695 IMMUNOFLUORESCENCE OF HUMAN REOVIRUS-LIKE AGENT OF INFANTILE DIARRHŒA
SIR,-Wyatt et al.’ described the successful cultivation ( human reovirus-like (R.v.L.) agent from stool filtrates in pr mary human embryonic kidney (H.E.K.) cell-culture. In Japan, R.V.L. agent is found in infantile diarrhoea accon panied by white stools (called hakuri in Japanese).2 The caus of the white stools is still unclear. We have tried to titral serum-antibody of infantile diarrhoea by indirect immum fluorescence (LF.), using primary H.E.K. cells infected wit antigen source. was prepared from white-diarrhoea sto( from an 11-month-old child admitted to the pacdiatric ward ( Nagoya University Hospital. Acute and convalescent sera wet collected from 7 other infants (aged 7-13 mo) with white-sto( diarrhrea. The stool filtrates of these 8 patients were examine by electron microscopy, and large quantities of R.V.L. ager were identified in all stools. 1.5 aliquots of the 2% stool fi trates were inoculated on primary H.E.K. monolayers in sma
R.V.L.
agent
as
A 2% stool filtrate3
initial choict of digoxin dose.
’Daily dose: first maintenance dose is given 6 h after loading dose. "Patients requiring reduced doses can be identified by routine -e measurement of serum-creatinine concentration and prediction of 10 creatinine clearance from nomogram of Siersbaek-Nielson et al.
tion of both loading2 6 and maintenance78 doses are necessary for greater degrees of impairment. The short interval between loading and maintenance dose allows the former to beBI small, so reducing the risk of early gastrointestinal symptoms. Department of Pharmacology and Therapeutics, Middlesex Hospital Medical School, London W1P 7PN
SYLVIA M. DOBBS
culture bottles. These infected cell-cultures were maintaine with Eagle M.E.M. at 37°C. Although inoculated monolaye; showed slight degenerative changes, clear cytopathic effect WI not observed. On the 14th day after inoculation the cultun were frozen and thawed once, and the cell-fluid mixture WI inoculated into other primary H.E.K. cell-cultures. At this tin infected cells were removed from the bottles with try] sin-E.D.T.A. and concentrated by centrifugation; and aceton fixed preparations were made for l.F. Convalescent serum froi 1 patient and F.I.T.c.-conjugated rabbit anti-human IgM we] used for staining. To check the successful passage of the agen Of. was done after every passage. Viral antigens, seen as fine granules in the cytoplasm, we: observed after each passage. After the fifth passage, the pe centage of I.F.-positive cells was estimated to be 3-4%.
Wyatt, R. G., Gill, V. W., Sereno, M. M., Kalica, A. R., Vankirk, D. H., Chanock, R. M., Kapikian, A. Z. Lancet, 1976, i, 98. 2. Konno, T., Suzuki, H., Ishida, N. ibid. 1975, i, 918. 3. Bishop, R. F., Davidson, G. P., Holmes, I. H., Ruck, B. J. ibid. 1974, i, 149. 4. Kapikian, A. Z., Kim, H. W., Wyatt, R. G., Rodriguez, W. J., Ross, S., Cline, W. L., Parrott, R. H., Chanock, R. M. Science, 1974, 185, 1049. 1.
ONDINE’S CURSE
SIR,-Dr Swift (Sept. 11,
p. 588) says he would welcome the case of Ondine’s curse he describes. At this point in our knowledge of sleep-linked respiratory periodicity may I suggest that the child should have a permanent tracheostomy with the valve open at night (and during the afternoon sleep if she has one) and closed by day. Quite a lot has been written on this subject-earlier this year, for example, Guilleminault et al. described sleep apnoea in comments on
eight children.11I If before a tracheostomy is done medical treatment can be tned I would suggest that clomipramine should be given in the erening (half the dose with the evening meal and half at bedtime This treatment
has been successful in adults with the
pickwickian syndrome 12 13 and has been tried with some success children with near-miss sudden-infant-death syndrome (unseems old enough for this treatment; in small babies with near-miss s.i.D.s. the problem is Tiore difficult because we do not know the effect of the drug un the maturing brain.
In
publishedl. Dr Swift’s patient
Laboratoire d’E.E G., G., Hôoital Broussais, Paris XIV, France
B. A. SCHWARTZ
6 Reuning, R.H., Sams, R. A., Notan, R. E. J. clin. Pharmac. 1973, 13, 127. 7 Jelif e, R. W., Brooker, G. Am. J. Med. 1974, 57, 63. 8 Dettli, L., Spring, P., Ryter, S. Acta Pharmac. Tox. 1971, 29, suppl. 3, 211. d, H. Cattell, M., Modell, W., Grainer, T., Guevara, R. J. Pharmac. exp. Ther 1950, 98, 337 10 Siersbaek-Nielson, K, Hansen, J. M., Kampmann, U., Kristensen, M. 9 Gol
Lancet, 1971, i, 1133. ault, C., Eldridge, F. L., Simmons, F. B., Dement, W. C. Pediatrics, 1976, 58, 23. 12 Schwartz, B A, Granelet-Eprinchard, M.-F. Revue E.E.G. Neurophysiol. 1974, 4, 79 13 Schwartz, B A, Rochemaure, J. Nouv Presse méd 1973, 2, 1520. 11 Guillemin
Fig. 1-IgG activities in sera of 7 patients.
696 Our methods for measuring total paracetamol and free paracetamol in plasma are based on the methods of Welch and Conner and Chafetz et al.,2 respectively. We did measurements in eighteen cases of paracetamol overdose; there were 28 results in all because paracetamol levels were determined several times in some cases. One individual was admitted on three separate occasions. The results were:
*The data for free and total skewed to the left.
Fig. 2-IgM activities in sera of 7 patients.
Using the method described above, i.F. activities of 7 patients’ sera were titrated. H.E.K. cells infected with R.V.L. agent (after 5th passage) were used as antigen source. Serially diluted acute and convalescent sera and F.I.T.c.-conjugated rabbit anti-human IgG and IgM were used for indirect I.F. The results were shown in figs. 1 and 2. In acute sera, no IgG activities were detected at 4-fold dilution. However,1 patient’s serum had IgM activity at this dilution. In the convalescent phase, all 7 patients’ sera developed IgM response, but only 3 patients’ sera developed IgG response. Thus, the IgG-antibody response may not occur or may be delayed in some cases. This might suggest that the invasion of R.V.L. agent is localised at intestinal epithelium, so that only a very small amount of viral antigen is recognised by immunoTSUNEO MORISHIMA SHOICHI NAGAYOSHI TAKAO OZAKI SHIN ISOMURA SAKAE SUZUKI
Department of Pædiatrics, Nagoya University, 65 Tsurumaicho, Showaku, Nagoya, Japan
PLASMA-PARACETAMOL (ACETAMINOPHEN) MEASUREMENT
S:R,—There are several ways of measuring plasma-paracetamol. 1-6 However, some methods measure only "free" (unconjugated) paracetamol, while others measure the "total" paracetamol (free plus conjugated drug). Mrochek et aU established that the serum concentration of the glucuronide
conjugate was higher than that of the free drug 2 h after ingestion in the two subjects studied. Thus many of the methods used for measuring paracetamol may yield results which represent only a fraction of the paracetamol present in the plasma. Routh, J. I., Shane, N. A., Arredondo, E. G., Paul, W. D. Clin. Chem. 1968, 14, 882. 2. Chafetz, L., Daly, R. E., Schriftman, H., Lomner, J. J. J. pharm. Sci. 1971, 60, 463. 3. Gwilt, J. R., Robertson, A., McChesney, E. W. J. Pharm. Pharmac. 1963, 15, 440. 4. Prescott, L. F. ibid. 1971, 23, 807. 5. Welch, R. M., Conney, A. H. Clin. Chem., 1965, 11, 1064. 6. Brodie, B. B., Axelrod, J. J. Pharmac. exp. Ther. 1948, 94, 22. 7. Mrochek, J. E., Katz, S., Christie, W. H., Dinsmore, S. R. Clin. Chem. 1974, 20, 1086. 1.
paracetamol
were not
normally distnbuted, bemg
The results clearly demonstrated a considerable variation in the degree of conjugation of paracetamol, depending on the individual, the time after drug ingestion, the amount of drug ingested, and so on. Since those clinicians who treat their patients with cysteamine base their decision on whether to use cysteamine or not on the plasma-paracetamol concentration, it is clearly important to indicate which method is used, as was pointed out by Martin and Powell,s but it is even more important to indicate whether the figure given for plasma-paracetamol is for free or total drug. A result for free paracetamol can be obtained in about 15 min whereas a result for total paracetamol takes more than an hour because hydrolysis is required and this takes half an hour. Department of Pathology, Sefton General Hospital, 2HE
Liverpool L15
THOMAS A. WHITE
CYSTIC FIBROSIS IN SUMMER
SIR,—This summer has been a difficult one for the parents of many children with cystic fibrosis. The unusually hot weather has increased the risk of excessive salt loss in the sweat with the risk of hyponatraemia and hypokalaemia. However, there may be an added factor this year that has exacerbated the situation besides the heat. This is the first year that low-solute milks have been given as the norm to infants in Britain, following the concern over hypematraemia and the Government’s advice. Moreover, health visitors are now alert to the dangers of giving too much salt to normal young babies and advise mothers accordingly. There have been at least two infants with cystic fibrosis attending the University Hospital of Wales, where the use of low-solute milks was probably a factor in their becoming ill with heat prostration. It may well be advisable, especially in the summer, for infants with cystic fibrosis to be given cow’s milk or a cow’s milk equivalent rather than a low-solute milk Department of Child Health, Welsh National School of Medicine, Heath Park, Cardiff CF4 4XW
J.
R. SIBERT
PRIMARY LIVER-CELL CARCINOMA IN ACCRA
SIR,-Edington and Greenwood’s letter on primary livercell carcinoma in the savannah country of Northern Nigeria’ has prompted us to report our experience in Accra. It shows that cancer registries or surveys, particularly in Africa, which are based on histologically diagnosed cases only will grosslv underestimate the frequency of deep-seated cancers. In 1970 routine testing for alpha-fetoprotein in liver diseases was introduced to the Korle Bu Teaching Hospital. Accra, and this service was made available to the major hospi8.
Martin, P. J., Powell, J. P. Lancet, 1976, i, 536.
SIR,-Wyatt et al.’ described the successful cultivation ( human reovirus-like (R.v.L.) agent from stool filtrates in pr mary human embryonic kidney (H.E.K.) cell-culture. In Japan, R.V.L. agent is found in infantile diarrhoea accon panied by white stools (called hakuri in Japanese).2 The caus of the white stools is still unclear. We have tried to titral serum-antibody of infantile diarrhoea by indirect immum fluorescence (LF.), using primary H.E.K. cells infected wit antigen source. was prepared from white-diarrhoea sto( from an 11-month-old child admitted to the pacdiatric ward ( Nagoya University Hospital. Acute and convalescent sera wet collected from 7 other infants (aged 7-13 mo) with white-sto( diarrhrea. The stool filtrates of these 8 patients were examine by electron microscopy, and large quantities of R.V.L. ager were identified in all stools. 1.5 aliquots of the 2% stool fi trates were inoculated on primary H.E.K. monolayers in sma
R.V.L.
agent
as
A 2% stool filtrate3
initial choict of digoxin dose.
’Daily dose: first maintenance dose is given 6 h after loading dose. "Patients requiring reduced doses can be identified by routine -e measurement of serum-creatinine concentration and prediction of 10 creatinine clearance from nomogram of Siersbaek-Nielson et al.
tion of both loading2 6 and maintenance78 doses are necessary for greater degrees of impairment. The short interval between loading and maintenance dose allows the former to beBI small, so reducing the risk of early gastrointestinal symptoms. Department of Pharmacology and Therapeutics, Middlesex Hospital Medical School, London W1P 7PN
SYLVIA M. DOBBS
culture bottles. These infected cell-cultures were maintaine with Eagle M.E.M. at 37°C. Although inoculated monolaye; showed slight degenerative changes, clear cytopathic effect WI not observed. On the 14th day after inoculation the cultun were frozen and thawed once, and the cell-fluid mixture WI inoculated into other primary H.E.K. cell-cultures. At this tin infected cells were removed from the bottles with try] sin-E.D.T.A. and concentrated by centrifugation; and aceton fixed preparations were made for l.F. Convalescent serum froi 1 patient and F.I.T.c.-conjugated rabbit anti-human IgM we] used for staining. To check the successful passage of the agen Of. was done after every passage. Viral antigens, seen as fine granules in the cytoplasm, we: observed after each passage. After the fifth passage, the pe centage of I.F.-positive cells was estimated to be 3-4%.
Wyatt, R. G., Gill, V. W., Sereno, M. M., Kalica, A. R., Vankirk, D. H., Chanock, R. M., Kapikian, A. Z. Lancet, 1976, i, 98. 2. Konno, T., Suzuki, H., Ishida, N. ibid. 1975, i, 918. 3. Bishop, R. F., Davidson, G. P., Holmes, I. H., Ruck, B. J. ibid. 1974, i, 149. 4. Kapikian, A. Z., Kim, H. W., Wyatt, R. G., Rodriguez, W. J., Ross, S., Cline, W. L., Parrott, R. H., Chanock, R. M. Science, 1974, 185, 1049. 1.
ONDINE’S CURSE
SIR,-Dr Swift (Sept. 11,
p. 588) says he would welcome the case of Ondine’s curse he describes. At this point in our knowledge of sleep-linked respiratory periodicity may I suggest that the child should have a permanent tracheostomy with the valve open at night (and during the afternoon sleep if she has one) and closed by day. Quite a lot has been written on this subject-earlier this year, for example, Guilleminault et al. described sleep apnoea in comments on
eight children.11I If before a tracheostomy is done medical treatment can be tned I would suggest that clomipramine should be given in the erening (half the dose with the evening meal and half at bedtime This treatment
has been successful in adults with the
pickwickian syndrome 12 13 and has been tried with some success children with near-miss sudden-infant-death syndrome (unseems old enough for this treatment; in small babies with near-miss s.i.D.s. the problem is Tiore difficult because we do not know the effect of the drug un the maturing brain.
In
publishedl. Dr Swift’s patient
Laboratoire d’E.E G., G., Hôoital Broussais, Paris XIV, France
B. A. SCHWARTZ
6 Reuning, R.H., Sams, R. A., Notan, R. E. J. clin. Pharmac. 1973, 13, 127. 7 Jelif e, R. W., Brooker, G. Am. J. Med. 1974, 57, 63. 8 Dettli, L., Spring, P., Ryter, S. Acta Pharmac. Tox. 1971, 29, suppl. 3, 211. d, H. Cattell, M., Modell, W., Grainer, T., Guevara, R. J. Pharmac. exp. Ther 1950, 98, 337 10 Siersbaek-Nielson, K, Hansen, J. M., Kampmann, U., Kristensen, M. 9 Gol
Lancet, 1971, i, 1133. ault, C., Eldridge, F. L., Simmons, F. B., Dement, W. C. Pediatrics, 1976, 58, 23. 12 Schwartz, B A, Granelet-Eprinchard, M.-F. Revue E.E.G. Neurophysiol. 1974, 4, 79 13 Schwartz, B A, Rochemaure, J. Nouv Presse méd 1973, 2, 1520. 11 Guillemin
Fig. 1-IgG activities in sera of 7 patients.
696 Our methods for measuring total paracetamol and free paracetamol in plasma are based on the methods of Welch and Conner and Chafetz et al.,2 respectively. We did measurements in eighteen cases of paracetamol overdose; there were 28 results in all because paracetamol levels were determined several times in some cases. One individual was admitted on three separate occasions. The results were:
*The data for free and total skewed to the left.
Fig. 2-IgM activities in sera of 7 patients.
Using the method described above, i.F. activities of 7 patients’ sera were titrated. H.E.K. cells infected with R.V.L. agent (after 5th passage) were used as antigen source. Serially diluted acute and convalescent sera and F.I.T.c.-conjugated rabbit anti-human IgG and IgM were used for indirect I.F. The results were shown in figs. 1 and 2. In acute sera, no IgG activities were detected at 4-fold dilution. However,1 patient’s serum had IgM activity at this dilution. In the convalescent phase, all 7 patients’ sera developed IgM response, but only 3 patients’ sera developed IgG response. Thus, the IgG-antibody response may not occur or may be delayed in some cases. This might suggest that the invasion of R.V.L. agent is localised at intestinal epithelium, so that only a very small amount of viral antigen is recognised by immunoTSUNEO MORISHIMA SHOICHI NAGAYOSHI TAKAO OZAKI SHIN ISOMURA SAKAE SUZUKI
Department of Pædiatrics, Nagoya University, 65 Tsurumaicho, Showaku, Nagoya, Japan
PLASMA-PARACETAMOL (ACETAMINOPHEN) MEASUREMENT
S:R,—There are several ways of measuring plasma-paracetamol. 1-6 However, some methods measure only "free" (unconjugated) paracetamol, while others measure the "total" paracetamol (free plus conjugated drug). Mrochek et aU established that the serum concentration of the glucuronide
conjugate was higher than that of the free drug 2 h after ingestion in the two subjects studied. Thus many of the methods used for measuring paracetamol may yield results which represent only a fraction of the paracetamol present in the plasma. Routh, J. I., Shane, N. A., Arredondo, E. G., Paul, W. D. Clin. Chem. 1968, 14, 882. 2. Chafetz, L., Daly, R. E., Schriftman, H., Lomner, J. J. J. pharm. Sci. 1971, 60, 463. 3. Gwilt, J. R., Robertson, A., McChesney, E. W. J. Pharm. Pharmac. 1963, 15, 440. 4. Prescott, L. F. ibid. 1971, 23, 807. 5. Welch, R. M., Conney, A. H. Clin. Chem., 1965, 11, 1064. 6. Brodie, B. B., Axelrod, J. J. Pharmac. exp. Ther. 1948, 94, 22. 7. Mrochek, J. E., Katz, S., Christie, W. H., Dinsmore, S. R. Clin. Chem. 1974, 20, 1086. 1.
paracetamol
were not
normally distnbuted, bemg
The results clearly demonstrated a considerable variation in the degree of conjugation of paracetamol, depending on the individual, the time after drug ingestion, the amount of drug ingested, and so on. Since those clinicians who treat their patients with cysteamine base their decision on whether to use cysteamine or not on the plasma-paracetamol concentration, it is clearly important to indicate which method is used, as was pointed out by Martin and Powell,s but it is even more important to indicate whether the figure given for plasma-paracetamol is for free or total drug. A result for free paracetamol can be obtained in about 15 min whereas a result for total paracetamol takes more than an hour because hydrolysis is required and this takes half an hour. Department of Pathology, Sefton General Hospital, 2HE
Liverpool L15
THOMAS A. WHITE
CYSTIC FIBROSIS IN SUMMER
SIR,—This summer has been a difficult one for the parents of many children with cystic fibrosis. The unusually hot weather has increased the risk of excessive salt loss in the sweat with the risk of hyponatraemia and hypokalaemia. However, there may be an added factor this year that has exacerbated the situation besides the heat. This is the first year that low-solute milks have been given as the norm to infants in Britain, following the concern over hypematraemia and the Government’s advice. Moreover, health visitors are now alert to the dangers of giving too much salt to normal young babies and advise mothers accordingly. There have been at least two infants with cystic fibrosis attending the University Hospital of Wales, where the use of low-solute milks was probably a factor in their becoming ill with heat prostration. It may well be advisable, especially in the summer, for infants with cystic fibrosis to be given cow’s milk or a cow’s milk equivalent rather than a low-solute milk Department of Child Health, Welsh National School of Medicine, Heath Park, Cardiff CF4 4XW
J.
R. SIBERT
PRIMARY LIVER-CELL CARCINOMA IN ACCRA
SIR,-Edington and Greenwood’s letter on primary livercell carcinoma in the savannah country of Northern Nigeria’ has prompted us to report our experience in Accra. It shows that cancer registries or surveys, particularly in Africa, which are based on histologically diagnosed cases only will grosslv underestimate the frequency of deep-seated cancers. In 1970 routine testing for alpha-fetoprotein in liver diseases was introduced to the Korle Bu Teaching Hospital. Accra, and this service was made available to the major hospi8.
Martin, P. J., Powell, J. P. Lancet, 1976, i, 536.
