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The etiology of blackwater fever
201
THE
ETIOLOGY
OF
BLACKWATER
FEVER.
BY GEORGE
C. LOW,
M.A., M.D., M,R.C.P.
Senivr Pkysician, Hospital~ .for Tropical Diseases. Lecturer and Co~tsultiuq. Patl~ologist, Loqzdon Schoob q/" T~'opical Med~ci'~e. AND J. T. DUNCAN, F.R.C.S.L, L.I~.C.P., D.T.~¢I.& H. (Camb.). Assistant Director of Tro])ic~l Pathol~Jgy, I~cndon Schcol (~f Troj)ical Medicine.
o
[From the Hospital for Tropical Diseases.]
The claims made by BLANCHARD and LEF]aOU t h a t they had discovered spiroch~etes in the blood of three cases of blackwater fever in the Congo, and that they had been able to prove that the organism was pathogenic to guinea-pigs, in w h i c h various h~emorrhages and h~ematuria were produced, have led us to examine a typical case of this disease which was admitted to the H o s p i t a l for Tropical Diseases recently. T h e patient, who recovered, was a m a n thirty-two years of age who had suffered frequently from malaria in Assam, and once from blackwater fever (9,8th January, 1923). A characteristic second attack came on nineteen days after his arrival in England. The patient was seen at 2 p.m. on 25th May, 1923, the second day of his illness. H e presented every sign of a very severe attack of blackwater fever, and his condition at this time appeared to be serious. A quantity of 14 c.c. of blood was withdrawn from the left median basilie vein. 10 c.c. of IGhis was received into a tube containing 1 c.c. of sterilized 90 per cent. sodium citrate solution, and this citrated blood was afterwards subjected to triple centrifugation for examination for spiroch~etes.
202
THE
ETIOLOGY
OF BLACKWATER
FEV:ER,
Auto-h~nolytic Test.--1 e.c. of blood was received into each of two small test for seven incubator, hmmolysis
tubes. minutes, and kept in either
One of these was immediately placed in crushed ice and then transferred with the second tube to an at 37 ° C. for two hours. There was no evidence of tube. Coagulability of the Blood.--The actual coagulation time was not recorded, but coagulation was delayed. The red cells sedimented, leaving a layer of clear plasma equal to about 30 per cent. of the total column of blood before clotting occurred. This was probably partly due to the delay in clotting and partly to reduction in the surface tension of the plasma. Twenty-four hours after clotting there was no apparent contraction of the clot and no expression of sermn, although the blood had been kept at 37 ° C. for several hours. Cholmmia was very marked. The presence of hmmoglobin was not detected in the citrated plasma when examined with the spectroscope. Examination for Spiroch~etes.--lO c.c. of citrated blood was subjected to triple centrifugation following exactly the technique of MARTIN, LEI3~lJ~ and ROUBAUD aS detailed by BLANCtIARD and LEFI~OTJ. The deposits resulting from the second and third operations were similar to those described by these workers. The deposit from the third eentrifugation consisted of an abundance of blood platelets with rarely a red cell or a leucocyte. E x a m i n a t i o n of the second and third deposits by dark ground illumination did not reveal the presence of any spiroch~etes, but numerous spirochaete-like filaments were observed. These filaments varied in length from about 4tx to 10~. Most of t h e m showed a linear beading and some presented terminal thickenings. Those that were free moved in the microscopic field with a motion entirely unlike that of a spiroehmte. Many of the filaments were attached to the platelets, but the actual development of a filament from a platelet was not observed, although there is no doubt that they arise in this way. A great increase in the number of filaments occurred when the fresh microscopic preparation was allowed to stand for a time. Smears made from the second and third deposits and stained by Giemsa's method showed filaments similar in appearance to those observed in the fresh preparations. No true spiroeh~etes were found in preparations stained by the methods of Giemsa and F o n t a n a ,
T H E E T I O L O G Y OF BLACKWATEI~ F E V E R .
203
The remainder of the deposit from the final cen~rifugation was resuspended in 5 e.c. of the citrated plasma* from which it had been separated, and with this suspension four guinea-pigs were inoculated as follows : Guinea-pig ,, ,, ,,
(a) (b) (c) (d)
10 1'0 1"0 2"0
c.c. of suspension into peritoneum. c.e . . . . . c.c. ,, subcutaneously. c.c. ,, ,,
None of these animals developed any sign of infection and the daily temperature records were normal. I n no case was blood found in the urine expressed from the bladder. Blood from the ear, in thick and in thin smears, was examined regularly for spirochsetes, but none were found. On the sixth day, guinea-pig (a) was chloroformed and examined. All the organs appeared to be normM, and there was nowhere any evidence of hsemorrhage or of ieterus. The urine contained no blood. Stained smears from the liver, spleen, lungs and kidneys showed no spirochmtes. Microscopical sections of the various organs showed no pathological change, and sections of organs treated by Levaditi's method for spirochsetes were also negative. A portion of the liver was ground up with 3 c.e. of isotonic saline, and 2 c.e. of this suspension injected into the peritoneum of a fifth guinea-pig (e). The blood of guinea-pig (a), withdrawn from the heart before death, was citrated and examined by the method of triple centrifugation, with results similar to those obtained in the patient's blood. Guinea-pigs (b), (c), (d) and (e) remained normal; the blood and urine examinations continued to yield negative results. Animals (b) and (d) were killed off at intervals of six days and examined as in the case of guinea-pig (a), but in neither case was the presence of pathological changes or of spirochsetes observed. Observations on guinea-pigs (c) and (e) are being continued, but up to date have been negative. It will thus be seen that no spiroch~etes have been detected either in the blood of the patient or in the experimental animals, and in the latter * In view of BLANCHARD slid LEI~ROU'S claim to have demonstrated protective (mtti-spiroeh~etM) properties in the serum of one of their patients, it would seem desirable to avoid the use of such serum or plasma in attempts to infect experimental aItimals, and to substitute sterilized salt solution or normal plasma.
204
T H E E T I O L O G Y OF BLACKWATER, F E V E R ,
no lesions of any sort h a v e b e e n m e t with. T h e s e results correspond w i t h those of Dr. J. G. T~o?aso~, m e n t i o n of w h i c h has been m a d e in an editorial in the B r i t i s h M e d i c a l J o u r n a l of 9th J u n e , 1923. Dr. T u o u s o ~ e x a m i n e d six patients and failed to find any evidence of spirochmtes in any of t h e m . T h e editorial abstract suggests t h e possibility of a superimposed spirochmtal jaundice as e x p l a i n i n g t h e spiroehmtes found in the F r e n c h a u t h o r ' s cases. As the blood was taken on the second day of the ilbless in our case, t h e possibility of finding t h e parasite necessarily was s t r o n g e r t h a n if the t h e blood had been t a k e n at a later date N o T E . - - S i n c e this paper was s u b m i t t e d for publication, t h e examination of guinea-pigs (c) and (e) has been completed. T h e m e t h o d s of e x a m i n a t i o n were similar to those e m p l o y e d in t h e cases of t h e other animals, and t h e results w e r e negative.
REFERENCES. 1, BLANCHARD, M., LEFROU G., LAIGI~ET, - - . 1922. Note pr61iminMre sen des c~s d'iet~res 6pid6miques observ6s • Erazzaville (A,E.F.). B~dl, 8oe. P~zth. Exot., p, 385. 2. BL~_NCHARD,M.,andL]~FI~ou, G. 1922. " P r e s e n c e de spiroehStes dans le sang d'europden atteints de fi~vre bilieuse hemoglobinurique. Le probl~me etiologique de eette
spirochgtose." Note pr61iminuire ~ l'Academie des Sciences, S6unce du 9 Oetobre. B~dl. Soc. Path. Ezol. VoL xv, pp. 699-722. 3. ]3LANCI~ARD:M.. ,~nd LEFROU, (~. Note by ~EBNIL Ol~discovery of spirochmtes in a third case of blackwal~erfever by B[~ANCHARDand LLL.'ROU. Ibid. Vol. xvi., p. 297.
THE
ETIOLOGY
OF
BLACKWATER
FEVER.
BY GEORGE
C. LOW,
M.A., M.D., M,R.C.P.
Senivr Pkysician, Hospital~ .for Tropical Diseases. Lecturer and Co~tsultiuq. Patl~ologist, Loqzdon Schoob q/" T~'opical Med~ci'~e. AND J. T. DUNCAN, F.R.C.S.L, L.I~.C.P., D.T.~¢I.& H. (Camb.). Assistant Director of Tro])ic~l Pathol~Jgy, I~cndon Schcol (~f Troj)ical Medicine.
o
[From the Hospital for Tropical Diseases.]
The claims made by BLANCHARD and LEF]aOU t h a t they had discovered spiroch~etes in the blood of three cases of blackwater fever in the Congo, and that they had been able to prove that the organism was pathogenic to guinea-pigs, in w h i c h various h~emorrhages and h~ematuria were produced, have led us to examine a typical case of this disease which was admitted to the H o s p i t a l for Tropical Diseases recently. T h e patient, who recovered, was a m a n thirty-two years of age who had suffered frequently from malaria in Assam, and once from blackwater fever (9,8th January, 1923). A characteristic second attack came on nineteen days after his arrival in England. The patient was seen at 2 p.m. on 25th May, 1923, the second day of his illness. H e presented every sign of a very severe attack of blackwater fever, and his condition at this time appeared to be serious. A quantity of 14 c.c. of blood was withdrawn from the left median basilie vein. 10 c.c. of IGhis was received into a tube containing 1 c.c. of sterilized 90 per cent. sodium citrate solution, and this citrated blood was afterwards subjected to triple centrifugation for examination for spiroch~etes.
202
THE
ETIOLOGY
OF BLACKWATER
FEV:ER,
Auto-h~nolytic Test.--1 e.c. of blood was received into each of two small test for seven incubator, hmmolysis
tubes. minutes, and kept in either
One of these was immediately placed in crushed ice and then transferred with the second tube to an at 37 ° C. for two hours. There was no evidence of tube. Coagulability of the Blood.--The actual coagulation time was not recorded, but coagulation was delayed. The red cells sedimented, leaving a layer of clear plasma equal to about 30 per cent. of the total column of blood before clotting occurred. This was probably partly due to the delay in clotting and partly to reduction in the surface tension of the plasma. Twenty-four hours after clotting there was no apparent contraction of the clot and no expression of sermn, although the blood had been kept at 37 ° C. for several hours. Cholmmia was very marked. The presence of hmmoglobin was not detected in the citrated plasma when examined with the spectroscope. Examination for Spiroch~etes.--lO c.c. of citrated blood was subjected to triple centrifugation following exactly the technique of MARTIN, LEI3~lJ~ and ROUBAUD aS detailed by BLANCtIARD and LEFI~OTJ. The deposits resulting from the second and third operations were similar to those described by these workers. The deposit from the third eentrifugation consisted of an abundance of blood platelets with rarely a red cell or a leucocyte. E x a m i n a t i o n of the second and third deposits by dark ground illumination did not reveal the presence of any spiroch~etes, but numerous spirochaete-like filaments were observed. These filaments varied in length from about 4tx to 10~. Most of t h e m showed a linear beading and some presented terminal thickenings. Those that were free moved in the microscopic field with a motion entirely unlike that of a spiroehmte. Many of the filaments were attached to the platelets, but the actual development of a filament from a platelet was not observed, although there is no doubt that they arise in this way. A great increase in the number of filaments occurred when the fresh microscopic preparation was allowed to stand for a time. Smears made from the second and third deposits and stained by Giemsa's method showed filaments similar in appearance to those observed in the fresh preparations. No true spiroeh~etes were found in preparations stained by the methods of Giemsa and F o n t a n a ,
T H E E T I O L O G Y OF BLACKWATEI~ F E V E R .
203
The remainder of the deposit from the final cen~rifugation was resuspended in 5 e.c. of the citrated plasma* from which it had been separated, and with this suspension four guinea-pigs were inoculated as follows : Guinea-pig ,, ,, ,,
(a) (b) (c) (d)
10 1'0 1"0 2"0
c.c. of suspension into peritoneum. c.e . . . . . c.c. ,, subcutaneously. c.c. ,, ,,
None of these animals developed any sign of infection and the daily temperature records were normal. I n no case was blood found in the urine expressed from the bladder. Blood from the ear, in thick and in thin smears, was examined regularly for spirochsetes, but none were found. On the sixth day, guinea-pig (a) was chloroformed and examined. All the organs appeared to be normM, and there was nowhere any evidence of hsemorrhage or of ieterus. The urine contained no blood. Stained smears from the liver, spleen, lungs and kidneys showed no spirochmtes. Microscopical sections of the various organs showed no pathological change, and sections of organs treated by Levaditi's method for spirochsetes were also negative. A portion of the liver was ground up with 3 c.e. of isotonic saline, and 2 c.e. of this suspension injected into the peritoneum of a fifth guinea-pig (e). The blood of guinea-pig (a), withdrawn from the heart before death, was citrated and examined by the method of triple centrifugation, with results similar to those obtained in the patient's blood. Guinea-pigs (b), (c), (d) and (e) remained normal; the blood and urine examinations continued to yield negative results. Animals (b) and (d) were killed off at intervals of six days and examined as in the case of guinea-pig (a), but in neither case was the presence of pathological changes or of spirochsetes observed. Observations on guinea-pigs (c) and (e) are being continued, but up to date have been negative. It will thus be seen that no spiroch~etes have been detected either in the blood of the patient or in the experimental animals, and in the latter * In view of BLANCHARD slid LEI~ROU'S claim to have demonstrated protective (mtti-spiroeh~etM) properties in the serum of one of their patients, it would seem desirable to avoid the use of such serum or plasma in attempts to infect experimental aItimals, and to substitute sterilized salt solution or normal plasma.
204
T H E E T I O L O G Y OF BLACKWATER, F E V E R ,
no lesions of any sort h a v e b e e n m e t with. T h e s e results correspond w i t h those of Dr. J. G. T~o?aso~, m e n t i o n of w h i c h has been m a d e in an editorial in the B r i t i s h M e d i c a l J o u r n a l of 9th J u n e , 1923. Dr. T u o u s o ~ e x a m i n e d six patients and failed to find any evidence of spirochmtes in any of t h e m . T h e editorial abstract suggests t h e possibility of a superimposed spirochmtal jaundice as e x p l a i n i n g t h e spiroehmtes found in the F r e n c h a u t h o r ' s cases. As the blood was taken on the second day of the ilbless in our case, t h e possibility of finding t h e parasite necessarily was s t r o n g e r t h a n if the t h e blood had been t a k e n at a later date N o T E . - - S i n c e this paper was s u b m i t t e d for publication, t h e examination of guinea-pigs (c) and (e) has been completed. T h e m e t h o d s of e x a m i n a t i o n were similar to those e m p l o y e d in t h e cases of t h e other animals, and t h e results w e r e negative.
REFERENCES. 1, BLANCHARD, M., LEFROU G., LAIGI~ET, - - . 1922. Note pr61iminMre sen des c~s d'iet~res 6pid6miques observ6s • Erazzaville (A,E.F.). B~dl, 8oe. P~zth. Exot., p, 385. 2. BL~_NCHARD,M.,andL]~FI~ou, G. 1922. " P r e s e n c e de spiroehStes dans le sang d'europden atteints de fi~vre bilieuse hemoglobinurique. Le probl~me etiologique de eette
spirochgtose." Note pr61iminuire ~ l'Academie des Sciences, S6unce du 9 Oetobre. B~dl. Soc. Path. Ezol. VoL xv, pp. 699-722. 3. ]3LANCI~ARD:M.. ,~nd LEFROU, (~. Note by ~EBNIL Ol~discovery of spirochmtes in a third case of blackwal~erfever by B[~ANCHARDand LLL.'ROU. Ibid. Vol. xvi., p. 297.