Part 37

The prognosis of neurosyphilis is not worse than that of the chronic diseases in general. In fact, the prognosis of neurosyphilis _quoad vitam_ is either good or dubious, certainly not bad. The surprising reversals of form which the spirochete shows in certain remissions are always to be awaited. Treatment of neurosyphilis has certainly effected amazing results, not so much by way of Ehrlich’s _therapia sterilisans magna_ as by means of systematic intensive treatment. Even paretic neurosyphilis (general paresis) seems to have been cured. Preparetic phases are theoretically hopeful. Nor is it so certain that paretic neurosyphilis will ultimately prove a perfectly distinct species of neurosyphilis. General paresis seems to us at least to be more closely related to diffuse neurosyphilis than is tabes dorsalis to diffuse neurosyphilis. In any particular case, moreover, =during a good part of the early months or years=, =it is difficult or impossible to tell the paretic from the non-paretic forms of diffuse neurosyphilis by any combination of clinical observations and tests=. In the instance of more protracted neurosyphilis, e.g., tabetic, the outlook for vascular complications is such that antisyphilitic treatment directed at prevention of these complications is scientifically warrantable, even if the tabetic process itself proves unassailable. The old distinction of syphilis and parasyphilis, so striking and apparently satisfactory when introduced by Fournier, seems to be a false distinction which should be dropped. Therapeutically, we should approach all cases of neurosyphilis without bias or nihilistic prejudgments.

(3) RE _universal applicability of syphilis tests in nervous and mental cases_.

The importance of putting every neurosis or psychosis through syphilis tests is not based alone on the frequency of neurosyphilis, though neurosyphilis is surely frequent enough. The importance of universally applying these tests is established by the experience of lingering doubts both in the physician’s mind and (nowadays increasingly) in the patient’s and friends’ minds, so long as these tests are not applied. Nor should the positive serum Wassermann reaction fail to be followed by lumbar puncture and appropriate tests. The general practitioner confronting neuroses or psychoses—and what practitioner does not?—must not expect valuable results from consultation with neurologists and psychiatrists when he does not carry to these specialists the results of at least the serum W. R. in his patient. Not only are practitioners, specialists, and patients subject to discomfiture on the eventual and delayed proof of syphilis or neurosyphilis, but valuable time has been lost to treatment. How often the physician of yore (and really not so long since) had to be regarded as an eccentric virtuoso if he tested urine as routine! Well, for routine use in nervous and mental diseases, the Wassermann serum reaction is at least as important as urinalysis. Nor would we cease our homily with the general practitioner. We know neurologists and psychiatrists who use the Wassermann test _only when it is likely to be positive_! But they are dying out.

APPENDIX A

In appendix A a brief outline is given of the six tests (W. R. on blood serum and spinal fluid, cell count, globulin test, albumin test, gold sol test). This is not intended as a complete working manual but rather as indicating the methods used in diagnosis in the cases presented herein. For more complete details the reader may be referred to textbooks on the subject of serology, among which may be mentioned Kaplan: “Serology of the Nervous System”; Plaut, Rehm and Schottmüller: “Leitfaden zur Untersuchungen der Zerebrospinalflüssigkeit”; Kolmer: “Infection, Immunity and Specific Therapy,” and, for the Wassermann technique, an article by Dr. W. A. Hinton in M. J. Rosenau’s “Preventive Medicine and Hygiene.”

Our own W. R’s. have been performed at the Wassermann laboratory of the Massachusetts State Board of Health (formerly the Neuropathological Testing Laboratory, Harvard Medical School), under the supervision of Dr. W. A. Hinton. The other tests are performed at the Psychopathic Hospital. It is very important that a close relationship should exist between the clinician and the Wassermann laboratory if the most is to be obtained from the reactions. This relationship has been effectively close between the authors and the above-mentioned laboratory; and has enabled us to get very much clearer ideas about certain cases than could otherwise have been obtained.

=Cell Count.= In order to obtain the number of cells per cmm., the examination should be made of the fresh fluid as soon as possible after this is withdrawn. The most convenient counting chamber for this purpose is the so-called Fuchs-Rosenthal counting chamber, the ruled spaces of which contain slightly over 3 cmm. (an ordinary blood cell counting chamber may be used). According to the method used by us the cells are stained in a pipette with Unna’s polychrome methylene blue. Using a white-counting pipette, stain is drawn up to the first or second marking and the remainder of the pipette filled with spinal fluid. This makes no change in the dilution for practical purposes. After two or three minutes the staining is satisfactory and the counting may be done. With this stain a differential count may be made. Plasma cells stain a lavender as contrasted to the blue of the lymphocytes. The characteristic halo surrounding the eccentric nucleus is visible. The blood cells do not assume color with this stain; hence it is unnecessary to add any acetic acid.

For permanent preparations, and more accurate differential counts of the spinal fluid, the Alzheimer method may be used. The technique is given in a paper by H. A. Cotton and J. B. Ayer as follows:[151]

1. Lumbar puncture in the usual manner.

2. 96% alcohol, in proportion to twice the amount of cerebrospinal fluid, is added drop by drop and well mixed.

3. Centrifuge the mixture for one hour at high speed in a glass tube with conical end. (An ordinary electric urinary centrifuge apparatus can be employed, the tube to be well stoppered to prevent evaporation.)

4. The supernatant fluid is poured off, leaving a small coagulum in the bottom of the tube.

5. Add absolute alcohol—alcohol and ether—ether, each separately for one hour, to dehydrate and harden coagulum.

6. The coagulum can now be gently loosened from the bottom of the tube by a long needle. The tube is then inverted, and the coagulum allowed to fall into the hand by a quick tap on the end of the tube. Care must be taken not to squeeze or handle the coagulum. The hand is placed over a small homeopathic vial, containing thin celloidin, and the coagulum allowed to drop into the celloidin, where it remains over night (twelve hours usually).

7. Coagulum is placed in thick celloidin which is allowed to evaporate slowly. It is then mounted on blocks and sections cut 14µ in thickness.

8. The sections are stained and mounted according to the following procedure:

(_a_) Remove celloidin by absolute alcohol and ether.

(_b_) 80% alcohol.

(_c_) Water.

(_d_) Sections are carried on glass or platinum needle into a dish of Pappenheim’s pyronin-methyl green stain and kept in a water bath at 40° C. five to seven minutes.

(_e_) Quickly cool dish in running water.

(_f_) Wash off superfluous stain in plain water.

(_g_) Absolute alcohol to differentiate—until no more stain comes away from section.

(_h_) Clear in Bergamot oil.

(_i_) Mount in balsam.

The normal cell count may be stated as being up to 6 cells per cmm.; from 6 to 12 cells may be considered as suggestive of pathological condition and more than 12 cells per cmm. as definitely pathological. The type of cell in syphilitic diseases is preponderantly the small lymphocyte. A low percentage, that is, very rarely over 20%, of large lymphocytes, endothelial phagocytic cells, polymorphonuclear leucocytes and plasma cells may also be found. The finding of plasma cells in any number in the spinal fluid is suggestive although not conclusive evidence for the diagnosis of paretic neurosyphilis.

=Globulin= is an albumin which is precipitated by half saturation with a salt. A very simple and satisfactory test is known as the Nonne-Appelt test, which has been modified by Ross-Jones. Into a test tube of small diameter, run 1 cc. of spinal fluid. Place under this fluid with a pipette, 1 cc. of a saturated solution of ammonium sulphate ((NH_{4})_{2}SO_{4}). If any globulin is present a white, sharply-defined ring will form at the junction of the two fluids. According to our readings, a ring that is just visible with the aid of a black background is called 1+, a ring that is just visible without the black background, 2+; a ring easily perceptible, 3+ and a relatively very heavy ring, 4+. On shaking the tube, if globulin is present, the fluid will show turbescence.

Another simple globulin test used in our laboratory as a check on the Nonne-Appelt test is the Pandy test. A few cc. of a clarified 10% solution of phenol are placed in a watch glass. One drop of spinal fluid is run into this solution. A milky turbescence indicates globulin.

The presence of globulin in the spinal fluid is always an indication of abnormality of the cerebrospinal axis. There is nothing differential in this finding as it occurs in all inflammatory processes. However, it is characteristically present in most cases of neurosyphilis (exception to the rule: the pure vascular type does not show globulin in a very high per cent).

=Albumin Test.= Albumin in small quantities is present in all spinal fluids. Increase over the normal amount occurs in pathological conditions such as most cases of neurosyphilis, especially in those in which globulin is found. Any albumin precipitant may be used for rough clinical calculation, comparing the amount of precipitate with that from the normal fluid. Our method is to place 1 cc. of spinal fluid in a small test tube of about 5 mm. diameter and to precipitate the albumin by the addition of 3 drops of 33⅓% of trichloracetic acid. This test has its chief value as confirmatory of the globulin test, since in the vast majority of instances where globulin is found there will also be found an increase in albumin.

The =Gold Sol Reaction= is an empirical test discovered by Carl Lange in the utilization of the work of Zsigmondi with solutions of colloidal gold and albumins. Briefly the details of the test are as follows:

Ten tubes are set up in a rack. To the first tube 1.8 cc. of a 0.4% of salt solution is added and to each of the following tubes 1 cc. of this solution. Then to the first tube containing 1.8 cc. of salt solution one adds 0.2 cc. of the spinal fluid to be tested. This gives a dilution of 1 to 10. From this tube 1 cc. is pipetted into the second tube and this process continued through the ten tubes. This gives dilutions of spinal fluid of 1 to 10, 1 to 20, 1 to 40, etc., to 1 to 5120 in the last tube. Then 5 cc. of colloidal gold solution is added to each tube. A positive reaction is indicated by the precipitation or throwing down of the colloidal gold into its metallic form. This produces a change in color. This precipitation may be partial or complete and the amount of precipitation is indicated by the color and is read as follows:

The unchanged fluid is called 0; a slight change giving a red-blue as 1; a further change giving a blue-red as 2; a straight blue as 3; a lavender or violet as 4; and the colorless fluid representing complete precipitation as 5. The numbers are placed in a row, indicating the tube in which the color occurs. The fluid from a case of paretic neurosyphilis will give a complete precipitation beginning in the first tube and running through a number of tubes and then grading off. It may be indicated 5 5 5 5 4 3 1 0 0 0. The characteristic reaction of fluids from tabetic and diffuse neurosyphilis is less strong than from the paretic. The greater part of the reaction will take place, however, in the first five tubes, but as a rule it will not begin very strongly in the first two. A characteristic reaction is 1 2 3 3 2 1 0 0 0 0. Another reaction that may be considered characteristic of the tabetic or diffuse form is 3 3 3 2 1 0 0 0 0 0. Fluids from non-syphilitic cases as a rule give a reaction having its greatest intensity beyond the fifth tube, that is, in the high dilutions.

A reaction characteristic of brain tumor or tuberculous meningitis is 0 0 0 0 1 3 3 2 1 0.

The conclusions that may be drawn from the gold sol reaction have been summarized by one of the authors as follows:

1. Fluids from cases of general paresis will give a strong and fairly characteristic reaction, especially if more than one sample is tested, in the vast majority of cases.

2. Very rarely a general paresis fluid will give a reaction weaker than the characteristic one.

3. Fluids from cases of syphilitic involvement of the central nervous system other than general paresis often give a weaker reaction than the paretic, but in a fairly high percentage of cases give the same reaction as the paretics.

4. Non-syphilitic cases may give the same reaction as the paretics; these cases are usually chronic inflammatory conditions of the central nervous system.

5. When a syphilitic fluid does not give the strong “paretic reaction,” it is good presumptive evidence that the case is not general paresis; and this test offers a very valuable differential diagnostic aid between general paresis, tabes and cerebrospinal syphilis.

6. The term “syphilitic zone” is a misnomer, as non-syphilitic as well as syphilitic cases give reactions in this zone; but no fluid of a case with syphilitic central nervous system disease has given a reaction out of this zone (test thus valuable negatively). Any fluid giving a reaction outside of this zone may be considered non-syphilitic.

7. Light reactions may occur without any evident significance, while a reaction of no greater strength may mean marked inflammatory reaction.

8. Tuberculous meningitis, brain tumor and purulent meningitis fluids characteristically, though not invariably, give reactions in higher dilutions than syphilitic fluids.

9. The unsupplemented gold sol test is insufficient evidence on which to make any diagnosis, but used in conjunction with the Wassermann reaction, chemical and cytological examinations, it offers much information looking toward the differential diagnosis of general paresis, cerebrospinal syphilis, tabes dorsalis, brain tumor, tuberculous meningitis, purulent meningitis.

10. We believe that no cerebrospinal fluid examination is complete for clinical purposes without the gold sol test.

The =Wassermann reaction= as carried out in the Wassermann Laboratory is based on the principles of the original method—the only essential modification consists in the employment of cholesterinized alcoholic extracts of human hearts as antigen instead of aqueous extracts of foetal livers from cases of congenital syphilis. Experience has shown that properly standardized antigens made from human hearts are much more sensitive in the detection of true cases of syphilis.

=Antigens.= Three antigens are used, each being an alcoholic extract of human heart which is saturated at room temperature with cholesterin. These antigens differ slightly in their sensitiveness. Before the test is made each antigen is diluted with 0.85% salt solution in the proportion of four parts of the cholesterinized antigen extract to sixteen parts of 0.85% salt solution. The amount to be used, the dosage, is carefully determined by testing each antigen against a large number of known positive and known negative specimens of blood. The dosage of the antigens employed is less than one-half the amount which inhibits hemolysis when the antigen is incubated for one hour with the hemolytic system which consists of complement, amboceptor and cells in the proper proportions. These antigens are designated as A, B, and C. Antigen A is the most sensitive. B and C are very similar to each other quantitatively and qualitatively.

=Specimens to be tested.= The serum which separates from the clot is withdrawn, centrifugalized if necessary, and then heated at 55 degrees for thirty minutes. 0.1 cc. of serum is used in the test and 0.2 cc. of each specimen is used as a control to exclude the presence of anti-complementary substances. Spinal fluids are tested in two ways. As a routine 0.5 cc. of the spinal fluid is used in the test and 1.0 cc. is used in the control; or when especially requested spinal fluids are titrated by using respectively 1.0, 0.7, 0.5, 0.3, and 0.1 cc. of the spinal fluid for each test and 1.0 cc. of spinal fluid for the control. Spinal fluids are not inactivated.

=Complement.= The complement is obtained from the serum of guinea pig’s blood. No complement is used when older than eighteen hours. A 10% solution and 0.85% salt solution is used in the test. The amount used is twice the minimum quantity necessary to hemolyze the sensitized cells.

=Sheep’s Corpuscles.= A 5% suspension of sheep’s corpuscles in 0.85% salt solution is prepared from defibrinated sheep’s blood. The corpuscles are washed three times and for each washing four to five times as much 0.85% salt solution is used as the original volume of the defibrinated blood.

=Amboceptor.= The amboceptor is prepared by injecting sheep’s corpuscles into a rabbit. The serum of this rabbit which contains amboceptor is diluted with 0.85% salt solution so that 0.25 cc. will hemolyze 0.5 cc. of a 5% suspension of sheep’s corpuscles. In the test twice the quantity or 0.5 cc. of amboceptor is used.

=Sensitized Cells.= The sensitized cells consist of equal parts of washed sheep’s corpuscles and diluted amboceptor. This mixture is incubated in a water bath at 37° C. for a half hour to effect the sensitization of the cells.

=Technique of the Wassermann Test.= One-tenth cubic centimeter of each inactivated specimen of serum and 0.5 cc. of each uninactivated specimen of spinal fluid is pipetted into a separate tube. A mixture is freshly prepared in salt solution, each cubic centimeter of which contains the proper amount of antigen A (the most sensitive antigen), and two units of a 10% solution of guinea pig serum (complement). One cubic centimeter of this mixture is pipetted into each test tube. These tubes are then incubated for forty minutes in a water bath at 37° C. At the end of this period, sensitized cells are added, and the tubes are again incubated in a water bath at 37° C. for one hour. Each specimen which shows any degree of inhibition of hemolysis is retested in the afternoon. For this second test antigen A is again used and in addition antigens B and C. A control is also made for each specimen retested to eliminate any possibility of the inhibition of hemolysis being due to anti-complementary substances in the serum or spinal fluid tested. The technique of the second test differs in no wise from that of the first, except for the use of a control in each retested specimen and the employment of three antigens instead of one. The degree of positiveness is noted for each retested specimen and compared with the degree of positiveness obtained for the corresponding specimen with the same antigen-complement-salt solution mixture in the morning’s test. The specimen is retested on the next day when discrepancies occur between the morning reading for antigen A and the afternoon reading for antigen A. From the above description it will be noted that the negative specimens have but a single test with one antigen only, while the positive specimens are retested, thus permitting a confirmation of any positive reaction. In this way attention is focalized on the positive specimens.

=Interpretation of Results.= Antigen C (the weakest of the three antigens) is used entirely for diagnostic purposes and any specimen showing the slightest degree of inhibition with this antigen and stronger degrees of inhibition with the other antigens is reported as positive. The specimens which are strongly or moderately positive with antigens A and B and negative with antigen C are reported as doubtful. In testing spinal fluids by the titration method, antigen C is used and the readings are based upon the degree of inhibition of hemolysis noted. The intensity of this inhibition is indicated by Arabic numerals: “5” indicates complete inhibition, while “1” means a faint cloudiness, hence a weak reaction. Intermediate numbers show relative intensity varying between complete inhibition “5” (strong positive) and slight inhibition “1” (weak positive); “—” equals no inhibition (negative).

Although it is commonly believed that the recent administration of antisyphilitic treatment will affect the reaction by making it negative, this is not our experience, and it is, therefore, not necessary that treatment be withdrawn for a short period before the specimen is submitted for examination.

The reaction as carried out in this laboratory has the following diagnostic significance: =Positive indicates syphilis=, except very rarely in acute febrile conditions such as malaria and pneumonia. =Negative does not exclude syphilis.= In obscure conditions a series of less than three negatives has little diagnostic significance. =Doubtful suggests syphilis.= It is therefore advisable to submit three or more specimens in such a case, and interpret a persistently or predominatingly doubtful reaction as indicative of syphilitic infection.

=Bruck Test.= A new serum test for syphilis has recently been described by C. Bruck.[152] Following are recent results in our laboratory with this test.[153]

This new test for the diagnosis of syphilis by C. Bruck has aroused much interest. The scientific standing of Bruck and the simplicity of the technique led us to overcome our prejudice, that has been the offspring of the numerous tests that have been offered of late. Bruck states that since the discovery of the complement fixation test for syphilis by Wassermann, Neisser and himself in 1906, he has been trying to find a simple chemical reaction that would take the place of the complicated technique of the Wassermann reaction. This method, as he has published it, was worked out and is being used at the front, in the present war, where complete laboratory equipment is not available.

Commencing our experiments with a great deal of scepticism, we were much surprised at the results obtained, which are given below. Whatever may be the final status of the test in the determination of syphilis, we feel that there is a great deal of interest in the fact that this simple chemical reaction does pick out certain differences in the composition of blood sera and that apparently a large number of syphilitic sera differ in their chemical composition percentage from the majority of non-syphilitic sera.

The technique, while exceedingly simple, offers many chances for errors and individual variations so that we have thought it well to give directions and cautions at some length.