THE WIDAL REACTION IN THE DIAG-

NOSIS OF TYPHOID FEVER : A RESUME

OF WHAT IS KNOWN ON THE SUBJECT.

By P. S. CHANDRA SEKAR, B.A.t M.D.

Historical.

The he are few diseases in which the presence of some unfailing pathognomonic sign would be more useful than in typhoid fever. There is not a single sign or group of signs which can be said to be pathognomonic of* the disease and

invariably present in all the cases. Although the clinical course of the disease renders the

diagnosis easy in the larger number of uncom- plicated cases when the disease is well advanced, the masked and insidious onset renders the

diagnosis in the earlier stages very difficult and doubtful. Even when the disease is more

advanced, difficulties in diagnosis often arise on account of the complications and also the close resemblance which the disease sometimes bears to many other fevers, e.g., pneumonia, acute

tuberculosis, &c.

430 THE INDIAN MEDICAL GAZETTE. [Nov. 1900.

The diagnosis may be uncertain in very mild cases, in abortive cases, in very acute cases

where there is a severe typhoid intoxication and in cases complicated with lesions other than

those of typhoid fever. Till about the middle of

1896, there were three other methods of diagnosis to help the clinician to confirm his clinical

diagnosis. These were, firstly, Ehrlich's diazo- reaction ; secondly, the blood count; and thirdly, the bacteriological examination of the stools, and urine for the detection of typhoid bacilli. The

relative merits of these methods will be dis-

cussed later on. But it is enough to state here that the first method is not pathognomonic of typhoid fever as it is found in many other

diseases besides typhoid, that the second method is of very little value in complicated cases and that the third method presents considerable difficulties even in the hands of experts owing to the great resemblance (both morphological and cultural) between the typhoid bacillus and

the colon bacillus which is constantly found in the stools.

In June 189G a most important advance was made in the method of diagnosing typhoid fever, viz., the discovery of the Widal reaction. Widal of Paris observed that when typhoid

serum (i.e., the serum derived from a typhoid patient) was mixed with a culture of typhoid bacilli and examined microscopically, certain characteristic changes took place. The bacilli which were extremely motile in the culture before admixture with the typhoid serum, began to lose their motility and ran together and become agglomerated into clumps of bacilli. Or to put briefly, the changes consisted in the "ag- glomeration

"

and "immobilization" of the bacilli. When lie mixed the typhoid serum and the culture of the typhoid bacilli in a test tube, the same changes were found to take place in the bacilli, and they were rendered manifest to the naked eye after a few hours by the formation of a sediment (consisting of the agglomerated bacilli) and the clearing of the supernatant fluid which was no longer turbid as the bacilli were no more distributed in the latter.

Since YVidal announced his discovery in June 1896, the question has been enthusiastically taken up all over the world by a host of obser- vers who have recorded their experiences in the various scientific journals, and in their communi- cations to the various medical societies. In this

way has arisen an enormous literature which I

intend to summarise in this paper. This test is one of the many instances of the

practical aid given to medicine by purely bac- teriological research. I his test may be consider-

ed as a b\Te-product in the purely scientific

study of the causation of immunity. In 1889 Charrin and Roger observed that when

the serum of an animal which had been injected ,?

with livinc or dead cultures of the bacillus ,

pyocyaneus, was mixed with the living culture of the same bacillus in a test tube, the turbid mixture began to clear in a short time and ulti- mately resolved itself into a flaky precipitate which settled at the bottom of the tube and a

supernatant clear fluid. (It' is needless to say that the turbidity of a culture is due to

the distribution of innumerable bacilli which

intercept the light.) They also noticed that when the living culture was mixed with serum

, O

of a non-immunised animal, the turbidity was uniform and never disappeared (i.e., the reaction was absent).

In 1894 Issacff and Ivanoff made a similar observation on a vibrio described by the latter.

Stein was the first to investigate the immu- nising powers of the serum obtained from con- valescent typhoid patients upon animals, and he found that animals could be rendered immune to the typhoid bacilli, by the injection of such serum from the typhoid convalescents, in the

majority of cases. Pfeiffer in following up this line of work, discovered his "

specilic immunity reaction."

In 1894 Pfeiffer observed that if living cholera or typhoid microbes be injected into the

peritoneal cavity of a guinea-pig previously immunised against the respective microbes, the living microbes underwent first immobilisation and agglomeration and subsequently granular degeneration ending in total disappearance. He studied these changes by examining the perito- neal exudation from time to time after the

injection. But when he injected bacilli of a different species from that used in previously immunising the guinea-pig, he noticed that the

changes did not occur. The next step he took was to mix a living typhoid culture with

the serum of an animal immunised against typhoid, and inject the mixture into the peri- toneal cavity of a non-immunised animal. The

results were, of course, as he expected, viz., clumping, granular degeneration and disappear- ance of the bacilli. As he found the peculiar changes to be produced in a species of bacilli only by the serum of an animal immunised

against that particular species, he considered

the reaction as a specific one. The clumping, granular degeneration and ultimate absorption of the microbes observed when specific living organisms were injected into the peritoneal cavity of an animal immunised against that

organism came to be known as " Pfeitier's

Reaction." After this observation was made by Pfeiffer,

this particular reaction came to be used as

a test for doubtful cultures of the typhoid bacillus. Pfeiffer and Ivolle advocated this

test to distinguish the typhoid bacillus from the closely resembling colon bacillus. Shortly afterwards Metchnikoff and Bordet showed that the peculiar phenomena observed b}T Pfeiffer

Nov. 1900.] EARLY EXPERIMENTS ON SERUM TESTS. 431

Could be demonstrated outside the animal body, by examining microscopically a hanging drop piepaiation of the serum of an animal immu- nised against the specific microbe, to which lias been added an emulsion of the doubtful culture.

^ (One drop of serum plus two drops

of emulsion.) Ilieyfound that if the microbes weie the specific ones, they clumped together; and the effect of adding a large proportion of the immune serum was found to be a granular degeneration of the bacilli. These changes were further noted to be absent when the sus- pected microbes happened to be other than those against which the serum-yielding animal had been immunised.

I he next step forward was taken by Gruber and Durham, who on mixing in a test tube the culture of the microbe to be tested with a more 01 less diluted serum from an animal immunised against the microbes to be identified found that the fluid in the test tube became clear owing to the formation and subsidence of a precipitate whenever the microbes belonged to a species same as that previously used for immunising the animal. Between November 1S94 and May 1895, Oruber and Durham were engaged in a series

experiments to test the limits of Pfeiffer's method of diagnosing microbes and to point out the use of the method for various objects.

Gruber in an article in the Wiener Medicinische yochenschrift<? for March 1896, criticized Pfeif-

j^i's conclusions and attempted to show that

"feiffer's reaction was by no means specific, and that when the reaction was positive, the diag- nosis was still doubtful, as the reaction was

quantitative only and not qualitative, so far as the cholera vibrion was concerned. Gruber, however, expressed the opinion that it was of greater service with regard to the typhoid bacil- lus and concluded that those investigations would render great assistance in the clinical diagnosis of cholera and typhoid fever. Durham contributed a paper embodying

the result of the experiments carried on by Gruber and himself in 1894-95 to the Journal of Pathology. He states " Hitherto my efforts to produce clumps by means other than the

fluids obtained from immunised animals have failed. Whilst the blood or serum and the peri- toneal fluid of immunised animals have this

curious power, the fluids which have been

artificially prepared (by culture, &c.) are not

able to cause the formation of clumps. Neither

does the fluid of the normal animal nor of

animals immunised against distantly related

organisms possess this remarkable power." Some of the conclusions in his paper are very

interesting as bearing upon the subject of this

paper. Out of nineteen races of typhoid bacilli not one

failed to react positively with the typhoid serum, aud out of ten races of the colon bacillus (the one

that is usually confounded with the typhoid bacillus) not one reacted with the typhoid serum (from animals immunised against typhoid).

In spite of these advances, in the beginning of 1896 the bacteriological examination of

typhoid fever still remained difficult 011 account of the initial difficulty of isolating the bacilli from the stools. Further, it required at least three days to isolate the bacillus. But after the isolation of bacilli, it was,

possible to diagnose with certainty whether they were really typhoid bacilli or not. There were at that time four methods of verifying the

species of the bacilli isolated. The first method consisted in cultivating the

suspected microbe in the serum of an animal immunised against typhoid fever (this can be called for brevity's sake typhoid serum) and

noting whether, fit the end of the experiment, the serum was clear or not and a precipitate had formed or not. This was the method of Charriri and Roger. The second consisted in the injection of the

suspected microbe after the addition of typhoid serum to it into the peritoneal cavitj7, of a

guinea-pig, and the subsequent examination of the peritoneal exudation for immobilisation and

granular degeneration (glabrification). This was the method of Pfeifl'er. The third consisted in the microscopical

examination of a hanging drop preparation of the suspected microbe in the typhoid serum.

This was the method of Metchnikoff and Bordet. And the fourth method, that of Gruber and

Durham, consisted in mixing the culture of the suspected microbe with the typhoid serum (not growing the microbe as was done by Charrin and Roger) in a test tube and observing whether the liquid became clear after the formation of a sediment.

It must be borne in mind that all these

observers, as they were bent upon verifying the nature of suspected microbes, started with the serum of an animal immunised against the

typhoid bacilli as a known factor and used the typhoid serum to diagnose the species of sus- pected bacilli.

Widal, however, in 1896 conceived the happy idea of reversing all the experiments which had been made up to then ; and he started with a culture of genuine typhoid bacilli as the known factor and proceeded to diagnose the nature of the unknown serum and consequently the nature of the infection in the individual yielding the serum ; or, to put it in other words, he began to use typhoid cultures to determine whether a patient was suffering from typhoid fever or not. He mixed the serum from cases of suspected typhoid fever with a genuine typhoid culture and watched for the phenomena mentioned by Charrin and Roger, Metchnikoff and Bordet, Pfeiffer, and Gruber and Durham.

432 THE INDIAN MEDICAL GAZETTE. [Nov. 1900.

Widal found that the serum of typhoid patients soon after the beginning of the fever (four or five days after the commencement) was able to produce iti a pure culture of the typhoid bacilli immobilisation and agglomeration of the microbes, whereas the serum from healthy people and non-typhoid patients had no such effect. As he found that the reaction was

specific and took place rapidly, he announced on the 26th of June 1896, that the method of serum diagnosis of typhoid fever was a very reliable one.

In his original communication he described several processes.

Later on Widal and Sigard observed that dead cultures of the typhoid bacilli were quite as efficacious as living cultures of the typhoid bacilli in the production of the reaction. The bacilli when killed by heat, by a temperature of between 57?c. and 60?c. or preferabty by adding a drop of formalin to the culture (1?150), were found to retain their agglutinating properties (which were embalmed as it were) for at least three months.

The use of dead bacilli in the diagnosis of

typhoid fever brings the test within reach of

practitioners far removed from bacteriological laboratories. Wright has taken much pains to popularise the use of the dead bacilli.

Widal further found that the agglutinating property was not confined to the liquid fresh

serum and blood, but was also present in

solutions of the dried blood or serum. But Wyatt Johnston of Montreal seems to

have discovered the property of dried blood

solutions to agglutinate the bacilli, before

Widal made the similar observation on dried blood. As the reaction appeared to depend probably

upon the presence of substance analogous to

the ordinary toxines and as many of the latter

preserve their characteristics in the dry state, it occurred to Johnston that the dried blood might

also retain the agglutinating properties, and his expectations have been completely realized by his extensive series of observations. The serum reaction having been proved to be

specific in nature, the method of sero-diagnosis has been extended to many other diseases in recent times. The following is a list of the diseases to which

sero-diagnosis has been extended :? Cholera. Relapsing fever. Diphtheria. Scarlet fever.

Colo-bacillary infection. Tetanus. Malta fever. Streptococcus infection. Plague. And some other diseases. Y ellow fever.

From the contributions of Grur.baum and Durham to the Lancet, it appears that Grun- baum had been working on nearly the same lines as Widal at the same time and had obtain- ed analogous results. But Grunbaum cannot

certainly claim for himself the credit which is

justly attached to Widal on account of his dis- covery. Durham states in the British Medical Journal

of 3rd September 1898, that the reaction was

first observed in the Hygienic Institute of Vienna by Grunbaum working at the suggestion ot Gruber, and that the subject was first brought forward by Gruber at the Medical Congress of Wiesbaden in April 1896. Grunbaum, writing to the Lancet of 28th

November 189G, states that he had anticipated Widal in the study of the serum reaction, and after briefly referring to Widal's short communication of the 26th June 1896, sets

forth his claims of priority to the discovery. He writes that his method was being employed in Vienna from March 1896 onwards, and that Gruber had mentioned it in the Medical Congress on the 16th of April 1896, i.e., two months before Widal announced his discovery. From Grunbaum's own communication to

the Lancet of 19th September 1896 (p. 806) it is evident that the suggestion thrown out to-

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Nov. 1900.] GALL-BLADDER AND LIVER ABSCESS.

Grunbaum by Gruber was to investigate the value of the reaction in identifying by its aid the bacteria isolated from the stools of typhoid patients, and not in diagnosing typhoid serum with a genuine typhoid culture; and it was

the investigation of this point with which Grunbaum was concerned. No doubt Grunbaum

independently made the same observations as

Widal, but he was not working with the idea of establishing a sero-diagnosis of typhoid fever. But Widal, in a moment of inspiration, conceived the idea that the reaction is one more of in- fection than of immunity, and this view made him test the blood in the earlier stages of the disease and establish the sero-diagnosis of the disease. As everybody before Widal thought the reaction to be one of immunity a condition which comes into existence only in the later

stages of the disease, the thought of utilising the o ' o o

reaction for diagnostic purposes never struck

any one of them. The idea that the reaction

belonged to the stage of infection (whether attended or not with more or less of immunity from the beginning of the infection) was absolute- ly essential before the reaction could be utilised

as a means of diagnosis (especially in the earlier stages). And there is no denying the fact that

this idea first occurred to Widal. As all the other observers were very much engrossed with the

idea of immunity (they were really studying the causation of immunity) they failed to grasp the diagnostic value of the reaction. It is indeed

most remarkable that some of the observers

though on the verge of discovering the diagnostic value of the reaction in typhoid fever, missed the discovery for the reason assigned above.

(To be continued.)