Intrathecal administration of interferon in neonatal herpes

736

B r i e f c l i n & a l and laboratory observations

pecially those formulas reconstituted with water containing 1 ppm of fluoride. DISCUSSION A study conducted by Hamberg, 4 in which 0.5 mg/ day of fluoride was added to the diet, revealed a signific a n t r e d u c t i o n in the a m o u n t of dental caries in children receiving supplementation when compared to control groups. He concluded that supplemental fluoride should be given to all children living in areas with water containing less than 0.6 ppm. McClure 5calculated the fluoride intake of children one to three years of age to be 0.417-0.825 mg/day based on an estimated 390-560 ml/day intake of water with 1 ppm and food containing 0.027-0.265 mg/day. However, Walker and associates 6found the volume of water ingested by those less than 24 months of age to be considerably less than the a m o u n t used in McClure's calculations. Our findings are in agreement with the data of Walker's group that children ingest much less water than is generally assumed. The present study of 50 children less than two years of age residing in an area with i ppm fluoride in the water supply demonstrated that 68% were not receiving the recommended daily fluoride requirements. Thus the assumption that fluoridated water sources supply the

Intrathecal administration of interferon in neonatal herpes E. De Clereq, M.D.,* V. G. Edy, Ph.D., H. De Vlieger, M.D., R. Eeekels, M.D., and J. Desmyter, M.D., Leuven, Belgium GENERALIZED HERPES SIMPLEX INFECTION in the neonate is a severe, often fatal, virus infection leaving the few s u r v i v o r s with neurologic sequelae. 1Several authors have described possible beneficial results in neonatal herpetic infection with systemic use of idoxu r i d i n e ( 5 - i o d o - 2 ' - d e o x y u r i d i n e ) .25 The u l t i m a t e usefulness of these compounds remains to be established. From the Rega Institute for Medical Research and the Department of Pediatrics, Katholieke Universiteit Leuven. *Reprintaddress;R ega Institutefor Medical Research, Minderbroedersstraat10, B-3000Leuven, Belgium.

The Journal o f Pediatrics May 1975

necessary requirements to children is challenged. The administration of fluoride supplements to all children during the years of tooth formation, including those previously thought to have been protected by addition of fluoride to the water supply, m u s t therefore be recommended to ensure optimal prophylaxis against the formation of dental caries. REFERENCES

1. McClure FJ: Water fluoridation: The search and the victory, Washington, D. C., 1970, U.S. Government Printing Office, pp 109-138. 2. Committee On Nutrition: Fluoride as a nutrient, Pediatrics 49:456, 1972. 3. American Society of Dentistry for Children Guidelines: Adopting a simple preventive dental program for children in your office, 1972. 4. Hamberg L : Controlled trial of fluoride in vitamin drops for prevention of caries in children, Lancet 1:441, 1971. 5. McClure FJ: Water fluoridation: The search and the victory, Washington, D.C., 1970, U.S. Government Printing Office, pp 191-197. 6. Walker JS, Margolis FJ, Luten HL Jr, Well ML, and Wilson HL: Water intake of normal children, Science 140:890, 1963. 7. Ross Laboratories: Personal communication. 8. Mead Johnson Research Center: Personal communication.

The possibility of high-dose, long-term administration of interferon in man has only recently b e e n established in adult cancer patients 6 (intramuscular injections of 1-3 • 106 u n i t s of i n t e r f e r o n three times weekly). I n the p r e s e n t study i n t e r f e r o n was adAbbreviations used IUdR: idoxuridine CSF: cerebrospinal fluid WBC: white blood cell UV: ultraviolet ministered intrathecally to a newborn infant with disseminated herpes simplex infection for six consecutive days, at 6 x 105 units twice a day for two days followed by 6 x 105 units once a day for four days. Idoxuridine was also given intravenously. CASE REPORT

A term male infant (birth weight, 3.3 kg) born on March 20, 1974, to a 20-year-old gravida 1, para 1 woman, whose pregnancy had been uneventful. The infant did well until the age of seven days when he developed vesiculopustular lesions on the trunk. Between the seventh and sixteenth day he became

Volume 86 Number 5

Brief clinical and laboratory observations

Table I. I n t e r f e r o n titers in cerebrospinal fluid and s e r u m of infant treated intrathecally with h u m a n interferon*

Time prior or after interferon administration

Interferon titer (lOglo international reference units/ml)

CSF CSF CSF CSF CSF CSF CSF CSF CSF CSF

36 hr prior Immediately before 12 hr after first dose 12 hr after second dose 12 hr after third dose 24 hr after fourth dose 24 hr after fifth dose 24 hr after sixth dose 24 hr after seventh dose 24 hr after eighth doset

( 1.6 ( 1.6 3.2 3.9 3.3 2.9 3.1 3.7 3.7 3.3

Serum Serum Serum

48 hr before first dose 1 hr after seventh dose 24 hr after eighth dose1"

( 1A 2.1 1.4

Sample

*Interferon administered at 600,000 units/dose; doses 1 through 4 administered with approx. 12 hr interval, doses 5 through 8,administered with approx. 24 hr interval. tThese samples were obtained 6 hr postmortem.

continued by the intravenous route, interferon was administered intrathecally. Eight doses of 600,000 units (in 2 ml vol) of interferon were given: the first dose on the twenty-first day, the first four doses every 12 hours, the last four doses every 24 hours. No immediate side effects were observed, except a rise in temperature (from ~ 36~ to 38~176 2 to 4 hours after injection. At the twenty-third day, two days after interferon treatment was started, CSF data were: glucose, 30 mg/dl; 37 WBC/mm 3. Four days later they were: protein, 388 mg/dl; glucose, 30.5 mg/dl; 211 WBC (70% lymphocytes)/mm 3. Blood cell counts remained essentially normal during the period of observation. New skin vesicles continued to appear over various parts of the body; Cheyne-Stokes respiration was noted from the twentyfifth day; the child died on the twenty-eight day. At autopsy the following pathologic findings were recorded : necrosis of the whole brain cortex and of the corpus callosum, corresponding to the type generally seen in herpes encephalitis; normal appearance of meninges and spinal cord; atelectasiS of left lung, focal necrosis (with coagulation) of liver parenchyma, a small thymus (2 gm; normal weight 8 _+ 5 gm at this age) and a rather large spleen (15 gin; normal weight 12 _+ 4 gm at this age). Herpesvirus was recovered from brain and liver tissue, but not from blood, spleen, lung, and kidneys. MATERIALS

lethargic. The infant was then transferred to our Department of Pediatrics. He was comatose, cyanotic, hypotonic, and hypothermic. Respirations were irregular and superficial, with apneic spells during clinical examination. Reflexes were essentially absent. Vesiculopustular lesions, some umbilicated, were noted on the right side of the trunk, right arm, and right hand. When the cell cultures inoculated with specimens from skin vesicles showed a cytopathic effect characteristic of herpes simplex virus, treatment with IUdR was started on the nineteenth day. IUdR was administered intravenously by continuous drip-at 100 mg/kg/day for five days. At the time IUdR treatment was started, cerebrospinal fluid data were: protein, 11.8 mg/dl; glucose, 56.2 mg/dl; 22 WBC (all lymphocytes)/mm 3. This CSF sample was also positive for herpes simplex virus as revealed by cytopathogenicity in WI 38 diploid cells. The virus was identified as herpesvirus hominis type 2, suggesting the mother's genital tract as the source of infection] There was temporary involvement in the child's condition from the eighteenth to the twenty-first day: most of the reflexes, including the sucking reflex, returned, and the patient was less lethargic. Seizures occurred during this period, and, from the twenty-first day, reflexes disappeared again. New vesicles continued to appear on the trunk. The critical state of the infant, the continuous appearance of new skin vesicles, the presence of virus in the CSF indicating massive virus multiplication inthe central nervous system, and the availability of high-titered, albeit crude, human leukocyte interferon (originally supplied by Dr. K. Cantell to our laboratory for stability studies) p r o m p t e d us to risk a therapeuti c attempt with interferon. Since IUdR treatment was

737

AND METHODS

T h e samples to be assayed for interferon were either U V - i r r a d i a t e d or dialyzed against 0.1M KC1-HC1 buffer, p H 2.0, for five days at 4~ T h e acid-treated samples were t h e n neutralized by dialysis against p h o s p h a t e buffered saline for 24 hours; protein precipitated upon the acid t r e a t m e n t was r e m o v e d by centrifugation. U V irradiated and acid-treated samples yielded similar int e r f e r o n t i t e r s . I n t e r f e r o n was a s s a y e d b y a d y e uptake method 8in human embryo fibroblasts c h a l l e n g e d with M e n g o virus. T h r e e laboratory standard i n t e r f e r o n preparations were included in the assays. T h e s e standards had t h e m s e l v e s b e e n calibrated against the British R e s e a r c h Standard B for h u m a n i n t e r f e r o n (69/19); all titers are expressed in terms of this standard. H e r p e s v i r u s was isolated on h u m a n diploid Wistar-38 cells. T h e cytopathic effect, n o t e d after 1 to 3 days, was indicative o f herpes simplex. In the initial isolates, the p r e s e n c e of herpesvirus was c o n f i r m e d i m m e d i a t e l y by electron microscopy. Since the s i m u l t a n e o u s p r e s e n c e of h u m a n interferon may have h a m p e r e d virus isolation f r o m certain samples, isolation was also a t t e m p t e d in a line of Morris rat h e p a t o m a cells, which was develo p e d in this laboratory and s h o w e d good sensitivity to h e r p e s v i r u s hominis. N o additional viruses w e r e isolated in this cell line. T h e distinction b e t w e e n two types of h e r p e s v i r u s h o m i n i s was m a d e according to criteria established by F i g u e r o a and Rawls. 9

738

Brief clinical and laboratory observations

RESULTS

AND DISCUSSION

In contrast with a previously reported case of herpes simplex encephalitis, 1~ no endogenous interferon was detected in the CSF or in the serum before exogenous interferon treatment was started (Table I). The interferon titers measured in the CSF at different times after intrathecal interferon treatment began (Table I), however, clearly show that in man relatively high interferon levels can be maintained in the CSF by direct a d m i n i s t r a t i o n of i n t e r f e r o n into the c e r e b r o s p i n a l space. The interferon titers recovered in the CSF at 12 or 24 hours after the interferon injections varied from 800 to 8,000 units/ml. This corresponds to 0.13-1.3% of the interferon dose that was injected each time. That interferon disappears rather slowly from the CSF has also b e e n demonstrated in rabbits injected intrathecally (or intraventricularly) with interferon H or inducers thereof [poly (I) poly(C)], t2 Ho and associates 11 assessed that interferon disappeared much slower from the CSF than from the bloodstream (half-life 1 hour and 11 minutes, respectively) and that about 1-5% of the initial (30 minutes) interferon titer could be recovered in the CSF 24 hours after intracisternal or intraventricular administration of interferon. Our data on the clearance of interferon in the CSF space of man are consistent with those of rio and associates 11in rabbits. A t two occasions after interferon administration (Table I), interferon titers were also determined in the serum. These titers were significantly lower (40 and 80 times, respectively) than the corresponding titers in the CSF, suggesting that interferon did not freely cross the blood-brain barrier, or, if it freely passed this barrier, that it was more rapidly inactivated in the bloodstream than in the CSF. The second possibility, however, is unlikely, because human interferon is not particularly more sensitive to inactivation by s e r u m than by CSF 13 and, if it were, one may expect that, owing to the concentration gradient generated by the selective degradation of interferon in the bloodstream, interferon would continuously flow from the CSF to the bloodstream; this would eventually lead to an equal distribution of interferon between the two compartments, if interferon c o u l d f r e e l y pass the C S F - b l o o d b a r r i e r . Being a macromolecuie interferon is not supposed to diffuse freely, and recent (unpublished) data of Habif, Lipton, and Cantell indicate that interferon does indeed diffuse poorly from the CSF into the bloodstream and vice versa (in monkeys administered h u m a n interferon by the intrathecal and intravenous or intramuscular route, respectively): for example, 24 hours after the intrathecal injection of 10 • 10 6 units of interferon per monkey, 6,000 units/ml of interferon were detected in the CSF

The Journal of Pediatrics May 1975

and only 200 units/ml in blood; similarly, the interferon titers found in the blood 3 hours after intravenous injection and 12 hours after intramuscular injection of 30 • 10 6 units of interferon pei" m o n k e y were about 40 times higher than those detected in the CSF. Habif, Lipton, and Cantell's findings in monkeys as well as ours in man and those of Cathala and Baron 12in rabbits support the concept that interferon does not freely cross the blood-brain barrier. Although treatment with I U d R of disseminated herp e s v i r u s i n f e c t i o n s o f n e w b o r n i n f a n t s has y i e l d e d p o s s i b l y p r o m i s i n g r e s u l t s , 2-5 I U d R did n o t prove efficacious in other instances of herpetic encephalitis in man. 14 It should be pointed out that no double-blind studies have been done on the efficacy of I U d R in generalized herpetic infections of the newborn h u m a n infant, and that in a controlled study on the efficacy of I U d R in d i s s e m i h a t e d h e r p e s i n f e c t i o n in n e w b o r n mice, IUdR, adrrlinistered intraperitoneally, failed to reduce the mortality rate. 15 Despite intensive I U d R therapy (total dose: 500 mg/ kg, intravenously) and interferon therapy (total dose: 4.8 • 106 units, intrathecally), the infant did not survive the infection, and virus was recovered from the brain postmortem. It may be questioned, therefore, whether interferon delivered into the CSF space gained access to the relevant neural tissue in which virus replicated. If interferon is prevented from penetrating into the nervous tissue, one may not expect a beneficial result with intrathecal administration of interferon in viral encephalitis which is not associated with leptomeningeal inflammation. A similar conclusion was reached by Ho a n d associates 11 who f o u n d that i n t e r f e r o n i n j e c t e d directly into the CSF space was ineffective in protecting rabbits against rabies. The isolation of herpesvirus from postmortem brain material might also be interpreted in terms of an abnormal low sensitivity of the virus toward exogenously applied i n t e r f e r o n . This p o s s i b i l i t y m a y be d i s m i s s e d , however, since experiments in human diploid cells revealed that the interferon sensitivity of this particular virus was of the same order as that of previously isolated herpesviruses (J. Desmyter, unpublished data). CONCLUSIONS The case reported herein presumably represents the -first therapeutic attempt with human interferon in a l i f e - t h r e a t e n i n g a c u t e virus i n f e c t i o n ( d i s s e m i n a t e d herpesvirus hominis infection) in the newborn infant. No beneficial effect to the infant could be noted from the intrathecal administration o f interferon as used here. Yet, the following conclusions seem to be in order.

Volume 86 Number 5

1. Intrathecal administration of h u m a n leukocyte interferon (8 x 600,000 units) into the n e w b o r n infant did not lead to overt toxicity. Even with the crude interferon available for this study, the only adverse effect noticed was a moderate rise of body temperature. 2. Relatively high interferon titers were maintained in the CSF (800-8,000 units/ml) at 12-24 hours after intrathecal administration of 600,000 units of interferon. 3. Interferon did not freely cross the blood-brain barrier since interferon levels in the serum were approximately 40- to 80-fold lower than the corresponding CSF titers. 4. The intrathecal route may not be the optimal single route for delivering interferon to all parts of the central nervous system in which virus is actively multiplying, i n a s m u c h as virus was recovered from b r a i n tissue postmortem. We are indebted to Dr. K. Cantell for providing the human leukocyte interferon, to Drs. W. E. Stewart II, M. Casteels-Van Daele, E. Eggermont, and L. Corbeel for helpful suggestions, to Dr. W.T. Liu for electron microscopic examination of the virus samples, to Dr. B. Van Damme for performing the autopsy studies, to Francine Cornette and Gaby Lenaerts for technical assistance, and to Janine Putzeys for editorial help. REFERENCES 1. Nahmias AJ, Alford CA, and Korones SB: Infection of the newborn with Herpesvirus hominis, Adv Pediatr 17:185, 1970. 2. Partridge JW, and Millis RR: Systemic herpes simplex infection in a newborn treated with intravenous idoxuridine, Arch Dis Child 43:377, 1968. 3. Golden B, Bell WE, and McKee AP: Disseminated herpes simplex with encephalitis in a neonate, J A M A 209:1219, 1969. 4. Tuffii GA, and Nahmias AJ: Neonatal herpetic infection.

Hepatotoxicity of rifampicin and isoniazid in children M. Casteels-Van Daele, M.D.,*

Brief clinical and laboratory observations

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

739

Report of two premature infants treated with systemic use of idoxuridine, Am J Dis Child 118:909, 1969. Charnock EL, and Cramblett HG: 5-Iodo-2'-deoxyuridine in neonatal herpes virus hominis encephalitis, J PEDIATR 76:459, 1970. Strander H, Cantell K, CarlstrOm G, and Jakobsson PA: Clinical and laboratory investigations on man : Systemic administration of potent interferon to man, J Natl Cancer Inst 51:733, 1973. Nahmias AJ, Dowdle WR, Josey WE, Naib ZM, Painter LM, and Luce C: Newborn infection with herpes virus hominis types 1 and 2, J PEDIATR75:1194, 1969. Finter NB: Dye uptake methods of assessing viral cytopathogenicity and their application to interferon assays, J Gen Virol 5:419, 1969. Figueroa MA, and Rawls WE: Biologicalmarkers for differentiation of herpes strains of oral and genital origin, J Gen Virol 4:259, 1968. Bellanti JA, Catalano LW Jr, and Chambers RW: Herpes simplex encephalitis : Virologic and serologic study of a patient treated with an interferon inducer, J PED1ATR 78:136, 1971. Ho M, Nash C, Morgan CW, Armstrong JA, Carroll RG, and Postic B: Interferon administered in the cerebrospinal space and its effect on rabies in rabbits, Infect Immun 9:286, 1974. Cathala F, and Baron S: Interferon in rabbit brain, cerebrospinal fluid and serum following administration of polyinosinicrpolycytidylic acid, J Immunol 104:1355, 1970. Cesario TC, Mandell A, and Tilles JG: Inactivation of human interferon by body fluids, Proc Soc Exp Biol Med 144:1030, 1973. Fishman MA, Haymond MW, and Middelkamp JN: Failure of idoxuridine treatment in herpes simplex encephalitis, Am J Dis Child 122:250, 1971. Kern ER, Overall JC Jr, and Glasgow LA: Herpes virus hominis infection in newborn mice. I, An experimental model and therapy with iododeoxyuridine, J Infect Dis 128:290, 1973.

THERE HAVE BEEN REPORTS of hepatotoxicity related to therapy with rifampicin, especially in association with isoniazid, in adults. 1, 2 As far as we know, there have been no reports of serious and life-threatening side effects in children in conjunction with this therapy. This report is of a boy who developed hepatic coma while receiving antituberculous therapy.

L. Igodt-Ameye, M.D., L. Corbeel, M.D., and R. Eeekels, M.D., Leuven,Belgium From the Department of Pediatrics, University of Leuven. *Reprint address: Department of Pediatrics, St. Raphael Academisch Ziekenhuis, Universityof Leuven, B-3OOOLeuven, Belgium.

CASE REPORT Ph., a 13-year-old boy, was admitted to our department in a comatose state. Two weeks before admission pleurisy of tuberculous origin was diagnosed. The patient was treated with isoniazid 500 mg/day (12 mg/kg), ethambutol 800 mg (20 rag/ kg), and rifampicin 450 mg/day (11 mg/kg). On the seventh