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THE BI-COLOURED GUAIAC TEST FOR THE CEREBRO-SPINAL FLUID.
339 wants to get the maximum amount of benefit in the minimum amount of time. Often in the early stages he feels so well that he cannot see why so much fuss is made over him, and this makes him all the less inclined to be patient and persevering. My own belief, however, is that continuity in the administration of iodide and mercury is of more importance A diagnosis of than courses of organic arsenic. aortic syphilis also implies unfitness for strenuous work and for violent exercise of any kind. Moreover, it imposes on its subject a need for strict temperance in the use of alcohol. If the sole reason for an interest in the subject that we have been discussing lay in the opportunity that it offers for successful treatment then, indeed, our time would have been largely wasted. There is, however, a far stronger motive impelling us to study this disease and to make known the outcome of our studies, and that is a desire to emphasise the urgent need for prevention and early treatment of syphilitic infection. If it is a melancholy reflection that in this disease we see a wholly needless wastage of valuable lives we may perhaps derive consolation from seeing in it also a supreme example of the immense power of enlightened public opinion as an engine of preventive
naturally
medicine. REFERENCES.
1. Allan, G. A.: Glasg. Med. Jour., 1924, cii., 81. 2. Allbutt, T. C. : St. George’s Hosp. Reports, 1868, iii., 56. 3. Horder, T. J.: THE LANCET, 1926, i., 695. 4. Anderson, J.: Ibid., 1915, i., 648. 5. Beretervide, E. A., and J. J. : Arch. de méd. des enfants Paris, 1924, xxvii., 257. 6. Bruce, J. M.: THE LANCET, 1911, ii., 69, 144, 205. 7. Nixon, J. A.: St. Bart.’s Hosp. Rep., 1911, xlvii., 43. 8. Clarke, R. C., Coombs, C. F., Hadfield, G., and Todd, A. T. Quart. Jour. Med., 1927, xxi., 51. 9. Clawson, B. J., and Bell, E. T.: Arch. Pathol. and Labor Med., 1927, ii., 922. 10. Coffen, T. H.: Amer. Heart Jour., 1929, v., 99. 11. Coombs, C. F., and Herapath, C. E. K. : Clin. Jour., Nov.
12. 13. 14. 15.
1926, lv., 529. Coombs, C. F., and Herapath, C. E.K. : Bristol Med.-Chir, Jour., 1924, xli., 154. Coombs, C. F., and Hadfield, G.: THE LANCET, 1926, i., 14. Corrigan D.: Edin. Med. and Surg. Jour., 1832, x., 225. Corvisart, J. N.: Essais sur les maladies du cœur, Paris, 1811.
J. M., and Faulds, J. S. : Brit. Med. Jour., 1929, ii., 285. 17. Cummer, C. L., and Dexter, R. : Jour. Amer. Med. Assoc., 1912, ii., 419. 18. Cutting, R. A., Loria, F. L., and Pickell, F. W.: Annals of Surgery, 1930, xci., 269. 19. Donnison, C. P.: Personal communication. 20. Fraser, F. R.: THE LANCET, 1927, i., 529, 589, 643. 21. Fordyce, A. D. : Brit. Med. Jour., 1930, i., 530. 22. Gallavardin, L. : Jour. de médicine de Lyon, Sept. 22nd, 1922. 23. Hall, F. de H. : THE LANCET, 1913, i., 803, 869, 945. 24. Hare, D., and Ross, J.: Ibid, 1929., ii., 806. 25. Harmer, I. M.: Heart, xii., 371. 26. Harris, I.: Brit. Med. Jour., 1928, i., 840. 27. Heimann, H. L.: Personal communication (see also Brit. Med. Jour., 1929, i., 344). 28. Heubner: Die Luetischen Erkrankungen der Hirnarterien, 1874. 29. Hodgson, J. : A Treatise on Diseases of the Arteries and Veins, London, 1815. 30. Juster, I. R., and Pardee, H. E. B. : Amer. Heart Jour., 1929, v. 84. 31. Keefer, C. S., and Resnik, W. H. : Archives of Internal Medicine, 1926, xxxvii., 264. 32. Keith, A., and Miller, C. H.: THE LANCET, 1906, ii., 1429. 33. Klotz, O. M.: Amer. Jour. Med. Sciences, 1918, i., 92. 34. Laignel-Lavastine, and Vinhit : Presse médicale, 1913, xxi., 607. 35. Levine, S. A. : Medicine Monographs, xvi. 36. Grant, R. T., Wood, J. E., and Jones, T. D. : Heart, 1928, 16.
Cowan,
xiv., 247.
37. Lian, C., Gilbert-Dreyfus, and Puech, P. : Bull. et. mém. de la soc. méd. des hôp. de Paris, 1927, li., 26. 38. Longcope, W. T.: Amer. Jour. Med. Sci., 1913, ii., 513. 39. Marañon, G.: El problema de la aortitis, Madrid, 1928. 40. Oberndorfer: Münch. med. Woch., 1913, ix., 505. 41. Parkinson, J., and Bedford, D, E. : Quart. Jour. Med., 1926, xix., 113. 42. Phillips, S.: THE LANCET, 1897, i., 223. 43. Reid, W. D.: Boston Med. and Surg. Jour., 1920, ii., 67. 44. Ricord, P. : Gazette des Hôpitaux, August, 1845, xvi., 101. 45. Rolleston, H. D.: Cardiovascular Diseases Since Harvey’s Discovery, Cambridge, 1928. 46. Scott, R. W., and Saphir, O. : Amer. Jour. Pathol., 1927, iii., 527. 47. Stern, R. M. : Brain, 1929, lii., 295. (Continued at foot of next column.)
THE
BI-COLOURED
GUAIAC TEST
FOR THE CEREBRO-SPINAL FLUID.
BY J. GODWIN
GREENFIELD, M.D. EDIN.,
F.R.C.P. LOND., PATHOLOGIST TO THE NATIONAL
HOSPITAL, QUEEN-SQUARE ;
AND
RUBY 0.
STERN, M.B. LOND.,
CLINICAL ASSISTANT AT THE HOSPITAL.
guaiac test on the cerebro-spinal introduced in 1929 by de Thurzo, who, working in the laboratory of the National Hospital for Diseases of the Nervous System, applied this now colloidal reaction to the cerebro-spinal fluid and investigated its behaviour with a number of pathological fluids. The object of this paper is to record the results we have obtained in a further series of fluids examined in the same laboratory, and to assess the practical value of the test. THE bi-coloured
fluid
was
Description of the Test. The
colloidal system used in the bi-coloured reaction is a suspension of guaiac resin with two coloured solutions of dyes, naphthol green and brilliant fuchsin. The brilliant fuchsin tends to be precipitated along with the resin in certain pathological fluids, and thus we obtain colour changes from red to green through an intermediate, but The B.G. solution itself obvious grey coloration. is easy to prepare and gives remarkably constant reactions. Standardisation of the solution is there-
guaiac (B.G.)
fore unnecessary. Theoretical considerations of the reaction will not be discussed here, for de Thurzo has dealt with them in his paper.’ It is sufficient to state that he ascribes the reaction to a particular sensitiveness of the colloidal system used to one fraction of the protein content of the cerebro-spinal fluid, euglobulin. A relative increase in this fraction, as found in different forms of neurosyphilis, causes precipitation of two components of the colloidal system and a characteristic change in colour. The Lange colloidal gold reaction appears to be produced in the same way. We have performed this reaction also on all the fluids in our series for the purpose of comparing the two reactions. PREPARATION OF THE
SUSPENSION.
Two solutions are required: Solution 1 consists of 0-2 per cent. sodium chloride withc.cm. ofper cent
References
continued
from previous
column.
48. Stolkind, E. J.: Brit. Jour. Child. Dis., 1920, xvii., 126. 49. Sumbal, J. : Bratislavske Lekarske Listy 1928, p. 267 (abstr. Brit. Med. Jour. Epit., Sept. 22nd, 1928). 50. Smith, D. C., and Kimbrough, R. D.: Southern Med. Jour., 1928, xxi., 634. 51. Symmers, D.: Jour. Amer. Med. Assoc., 1916, lxvi., 1457. 52. Turnbull, H. M.: Quart. Jour. Med., 1915, viii., 201. 53. Turner, K. B., and White, P. D. : Arch. Int. Med., 1927, xxxix., 1. 54. Vela : Quoted by Marañon (q.v., supra., 39). 55. Virchow, R.: Arch. für Pathol. Anat. und Physiol., 1858, xv. 217. 56. von Glahn W. C., and Wilshusen, H. F.: Proc., New York Path. Soc., 1924, xxiv., 71. 57. Welch, F. H.: Med.-Chir. Trans., 1876, lix., 59. 58. Wilks, S.: Trans. Path. Soc. Lond., 1857, viii., 56. 59. Wittgenstein, A.: Münch. med. Woch., 1924, lxxi., 1354. 60. Worster-Drought, C., and Danvers-Atkinson, F. C.: Clin. Jour., 1929, lviii., 355. 61. Wyckoff, J. A., and Lingg, C. : Amer. Heart Jour., 1926, i., 446. 62. Waldo, H., and Herapath, C. E. K. : THE LANCET, 1922,
i., 271.
63. Wearn : New Eng. Jour.
Med., 1928, cxcviii., 726.
340 The results indicate that the B.G. reaction closely resembles the Lange reaction in its behaviour towards syphilitic fluids. The only fluids which produced complete precipitation in any tube with a clear green supernatant fluid in the B.G. reaction
sodium carbonate added to each 100 c.cm. of saline. The sodium carbonate solution keeps indefinitely. De Thurzo suggests that the saline should be prepared freshly for each test from a stock 10 per cent. solution diluted, but we have found it advisable to make up the 0-2 per cent. solution from the dry salt each time. Solution2 is the bi-coloured guaiac suspension, which is prepared as follows : 0.22 c.cm. of 20 per cent. tincture of guaiac (B.P.C.) in 9 c.cm. of absolute alcohol are added to a flask containing 40 c.cm. of distilled water. This mixing of the suspension must be done very slowly and with constant shaking of the flask. The best method of mixing these two solutions is to pipette the guaiac tincture into the water from a 1 c.cm. pipette, allowing not more than 0-5 c.cm. to be delivered at a time, and shaking well between the delivery of each few drops. As the success of the test depends on the thorough mixing of these two solutions, it is important not to hurry this step. 2 c.cm. of a watery solution of naphthol green (Gurr) are then slowly added to the suspension. The flask should be shaken after this is added. Finally, 0-3 c.cm. of an alcoholic solution of brilliant fuchsin (basic fuchsin, Gurr), is added, and the flask again shaken. It is then left to stand for 30 minutes
were, with a very few exceptions, those from cases of general paralysis. The exceptions were: a case of polyneuritis with a protein content of 1 per cent., cases of tabes dorsalis, and three cases of disseminated sclerosis. Reference to these cases will be made later. We found that, with the exception again of certain cases of disseminated sclerosis and of cases with an exceptionally high protein content, precipitation represented by figures 2, 3, and 4 occurred only in neurosyphilis. The increase in
two
protein content necessary to produce a very slight precipitation of the B.G. suspension produced a very definite " shift to the right," or meningitic curve, in the Lange colloidal gold suspension. For example, a fluid from a case of polyneuritis with a protein content of 0-45 per cent. gave a Lange reaction of 0001123333, but a completelynegative
before use. A good suspension should be slightly opalescent and of a dark cherry red colour when freshly prepared. The colour fades to a light cherry red by the end of half an hour, and after twelve hours the colour is a rather dirty shade of pink. The suspension can only be used within three hours of preparation, as after this time the sensitivity alters, and anomalous results are obtained.
B.G. reaction. Whatever the theoretical basis of the reaction may be-i.e., whether the colour change is due to TECHNIQUE OF THE TEST. an increase in the proportion of euglobulin or notEach test is carried out thus : ten test-tubes, it is clear that the B.G. reaction is decidedly more 4 inches in length, are set out in a rack. 0-5 c.cm. sensitive to syphilitic fluids, however low their of cerebro-spinal fluid is put into the first and second protein content, than to non-syphilitic fluids with a tubes, leaving the pipette in the second tube. very high protein content. The B.G. suspension 0-5 c.cm. of solution 1 is added to each of the tubes thus seems to be more selective in its reaction than is 2 to 10. The cerebro-spinal fluid in tube 2 is mixed the Lange gold-sol. with the saline, and 0-5 c.cm. is transferred to tube 3. GENERAL PARALYSIS. Mixing takes place as in tube 2, and 0-5 c.cm. is The majority of fluids from cases of general transferred to tube 4, and so on to tube 10, from which 0-5 c.cm. is removed and. rejected. The I paralysis gave a reading of the type 244444321. It was rare to find complete precipitation in the first dilutions of cerebro-spinal fluid are 0,5 c.cm. of the B.G. tube-i.e., with undiluted cerebro-spinal fluid. The 1/1, !, i.... to 1/512. is to each tube. The rack is well strongest reaction was seen in the third tube in suspension added shaken and set aside. The results are read after practically every case. Not uncommonly a slight 12 to 18 hours by transmitted light, preferably by reaction in the first tube was followed by complete electric light. precipitation in the next six, a reaction of the type In only eight out of our 52 fluids In reading the test the alterations in colour are repre- 1444444211. sented by numbers : 0 =No change. 1 == Slight precipitation, from cases of general paralysis was there less than some turbidity ; no change in colour. 2 =Red precipitation ; complete precipitation in at least one tube-i.e., upper fluid greyish or greyish-red. 3=Red precipitation ; about 14-5 per cent. failed to give the paretic reaction. upper fluid greyish-green to dull green. 4=Red precipiOf these fluid vivid is no difficulty eight fluids, only three which were examined green. There tation ; upper about reading the changes in colour indicated by figures on several occasions, gave at any time complete 3 and 4. The only difficulty arises occasionally in deciding ’, with the Lange reaction. whether the change in colour is represented better by the precipitation Table I. the readings of a few fluids which gives 1 2. This vanishes with or soon ’ , experience figures difficulty ’ in reading the test. were examined several times. Most of the patients Results. from whom the fluids were taken were treated by Our series comprised fluids from patients with malaria after the first examination of the fluid. the following diseases (the figure after the disease It is interesting to note that in this series of cases the indicates the number of cases of the particular colloidal reactions were very seldom altered by disease in which we examined the fluid) : general treatment, although the cell count fell rapidly in paralysis (52), tabes dorsalis (23), cerebro-spinal every case, in some almost to within normal limits. Lange reaction is given for comparison with the syphilis (11), disseminated sclerosis (21), cerebral The tumour (7), spinal compression (6), cerebral abscess (2), B.G. reaction. No relation between clinical improvement and alteration in the colloidal reactions could syphilitic meningitis (2), chronic meningitis (2), be established. Progress seemed to be related rather cerebral thrombosis, polyneuritis (2), hemiplegia, acute meningitis, Friedreich’s ataxia, osteitis of to the number of cells in the fluid, for in those cases orbit, local meningitis, cerebral malaria, third nerve which did not improve after malarial therapy, the palsy and arthritis, one case of each. The fluids cell count remained high. It may be of interest of many of the syphilitic cases, especially those of also to point out that the course of novarsenobillon which was given to many patients after malaria were examined on several occasions. general The Lange reaction was done first on every fluid, treatment, likewise had no effect on the colloidal and only those which gave a reading of (2) in at reactions. TABES DORSALIS. least one tube were used for the B.G. reaction. It The results of the test in tabes conformed less to was found that fluids giving a reading of only (1) in the Lange reaction did not give any colour change one type than those in general paralysis, but again with the B.G. reaction, and except for control cases the reaction approximated to that given by the Lange test. An average reading was 0012321000. Out they have been excluded from this series.
’i
therefore
paralysis,
341
a
of 23 cases, three were completely negative. Of these typical luetic reading, but the Wassermann was the Lange reaction was negative in one case, though negative ; whilst in the third, the Lange reading was the Wassermann was positive in cerebro-spinal very low. One case which was diagnosed as a pure in the second, the Lange gave tabes, gave the typical paretic reading of 1344444431, fluid and blood ; with a Lange reading of 5555555554. Later clinical TABLE I. examination revealed mental changes consistent with early paresis. One other case of tabes which gave a paretic response to the B.G. test and complete precipitation in the Lange reaction is still under
observation. CEREBRO-SPINAL SYPHILIS.
Slight precipitation up to the amount expressed by the figure 2 was found in most cases of cerebro-spinal syphilis. Two typical readings were When the Wasserreaction was negative the B.G. reaction was negative also. With one exception no fluid in this group gave a reading higher than is expressed by a 3. The exception was a case in which the diagnosis may have to be reconsidered. The patient is a young woman of 28, whose symptoms and signs point either to cerebro-spinal syphilis (there is a suspicious history of two stillbirths), or to disseminated sclerosis. The Wassermann is negative in the cerebrospinal fluid and blood, whilst the Lange reading is of the paretic type, 5543321000, and the B.G. reaction is 2444442000. On these laboratory findings’it seems more probable that the case is one of disseminated sclerosis. 1112110000 and x001211000.
mann
DISSEMINATED SCLEROSIS.
* mg.
per 100 c.cm.
M. =malarial treatment; N.A.B. -=novarsenobillon ; Tryp. tryparsamide; Sulf. =sulfosin. (x in the B.G. reaction indicates that as insufficient cerebro-spinal fluid was available, the first tube was left empty.) B.a. blood admixture. =*=
In view of the interest which is being centred on this disease at the present time, we propose to discuss in some detail the results we obtained in the 21 cases which we examined by the B.G. test, and to refer also to the Lange reaction in these cases. The duration of symptoms in the cases examined varied from two weeks to 15 years. No case was in the stage of remission so far as could be ascertained. Lumbar puncture was usually done within a week of admittance to hospital, and inquiries as to progress of symptoms after the fluid had been examined invariably elicited the reply that patients felt better since they had been in hospital. The duration of symptoms bore no relation whatsoever to the colloidal reactions of the cerebro-spinal fluid. Whereas the most acute case, with a history of two weeks’ diplopia and paraplegia, gave a Lange reaction of 4544443221 and a B.G. reaction of 1323421000, two other acute cases, one of six weeks and one of eight weeks’ duration, gave readings of 0111000000 and no change respectively in the Lange reaction and no change in the B.G. reaction. On the other hand, a chronic case of ten years’ standing gave a Lange reading of 4555553210 and a B.G. reaction of 1444421000. There was no suggestion of a fresh acute attack in this case. It was felt that no useful purpose would be served by testing the E.G. reaction on the large number of completely negative fluids from patients with disseminated sclerosis which we examined by the Lange reaction. The total number of fluids we examined from patients with this disease during this investigation was 53. Of these, 27 gave a Lange reaction containing one or more readings of 2, and 19 were tested by the B.G. reaction, there being insuffieient fluid available in the other eight cases to carry out the test. Twenty-six cases gave a completely negative Lange reaction. Two of these were tested by the B.G. reaction as controls. Table II. gives our findings .n the 21 cases tested. It will be noted that in six It will ases the reaction is completely negative. dso be noted that the colloidal reactions are indepenlent of the duration of the disease. As the examination of the fluids was carried out soon after admission to hospital it is not possible to lay what effect treatment may have on the colloidal
342 reactions may be obtained in spite of great protein increase. The ease and certainty with which a good bi. coloured guaiac suspension can be prepared from stock solutions gives the test a definite advantage over the Lange reaction, since good colloidal gold solutions may be difficult to prepare, and there is no satisfactory method of testing them except by their reaction to known specimens of cerebro-spinal fluid. The Lange reaction is therefore only suited to labora. tories where a large number of specimens of cerebro. spinal fluid are examined. While we have not in this investigation examined the B.G. reaction in relation to the better known mastic and benzoin reactions, previous experience indicated that the B.G. reaction is more sensitive and more easy to read, and agrees more closely in its results with the Lange reaction. The disadvantages of the test are : (1) that a comparatively large quantity, 1 c.cm. of cerebrospinal fluid, is required for the test; and (2) that it is not possible to judge what the final reaction will be under 12 hours, whereas with the Lange reaction a fair estimate may be made after half an hour. For these reasons we do not suggest that this test should replace the Lange test in the routine examination of the cerebro-spinal fluid. But when difficulty arises in the preparation of a good colloidal gold reagent, the bi-coloured guaiac test may be substituted, or may, owing to its more standard nature, be used as a control for the Lange test.
reactions. A single observation is of no value, but as a matter of interest, the patient A. B. (Case 20), whose condition was of long standing, and who had had several courses of arsenic injections, gave one of the strongest readings of the series, both in the Lange and B.G. reaction. The B.G. reaction does not seem to be so sensitive as the Lange reaction in this disease. In only five cases, less than 25 per
TABLE II.
We desire to thank the Medical Staff of the National * In mg. per 100 w.
c.cm.
=weeks ; m. = months ; Abbreviations
as
Hospital, Queen-square, for permission to refer to the clinical records of cases in the hospital.
y. = years.
in Table I.
REFERENCES. 1. Thurzo, E. de : The Bi-coloured Guaiac Test, Brain, 1929, lii., 196. 2. Same author: Les Nouvelles Méthodes sur les Réactions collöidales du Liquide céphalo-rachidien, Paris, 1927.
there complete precipitation in any tube, three cases was there a typical paretic The response. proportion of cases giving a paretic in the response Lange reaction was also not very high. In the series of 21, 7, or 33 per cent., showed this reading.
cent.,
was
and in
only
OTHER DISEASES
THE ÆTIOLOGY OF CHOLECYSTITIS: BACTERIOLOGICAL OBSERVATIONS.
OF THE NERVOUS SYSTEM.
Brief reference must be made to other conditions in which a positive reaction may be given by the B.G. test. These are conditions in which the protein content of the fluid is exceptionally high. In a case of polyneuritis, with a protein content of 1 per cent. and a Lange reaction of 4555422222, the B.G. reaction was 4411000000. Certain cases of spinal compression with a very high total protein gave partial precipitation, the first two or three tubes being completely unchanged, and the precipitation taking place in the fifth and sixth tubes. In other cases only the first or the first two tubes were affected. These curves therefore did not resemble those of tabes or general paralysis. In many other cases with a highly albuminous fluid a completely negative reaction was obtained.
liI.
SUMMARY
AND
CONCLUSIONS.
The cerebro-spinal fluids of 140 patients with diseases of the nervous system have been examined by the bi-coloured guaiac reaction. A Lange colloidal gold test has also been done on each of the fluids for comparison with the new reaction. Our results justify the statement that the bicoloured guaiac test is of the same practical value as the Lange test in the laboratory diagnosis of neurosyphilis. The bi-coloured guaiac test runs parallel with the Lange reaction in that certain cases of disseminated sclerosis give positive results. It differs from the Lange reaction in that negative
BY BRYAN WILLIAMS, M.B.
EDIN.,
SCHOLAR, AND ASSISTANT IN THE BACTERIOLOGY DEPARTMENT, UNIVERSITY OF EDINBURGH;
VANS DUNLOP
AND
D. G. S. MCLACHLAN, M.B. EDIN., LECTURER IN BACTERIOLOGY IN THE UNIVERSITY OF EDINBURGH.
BEFORE
gall-stones were supposed to be simple chemical precipitation of In as a result of hypercholesterolaemia. showed the however, Naunyn importance 1892
produced by
cholesterol that year, of infection in the formation of stones, and advanced his theory of a " lithogenous catarrh " of the gallbladder. Since then, the importance of infection in gall-stone formation has been generally admitted, and Moynihan has picturesquely described how a gall-stone may be a tombstone erected to the memory of an organism still living inside. Earlier investigations were directed rather towards the occurrence of organisms in the bile, and members of the coliform group were the chief ones isolated. Owing to the prevalence of typhoid fever at the end of the last century, much attention was paid to typhoid cholecystitis. More recently, the organisms of the enteric group have been encountered much less frequently in this condition. As will be seen below, Bacillus paratyphosus B was isolated only once in the present series of 106 cases.
naturally
medicine. REFERENCES.
1. Allan, G. A.: Glasg. Med. Jour., 1924, cii., 81. 2. Allbutt, T. C. : St. George’s Hosp. Reports, 1868, iii., 56. 3. Horder, T. J.: THE LANCET, 1926, i., 695. 4. Anderson, J.: Ibid., 1915, i., 648. 5. Beretervide, E. A., and J. J. : Arch. de méd. des enfants Paris, 1924, xxvii., 257. 6. Bruce, J. M.: THE LANCET, 1911, ii., 69, 144, 205. 7. Nixon, J. A.: St. Bart.’s Hosp. Rep., 1911, xlvii., 43. 8. Clarke, R. C., Coombs, C. F., Hadfield, G., and Todd, A. T. Quart. Jour. Med., 1927, xxi., 51. 9. Clawson, B. J., and Bell, E. T.: Arch. Pathol. and Labor Med., 1927, ii., 922. 10. Coffen, T. H.: Amer. Heart Jour., 1929, v., 99. 11. Coombs, C. F., and Herapath, C. E. K. : Clin. Jour., Nov.
12. 13. 14. 15.
1926, lv., 529. Coombs, C. F., and Herapath, C. E.K. : Bristol Med.-Chir, Jour., 1924, xli., 154. Coombs, C. F., and Hadfield, G.: THE LANCET, 1926, i., 14. Corrigan D.: Edin. Med. and Surg. Jour., 1832, x., 225. Corvisart, J. N.: Essais sur les maladies du cœur, Paris, 1811.
J. M., and Faulds, J. S. : Brit. Med. Jour., 1929, ii., 285. 17. Cummer, C. L., and Dexter, R. : Jour. Amer. Med. Assoc., 1912, ii., 419. 18. Cutting, R. A., Loria, F. L., and Pickell, F. W.: Annals of Surgery, 1930, xci., 269. 19. Donnison, C. P.: Personal communication. 20. Fraser, F. R.: THE LANCET, 1927, i., 529, 589, 643. 21. Fordyce, A. D. : Brit. Med. Jour., 1930, i., 530. 22. Gallavardin, L. : Jour. de médicine de Lyon, Sept. 22nd, 1922. 23. Hall, F. de H. : THE LANCET, 1913, i., 803, 869, 945. 24. Hare, D., and Ross, J.: Ibid, 1929., ii., 806. 25. Harmer, I. M.: Heart, xii., 371. 26. Harris, I.: Brit. Med. Jour., 1928, i., 840. 27. Heimann, H. L.: Personal communication (see also Brit. Med. Jour., 1929, i., 344). 28. Heubner: Die Luetischen Erkrankungen der Hirnarterien, 1874. 29. Hodgson, J. : A Treatise on Diseases of the Arteries and Veins, London, 1815. 30. Juster, I. R., and Pardee, H. E. B. : Amer. Heart Jour., 1929, v. 84. 31. Keefer, C. S., and Resnik, W. H. : Archives of Internal Medicine, 1926, xxxvii., 264. 32. Keith, A., and Miller, C. H.: THE LANCET, 1906, ii., 1429. 33. Klotz, O. M.: Amer. Jour. Med. Sciences, 1918, i., 92. 34. Laignel-Lavastine, and Vinhit : Presse médicale, 1913, xxi., 607. 35. Levine, S. A. : Medicine Monographs, xvi. 36. Grant, R. T., Wood, J. E., and Jones, T. D. : Heart, 1928, 16.
Cowan,
xiv., 247.
37. Lian, C., Gilbert-Dreyfus, and Puech, P. : Bull. et. mém. de la soc. méd. des hôp. de Paris, 1927, li., 26. 38. Longcope, W. T.: Amer. Jour. Med. Sci., 1913, ii., 513. 39. Marañon, G.: El problema de la aortitis, Madrid, 1928. 40. Oberndorfer: Münch. med. Woch., 1913, ix., 505. 41. Parkinson, J., and Bedford, D, E. : Quart. Jour. Med., 1926, xix., 113. 42. Phillips, S.: THE LANCET, 1897, i., 223. 43. Reid, W. D.: Boston Med. and Surg. Jour., 1920, ii., 67. 44. Ricord, P. : Gazette des Hôpitaux, August, 1845, xvi., 101. 45. Rolleston, H. D.: Cardiovascular Diseases Since Harvey’s Discovery, Cambridge, 1928. 46. Scott, R. W., and Saphir, O. : Amer. Jour. Pathol., 1927, iii., 527. 47. Stern, R. M. : Brain, 1929, lii., 295. (Continued at foot of next column.)
THE
BI-COLOURED
GUAIAC TEST
FOR THE CEREBRO-SPINAL FLUID.
BY J. GODWIN
GREENFIELD, M.D. EDIN.,
F.R.C.P. LOND., PATHOLOGIST TO THE NATIONAL
HOSPITAL, QUEEN-SQUARE ;
AND
RUBY 0.
STERN, M.B. LOND.,
CLINICAL ASSISTANT AT THE HOSPITAL.
guaiac test on the cerebro-spinal introduced in 1929 by de Thurzo, who, working in the laboratory of the National Hospital for Diseases of the Nervous System, applied this now colloidal reaction to the cerebro-spinal fluid and investigated its behaviour with a number of pathological fluids. The object of this paper is to record the results we have obtained in a further series of fluids examined in the same laboratory, and to assess the practical value of the test. THE bi-coloured
fluid
was
Description of the Test. The
colloidal system used in the bi-coloured reaction is a suspension of guaiac resin with two coloured solutions of dyes, naphthol green and brilliant fuchsin. The brilliant fuchsin tends to be precipitated along with the resin in certain pathological fluids, and thus we obtain colour changes from red to green through an intermediate, but The B.G. solution itself obvious grey coloration. is easy to prepare and gives remarkably constant reactions. Standardisation of the solution is there-
guaiac (B.G.)
fore unnecessary. Theoretical considerations of the reaction will not be discussed here, for de Thurzo has dealt with them in his paper.’ It is sufficient to state that he ascribes the reaction to a particular sensitiveness of the colloidal system used to one fraction of the protein content of the cerebro-spinal fluid, euglobulin. A relative increase in this fraction, as found in different forms of neurosyphilis, causes precipitation of two components of the colloidal system and a characteristic change in colour. The Lange colloidal gold reaction appears to be produced in the same way. We have performed this reaction also on all the fluids in our series for the purpose of comparing the two reactions. PREPARATION OF THE
SUSPENSION.
Two solutions are required: Solution 1 consists of 0-2 per cent. sodium chloride withc.cm. ofper cent
References
continued
from previous
column.
48. Stolkind, E. J.: Brit. Jour. Child. Dis., 1920, xvii., 126. 49. Sumbal, J. : Bratislavske Lekarske Listy 1928, p. 267 (abstr. Brit. Med. Jour. Epit., Sept. 22nd, 1928). 50. Smith, D. C., and Kimbrough, R. D.: Southern Med. Jour., 1928, xxi., 634. 51. Symmers, D.: Jour. Amer. Med. Assoc., 1916, lxvi., 1457. 52. Turnbull, H. M.: Quart. Jour. Med., 1915, viii., 201. 53. Turner, K. B., and White, P. D. : Arch. Int. Med., 1927, xxxix., 1. 54. Vela : Quoted by Marañon (q.v., supra., 39). 55. Virchow, R.: Arch. für Pathol. Anat. und Physiol., 1858, xv. 217. 56. von Glahn W. C., and Wilshusen, H. F.: Proc., New York Path. Soc., 1924, xxiv., 71. 57. Welch, F. H.: Med.-Chir. Trans., 1876, lix., 59. 58. Wilks, S.: Trans. Path. Soc. Lond., 1857, viii., 56. 59. Wittgenstein, A.: Münch. med. Woch., 1924, lxxi., 1354. 60. Worster-Drought, C., and Danvers-Atkinson, F. C.: Clin. Jour., 1929, lviii., 355. 61. Wyckoff, J. A., and Lingg, C. : Amer. Heart Jour., 1926, i., 446. 62. Waldo, H., and Herapath, C. E. K. : THE LANCET, 1922,
i., 271.
63. Wearn : New Eng. Jour.
Med., 1928, cxcviii., 726.
340 The results indicate that the B.G. reaction closely resembles the Lange reaction in its behaviour towards syphilitic fluids. The only fluids which produced complete precipitation in any tube with a clear green supernatant fluid in the B.G. reaction
sodium carbonate added to each 100 c.cm. of saline. The sodium carbonate solution keeps indefinitely. De Thurzo suggests that the saline should be prepared freshly for each test from a stock 10 per cent. solution diluted, but we have found it advisable to make up the 0-2 per cent. solution from the dry salt each time. Solution2 is the bi-coloured guaiac suspension, which is prepared as follows : 0.22 c.cm. of 20 per cent. tincture of guaiac (B.P.C.) in 9 c.cm. of absolute alcohol are added to a flask containing 40 c.cm. of distilled water. This mixing of the suspension must be done very slowly and with constant shaking of the flask. The best method of mixing these two solutions is to pipette the guaiac tincture into the water from a 1 c.cm. pipette, allowing not more than 0-5 c.cm. to be delivered at a time, and shaking well between the delivery of each few drops. As the success of the test depends on the thorough mixing of these two solutions, it is important not to hurry this step. 2 c.cm. of a watery solution of naphthol green (Gurr) are then slowly added to the suspension. The flask should be shaken after this is added. Finally, 0-3 c.cm. of an alcoholic solution of brilliant fuchsin (basic fuchsin, Gurr), is added, and the flask again shaken. It is then left to stand for 30 minutes
were, with a very few exceptions, those from cases of general paralysis. The exceptions were: a case of polyneuritis with a protein content of 1 per cent., cases of tabes dorsalis, and three cases of disseminated sclerosis. Reference to these cases will be made later. We found that, with the exception again of certain cases of disseminated sclerosis and of cases with an exceptionally high protein content, precipitation represented by figures 2, 3, and 4 occurred only in neurosyphilis. The increase in
two
protein content necessary to produce a very slight precipitation of the B.G. suspension produced a very definite " shift to the right," or meningitic curve, in the Lange colloidal gold suspension. For example, a fluid from a case of polyneuritis with a protein content of 0-45 per cent. gave a Lange reaction of 0001123333, but a completelynegative
before use. A good suspension should be slightly opalescent and of a dark cherry red colour when freshly prepared. The colour fades to a light cherry red by the end of half an hour, and after twelve hours the colour is a rather dirty shade of pink. The suspension can only be used within three hours of preparation, as after this time the sensitivity alters, and anomalous results are obtained.
B.G. reaction. Whatever the theoretical basis of the reaction may be-i.e., whether the colour change is due to TECHNIQUE OF THE TEST. an increase in the proportion of euglobulin or notEach test is carried out thus : ten test-tubes, it is clear that the B.G. reaction is decidedly more 4 inches in length, are set out in a rack. 0-5 c.cm. sensitive to syphilitic fluids, however low their of cerebro-spinal fluid is put into the first and second protein content, than to non-syphilitic fluids with a tubes, leaving the pipette in the second tube. very high protein content. The B.G. suspension 0-5 c.cm. of solution 1 is added to each of the tubes thus seems to be more selective in its reaction than is 2 to 10. The cerebro-spinal fluid in tube 2 is mixed the Lange gold-sol. with the saline, and 0-5 c.cm. is transferred to tube 3. GENERAL PARALYSIS. Mixing takes place as in tube 2, and 0-5 c.cm. is The majority of fluids from cases of general transferred to tube 4, and so on to tube 10, from which 0-5 c.cm. is removed and. rejected. The I paralysis gave a reading of the type 244444321. It was rare to find complete precipitation in the first dilutions of cerebro-spinal fluid are 0,5 c.cm. of the B.G. tube-i.e., with undiluted cerebro-spinal fluid. The 1/1, !, i.... to 1/512. is to each tube. The rack is well strongest reaction was seen in the third tube in suspension added shaken and set aside. The results are read after practically every case. Not uncommonly a slight 12 to 18 hours by transmitted light, preferably by reaction in the first tube was followed by complete electric light. precipitation in the next six, a reaction of the type In only eight out of our 52 fluids In reading the test the alterations in colour are repre- 1444444211. sented by numbers : 0 =No change. 1 == Slight precipitation, from cases of general paralysis was there less than some turbidity ; no change in colour. 2 =Red precipitation ; complete precipitation in at least one tube-i.e., upper fluid greyish or greyish-red. 3=Red precipitation ; about 14-5 per cent. failed to give the paretic reaction. upper fluid greyish-green to dull green. 4=Red precipiOf these fluid vivid is no difficulty eight fluids, only three which were examined green. There tation ; upper about reading the changes in colour indicated by figures on several occasions, gave at any time complete 3 and 4. The only difficulty arises occasionally in deciding ’, with the Lange reaction. whether the change in colour is represented better by the precipitation Table I. the readings of a few fluids which gives 1 2. This vanishes with or soon ’ , experience figures difficulty ’ in reading the test. were examined several times. Most of the patients Results. from whom the fluids were taken were treated by Our series comprised fluids from patients with malaria after the first examination of the fluid. the following diseases (the figure after the disease It is interesting to note that in this series of cases the indicates the number of cases of the particular colloidal reactions were very seldom altered by disease in which we examined the fluid) : general treatment, although the cell count fell rapidly in paralysis (52), tabes dorsalis (23), cerebro-spinal every case, in some almost to within normal limits. Lange reaction is given for comparison with the syphilis (11), disseminated sclerosis (21), cerebral The tumour (7), spinal compression (6), cerebral abscess (2), B.G. reaction. No relation between clinical improvement and alteration in the colloidal reactions could syphilitic meningitis (2), chronic meningitis (2), be established. Progress seemed to be related rather cerebral thrombosis, polyneuritis (2), hemiplegia, acute meningitis, Friedreich’s ataxia, osteitis of to the number of cells in the fluid, for in those cases orbit, local meningitis, cerebral malaria, third nerve which did not improve after malarial therapy, the palsy and arthritis, one case of each. The fluids cell count remained high. It may be of interest of many of the syphilitic cases, especially those of also to point out that the course of novarsenobillon which was given to many patients after malaria were examined on several occasions. general The Lange reaction was done first on every fluid, treatment, likewise had no effect on the colloidal and only those which gave a reading of (2) in at reactions. TABES DORSALIS. least one tube were used for the B.G. reaction. It The results of the test in tabes conformed less to was found that fluids giving a reading of only (1) in the Lange reaction did not give any colour change one type than those in general paralysis, but again with the B.G. reaction, and except for control cases the reaction approximated to that given by the Lange test. An average reading was 0012321000. Out they have been excluded from this series.
’i
therefore
paralysis,
341
a
of 23 cases, three were completely negative. Of these typical luetic reading, but the Wassermann was the Lange reaction was negative in one case, though negative ; whilst in the third, the Lange reading was the Wassermann was positive in cerebro-spinal very low. One case which was diagnosed as a pure in the second, the Lange gave tabes, gave the typical paretic reading of 1344444431, fluid and blood ; with a Lange reading of 5555555554. Later clinical TABLE I. examination revealed mental changes consistent with early paresis. One other case of tabes which gave a paretic response to the B.G. test and complete precipitation in the Lange reaction is still under
observation. CEREBRO-SPINAL SYPHILIS.
Slight precipitation up to the amount expressed by the figure 2 was found in most cases of cerebro-spinal syphilis. Two typical readings were When the Wasserreaction was negative the B.G. reaction was negative also. With one exception no fluid in this group gave a reading higher than is expressed by a 3. The exception was a case in which the diagnosis may have to be reconsidered. The patient is a young woman of 28, whose symptoms and signs point either to cerebro-spinal syphilis (there is a suspicious history of two stillbirths), or to disseminated sclerosis. The Wassermann is negative in the cerebrospinal fluid and blood, whilst the Lange reading is of the paretic type, 5543321000, and the B.G. reaction is 2444442000. On these laboratory findings’it seems more probable that the case is one of disseminated sclerosis. 1112110000 and x001211000.
mann
DISSEMINATED SCLEROSIS.
* mg.
per 100 c.cm.
M. =malarial treatment; N.A.B. -=novarsenobillon ; Tryp. tryparsamide; Sulf. =sulfosin. (x in the B.G. reaction indicates that as insufficient cerebro-spinal fluid was available, the first tube was left empty.) B.a. blood admixture. =*=
In view of the interest which is being centred on this disease at the present time, we propose to discuss in some detail the results we obtained in the 21 cases which we examined by the B.G. test, and to refer also to the Lange reaction in these cases. The duration of symptoms in the cases examined varied from two weeks to 15 years. No case was in the stage of remission so far as could be ascertained. Lumbar puncture was usually done within a week of admittance to hospital, and inquiries as to progress of symptoms after the fluid had been examined invariably elicited the reply that patients felt better since they had been in hospital. The duration of symptoms bore no relation whatsoever to the colloidal reactions of the cerebro-spinal fluid. Whereas the most acute case, with a history of two weeks’ diplopia and paraplegia, gave a Lange reaction of 4544443221 and a B.G. reaction of 1323421000, two other acute cases, one of six weeks and one of eight weeks’ duration, gave readings of 0111000000 and no change respectively in the Lange reaction and no change in the B.G. reaction. On the other hand, a chronic case of ten years’ standing gave a Lange reading of 4555553210 and a B.G. reaction of 1444421000. There was no suggestion of a fresh acute attack in this case. It was felt that no useful purpose would be served by testing the E.G. reaction on the large number of completely negative fluids from patients with disseminated sclerosis which we examined by the Lange reaction. The total number of fluids we examined from patients with this disease during this investigation was 53. Of these, 27 gave a Lange reaction containing one or more readings of 2, and 19 were tested by the B.G. reaction, there being insuffieient fluid available in the other eight cases to carry out the test. Twenty-six cases gave a completely negative Lange reaction. Two of these were tested by the B.G. reaction as controls. Table II. gives our findings .n the 21 cases tested. It will be noted that in six It will ases the reaction is completely negative. dso be noted that the colloidal reactions are indepenlent of the duration of the disease. As the examination of the fluids was carried out soon after admission to hospital it is not possible to lay what effect treatment may have on the colloidal
342 reactions may be obtained in spite of great protein increase. The ease and certainty with which a good bi. coloured guaiac suspension can be prepared from stock solutions gives the test a definite advantage over the Lange reaction, since good colloidal gold solutions may be difficult to prepare, and there is no satisfactory method of testing them except by their reaction to known specimens of cerebro-spinal fluid. The Lange reaction is therefore only suited to labora. tories where a large number of specimens of cerebro. spinal fluid are examined. While we have not in this investigation examined the B.G. reaction in relation to the better known mastic and benzoin reactions, previous experience indicated that the B.G. reaction is more sensitive and more easy to read, and agrees more closely in its results with the Lange reaction. The disadvantages of the test are : (1) that a comparatively large quantity, 1 c.cm. of cerebrospinal fluid, is required for the test; and (2) that it is not possible to judge what the final reaction will be under 12 hours, whereas with the Lange reaction a fair estimate may be made after half an hour. For these reasons we do not suggest that this test should replace the Lange test in the routine examination of the cerebro-spinal fluid. But when difficulty arises in the preparation of a good colloidal gold reagent, the bi-coloured guaiac test may be substituted, or may, owing to its more standard nature, be used as a control for the Lange test.
reactions. A single observation is of no value, but as a matter of interest, the patient A. B. (Case 20), whose condition was of long standing, and who had had several courses of arsenic injections, gave one of the strongest readings of the series, both in the Lange and B.G. reaction. The B.G. reaction does not seem to be so sensitive as the Lange reaction in this disease. In only five cases, less than 25 per
TABLE II.
We desire to thank the Medical Staff of the National * In mg. per 100 w.
c.cm.
=weeks ; m. = months ; Abbreviations
as
Hospital, Queen-square, for permission to refer to the clinical records of cases in the hospital.
y. = years.
in Table I.
REFERENCES. 1. Thurzo, E. de : The Bi-coloured Guaiac Test, Brain, 1929, lii., 196. 2. Same author: Les Nouvelles Méthodes sur les Réactions collöidales du Liquide céphalo-rachidien, Paris, 1927.
there complete precipitation in any tube, three cases was there a typical paretic The response. proportion of cases giving a paretic in the response Lange reaction was also not very high. In the series of 21, 7, or 33 per cent., showed this reading.
cent.,
was
and in
only
OTHER DISEASES
THE ÆTIOLOGY OF CHOLECYSTITIS: BACTERIOLOGICAL OBSERVATIONS.
OF THE NERVOUS SYSTEM.
Brief reference must be made to other conditions in which a positive reaction may be given by the B.G. test. These are conditions in which the protein content of the fluid is exceptionally high. In a case of polyneuritis, with a protein content of 1 per cent. and a Lange reaction of 4555422222, the B.G. reaction was 4411000000. Certain cases of spinal compression with a very high total protein gave partial precipitation, the first two or three tubes being completely unchanged, and the precipitation taking place in the fifth and sixth tubes. In other cases only the first or the first two tubes were affected. These curves therefore did not resemble those of tabes or general paralysis. In many other cases with a highly albuminous fluid a completely negative reaction was obtained.
liI.
SUMMARY
AND
CONCLUSIONS.
The cerebro-spinal fluids of 140 patients with diseases of the nervous system have been examined by the bi-coloured guaiac reaction. A Lange colloidal gold test has also been done on each of the fluids for comparison with the new reaction. Our results justify the statement that the bicoloured guaiac test is of the same practical value as the Lange test in the laboratory diagnosis of neurosyphilis. The bi-coloured guaiac test runs parallel with the Lange reaction in that certain cases of disseminated sclerosis give positive results. It differs from the Lange reaction in that negative
BY BRYAN WILLIAMS, M.B.
EDIN.,
SCHOLAR, AND ASSISTANT IN THE BACTERIOLOGY DEPARTMENT, UNIVERSITY OF EDINBURGH;
VANS DUNLOP
AND
D. G. S. MCLACHLAN, M.B. EDIN., LECTURER IN BACTERIOLOGY IN THE UNIVERSITY OF EDINBURGH.
BEFORE
gall-stones were supposed to be simple chemical precipitation of In as a result of hypercholesterolaemia. showed the however, Naunyn importance 1892
produced by
cholesterol that year, of infection in the formation of stones, and advanced his theory of a " lithogenous catarrh " of the gallbladder. Since then, the importance of infection in gall-stone formation has been generally admitted, and Moynihan has picturesquely described how a gall-stone may be a tombstone erected to the memory of an organism still living inside. Earlier investigations were directed rather towards the occurrence of organisms in the bile, and members of the coliform group were the chief ones isolated. Owing to the prevalence of typhoid fever at the end of the last century, much attention was paid to typhoid cholecystitis. More recently, the organisms of the enteric group have been encountered much less frequently in this condition. As will be seen below, Bacillus paratyphosus B was isolated only once in the present series of 106 cases.