Isolation of varicella-zoster virus from blood

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and edematous or nodular. The vocal cords were rarely abnormal, which explains why airway obstruction rather than vocal changes usually occurs. In a few patients, laryngeal sarcoidosis was observed in the absence of other systemic manifestations of the disease. It is noteworthy that progression of the extrathoracic manifestations of our patient's disease occurred at a time when pulmonary involvement appeared to be stable. This coincides with the experience previously reported in adults. 6 Recognition of this phenomenon should encourage upper airway examination of children with increasing respiratory distress associated with sarcoidosis. Diminution of t h e laryngeal edema by large dose prednisone therapy prevented an anticipated tracheotomy. The recurrence of dyspnea while on a high-dose, alternate-day prednisone regimen, however, necessitated surgical excision of polypoid granulomatous tissue to prevent upper respiratory tract obstruction. Localization of the subglot-

Isolation of varicellazoster virus from blood Sandor Feldman, M.D.,* and Elizabeth Epp, B.A., Memphis, Tenn. V A R I C E L L A is a common childhood infection, manifest as cutaneous lesions, little is known about the hematogenous dissemination of v-z virus. The frequent occurrence of viscerally disseminated lesions in the immunosuppressed host 1 indicates that varicella has a viremic phase; however, antemortem isolation of v-z virus from blood has been reported only once. In 1966 Gold ~ isolated this virus from the blood of a child with hepatoma and varicella but further attempts to isolate bloodborne v-z virus in this and other patients were unsuccessful. Gold's report does not state the type of blood specimen used, the culture technique, or the relationship of blood cultures to the clinical status of the patient. A simple and reliable method for isolating v-z virus ALTHOUGH

From the Infectious Diseases Service, St. Jude Children's Research Hospital. Supported by Cancer Research Center Grant CA 08480from the National Cancer Institute, by a grant from the Washington County, Tennessee Cancer Society, and by A LSA C. *Reprint address: Infectious Diseases Service, St. Jude Children's Research Hospital, P.O. Box 318, Memphis, Tenn. 38101.

B r i e f clinical and laboratory observations

265

tic lesions by xeroradiography 7 prior to surgery facilitated therapy by accentuating the margins of nodular masses accessible to extirpation. REFERENCES 1. Jasper PL, and Denny FW: Sarcoidosis in children, J PEDIATR73:499, 1968. 2. McGovern JP, and Merritt DH: Sarcoidosis in children, Adv Pediatr 8:97, 1956. 3. Kendig EL: Sarcoidosis among children, J PEDIATR61:269, 1962. 4. Siltzbach LE and Greenberg GM: Childhood sarcoidosis: A study of 18 patients, N Engl J Med 279:1239, 1968. 5. Reisner D: Boeck's sarcoid and systemic sarcoidosis: A study of 35 cases, Am Rev Tuberc 49:437, 1944. 6. Devine KD: Sarcoidosis and sarcoidosis of the larynx, Laryngoscope 55i533, 1965. 7. Holinger PH, Lutterbeck EF, and Bulger R: Xeroradiography of the larynx, Ann Otol Rhinol Laryngol 81:1, 1972.

from blood would be useful in studying the pathogenesis of variceUa as well as evaluating the efficacy of antiviral agents in treatment of the infection. Such a method is reported here. MATERIALS

AND METHODS

Clinical specimens. Specimens for the isolation of virus were obtained from two children with acute leukemia and varicella. These patients developed varicella pneumonitis on days 2 and 4 of infection, respectively; both were treated with IDUR and survived. Abbreviations used: v-z virus: varicella-zoster virus IDUR: idoxuridine PBS: phosphate-buffered saline CPE: cytopathic effect BHK: baby hamster kidney From each patient approximately 0.1 ml of vesicle fluid was aseptically aspirated with a sterile, disposable 27gauge needle attached to a 1.0 ml syringe. Two to 3 ml of blood, obtained by venipuncture, were drawn into a syringe containing 0.1 m l of heparin (1,000 units/ml). All specimens were inoculated into tissue cultures within 2 hours. Culture system. Human embryonic lung fibroblasts were used to isolate v-z virus from clinical specimens. Fibroblasts were prepared from the lungs of a spontaneously aborted fetus of less than 20 weeks' gestation; the procedure was similar to that of Lennette and Schmidt. 3

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Brief clinical and laboratory observations

The Journal of Pediatrics February 1976

Table I. Detection of varicella-zoster virus in blood and vesicle fluid

Progression

Patient 1

Patient 2

of varicella

Vesicle

(day)

fluid

Blood

1

ND + + + + + +

ND + + +*

2 3 4 5 6 7 8 9

Vesicle fluid ND + + + +

Blood ND +*

the culture. If after 3 weeks no CPE was observed, the specimen was considered negative and discarded. When 50-75% CPE was observed in the culture inoculated with blood, the infected cells were removed by trypsin and versene. Half of the sample was inoculated onto a monolayer of BHK cells and the remainder onto a monolayer of human embryonic lung fibroblasts. Both were observed for CPE. RESULTS

ND ND ND

i0

ll + = positivefor v-z virus; = negative; ND = not done. *Intravenous IDUR therapy (2.4 gm/m2/dayin 4 divided doses for 5 to 7 days) administered for varicella pneumonia, after blood sample was obtained for culture. The cells were grown as monolayers in 25 cm ~ plastic tissue-culture flasks containing Eagle's minimal essential medium and Earle's salts supplemented with glutamine as well as 10% fetal bovine serum, 100 units of penicillin/ml, 100/~g of streptomycin/ml, and 2.5/xg of amphotericin B/ ml. At each passage the cells were exposed for 30 seconds to a 5% carbon dioxide--95% air mixture at a flow rate of 4 1/minute. The cells were then maintained on the same medium except that the concentration of fetal bovine serum was changed to 4%. Confluent monolayers (passages 3 through 9) were used for isolation of virus. After the monolayer was rinsed with 5 ml o f PBS, pH 7.2, it was inoculated with either vesicle fluid diluted with 0.5 ml PBS or with 2 to 3 ml of heparinized blood and incubated for 1 hour at 35~ Four milliliters of maintenance media was then added. Eighteen to 24 hours later the monolayers inoculated with blood were washed five times with 5 ml of PBS, and 4 ml of maintenance media was added. Cultures were observed twice weekly for 3 weeks for cytopathic effect. If after 3 weeks no CPE was observed, vesicle fluid specimens were considered negative. At the end of 3 weeks the cells from flasks inoculated with blood samples showing no CPE were removed. After the medium was decanted, a 1 ml mixture of 0.25% trypsin and 0.05% versene in Earle's balanced salt solution (without magnesium sulfate and calcium chloride) was added and incubated for 5 minutes. The cells were centrifuged at 1000 rpm for 10 minutes; then the cell pellet was diluted, resuspended in 1 ml of maintenance media and inoculated into a new monolayer. After 1 hour of incubation 4 ml of maintenance media was added to

AND DISCUSSION

Presented in Table I are the number of isolations attempted from vesicle fluid and blood, the presence or absence of virus in relation to onset of infection, and the administration of systemic IDUR, Varicella-zoster virus was isolated from the blood of both patients but only before the initiation of treatment with systemic IDUR. The efficacy of this antiviral treatment, however, cannot be evaluated, since only two patients were studied. On the four instances when virus was isolated from blood, it was also found in vesicle fluid. The presence of virus in vesicle fluid, however, does not necessarily indicate viremia. Focal cytopathic effects in lung fibroblasts were observed between 5 and 10 days after the monolayers were inoculated with vesicle fluid or blood. These effects consisted of small scattered groups of swollen, rounded cells distinct from the surrounding fibroblasts, and were similar to those previously described for v-z virus. 4 Similar cytopathic changes involving 75% of the monolayers were observed 4 to 10 days after the initial focal changes. Varicella-zoster virus was differentiated from herpes simplex by the following criteria: (1) lack of cytopathic effect on BHK cells and (2) slower growth with focal changes in human embryonic lung fibroblasts. The four viral isolates from blood were obtained on the initial inoculation only, and each isolate produced infection when subsequently passed to other human embryonic lung fibroblast monolayers. None of the isolates produced a CPE in BHK cells. Lang and Noren ~ in their study of cytomegaloviremia using WI-38 human embryonic lung fibroblasts noted that anti-coagulated leukocyte-rich plasma was sometimes toxic to cells. By contrast, we found neither morphologic changes nor decreased cell survival during the 14 isolation attempts, indicating that heparinized whole blood was not cytotoxic. This study of blood-borne v-z virus in two children with disseminated varicella and leukemia demonstrates a simple method of virus isolation requiring only 2 to 3 ml of heparinized whole blood. The materials required for this procedure are readily available in most diagnostic virology laboratories and the culturing techniques are similar to those used in isolation of virus from vesicle

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B r i e f clinical and laboratoty observations

fluid. A l t h o u g h h u m a n e m b r y o n i c lung fibroblasts were used to culture v-z virus, we see no r e a s o n o t h e r cell lines susceptible to the virus could n o t be used. Isolation o f virus from b l o o d will m a k e possible investigation o f the viremic p h a s e o f varicella as well as p r o v i d e additional criteria for the evaluation o f antiviral c h e m o therapeutic agents in v-z virus infection.

ADDENDUM Since the p r e p a r a t i o n of this m a n u s c r i p t we h a v e isolated v-z virus from b l o o d o n nine a d d i t i o n a l occasions from four other patients, using h u m a n foreskin fibroblasts rather t h a n h u m a n e m b r y o n i c lung fibroblasts as the culture system.

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REFERENCES 1. Hughes WT, Feldman S, and Cox F: Infectious diseases in children with cancer, Pediatr Clin North Am 21:583, 1974. 2. Gold E: Serologic and virus-isolation studies on patients with varicella or herpes zoster infections, N Engl J Med 174:181, 1966. 3. Lennette EH, and Schmidt N J: Diagnostic procedures for viral and rickettsial infections, ed 4, New York, 1969, American Public Health Association, Inc., p 98. 4. Lennette EH, and Schmidt NJ: Diagnostic procedures for viral and rickettsial infections, ed 4, New York, 1969, American Public Health Association, Inc., p 740. 5. Lang D, and Noren B: Cytomegaloviremia following congenital infection, J PEDIATR 73:812, 1968.

The authors gratefully acknowledge Dr. James Nakano of the Center for Disease Control, Atlanta, Georgia, for confirming the isolates of varicefia-zoster virus.

Retinopathy in juvenile dermatomyositis L. S. Fruman, C. G. Ragsdale, M.D., D. B. Sullivan, M.D., and R. E. Petty, M.D.,* A n n Arbor, Mich.

C Y T O I D B O D I E S, the microscopic foci o f e d e m a t o u s or degenerated retinal nerve fibers which form so-called cotton wool exudates, are t h o u g h t to result f r o m d a m a g e to retinal capillaries. 1 T h e y are seen in a p p r o x i m a t e l y 10% of patients with systemic lupus erythematosus, p a r t i c u larly in those with severe active disease or central n e r v o u s system involvement.'-' T h e occurrence o f such exudates in other r h e u m a t i c diseases is unusual. In the m i x e d c o n n e c tive tissue disease s y n d r o m e in which there are features o f SLE, dermatomyositis, a n d scleroderma, n o retinal changes were reported either in the original series o f 20 patients ~ or in the one r e p o r t e d childr T h e r e are few reported cases o f r e t i n o p a t h y in c h i l d h o o d d e r m a t o m y o sitis? -~1 This p a p e r reports a case a n d reviews the literature regarding the occurrence o f r e t i n o p a t h y in c h i l d r e n with dermatomyositis. From the Section of Pediatric Rheumatology, Department of Pediatrics and Communicable Diseases, University of Michigan Medical Center. *Reprint address:R6056 Kresge II, Department of Pediatrics, Universit~ of Michigan Medical Center; Ann Arbot, Mich. 48104.

CASE REPORT A six-year-old boy was admitted to another hospital with a four-week history of progressive muscular weakness and pain. The illness had been characterized at onset by fever, sore throat, perioral "blisters," generalized muscular pain, and a transient rash over face and chest. Serum muscle enzyme values were elevated. An electromyogram revealed short duration and low amplitude motor units without fibrillations; proximal muscle groups were most severely involved. In biopsied tissue of the left quadriceps femoris, histiocytic and lymphocytic infiltrations without vasculitis were seen.

Abbreviations used ANA: antinuclear antibody ENA: extractable nuclear antigen LE: lupus erythematosus EMG: electromyogram At the time of transfer to C. S. Mott Children's Hospital one week later, the child appeared acutely ill. He was afebrile; the blood pressure was 106/72. Periorbital edema, violaceous discoloration of the eyelids, and atrophic erythematous changes over the extensor surfaces of fingers and elbows were typical of acute dermatomyositis. A fluffy exudate was seen near the superior temporal disc margin of each eye. The examination of the optic fundi was otherwise normal. There were severe generalized muscular weakness, tenderness, and brawny indurations which were most marked in axial and limb girdle muscles. The patient had dysphagia and nasal speech and could not sit or stand without assistance. There was no respiratory embarrassment. Except for this profound weakness, the neurologic examination was normal. Laboratory determinations included hemoglobin 9.1 gm/dl,