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Cataphoretic Velocity of Streptococci Isolated in Cases of Encephalitis and of Other Diseases of the Nervous System
Published in The Journal of Infectious Diseases, Vol. 52, No. 2, March 1933, pp. 167-184.
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In this report we record the results obtained in a study of the cataphoretic velocity of streptococci as isolated from atria of infection of patients suffering from encephalitis and other diseases of the nervous system, and from corresponding inoculated animals, and of the effect of the serums of the patients on the respective streptococci isolated.
Methods of Study
The material for study was obtained, as in previous work,1 from various atria of infection in such a way as to exclude, so far as possible, contamination from extraneous sources. The nasopharynx was swabbed without touching the tongue with a cotton-wrapped, wire swab, bent to a suitable angle. Pus and other material for cultures were expressed and scooped from tonsils with a small laryngeal mirror 1.5 cm. in diameter, bent to an angle of about 45 degrees. The material obtained from the nasopharynx and the tonsil was suspended as a routine in 2 cc. of gelatin (0.2 per cent)-Locke’s solution, for cultures and animal inoculations. Extirpated tonsils were washed in large amounts of salt solution, and cultures were made from material obtained from the depths with pipets after searing the surface, or from emulsions made in salt solution by grinding with sand in a mortar. Pus from the depths of pyorrhea pockets was aspirated into small pipets after disinfecting the surface with alcohol. Pulpless teeth were extracted in a sterile manner; the apical fourth was immediately severed with bone-cutting forceps, washed thoroughly by shaking vigorously for some time in 2 cc. of gelatin-Locke’s solution containing quartz sand, according to Baden’s method, and cultures were made from the washings and by placing the apex itself in dextrose brain broth. Exudates in cases of prostatitis were obtained from the urine or collected in a sterile manner after massage of the prostate gland, and swabbings were made from the uterine cervix in cases of endocervicitis. Cultures were also made from these and from washings from the sinuses in cases of maxillary sinusitis. Cultures for cataphoretic studies and inoculation of animals were made chiefly in the mediums of choice for studies of elective localization (tall tubes of dextrose brain broth and soft 0.2 to 0.7 per cent dextrose brain agar), which afford a gradient of oxygen tension, relatively low oxidation-reduction potential and other favorable conditions for the rapid growth of highly sensitive parasitic organisms, especially green-producing streptococci. Assuming that even these culture mediums might not suffice, intracerebral and other inoculations of animals and direct cataphoretic determinations in distilled water were made with material obtained from some of the foci (chart 1). No attempt was made, as a routine, to isolate pure cultures of the presumed causative organism by means of aerobic plates, because it has been found that these do not grow when plated directly, but usually do grow out in dextrose brain broth and in soft dextrose brain agar. Shake cultures in series in tall tubes of soft dextrose brain agar were chiefly used to isolate pure cultures from the mixtures before injection. Intracerebral injection of small amounts of suspensions of material from various foci, or corresponding small amounts of the primary culture in dextrose brain broth, and intravenous injection of larger amounts of the cultures, were given in most instances to separate the parasitic organisms having peculiar neurotropic properties from the purely saprophytic and other types of organisms. The dextrose brain broth was a 0.2 per cent dextrose broth prepared from beef extract or from meat infusion and the commercial peptones. The hydrogen ion concentration was adjusted to ???????? 7.4 by adding 1.5 cc. of sodium hydroxide per liter, and Andrade’s indicator was added before cooling. The medium was placed in tall tubes measuring 20 by 1.5 cm., so that the column was not less than 12 cm. tall, and in each tube were then added several pieces of fresh calf or sheep brain equivalent to about 2.5 to 3 cc. in all, and several small pieces of marble to check the reaction. The dextrose brain agar was prepared in like manner with the addition of from 0.2 to 0.7 per cent agar. These were then autoclaved at 20 pounds’ (9 Kg.) pressure for twenty minutes. Streak cultures on blood agar plates were made as a routine with material obtained from patients and inoculated animals. All cultures were incubated at 35 C.
Cataphoretic studies2 of the streptococci were made with the Northrop-Kunitz-Mudd apparatus at approximately constant voltage and temperature. The dextrose brain broth medium proved especially useful in these studies. It often maintained characteristic velocities of the streptococci in individual cultures from the time abundant growth had occurred until the culture was 24 hours old, and through many generations of rapidly repeated subculture, whereas in corresponding cultures in ordinary broth, even in identical broth medium, exclusive of brain substance, the streptococci often lost their specific velocity promptly, sometimes in a single subculture. After many trial experiments in which the streptococci were repeatedly washed in distilled water and suspended in this menstruum, after determining the velocity of individual cultures in dextrose brain broth at frequent intervals, and after it was found that there was close agreement between the rate of migration of the streptococci in these tests and that of streptococci from atria of infection directly suspended in distilled water, and that of streptococci of the corresponding primary culture in dextrose brain broth also suspended in distilled water, the following simple procedure was adopted as reliable for routine work:
About 2 cc. of each 18 to 24 hour old culture in dextrose brain broth, containing a pure or almost pure culture of streptococci, was poured, under sterile precautions, into a scrupulously clean test tube, which was centrifugated at 1,500 revolutions per minute for ten minutes (10 inch [25.4 cm.] radius), and the supernatant broth was thoroughly drained off by keeping the tubes inverted for at least ten minutes on sterile filter paper or on freshly laundered towels. About 3 cc. of distilled water obtained in the same manner throughout the study from a large still, stored in 5 gallon (15 liter) bottles stoppered immediately after the bottle was filled, was then added in like manner to each tube and the sediment suspended by shaking the different tubes vigorously and equally. The tubes containing the suspensions were then filled with distilled water to within about 2 cm. from the top or about 17 cc. and allowed to stand for a short time. The content of each tube was then poured, in turn, into the cell, care being taken to eliminate bubbles. The current was then turned on, and the exact voltage and temperature, ranging usually, respectively, from 118 to 122 volts and from 24 to 27 C., were noted each day. After determining that the migration of the bacteria was even and not jerky and alike in opposite directions by reversing the current, the time required for each of from ten to twenty organisms, depending on the evenness of their rate of migration, to traverse 50 microns, the unit distance, was determined with a stopwatch. The time required in seconds and fourths of a second for each organism to traverse this unit distance was recorded, and from this the cataphoretic velocity was calculated in terms of microns per second per volt per centimeter, according to the formula:
Thus, most streptococci derived from cases of disease of the nervous system traversed the unit distance of 50 microns in the cataphoretic cell in four seconds, and the effective potential gradient as the cell was used was found to be 7.8 voIts per centimeter by actual measurement; hence microns per second per volt per centimeter equals 50/ (4 x 7.8), or 1.6.3
The operators in most instances were unaware of the diagnoses in the cases in which the cultures were obtained. Cultures from material from atria of infection of patients having different diseases, from old cultures and of standard suspensions of streptococci were often included on the same day in order to be sure that the results were reliable.
Shibley4 and others showed that agglutination of pneumococci and cataphoretic velocity-lowering action on pneumococci by antipneumococcus serum ran fairly parallel. In the course of our previous studies, specific agglutination was sometimes obtained with the serum of the patient and the streptococci isolated with which the disease was simulated; the streptococci were agglutinated in high dilution in the corresponding hyperimmune horse serums, but the agglutination was not constant enough to be of unmistakable diagnostic value. It occurred to us that possibly the serum of the patient might lower specifically the cataphoretic velocity of the streptococcus in question. Accordingly, a series of experiments was undertaken to determine the most reliable method by varying the density of the suspensions, the electrolyte content, the duration and degree of heating of the suspensions, and the methods of holding characteristic potential of stock suspensions of the different strains of streptococci. The following technic was adopted as reliable and is similar to that used by Thompson;5 the results recorded were obtained by its careful application.
Dilutions of serum were made as a routine, in series, in suspensions of the bacteria as follows: 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1,280, 1:2,560 and sometimes also 1:5,120 and 1:10,240, each tube containing 1.5 cc. The diluted suspensions of the respective streptococci were made from stock dense suspensions in glycerin (2 parts) and 25 per cent sodium chloride solution (1 part), which had been kept in the refrigerator. The dense suspensions were diluted with distilled water to about a fourth the density of dextrose brain broth cultures, or about 500,000,000 streptococci in each cubic centimeter, and used directly or after one washing. After thoroughly mixing the suspensions and serum, the setup was first placed in the incubator for one hour and a half, in the oven at 52 C. for two hours, and then in the refrigerator overnight. The following morning the tubes were inspected for evidence of macroscopic agglutination; the controls and each mixture containing various amounts of serum in turn were diluted with 14 cc. of distilled water, and poured into the cataphoretic cell, and the rate of migration was determined in the usual manner.
Results of Studies of Cataphoretic Velocity
The results of a long series of electrical measurements are summarized graphically in charts 1, 2, 3, 4 and 5. The figures following the legend for each graph, or in the blank space in the graph, represent, respectively, the number of strains or of cases studied, the number of cultures of these strains and the number of streptococci timed. In the graphs, in charts 1 to 5, each of the peaks of the shaded areas represents the percentage of all streptococci timed in the different groups that migrated at that particular velocity. Thus, in chart 1, the tall peak in the second graph indicates that 33 per cent of 1,418 streptococci timed in cultures from atria of infection in the group of epidemic encephalitis migrated the unit distance (50 microns) in four seconds or at the rate of 1.60 microns per second per volt per centimeter, and so forth. The consistently high incidence of streptococci having “neurotropic” velocity (about four seconds or 1.60 microns per second per volt per centimeter) in distilled water as swabbed directly and as grown in dextrose brain broth from atria of infection, and after animal passage from pipettings of the brains of animals that reacted positively, is well shown in each of the diseases of the nervous system studied. The green-producing streptococci isolated, which had a fairly constant neurotropic velocity, produced in animals symptoms and lesions that simulated in important respects the diseases in question, confirming and extending previous work.1 The cataphoretic velocity of streptococci isolated in the neuro-myositis-arthritis group (chart 3, graphs 5 and 6) was transitional between that of streptococci found in studies of uncomplicated diseases of the nervous system, including neuritis, and that of streptococci from patients with diseases of muscles and joints. The incidence of streptococci or neurotropic velocity was high, as was also the incidence of those of arthrotropic velocity. However, the incidence of streptococci of neurotropic velocity was only about half that of streptococci isolated in study of diseases of the nervous system only, and the incidence of streptococci of arthrotropic velocity was only about half that of streptococci in study of diseases of the joints only.
The cataphoretic velocity of arthrotropic streptococci derived from patients with chronic infectious arthritis unassociated with evidence of nerve involvement was particularly high in cultures from atria of infection, and in those after animal passage (chart 3, graphs 7 and 8). In a number of cases of chronic infectious arthritis having marked symptoms suggesting involvement of the sympathetic nervous system, or the type of arthritis often considered to be of neurogenic origin, the streptococci had marked arthrotropic neurotropic velocities. In sharp contrast to the mobilities of the streptococci derived from patients with diseases of the nervous system and of joints were the mobilities of those obtained in groups of cases of tumor of the brain and syphilis of the nervous system (chart 2, graph 9), from two groups of normal controls (chart 1, graph 12; chart 2, graph 10), from patients with pernicious and splenic anemia and leukemia (chart 1, graph 11), from patients with tuberculosis of bones and joints (chart 3, graph 9) and from patients suffering from miscellaneous diseases not of the nervous system (chart 3, graph 10). Among the latter group were patients suffering from pemphigus, dermatitis, prostatitis, ulcer of the stomach, habit spasm, thrombo-angiitis obliterans, diabetes, angina pectoris and goiter. In contrast, also, are the results (chart 4) obtained with streptococci isolated from the stools, grown from single colonies in dextrose brain broth and in blood agar shake cultures. The streptococci in each of the groups of cases in which cultures were made from the stools were considered Streptococcus faecalis. Of the twelve cases of epidemic encephalitis in which stools were cultured, two yielded streptococci of predominatingly neurotropic mobility. The cataphoretic velocity resembled that of streptococci from the nasopharynges of normal persons, as did also that of streptococci from patients with ulcerative colitis, from those with miscellaneous diseases and from those with pernicious and splenic anemia and leukemia. On the contrary, a high percentage of the streptococci isolated from the stools and grown in single colonies on blood agar plates, and transplanted into dextrose brain broth, in cases of chronic infectious arthritis and from the joints of animals in which arthritis developed, possessed characteristic arthrotropic velocity. In order to determine, if possible, whether the velocity was characteristic of streptococci as found in persons suffering from encephalitis and arthritis, respectively, thirty-six rabbits were given injections, intracerebrally and into the right knee joints, of from 0.1 to 0.2 cc. in each place, of suspensions in gelatin-Locke’s solution of nasopharyngeal swabbings derived from each of twenty patients with encephalitis. Moreover, thirty-six rabbits were given injections, in like manner, of suspensions of nasopharyngeal swabbings from each of twenty persons with chronic infectious arthritis. The cataphoretic velocity of the streptococci in the primary dextrose brain broth culture was chiefly neurotropic or arthrotropic, respectively, in each of these cases. The mortality from encephalitis in the former group was 67 per cent; in the latter, 30 per cent. The incidence and degree of arthritis, and the incidence of successful isolation of the streptococci from arthritic joints of animals, were far greater in the group given injections of material from patients with arthritis than in that given injections of material from patients with encephalitis. The numbers of streptococci, as controlled by blood agar plating, were approximately the same in the material injected from the two groups of patients. The cataphoretic velocities were determined for the streptococci in the primary cultures in dextrose brain broth, as isolated from the brains of eleven rabbits that died among those which received injections of material from patients with encephalitis, and of nine of those that died following injection of material from patients with arthritis. All but one strain of each group were chiefly of neurotropic velocity; the two exceptions were of mixed velocities. The cataphoretic velocities of the streptococci as isolated from the joints in both groups were likewise determined. The velocities of eleven of the twelve strains derived from patients with encephalitis, and of nineteen of twenty-one derived from patients with arthritis, were chiefly arthrotropic. The three not arthrotropic were of mixed mobilities.
Seasonal Changes in the Cataphoretic Velocity of the Streptococcus
We have studied the cataphoretic velocity of streptococci isolated in cases of encephalitis as opportunity was afforded for the last three years. A change in the velocity of streptococci isolated from the throats of normal persons and persons ill with various diseases toward that characteristic of the streptococci isolated in influenza occurred with the appearance of influenza during the winter and early months of each of the three years (chart 5). Of this we were not aware until the most severe of the three epidemics appeared during the winter and spring of 1932. The seasonal changes in the velocity of the streptococci isolated from the nasopharynx and other atria, of infection, before and after animal passage, in cases of chronic epidemic encephalitis in contrast with those of the streptococci isolated from the nasopharynx during attacks of influenza and during convalescence from influenza are summarized in chart 5. The characteristic slow velocity of the streptococci isolated in cases of influenza at the time of attack (graph 1) and the greater velocity, especially high in the neurotropic column, during convalescence from influenza (graph 2) may be noted. The velocity of the streptococci from cases of encephalitis noted during the winter of 1929-1930 resembled only slightly that of the streptococci from influenza, and the epidemic of influenza was the mildest of the three. In graph 3 may be noted a decided shifting toward the slow influenzal type of velocity between March 27 and May 20, 1931, and the epidemic of influenza was more severe than the one a year previously. This slow rate of migration had disappeared between June 20 and Oct. 5, 1931 (graph 4). The velocity became typically “neurotropic.” The decided rise at the velocity of two seconds, or 3.2 microns per second per volt per centimeter, coincided with the prevalence of cases of acute poliomyelitis and encephalitis in and about Rochester during these months. The streptococci isolated in these acute cases have been found predominantly of this velocity. The epidemic of influenza during the winter and spring of 1932 was the most severe of the three, and the shifting toward the influenza! type of velocity was correspondingly most pronounced in strains isolated from cases of encephalitis, and this change was retained through one animal passage (graphs 5 and 7). Following this epidemic, the velocity of the streptococci from the nasopharynx in cases of encephalitis again became mainly neurotropic as in nonepidemic times and as occurred in 1931, but this year with an abnormally high incidence of streptococci having arthrotropic velocity (graphs 6 and 8). In none of the cases of encephalitis included in this series was influenza or arthritis present at the time of study, or shortly before. Streptococci as isolated from the throats of normal persons following this epidemic also revealed an abnormally high incidence of arthrotropic and neurotropic types of velocity, and concomitantly there occurred a noticeable increase in the general population of cases of acute arthritis, myositis, neuritis and various combinations of these. The cataphoretic velocity and elective localizing power in animals following intravenous injection of the streptococci isolated from the nasopharynx and other foci in these cases usually corresponded to the velocity and particular type of localization in patients from whom the streptococci were isolated. If joints and muscles were chiefly involved in the patients, the velocity was particularly arthrotropic, and lesions in joints and muscles of the animals were predominant; if nerves or nerve sheaths were chiefly involved in the patients, the velocity was chiefly neurotropic, and lesions in nerves and nerve sheaths were chiefly found in the animals.
The streptococci which had neurotropic velocity on isolation tended to lose this property promptly when cultivated on the ordinary artificial mediums in the usual way, but this varied within wide limits in different strains and at different seasons of the year. The instability of these strains as measured by their cataphoretic velocity and pathogenicity was more marked during the colder weather, especially during and after the occurrence of epidemic waves of influenza. The cataphoretic velocity characteristic of neurotropic streptococci was best preserved in cultures in dextrose brain broth, especially if four or more subcultures were made daily, or in latent life in old cultures of meat infusion containing the meat mash, or in latent life in old cultures on blood agar slants sealed with paraffined corks. Preservation of the brains of animals that died of meningo-encephalomyelitis in 50 per cent glycerin maintained viability and specificity often for many months. However, no methods thus far used have sufficed to prevent changes in the magnitude of the negative electrical charge of streptococci and changes in other properties which in part were seasonal or due to causes wholly obscure. Thus, twenty strains of streptococci from patients with encephalitis, all of them kept in latent life for from three to seven years in sealed tubes containing meat infusion or on sealed blood agar slants without transfer were inoculated into dextrose brain broth for determination of cataphoretic velocity; as controls, these same strains were streaked on blood agar plates in April, 1930, and from the same tubes similar cultures were made again one year later. In both instances, on the days the readings were made, control determinations were made on strains the mobilities of which were known, to be certain that the readings were reliable. Of the twenty subcultures in dextrose brain broth, made in 1930, only five (25 per cent) were mainly of neurotropic mobility; five were mainly of arthrotropic mobility, and ten were of other mobilities, whereas of the twenty cultures in dextrose brain broth made in 1931, fifteen (75 per cent) were mainly of neutrotropic mobility, one of arthrotropic mobility and four of other mobilities. Four of the five strains that were neurotropic in 1930 were also neurotropic in 1931, as determined by experiments on animals and by physical measurements.
On the basis of the loss of and return of electrical values characteristic of neurotropic streptococci as preserved in the test tube, the mortality, over a period of six years, of animals given injections of material derived from atria of infection was determined seasonally. The results are summarized graphically in chart 6. The rise in mortality in early spring (April) should be noted, corresponding roughly to the usual high incidence of the occurrence of encephalitis following influenza in human beings at about this time. Also noteworthy are the following: the drop in May; the gradual rise during June, July and August, when the general incidence of neurotropic streptococci in the throats of human beings becomes abnormally high, and when epidemics of poliomyelitis often occur; the slight drop in September; the rise in October, the time when epidemics of poliomyelitis disappear, and when atypical cases of poliomyelitis and mild attacks of encephalitis commonly occur; the marked drop in November and December, when the more severe infections of the upper part of the respiratory tract begin to occur, and when streptococci haying velocity other than neurotropic become more prevalent.
Cataphoresis of Tissue Cells
In order that we might have a better understanding as to why streptococci that possess characteristic cataphoretic velocities localize electively in certain tissues, we have determined the cataphoretic velocities of parenchymatous cells of different organs and tissues of the animal body. Great differences were found in the cells of different organs, but in harmony with studies on elective localization, close agreement was found in the cells of the same organs of different species (man, monkey, rabbit, guinea-pig, mouse); that is, the sequence of electrical charges on parenchymatous cells of different tissues in one species is practically identical with that in another. This was especially true of the lung and brain. In the case of tissue cells of the monkey the migration rates in microns per second per volt per centimeter were about as follows: lung, 1.06; liver, 0.83 ; gastric mucosa, 1.28; cardiac muscle, 0.71; kidney, 1; spleen, 1.88; thyroid gland, 1.8; suprarenal gland, 1.06; parotid gland, 0.58; choroid plexus, 0.45; gallbladder. 1.9; cerebral cortex, 4; base of brain, 3.20, and medulla, 5.34. This phase of the work will be dealt with more fully in a forthcoming paper.
Studies of Serum
In chart 7 are given graphically the results of a representative experiment on the cataphoretic potential-lowering effect (evidenced by slowing of the migration rate) of various serums on the respective specific and control strains of streptococci. It will be seen by following the curves that the serum from a patient with encephalitis had a marked slowing effect on encephalitic streptococci in dilutions as high as 1:2,560 and no effect, except in low dilutions, on streptococci of arthritis and poliomyelitis; that the serum from a patient with arthritis had a marked effect in dilutions as high as 1:2,560 on the streptococci of arthritis, and little if any effect on streptococci of encephalitis and poliomyelitis, and that the serums from normal persons of about the same age had little if any effect on any of the three types of streptococci. Similar specific effects were noted with serums from patients suffering from different diseases of the nervous system, from those who had arthritis, and from those who had recovered from poliomyelitis, but not with the serums from normal persons. The three suspensions of streptococci derived respectively from patients with encephalitis, arthritis and poliomyelitis, used in the experiment recorded in chart 7, and those listed in the table, were each freshly prepared at the time of the experiment from representative dense stock suspensions of a mixture of the respective specific strains. Freshly prepared autogenous and heterologous suspensions were also included in many of the experiments, and with similar results. The results obtained with different serums and the representative antigens derived from patients with encephalitis, arthritis and poliomyelitis are summarized in the table. It will be seen that the action of each of the serums derived from patients with the different diseases studies on the streptococci isolated from patients with the disease in question was specific in that it occurred in extremely high dilutions, also in that a similar action was observed in moderately high dilutions on streptococci isolated in the more closely related diseases; this was not the case with serums from normal persons. Thus, in the cases of chronic encephalitis the specific effect of the serums was noted only on the streptococci isolated in encephalitis, with one exception, whereas in the cases of acute encephalitis in which the effect on the encephalitic streptococci was particularly marked (1:5,120) the serums affected moderately also the closely related streptococci from patients with poliomyelitis, but not at all, or little or no more than normal serum, the streptococci from patients with arthritis. Likewise, when the effect of the serums from patients convalescent from poliomyelitis was particularly marked on the streptococci derived from patients with that disease, these serums affected moderately the streptococci from patients with encephalitis, but not those from patients with arthritis. On the other hand, in diseases in which the nervous system and muscles or joints were involved (neuromyositis and arthritis), the serums affected both the streptococci from the patients with encephalitis and those from the patients with arthritis, and finally the serums from patients with arthritis affected markedly only the streptococci from patients with that disease. The cataphoretic velocities of the streptococci from the nasopharynx or other foci of infection at the time or shortly before the serums were drawn from the patients were mainly neurotropic in value in diseases of the nervous system, mixed neurotropic and arthrotropic in diseases affecting the nerves, muscles and joints (neuromyositis and arthritis) and mainly arthrotropic in chronic infectious arthritis and other forms of arthritis and myositis. The respective streptococci over which the serum had a specific velocity-slowing effect removed this property in the usual absorption experiments.
The serums from a few patients with multiple sclerosis affected specifically in high dilution the homologous composite suspensions of streptococci from patients with multiple sclerosis and in lower but significant dilution the streptococci found in cases of encephalitis. The serums from several patients convalescent from herpes zoster affected only the streptococci having neurotropic velocity found in cases of herpes zoster.
The neurotropic type of velocity of the streptococcus isolated from the nasopharynx and the specific action of the serum in high dilution against neurotropic strains of streptococci, in diseases of the nervous system, have made clear the type of infection at hand in some of the cases in each group of diseases studied. Such observations aided in the differential diagnosis in one case in which the patient had had recurring attacks of either influenza or encephalitis, in one of suspected labyrinthitis with vertigo, in one of hysterical hiccough, in one of hysterical spasms of abdominal muscles, in one of spasms of muscles of the upper extremities, in two cases of marked postinfluenzal exhaustion, and in two in which it was uncertain whether the symptoms of sciatica were due to spondylitis or to true neuritis. The presence of neurotropic flora in the nasopharynx at the time of the attack and somewhat later, a high antibody titer of the serum for the poliomyelitic streptococcus, and at the same time a low titer for the streptococcus of encephalitis and for that of arthritis, seemingly made clear the true nature of the infection in two cases in which there were mild attacks of either encephalitis or high poliomyelitis, and in one case of obscure, intense headache associated with fever, lasting for about a week, followed by exhaustion, then by recovery, without at any time development of paralysis. The absence of the neurotropic type of streptococci and the lack of a specific velocity-slowing effect of the serums on the bacterial cells have eliminated definitely the possibility of encephalitis in the early differential diagnosis of several cases of tumor of the brain.
Vaccine and Serum Therapy
The use of autogenous or stock vaccines of streptococci true to type in the manner previously reported6 again proved of decided benefit in a considerable number of cases in which patients suffered from the different diseases of the nervous system. Repeatedly, patients have expressed themselves as feeling better when they were given the vaccine, worse when it was discontinued, and again better when the injections were resumed. In some cases this result has been noted repeatedly, and was certainly not always psychogenic. In others, there was no noticeable change even when the vaccine was used from three to six months. In no case did the vaccine seem to do harm. The encephalitic antistreptococcus serum in the form of antibody euglobulin solution has again seemingly been of benefit in some cases, especially in cases of acute encephalitis and in epidemic hiccough.1 Recently, six patients with epidemic hiccough who had resisted all other measures recovered promptly after being given the encephalitic antibody euglobulin solution.
As shown in chart 1, graphs 1 and 2, the streptococci isolated from atria of infection in cases of encephalitis were almost always mainly neurotropic. This was also true of those cases in which swabbing was done repeatedly. The patients included in chart 1 had not previously been vaccinated, or else had received vaccine for only a short time. Identical studies of streptococci derived from the nasopharynx were made in eighteen cases of encephalitis in which the patients had been given the stock encephalitic antistreptococcus vaccine once or twice weekly for from one to four years previously, to see if prolonged immunization might rid the nasopharynx of the neurotropic type of streptococcus. Only five of the eighteen vaccinated patients were found to be carriers of the neurotropic type of streptococcus; in the remainder, the streptococci isolated had normal velocity. In some of these, the swabbings were repeated, and in each instance with the same result. The conditions of all of the patients who now had normal flora had improved, and some were much improved while using the vaccine. Of the five patients who were still carriers of the neurotropic streptococcus, one showed slight improvement; the remainder were not improved. The potential-lowering power on homologous streptococci of the serum of the patient tested was very marked, and in a number the serum agglutinated in dilution as high as 1:1,280 streptococci from patients with encephalitis.
Similar results were obtained in three cases of spasmodic torticollis and in two of chronic poliomyelitis. Various other methods have been tried to rid the nasopharynx of the neurotropic type of streptococcus; antiseptic gargles, sprays and nasal insufflation, even spraying the nasopharynx with the encephalitic antistreptococcus serum and with dried serum diluted with powdered acacia and bismuth subcarbonate snuffed into the nose, were all ineffective. Tonsillectomy and extraction of infected teeth sufficed in a few cases. Only prolonged immunization with the vaccine was found effective in a series of cases of long standing. In cases of acute encephalitis, epidemic hiccough, herpes zoster, post-influenzal exhaustion, neuritis and sciatica, the neurotropic type of streptococcic flora in the nasopharynx, found constantly during the acute stage, disappeared and was displaced by a normal flora coincidentally with recovery occurring with or, without the use of vaccine or antiserum.
In two cases of recurring herpes of the skin, the cataphoretic velocity of the streptococci from the nasopharynx was found normal during the quiescent interval shortly before an attack. With the appearance of an attack, the velocity of the streptococci from the nasopharynx became neurotropic in both cases, and lesions of the skin resembling herpes developed in rabbits given injections of the streptococci at this time. The cataphoretic velocity of the nasopharyngeal streptococci from both rabbits became normal within a week after the attack subsided.
Summary and Conclusions
The results of a study of the cataphoretic velocities of the streptococci isolated from atria of infection of persons suffering from encephalitis and other diseases of the nervous system, and from animals in which characteristic symptoms developed, or which died, following inoculation of the streptococci, are reported. The streptococci as isolated from the nasopharynx and other atria of infection, especially the apexes of pulpless teeth, and sometimes from the blood and involved organs of persons suffering from encephalitis and other diseases of the nervous system possess a characteristic neurotropic cataphoretic velocity. The velocity of the streptococci as isolated in cases of encephalitis shifted toward the slow velocity of the streptococci from influenza during epidemic waves of influenza. The marked neurotropic type of velocity of the streptococci found during convalescence from influenza suggests, perhaps, why encephalitis and other diseases of the nervous system, such as epidemic hiccough, polio-encephalomyelitis, radiculitis and neuritis, are so prone to occur following attacks of influenza or epidemics of influenza.
The serums of almost all patients having encephalitis and certain other diseases of the nervous system had specific velocity-slowing effects on the respective strains of streptococci isolated in cases of encephalitis and having neurotropic velocity.
Cataphoretic measurements of the streptococci isolated from atria of infection and measurements of the specific slowing effect of the serum of the patient have proved of value in the differential diagnosis in puzzling cases.
Prolonged use of vaccines containing streptococci having chiefly neurotropic velocity, as isolated especially from animals given injections of material derived from patients having encephalitis and other diseases of the nervous system, has been followed by improvement in symptoms and concomitant disappearance of neurotropic streptococci from the nasopharynx in some cases.
The results of the studies by the new methods support the hypothesis that streptococci having a common physical characteristic, namely, neurotropic cataphoretic velocity, are directly or indirectly etiologic in the diseases of the nervous system studied.
References Cited:
- Rosenow, E. C.: (a) J. Infect. Dis. 34:329, 1924; (b) Arch. Neurol. & Psychiat. 15:712, 1926; (c) ,16:21, 1926; (d) 19:424, 1928.
- The terms electrophoretic potential difference ( P. D.), cataphoretic potential, electrophoretic potential, cataphoretic velocity and cataphoretic mobility are often used! synonymously. In this paper, the electrophysical measurements are recorded in microns per second per volt per centimeter. The equation, which appears later in the paper, is:
For this reason we favor the use of the designation “cataphoretic velocity” as expressive of our measurements, but we use the terms “mobility” and “potential” in some instances.
Through an inadvertent error, the migration rates or velocities of the streptococci in our preliminary report (Proc. Soc. Exper. Biol. & Med. 27: 442, 1930) were calculated incorrectly, for we considered, on misinformation, that the 1 unit distance traversed by the microbes was 32 microns instead of 50 microns. AII velocities recorded in that report may be corrected and expressed in the standard cataphoresis unit, microns per second per volt per centimeter, by multiplying the velocities recorded by 50/32 and dividing this by 7.8 volts per centimeter, the effective potential gradient. - Dr. E. J. Baldes, of the Section on Biophysics, determined the effective potential gradient of the cell as used, and gave other help.
- J. Exper. Med. 40:453, 1924; 44:667, 1926.
- Am. J. Hyg. 14:244, 1931.
- Rosenow, E. C., and Nickel, A. C.: J. Lab. & Clin. Med. 14: 504, 1929.