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ANTISERUM AND SULPHAPYRIDINE IN MENINGOCOCCAL INFECTIONS IN MICE
999 to combined treatment with
ANTISERUM AND SULPHAPYRIDINE
meningococcus
IN MENINGOCOCCAL INFECTIONS IN MICE
BY C. RUSSELL
AMIES, M.D. Lond.
BACTERIOLOGIST-IN-CHARGE, SERUM DEPARTMENT, LISTER INSTITUTE, ELSTREE
THE spectacular results of using sulphonamides in cerebrospinal meningitis are founded on laboratory studies of experimental meningococcal infection of
mice.
sulphapyridine
and anti-
serum.
TECHNIQUE
Mice
were
inoculated with
a
highly
virulent strain
of meningococcus suspended in mucin. One group received the test dose of organisms alone, while other groups received also serum, or sulpha pyridine, or a combination of these. The interval between the inoculation of the meningococci and the institution of treatment was varied in different experiments, while the dosage of the drug or of the serum was kept constant.
Two group-i meningococcus strains, here designated 198 and 220, were chosen for this work because of their excepp-aminobenzenesulphonamide (sulphanilamide) would tional virulence to mice. Both of them were originally protect mice against experimentally induced meningo- obtained from Dr. W. M. Scott, of the Ministry of Health, Proom (1937) demonstrated No. 198 in coccal septicaemia. May, 1939, and No. 220 in February, 1940. that this preparation was effective when given by Each had been recently isolated from a case of cerebroand that it could protect against a million fatal mouth, fever. They were maintained on nutrient agar conspinal doses of meningococci, provided that the administrataining 2 per cent. of bacteriological peptone (Evans Sons tion of the drug was begun immediately after the Lescher and Webb), 0-5 per cent. of glucose, and 5 per cent. inoculation of the organisms. When treatment was of defibrinated horse blood. Subcultures were made at delayed for several hours, however, much less protec- intervals of twelve hours, and the strains were passed tion was obtained. through mice at least once a week. In the treatment of an established infection in mice A five-hour culture was used for infecting the mice. both Branham and Rosenthal (1937) and Brown (1937) After the water of condensation had been removed and that the administration of combined reported sulph- discarded, the growth was thoroughly dispersed in plain anilamide and antimeningococcal serum gave better nutrient broth and then further diluted with the same results than either drug or serum alone. medium until the suspension matched the opacity of a Cerebrospinal fever in man was at first treated with standard suspension of barium sulphate. The number the drug and the serum, and the results were far of organisms required to produce this degree of opacity beyond expectation (McIntosh, Wilcox, and Wright was approximately 600 million. No great importance is 1937, Mitchell and Trachsler 1937, Jewesbury 1938). attached to the estimate of the number of cocci injected, Successful results obtained with sulphonamides withbecause the usual methods of enumerating bacteria are out serum were later reported by Schwentker, Gelman, associated with a large experimental error. In each and Long (1937), Crawford and Fleming (1938), experiment the activity of the suspensions was tested by Somers (1939), and Bryant and Fairman (1939). Of determining the minimal lethal dose (M.L.D.) by titration particular interest, however, are the papers by on groups of 5 mice, and in this way it was possible to Banks (1938, 1939), both because of the relatively express the test dose in terms of the number of M.L.D. large number of cases treated and because of the care- injected. All dilutions were made in plain broth, except fully controlled manner in which his investigations the final dilution, which was prepared in a 5 per cent. In one series of 65 cases serum were carried out. solution of mucin. This mucin was obtained from the (" meningococcus antitoxin " P. D. and Co.) was given Wilson Laboratories, Chicago, under the designation intravenously in large amounts and sulphanilamide Granular Mucin Type 1701 and was prepared according in various dosages, and there were 8 deaths. A comto the method of Miller and Castles (1936). parable series of 72 cases was treated by chemotherapy The infecting dose was injected intraperitoneally in a 1 in and there death. alone was This large dosage, of 1-0 c.cm. The dose of sulphapyridine was volume remarkable result may be regarded partly as a per20 mg. suspended in 0-5 c.cm. of physiological saline. sonal triumph for Dr. Banks and his nursing staff, This was injected into the lumen of the oesophagus with because the results obtained elsewhere are far less remarkable. Banks (1940), for example, gives some a wide-bore needle, of which the point had been removed and the end ground to a smooth bevel. The serum used figures, supplied by Dr. E. L. Sturdee, of the results in each experiment was taken from a batch of multivalent obtained by chemotherapy alone in hospitals throughantimeningococcus globulin prepared for normal issue. A in out England and Wales 1937-39. There were 100 of 0-004 c.cm. of this solution of globulin protected patients, and 29 died. Harries (1940) has described volume mice against 1000 minimal infective doses of group-i another 100 cases treated with sulphapyridine only, meningococci. The serum was injected intravenously. with 8 deaths. The mice weighed 17-21 g. A pure-line strain not being The following figures may serve as a basis for to use about 20 mice in each available, it was comparison with these. Before the introduction of test group. The necessary time between infection and treatment serum therapy the case-mortality of cerebrospinal varied in different experiments between four and thirteen fever was 60-80 per cent. Treatment with adequate hours. With the two strains of meningococci employed amounts of serum at the earliest possible moment invasion of the blood-stream could be demonstrated two will reduce this figure to a third or even less, hours after the injection of the test dose of organisms.
Buttle, Gray, and Stephenson (1936) showed that
provided that the serum contains specific antibodies for the prevailing type of meningococcus. Wadsworth (1932), for example, obtained a case-mortality of 18-4 per cent. among 742 patients of all ages ; and Banks (1938) has reported a series of 38 cases with
6 deaths. The question at issue is whether antimeningococcus serum can still be usefully employed as an adjuvant to chemotherapy, or whether, as Banks insists, it should be entirely dispensed with. The Ministry of Health (1940) appears to favour the latter view. Laboratory studies, which can be conducted under controlled conditions impossible in clinical practice, do not support this contention. The practical importance of this problem at the present time justifies the publication of the following experiments which, it is hoped, may induce clinicians to give a further trial
RESULTS
The results of thirteen
and two prosummarised in table I. The inclusion of several variable factors renders these numerical data unsuitable for statistical analysis, but it is admissible to givethe totals obtained by adding the figures of each experiment, provided that it is clearly recognised that these totals (table II) must be regarded as an indication of the trend of the results
phylactic experiments
therapeutic
are
and not as an accurate analysis. In every experiment the number of deaths was least in the group of mice that received both sulphapyridine and serum. In some cases the superiority of the combined treatment is admittedly not great, and the differences are not statistically significant. In
.
1000 TABLE I RESULTS OF TREATMENT OF EXPERIMENTAL MENINGOCOCCAL INFECTION OF MICE
P
=
prophylaxis.
In the two
prophylactic experiments the amount of
experiments 2, 6, 9, 10, and 11, however, the results indicate that the drug and serum exerted a synergic action, the combined effect being considerably greater than that produced by either alone. The case-mortality should not be taken as the sole criterion of success. Further information can be obtained by a comparison of the average duration of illness in the mice (see table III). COMMENTS
The results, taken as a whole, clearly demonstrate the value of giving serum besides sulphapyridine to cure experimental meningococcal septicaemia in mice. Such a procedure is, moreover, entirely rational. Without treatment the host’s natural defences, inhibited by the mucin, cannot prevent rapid multiplication of the invading organism. Sulphapyridine prevents this multiplication and so gives the host time to reorganise its immunological mechanism. If the administration of the drug is unduly delayed, the overwhelming number of organisms already present precludes the possibility of recovery, because the toxins liberated by autolysis of the meningococci will exert a lethal effect after the cocci have ceased to proliferate. On the other hand, when the combined treatment is given, sulphapyridine holds the organisms in check while the specific antibodies injected, together with such natural defences as the host may succeed in mobilising, are rendered immediately available for killing the invading organism and neutralising its toxins. These fundamental principles are valid whether the host is a man or a mouse. Clinical
serum
injected
was
0’01
c.cm.
highly virulent group-I strain of meningococcus suspended in mucin. At various periods after infection the mice received sulphapyridine, or antimeningococcus serum, or a combination of these. The combination of drug and serum gave better protection than that obtained with the drug alone. The average case-mortality in mice treated with sulphaC TABLE III-VALUE OF COMBINED TREATMENT IN CUTTIKG SHORT THE ILLNESS
(Figures taken from protocol of expt. 13)
was 59 per cent., whereas the giving oi besides the drug reduced it to 26 per cent. It is suggested that the use of antimeningococcus serum, given intravenously, as an adjuvant to chemotherapy in human cerebrospinal fever deserves further clinical trial.
pyridine only serum
TABLE II-ANALYSIS OF RESULTS SHOWN IN TABLE I
REFERENCES
Banks, H. S. (1938) Lancet, 2, 7. (1939) Ibid, 2, 921. (1940) Ibid, Jan. 6, p. 42. Branham, S. E. and Rosenthal, S. M. (1937) Publ. Hlth Rep., Wash. 52, 685. Brown, T. M. (1937) Johns Hopk. Hosp. Bull. 61, 272. Bryant, J. and Fairman, H. D. (1939) Lancet, 1, 923. Buttle, G. A. H., Gray, W. H. and Stephenson, D. (1936) Ibid, 1, 1286. Crawford, T. and Fleming, G. B. (1938) Ibid, 1, 987. Harries, G. E. (1940) Ibid, March 16, p. 522. Jewesbury, E. C. O. (1938) Ibid, 1, 1262. McIntosh, R., Wilcox, D. A. and Wright, F. H. (1937) J. Pediat. —
—
experience, however, does not favour serum to sulphonamides ; yet the
the addition of results of the experiments here recorded seem to call for further trial of a method so essentially rational. SUMMARY
A
meningococcal septicaemia, normally fatal, was produced in mice by intraperitoneal injection of a ,
11, 167. Miller, C. P. and Castles, R. (1936) J. infect. Dis. 58, 263. Ministry of Health (1940) Memorandum 234, London. Mitchell, A. G. and Trachsler, W. H. (1937) J. Pediat. 11, 183.
Proom, H. (1937) Lancet, 1, 16. Schwentker, F. F., Gelman, S. and Long, P. H. (1937) J. Amer. med. Ass. 108, 1407. Somers, R. B. U. (1939) Lancet, 1, 921.
Wadsworth,
A.
(1932) Trans. Ass. Amer. Phys. 47,
161.
ANTISERUM AND SULPHAPYRIDINE
meningococcus
IN MENINGOCOCCAL INFECTIONS IN MICE
BY C. RUSSELL
AMIES, M.D. Lond.
BACTERIOLOGIST-IN-CHARGE, SERUM DEPARTMENT, LISTER INSTITUTE, ELSTREE
THE spectacular results of using sulphonamides in cerebrospinal meningitis are founded on laboratory studies of experimental meningococcal infection of
mice.
sulphapyridine
and anti-
serum.
TECHNIQUE
Mice
were
inoculated with
a
highly
virulent strain
of meningococcus suspended in mucin. One group received the test dose of organisms alone, while other groups received also serum, or sulpha pyridine, or a combination of these. The interval between the inoculation of the meningococci and the institution of treatment was varied in different experiments, while the dosage of the drug or of the serum was kept constant.
Two group-i meningococcus strains, here designated 198 and 220, were chosen for this work because of their excepp-aminobenzenesulphonamide (sulphanilamide) would tional virulence to mice. Both of them were originally protect mice against experimentally induced meningo- obtained from Dr. W. M. Scott, of the Ministry of Health, Proom (1937) demonstrated No. 198 in coccal septicaemia. May, 1939, and No. 220 in February, 1940. that this preparation was effective when given by Each had been recently isolated from a case of cerebroand that it could protect against a million fatal mouth, fever. They were maintained on nutrient agar conspinal doses of meningococci, provided that the administrataining 2 per cent. of bacteriological peptone (Evans Sons tion of the drug was begun immediately after the Lescher and Webb), 0-5 per cent. of glucose, and 5 per cent. inoculation of the organisms. When treatment was of defibrinated horse blood. Subcultures were made at delayed for several hours, however, much less protec- intervals of twelve hours, and the strains were passed tion was obtained. through mice at least once a week. In the treatment of an established infection in mice A five-hour culture was used for infecting the mice. both Branham and Rosenthal (1937) and Brown (1937) After the water of condensation had been removed and that the administration of combined reported sulph- discarded, the growth was thoroughly dispersed in plain anilamide and antimeningococcal serum gave better nutrient broth and then further diluted with the same results than either drug or serum alone. medium until the suspension matched the opacity of a Cerebrospinal fever in man was at first treated with standard suspension of barium sulphate. The number the drug and the serum, and the results were far of organisms required to produce this degree of opacity beyond expectation (McIntosh, Wilcox, and Wright was approximately 600 million. No great importance is 1937, Mitchell and Trachsler 1937, Jewesbury 1938). attached to the estimate of the number of cocci injected, Successful results obtained with sulphonamides withbecause the usual methods of enumerating bacteria are out serum were later reported by Schwentker, Gelman, associated with a large experimental error. In each and Long (1937), Crawford and Fleming (1938), experiment the activity of the suspensions was tested by Somers (1939), and Bryant and Fairman (1939). Of determining the minimal lethal dose (M.L.D.) by titration particular interest, however, are the papers by on groups of 5 mice, and in this way it was possible to Banks (1938, 1939), both because of the relatively express the test dose in terms of the number of M.L.D. large number of cases treated and because of the care- injected. All dilutions were made in plain broth, except fully controlled manner in which his investigations the final dilution, which was prepared in a 5 per cent. In one series of 65 cases serum were carried out. solution of mucin. This mucin was obtained from the (" meningococcus antitoxin " P. D. and Co.) was given Wilson Laboratories, Chicago, under the designation intravenously in large amounts and sulphanilamide Granular Mucin Type 1701 and was prepared according in various dosages, and there were 8 deaths. A comto the method of Miller and Castles (1936). parable series of 72 cases was treated by chemotherapy The infecting dose was injected intraperitoneally in a 1 in and there death. alone was This large dosage, of 1-0 c.cm. The dose of sulphapyridine was volume remarkable result may be regarded partly as a per20 mg. suspended in 0-5 c.cm. of physiological saline. sonal triumph for Dr. Banks and his nursing staff, This was injected into the lumen of the oesophagus with because the results obtained elsewhere are far less remarkable. Banks (1940), for example, gives some a wide-bore needle, of which the point had been removed and the end ground to a smooth bevel. The serum used figures, supplied by Dr. E. L. Sturdee, of the results in each experiment was taken from a batch of multivalent obtained by chemotherapy alone in hospitals throughantimeningococcus globulin prepared for normal issue. A in out England and Wales 1937-39. There were 100 of 0-004 c.cm. of this solution of globulin protected patients, and 29 died. Harries (1940) has described volume mice against 1000 minimal infective doses of group-i another 100 cases treated with sulphapyridine only, meningococci. The serum was injected intravenously. with 8 deaths. The mice weighed 17-21 g. A pure-line strain not being The following figures may serve as a basis for to use about 20 mice in each available, it was comparison with these. Before the introduction of test group. The necessary time between infection and treatment serum therapy the case-mortality of cerebrospinal varied in different experiments between four and thirteen fever was 60-80 per cent. Treatment with adequate hours. With the two strains of meningococci employed amounts of serum at the earliest possible moment invasion of the blood-stream could be demonstrated two will reduce this figure to a third or even less, hours after the injection of the test dose of organisms.
Buttle, Gray, and Stephenson (1936) showed that
provided that the serum contains specific antibodies for the prevailing type of meningococcus. Wadsworth (1932), for example, obtained a case-mortality of 18-4 per cent. among 742 patients of all ages ; and Banks (1938) has reported a series of 38 cases with
6 deaths. The question at issue is whether antimeningococcus serum can still be usefully employed as an adjuvant to chemotherapy, or whether, as Banks insists, it should be entirely dispensed with. The Ministry of Health (1940) appears to favour the latter view. Laboratory studies, which can be conducted under controlled conditions impossible in clinical practice, do not support this contention. The practical importance of this problem at the present time justifies the publication of the following experiments which, it is hoped, may induce clinicians to give a further trial
RESULTS
The results of thirteen
and two prosummarised in table I. The inclusion of several variable factors renders these numerical data unsuitable for statistical analysis, but it is admissible to givethe totals obtained by adding the figures of each experiment, provided that it is clearly recognised that these totals (table II) must be regarded as an indication of the trend of the results
phylactic experiments
therapeutic
are
and not as an accurate analysis. In every experiment the number of deaths was least in the group of mice that received both sulphapyridine and serum. In some cases the superiority of the combined treatment is admittedly not great, and the differences are not statistically significant. In
.
1000 TABLE I RESULTS OF TREATMENT OF EXPERIMENTAL MENINGOCOCCAL INFECTION OF MICE
P
=
prophylaxis.
In the two
prophylactic experiments the amount of
experiments 2, 6, 9, 10, and 11, however, the results indicate that the drug and serum exerted a synergic action, the combined effect being considerably greater than that produced by either alone. The case-mortality should not be taken as the sole criterion of success. Further information can be obtained by a comparison of the average duration of illness in the mice (see table III). COMMENTS
The results, taken as a whole, clearly demonstrate the value of giving serum besides sulphapyridine to cure experimental meningococcal septicaemia in mice. Such a procedure is, moreover, entirely rational. Without treatment the host’s natural defences, inhibited by the mucin, cannot prevent rapid multiplication of the invading organism. Sulphapyridine prevents this multiplication and so gives the host time to reorganise its immunological mechanism. If the administration of the drug is unduly delayed, the overwhelming number of organisms already present precludes the possibility of recovery, because the toxins liberated by autolysis of the meningococci will exert a lethal effect after the cocci have ceased to proliferate. On the other hand, when the combined treatment is given, sulphapyridine holds the organisms in check while the specific antibodies injected, together with such natural defences as the host may succeed in mobilising, are rendered immediately available for killing the invading organism and neutralising its toxins. These fundamental principles are valid whether the host is a man or a mouse. Clinical
serum
injected
was
0’01
c.cm.
highly virulent group-I strain of meningococcus suspended in mucin. At various periods after infection the mice received sulphapyridine, or antimeningococcus serum, or a combination of these. The combination of drug and serum gave better protection than that obtained with the drug alone. The average case-mortality in mice treated with sulphaC TABLE III-VALUE OF COMBINED TREATMENT IN CUTTIKG SHORT THE ILLNESS
(Figures taken from protocol of expt. 13)
was 59 per cent., whereas the giving oi besides the drug reduced it to 26 per cent. It is suggested that the use of antimeningococcus serum, given intravenously, as an adjuvant to chemotherapy in human cerebrospinal fever deserves further clinical trial.
pyridine only serum
TABLE II-ANALYSIS OF RESULTS SHOWN IN TABLE I
REFERENCES
Banks, H. S. (1938) Lancet, 2, 7. (1939) Ibid, 2, 921. (1940) Ibid, Jan. 6, p. 42. Branham, S. E. and Rosenthal, S. M. (1937) Publ. Hlth Rep., Wash. 52, 685. Brown, T. M. (1937) Johns Hopk. Hosp. Bull. 61, 272. Bryant, J. and Fairman, H. D. (1939) Lancet, 1, 923. Buttle, G. A. H., Gray, W. H. and Stephenson, D. (1936) Ibid, 1, 1286. Crawford, T. and Fleming, G. B. (1938) Ibid, 1, 987. Harries, G. E. (1940) Ibid, March 16, p. 522. Jewesbury, E. C. O. (1938) Ibid, 1, 1262. McIntosh, R., Wilcox, D. A. and Wright, F. H. (1937) J. Pediat. —
—
experience, however, does not favour serum to sulphonamides ; yet the
the addition of results of the experiments here recorded seem to call for further trial of a method so essentially rational. SUMMARY
A
meningococcal septicaemia, normally fatal, was produced in mice by intraperitoneal injection of a ,
11, 167. Miller, C. P. and Castles, R. (1936) J. infect. Dis. 58, 263. Ministry of Health (1940) Memorandum 234, London. Mitchell, A. G. and Trachsler, W. H. (1937) J. Pediat. 11, 183.
Proom, H. (1937) Lancet, 1, 16. Schwentker, F. F., Gelman, S. and Long, P. H. (1937) J. Amer. med. Ass. 108, 1407. Somers, R. B. U. (1939) Lancet, 1, 921.
Wadsworth,
A.
(1932) Trans. Ass. Amer. Phys. 47,
161.