Popular Science Monthly/Volume 46/February 1895/The Serum Treatment of Diphtheria

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IT is almost seventy-four years since Bretonneau submitted to the Paris Academy of Medicine a report on croup and malignant sore throat, in which he maintained that a number of differently named diseases that were characterized by a membranous inflammation of the fauces and upper part of the air passages constituted but one specific disease, for which he proposed the name diphtheria. But the confusion he hoped to dissipate by the use of a generic term was maintained until recent years, notwithstanding the familiar employment of that term in medical nomenclature. There are few to whom its sound is unassociated with dread, for old and young, rich and poor, are alike susceptible to its infection.

In 1883 Prof. Edwin Klebs discovered, and in 1884 Prof. F. Loeffler succeeded in isolating and cultivating, the micro-organism now known as the Klebs-Loeffler bacillus, that is generally accepted as the productive agent of diphtheria. These bacilli, inoculated upon an abraded mucous membrane of animals susceptible to diphtheria, produce false membranes, systemic disturbances, and even death; they are found in the nasal and throat secretions and in the diphtheria membrane; microscopically they occur in the form of straight or curved rods that stain, with aniline dyes, most intensely at the ends, and thus present a dumb-bell appearance. Imbedded in organic matter and protected from the light, the bacilli may keep alive for many months outside of the animal organism. Uncleanliness, accumulations of dirt, and particularly dark, damp rooms favor the preservation of the bacilli and the propagation of the disease. This bacillus has never been found as the morbid agent in any other similar affection, or in a spontaneous disease among lower animals; nor is there any existing evidence to support the idea that true diphtheria may be conveyed from sick animals to human beings.

The diphtheria bacillus may be found in the air passages, especially the nose, of healthy persons, in whom it has produced no disturbance whatever, nor does it cause any indication of sickness until it has settled down for good. Lesions of the mucous membrane of the nose or throat, small eruptions in the air passages, chronically enlarged tonsils, and especially catarrhal changes consequent upon cold, damp weather, favor the development of the bacillus. If these membranes are normal, the bacillus finds insufficient material in which to multiply, and the poisonous products that it forms are thrown out with the mucus ordinarily produced; but if there is any lesion or disorder of the membranes, then these poisonous products act at first locally and cause such disturbances of the usual processes of the affected region that the bacilli multiply vigorously and form such quantities of their poison that it is absorbed into the system and the disease follows.

From the moment the bacilli are found on the membranes the person should be regarded as infected, and the term diphtheria should be reserved exclusively for those affections in which this particular bacillus is found. Not infrequently cases are met with that present the physical features of diphtheria, but a bacteriological examination will show that the condition is due to other organisms, such as pneumococci, streptococci, and staphylococci. Indeed, in 1887 Loeffler announced that, in addition to the organism identified with his name and Klebs's, there occurred in diphtheritic membranes another organism resembling it in many essential respects, but differing from it in its disease-producing power, and to this organism he gave the name of pseudo-diphtheria bacillus; these bacilli are shorter and more uniform in size than the typical Klebs-Loeffler bacillus.

The importance of distinguishing between these forms of throat inflammations is apparent when it is recalled that the specific character of the disease not only determines the necessity of isolating the affected person but also the method of treatment that should be followed. New York was the first city in which a municipality endeavored to make that distinction, and what is now known as the "New York plan" was inaugurated by Prof. Hermann M. Biggs, and his associate, Dr. William Hallock Park, of the Board of Health. In New York, as in most cities, physicians are required to report the cases of diphtheria they are called upon to treat; if the environment of the affected person renders it desirable to transfer the individual to a hospital for contagious diseases, it is a matter of importance to decide that the disease is diphtheria. Some of the material from the suspected person's throat is obtained by rubbing the latter with a rod having a piece of sterilized cotton wound around its end, and then the cotton is rubbed over a sterilized mixture of blood-serum contained in a closed test-tube, so as to transfer any microorganisms from the cotton to the serum. The test-tube is placed in an incubating oven and within twenty-four hours the character of the growth of micro-organisms may be decided. In one year Dr. Park and his assistant, Mr. A. Beebe, made bacteriological examinations of 5,611 cases of suspected diphtheria, and the bacilli of true diphtheria were only found in 3,255 cases, or fifty-eight per cent, twenty-seven per cent of the cases being pseudo-diphtheria, and fifteen per cent being of a doubtful character. This seems to prove conclusively that every case suspected of being diphtheria must be examined bacteriologically by an expert.

The affected person should be regarded as infectious from the moment the diphtheria bacilli are found to be present upon the mucous membranes, and, while the bacilli usually disappear with or soon after the disappearance of the local signs, yet in some cases they may remain in an active and virulent state for weeks and even months after the patient has apparently recovered. Therefore no patient should be discharged as recovered until at least two or three bacteriological examinations, made at different times, have failed to reveal the presence of the Klebs-Loeffler bacillus in the secretions of the air passages.

Diphtheria is most rapidly communicated by direct contact between the sick and healthy persons. Coughing, sneezing, spitting, kissing, holding the patient's hands, the use of utensils in contact with the patient, drink or food in the sick-room, and clothing, books, and toys may be the media for carrying the bacilli. From this it may be appreciated that the diphtheria patient must be isolated either in a separate room in the house or in an isolation ward, and that there must be thorough disinfection of all articles in contact with the patient, as well as a local antibacillary treatment of the nose and throat to remove the bacilli and thus limit the possibility of their dissemination.

When diphtheria is prevalent the best prophylactic measures are cleanliness, dryness, ventilation, and proper light in dwelling rooms; cleanliness of the nose and mouth that may be secured by thorough daily irrigation with a one-per-cent solution of common salt or a two-and-half-per-cent solution of common baking soda, used as hot as can be borne, with careful brushing of the teeth and medical care of the tonsils if enlarged or diseased; and, finally, cold ablutions of the neck.

The difficulty heretofore referred to of distinguishing, by the naked-eye appearances, the diphtheritic from the pseudo-diphtheritic inflammation, as well as the formerly existing confusion regarding the identity of all such inflammations, at first inspired doubt in the minds of many investigators that the Klebs-Loeffler bacillus caused diphtheria, especially as it could be injected into animals without producing diphtheritic paralysis. But in 1888 Roux and Yersin found that such injections would kill animals if the bacilli were introduced in sufficient number and sufficient degree of virulence; and, further, that the growth of the bacilli in culture fluids produced poisonous substances, called toxines, so that if the cultures were passed through a porcelain filter in order to remove all the living germs, the filtered liquid would produce exactly the same symptoms, and consequently the microbe only acted through its toxines.

The toxine is produced by the cultivation of the virulent diphtheritic bacillus in broth, in contact with the air. Flat-bottomed flasks that have lateral tubes, and that contain a thin layer of a two-per-cent peptonized alkaline broth, are sterilized in an incubating oven, and then a fresh culture of very virulent diphtheritic bacillus is added to the broth. The flasks are kept in the oven at a temperature of 98º F., and by means of the lateral tubes moist air that has been passed through a wash-bottle is constantly kept passing over the broth; within from three to four weeks a culture that is rich in toxines is obtained, a thick layer of bacilli covering the bottom of the flask. All diphtheritic bacilli do not furnish the same quantity of toxine in cultures, nor is the power of the toxine the same in cultures that are apparently made under the same conditions. When the cultures are completed they are filtered by a Chamberland filter, and the clear liquid is kept at the ordinary temperature in well-filled, stoppered bottles, that are protected from the light. A dose of one tenth of a cubic centimetre, about a drop and a half, of this toxine usually kills a guinea pig weighing five hundred grammes, or one pound, within from forty-eight to sixty hours.

Pasteur's success in producing immunity to chicken cholera, anthrax, and rabies by the inoculation of toxines produced by those diseases has stimulated other investigators to seek out methods for producing immunity to the infectious diseases affecting man. Prof. Carl Fraenkel first immunized guinea pigs against diphtheria by injecting them, with great care, with diphtheria toxine modified by heating it at a temperature of 70º C. (158º F.). Subsequently Behring commenced his experiments regarding the production of immunity, and injected animals with a mixture of the toxines and iodine trichloride, though to-day he injects very small doses of pure toxine at sufficient intervals for the animals to rest comfortably. Brieger and Wassermann produced immunity by injecting a culture of diphtheria bacilli in a broth of thymus gland, after having heated it at 65º to 70º C. (149º to 158º F.) for a quarter of an hour, they having assumed that the thymus extract exercised an antitoxic influence on the specific diphtheria toxine. Roux and Vaillard immunized animals by a mixture of three parts of toxine and one part of Gram's solution of iodine, the substances being mixed a few moments before they were injected beneath the animal's skin. Roux found that a rabbit of medium weight easily supported an injection of half a cubic centimetre of that liquid, and after a few days the injection could be renewed and so continued during a few weeks, when the injection would be increased in quantity or the pure toxine might be administered. He also found that it was necessary to frequently weigh the animals and to interrupt the injections when they lost weight, otherwise a depraved condition of the animal's system developed, that might terminate fatally. Animals thus immunized may be injected with a dose of toxine, or a quantity of culture of virulent bacillus that would ordinarily be fatal with but little if any unpleasant effect.

In 1890 Behring demonstrated the fact that blood-serum taken from an immunized animal was capable not only of producing immunity from the same infectious principle in another animal, but, further, that it possessed the power of curing an infection already in progress. This latter remedial employment of serum containing some antitoxine is called serum therapy. The serum is called an antitoxine serum because it contains some agent that antagonizes the toxine.

Besides the serum, Ehrlich, Roux, and others found that the milk of goats and cows that had been immunized was a source of antitoxine, though such milk was much less active than the serum.

The investigators found that of all the animals capable of furnishing large quantities of antidiphtheritic serum the horse was most easily immunized. Roux frequently found horses in which the injection of from two to five cubic centimetres of strong toxine beneath the skin provoked only a transient fever and a local swelling that quickly disappeared. The cow and the ass were found to be much more susceptible to the action of the toxine. Behring held that the antitoxic properties of the serum furnished by an immunized animal were greater in proportion to that animal's sensitiveness to the action of the toxine. But Roux did not consider this an established fact, and since 1892 had employed horses for immunization against diphtheria, because horse serum was not harmful when injected into lower animals or man, and from the jugular vein of a horse large quantities of blood might be obtained from which a perfectly clear serum could be separated.

The horses selected for the purpose of supplying serum should, Roux states, be ordinary coach horses from six to nine years old, well nourished but incapacitated for work on account of some injury of the limbs. Such horses must be carefully examined to determine the absence of lesions of the internal organs, especially of the kidneys, while the absence of tuberculosis or glanders must invariably be determined by a failure of the animal to react to an injection of tuberculin or mallein.[1] Roux reported the details of the process in a horse seven years old, weighing four hundred kilogrammes, that was injected beneath the skin of the neck or behind the shoulder with toxine, one tenth of a cubic centimetre of which sufficed to kill a guinea pig weighing five hundred grammes in forty-eight hours.

Day. Injected.
1st ¼ c.c. of toxine with ten per cent iodine. No local or general reaction.
2d, 4th, 6tb, 8th ½ c.c. Do. Do.
13th, 14th. 1 c.c. Do. Do.
17th ¼ c.c. of pure toxine. Slight reaction.
22d 1 c.c. "" ""
23d 2 c.c. "" ""
25th 3 c.c. ""
28th, 30th, 32d, 36th 5 c.c. ""
39th, 41st 10 c.c. ""
43d, 46th, 48th, 50th 30 c.c. of pure toxine. Œdema that disappeared in twenty-four hours.
53d, 57th, 63d, 65th, 67th 60 c.c. of pure toxine.
72d 90 c.c. ""
80th 250 c.c. ""

In two months and twenty days this horse received more than eight hundred cubic centimetres, or twenty-five ounces, of toxine with no worse symptoms than transient local swelling and temporary rise of temperature about one degree centigrade. Serum was obtained from this horse by bleeding it on the eighty-seventh day, and immediately thereafter two hundred cubic centimetres of toxine were injected into the vein with but moderate subsequent fever. The latter procedure is less efficacious than injecting smaller doses of toxine from time to time and allowing the animal to rest for twenty days before being bled again. Roux has horses from which blood has been taken more than twenty times with a large trocar, yet the vein is as supple as in the beginning.

The serum obtained from the horse above referred to had a preventive power above fifty thousand—that is to say, a guinea pig was unharmed by an inoculation of half a cubic centimetre (fifteen drops) of a recent virulent culture of diphtheria bacillus if it was injected one hour before with a quantity of serum equal to one fifty-thousandth part of its weight. If this antidiphtheritic serum is mixed with diphtheritic toxine, either in a test-tube or before injection into the organism, the toxine is rendered harmless.

The serum is obtained by abstracting blood from the jugular vein of the horse by means of a small hollow needle. All the instruments employed are carefully sterilized and kept in a five per-cent solution of carbolic acid until they are used. The blood is received in wide-mouthed bottles, holding about two quarts, that have paper tied over the mouths, and that have been carefully sterilized. The horse is blindfolded, its extremities fastened to prevent struggling, a noose is passed around its upper lip, the neck is then made tense, the hair clipped from the skin where the hollow needle is to be introduced, and the entire locality thoroughly scrubbed with a five-per-cent carbolic-acid solution. A small incision is then made through the skin of the neck, and the needle, with the point directed downward, is passed into the jugular vein; a tube connected with the needle is pushed through the paper covering the bottle, and from one and a half to two gallons of blood are withdrawn. The blood is allowed to coagulate and the bottles are placed in an ice chest, where they remain until the serum, amounting to from five to six pints, has separated from the other constituents of the blood. In twenty-four hours, as a rule, the serum is withdrawn from the bottles by means of peculiarly shaped tubes devised by Pasteur, and it is transferred to a flask containing a small piece of camphor that is intended to preserve it.

The serum may be filtered through a porcelain filter if there is reason to believe it was contaminated during its withdrawal, or if it is desired to keep it for some time. As at present prepared the serum has a tendency to lose its remedial influence after it has been kept for a time, and especially if it has been exposed to variations in temperature or to light.

Numerous experiments on animals inoculated with virulent cultures of diphtheria bacilli showed that the quantity of serum necessary to save life varied according to weight, to dose of toxine, to quality of toxine, and to the time of intervention. The serum is preservative and therapeutic, not only when opposed to the toxine but also against the living virus. Roux has frankly acknowledged that these properties of antidiphtheritic serum were discovered by Behring, and upon them depends the serum treatment of diphtheria. The specific action of the serum depends upon a complex substance known as "antitoxine."

An animal injected with the antidiphtheritic serum becomes refractory to diphtheria almost immediately; but the immunity does not last, as it diminishes from time to time, disappearing in some days or weeks according to the strength and the quantity of the serum administered.

To give an idea of the strength of the serum used in his experiments on children and animals, Roux stated that one tenth of a cubic centimetre, about a drop and a half, of the toxine he employed would kill a guinea pig weighing five hundred grammes, or one pound, in forty-eight hours; but a mixture of one tenth of a cubic centimetre of this serum with nine tenths of a cubic centimetre of those toxines did not even cause a local effect in the guinea pig that received it. In other words, a dose of toxine that would suffice to kill nine average guinea pigs was rendered inert by one ninth of its quantity of serum. Experiments on animals have shown that where the toxine is introduced first it is necessary to give more of the serum, and after a certain delay the serum exercises no antagonistic effect on the toxine that was administered.

The preceding facts have served to bring us to the consideration of the use of the serum in the treatment of diphtheria. The prevalence of the latter disease may be judged from the reports of the United States census of 1880 and of 1890, the statistics of the former year showing that diphtheria and croup caused 77·96 deaths per thousand deaths, and those of the latter year showing that they caused 49·54 deaths per thousand deaths. This experience of a lessening of the mortality from diphtheria has not been confirmed by the statistics of New York city, in which there was a rise and fall from 1883 to 1892 inclusive:

Mortality per Thousand of Deaths.

1883 48·60 1888 63·54
1884 52·46 1889 59·55
1885 61·09 1890 44·46
1886 72·15 1891 45·12
1887 78·49 1892 47·55

In fact, it may be noticed that the year the census statistics were obtained the mortality in the city of New York was less than in any other year of the series. This oscillation has occurred in other countries. Thus, in England, during the decade 1861 to 1870 the diphtheria mortality was one hundred and eighty-seven per million living; during the decade 1871 to 1880 it fell to one hundred and twenty-one per million living; while from 1881 to 1890 it increased to one hundred and fifty-nine per million; and this increase was the more conspicuous because both the general death-rate and the death-rate from infectious diseases had been constantly diminishing during the three decades mentioned. With this evident increase in diphtheria mortality—an increase that is the more noticeable when the general activity in sanitation during recent years is recalled—the necessity for some remedial agent is apparent. Like epidemics of other infectious diseases, diphtheria epidemics show various characters; sometimes being very mild, sometimes very severe, with a high death-rate. This variation seems to be due to differences in the number and virulence of the bacilli, the result of unknown causes, to the association of other bacteria with the Klebs-Loeffler bacillus, and to unrecognized individual tendencies.

The serum treatment of diphtheria is being generally tried throughout Europe and America, and the evidence seems conclusive that it is of benefit. In a series of almost fifteen hundred cases of diphtheria that has been collated from the reports of a number of observers, treated by antitoxine serum, the mortality averaged 22·99 per cent, the maximum being 44·9, the minimum 5·5 per cent. This in itself is striking, for the usual diphtheria mortality is over 50 per cent. Roux, Martin, and Chaillon reported four hundred and forty-eight cases treated in the Paris Hospital for Children's Diseases, from February 1 to July 24, 1894, with a mortality of 24·33, while during the same time five hundred cases of diphtheria were treated in the usual manner at the Trousseau Hospital in the same city, and the mortality was 63·2 per cent. W. Koerte reported one hundred and twenty-one cases treated in the Berlin Urban Hospital between January 20 and October 27, 1894, with the serum, in which there was a mortality of 33·1 per cent, while of one hundred and six cases treated during a period of that time without the serum—none being obtainable—53·8 per cent died.

The injection, which is administered slowly in quantities of twenty cubic centimetres (a little more than five drachms) beneath the skin of the flank, is not painful; and if it is made antiseptically, no ill effect follows, and the dose is absorbed within an hour. In twenty-four hours a second injection of from ten to twenty cubic centimetres may be given, and the two injections ordinarily suffice to cure. The temperature usually falls after the injection, although in grave cases the fever may persist. The pulse becomes normal more promptly than the temperature.

The general condition remains good, as a rule; and the false membranes usually cease growing after the first injection, becoming detached within seventy-two hours. Roux gave a child a thousandth part of its weight in serum, though in severe cases he increased the quantity to a hundredth part of the weight. The treatment should be instituted as soon as possible after the infection, as those children treated with serum on the first or second day of infection have all recovered. While the experiments on animals showed that injections of the serum did not exercise a permanent influence in immunizing the animal, there can be no doubt that such injections would exercise a prophylactic effect if administered to those that have been exposed to diphtheria.

One of the great obstacles to the general employment of the serum is the cost of its manufacture. In this country from five to ten dollars is asked for a small quantity that sells on the continent of Europe for not more than one fourth of those sums. The British Institute for Preventive Medicine finds that the serum for a single case costs, to be manufactured, from fifteen to twenty-five cents. Public subscriptions have been started in various large cities in the world for the purpose of securing funds to establish and maintain laboratories for the manufacture of the serum. Roux estimated that for a population such as Paris has (two millions and a half) a serum laboratory would require twenty horses, three grooms, two bacteriologists, and two laboratory assistants, bringing the expenses of maintenance to eight thousand dollars a year, a sum that would be insufficient in this country, where the salaries, etc., would have to be so much higher.

Prom what has been said it may be deduced that the production of antitoxine serum is a matter of time, that it must be made with the greatest care, and that each lot must be tested to determine the degree of its antitoxic power. Only by such tests can its efficiency be determined, for there is nothing in the gross appearance of the yellowish fluid to indicate whether it will or will not exercise therapeutic influence. As no other remedy should be employed in conjunction with it, the dire results to the patient of administering a worthless serum may be appreciated. The Board of Health of New York has found specimens of serum, alleged to be antitoxic, exposed for sale, bacteriological tests of which demonstrated its worthlessness. This can only be prevented by the enactment of State laws that punish by heavy fine the sale of, or allow the recovery of heavy penalties for the administration of, any antitoxic serum that is not approved by the State Board of Health. The importance of exercising such control is appreciated abroad, where, in France, a bill is in preparation for introduction in the Chambers providing that no antidiphtheritic serum but that prepared under Roux's observation, or tested in his laboratory and found equal in curative influence to that prepared by him, shall be sold or administered. In Italy no antidiphtheritic serum but that prepared by Roux, Behring, or Aronson is admitted into the country. A good antidiphtheritic serum is not only harmless but is a remedial agent; a poor or spurious serum may be poise nous in itself as well as being worthless for controlling the disease.

Our present knowledge of diphtheria depends upon the discovery of its specific microbe by Klebs and Loeffler, on the proof that this micro-organism causes diptheria, on the isolation of the diphtheria toxine by Roux and Yersin, and on the discovery of the antitoxine by Behring. An immense field of research spreads out before us; for example, all but the last-mentioned fact is as true of typhoid fever as of diphtheria, and it is probably a matter of but a few months when physicians will be at work determining the scope of usefulness of a typhoid antitoxine.

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  1. Tuberculin is a sterilized and filtered solution, in glycerin, of a culture of the tubercle bacillus, and mallein a similar preparation of the glanders bacillus.