DIAGN MICROBIOL INFECT DIS 149 1988;10:149-158

VIROLOGY

Laboratory Studies of Acute Varicel la and

Varicel la Immune 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 spec- imens 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-to- positivity 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 vaccine- induced 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 pri- mary (varicella) and reactivation (herpes zoster) forms. Varicella is the most common 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 com- pleted 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

150 H .H . Balfour, Jr., et al.

Virology Laboratory, we had a unique oppor tuni ty to evaluate our VZV culture and serum ant ibody methods.

MATERIALS AND METHODS

Patients

Otherwise normal ch i ld ren aged 12-24 mo who par t ic ipated in field trials of Oka Merck strain var icel la vaccine be tween August 1984 and August 1986 were bled at enrol lment , 6-12 wk later, and somet imes 1 yr later. Between February and June 1987, o therwise normal ch i ld ren aged 5-16 yr wi th acute varicel la were enrol led in a randomized , p lacebo-control led, double-b l ind s tudy of oral acyclovir pediat r ic suspens ion (40 mg/ml) for t reatment of acute varicella. Lesion aspirates for cul ture were col lected from these ch i ld ren on the first day of their exanthem, which was the day of enrol lment in the study. Sera were also obtained then and again 4 wk later for de terminat ion of VZV ant ibody titers. Also, sera submit ted from pat ients and personnel of the Univers i ty of Minnesota Hospi tal and Clinic for rout ine VZV an- t ibody testing were u t i l ized in comparat ive evaluat ions of the ant ibody methods. These studies were approved by the Universi ty of Minnesota Commit tee on the Use of Human Subjects in Research, and informed consent was obtained from the subjects and/or their guardians before par t ic ipat ion.

VZV Cultures

Up to six vesicular lesions were cul tured per spec imen using a tubercul in syringe. Each vesicle was gently punc tured with a 25-gauge needle kept near ly paral le l to the skin surface wi th bevel facing downward . The base of each lesion was gently scraped as fluid was being aspi ra ted into the syringe. The syringe then was sent as soon as possible (usually wi th in 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 med ium wi th Earle 's Salts, (later modif ied 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 penic i l l in G potassium). This m e d i u m was aspira ted twice into the syringe and reinjected into the tube. After 10 min at room temperature, 0.1 ml (later modif ied to 0.2 ml; see Results) was inocula ted into each of two tubes of human foreskin fibroblast cells. These cells were der ived from c i rcumcised tissue col lected in our nursery and were used be tween the 12th-25th cell cul ture passage. The inocula ted cell cul tures were incubated at 36°C in a 5% CO2 atmosphere and observed dai ly for characteris t ic 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 unti l tested. Sera col lected at enro l lment were tested s imul taneous ly wi th second specimens obtained 4-12 wk later and, in some instances, 1 yr later. Manufacturers ' ins t ruct ions were fol lowed precisely when commercia l kits were used. The fluorescent an t ibody to membrane antigen test (FAMA) discovered by Wi l l iams et al. (1974) has been our "gold s tandard" since 1977. Therefore, the other serological methods we invest igated were compared direct ly wi th FAMA. For b l inded studies, test sera were assigned a code number by a technologis t who d id not perform the VZV ant ibody tests. Results were evaluated after sera had been comple te ly tested by both methods. Diagnosis of an acute VZV infection was based on a fourfold or greater rise in FAMA titers or a Whit taker ELISA seroconvers ion ratio (convalescent predict ive index d iv ided by acute predic t ive index) 91.65.

Varicella Diagnosis and Immune Status Testing 151

FAMA Test

Our "s tandard" FAMA test is that of Wil l iams et al. (1974), wi th minor modifications. The FAMA antigen was the KMcC strain of VZV (a candidate vaccine strain deve loped by Merck). Sera usual ly were tested beginning at a 1:2 di lut ion. Fluorescence was graded from 1+ to 4 + . A 1+ fluorescence was defined as the lowest in tensi ty of specific apple-green membrane fluorescence on the VZV-infected cells. The fluores- cent intensi t ies of pat ients ' sera were graded by compar ing them with t i tered control sera. Titers were expressed as the reciprocal of the last d i lu t ion showing a 1+ fluorescence. Subjects wi th FAMA titers t>4 were considered to be immune or to have vacc ine- induced antibody.

Viran FAMA Test

The Viran FAMA, a commercia l ly available kit, is a modif icat ion of the s tandard FAMA. Cells infected with the 1B strain of VZV were fixed to masked slides. Ant ibody was detected indi rec t ly using goat an t ihuman IgG (antiheavy and light chains) con- jugated with f luorescein isothiocyanate. Sera were tested starting at a 1:2 di lut ion. Fluorescence was graded from 1 + to 4 + . Titers were expressed as the reciprocal of the last d i lu t ion showing a 1+ fluorescence. The same criteria for grading fluores- cence as the s tandard FAMA were used.

Merck Enzyme-linked Immunosorbent Assay

The Merck Sharp and Dohme Research Laboratories enzyme- l inked immunosorben t assay (ELISA) is a modif icat ion of the method of Shehab and Brunell (1983). This ELISA employed the KMcC strain of VZV attached to 96-well flat bot tom (Nunc) microplates to which 1:100 serum di lut ions were added. After incubat ion, alkaline phosphatase conjugated goat an t ihuman IgG was added fol lowed by p-ni t rophen- y lphospha te as the color substrate. Color formation was measured spectrophoto- metr ical ly at 405 nm. To derive ant ibody units from OD readings, a s tandard curve was constructed using a high-t i tered serum from a convalescent herpes zoster pat ient tested in serial twofold di lut ions starting at 1:10,000. Merck units of <2.5 were considered negative and units of I>2.5 were considered posi t ive for VZV antibody.

Whittaker M.A. Bioproducts ELISA

This VZV ELISA system is a commercia l ly available kit (Varicella-Stat, Whit taker M. A. Bioproducts Inc, Walkersvi l le , MD) that employs a modif icat ion of the Voller and Bidwell method (Voller and Bidwell , 1975) for detect ion of VZV-specific ant ibody. Ellen strain VZV grown in cell culture was bound to 8-well strips that were p laced in a 96-well holder (12 strips/plate). Sera d i lu ted at 1:21 were added to the wells. An t ibody was detected by the indirect method using an alkal ine phosphatase con- jugated an t ihuman IgG and phenophtha le in monophospha te as substrate. Plates were read spect rophotometr ica l ly (550 nm), and a predic ted index was calculated ut i l iz ing the s tandard line and l inear regression. A predic ted index />1.00 was considered immune for VZV.

Statistics

For compar ison of proport ions, the X 2 test wi th Yates' correct ion was employed. Time-to-event analysis was done by the Wilcoxon rank-sum test; p values ~<0.05 were considered to be significant.

152 H . H . Balfour, Jr., et ah

TABLE 1. Recove ry of VZV f rom O t h e r w i s e Hea l thy Ch i ld r en on First Day of Var ice l la

Culture method No. cultured No. positive a Mean days-to-positive ~

Original 57 29 (51%) 5.6 Modified 45 36 (80%) 3.8

Total 102 65 (64%) 4.6

°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

Only 29 (51%) of the l es ion cu l tu res f rom the first 57 c h i l d r e n en ro l l ed in the acyc lov i r t r ea tment p ro toco l were pos i t ive (Table 1). Because that v i ra l i so la t ion rate was l ower than an t ic ipa ted , an in t e r im r e v i e w of the data was c o n d u c t e d and the m e t h o d al tered. It was d e c i d e d that the con ten t s of the syr inge con t a in ing ves i cu la r f luid and scrapings s h o u l d be e x p e l l e d into 0.5 ml of m a i n t e n a n c e m e d i u m ins tead of 1 ml, and that 0.2 ml ra ther than 0.1 ml shou ld be i n o c u l a t e d into each of two tubes con t a in ing SF cells. Af ter the m e t h o d was modi f i ed , 36 (80%) of 45 subsequen t cu l tu res were pos i t ive . Th is r ecove ry rate was s igni f icant ly h igher t han that of the first g roup 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, cu l tures co l l ec t ed f rom the last 45 c h i l d r e n also b e c a m e pos i t ive s igni f icant ly sooner (p < 0.001, W i l c o x o n rank s u m test).

Comparison of Standard FAMA and Viran FAMA Tests

Cons ide r ing the resul ts as i m m u n e or not i m m u n e ( i m m u n e status), the sens i t iv i ty of the Viran c o m p a r e d w i t h F A M A was 93.3% (42/45); the speci f ic i ty was 81.3% (13/16). A b l i n d e d s tudy us ing s p e c i m e n s f rom 61 pa t ien ts was p e r f o r m e d to c o m p a r e the Viran and F A M A ti ter va lues (Table 2). Three s p e c i m e n s were d i s c r epan t for

TABLE 2. B l i nded C o m p a r i s o n of S tandard F A M A and Viran F ixed-ce l l F A M A in 61 Pat ients

Viran titers

<2 2 4 8 16 32 64 128 256 512

Standard FAMA Titers

<2 13 2 4 1 ° 8 I a 16 1 ° 32 64 128 256 512

6 1 3 3 1 1

2 3 3 1 1 1 4

1 2 1

°Results were descrepant for immune status.

Varicella Diagnosis and Immune Status Testing 153

TABLE 3. Blinded Comparison of FAMA and Merck ELISA Antibody Quanti tat ion in 106 Children Participating in Oka Merck Varicella Vaccine Trial

Merck ELISA units

<2.5 2.5-5 5.5-10 11-20 21-40 41-80 81-160 161-320

Standard FAMA Titers <2 16

2 2 4 3 ° 8 2 a 16 I a 32 64 128

I a

1 2 6 5 1 6 7 4

4 20 2 8

°Results were discrepant for immune status.

immune status. Although there was excellent concordance when 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, when the 16 lowest positive Viran titers were retested, seven were at least a fourfold di lut ion different than the previous results. This led us to question the reproducibil i ty 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 inded 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 immune 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 ant ibody induced 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 post immunizat ion samples (Table 4). The Whittaker ELISA antibody method therefore was abandoned for vaccine field studies, but was evaluated further for detection of naturally induced VZV antibodies using specimens routinely sub- mitted 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 un- known. Five samples were discrepant as to immune 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 deter- minat ion of immune status, the sensitivity of the Whittaker ELISA was 95% (83/87); its specificity was 92% (12/13).

A bl ind comparison of the standard FAMA and Whittaker ELISA was done uti- lizing 98 paired acute and convalescent sera from children with acute varicella enrolled in a placebo-controlled acyclovir treatment protocol. For this comparison,

154 H.H. Balfour, Jr., et al.

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 <2 - 0.15 10/18/84 32 1.13 10/4/85 8 0.65

2 9/16/84 <2 - 0.09 10/30/84 16 - 0.09 10/22/85 8 0.79

3 9/1/84 <2 - 0.09 10/23/84 8 - 0.09 9/24/85 4 - 0.18

4 6/11/85 <2 0.09 7/23/85 32 0.95

5 6/18/85 <2 0.06 8/6/85 32 0.98

6 7/8/85 <2 0.06 9/8/85 32 1.44

a>l.00 is positive.

sera were tested by the FAMA method beginning at a di lut ion of 8. Paired sera from all of the subjects fulfilled the criteria for acute infection in the ELISA system (sero- conversion 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 s imultaneously measured convalescent titer was 1,024. That patient 's ELISA seroconversion index was 1.80. Concordance of antibody quan- titation was quite good, as shown 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 immunocompromised 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., unpub l i shed observation). However, we believe that with appropriate collection, transport, inoculation, and microscopic examination, VZV can be isolated from at least 80% of otherwise healthy chi ldren with acute varicella.

Our VZV culture data are un ique because no large-scale virologic study of varicella treatment in otherwise healthy children has been published. The enrol lment criteria and study design of the placebo-controlled acyclovir treatment trial permitted us to obtain vesicular cultures wi th in 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 inocu lum from the lesion provided a better opportuni ty for extracellular VZV to attach and penetrate the huma n foreskin fibroblast cells before the virus lost infectivity. To obtain the specimen itself, we recommend that personnel collecting the culture gently scrape the base of the

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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 di- agnosis 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 immuno- fluorescence (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 immunoflu- orescence 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 Whit- taker 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 neg- atives 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 pos- itives might erroneously permit an employee to continue to work during the incu- bation 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 in- creased 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.

158 H.H. Balfour, Jr., et al.

Although we only t e s t ed /8 chi ldren for immune status, ELISA indices in chi ldren should be higher than adults, because chi ldren 's experiences with primary VZV almost always has been more recent. In that regard, the ELISA indices among 98 chi ldren during the convalescent phase of varicella were very high (Table 6). There- fore, we have reason to believe that the Whittaker ELISA will perform adequately for detection of immune 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 immu- nofluorescence. 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 varicella- zoster 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 immunosorbent assay 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.