Laboratory studies of acute varicella and varicella immune status

DIAGNMICROBIOLINFECTDIS 1988;10:149-158

149

VIROLOGY Laboratory Studies of Acute Varicella and Varicella I m m u n e Status Henry H. Balfour, Jr., Charlene K. Edelman, Cynthia L. Dirksen, Donna R. Palermo, Carmen S. Suarez, Joan Kelly, Jan T. Kentala, and Deborah D. Crane

We evaluated varicella-zoster virus (VZV) culture and serum antibody methods utilizing specimens from 620 children enrolled in protocols for prevention or treatment of varieella and samples routinely submitted to the Clinical Virology Laboratory. In a foreskin fibroblast tube culture system, we initially isolated VZV from only 29 (51%) of 57 children cultured on the first day of varieella. After modifying the method, the proportion of culture-positive children increased significantly to 36 (80%) of 45 (p < 0.005 by corrected X2), and the median days-topositivity were significantly shortened from 5.6 to 3.8 days (p < 0.001, Wilcoxon rank-sum test). The Viran fluorescent antibody to membrane antigen (FAMA) assay was difficult to read and not reproducible. The standard FAMA was more sensitive than the Merck ELISA antibody test for detecting vaccine-induced antibody. The Whittaker ELISA did not detect vaccineinduced antibody but was comparable to FAMA for immune status testing (sensitivity, 95%; specificity, 92%) and for diagnosis of acute varicella.

INTRODUCTION Varicella-zoster virus (VZV) causes substantial worldwide morbidity in both its primary (varicella) and reactivation (herpes zoster) forms. Varicella is the most c o m m o n exanthematous disease of childhood in the United States with approximately 3.5 million cases occurring each year (Preblud, 1986). Although varicella is usually mild, otherwise healthy children may experience a difficult clinical course. Approximately 4,000 youngsters are hospitalized in the United States each year due to complications of primary VZV infection (Preblud, 1986). Because of this, and because varicella is routinely severe and frequently life-threatening in immunocompromised children (Feldman and Lott, 1987), strategies for prevention or treatment would be desirable. We have been involved in both the preventive and therapeutic aspects of varicel|a control. Between 1984 and 1986, our group conducted field studies of Oka Merck strain live attenuated varicella vaccine (Englund et al., 1988) and in 1987 we completed a randomized, placebo-controlled, double-blind varicella treatment protocol to assess the efficacy of oral acyclovir pediatric suspension. Utilizing specimens for those studies involving 620 children and samples routinely submitted to the Clinical From the Department of Laboratory Medicine and Pathology, and Department of Pediatrics, University of Minnesota Health Sciences Center, Minneapolis, Minnesota. Address reprint requests to: Dr. H. H. Balfour, Jr., Box 437 UMHC, University of Minnesota, Minneapolis, MN 55455. Received May 12, 1988; revised and accepted July 22, 1988. © 1988 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10017

0732-8893/88/$03.50

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Virology Laboratory, we had a unique o p p o r t u n i t y to evaluate our VZV culture and serum antibody methods.

MATERIALS AND METHODS Patients Otherwise normal c h i l d r e n aged 12-24 mo who participated in field trials of Oka Merck strain varicella vaccine b e t w e e n August 1984 and August 1986 were bled at enrollment, 6-12 w k later, and sometimes 1 yr later. Between February and June 1987, otherwise normal c h i l d r e n aged 5-16 yr w i t h acute varicella were enrolled in a r a n d o m i z e d , placebo-controlled, d o u b l e - b l i n d study of oral acyclovir pediatric s u s p e n s i o n (40 mg/ml) for treatment of acute varicella. Lesion aspirates for culture were collected from these c h i l d r e n on the first day of their exanthem, w h i c h was the day of enrollment in the study. Sera were also obtained then and again 4 w k later for d e t e r m i n a t i o n of VZV a n t i b o d y titers. Also, sera submitted from patients and personnel of the University of Minnesota Hospital and Clinic for routine VZV ant i b o d y testing were utilized in comparative evaluations of the a n t i b o d y methods. These studies were a p p r o v e d by the University of Minnesota Committee on the Use of H u m a n Subjects in Research, and informed consent was obtained from the subjects and/or their guardians before participation.

VZV Cultures Up to six vesicular lesions were cultured per s p e c i m e n using a tuberculin syringe. Each vesicle was gently p u n c t u r e d with a 25-gauge needle kept nearly parallel to the skin surface w i t h bevel facing d o w n w a r d . The base of each lesion was gently scraped as fluid was being aspirated into the syringe. The syringe then was sent as soon as possible (usually w i t h i n 15 min) to the Clinical Virology Laboratory, where its contents were injected into a tube containing 1.0 ml of Eagle's m i n i m u m essential m e d i u m w i t h Earle's Salts, (later modified to 0.5 ml; see Results) 0.5% gelatin and antibiotics (2.5 mcg/ml amphotericin B, 0.05 mg/ml gentamicin sulfate, and 50 units/ml p e n i c i l l i n G potassium). This m e d i u m was aspirated twice into the syringe and reinjected into the tube. After 10 m i n at room temperature, 0.1 ml (later modified to 0.2 ml; see Results) was i n o c u l a t e d into each of two tubes of h u m a n foreskin fibroblast cells. These cells were derived from c i r c u m c i s e d tissue collected in our nursery and were used b e t w e e n the 1 2 t h - 2 5 t h cell culture passage. The inoculated cell cultures were incubated at 36°C in a 5% CO2 atmosphere and observed daily for characteristic VZV cytopathic effect for up to 2 wks.

General Methodology for VZV Antibody Tests Sera were separated from clots at room temperature and stored at - 70°C until tested. Sera collected at e n r o l l m e n t were tested s i m u l t a n e o u s l y w i t h second s p e c i m e n s obtained 4 - 1 2 w k later and, in some instances, 1 yr later. Manufacturers' instructions were followed precisely w h e n commercial kits were used. The fluorescent a n t i b o d y to m e m b r a n e antigen test (FAMA) discovered by W i l l i a m s et al. (1974) has been our "gold standard" since 1977. Therefore, the other serological methods we investigated were c o m p a r e d directly with FAMA. For b l i n d e d studies, test sera were assigned a code n u m b e r by a technologist w h o d i d not perform the VZV antibody tests. Results were evaluated after sera had been c o m p l e t e l y tested by both methods. Diagnosis of an acute VZV infection was based on a fourfold or greater rise in F A M A titers or a Whittaker ELISA seroconversion ratio (convalescent predictive i n d e x d i v i d e d by acute predictive index) 91.65.

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151

FAMA Test Our "standard" F A M A test is that of W i l l i a m s et al. (1974), with m i n o r modifications. The F A M A antigen was the KMcC strain of VZV (a candidate vaccine strain d e v e l o p e d by Merck). Sera u s u a l l y were tested beginning at a 1:2 dilution. Fluorescence was graded from 1 + to 4 + . A 1 + fluorescence was defined as the lowest intensity of specific apple-green m e m b r a n e fluorescence on the VZV-infected cells. The fluorescent intensities of patients' sera were graded by comparing t h e m with titered control sera. Titers were expressed as the reciprocal of the last dilution showing a 1 + fluorescence. Subjects with F A M A titers t>4 were considered to be i m m u n e or to have v a c c i n e - i n d u c e d antibody.

Viran FAMA Test The Viran FAMA, a c o m m e r c i a l l y available kit, is a modification of the standard FAMA. Cells infected with the 1B strain of VZV were fixed to masked slides. A n t i b o d y was detected i n d i r e c t l y using goat a n t i h u m a n IgG (antiheavy and light chains) conjugated with fluorescein isothiocyanate. Sera were tested starting at a 1:2 dilution. Fluorescence was graded from 1 + to 4 + . Titers were expressed as the reciprocal of the last dilution showing a 1 + fluorescence. The same criteria for grading fluorescence as the standard F A M A were used.

Merck Enzyme-linked Immunosorbent Assay The Merck Sharp and Dohme Research Laboratories e n z y m e - l i n k e d i m m u n o s o r b e n t assay (ELISA) is a modification of the method of Shehab and Brunell (1983). This ELISA e m p l o y e d the KMcC strain of VZV attached to 96-well flat bottom (Nunc) microplates to w h i c h 1:100 serum dilutions were added. After incubation, alkaline p h o s p h a t a s e conjugated goat a n t i h u m a n IgG was a d d e d followed by p-nitropheny l p h o s p h a t e as the color substrate. Color formation was measured spectrophotometrically at 405 nm. To derive antibody units from OD readings, a standard curve was constructed using a high-titered serum from a convalescent herpes zoster patient tested in serial twofold dilutions starting at 1:10,000. Merck units of <2.5 were considered negative and units of I>2.5 were considered positive for VZV antibody.

Whittaker M.A. Bioproducts ELISA This VZV ELISA system is a c o m m e r c i a l l y available kit (Varicella-Stat, Whittaker M. A. Bioproducts Inc, Walkersville, MD) that e m p l o y s a modification of the Voller and Bidwell m e t h o d (Voller and Bidwell, 1975) for detection of VZV-specific antibody. Ellen strain VZV grown in cell culture was b o u n d to 8-well strips that were placed in a 96-well h o l d e r (12 strips/plate). Sera diluted at 1:21 were a d d e d to the wells. A n t i b o d y was detected by the indirect m e t h o d using an alkaline p h o s p h a t a s e conjugated a n t i h u m a n IgG and p h e n o p h t h a l e i n m o n o p h o s p h a t e as substrate. Plates were read spectrophotometrically (550 nm), and a p r e d i c t e d index was calculated utilizing the standard line and linear regression. A predicted index />1.00 was considered i m m u n e for VZV.

Statistics For comparison of proportions, the X 2 test with Yates' correction was employed. Time-to-event analysis was done by the Wilcoxon rank-sum test; p values ~<0.05 were considered to be significant.

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TABLE 1. R e c o v e r y of VZV f r o m O t h e r w i s e H e a l t h y C h i l d r e n on First Day of Varicella Culture method

No. cultured

No. positive a

Mean days-to-positive ~

57 45

29 (51%) 36 (80%)

5.6 3.8

102

65 (64%)

4.6

Original Modified Total

°Proportion of positive cultures significantly greater for modified method: corrected ×2 = 7.96, p < 0.005. bTime-to-positive culture significantly faster for modified method by Wilcoxon rank sum test, p < 0.001.

RESULTS VZV Cultures O n l y 29 (51%) of t h e l e s i o n c u l t u r e s f r o m the first 57 c h i l d r e n e n r o l l e d in the a c y c l o v i r t r e a t m e n t p r o t o c o l w e r e p o s i t i v e (Table 1). B e c a u s e that viral i s o l a t i o n rate was l o w e r t h a n a n t i c i p a t e d , an i n t e r i m r e v i e w of the data was c o n d u c t e d a n d t h e m e t h o d altered. It was d e c i d e d that the c o n t e n t s of the syringe c o n t a i n i n g v e s i c u l a r fluid a n d s c r a p i n g s s h o u l d be e x p e l l e d into 0.5 m l of m a i n t e n a n c e m e d i u m i n s t e a d of 1 ml, a n d that 0.2 m l rather t h a n 0.1 m l s h o u l d be i n o c u l a t e d into e a c h of t w o tubes c o n t a i n i n g SF cells. After the m e t h o d was m o d i f i e d , 36 (80%) of 45 s u b s e q u e n t c u l t u r e s w e r e positive. T h i s r e c o v e r y rate w a s s i g n i f i c a n t l y h i g h e r t h a n that of the first g r o u p of c h i l d r e n (corrected X 2, 7.96; p < 0.005). As s h o w n in Table 1, c u l t u r e s c o l l e c t e d f r o m the last 45 c h i l d r e n also b e c a m e p o s i t i v e s i g n i f i c a n t l y s o o n e r (p < 0.001, W i l c o x o n r a n k s u m test).

Comparison of Standard FAMA and Viran FAMA Tests C o n s i d e r i n g the results as i m m u n e or not i m m u n e ( i m m u n e status), the s e n s i t i v i t y of the V i r a n c o m p a r e d w i t h F A M A was 93.3% (42/45); the specificity was 81.3% (13/16). A b l i n d e d s t u d y u s i n g s p e c i m e n s f r o m 61 p a t i e n t s was p e r f o r m e d to c o m p a r e the V i r a n a n d F A M A titer v a l u e s (Table 2). T h r e e s p e c i m e n s w e r e d i s c r e p a n t for

TABLE 2. B l i n d e d C o m p a r i s o n of S t a n d a r d F A M A a n d Viran F i x e d - c e l l F A M A in 61 Patients Viran titers <2 Standard FAMA Titers <2 13 2 4 1° 8 Ia 16 1° 32 64 128 256 512

2

4

8

16

6 3

1 3 2 1 1

1

°Results were descrepant for immune status.

32

64

1 3

1 1

2

128

3 4 1

256

512

153

Varicella Diagnosis and I m m u n e Status Testing

TABLE 3. Blinded Comparison of FAMA and Merck ELISA A n t i b o d y Quantitation in 106 Children Participating in Oka Merck Varicella Vaccine Trial Merck ELISA units <2.5

2.5-5

Standard FAMA Titers <2 16 2 2 4 3° 8 2a 16 Ia 32 64 128

5.5-10

11-20

21-40

2 5 7 4 2

1 4 20 8

41-80

81-160

161-320

Ia 1 6 6

°Results were discrepant for immune status. i m m u n e status. Although there was excellent concordance w h e n FAMA and Viran values were ~2, positive titers by the Viran method were 2- to 8-fold lower than the standard FAMA results. In addition, w h e n the 16 lowest positive Viran titers were retested, seven were at least a fourfold dilution different than the previous results. This led us to question the reproducibility of the method. Another problem with the Viran test was that the cell preparations were too heavy. Distinguishing truly positive fluorescing cells in clumps from nonspecific conjugate trapping was difficult.

Comparison of Standard FAMA and Merck ELISA Antibody Results The Merck ELISA test was compared quantitatively in a b l i n d e d study with the FAMA analyzing sara from 106 children who participated in an Oka Merck varicella vaccine trial (Table 3). Seven specimens were discrepant as to i m m u n e status. One specimen that had a Merck value of eight units was ~ 2 by FAMA, possibly representing a false negative FAMA result. On the other hand, six specimens that were negative by the Merck ELISA had FAMA titers between 4 and 16. Thus, the FAMA appeared to be more sensitive than the Merck ELISA for our vaccinated children.

Comparison of FAMA and Whittaker ELISA Antibody Results We were disappointed to find that the Whittaker ELISA was unable to detect antibody i n d u c e d by Oka varicella vaccine. Of the first six vaccinees tested, the Whittaker ELISA was weakly positive with two of the six convalescent sera and with zero of three oneoyr p o s t i m m u n i z a t i o n samples (Table 4). The Whittaker ELISA antibody method therefore was abandoned for vaccine field studies, but was evaluated further for detection of naturally i n d u c e d VZV antibodies using specimens routinely submitted to our Clinical Virology Laboratory (Table 5). Of the 100 patients tested, 81 were adults, 18 were children (age range, 2-17 yr), and one patient's age was unknown. Five samples were discrepant as to i m m u n e status. Four had FAMA titers of 4 or 8 but were negative by ELISA. One serum was negative by FAMA but had a positive Whittaker ELISA index. In comparison with the standard FAMA for determ i n a t i o n of i m m u n e status, the sensitivity of the Whittaker ELISA was 95% (83/87); its specificity was 92% (12/13). A b l i n d comparison of the standard FAMA and Whittaker ELISA was done utilizing 98 paired acute and convalescent sera from children with acute varicella enrolled in a placebo-controlled acyclovir treatment protocol. For this comparison,

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TABLE 4. Comparison of FAMA and Whittaker ELISA Antibody Results in Six Children Given Oka Merck Varicella Vaccine Vaccinee

Dates tested

FAMA titer

Whittaker ELISA Predictive Index°

1

8/20/84 10/18/84 10/4/85

<2 32 8

- 0.15 1.13 0.65

2

9/16/84 10/30/84 10/22/85

<2 16 8

- 0.09 - 0.09 0.79

3

9/1/84 10/23/84 9/24/85 6/11/85 7/23/85 6/18/85 8/6/85

<2 8 4 <2 32 <2 32

- 0.09 - 0.09 - 0.18 0.09 0.95 0.06 0.98

7/8/85 9/8/85

<2 32

0.06 1.44

4 5 6

a>l.00 is positive.

sera were tested by the FAMA method b e g i n n i n g at a d i l u t i o n of 8. Paired sera from all of the subjects fulfilled the criteria for acute infection in the ELISA system (seroconversion ratios ranged from 1.8 to 280). All but one of the 98 patients demonstrated a fourfold or greater rise in FAMA titers. The exception was a 9-yr-old girl whose acute FAMA titer was 512; her simultaneously measured convalescent titer was 1,024. That patient's ELISA seroconversion index was 1.80. Concordance of antibody quantitation was quite good, as s h o w n in Table 6, with the exception that five of seven acute sera positive by FAMA were antibody-negative by ELISA.

DISCUSSION Historically, VZV has been relatively difficult to isolate from patients. For example, VZV was cultured from only 59 (63%) of 94 i m m u n o c o m p r o m i s e d adults with early acute herpes zoster enrolled in a multicenter, placebo-controlled acyclovir treatment study (Balfour et al., 1983). Several of the 20 study centers collaborating in that trial were unable to obtain any positive cultures from their zoster patients (Balfour, H. H , Jr., u n p u b l i s h e d observation). However, we believe that with appropriate collection, transport, inoculation, and microscopic examination, VZV can be isolated from at least 80% of otherwise healthy children with acute varicella. Our VZV culture data are u n i q u e because no large-scale virologic study of varicella treatment in otherwise healthy children has been published. The e n r o l l m e n t criteria and study design of the placebo-controlled acyclovir treatment trial permitted us to obtain vesicular cultures w i t h i n a day after onset of the exanthem. By modifying our standard method so that more vesicular fluid was inoculated per tissue culture tube, we significantly increased the rate of viral recovery. The improved VZV isolation rate likely occurred because a larger volume of concentrated i n o c u l u m from the lesion provided a better opportunity for extracellular VZV to attach and penetrate the h u m a n foreskin fibroblast cells before the virus lost infectivity. To obtain the specimen itself, we r e c o m m e n d that personnel collecting the culture gently scrape the base of the

9 3 Ia 3a 5

1.00-1.25

1 6

ia

1.26-1,50

1 1 8

1.51-2.00

33

2.01-3,00

14

1

3,01-4.00

7

1

4.01-5.00

Tests on 100 Consecutive

W h i t t a k e r ELISA I n d i c e s

FAMA and Whittaker ELISA Antibody

°Results were discrepant for immune status.

S t a n d a r d F A M A Titers <2 2 4 8 ->16

0-0.99

T A B L E 5. C o m p a r i s o n o f S t a n d a r d Routine VZV Antibody Testing

3

5.01---6.00

for

6.01-8;00

Sera Submitted

~.~°

0~

91

Ia

1

Ia

1.26-1.50

of S t a n d a r d F A M A

1.00-1.25

°Results for convalescent sera.

Standard FAMA Titers <8 8 16 32 64 128 256 512 1024 ->2048

0.-0.99

with Varicella

T A B L E 6. B l i n d C o m p a r i s o n

4a

5a

1.51-2.00

2a

7a 17 a 2o

2a

Ia

4.01-5.00

5 .0 1 - 6 .0 0

Ia

6.01-7.00

Tests on Paired Sera From 98 Children

3.01-4.00

19 a 22 a 14 °

2.01-3.00

W h i t t a k e r ELISA i n d i c e s

and Whittaker ELISA Antibody

©

Varicella Diagnosis and Immune Status Testing

157

lesions with a 25-gauge needle as the vesicular fluid is being aspirated. The cells at the lesion base are laden with VZV, as aptly demonstrated by the Tzanck test utilized to diagnose herpesviral exanthems (Tzanck, 1947). Of course, it is unnecessary to order a viral culture on every child to confirm the diagnosis of chickenpox. We cultured all children who were participating in an acyclovir treatment study in order to verify the clinical diagnosis and validate the results. Analyses of the safety and efficacy of acyclovir in that trial are now underway. Varicella cultures could be valuable for solving certain medical dilemmas, such as what to do when a child on an oncology or transplant ward develops a few vesicular lesions or lesions with an atypical distribution. In such instances, the precise diagnosis is needed to determine whether other patients will require VZV prophylaxis and/or treatment. Also, epidemiologic investigations could utilize varicella cultures to establish the exact period of communicability and modes of transmission. Although airborne spread of varicella within a hospital has been demonstrated (Leclair et al., 1980), it is still not clear that the respiratory route is the major avenue for transmission of chickenpox within communities. Direct detection of VZV antigens by immunofluorescence (Drew and Mintz, 1980; Sadick et al., 1987), ELISA (Ziegler, 1984), or enzyme immunofiltration (Cleveland and Richman, 1987) is more rapid than culture and may replace viral isolation in the future for routine clinical diagnosis. The standard FAMA is a highly specific and sensitive antibody test. However, it is not ideal for a diagnostic virology laboratory because weekly preparations of fresh VZV-infected cells must be made and maintained. The anticomplement immunofluorescence test may be more practical, and it has been reported to be nearly as sensitive and specific as the FAMA (Preissner et al., 1982). The commercially available Whittaker ELISA is ideal for a diagnostic laboratory. In addition, it closely matched the FAMA results except for vaccinees. The manufacturer is planning to change the ELISA test antigen so that the assay will recognize antibody elicited by Oka strain varicella vaccine. The Merck ELISA appears to be comparable to the Whittaker assay in terms of sensitivity, but the Merck assay is not commercially available. In a recent study (Demmler et al., 1988) of the Whittaker ELISA versus FAMA utilizing 179 sera, most of which were from hospital personnel, the ELISA was reported to have a sensitivity of 86.2% and a specificity of 98.6%. These results are comparable to ours except that the proportion of false negative (1 minus sensitivity) ELISA results (15/109 or 13.8%) was higher than ours (4/87 or 4.6%). The relatively high proportion of false negatives in the ELISA test led Demmler et al. to recommend retesting ELISA-negative sera by FAMA. This may be advisable to reduce false negatives in order that employees not be excluded from work unnecessarily. However, a more important problem, as those authors point out, is false positive (1 minus specificity) ELISA results especially when screening hospital employees. False positives might erroneously permit an employee to continue to work during the incubation period of chickenpox, thus setting up the potential for nosocomial spread. The FAMA does appear to be more sensitive than the Whittaker ELISA for detection of antibody induced early during acute varicella. Seven of 98 children tested on the first day of varicella were seropositive by FAMA, whereas only 2/98 were seropositive by ELISA. The one apparently false negative FAMA result was from a 72-yr-old man with chronic myelomonocytic leukemia whose VZV antibody test was ordered at the time his malignancy was diagnosed but prior to initiation of chemotherapy. Because it has been inferred that varicella immunity wanes with time, as reflected by the increased frequency of herpes zoster in the elderly, it is possible that this patient's sera contained some but not all of the VZV antibodies elicited after natural infection many years ago. VZV antibodies that the ELISA method could detect still persisted, whereas the FAMA response had been lost with time.

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Although we only t e s t e d / 8 children for i m m u n e status, ELISA indices in children should be higher than adults, because children's experiences with primary VZV almost always has been more recent. In that regard, the ELISA indices among 98 children during the convalescent phase of varicella were very high (Table 6). Therefore, we have reason to believe that the Whittaker ELISA will perform adequately for detection of i m m u n e status as well as diagnosis of acute varicella in children. This study was supported in part by grants from Merck Sharp and Dohme Research Laboratories, Burroughs Wellcome Co., and the Minnesota Medical Foundation. Presented in part at the annual meeting of the Central Society for Clinical Research~ Chicago, Illinois, November 13, 1987.

REFERENCES Balfour HH, Jr., Bean B, Laskin OL, Ambinder RF, Meyers JD, Wade JC, Zaia JA, Aeppli D, Kirk LE, Segreti AC, Keeney RE, the Burroughs Wellcome Collaborative Acyclovir Study Group (1983) Acyclovir halts progression of herpes zoster in immunocompromised patients. N Engl J Med 308:1448-1453. Cleveland PH, Richmond DD (1987) Enzyme immunofiltration staining assay for immediate diagnosis of herpes simplex virus and varicella-zoster virus directly from clinical specimens. J Clin Microbiol 25:416-420. Demmler GJ, Steinberg SP, Blum G, Gershon AA (1988) Rapid enzyme-linked immunosorbent assay for detecting antibody to varicella-zoster virus. J Infect Dis 157:211-212. Drew WL, Mintz L (1980) Rapid diagnosis of varicella-zoster virus infection by direct immunofluorescence. Am J Clin Pathol 73:699-701. Englund JA Suarez CS, Kelly J, Tate DY, Balfour HH, Jr (in press) Placebo-controlled trial of varicella vaccine given with or following measles, mumps, and rubella vaccine. J Pediatr. Feldman S, Lott L (1987) Varicella in children with cancer: Impact of antiviral therapy and prophylaxis. Pediatrics 80:465-472. Leclair JM, Zaia JA, Levin MJ, Congdon RG, Goldmann DA (1980) Airborne transmission of chickenpox in a hospital. N Engl J Med 302:450-453. Preblud SR (1986) Varicella: Complications and costs. Pediatrics 78:728-735. Preissner CM, Steinberg SP, Gershon AA, Smith TF (1982) Evaluation of the anticomplement immunofluorescence test for detection of antibody to varicella-zoster virus. J Clin Microbiol 16:373-376. Sadick NS, Swenson PD, Kaufman RL, Kaplan MH (1987) Comparison of detection of varicellazoster virus by the Tzanck smear, direct immunofluorescence with a monoclonal antibody, and virus isolation. J Am Acad Dermatol 17:64-69. Shehab Z, Brunell PA (1983) Enzyme-linked immunosorbentassay for susceptibility to varicella. J Infect Dis 148:472-476. Tzanck A (1947) Le cytodiagnostic immediat en dermatologie. Bull Soc Franc Derm Syph 7:68. Voller A, Bidwell DE (1975) A simple method for detecting antibodies to rubella. Br J Exp Path 56:338-339. Williams V, Gershon A, Brunell PA (1974) Serologic response to varicella-zoster membrane antigen measured by indirect immunofluorescence. J Infect Dis 130:669-672. Ziegler T (1984) Detection of varicella-zoster viral antigens in clinical specimens by solid-phase enzyme immunoassay. J Infect Dis 150:149-154.