens back. Sens i t i vi t y and s peci f i ci t y
wi t h bi ops y s pe c i me ns are about t h e s ame as wi t h
smear s. Pos i t i ve bi ops y r esul t s show mul t i nu-
cl eat ed gi ant cel l s , gi ant ke r a t i noc yt e s , and t ypi cal
nucl ear changes. So me o f t he ear l i er a n d mo s t
charact eri st i c changes are i n t he hair f ol l i cl es
r at her t han t he s ur f ace e pi de r mi s .
An o t h e r mor phol ogi c t est is el ect r on mi cr os -
copy. I t is expens i ve and r equi r es cons i der abl e
exper i ence. I f t he l abor at or y is set up to d o it, t he
resul t s can be obt a i ne d i n 2 hour s . Sensi t i vi t y a nd
speci fi ci t y vary wi t h exper i ence. I t does not di f -
f er ent i at e be t we e n her pes s i mpl e x and var i cel l a
zost er vi ruses. El e c t r on mi c r o s c o p y is us ual l y o f
l i mi t ed val ue i n mos t cl i ni cal si t uat i ons.
I mmu n o mo r p h o l o g i c t echni ques 7 are ve r y g o o d
and are b e c o mi n g mor e popul ar . Ei t her s me a r s or
hi st ol ogi c s ect i ons can be used. Ma ny di f f er ent
t echni ques have been des cr i bed. T h e per oxi das e-
ant i per oxi dase a nd t he a vi di n- bi ot i n met hods ar e
used mos t of t en. The r e a ge nt s are c omme r c i a l l y
avai l abl e so t hat the l a bor a t or y n o l onger has t o
raise i t s own ant i bodi es . Fi xe d t i ssues and s mear s
( f or mal i n or acet one, et c. ) can be used. T h e t ech-
ni cal di ffi cul t i es of d o i n g t he t est ar e hi gh, but t he
mor e exper i ence t he l a bor a t or y has the eas i er i t is
to do it. I nt er pr et at i onal di f f i cul t i es are l ow be-
cause t he t est r esul t s ar e us ual l y d e t e r mi n e d to b e
ei t her posi t i ve or negat i ve. Tes t r esul t s ar e us ual l y
avai l abl e wi t hi n 4 t o 5 hour s wi t h s mear s and
4 t o 5 hour s wi t h bi ops i es af t er t he y have been c u t
and sect i oned. T h e tests are r e l a t i ve l y i nexpens i ve.
Test r esul t s are pos i t i ve i n about t wo t hi rds o f
s peci mens and t her e is a hi gh de gr e e of speci fi ci t y.
Pot ent i al l y t hese tests can di s t i ngui s h be t we e n
her pes si mpl ex t ype 1 and t ype 2 and var i cel l a
zost er vi r es , but not al l of t he c o mme r c i a l ant i -
220 Solomon
Journal of the
American Academy of
Dermatology
bodies will accurately distinguish between herpes
types 1 and 2. These tests use chromogens that are
activated by the immunologic reactants so that the
results of the tests are easy to interpret. Most sys-
tems allow fixing in permanent mounting media
to give better cytologic detail and provide a per-
manent record.
Immunofluorescence 7'8 is also a good technique
that is used by many. The main disadvantages are
(1) requirement for a fluorescence microscope, (2)
biopsy materials are required to be frozen or fixed
in special media, (3) considerable experience is
required to interpret specimens, and (4) additional
time is required for eye accommodation. Gener-
ally, antigen in tissue reacts wi t h fluorescein-
tagged antibody that is directed against viral an-
tigens in the direct test. In the indirect test, anti-
body to the virus is added to virus-containing
tissues or cells and this primary antibody is then
detected and amplified by a fluorescein-tagged
secondary antibody to the primary antibody.
Fluorescein-tagged monoclonal antibodies to her-
pes simplex types 1 and 2 and varicella zoster virus
are now available commercially.
Serologic tests play only a small role in the
diagnosis of herpes simplex infections. 9 They are
used primarily to document primary infections
when antibodies are absent at the onset of the clin-
ical or subclinical infection and appear during its
course. A fourfold or greater antibody rise is usu-
ally required for a diagnosis of a primary infection.
In recurrent herpes simplex infections, <5% of
the patients have significant rises in antibody
levels. Essentially all patients with recurrent
herpes have detectible serum antibodies so that a
negative test result is strong evidence against a
diagnosis of recurrent herpes. In addition, in some
serologic tests, antibodies to herpes type 1 and
herpes type 2 cross-react, and these antibodies may
also cross-react with antibodies to varicella zoster
virus.
The complement fixation test for serum anti-
bodies has been the standard and most widely
used, but it is less sensitive than many of the other
tests. Neutralization, direct hemagglutination,
and passive hemagglutination tests are available.
Enzyme-linked immunosorbent assay (ELISA)
and western blot techniques may become widely
available soon. With monoclonal antibodies the
ELISA test can determine antibody immunoglob-
ulin subtype levels. Western blot analysis can de-
termine antibody levels to specific herpes simplex
virus polypeptide antigens. Another promising test
is the detection of low levels of herpes virus an-
tigen in vesicle fluid using techniques such as
ELISA,10 agar gel immunodiffusion, countercur-
rent immunoelectrophoresis, and deoxyribonu-
cleic acid (DNA) probes.11.12
Virus isolation in tissue culture is currently the
reference method or gold standard for virus iden-
tification and diagnosis. 1.2.5,6 However, viral iso-
lation is not always 100% sensitive and 100% spe-
cific. Virus may be present in the patient but not
show up in the cultures for various reasons in-
cluding (1) the virus may not survive inappropriate
transport conditions, (2) the sample may have been
taken from the wrong lesion or area, (3) there may
have been too little virus in the lesions (i.e.,
crusted, almost healed lesions), and (4) antiviral
therapy may have eliminated the virus.
Recognition of herpesvirus in tissue cultures
usually depends on the appearance of a cytopathic
effect (viral acantholysis, multinucleated giant
cells, intranuclear inclusions, nuclear changes,
etc.). This cytopathic effect is usually seen with
herpes simplex virus in 24 to 48 hours but may
take as long as 7 days. Varicella zoster virus may
take 1 to 3 weeks to produce characteristic
changes. Newer techniques 13 to speed up diagnosis
include adding tagged antibody to cultures to de-
tect viral antigen before a cytopathic effect appears
and adding antiviral drugs that specifically in-
hibit growth of certain types of viruses. For in-
stance, E-5(2-bromovinyl)-2' deoxyuridine inhib-
its the growth of herpes simplex virus type 1 more
than herpes simplex virus type 2 to the point where
it can be used in diagnosis. Isolation of virus in
tissue cultures and subsequent identification by
specific fluorescein-tagged monoclonal antibodies
results in greater specificity and sensitivity.
Restriction endonuclease analysis is a technique
that identifies viruses by their DNA composition.
It has great potential for becoming an important
adjunct in the clinical laboratory diagnosis of her-
pesvirus infections in the near future.
There are several reasons for typing herpes sim-
Volume 18
Number 1, Part 2
January 1988
Tests for herpes simplex and varicella zoster 221
pl ex vi r us e s i nt o t ype 1 a nd t ype 2. One r eas on is
t hat t y p e 2 geni t al i nf ect i ons t end to r ecur mo r e
of t en t ha n t ype 1, s o this bear s on pr ognosi s. The
t wo t ype s di f f er i n t hei r sensi t i vi t y t o vari ous an-
t i vi r al dr ugs . Typi ng ma y hel p to det er mi ne
wh e t h e r a par t i cul ar her pet i c epi s ode is a ne w or
a r e c ur r e nt i nf ect i on, a l t hough rest ri ct i on endo-
n u c l e a s e anal ysi s wo u l d be mo r e specific f or i den-
t i f yi ng di f f er ent t ypes or st rai ns of virus.
I n t h e maj or i t y o f pat i ent s t he di agnosi s ma y be
ma d e cl i ni cal l y. I f t he di sease is not charact eri st i c
or t he pa t i e nt want s f ur t her pr oof , a Tzanck s mear
can be d o n e . I f t he Tz a nc k s mear is negat i ve and
t hi s d o e s n o t agr ee wi t h t he cl i ni cal i mpr es s i on
and f ur t he r e vi de nc e is desi r ed, ei t her for or agai nst
t he he r pe t i c i nf ect i on, a cul t ur e can be done or
i mmu n o f l u o r e s c e n t or i mmunope r oxi da s e st udi es
can be done. I f t he Tz a nc k s me a r is posi t i ve and
i t is i mp o r t a n t t o k n o w i f her pes si mpl ex or var-
i cel l a z os t e r is pr es ent , cul t ur es or ot her st udi es
mu s t b e done. I n mo s t cases I woul d do a Tz a nc k
s me a r a nd pos s i bl y f ol l ow t hi s wi t h cul t ures or
i mmu n o p e r o x i d a s e or i mmunof l uor e s c e nt stains.
( Gues t edi t or : S o me aspect s o f l aborat ory exami -
nat i on n o t i n t e n d e d to be cover ed by Dr. Sol o-
mo n ' s pr e s e nt a t i on mi ght be ment i oned: (1) Fast ,
i ne xpe ns i ve , h i g h - v o l u me t est i ng of viral i sol at es
t o a bat t er y of ant i vi r al dr ugs is bei ng devel oped
t o f aci l i t at e c h o o s i n g t he mo s t ef f ect i ve therapy' 4;
(2) a n u mb e r of t est s exi st t o det er mi ne t he pro-
duc t i on o f t h y mi d i n e ki nas e by her pesvi r uses;
p r o d u c t i o n o f t hymi di ne ki nas e act i vi t y has a
di r ect be a r i ng on whi c h ant i vi ral drug wi l l be
effective15'~6; and (3) tests f or ant i vi ral dr ug levels
i n b l o o d and ot her fl ui ds are be c omi ng mor e i m-
por t a nt i n mo n i t o r i n g t her apeut i c levels and con-
s i der at i on of dr ug r esi st ance. ,7 Ma na ge me nt o f vi-
ral i nf ect i ons is get t i ng to be mor e like manage-
me n t o f bact er i al i nf ect i ons . )
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