80 such less commonly encountered virus infections of the skin as molluscum contagiosum and contagious pustular dermatitis (orf) which are both caused by pox viruses which cannot be isolated in cell-culture. In our opinion, if electron microscopy is available, the routine examination of stools of any child with gastroenteritis should now include, not only attempts to detect pathogenic bacteria, but also attempts to detect viruses since rotaviruses may be present in up to 70% of stools from children with gastroenteritis1 and adenoviruses which often fail to grow in cell cultures have also been detected in fsecal extracts.22 The electron microscope cannot distinguish between viruses that are morphologically identical-e.g., herpes simplex and varicella/zoster-but this may not matter therapeutically. Generally further virus identification is carried out in cellculture or in fertile hens’ eggs and may take a few days. However, the clinical picture, including information related to the patient’s immunological competence, together with identification of the group to which a virus detected in a clinical specimen belongs, will usually suggest a firm provisional diagnosis, enabling the clinician to consider treating his patient or protecting contacts. During the past three years, the electron microscope in this department has been in daily use for diagnostic purposes; the days are long over when the electron microscope was regarded as a sophisticated piece of research equipment which could only be operated by a full-time research worker. Virology Department, St. Thomas’ Hospital and Medical School, London SE1 7EH.
hope that the same effect could be achieved by the use of 0-05%’Liquoid’ broth, which would not have the disadvantage of inhibiting the gram-positive organisms. Consequently, for a six-month period, a comparison was made between liquoid broth and bile-salt broth in the diagnosis of typhoid fever. Results showed that, in 37 consecutive cases, the typhoid bacillus was recovered as follows: liquoid broth and bile-salt broth 22; liquoid broth only 1; bile-salt broth only 14. I am unable to explain these observations, but the practical lesson seems clear-if typhoid fever specifically is
suspected, then part of the blood should be inoculated into bile-salt broth in addition to any other media. Microbiology Department, Macclesfield Hospital, West Park Branch,
Prestbury Road,
W. D. FOSTER.
Macclesfield SK10 3BL.
J. E. BANATVALA
FATTY-ACID OXIDATION IN CYSTIC FIBROSIS SiR,—The nature of the metabolic defect in cystic fibrosis (c.F.) is unknown. However, Rosenlund et al.1 found a deficiency of certain essential fatty acids in the sera of patients with C.F., which to some extent confirms the findings of others. The observed deficiency of certain fatty acids might be a consequence of reduced intestinal absorption or of some defect in their metabolism. We
I. L. CHRYSTIE A. J. E. FLOWER.
therefore undertook to study dation) in patients with c.F.
fatty-acid oxidation (P-oxiPeripheral-blood leucocytes
FATTY-ACID OXIDATION (P-OXIDATION) IN LEUCOCYTES FROM HEALTHY CONTROLS, AFFECTED INDIVIDUALS (HOMOZYGOTES), AND CARRIERS (HETEROZYGOTES) OF C.F.
LABORATORY DIAGNOSIS OF TYPHOID FEVER
,
SIR,-Dr Watson (June 21, p. 1377) is right to call attention to the importance of the technical details of bloodculture in the diagnosis of typhoid fever. The importance of a high dilution of the blood in culture medium has been clearly demonstrated in the experimental situation,3 and the value of bile-salt broth has been known for many years.4 Perhaps some observations made in the New Mulago Hospital, Kampala, ten years ago may be of interest. In that hospital most cases of typhoid fever were diagnosed Our practice was to take on a positive blood-culture. 10 ml. venous blood and inoculate 5 ml. into each of 50 ml. glucose broth and 50 ml. bile-salt broth. In 61 consecutive cases the typhoid bacillus was recovered as follows: glucose and bile-salt broth, 30; glucose broth only, 6; bilesalt broth only, 25. The value of bile-salt broth is clearly demonstrated. However, during the same period of time, other salmonella species (mostly S. enteritidis) were isolated from blood-cultures In this case bile-salt broth showed no on 19 occasions. superiority, for the organisms were recovered from both media on 15 occasions and only from the glucose or bilesalt broth on 2 occasions each. But, bile-salt broth being highly inhibitory towards a number of important pathogens which might occur in the blood-stream, that bottle is wasted, except as an aid to the diagnosis of typhoid fever. It is supposed that bile salts exert their beneficial effect by neutralising antibacterial substances in the serum, and it seemed reasonable to ,
I
used since the metabolic defect in a number of disorders has been demonstrated in these cells,3 which avoids the need for surgical biopsy if skin fibroblasts are used. Leucocytes were isolated from 5-10 ml. of heparinised venous blood as described previously.4 [14C]-palmitate was used as the substrate, and after 2 hours’ incubation at 37 °C carbon-14 dioxide trapping and subsequent scintillation counting was used to measure the rate of oxidation.Ó Values obtained with this technique are unrelated to age Ó or sex. There was no significant difference in the results obtained in controls and either affected individuals 6r heterozygous carriers (see accompanying table). All the values in the affected individuals lay within the normal range. Unless for some special reason the defect is not expressed in leucocytes, these results indicate that the metabolic pathway involved in fatty-acid oxidation (p-oxidation) is intact in c.F. It therefore seems unlikely that the reported
were
1.
Davidson, G. P., Bishop, R. F., Townley, R. R. W., Holmes, I. H., Ruck, B. J. Lancet, 1975, i, 242. 2. Flewett, T. H., Bryden, A. S., Davies, H., Morris, C. A. ibid. p. 4. 3. Roome, A. P. C. H., Tozer, R. A. J. clin. Path. 1968, 21, 719. 4. Wilson, G. S., Miles, A. A. Topley and Wilson’s Principles of Bacteriology and Immunology; p. 1838. London, 1964.
I
1.
Rosenlund,
M. L.,
Kim, H. K., Kritchevsky, D. Nature, 1974, 251,
719. 2. 3. 4.
Kuo, P. T., Huang, N. N. J. clin. Invest. 1965, 44, 1924. Hsia, D. Y. Y. Clin. Genet. 1970, 1, 5. Emery, A. E. H., King, B., Brock, D. J. H. J. neurol. Sci. 1971, 14,
5.
King, B., Emery, A.
463. E. H. ibid.
1973, 20,
297.
81
deficiency of certain essential fatty acids in result of a defect in fatty-acid metabolism.
C.F.
is the
We thank Dr J. A. Raeburn for providing samples from of his patients, and Mrs L. Bain for technical assistance. University Department of Human
Genetics, Hospital, Edinburgh. Department of Pediatrics, Western General Hospital, Edinburgh. University Department of Child Life and Health, Royal Hospital for Sick Children, Edinburgh.
some
Western General
A. E. H. EMERY. K. FARRELL A. J. KEAY.
W. M. MCCRAE.
EQUIVOCAL AMNIOTIC A.F.P. LEVELS ASSOCIATED WITH OPEN SPINA BIFIDA followed by alpha-fetoprotein estimation is now well established as a means of (A.F.P.) diagnosing open neural-tube lesions in high-risk pregnancies. Between 16 and 20 weeks’ pregnancy, the test is generally regarded as highly sensitive, with a wide separation between the upper limit for normal values and the lowest A.F.P. levels associated with open neural-tube lesions.l,2 The following case should, however, sound a note of caution.
SIR,-Amniocentesis
A 31-year-old woman, who gave birth to a female anencephalic in January, 1971, and a normal female in February, 1972, became pregnant again (last menstrual period July 20, 1974). She was advised to have the pregnancy monitored because the risk of recurrence was considered to be about 1 in 20. An ultrasound scan at 17 weeks showed no abnormalities, and an amniocentesis yielded amniotic fluid containing 78 g. per ml. of A.F.P. measured by the Rocket technique3 (upper limit of normal 45 tj.g. per ml.). The serum level, estimated by radioimmunoassay at the same time, was 110 ng. per ml. (this is at the 95th percentile level). Because of the high serum result the amniocentesis was repeated at l8! weeks, this time yielding amniotic fluid containing 50 ;j.g. per ml. (upper level of normal 35 !g. per ml.). Serum taken simultaneously contained 120 ng. A.F.P. per ml. (at the 95th percentile). As the pregnancy appeared to be clinically normal and the amniotic A.F.P. level was falling, and also because the A.F.P. levels in the two amniotic fluids estimated in another laboratory were thought to be only 70 and 31 fig., the pregnancy was allowed to run its normal course. A moderately well-nourished 2-9 kg. female infant was born at term, with a large open myelocele extending from T10 to the lumbar region associated with kyphosis and with almost complete paralysis of the legs and sphincters but no hydrocephalus. In addition, there was an exomphalos containing small intestines. The infant died aged 5 days.
Although the A.F.P. levels were well above the normal values, they were considerably lower than are usually associated with open neural-tube lesions. In addition, the exomphalos might also have led to an increase in the amniotic levels.4 With this case in mind, it might be advisable to view any level above the usual normal range with extreme suspicion and not to be too dogmatic about the ability to detect widely open neural-tube malformations by means of A.F.P. levels in the amniotic fluid between 16 and 20 weeks. Department of Child Health, Welsh National School of Medicine, K. M. LAURENCE Heath Park, SHEILA M. WALKER. Cardiff CF4 4XN. MARY LLOYD General Hospital, B. L. GRIFFITHS. Neath SA11 3SU. 1. 2.
Laurence, K. M. Lancet, 1974, ii, 939. Laurence, K. M. Develop. Med. Child. Neurol. 1974, 16, suppl. 32,
3.
Allen, L. P., Ferguson-Smith, M. A., Donald, I., Sweet, E., Gibson, A. A. M. Lancet, 1973, ii, 522. De Bruijn, H. W. A., Huisjes, H. J. ibid. 1975, i, 525.
BONE-MARROW CELLS RESISTANT TO CHLORAMPHENICOL IN CHLORAMPHENICOLINDUCED APLASTIC ANÆMIA
SIR,-Dr Kern and his colleagues (May 24, p. 1190) important points concerning the effect of
raise many
on human bone-marrow cultures in semi-solid agar, and they suggest that it would be premature to conclude that bone-marrow precursors of patients with chloramphenicol-induced aplastic anxmia are resistant to chloramphenicol in vitro. They found that total inhibition of colony growth for patients and controls occurred over a wide range of chloramphenicol concentrations. It is notoriously difficult to measure the point of total inhibition in pharmacological experiments and we have followed the example of the pharmacologists by estimating the E.D’50’ the concentration of drug required to inhibit response by 50%: the response in this case being colony formation. 1, The accompanying table shows the E.D.óo concentration of chloramphenicol
chloramphenicol
EFFECT OF CHLORAMPHENICOL ON COLONY FORMATION BY HUMAN BONE-MARROW CELLS FROM NORMAL CONTROLS AND PATIENTS WITH
OR
RECOVERED
FROM
APLASTIC
ANEMIA
FOLLOWING
EXPOSURE TO CHLORAMPHENICOL
poor
serum test.
Dr Kern and others’ data.
for normal colony formation, the approximate relevant in our two patients, and the estimated E.D.óo from the data presented by Dr Kern. Control 6 was run in poor serum to attempt to mimic low colony formation found in chloramphenicol-induced aplastic anaemia. The estimated E.D.óo for both Dr Kern’s patients and our own falls outside the values for our controls in all instances suggesting that colony-forming cells in these patients are relatively resistant to chloramphenicol. The E.D’97oó for controls is also given in the table: it indicates the wide variation when attempting to estimate near complete inhibition and the relative resistance of some of the cells in normal marrow. Some of the data in our paper may be explained if early in culture the cells which will form clusters (groups of 4-49 cells) are more resistant to chloramphenicol than the cells which will go on to form colonies (groups of 50 or more cells), the proportion of clusters/colonies being greater in chloramphenicol-induced aplastic anaemia than in controls. It does not explain the relative resistance of colony-forming cells to chloramphenicol outlined in the table. Our own and Dr Kern’s data suggest a shift, towards resistance to chloramphenicol in chloramphenicol-induced
figures
p. 117.
4.
1. 2.
Finney, D. J. 1971 Probit Analysis. Cambridge. Howell, A., Chinn, S., Andrews, T. M., Watts, R. W. E. Clin. Sci. molec. Med. 1974, 46, 619.
hope that the same effect could be achieved by the use of 0-05%’Liquoid’ broth, which would not have the disadvantage of inhibiting the gram-positive organisms. Consequently, for a six-month period, a comparison was made between liquoid broth and bile-salt broth in the diagnosis of typhoid fever. Results showed that, in 37 consecutive cases, the typhoid bacillus was recovered as follows: liquoid broth and bile-salt broth 22; liquoid broth only 1; bile-salt broth only 14. I am unable to explain these observations, but the practical lesson seems clear-if typhoid fever specifically is
suspected, then part of the blood should be inoculated into bile-salt broth in addition to any other media. Microbiology Department, Macclesfield Hospital, West Park Branch,
Prestbury Road,
W. D. FOSTER.
Macclesfield SK10 3BL.
J. E. BANATVALA
FATTY-ACID OXIDATION IN CYSTIC FIBROSIS SiR,—The nature of the metabolic defect in cystic fibrosis (c.F.) is unknown. However, Rosenlund et al.1 found a deficiency of certain essential fatty acids in the sera of patients with C.F., which to some extent confirms the findings of others. The observed deficiency of certain fatty acids might be a consequence of reduced intestinal absorption or of some defect in their metabolism. We
I. L. CHRYSTIE A. J. E. FLOWER.
therefore undertook to study dation) in patients with c.F.
fatty-acid oxidation (P-oxiPeripheral-blood leucocytes
FATTY-ACID OXIDATION (P-OXIDATION) IN LEUCOCYTES FROM HEALTHY CONTROLS, AFFECTED INDIVIDUALS (HOMOZYGOTES), AND CARRIERS (HETEROZYGOTES) OF C.F.
LABORATORY DIAGNOSIS OF TYPHOID FEVER
,
SIR,-Dr Watson (June 21, p. 1377) is right to call attention to the importance of the technical details of bloodculture in the diagnosis of typhoid fever. The importance of a high dilution of the blood in culture medium has been clearly demonstrated in the experimental situation,3 and the value of bile-salt broth has been known for many years.4 Perhaps some observations made in the New Mulago Hospital, Kampala, ten years ago may be of interest. In that hospital most cases of typhoid fever were diagnosed Our practice was to take on a positive blood-culture. 10 ml. venous blood and inoculate 5 ml. into each of 50 ml. glucose broth and 50 ml. bile-salt broth. In 61 consecutive cases the typhoid bacillus was recovered as follows: glucose and bile-salt broth, 30; glucose broth only, 6; bilesalt broth only, 25. The value of bile-salt broth is clearly demonstrated. However, during the same period of time, other salmonella species (mostly S. enteritidis) were isolated from blood-cultures In this case bile-salt broth showed no on 19 occasions. superiority, for the organisms were recovered from both media on 15 occasions and only from the glucose or bilesalt broth on 2 occasions each. But, bile-salt broth being highly inhibitory towards a number of important pathogens which might occur in the blood-stream, that bottle is wasted, except as an aid to the diagnosis of typhoid fever. It is supposed that bile salts exert their beneficial effect by neutralising antibacterial substances in the serum, and it seemed reasonable to ,
I
used since the metabolic defect in a number of disorders has been demonstrated in these cells,3 which avoids the need for surgical biopsy if skin fibroblasts are used. Leucocytes were isolated from 5-10 ml. of heparinised venous blood as described previously.4 [14C]-palmitate was used as the substrate, and after 2 hours’ incubation at 37 °C carbon-14 dioxide trapping and subsequent scintillation counting was used to measure the rate of oxidation.Ó Values obtained with this technique are unrelated to age Ó or sex. There was no significant difference in the results obtained in controls and either affected individuals 6r heterozygous carriers (see accompanying table). All the values in the affected individuals lay within the normal range. Unless for some special reason the defect is not expressed in leucocytes, these results indicate that the metabolic pathway involved in fatty-acid oxidation (p-oxidation) is intact in c.F. It therefore seems unlikely that the reported
were
1.
Davidson, G. P., Bishop, R. F., Townley, R. R. W., Holmes, I. H., Ruck, B. J. Lancet, 1975, i, 242. 2. Flewett, T. H., Bryden, A. S., Davies, H., Morris, C. A. ibid. p. 4. 3. Roome, A. P. C. H., Tozer, R. A. J. clin. Path. 1968, 21, 719. 4. Wilson, G. S., Miles, A. A. Topley and Wilson’s Principles of Bacteriology and Immunology; p. 1838. London, 1964.
I
1.
Rosenlund,
M. L.,
Kim, H. K., Kritchevsky, D. Nature, 1974, 251,
719. 2. 3. 4.
Kuo, P. T., Huang, N. N. J. clin. Invest. 1965, 44, 1924. Hsia, D. Y. Y. Clin. Genet. 1970, 1, 5. Emery, A. E. H., King, B., Brock, D. J. H. J. neurol. Sci. 1971, 14,
5.
King, B., Emery, A.
463. E. H. ibid.
1973, 20,
297.
81
deficiency of certain essential fatty acids in result of a defect in fatty-acid metabolism.
C.F.
is the
We thank Dr J. A. Raeburn for providing samples from of his patients, and Mrs L. Bain for technical assistance. University Department of Human
Genetics, Hospital, Edinburgh. Department of Pediatrics, Western General Hospital, Edinburgh. University Department of Child Life and Health, Royal Hospital for Sick Children, Edinburgh.
some
Western General
A. E. H. EMERY. K. FARRELL A. J. KEAY.
W. M. MCCRAE.
EQUIVOCAL AMNIOTIC A.F.P. LEVELS ASSOCIATED WITH OPEN SPINA BIFIDA followed by alpha-fetoprotein estimation is now well established as a means of (A.F.P.) diagnosing open neural-tube lesions in high-risk pregnancies. Between 16 and 20 weeks’ pregnancy, the test is generally regarded as highly sensitive, with a wide separation between the upper limit for normal values and the lowest A.F.P. levels associated with open neural-tube lesions.l,2 The following case should, however, sound a note of caution.
SIR,-Amniocentesis
A 31-year-old woman, who gave birth to a female anencephalic in January, 1971, and a normal female in February, 1972, became pregnant again (last menstrual period July 20, 1974). She was advised to have the pregnancy monitored because the risk of recurrence was considered to be about 1 in 20. An ultrasound scan at 17 weeks showed no abnormalities, and an amniocentesis yielded amniotic fluid containing 78 g. per ml. of A.F.P. measured by the Rocket technique3 (upper limit of normal 45 tj.g. per ml.). The serum level, estimated by radioimmunoassay at the same time, was 110 ng. per ml. (this is at the 95th percentile level). Because of the high serum result the amniocentesis was repeated at l8! weeks, this time yielding amniotic fluid containing 50 ;j.g. per ml. (upper level of normal 35 !g. per ml.). Serum taken simultaneously contained 120 ng. A.F.P. per ml. (at the 95th percentile). As the pregnancy appeared to be clinically normal and the amniotic A.F.P. level was falling, and also because the A.F.P. levels in the two amniotic fluids estimated in another laboratory were thought to be only 70 and 31 fig., the pregnancy was allowed to run its normal course. A moderately well-nourished 2-9 kg. female infant was born at term, with a large open myelocele extending from T10 to the lumbar region associated with kyphosis and with almost complete paralysis of the legs and sphincters but no hydrocephalus. In addition, there was an exomphalos containing small intestines. The infant died aged 5 days.
Although the A.F.P. levels were well above the normal values, they were considerably lower than are usually associated with open neural-tube lesions. In addition, the exomphalos might also have led to an increase in the amniotic levels.4 With this case in mind, it might be advisable to view any level above the usual normal range with extreme suspicion and not to be too dogmatic about the ability to detect widely open neural-tube malformations by means of A.F.P. levels in the amniotic fluid between 16 and 20 weeks. Department of Child Health, Welsh National School of Medicine, K. M. LAURENCE Heath Park, SHEILA M. WALKER. Cardiff CF4 4XN. MARY LLOYD General Hospital, B. L. GRIFFITHS. Neath SA11 3SU. 1. 2.
Laurence, K. M. Lancet, 1974, ii, 939. Laurence, K. M. Develop. Med. Child. Neurol. 1974, 16, suppl. 32,
3.
Allen, L. P., Ferguson-Smith, M. A., Donald, I., Sweet, E., Gibson, A. A. M. Lancet, 1973, ii, 522. De Bruijn, H. W. A., Huisjes, H. J. ibid. 1975, i, 525.
BONE-MARROW CELLS RESISTANT TO CHLORAMPHENICOL IN CHLORAMPHENICOLINDUCED APLASTIC ANÆMIA
SIR,-Dr Kern and his colleagues (May 24, p. 1190) important points concerning the effect of
raise many
on human bone-marrow cultures in semi-solid agar, and they suggest that it would be premature to conclude that bone-marrow precursors of patients with chloramphenicol-induced aplastic anxmia are resistant to chloramphenicol in vitro. They found that total inhibition of colony growth for patients and controls occurred over a wide range of chloramphenicol concentrations. It is notoriously difficult to measure the point of total inhibition in pharmacological experiments and we have followed the example of the pharmacologists by estimating the E.D’50’ the concentration of drug required to inhibit response by 50%: the response in this case being colony formation. 1, The accompanying table shows the E.D.óo concentration of chloramphenicol
chloramphenicol
EFFECT OF CHLORAMPHENICOL ON COLONY FORMATION BY HUMAN BONE-MARROW CELLS FROM NORMAL CONTROLS AND PATIENTS WITH
OR
RECOVERED
FROM
APLASTIC
ANEMIA
FOLLOWING
EXPOSURE TO CHLORAMPHENICOL
poor
serum test.
Dr Kern and others’ data.
for normal colony formation, the approximate relevant in our two patients, and the estimated E.D.óo from the data presented by Dr Kern. Control 6 was run in poor serum to attempt to mimic low colony formation found in chloramphenicol-induced aplastic anaemia. The estimated E.D.óo for both Dr Kern’s patients and our own falls outside the values for our controls in all instances suggesting that colony-forming cells in these patients are relatively resistant to chloramphenicol. The E.D’97oó for controls is also given in the table: it indicates the wide variation when attempting to estimate near complete inhibition and the relative resistance of some of the cells in normal marrow. Some of the data in our paper may be explained if early in culture the cells which will form clusters (groups of 4-49 cells) are more resistant to chloramphenicol than the cells which will go on to form colonies (groups of 50 or more cells), the proportion of clusters/colonies being greater in chloramphenicol-induced aplastic anaemia than in controls. It does not explain the relative resistance of colony-forming cells to chloramphenicol outlined in the table. Our own and Dr Kern’s data suggest a shift, towards resistance to chloramphenicol in chloramphenicol-induced
figures
p. 117.
4.
1. 2.
Finney, D. J. 1971 Probit Analysis. Cambridge. Howell, A., Chinn, S., Andrews, T. M., Watts, R. W. E. Clin. Sci. molec. Med. 1974, 46, 619.
