VOLUME 18

Original Contributions

BLOOD CULTURES AS AN AID IN THE EARLY DIAGNOSIS OF TYPHOID FEVER. *

BY LEON K. BALDAUF, A. B., M. D., Department Pathology and Bacteriology, University of Louisville,

LOUISVILLE, KY.

Within the last few years and in rapid succession numerous articles have appeared relating to blood culture work in typhoid fever. Series of large numbers of cases have been reported and while these communications have emphasized mainly the constant finding of bacteria in the circulating blood in the active stage of the disease, the attempt has been made constantly to simplify the technique and to select a culture medium which would be effectual and reliable. The results have been extremely satisfactory and now blood culture work is not limited to those in a well equipped hospital, but may be made use of by others. During my last months at the City Hospital in St. Louis Dr. J. P. Simonds and I attempted, as far as possible, to make systematic and routine examinations of the blood of the patients with suspected typhoid fever. While our series was very small and compares numerically rather unfavorably with the published reports, it is satisfactory otherwise. While we were much interested in determining the frequency

*Read before the Medico-Chirurgical Society of Louisville.

NUMBER IO

of bacillemia in typhoid infections, we were much more concerned with the isolation of different organisms from several individulas. We believe with others that there are numerous strains of the typhoid bacillus and that the severity of the symptoms is dependent in many cases upon the variety of the organism. This idea, of course, is not original. We are familiar with Durham's explanation of intercurrent relapses, with his normal or isozymic infection where the infection is due to a number of varieties and sub-varieties, no unit predominating and no relapses occurring; with his abnormal or anisozymic infection where there is a small protection against one form which may predominate and which may give rise to another infection.

The discussion of technique and methods about which at present there is the greatest interest, resolves itself into a consideration of the following: (1) The simplification of the technique; (2) The prevention of blood coagulation; (3) The lytic action of the serum; (4) The quantity of blood to be used; (5) The amount of dilution necessary; (6) The character of the medium.

Those who have been making blood cultures for several years know the great difficulties under which the work was formerly done. A procession was started to the ward with a number of plates, a

1 A System of Medicine-Osler and McCrae, 1907, Vol. 2. p. 173.

As a

number of boullion flasks and a mass of melted media in a large jacketed vessel containing a quantity of water. The work had to be done quickly because of the danger of sudden chilling of the media and the fear of clogging the hypodermic needle. We also remember the effect which this unnecessary confusion produced on the patient. The adoption of a method and technique then which would eliminate these objectionable features was not only essential but necessary. distinct advance, Conradi in 1906 introduced the use of ox-bile medium. The advantages which have been claimed for this medium are the following: (1) That it causes immediate haemolysis of the red blood cells, prevents the formation of a blood clot and at the same time furnishes a haemoglobin medium; (2) That it has no bacteriolytic action; (3) That it is an excellent culture medium; (4) That it simplifies the technique and reduces the amount of apparatus to a minimum. Since then slight modifications have been proposed, but the solutions in the main have remained essentially the same. In June, 1907, Epstein' from the Mount Sinai Hospital reported his results with ammonium oxalate solution. This consists of Ammonium Oxalate 2 grams. NaCl 6 grams, Distilled Water 1000 cc., the ammonium oxalate having been added to prevent the blood coagulation. Blood clotting in blood culture work must be avoided for several reasons. If the work is delayed for any length of time the formation of blood clot within the barrel of the syringe or in the hypodermic needle is very likely to occur. Again, clotting of the blood prevents an even distribution of the blood in the medium and in many cases is supposed to retard the growth of the organism on plated media by en

Epstein, A.-The Use of Ammonium Oxalate in Blood Culture Technique.-The American Journal of the Medical Sciences, 1907-Vol. 134, p. 424.

gulfing the organism in a mass of fibrin. Mueller and Graef have recently shown, however, that the fluidity of the blood is not essential as far as ultimate development of the organism is concerned, and have demonstrated a multiplication of bacteria in masses of blood clot.

In discussing bacteriolytic action, both the medium and the amount of blood withdrawn should be considered. The Conradi medium has been praised on account of its supposed anti-bacteriolytic action, while the use of small quantities of blood has been advocated by those who fear a bacteriolytic action of the serum. Others, while using large quantities of blood, have attempted to reduce the bacteriolysis by diluting the blood with large quantities of media. The claim that numerous organisms failed to grow because of the bacteriolytic action of the serum in vitro is not supported, however, by experiment. Eppenstein and Korte* have proved definitely that the organisms have evidently become immune to this bacteriocidal action. Their conclusions are further substantiated by the ability of the organism to multiply in defibrinated, oxalated, laked and clotted blood. It has been further shown, that the quantity of blood used is not an important consideration. Maybee and Taft' from the Boston City Hospital, while using only ten to twenty drops of blood which they removed from the lobe of the ear obtained results comparable with those of Epstein. who used several cubic centimeters.

As to the choice of media, Epstein to

3 Mueller and Graef-Wert der Blutuntersuchung fuer die Typhusdiagnose.-Centralbl. f. Bakt. etc., Abt. Originale Bd. xliii, Heft 8-856.

Eppenstein und Korte-Ueber das Verhalten der im Blute der Typhus Bazillen gegenueber der bakteriziden Wirkung des Blutes. Muenchener Med. Wochen. 1906. No. 24, p. 1149.

Maybee and Taft-The Early Diagnosis of Typhoid Fever by Blood Cultures from the Ear. Boston Medical and Surgical Journal. 1908. Vol. 158No. 23, p. 863.

Epstein, A.-Blood Cultures in Typhoid Fever, The Amer. Jour. of the Med. Sciences. 1908. Vol. 136, p. 190.

determine the relative value of different media experimented with: (1) plain bouillon; (2) 2% glucose bouillon: (3) plain agar; (4) plain agar and serum; (5) 2% glucose agar; (6) 2% glucose agar and serum; (7) neutral plain agar; (8) 5% glycerine agar; (9) Conradi medium; (10) Kayser medium; (11) ammonium oxalate solution. The best results were obtained with 2% glucose bouillon, 2% glucose agar and ammonium oxalate solution. With him the bile media were not as reliable. In our work at the City Hospital, we have used almost exclusively ammonium oxalate solution and Conradi's ox-bile medium. We selected these solutions because as we have said before they minimized the work and were extremely satisfactory otherwise. We withdrew the blood from one of the large veins at the bend of the elbow. The arm was cleansed with green soap and alcohol, an Esmarch bandage was applied around the forearm and with a glass hypodermic syringe with a capacity of 15 cc. from 5 to 10 cc. of blood were removed. In the case of the Conradi medium, the blood was immediately added to the ox-bile mixture, incubated at 37 C. for 7 or 8 hours and then sub-cultures made. With the oxalate solution plates were made on reaching the laboratory. Within 24 hours we could give a positive report, and where we isolated an organism which was motile, grew diffusely in bouillon and did not produce gas on glucose agar, we felt certain that we were dealing with a typhoid organism even though we did not obtain. a typical reaction with an agglutinating serum. It is a well-known fact that certain typhoid organisms do not agglutinate well.

Sciences for June, 1907, Coleman and Buxton' reviewed the reports of 1602 cultures, which, including their own, they had collected. The analysis of the cases by week was as follows:

Of 224 examinations in the first week of the disease, 200, i. e. 89%, were positive; of 484 examinations made in the second week of the disease, 353 or 73% were positive; of 268 examinations made in the third week of the disease, 178 or 60% were positive; of 103 examinations made in the fourth week of the disease, 39 or 38% were positive; of 58 examinations made after the fourth week of the disease, exclusive of the relapses, 15 or 26% were positive. Their analysis showed that positive results were obtained more frequently at the end of the first week and that there was a gradual decrease from that time on.

As to the relation of the bacillemia to the course of the disease, evidently the organisms invade the blood in greatest numbers in the early stage of the disease. As defervescence takes place the bacilli disappear from the blood, the duration of the fever being practically measured by the persistence of the organism.

There seems to be no relation between the bacillemia and the type of the disease. The organisms were recovered with equal frequency from mild as well as severe cases, but persisted longer in the severe

cases.

Relapses: Of these 1602 cases, 33 represented relapses. The typhoid bacillus was recovered in 30 or 90%. In 391 cases studied with relation to the serum reaction in 94 the bacillus was found before the Widal Reaction was positive. This is readily understood when we remember that the serum reaction may not appear before the ninth or tenth days and

Coleman and Buxton. The Bacteriology of the Blood in Typhoid Fever. The Amer. Jour. of the Med. Sciences. 1907. Vol. 133, p. 896.

then may be present for only 2 or 3 days. From the summary of the cases they conclude, "that the typhoid bacillus is present in the blood of every case of typhoid fever, throughout its course." Second, "that the bacillemia in typhoid fever does not constitute a true septicaemia, but it represents an overflow of bacilli from the lympho-poeitic organs." Third, "that the clinical picture of typhoid fever results only from infection of the lympho-poeitic organs by the typhoid bacillus, with the invasion of the blood stream and destruction there of vast numbers of bacilli." Fourth, "the endo-toxins of the typhoid bacillus are not cumulative in action and convalescence from typhoid fever per se is established within a few days after the disappearance of the bacilli from the blood." Coleman and Buxton do not mention the frequency of paratyphoid infection, but Epstein reports that in 154. cultures "taken promiscuously, some for diagnosis and others for research, 110 gave growths of the typhoid bacillus and 12 paracolon (so-called paratyphoid) bacilli." In the light of these findings then, what bearing do they have on the nature of the disease? This question is admirably discussed by Richardson" of the Massachusetts General Hospital. The typhoid bacillus, unlike the diphtheria bacillus, does not produce a soluble toxin. Its toxin is introcellular and belongs to the class of endotoxins. The immunity which follows an infection is not antitoxic, but is antibacillary. Typhoid intoxication, therefore, is dependent upon bacillary destruction. It is not the bacillary infection which gives rise to the symptoms of typhoid fever, it is the active destruction of the bacilli which is responsible. Patients exposed to the disease may harbor the bacilli in the circulating

Richardson, M. W.-Fact and Speculation Concerning the Nature of Typhoid Fever. Boston Med. and Surgical Jour. 1908. Vol. 158-No. 19, p. 688. *

blood and still show no effects. This has been recently demonstrated, and in an article in the Muenchener Medizinische Wochenschrift Busse reports two interesting cases: In two other rather obscure cases with atypical toxic and intestinal symptoms, typhoid bacilli were cultivated from the circulating blood. The existence of typical miliary tuberculosis and tuberculous intestinal ulcers was disclosed at autopsy. Microscopically the lymph glands, spleen and intestines presented typical tuberculous. lesions and showed no evidence of typhoid infection. This may explain the peculiar localized typhoid infections which often occur when no previous history of typhoid fever can be obtained. A few organisms may circulate in blood. The destruction of a few bacilli is not sufficient to give rise to symptoms and probably eliminated in the bile, they now reach the medium in which they can multiply rapidly and cholecystitis and cholelithiasis follow. According to Richardson then, while a typhoid bacillemia is essential the typhoid intoxication is dependent upon a destruction of these bacilli. In an article in the Boston Medical and Surgical Journal, he concludes that the intensity of the disease varies with the rate of destruction and the efficiency in elimination; rapid destruction of a few bacilli will result in a short, severe attack, ending probably by a crisis; rapid destruction of a large number of bacilli will give rise to a severer form and probably of longer duration, the fulminating type which is frequently fatal; slow destruction of a moderate number will give rise to a moderate intoxication of longer duration, the true slow fever. Where the gradual increasing immunity does not check the

Busse, Otto-Ueber das Vorkommen von Typhus Bazillen im Blute von nicht typhus kranken Personen. Muenchener Med. Woch. 1908. 55-No. 21, p. 1113.

growth of the bacilli, intoxication gradually increases and the patient may die.

DISCUSSION.

DR. J. B. MARVIN: I think it was a step far in advance, when the profession began to realize that typhoid fever is a bacillemia, and out of this grew the idea that the bacillus could always be detected in the blood.

The technique has been greatly simplified, as the essayist stated. I think he might have elaborated a little for the benefit of those of us who are not as expert as he is. It is a comparatively simple matter now to get blood from the lobe of the ear; we do not need such a large quantity.

Next, the modified ox-bile culture media can be easily handled by anybody who has the facilities of a laboratory. I have always thought that Epstein's media was intended more where you wanted to carry blood from the patient to the laboratory, or keep it for a while; that it was a mixture of oxylate of ammonia and salt solution. We might accomplish the same thing with citrate of soda solution, but for immediate work it is not necessary to use either one.

You could inoculate directly, could you not on the media? Conradi makes a culture media containing ox-bile with peptone and glycerine and incubates that for a while. This modification, which is some improvement, is to inoculate from the oxbile media onto this lactose-litmus-agar, and incubate for a while; then you can make a positive diagnosis inside of thirty hours. The technique has been very much simplified and it is a pretty rapid thing. The beauty about it is that this gives its best results-maybe 90 to 100 percent.--in the first week, the very time when we all halt and hesitate and are in doubt about the diagnosis of typhoid

fever. The Widal gives no results before the second week or even later. Now, here is a method which gives you the earliest and most positive diagnosis of typhoid fever that we have at the present time. Therefore, I think we have here a wonderful advance. The technique has been so simplified now that we can get enough blood by puncturing the ear, incubating 15 or 16 hours, then re-inoculating, and inside of 30 hours you get a most positive diagnosis of typhoid fever. I hit others and myself as well when I say that we have not had this test applied; we wait and hesitate about it. It ought to be done in every case where a person has access to a laboratory and a competent bacteriologist.

DR. JNO. G. CECIL: I have thoroughly enjoyed this paper, but, not being a practical bacteriologist, I can hardly discuss it to any advantage. I am certainly glad to know that we have at our command an efficient and practical aid to diagnosis in typhoid fever. It certainly seems to be of great value, and it will oftentimes 1elieve us, as well as the patient and his family, of a great deal of uncertainty. I propose to use this method of diagnosis as I find myself able to do so.

Another point which impressed me as being of a great deal of importance, is the fact that the positive discovery of the bacilli in the blood of typhoid fever patients demonstrates the fallacy of the idea. which has been pretty generally entertained, that this disease is curable by means of intestinal antiseptics. Therefore, we will have to look to such workers as Dr. Baldauf and others for a vaccine, or serum, which will cure typhoid fever.

DR. LOUIS FRANK: It has been my good fortune ot have had opportunities for using this method in three cases. Dr. Davis made the tests. One of these cases Dr. Marvin will remember.