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Detection of antibody to rotavirus by counterimmunoelectrophoresis in human serum, colostrum, and milk
Voltnue 93
Brie! clinical and laboratory observations
Number 6
In infancy the combination of an increased rate of gain in weight with a decreased growth rate should make one suspect the possibility of Cushing syndrome. III If in add ition the typical moon face is present, the possibtlity is greatly strengthened. Confirmatory studies include absence of the thymic shadow on chest radiograph , impaired suppressibility of cortisol secretion by dexamethasone, and increased excretion of free cortisol in the urine. 1 I Loss of the normal diurnal pattern of cortisol secretion is a valuable diagnostic aid in adul ts but of little use in infants, in whom the diurnal pattern is not yet established. " The slowing of the growth rate and excessive gain in weight combined with the facial app eH mnce led to the presurn ptive diagnosis of Cushing syndrome in our patient. The 24-hollf excretion of cortisol calculated on the basis-of surface are a was increased, bu t not strikingly so. However, the thymic shadow, though small, was present , and dexameth asone readily suppressed the secretion of cortisol. The clinical findings seemed more severe than might be expected from the modest increase in secretion of cortisol. Possibly the secretion of cortisol had been greater than revealed by our tests in the hospital, since during both of the ad missions to the hospital the mothe r had stopped drinking. In adults, rapid reversal of alcohol-induced pseudo-Cu shing syndrome may occur if alcohol is withheld." Whatever the reason for the disproportionate severity of the clinical findings, the striking alteration in the patient's course after the mother had been persuaded to
967
stop drinking confirmed the relationship between the clinical findings and the alcohol in the breast milk. We remain advocates of breast -feeding and would not proscribe alcohol. It is the combin ation that is und esirable. REFERENCES I. Smals AG, Klopp e nborg PW , Nj o KT , K noben. JM , a nd Rul a nd OM : Alco hol ind uced Cus hing oid synd rom e, Br Med 12: 1298, 1976 , 2, Re es LH, Besser OM, an d Jeffc oate WJ : Alcohol induced pseudo-Cushing's syndrome, Lan cet 1:726, 1977. 3. Sm als A, and Kloppenborg P: Alcoh ol ind uced psuedoCush ing's syndrome, La ncel 2: 1369, 1977 . 4. Fraj rla R, a nd Angeli A: A lco hol ind uced pse udo-Cush ing's syndrome, Lancet 1: 1050, 1977. 5, Paton A: Alcohol induced Cushingo id synd rome , BI" Med J 2: 1504, 1976. 6. Jenkins IS, and Connelly .1 : Adr en ocortical respo nse to et hanol in ma n, Br Mcd J 2: 804, 1968. 7. Mendelson JI-I: Biolog ic co nco m ita n ts o r alco holism , N Engl J Med 283: 71, 1970. 8, Beller S, Roman L, DeCastro 0, and Herrera M : Effect of acute ethanol intake on plasma 11 .hydrol\yco~ticosteroid levels, Metabolism 19:664, 1970. 9. Mendelson JH, Ogotn M, and Mello N K : Ad renal fun ction and a lcoholism I. Serum cortiso l, Psych osorn Med 33: 145,
t971.
10.
Lorid an L, an d Senior B: Cush ing's syndrome in infan cy, J PWI ATR 75: 349, 1969. I I. Edd y RL, Jones A L, Gillilund PF . Ib ar ra 10, Thompson 1Q , an d McMu rry JF: Cushing's syndrome: A prospective study o r di agnostic methods, Am J Med 55:6 21, 1973. 12. Fr a nks RC : Diurnal vari a tion of pl asma 17 hydro xycort icosteroids in chil dren, 1 Clin End oc rinol 27:75, 1967.
Detection of antibody to rotavirus by counterimmunoelectrophoresis in human serum, colostrum, and milk O. A. Cook, B.Sc.," A, Zbitnew, M .Sc ., G, Dempster, M.D ., and J. W. Gerrard, M.D., Saskatoon, Sask. Canada
AT LE AST ONE STUDY has demonstrate d a significantly lower incidence of rotavirus infection in breast-fed versus bottle-fed infants, I a nd others" :I have indicated that most
From the Departmen t of Clin ical Mic robiology, University H ospital, and Departm ent of Pediatrics, University of Sask at chewan, >Reprlnt address: Department of Clinical Mtcrobiology, University Hospttat; S askatoon, Saska tchewan. Canada, S7 N OW8.
0022-3476/78 /120967 + 04$00 .40/0 \0 1978 The C. V. Mosby Co.
ill patien ts in their stud ies were not breast- fed. Woode et al' have demonstrated rotav irus neutralizing an tibody in bovine colostrum and tha t such colostr um, when fed to calves, protects them Cram subsequen t challen ge with virulent virus. Hum an colostrum conta ins antibodies to other enteric viruses, including polioviruses, some Coxsackie type B viruses, and some echov iruses.' ' L We speculated that rota virus antibodies might be found in human colostrum and milk, which might account to some
968
Brief clinical and laboratory observations
degree for its protective effects. This study was designed to investigate the prevalence of such an tibodies in human colostrum and milk. See related article, p. 9] 6.
Abbreviations used ClEP:
counterimmuncelectrophoresis
FA:
immunofluorescence
MATERIALS AND METHODS Clinical material. Specimens were collected from consenting maternity pa tien ts following delivery at University Hospital, Saskatoon. Saskatchewan. Cord serum was collected at delivery and maternal serum on the third day post partum. Colostrum was collected on the first day and samples of milk and of infant stool were obtained on the third day. A total of 32 maternal sera, 32 cord sera, 29 colostrum samples, 26 milk samples. and 21 infant stools were collected from 34 maternity patients. In 31 of these patients, the infants were being breast-fed. In addition , milk samples from human donors were provided by the mothers' milk bank, Saskatoon City Hospital. A total of 22 samples of milk from 20 donors was obtained. Length of time between parturition and collection of the milk varied from three days to one year. Preparation of clinical material. Colostrum and milk samples were first clarified by centrifugation at 10,000 rpm for 30 minutes at 4°C in a Sorvall RC-2B centrifuge. The fat layer at 'the top was carefully removed and the clear supernatant fluid was removed and stored at -20°e. All milk samples and some colostrum samples were concentrated approximately fivefold using Lyphogel (Gelman Scientific) before testing. Stools were emulsified with a small amount of 0.85% saline to make a heavy suspension and were then clarified by centrifugation at 17,000 rpm for 30 minutes at 4°C. The clear supernatant was removed and stored at -20°e. Counterimmunoelectrophoresis. CIEP assays for determination of rotavirus antibody were performed by the method of Middleton et aF; however, the pH of the barbital buffer was 8.6 and this buffer was diluted 1:5 for preparation of gels. To enhance visualization of the precipitate lines, following electrophoresis, slides were washed twice in 0.85% saline followed by distilled water and then air dried at 37°C. Slides were stained for one hour in 0.1% thiazine red in 1% acetic acid followed by thrice rinsing in a solution of 45 parts water: 45 parts methanol: 10 parts acetic acid. Rotavirus antigen was prepared from the fluid stoat of
The Journal of Pediatrics December 1978
an infant in which large numbers of typical rotavirus particles had been observed by negative staining electron microscopy. A 50% suspension of stool was prepared in borate-buffered saline (80 ml 1.5M NaCl, 100 ml O.5M boric acid, 24 ml l.OM NaOH , and distilled deionized water to make one liter) , pH 9.0. Further dilutions of this material were prepared in gel buffer. The optimum dilution of antigen for detection of antibody was determined by checkerboard titration using CIEP; the optimum was considered that dilution of antigen which detected the lowest concentration of antibody. Indirect immunofluorescence. The method was similar to those previously described." Calf rota virus (Norden Laboratories, Lincoln, Neb .) infected LLC-MK, cells were fixed using the method of Schmitz and Enders" and were overlaid with serum diluted 1:2, followed by staining with fluorescein-labeled antiserum to human immunoglobulins (Hyland Laboratories). Slides were examined by ultraviolet illumination; only those sera giving unequivocal fluorescence in the absence of staining in control cultures were recorded as positive. Reference antiserum. Rotavirus reference antiserum (rabbit anti neonatal calf diarrhea virus) was obtained from Norden Laboratories. Lincoln, Neb. RESULTS Specificity of the CIEP test. Specificity of the antigen was tested by titration of acute and convalescent phase sera from patients known to have had rotavirus infections. Sera from three such patients all showed rising titers of antibody to the antigen. In contrast, paired sera showing fourfold or greater rises in antibody titer to other viruses (four adenovirus, two coxsackievirus B2, one echovirus 6, and one echovirus II) failed to show rising titers of antibody to the rota virus antigen . Specificity of the reference antiserum was examined by testing in CIEP against similarly prepared human stool antigens from which a variety of viruses had been isolated, including adenoviruses 2, 3, and 7, reovirus 2, coxsackievirus type A. coxsackievirus type B, and echovi rus 9. No precipitate lines were observed with any of these antigens . Specificity of the reaction was further tested by mixing the rotavirus antigen with reference serum and incubating at room temperature for one hour. When this mixture was placed in cathodal wells and reacted with antibodycontaining material, no precipitate lines were observed. Antibody detection. The results of antibody detection from the maternity patients are as follows: Precipitate lines were observed in 30/32 (93.8%) of the maternal sera, 17/32 (53.1%) of the cord sera, 20129 (68.9%) of the colostrum samples, 5/26 (19.3%) of the milk samples, and
Brief clinical and laboratory observations
Volume 93 Number 6
1121 (4.8%) of the infant stools at 3 days of age. Antibody was detected by indirect FA in 29/32 (90.6%) of the maternal sera and in 24/32 (79.4%) of the cord sera. The results of the two tests are not significantly different for detection of rotavirus antibody in serum. Attempts to demonstrate antibody by indirect FA in colostrum, milk and infant stools were unsuccessful. In milk samples from the mothers' milk bank, antibodies were detected by CIEP in two of seven samples collected less than one week post-partum, but not in any of 13 samples collected more 'than one week postpartum.
DISCUSSION The results of this study indicate that under the conditions employed, CIEP is a useful and sensitive tool for detection of antibody to rotavirus in epidemiologic surveys. This is in contrast to the findings of Middleton et at' who reported the test useful for diagnostic serology and detection of antibody in sera of children, but that this technique rarely detected antibody in adult sera. We found CIEP as sensitive as some of the other reported techniques, such as indirect FA and complement fixation. Blacklow et al'' detected antibody by such techniques in 62 to 84% of sera from human beings more than 18 months of age. In the present study, 93.8% of the adults were found by CIEP to have circulating rotavirus antibody, as compared to 90.6% by indirect FA. A rapid decline in the ability to detect antibodies in milk occurs by the third day of lactation. Even fivefold concentration of the milk failed to reveal antibodies in milk of 72% of the patients who had demonstrable antibodies in colostrum. These findings are similar to those of Woode et al,' who noted a rapid decline in levels of neutralizing antibody to calf rotavirus in bovine colostrum within 24 to 48 hours of delivery. Subsequent to completion of our study, Thouless et al'" reported rotavirus neutralizing antibodies in human milk. These workers report a similar decline in levels of antibodies, which have all but disappeared by the fifth day after delivery. Simhon and Mata" recently reported rotavirus antibodies in the colostrum of 30% of a group of Costa Rican women. These workers used an enzyme-linked imrnunosorbent assay, which is reported to be a very sensitive technique." We detected antibodies in a much greater percentage (68.9%) of the colostrum samples tested in our study. In view of the sensitivity and simplicity of elEP over other methods, further work using this test is warranted, particularly since such a method would be useful in areas lacking access to more sophisticated equipment. Woode et al ' showed that feeding colostrum with high antibody titers to calves, followed by challenge with
969
virulent virus, was protective. Similar studies with lambs indicated that oral administration of serum or colostrum containing rotavirus antibody protected against experimental infection with Iamb rotavirus.':' Our study revealed rotavirus-specific antibody in the stools of at least one and probably two infants who had been breastIed-colostrum which contained antibody to rotavirus. This suggests that antibodies in milk do find their way to the intestinal lumen of the infant and are thus in a position to protect the infant against invading microorganisms. The fact that rotavirus antibodies disappear rapidly from milk suggests that the reduced incidence of rotavirus gastroenteritis in older breast-fed infants may be due to other antiviral factors in milk, such as that described by Matthews et al.H Donor milk banks, including the one in this city, usually sterilize milk by boiling as a standard procedure prior to feeding it to infants. This practice has the unfortunate consequence of destroying many of the protective proteins found in milk, including antibodies. Raptopoulou-Gigi et al" pointed out that pasteurization is effective for sterilizing milk, even with samples that were artificially heavily contaminated with bacteria. The pasteurization process destroys neither antibody nor tactoferrin, another important milk protein. In light of the ever-increasing numbers of reports of protective factors in milk, the practice of boiling milk should be discouraged in favor of sterilization by pasteurization so that the alleged benefits of feeding pooled human milk can be fully realized. We thank Dr. R. O.Sugarman and theobstetrics stafffor their cooperation in collection ofspecimens, Drs. E. I. Orodums and P. J. Middleton and Ms. M. Sarich for reviewing themanuscript and for helpfulcomments. and Miss Heather Blondeau for secretarial assistance. REFERENCES I.
Totterdell BM, Chrystie II." and Danatvala lE: Rotavirus infections in a maternity unit, Arch Dis Child 51:924, 1976.
2.
Bryden AS, Davies HA, Hadley RE, Flcwett TH, Morris CA, and Oliver P: Rotavirus enteritis in thewest midlands during 1974, Lancet 2:241, 1975. 3. Kapikian AZ, Kim HW, Wyatt RO, ClineWL, Arrobio .IO, Brandt CD, Rodriguez WJ, Sack DA, Chanock RM, and Parrott RH: Human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children, N Engl J Med 294:965, 1976.
4. Woode ON, Jones J, and Bridger J: Levels of colostral antibodies against neonatal calf diarrhea virus, Vet Rec 97:148,1975.
5. John TJ, and Devarajan LV: Poliovirus antibody in milk and sera of lactating women, Indian J Med Res 61: 1009, 1973.
6. Kenny JF, Boesman MI, and Michaels RH: Bacterial lind
970
7.
8.
9.
10.
II.
Brief clinical and laboratory observations
viral coproantibodies in breast-fed infants, Pediatrics 39:202, 1967. Middleton PJ, Petrie M, Hewitt CM, Szymanski MT, and Tam JS: Counterimmunoelectro-osmophoresis for the detection of infantile gastroenteritis virus (orbi-group) antigen and antibody, J Clin Patho129:191, 1976. Blacklow NR, Echeverria P, and Smith DH: Serological studies with reovirus-like enteritis agent, Infect Immun 13:1563, 1976. Schmitz H, and Enders G: Cytomegalovirus as a frequent cause of Guillain-Barre syndrome, J Med Virol 1:21, 1977. Thouless ME, Bryden AS, and Flewett TH: Rotavirus neutralization by human milk, Br Med J 2: 1390, 1977. Simhon A, and Mata L: Anti-rotavirus antibody in human colostrum, Lancet 1:39, 1978.
The Journal of Pediatrics December 1978
12.
Yolken R, Kim HW, Clem T, Wyatt RG, Kalica AR, Chanock RM, and Kapikian AZ: Enzyme-linked immunesorbent assay (ELISA) for detection of human reovirus-like agent of infantile gastroenteritis, Lancet 2:263, 1977. 13. Snodgrass DR, and Wells PW: Rotavirus infection in lambs: studies on passive protection, Arch Virol 52:201, 1976. 14. Matthews THJ, Nair CDG, Lawrence MK, and Tyrrell DAJ: Antiviral activity in milk of possible clinical importance, Lancet 2: 1387, 1976. 15. Raptopoulou-Gigi M, Marwick K, and McClelland DBL: Antimicrobial proteins in sterilised human milk, Br Med J 1: 12, 1977.
Cold weather and rhabdomyolysis Mark A. Raifman, M.D., Far Rockaway, N. Y., Moshe Berant, M.D., Hadera, Israel, and Carl Lenarsky, M.D., Far Rockaway, N. Y.
RHABDOMYOLYSIS is an uncommon condition which may have serious consequences, such as severe electrolyte imbalance, cardiac arrhythmias, renal failure, and respiratory failure.' The clinical presentation is frequently nonspecific. Reports on rhabdomyolysis do not list exposure to cold as one of the causes of this condition. This report describes three instances of rhabdomyolysis asso-
Laboratory investigation as well as family and past history failed to disclose any of the factors that are usually considered in a patient with an attack of rhabdomyo 'g'sis. However, a detailed history of the youngster's activities on the day of admission revealed that he had spent one and one-half hours standing at a bus stop. dressed in a short overcoat, thin trousers, tube socks, and sneakers. That day was dry, with temperatures around -SoC and winds of 20 miles per hour.
ciated with exposure to cold weather. CASE REPORTS Patient 1. A 15-year-old boy was referred to the emergency room at Peninsula Hospital Center as "rule out nephrotic syndrome," because of bilateral ankle edema and 2+ protein in the urine. Blood pressure on admission was 120170 mm Hg. The positive physical findings were limited to the lower extremities, with painful and tender symmetric swelling of the legs and ankles. The overlying skin was normal and was cool to touch. There was no evidence of any vascular incompetence and peripheral pulses were equal and palpated bilaterally. Urinalysis showed 2+ protein, was slightly benzidine positive, and had no cells or casts on microscopic examination. The color of the urine was not noted. Results of tests for blood urea nitrogen, creatinine, electrolytes, proteins, cholesterol, and total lipids were all normal. Serum phosphorus concentration was 5.8 mg/dl, and calcium 10.1 mg/dl; SOOT was 180 units, LDH 800 units, and CPK was initially 3,400 units and rose to 5,200 units on the following day. From the Department of Pediatrics, Peninsula Hospital Center. • Reprint address: Department of Pediatrics, Peninsula Hospitai Center, For Rockaway, NY 11691.
See related article, p, 1015. Abbreviations used: SOOT: serum glutamic oxaloacetic transaminase LDH: lactate dehydrogenase CPK: creatine phosphokinase Patient 2. A 4-year-old girl was referred with a diagnosis of "acute rheumatic fever." She had awakened that morning with swelling and severe pain in both feet and ankles. The patient was one of eight children of an indigent family, living in a shabby, unheated home. The evening before admission the child had been stepping into puddles covered with thin' ice and went to sleep without taking off her wet socks and sneakers. The temperature that night was below the freezing point. Physical examination showed her to be unable to stand because of the pain in her feet. Her ankles and her feet were swollen and exquisitely tender. The overlying skin appeared normal, without lesions, and was ice cold to touch. There were no other abnormal findings. The urine had a slightly pink tinge, contained 2+ protein with a positive benzidine test. Blood counts and renal function studies were all normal. The serum potassium concen-
0022-3476/78/120970+02$00.20/0 © 1978 The C. V. Mosby Co.
Brie! clinical and laboratory observations
Number 6
In infancy the combination of an increased rate of gain in weight with a decreased growth rate should make one suspect the possibility of Cushing syndrome. III If in add ition the typical moon face is present, the possibtlity is greatly strengthened. Confirmatory studies include absence of the thymic shadow on chest radiograph , impaired suppressibility of cortisol secretion by dexamethasone, and increased excretion of free cortisol in the urine. 1 I Loss of the normal diurnal pattern of cortisol secretion is a valuable diagnostic aid in adul ts but of little use in infants, in whom the diurnal pattern is not yet established. " The slowing of the growth rate and excessive gain in weight combined with the facial app eH mnce led to the presurn ptive diagnosis of Cushing syndrome in our patient. The 24-hollf excretion of cortisol calculated on the basis-of surface are a was increased, bu t not strikingly so. However, the thymic shadow, though small, was present , and dexameth asone readily suppressed the secretion of cortisol. The clinical findings seemed more severe than might be expected from the modest increase in secretion of cortisol. Possibly the secretion of cortisol had been greater than revealed by our tests in the hospital, since during both of the ad missions to the hospital the mothe r had stopped drinking. In adults, rapid reversal of alcohol-induced pseudo-Cu shing syndrome may occur if alcohol is withheld." Whatever the reason for the disproportionate severity of the clinical findings, the striking alteration in the patient's course after the mother had been persuaded to
967
stop drinking confirmed the relationship between the clinical findings and the alcohol in the breast milk. We remain advocates of breast -feeding and would not proscribe alcohol. It is the combin ation that is und esirable. REFERENCES I. Smals AG, Klopp e nborg PW , Nj o KT , K noben. JM , a nd Rul a nd OM : Alco hol ind uced Cus hing oid synd rom e, Br Med 12: 1298, 1976 , 2, Re es LH, Besser OM, an d Jeffc oate WJ : Alcohol induced pseudo-Cushing's syndrome, Lan cet 1:726, 1977. 3. Sm als A, and Kloppenborg P: Alcoh ol ind uced psuedoCush ing's syndrome, La ncel 2: 1369, 1977 . 4. Fraj rla R, a nd Angeli A: A lco hol ind uced pse udo-Cush ing's syndrome, Lancet 1: 1050, 1977. 5, Paton A: Alcohol induced Cushingo id synd rome , BI" Med J 2: 1504, 1976. 6. Jenkins IS, and Connelly .1 : Adr en ocortical respo nse to et hanol in ma n, Br Mcd J 2: 804, 1968. 7. Mendelson JI-I: Biolog ic co nco m ita n ts o r alco holism , N Engl J Med 283: 71, 1970. 8, Beller S, Roman L, DeCastro 0, and Herrera M : Effect of acute ethanol intake on plasma 11 .hydrol\yco~ticosteroid levels, Metabolism 19:664, 1970. 9. Mendelson JH, Ogotn M, and Mello N K : Ad renal fun ction and a lcoholism I. Serum cortiso l, Psych osorn Med 33: 145,
t971.
10.
Lorid an L, an d Senior B: Cush ing's syndrome in infan cy, J PWI ATR 75: 349, 1969. I I. Edd y RL, Jones A L, Gillilund PF . Ib ar ra 10, Thompson 1Q , an d McMu rry JF: Cushing's syndrome: A prospective study o r di agnostic methods, Am J Med 55:6 21, 1973. 12. Fr a nks RC : Diurnal vari a tion of pl asma 17 hydro xycort icosteroids in chil dren, 1 Clin End oc rinol 27:75, 1967.
Detection of antibody to rotavirus by counterimmunoelectrophoresis in human serum, colostrum, and milk O. A. Cook, B.Sc.," A, Zbitnew, M .Sc ., G, Dempster, M.D ., and J. W. Gerrard, M.D., Saskatoon, Sask. Canada
AT LE AST ONE STUDY has demonstrate d a significantly lower incidence of rotavirus infection in breast-fed versus bottle-fed infants, I a nd others" :I have indicated that most
From the Departmen t of Clin ical Mic robiology, University H ospital, and Departm ent of Pediatrics, University of Sask at chewan, >Reprlnt address: Department of Clinical Mtcrobiology, University Hospttat; S askatoon, Saska tchewan. Canada, S7 N OW8.
0022-3476/78 /120967 + 04$00 .40/0 \0 1978 The C. V. Mosby Co.
ill patien ts in their stud ies were not breast- fed. Woode et al' have demonstrated rotav irus neutralizing an tibody in bovine colostrum and tha t such colostr um, when fed to calves, protects them Cram subsequen t challen ge with virulent virus. Hum an colostrum conta ins antibodies to other enteric viruses, including polioviruses, some Coxsackie type B viruses, and some echov iruses.' ' L We speculated that rota virus antibodies might be found in human colostrum and milk, which might account to some
968
Brief clinical and laboratory observations
degree for its protective effects. This study was designed to investigate the prevalence of such an tibodies in human colostrum and milk. See related article, p. 9] 6.
Abbreviations used ClEP:
counterimmuncelectrophoresis
FA:
immunofluorescence
MATERIALS AND METHODS Clinical material. Specimens were collected from consenting maternity pa tien ts following delivery at University Hospital, Saskatoon. Saskatchewan. Cord serum was collected at delivery and maternal serum on the third day post partum. Colostrum was collected on the first day and samples of milk and of infant stool were obtained on the third day. A total of 32 maternal sera, 32 cord sera, 29 colostrum samples, 26 milk samples. and 21 infant stools were collected from 34 maternity patients. In 31 of these patients, the infants were being breast-fed. In addition , milk samples from human donors were provided by the mothers' milk bank, Saskatoon City Hospital. A total of 22 samples of milk from 20 donors was obtained. Length of time between parturition and collection of the milk varied from three days to one year. Preparation of clinical material. Colostrum and milk samples were first clarified by centrifugation at 10,000 rpm for 30 minutes at 4°C in a Sorvall RC-2B centrifuge. The fat layer at 'the top was carefully removed and the clear supernatant fluid was removed and stored at -20°e. All milk samples and some colostrum samples were concentrated approximately fivefold using Lyphogel (Gelman Scientific) before testing. Stools were emulsified with a small amount of 0.85% saline to make a heavy suspension and were then clarified by centrifugation at 17,000 rpm for 30 minutes at 4°C. The clear supernatant was removed and stored at -20°e. Counterimmunoelectrophoresis. CIEP assays for determination of rotavirus antibody were performed by the method of Middleton et aF; however, the pH of the barbital buffer was 8.6 and this buffer was diluted 1:5 for preparation of gels. To enhance visualization of the precipitate lines, following electrophoresis, slides were washed twice in 0.85% saline followed by distilled water and then air dried at 37°C. Slides were stained for one hour in 0.1% thiazine red in 1% acetic acid followed by thrice rinsing in a solution of 45 parts water: 45 parts methanol: 10 parts acetic acid. Rotavirus antigen was prepared from the fluid stoat of
The Journal of Pediatrics December 1978
an infant in which large numbers of typical rotavirus particles had been observed by negative staining electron microscopy. A 50% suspension of stool was prepared in borate-buffered saline (80 ml 1.5M NaCl, 100 ml O.5M boric acid, 24 ml l.OM NaOH , and distilled deionized water to make one liter) , pH 9.0. Further dilutions of this material were prepared in gel buffer. The optimum dilution of antigen for detection of antibody was determined by checkerboard titration using CIEP; the optimum was considered that dilution of antigen which detected the lowest concentration of antibody. Indirect immunofluorescence. The method was similar to those previously described." Calf rota virus (Norden Laboratories, Lincoln, Neb .) infected LLC-MK, cells were fixed using the method of Schmitz and Enders" and were overlaid with serum diluted 1:2, followed by staining with fluorescein-labeled antiserum to human immunoglobulins (Hyland Laboratories). Slides were examined by ultraviolet illumination; only those sera giving unequivocal fluorescence in the absence of staining in control cultures were recorded as positive. Reference antiserum. Rotavirus reference antiserum (rabbit anti neonatal calf diarrhea virus) was obtained from Norden Laboratories. Lincoln, Neb. RESULTS Specificity of the CIEP test. Specificity of the antigen was tested by titration of acute and convalescent phase sera from patients known to have had rotavirus infections. Sera from three such patients all showed rising titers of antibody to the antigen. In contrast, paired sera showing fourfold or greater rises in antibody titer to other viruses (four adenovirus, two coxsackievirus B2, one echovirus 6, and one echovirus II) failed to show rising titers of antibody to the rota virus antigen . Specificity of the reference antiserum was examined by testing in CIEP against similarly prepared human stool antigens from which a variety of viruses had been isolated, including adenoviruses 2, 3, and 7, reovirus 2, coxsackievirus type A. coxsackievirus type B, and echovi rus 9. No precipitate lines were observed with any of these antigens . Specificity of the reaction was further tested by mixing the rotavirus antigen with reference serum and incubating at room temperature for one hour. When this mixture was placed in cathodal wells and reacted with antibodycontaining material, no precipitate lines were observed. Antibody detection. The results of antibody detection from the maternity patients are as follows: Precipitate lines were observed in 30/32 (93.8%) of the maternal sera, 17/32 (53.1%) of the cord sera, 20129 (68.9%) of the colostrum samples, 5/26 (19.3%) of the milk samples, and
Brief clinical and laboratory observations
Volume 93 Number 6
1121 (4.8%) of the infant stools at 3 days of age. Antibody was detected by indirect FA in 29/32 (90.6%) of the maternal sera and in 24/32 (79.4%) of the cord sera. The results of the two tests are not significantly different for detection of rotavirus antibody in serum. Attempts to demonstrate antibody by indirect FA in colostrum, milk and infant stools were unsuccessful. In milk samples from the mothers' milk bank, antibodies were detected by CIEP in two of seven samples collected less than one week post-partum, but not in any of 13 samples collected more 'than one week postpartum.
DISCUSSION The results of this study indicate that under the conditions employed, CIEP is a useful and sensitive tool for detection of antibody to rotavirus in epidemiologic surveys. This is in contrast to the findings of Middleton et at' who reported the test useful for diagnostic serology and detection of antibody in sera of children, but that this technique rarely detected antibody in adult sera. We found CIEP as sensitive as some of the other reported techniques, such as indirect FA and complement fixation. Blacklow et al'' detected antibody by such techniques in 62 to 84% of sera from human beings more than 18 months of age. In the present study, 93.8% of the adults were found by CIEP to have circulating rotavirus antibody, as compared to 90.6% by indirect FA. A rapid decline in the ability to detect antibodies in milk occurs by the third day of lactation. Even fivefold concentration of the milk failed to reveal antibodies in milk of 72% of the patients who had demonstrable antibodies in colostrum. These findings are similar to those of Woode et al,' who noted a rapid decline in levels of neutralizing antibody to calf rotavirus in bovine colostrum within 24 to 48 hours of delivery. Subsequent to completion of our study, Thouless et al'" reported rotavirus neutralizing antibodies in human milk. These workers report a similar decline in levels of antibodies, which have all but disappeared by the fifth day after delivery. Simhon and Mata" recently reported rotavirus antibodies in the colostrum of 30% of a group of Costa Rican women. These workers used an enzyme-linked imrnunosorbent assay, which is reported to be a very sensitive technique." We detected antibodies in a much greater percentage (68.9%) of the colostrum samples tested in our study. In view of the sensitivity and simplicity of elEP over other methods, further work using this test is warranted, particularly since such a method would be useful in areas lacking access to more sophisticated equipment. Woode et al ' showed that feeding colostrum with high antibody titers to calves, followed by challenge with
969
virulent virus, was protective. Similar studies with lambs indicated that oral administration of serum or colostrum containing rotavirus antibody protected against experimental infection with Iamb rotavirus.':' Our study revealed rotavirus-specific antibody in the stools of at least one and probably two infants who had been breastIed-colostrum which contained antibody to rotavirus. This suggests that antibodies in milk do find their way to the intestinal lumen of the infant and are thus in a position to protect the infant against invading microorganisms. The fact that rotavirus antibodies disappear rapidly from milk suggests that the reduced incidence of rotavirus gastroenteritis in older breast-fed infants may be due to other antiviral factors in milk, such as that described by Matthews et al.H Donor milk banks, including the one in this city, usually sterilize milk by boiling as a standard procedure prior to feeding it to infants. This practice has the unfortunate consequence of destroying many of the protective proteins found in milk, including antibodies. Raptopoulou-Gigi et al" pointed out that pasteurization is effective for sterilizing milk, even with samples that were artificially heavily contaminated with bacteria. The pasteurization process destroys neither antibody nor tactoferrin, another important milk protein. In light of the ever-increasing numbers of reports of protective factors in milk, the practice of boiling milk should be discouraged in favor of sterilization by pasteurization so that the alleged benefits of feeding pooled human milk can be fully realized. We thank Dr. R. O.Sugarman and theobstetrics stafffor their cooperation in collection ofspecimens, Drs. E. I. Orodums and P. J. Middleton and Ms. M. Sarich for reviewing themanuscript and for helpfulcomments. and Miss Heather Blondeau for secretarial assistance. REFERENCES I.
Totterdell BM, Chrystie II." and Danatvala lE: Rotavirus infections in a maternity unit, Arch Dis Child 51:924, 1976.
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Bryden AS, Davies HA, Hadley RE, Flcwett TH, Morris CA, and Oliver P: Rotavirus enteritis in thewest midlands during 1974, Lancet 2:241, 1975. 3. Kapikian AZ, Kim HW, Wyatt RO, ClineWL, Arrobio .IO, Brandt CD, Rodriguez WJ, Sack DA, Chanock RM, and Parrott RH: Human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children, N Engl J Med 294:965, 1976.
4. Woode ON, Jones J, and Bridger J: Levels of colostral antibodies against neonatal calf diarrhea virus, Vet Rec 97:148,1975.
5. John TJ, and Devarajan LV: Poliovirus antibody in milk and sera of lactating women, Indian J Med Res 61: 1009, 1973.
6. Kenny JF, Boesman MI, and Michaels RH: Bacterial lind
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Brief clinical and laboratory observations
viral coproantibodies in breast-fed infants, Pediatrics 39:202, 1967. Middleton PJ, Petrie M, Hewitt CM, Szymanski MT, and Tam JS: Counterimmunoelectro-osmophoresis for the detection of infantile gastroenteritis virus (orbi-group) antigen and antibody, J Clin Patho129:191, 1976. Blacklow NR, Echeverria P, and Smith DH: Serological studies with reovirus-like enteritis agent, Infect Immun 13:1563, 1976. Schmitz H, and Enders G: Cytomegalovirus as a frequent cause of Guillain-Barre syndrome, J Med Virol 1:21, 1977. Thouless ME, Bryden AS, and Flewett TH: Rotavirus neutralization by human milk, Br Med J 2: 1390, 1977. Simhon A, and Mata L: Anti-rotavirus antibody in human colostrum, Lancet 1:39, 1978.
The Journal of Pediatrics December 1978
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Yolken R, Kim HW, Clem T, Wyatt RG, Kalica AR, Chanock RM, and Kapikian AZ: Enzyme-linked immunesorbent assay (ELISA) for detection of human reovirus-like agent of infantile gastroenteritis, Lancet 2:263, 1977. 13. Snodgrass DR, and Wells PW: Rotavirus infection in lambs: studies on passive protection, Arch Virol 52:201, 1976. 14. Matthews THJ, Nair CDG, Lawrence MK, and Tyrrell DAJ: Antiviral activity in milk of possible clinical importance, Lancet 2: 1387, 1976. 15. Raptopoulou-Gigi M, Marwick K, and McClelland DBL: Antimicrobial proteins in sterilised human milk, Br Med J 1: 12, 1977.
Cold weather and rhabdomyolysis Mark A. Raifman, M.D., Far Rockaway, N. Y., Moshe Berant, M.D., Hadera, Israel, and Carl Lenarsky, M.D., Far Rockaway, N. Y.
RHABDOMYOLYSIS is an uncommon condition which may have serious consequences, such as severe electrolyte imbalance, cardiac arrhythmias, renal failure, and respiratory failure.' The clinical presentation is frequently nonspecific. Reports on rhabdomyolysis do not list exposure to cold as one of the causes of this condition. This report describes three instances of rhabdomyolysis asso-
Laboratory investigation as well as family and past history failed to disclose any of the factors that are usually considered in a patient with an attack of rhabdomyo 'g'sis. However, a detailed history of the youngster's activities on the day of admission revealed that he had spent one and one-half hours standing at a bus stop. dressed in a short overcoat, thin trousers, tube socks, and sneakers. That day was dry, with temperatures around -SoC and winds of 20 miles per hour.
ciated with exposure to cold weather. CASE REPORTS Patient 1. A 15-year-old boy was referred to the emergency room at Peninsula Hospital Center as "rule out nephrotic syndrome," because of bilateral ankle edema and 2+ protein in the urine. Blood pressure on admission was 120170 mm Hg. The positive physical findings were limited to the lower extremities, with painful and tender symmetric swelling of the legs and ankles. The overlying skin was normal and was cool to touch. There was no evidence of any vascular incompetence and peripheral pulses were equal and palpated bilaterally. Urinalysis showed 2+ protein, was slightly benzidine positive, and had no cells or casts on microscopic examination. The color of the urine was not noted. Results of tests for blood urea nitrogen, creatinine, electrolytes, proteins, cholesterol, and total lipids were all normal. Serum phosphorus concentration was 5.8 mg/dl, and calcium 10.1 mg/dl; SOOT was 180 units, LDH 800 units, and CPK was initially 3,400 units and rose to 5,200 units on the following day. From the Department of Pediatrics, Peninsula Hospital Center. • Reprint address: Department of Pediatrics, Peninsula Hospitai Center, For Rockaway, NY 11691.
See related article, p, 1015. Abbreviations used: SOOT: serum glutamic oxaloacetic transaminase LDH: lactate dehydrogenase CPK: creatine phosphokinase Patient 2. A 4-year-old girl was referred with a diagnosis of "acute rheumatic fever." She had awakened that morning with swelling and severe pain in both feet and ankles. The patient was one of eight children of an indigent family, living in a shabby, unheated home. The evening before admission the child had been stepping into puddles covered with thin' ice and went to sleep without taking off her wet socks and sneakers. The temperature that night was below the freezing point. Physical examination showed her to be unable to stand because of the pain in her feet. Her ankles and her feet were swollen and exquisitely tender. The overlying skin appeared normal, without lesions, and was ice cold to touch. There were no other abnormal findings. The urine had a slightly pink tinge, contained 2+ protein with a positive benzidine test. Blood counts and renal function studies were all normal. The serum potassium concen-
0022-3476/78/120970+02$00.20/0 © 1978 The C. V. Mosby Co.