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BrysonJou rna l o f the

American Academy ofDermatology

moviny l deoxyur id ine and human immunog lobu l in inherpes s implex v irus-infected mice. Antiv iral Res 1987;7 :227 -35 .

54 . Gang emi JD Nacht iga l M Barnhar t D Krech L

Jan i P. Therapeu t ic e ff i cacy o f lyposome-encapsu la tedribavirin and muramyl t r ipept ide in experimental infec-

t ion with influenza or herpes s implex v irus . J Infe ct Dis1987;155:510-7.

ew d iagnos t ic t es t s fo r herpesvar icel la zos ter infect ions

s imp lex and

A l v i n R . S o l o m o n , M . D .Galveston TX

Laboratory tests for h erpetic infections can be divided into (1) morphologic,(2) immuno morp hologic, (3) serologic, and (4) virologic. Tzanck smearsare easy to do, inexpensive, and compare favorably with cultures andimm unofluorescence tests for specificity and sensitivity, but they requireconsiderable experie nce to interpret a ccurately and the y cannot differentiatebetween herpes typ e 1, herpes type 2, and varicella zoster infections.Biopsies are useful when clues to a diagnosis are being sought.Pero xidase -antipero xidase and avidin-biotin tests pre sent tec hnical difficultiesbut interpretational difficulties are low and the results are available in afew hours. They c an d istinguish between herpes type 1, herpes tyi~e 2, andvaricella zoster virus, as can imm unofluorescence using mono clonal antibodies.

Serologic tests a re use d p rimarily to distinguish betwe en primary an d recu rrentherpes simplex infe ction s. Virus isolation in tissue cu ltures is the go ld standardfor identifying herp es simple x virus but it is not 100 specific or 100sensitive. R estriction end onu ctea se analysis identifies types an d strains o f virusby their deoxyribo nucleic acid compo sition and it is very usefu l inepidemiologic studies. Ability to find virus by whateve r me thod is influencedby the stage of the lesion. As lesions age, less infectivity and antigen resultin less sensitivity o f th e tests. (J AM A CAD DERMATOL 1988;18:218-21 .)

I h a v e s o r t e d t h ro ug h th e m a n y d i f f e re n t l a b o -ra to ry tes ts used fo r d iagnos ing herpe t ic in fec t ionsto d is t i l l them down to the ones tha t would beusefu l to the p rac t ic ing dermato log is t . The tes tscan be d iv ided in to four bas ic types : (1 ) morpho-log ic , (2 ) immunom0rpholog ic , (3 ) se ro log ic , and(4) viro log ic. 1,2

From the D epartment of Dermatology University of Texas MedicalBranch.

Reprint requests to: Alvin R. Solom on M .D. Department of Der-matology University of Texas M edical Branch Galveston TX77550.

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I s t a r te d w i th t h e mo rp h o lo g ic t y p e o f d ia g n o s t i cme th o d o lo g ie s t h a t i s t y p if i e d b y th e T z a n c ks me a r, a t e c h n iq u e u s e d b y ma n y p h y s i c i a n s , e s -p e c i a l l y d e rma to lo g i s t s ) S o me o f t h e c h a ra c t e r -i st ic s a n d s p e c ia l f e a tu r e s o f t h e T z a n c k s m e a r a r e(1) the Tzanck smear i s a techn ique , no t a s ta in .S e v e ra l t y p e s o f s t a in s ma y b e u s e d , a n d (2 ) t h es p e c ime n i s o b t a in e d b y s c r a p in g th e l e s io n s , a ne a s y t e c h n iq u e w i th l o w mo rb id i ty ; ( 3 ) t h e t e c h -n i c a l d i f fi c u l ty o f d o in g th e T z a n c k p r e p a ra t i o n i slow; (4 ) the in te rpre ta t ive d i ff icu l ty w i th th e tech-n iq u e i s h ig h ly v a r i a b l e a n d d e p e n d s o n th e e x -p e r i e n c e o f t h e i n t e rp r e t e r - -d i f f i c u l t w i th i n a c -

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Volume 18Nu mb er 1 Part 2January 1988

ests for herpes simplex a nd varicel la zoster2 1 9

c u r a t e i n t e r p r e t a t i o n s i f t h e i n t e r p r e t o r i s n o t e x -p e r i e n c e d ; ( 5 ) t h e t e s t i s n e a r l y u n i v e r s a l l y

a v a i l a b l e . T h e o n l y e q u i p m e n t r e q u i r e d i s a m i -c r o s c o p e a n d a w a y t o d o t h e s ta i n s e i th e r m a n u a l l yo r a u t o m a t e d ( b l o o d f i l m s t a i n e r s ) ; ( 6) th e t i m e f o rd o i n g t h e t e s t i s v e r y s h o r t - - a f e w m i n u t e s ; ( 7 )t h e t e s t i s i n e x p e n s i v e ; ( 8 ) t h e s e n s i t i v i t y o f t h et e s t i s v e r y g o o d b u t t h e p e r c e n t p o s i t i v i t y o f t h et e s t v a r i e s w i t h t h e s t a g e o f t h e l e s i o n . P o s i t i v i t yi s 7 0 o r m o r e i n v e s i c l e s , a b o u t 5 5 i n p u s t u l e s ,a n d v e r y l o w i n c r u s t s , a l t h o u g h s o m e c r u s te d le -s i o n s a r e p o s i t i v e i n h e r p e s s i m p l e x i n f e c t i o n s ; (9 )t h e s p e c i f i c i t y i s h i g h , th e r e b e i n g a b o u t 3 f a l s ep o s i t i v e t e s t r e s u l t s i n e x p e r i e n c e d h a n d s ; (1 0 ) iti s a c o s t - e f f e c t i v e w a y o f d e t e r m i n i n g t h a t a h e rp e ss i m p l e x v i r u s o r v a r i c e l l a z o s t e r v i r u s i n f e c t i o ni s p r e s e n t b u t i t c a n n o t d i f f e r e n t i a t e b e t w e e nh e r p e s 1 a n d h e r p e s 2 o r v a r i c e l l a z o s te r v ir u sin fe c t i on s . 4-6

T h e T z a n c k t e st h a s a v a r y i n g d e g r e e o f u se rf r i e n d l i n e s s . T h e n o v i c e m a y w i s h t o r e l y o n fi n d-i n g m u l t i n u c l e a t e d g i a n t v i m c y t e s w i t h m o l d e d n u -c l e i b e c a u s e t h e s e a r e t h e m o s t p a t h o g n o m o n i c .T h e m o r e e x p e r i e n c e d p e r s o n r e c o g n iz e s a so r t o fh o m o g e n i z a t i o n o f n u c l e a r c h r o m a t i n o r g r o u n d

g l a s s a p p e a r a n c e t h a t o c c u rs i n i n f e c t e d e p i d e rm a lc e l l s a n d i n m o n o n u c l e a r c e l ls a s w e l l . I n t ra n u c l e a ri n c l u s i o n s ( C o w d r y t y p e A ) a r e n o t s e e n w i t h a n yd e g r e e o f r e g u l a ri t y w h e n m o n o c h r o m a t i c s t ai n sa r e u s e d . T h e t y p e o f s t a in t o u s e i s l a rg e l y a m a t t e ro f p e r s o n a l p r e f e r e n c e . I t e n d to u s e m e t h y l e n eb l u e . H e m a t o x y l i n - e o s i n s t a in c an . b e u s e d b u t i ti s m o r e d i f fi c u l t t o d o . T h e R o m a n o v s k y s t ai n s ,s u c h a s t h e G i e m s a a n d Wr i g h t s t a in s , g i v e g o o dr e s u l t s a n d a r e e a s y t o u s e i f y o u h a v e a n a u t o m a t i cb l o o d f i l m s t a in e r. T h e b e s t s t a i n t o u se f o r n u c l e a rm o r p h o l o g y a n d d e t a i l i s t h e P a p a n i c o l a o u s t a i n ,w h i c h m a k e s t h e s m e a r e a s i e r t o i n t e r p r e t . T h i ss t a i n c a n b e d o n e i n t h e o f f i c e b u t i t i s m o r e d i f -f i c u l t t o d o . O n e m u s t f i x t h e s m e a r i m m e d i a t e l yi n a l c o h o l . I t i s a m u l t i s t e p s t a i n , i t t a k e s l o n g e rt o d o , a n d t h e c o s t i s h i g h e r, b u t s p e c i f i c i t y a n da n d s e n s i t i v i t y a r e a b o u t t h e s a m e a s f o r t h e o th e r s .

T h e n e x t p r o c e d u r e i n th e m o r p h o l o g i c d i a g -n o s i s o f h e r p e s i s t h e b i o p s y. B i o p s y i s n o t o f t e nd o n e ( o r h e lp f u l ) w h e n c l a s s i c l e s io n s o f h e rp e ss i m p l e x , h e r p e s z o s t e r, o r v a r i c e l l a a re p r e s e n t . I ti s o f g r e a t e s t v a l u e w h e n t h e g r o s s m o r p h o l o g y o f

t h e l e s i o n s i s n o t d i a g n o s t i c o r c h a r a c t e r i s t i c a n do n e i s s e a r c h i n g f o r c l u e s t h a t m i g h t l e a d t o t h e

d i a g n o s i s . T h i s i s m o s t l i k e l y t o b e t h e c a s e i n t h ee v e r - i n c r e a s i n g n u m b e r o f i m m u n o c o m p r o m i s e dp a t i e n t s w h o s e h e r p e t i c l e s i o n s a r e b i z a r r e a n d f a rf r o m t h e c l as s i c c li n i c a l a p p e a r a n c e . T h e b i o p s yc a n b e d o n e b y s h a v e , p u n c h , o r w e d g e e x c i s i o nt e c h n i q u e s . I n t e r p r e t a t i o n o f t h e b i o p s y s p e c i m e nm a y b e e a s i e r t h a n i n t e r p r e t a ti o n o f T z a n c k s m e a r sb e c a u s e o n e i s a b l e t o e v a l u a t e h i s t o l o g ic a s w e l la s c y t o l o g i c f i n d i n g s . I t m a y t a k e 2 4 h o u r s t o g e tt h e s p e c i m e n s b a c k . S e n s i t i v i t y a n d s p e c i f ic i t yw i t h b i o p s y s p e c i m e n s a r e a b o u t t h e s a m e a s w i t hs m e a r s . P o s i t i v e b i o p s y r e s u l t s s h o w m u l t i n u -c l e a t e d g i a n t c e l l s , g i a n t k e r a t i n o c y t e s , a n d t y p i c a ln u c l e a r c h a n g e s . S o m e o f t h e e a r l i e r a n d m o s tc h a r a c t e r i s t i c c h a n g e s a r e i n t h e h a i r f o l l i c l e sr a t h e r t h a n t h e s u r f a c e e p i d e r m i s .

A n o t h e r m o r p h o l o g i c t e s t i s e l e c t r o n m i c r o s -c o p y. I t i s e x p e n s i v e a n d r e q u i r e s c o n s i d e r a b l ee x p e r i e n c e . I f t h e l a b o r a t o r y i s s e t u p t o d o i t , t h er e s u l ts c a n b e o b t a i n e d i n 2 h o u r s . S e n s i t i v i t y a n ds p e c i f i c i t y v a r y w i t h e x p e r i e n c e . I t d o e s n o t d i f -f e r e n t i a t e b e t w e e n h e r p e s s i m p l e x a n d v a r i c e l l az o s t er v i ru s e s . E l e c t r o n m i c r o s c o p y i s u s u a l l y o f

l i m i t e d v a l u e i n m o s t c l i n i c a l s i t u a t i o n s .I m m u n o m o r p h o l o g i c t e c h n i q u e s 7 a re v e r y g o o d

a n d a r e b e c o m i n g m o r e p o p u l a r. E i t h e r s m e a r s o rh i s to l o g i c s e c t i o n s c a n b e u s e d . M a n y d i f f e r e n tt e c h n i qu e s h a v e b e e n d e s c r i b e d . T h e p e r o x i d a s e -a n t i p e r o x i d a s e a n d t h e a v i d i n - b i o t i n m e t h o d s a r eu s e d m o s t o ft e n . T h e r e a g e n t s a r e c o m m e r c i a l l ya v a i l a b l e s o t h a t t h e l a b o r a t o r y n o l o n g e r h a s t or a i se it s o w n a n t i b o d i e s . F i x e d t i s s u e s a n d s m e a r s( f or m a l i n o r a c e t o n e , e t c . ) c a n b e u s e d . T h e t e c h -n i c a l d if f i c u l ti e s o f d o i n g t h e t e s t a r e h i g h , b u t t h em o r e e x p e r i e n c e t h e l a b o r a t o r y h a s t h e e a s i e r i t i st o d o i t . I n t e r p r e t a t i o n a l d i f f i c u l t i e s a r e l o w b e -c a u s e t h e t es t r e s u lt s a r e u s u a l l y d e t e r m i n e d t o b ee i t h e r p o s i t i v e o r n e g a t i v e . T e s t re s u l t s a r e u s u a l l ya v a i l a b l e w i t h i n 4 t o 5 h o u r s w i t h s m e a r s a n d4 t o 5 h o u r s w i t h b i o p s i e s a f t e r t h e y h a v e b e e n c u ta n d s e c t i o n e d . T h e t e s t s a r e r e l a t i v e l y i n e x p e n s i v e .Te s t r e s u l t s a r e p o s i t i v e i n a b o u t t w o t h i r d s o fs p e c i m e n s a n d t h e r e i s a h i g h d e g r e e o f s p e c i f i c i ty.P o t e n t i a l l y t h e s e t e s t s c a n d i s t i n g u i s h b e t w e e nh e r p e s s i m p l e x t y p e 1 a n d t y p e 2 a n d v a r i c e l l az o s te r v i r e s , b u t n o t a l l o f t h e c o m m e r c i a l a n t i -

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2 2 olomon

Journal of themer ican cademy o f

Dermato logy

bodies will accurately dist inguish between herpestypes 1 and 2 . T hese tests use chrom ogens that areactivated by the im muno logic reactants so that theresults o f the tests are easy to in terpret . M ost sys-tems a l low f ix ing in permanen t mount ing mediato give better cytologic detail and provide a per-ma nen t record .

Imm unofluorescen ce '8 is also a good te chniq uethat is used by many. The main disadvanta ges are(1) requiremen t for a fluorescence mic roscop e, (2)biopsy materials are required to be frozen o r f ixedin special media, (3) considerable experience isrequire d to in terpret specimens, a nd (4) addit ional

t ime is requ ired fo r eye accommodat ion . Gener-ally, antigen in t issue reacts with fluorescein-tagged antibody that is d irected against v iral an-t igens in the direct test. In the in direc t test, anti-body to the virus is added to virus-containingtissues or cells and this primary antibody is thendetecte d and amplified by a f luorescein-tag gedsecondary an t ibody to the p r imary an t ibody.F luorescein - tagged monoclonal an t ibod ies to her-pes simplex types 1 and 2 and varice lla zoster v irusare now available commercially.

Serologic tests p lay only a small ro le in the

diagnosis of herpes simplex infe ctions. 9 Th ey areused p r imar i ly to document p r imary in fectionswh en antibodies are absent at the onset o f the cl in-ical or subclinical infection and ap pear during i tscourse. A fourfold or greater antibod y rise is usu-ally required for a diagnosis of a prim ary infection.In recurrent herpes simplex infections, < 5 ofthe patients have significant r ises in antibod ylevels. Essen tially all patients wi th rec urre ntherpes have detectib le serum antibodies so that anegative test result is strong evidence against adiagnosis of recurrent herpes. In addit ion, in som eserolo gic tests, antibodies to herp es type 1 andherpes type 2 cross-react , and these antibodies ma yalso cross-react with antibodies to varicella zostervirus.

Th e com plem ent f ixation test for serum a nti-bod ies has been the s tandard and most widelyused, but i t is less sensit ive than m an y o f the othe rtests. N eutralization, d irect hem agglu tination ,and passive hem agglutination tests are availab le.Enzym e-l inked immunosorben t assay (ELISA )and western b lo t techn iques may become widely

avai lab le soon . With monoclonal an t ibod ies theE L IS A test can d e te rm in e an t ib o d y im m u n o g lo b -u l in sub type levels . W estern b lo t analys is can de-termine antibody levels to specific herpes simplexv irus po lypep t ide an t igens . Ano ther p rom ising tes ti s the detect ion o f low levels o f herpes v i rus an -t igen in vesicle f lu id using techniques such asELISA,10 agar gel immunodiffus ion , coun tercur-ren t immunoelect rophores is , and deoxyr ibonu-cleic acid (DNA) probes.11.12

Virus isolation in t issue culture is currently thereference method o r g o ld s tandard fo r v i rus iden-tification and diag nosis . 1.2.5,6 Ho we ve r, viral iso-

lat ion is not alway s 100 sensit ive and 100 spe-c i fic . Virus may be p resen t in the pat ien t bu t no tshow up in the cultures for various reasons in-c lud ing (1 ) the v i rus m ay no t su rv ive inappropr ia tet ransport cond i t ions , (2 ) the sample m ay hav e beentaken f rom the wrong les ion o r area , (3 ) there mayhave been too l i t t le v irus in the lesions (i .e. ,crusted, almost healed lesions), and (4) antiviraltherapy may have e l iminated the v i rus .

Recogn i t ion o f herpesv i rus in t i ssue cu l tu resusual ly depends on the appearance o f a cy topath iceffect (v i ra l acan tho lys is , mul t inucleated g ian t

cel l s , in t ranuclear inclus ions , nuclear changes ,etc.) . This cytopathic effect is usually seen withherpes s implex v i rus in 24 to 48 hours bu t maytake as long as 7 days. Varicella zoster v irus m aytake 1 to 3 weeks to p roduce character is t icchanges. Ne we r techniq ues 13 to speed u p d iagnosisinclude add ing tagged an t ibody to cu l tu res to de-tect v iral antigen before a cyto pathic effect appearsand adding antiviral drugs that specifically in-hibit growth of certain types of v iruses. For in-s tance , E-5(2-bromoviny l) -2 'deoxyur id ine inh ib -i ts the g rowth o f herpes s implex v i rus type 1 m orethan herpes s implex v i rus type 2 to the po in t w hereit can be used in diagnosis. Isolation of virus intissue cultures and subsequent identif ication byspeci f ic f luorescein - tagged m ono clona l an t ibod iesresults in greater specifici ty and sensit iv ity.

Rest r ic tion endonu clease analys is i s a techn iquethat iden ti f ies v i ruses by thei r DNA composi t ion .I t has g reat po ten t ia l fo r becoming an impor tan tad junct in the c l in ical laborato ry d iagnosis o f her-pesvirus infections in the near future.

There are several reasons fo r typ ing herpes s im-

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