377 combination 8 (73%) had residual deficits
on
follow-up,
although their illness was of comparable severity. In a group of 17 children whose treatment began with either ampicillin alone or chloramphenicol in combination with penicillin and a sulphonamide, and was later changed to chloramphenicol and ampicillin, respectively, the incidence of sequels was 35%. Therapy was often changed because of a complicated clinical course, yet this group had a lower incidence of sequelae than the group treated with ampicillin and chloramphenicol. These results suggest that this problem may indeed be relevant, at least in children. Yale
University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, U.S.A.
THOMAS MATTHEWS
CLOSTRIDIA IN NECROTISING ENTEROCOLITIS
SIR,-Howard et aLl suggested that C. butyricum was of primary importance in necrotising enterocolitis (N.E.C.). Their observation was mainly based on direct and indirect evidence of the presence of this organism in blood-cultures in 9 of 10 affected babies. Anaerobic stool-cultures, however, were positive for C. butyricum in only 2 babies "probably attributable to inappropriate methods". Using a previously described methodwe have examined stool-cultures from two groups of neonates for the presence of clostridia. The first group consisted of ten consecutive cases of N.E.C. The age of the neonates at the onset of disease varied from 2-30 days (mean 5-7). Stool-cultures were obtained during the acute phase of the illness once the diagnosis was established. Cultures were positive for clostridia in 7 patients. Only one species of clostridium was isolated per patient. C. butyricum was isolated in 6 patients and C. paraputrificum in another. The second group of patients consisted of 19 non-N.E.C. neonates. 7 infants had been admitted to the intensive-care unit for a variety of conditions including prematurity, hyaline membrane disease, and other pulmonary disorders. The others were normal, symptom-free newborns and were cared for in a different nursery. All specimens were obtained during the first week of life (mean 3.7days of age). Clostridia were isolated in 8 of these 19 neonates. C. butyricum was found in 7 babies. On one occasion it was isolated with C. paraputrificum and on another with C. perfringens. In one neonate C. paraputrificum was isolated in pure culture. There was no significant difference in the prevalence of C. butyricum between the intensive-care-unit babies and the other nursery babies (2/7 vs 5/12). 5 babies were exclusively breast-fed and C. butyricum was isolated in one of them. None of the 7 C. butyricum-carriers had N.E.C. Gothefors and Blenkharn3have reported that, at 2 weeks of age, the gut flora of preterm babies contained several species of clostridia (including C. butyricum). With a much younger population, we failed to isolate such a variety of clostridia per patient. C. butyricum could, therefore, be one of the first species of clostridia to colonise the gut of neonates and may be the predominant flora in the gut of neonates during their first days of life. We agree with Gothefors et al. that C. butyricum in blood-cultures of N.E.C. patients could arise from a transient secondary invasion. A quantitative study of the ftcal flora of neonates from their first hours of life to several weeks of age is merited. of Microbiology and Pediatrics,
Departments
HôpitalaMaisonneuve-Rosemont, Montréal, Québec, Canada 1.
M. LAVERDIÈRE A. ROBERT R. CHICOINE
D. SALET R. ROSENFELD
Howard, F., Flynn, D., Bradley, J., Noone, P., Szawatkowski, M. Lancet, 1977, ii, 1099. 2. Laboratory Methods in Anærobic Bacteriology. C.D.C. Laboratory Manual; p. 46. 1974. 3. Gothefors, L., Blenkharn, I. Lancet, 1978, i, 52.
FALSE-POSITIVE ACID-FAST SMEARS
SiR,—The report by Kelly’ of false-positive acid-fast smears
by Mycobacterium gordonae contamination of distilled interesting. Because of the high incidence of falsesmears positive previously reported from this laboratory,2 I have carefully monitored the acid-fast-smear results for the past year. 6550 specimens were received for mycobacterial culture in 1977; mycobacteria were recovered in 230 specimens (3.5%). M. tuberculosis was recovered in 155 specimens. Acidfast smears, which consists of initial specimen screening with caused
water was
the auramine-rhodamine fluorescent stain and confirmation of all positives with the Kinyoun stain, were performed on 3754 specimens, and 59 were positive. Smears were performed on 104 specimens in which M. tuberculosis was isolated and 50 (48%) were smear-positive. Other mycobacteria were isolated in 8 specimens with positive smears (seven M. kansasii, one M. avium-intracellulare), and no mycobacteria were recovered on 1 smear-positive specimen. The false-positive smear was unusual in that the bacilli stained irregularly with both the fluorochrome and Kinyoum staining methods. Most organisms had one to two bulbous expansions, which retained the stains, along the length of the bacillus. Because of this irregular staining pattern and the fact that the patient was believed to not have a mycobacterial infection, the staining reagents were examined. The same organisms were found in all of the reagents. Mycobacteria were not grown from cultures of the reagents; this was expected since the deionised water used for preparation of the reagents is routinely autoclaved. Kelly reported that his source of contamination was an improperly cleaned distilled water reservoir. At the time the falsepositive smear was observed in this laboratory, the recirculation pump in the deioniser was not working, permitting a portion of the water to remain undisturbed. It is likely that stasis is necessary for the organisms to multiply to detectable numbers. All water used in the mycobacteriology laboratory is now filter sterilised before use. False-positive acid-fast smears have been reduced to less than 1% compared with the 55% previously reported in a retrospective study from this laboratory. Although the exact reason for this discrepancy is not known, it cpuld be attributed to an unrecognised problem with contaminated reagents. I agree with Kelly’s recommendation that only membrane-filtered solutions should be used for processing mycobacterial specimens, and would add that all unusual observations made in the laboratory, such as a high incidence of false-positive smears, should be carefully investigated. Barnes Hospital, St. Louis, Missouri 63110, U.S.A.
PATRICK R. MURRAY
SYNTHESIS OF PROCOAGULANT ANTIHÆMOPHILIC FACTOR IN VITRO
SIR,-Dr Blecher and colleagues (June 24, p. 1333) reported an interesting and innovative investigation which, we were gratified to see, confirmed our original observation that leucocytes synthesise factor vin coagulant activity.3,4We are, however, at a loss to understand why they feel that the activity we described was tissue thromboplastin and was less likely to be factor vm coagulant activity than the activity they described. The fact that their experiments were more elegant than ours does not necessarily mean that we measured,adifferent activity. Our preparations shortened the clotting-time of 1. 2.
Kelly, M. T. Lancet, 1978, i, 510. Body, J. C., Marr, J. J. Ann. intern. Med. 1975, 82, 489. 3. Zacharski, L. R., Bowie, E. J. W., Titus, J. L., Owen, Meet. Mayo Clin. 1968, 43, 617. 4. Zacharski, L. R., Bowie, E. J. W., Titus, J.
44, 784.
C.
A., Jr. Proc. Staff
L., Owen, C. A., Jr. ibid. 1969,
on
follow-up,
although their illness was of comparable severity. In a group of 17 children whose treatment began with either ampicillin alone or chloramphenicol in combination with penicillin and a sulphonamide, and was later changed to chloramphenicol and ampicillin, respectively, the incidence of sequels was 35%. Therapy was often changed because of a complicated clinical course, yet this group had a lower incidence of sequelae than the group treated with ampicillin and chloramphenicol. These results suggest that this problem may indeed be relevant, at least in children. Yale
University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, U.S.A.
THOMAS MATTHEWS
CLOSTRIDIA IN NECROTISING ENTEROCOLITIS
SIR,-Howard et aLl suggested that C. butyricum was of primary importance in necrotising enterocolitis (N.E.C.). Their observation was mainly based on direct and indirect evidence of the presence of this organism in blood-cultures in 9 of 10 affected babies. Anaerobic stool-cultures, however, were positive for C. butyricum in only 2 babies "probably attributable to inappropriate methods". Using a previously described methodwe have examined stool-cultures from two groups of neonates for the presence of clostridia. The first group consisted of ten consecutive cases of N.E.C. The age of the neonates at the onset of disease varied from 2-30 days (mean 5-7). Stool-cultures were obtained during the acute phase of the illness once the diagnosis was established. Cultures were positive for clostridia in 7 patients. Only one species of clostridium was isolated per patient. C. butyricum was isolated in 6 patients and C. paraputrificum in another. The second group of patients consisted of 19 non-N.E.C. neonates. 7 infants had been admitted to the intensive-care unit for a variety of conditions including prematurity, hyaline membrane disease, and other pulmonary disorders. The others were normal, symptom-free newborns and were cared for in a different nursery. All specimens were obtained during the first week of life (mean 3.7days of age). Clostridia were isolated in 8 of these 19 neonates. C. butyricum was found in 7 babies. On one occasion it was isolated with C. paraputrificum and on another with C. perfringens. In one neonate C. paraputrificum was isolated in pure culture. There was no significant difference in the prevalence of C. butyricum between the intensive-care-unit babies and the other nursery babies (2/7 vs 5/12). 5 babies were exclusively breast-fed and C. butyricum was isolated in one of them. None of the 7 C. butyricum-carriers had N.E.C. Gothefors and Blenkharn3have reported that, at 2 weeks of age, the gut flora of preterm babies contained several species of clostridia (including C. butyricum). With a much younger population, we failed to isolate such a variety of clostridia per patient. C. butyricum could, therefore, be one of the first species of clostridia to colonise the gut of neonates and may be the predominant flora in the gut of neonates during their first days of life. We agree with Gothefors et al. that C. butyricum in blood-cultures of N.E.C. patients could arise from a transient secondary invasion. A quantitative study of the ftcal flora of neonates from their first hours of life to several weeks of age is merited. of Microbiology and Pediatrics,
Departments
HôpitalaMaisonneuve-Rosemont, Montréal, Québec, Canada 1.
M. LAVERDIÈRE A. ROBERT R. CHICOINE
D. SALET R. ROSENFELD
Howard, F., Flynn, D., Bradley, J., Noone, P., Szawatkowski, M. Lancet, 1977, ii, 1099. 2. Laboratory Methods in Anærobic Bacteriology. C.D.C. Laboratory Manual; p. 46. 1974. 3. Gothefors, L., Blenkharn, I. Lancet, 1978, i, 52.
FALSE-POSITIVE ACID-FAST SMEARS
SiR,—The report by Kelly’ of false-positive acid-fast smears
by Mycobacterium gordonae contamination of distilled interesting. Because of the high incidence of falsesmears positive previously reported from this laboratory,2 I have carefully monitored the acid-fast-smear results for the past year. 6550 specimens were received for mycobacterial culture in 1977; mycobacteria were recovered in 230 specimens (3.5%). M. tuberculosis was recovered in 155 specimens. Acidfast smears, which consists of initial specimen screening with caused
water was
the auramine-rhodamine fluorescent stain and confirmation of all positives with the Kinyoun stain, were performed on 3754 specimens, and 59 were positive. Smears were performed on 104 specimens in which M. tuberculosis was isolated and 50 (48%) were smear-positive. Other mycobacteria were isolated in 8 specimens with positive smears (seven M. kansasii, one M. avium-intracellulare), and no mycobacteria were recovered on 1 smear-positive specimen. The false-positive smear was unusual in that the bacilli stained irregularly with both the fluorochrome and Kinyoum staining methods. Most organisms had one to two bulbous expansions, which retained the stains, along the length of the bacillus. Because of this irregular staining pattern and the fact that the patient was believed to not have a mycobacterial infection, the staining reagents were examined. The same organisms were found in all of the reagents. Mycobacteria were not grown from cultures of the reagents; this was expected since the deionised water used for preparation of the reagents is routinely autoclaved. Kelly reported that his source of contamination was an improperly cleaned distilled water reservoir. At the time the falsepositive smear was observed in this laboratory, the recirculation pump in the deioniser was not working, permitting a portion of the water to remain undisturbed. It is likely that stasis is necessary for the organisms to multiply to detectable numbers. All water used in the mycobacteriology laboratory is now filter sterilised before use. False-positive acid-fast smears have been reduced to less than 1% compared with the 55% previously reported in a retrospective study from this laboratory. Although the exact reason for this discrepancy is not known, it cpuld be attributed to an unrecognised problem with contaminated reagents. I agree with Kelly’s recommendation that only membrane-filtered solutions should be used for processing mycobacterial specimens, and would add that all unusual observations made in the laboratory, such as a high incidence of false-positive smears, should be carefully investigated. Barnes Hospital, St. Louis, Missouri 63110, U.S.A.
PATRICK R. MURRAY
SYNTHESIS OF PROCOAGULANT ANTIHÆMOPHILIC FACTOR IN VITRO
SIR,-Dr Blecher and colleagues (June 24, p. 1333) reported an interesting and innovative investigation which, we were gratified to see, confirmed our original observation that leucocytes synthesise factor vin coagulant activity.3,4We are, however, at a loss to understand why they feel that the activity we described was tissue thromboplastin and was less likely to be factor vm coagulant activity than the activity they described. The fact that their experiments were more elegant than ours does not necessarily mean that we measured,adifferent activity. Our preparations shortened the clotting-time of 1. 2.
Kelly, M. T. Lancet, 1978, i, 510. Body, J. C., Marr, J. J. Ann. intern. Med. 1975, 82, 489. 3. Zacharski, L. R., Bowie, E. J. W., Titus, J. L., Owen, Meet. Mayo Clin. 1968, 43, 617. 4. Zacharski, L. R., Bowie, E. J. W., Titus, J.
44, 784.
C.
A., Jr. Proc. Staff
L., Owen, C. A., Jr. ibid. 1969,
