ABSTRACTS AND REPORT. 33 1

THE FINDING OF ALLEGED TUBERCLE BACILLI IN THE BLOOD.

A YEAR has elapsed now since Rosenberger read his initial paper before the Pathological Society of Philadelphia upon the presence of tubercle bacilli in the blood. This paper gave rise to no little interest in the medical world at large, because he asserted that it was easily possible to demonstrate the presence of tubercle bacilli in the blood of every case of tuberculosis by means so simple that it became a matter of surprise that previous efforts along the same line by many competent investigators should not long ago have been rewarded by equal success.

Rosenberger's original studies comprised fifty cases, divided as follows: Two cases of fibroid tuberculosis; one case of pneumo-thorax; fifteen cases of incipient tuberculosis ; twenty-three cases of advanced tuberculosis ; and three cases of the larnygeal form of the disease. He has since augmented this number, so that at the present time they embrace over 300 observations, including cases of lUpus. In all these cases he has met with the same suc­cess, finding the bacilli in the blood in every instance. This contribution was immensely important because, if correct, some of our preconceived ideas concerning tuberculosis must be revised, and because it would also afford the clinician a simple and almost infallible means of diagnosing the malady in its earliest incipiency, a matter which at the present time is too often perplexing, if not impossible. However, with the exception of two brief contributions upon the subject, Rosenberger's observations have not been confirmed.

Forsyth reports the study of twelve cases, with positive results in ten. Bernham and Byons read a paper before the Louisiana Medical Society

detailing their observations in ten cases-negative in every instance. Schroeder and Cotton, in Bulletin No. 116 of the United States Depart­

ment of Agriculture, Bureau of Animal Industry, published the results ot their studies of the blood of forty-eight cases of tuberculous cattle. Their findings were uniformly negative.

Mohler, chief of the pathological division of the above bureau, carrying on an independent investigation, examined the blood of eight tuberculous cattle, and with results no different from his associates.

Ravenel and Smith, at the sixty-third annual meeting of the Wisconsin State Medical Society, stated that they had examined the blood of eighteen cases and were unable to demonstrate tubercle bacilli in any of them.

Petty and Mendenhall studied ten cases. Only seven of these were tuberculous, and in all of them bacilli were demonstrable in the blood. One case of axillary adenitis, and presumably tuberculous in nature, but subsequently shown by pathologists to be sarcomatous, gave a negative result. The two remaining cases, it is to be inferred, were cases of enteric fever, but nevertheless, in both of them acid-fast organisms supposed to be tubercle bacilli were found in the blood.

33 2 ABSTRACT S AND REPORT.

In the British MedicalJournal, Hewat and Sutherland report the results of their investigation in twenty cases. I n one case two acid-fa st bacilli were found, but on a second examination of this patient's blood none could be demonstrated after a long search, so that they were led to believe that the presence of the two bacilli in the first examination was accidental. In none of the other cases were they able to demonstrate acid-fast organisms.

In Bulletin No. 57 from the Hygienic Laboratory, U. S. P. H. and M. H . S., Anderson reports the results of his studies, which were made on forty-eight cases of human tuberculosi ~ , together with thirteen guinea-pigs and eight rabbits experimentally infected. In only a single instance was he able to demonstrate acid-fast organisms in the blood by the . Rosenberger method, and inasmuch as this specimen of blood which was withdrawn from one of the cases of human tuberculosis was injected into a guinea-pig with negative results, Anderson is led to believe that the organisms found were not tubercle bacilli . It is of especial interest to note that even though there were sufficient tubercle bacilli in some of the blood obtained from rabbits from which cultures were successfully grown, none could be demonstrated in the blood-smears.

In this report is added a study of fifty-six cases in whose blood tubercle bacilli were sought for with the utmost pains. The cases were divided as follows :-

Five cases of suspected tuberculosis in which the physical examination was negative or questionable; ten cases in which physical examination was clearly positive, but in which the sputum was negative or had not been examined; four incipient cases, in which both the physical examination and sputum were conclusive; nine cases of moderately advanced tubercu­losis; twenty-two advanced cases; one moderately advanced case, and complicated with larnygeal tuberculosis; one case of tuberculous adenitis; one case of miliary tuberculosis in an infant; one case of bilateral senile pneumonia, and, lastly, two cases of epidemic cerebro-spinal meningitis.

Before the appearance of Rosenberger's paper a similar and independent investigation by the author was in progress at the Henry Phipps Institute, the technique employed differing but little from that of Rosenberger. The work had been abandoned, however, after the careful study of the blood of eight cases of undoubted tuberculosis was productive of negative results only. The technique used was as follows: 5 cc. of blood were withdrawn from any convenient vein and immediately added to an equal quantity of a 2 per cent solution of sodium citrate. The mixture was then centrifugalised for ten minutes in a rapidly revolving electric machine, the supernatant fluid pi petted off, distilled water added to the sediment, and the mixture vigorously shaken and recentrifugalised. The sediment thus laked was withdrawn with a pipette, thick smears made on clean glass slides, fixed and stained for five minutes in carbol-fuchsin, decolourised in 5 per cent. nitric acid solution, in 95 per cent. alcohol, and counterstained for five seconds in borated methylene-blue. Rosenberger at first employed the centrifuge, but subsequently discontinued its use, as he thought it gave less satisfactory results than simple sedimentation. On the other hand, Forsyth employed the centrifuge, which in his hands gave better results.

Rosenberger's technique, as given in his original article, was rigidly fol­lowed in a later series of cases. It requires that the blood, after being agitated with a citrate solution, be allowed to sediment for twenty-four hours in a refrigerator, at the end of which time the sediment is pipette<j off and thinly spread upon clean glass slides, which are then placed on a copper plate and subjected to a low degree of heat until dry. They are sub-

ABSTRACTS AND REPORT. 333

sequently laked by placing them in a jar of distilled water, and the resulting thin, almost colourless, film dried and fixed by rapidly passing through a Bunsen flame. They were next stained with carbol-fuchsin, Pappenheim's stain being employed as a decolouriser and counterstain.

In the first fifteen examinations this technique was followed exactly, but Pappenheim's solution was eventually discarded, because it gave rise to so many confusing artefacts. Indeed, in the large majority of the slides so stained these were met with, and were sometimes so deceptive in their resemblance to tubercle bacilli that co-workers in the laboratory who were asked to examine them frequently differed in their judgment. The fact, however, that, after the Pappenheim stain was abandoned and decolorisation in acid alcohol with counterstaining in borated methylene-blue substituted for it, no such bodies were encountered would seem to prove that they were artefacts.

In the initial part of the investigation six slides were made from the blood of each case, and each critically examined field by field, but after seven cases had been so studied and found negative it was deemed sufficient that two slides be prepared and from thirty minutes to two hours devoted to the study of each of them.

Singularly enough, in the few cases in which acid-fast organisms were demonstrable they were found in less than fifteen minutes. In one case only three minutes were required; in another it required only eight minutes' search to discover the first group of bacilli, and a second group was discovered on the same slide after twelve more minutes. In studying another slide only thirteen minutes were required to find a group of organisms, which were the only ones met with, although another half-hour was expended in attempting to locate more. Bacilli were found in six minutes in another case, and a few others afler twenty-three more minutes. Lastly, one and one-half minutes was all the time required to demonstrate them in another case.

The cases which gave positive results were five in number, and, remarkable though it may seem, with two exceptions were not cases of tuberculosis. As a matter of fact, if a case of miliary tuberculosis ill an infant and one suspicious case of incipient tuberculosis be excluded, every case of tubercu­losis examined gave a uniformly negative result.

The five cases referred to as being positive were as follows: One suspected case of incipient tuberculosis, one case of bilateral senile pneumonia, and two cases of epidemic cerebro-spinal meningitis. The finding of acid­fast organisms in the blood of the cases of cerebro-spinal meningitis was interesting, as showing how readily the clinician could be led astray in his diagnosis by such laboratory findings.

Case I.-W. B., a student in one of the colleges in the suburbs of Philadelphia, was admitted to the Bryn Mawr Hospital with a history of headache and malaise for three or four days prior to admission. The head­ache was very intense, pyrexia was present, the pupils were dilated, and he complained of some abdominal pain. At times he talked incoherently. His neck was only slightly rigid, with no pain on movement. There was amesthesia of the cornea. No rose spots were demonstrable. The attend­ing physician suspected enteric fever, although a tentative diagnosis of meningitis was made. A Widal test was negative, and a leucocyte count of 10,200 was obtained. A lumbar puncture being refused, J 0 cc. of blood were withdrawn from an arm vein, 5 ce. of which were utilised in making a blood culture for the purpose of demonstrating, if possible, typhoid bacilli, the other 5 cc. being mixed with citrate solution to be examined for

334 ABSTRACTS AND REPORT.

tubercle bacilli, after the method of Rosenberger. On the following day the patient's symptoms had become intensified. He was now very delirious; there was marked rigidity of the neck, with expressions of pain on movement, and herpes labial is appeared. Lumbar puncture was still refused. Examination of the blood on this day showed acid-fast bacilli, and in the absence of any epidemic of cerebro-spinal meningitis, a diagnosis of tuberculous meningitis with miliary tuberculosis was made.

Case 2.-P. B., a college chum and room-mate of W. B., was admitted to the same hospital on the day that the diagnosis of Case I was supposed to have been cleared up. He already had marked meningitic symptoms­delirium, retraction of the neck, and the characteristic decubitus assumed by such victims. Kernig's sign was marked. There were no rose spots, and the spleen was not enlarged. Becoming ill so soon after his room-mate, and with similar symptoms, it was suspected that both young men were the victims of one and the same disease-epidemic cerebro-spinal meningitis­and a lumbar puncture was performed. The fluid obtained was purulent, and upon microscopical examination showed innumerable diplococci, with the morphological and tinctorial characteristics of the meningococcus. No acid-fast bacilli were found in this fluid, though they were diligently sought for. A lumbar puncture was now made in Case I, and meningococci found, but again no acid-fast organisms. As a matter of interest, however, 5 cc. of blood of Case 2 were secured and examined for tubercle bacilli, and to the writer's surprise a few acid-fast organisms were demonstrated. True, there is a possibility that both these patients were tuberculous, though it must be admitted that it would be a pecaliar coincidence. We must also remember that Rosenberger claims to have demonstrated acid-fast bacilli in the blood of apparently healthy individuals. Might the blood of these two individuals have revealed acid-fast bacilli had such an examina­tion been made prior to their falling ill ?

Petty and Mendenhall found acid-fast organisms in the blood of two ,of their patients who were suffering from typhoid fever. Be that as it may, the point is, that even though acid-fast bacilli were demonstrable in the blood, it could scarcely be regarded as definite proof of the existence of tuberculosis. Had Case No.2 not contracted the disease, W. B. dying, as he did, after a protracted (Case 2 died in forty-eight hours) illness, would have heen considered a case of tuberculous meningitis with miliary tuberculosis, and a death certificate issued accordingly, when, as a matter of fact, he was not a victim of either disease. It might be added that an autopsy, though urgently sought for, was refused in both instances. Unfortunately, too, no autopsy was permitted in the case of bilateral pneumonia in whose blood acid-fast bacilli were also found. The patient was a woman of sixty-five years, whose clinical symptoms were classical of pneumonia. Repeated examinations of her sputum failed to reveal any tubercle bacilli; the pneumococci were founn in abundance.

The fifty cases of tuberculosis, the study of which forms the basis of the present paper, were ward and dispensary patients at the Henry Phipps Institute of Philadelphia. In every case the blood was obtained by intro­ducing a hypodermic needle in a vein, and the preparations made according to the technique already given. Careful examinations were made, and with a single exception were uniformly negative.

The one patient in whose blood acid-fast organisms were found presented no physical signs whatsoever. Her history, slight cough extending over a period of a few months, with loss of weight and a feeling of fatigue, however, were regarded as suspicious. She did not respond to the tuberculin test. In

ABSTRACTS AND REPORT. 335

a ~mear made from her blood two separate groups, one of five and one of three, acid-fast organisms were found. They were by no means typical of the tubercle bacillus. In fact, in no case have the organisms been morpho­logically typical. In some instances they appeared much larger and broader, With round ends and staining a pale pink colour; in other instances they were short and stained intensely. None showed irregularity of staining, and in no case was clumping observed. In the case of W. B. the bacilli found were chiefly of the large, broad type, and peculiar in that one group of the organisms were in chain formation, two chains lying perfectly parallel, one consisting of five elements and the other of four.

Inoculation Experi7llents.-In order that the character of the organisms might be more accurately investigated, inoculation of guinea-pigs with the blood was used as a control. Approximately 2 cc. of the blood from each of the thirty-seven of the tuberculous cases were inoculated into guinea-pigs. In the first twenty-one animals the injection was made intra peritoneally, but as it was suggested that the peritoneal fluids might possibly exert more bactericidal influence, the subcutaneous method was substituted in the remainder. Two pigs were inoculated from each case, seventy-four animals being thus utilised. Of these, seventeen died prematurely under three weeks. Of the remainder, thirteen survived the injection from two to seven months, fourteen from one to two months, and five approximated three weeks. All were carefully autopsied, and in no single case was there microscopical evidence of tuberculosis, glandular or otherwise. Theblood from the two cases of epidemic cerebra-spinal meningitis and that frolll the case of bilateral pneumonia, in which the acid-fast organisms were de­monstrated in the blood-smears, was, unfortunately, not injected into guinea-pigs. That from the infant, on the other hand, was inoculated into the animals, but autopsy on the latter did not reveal evidence of tuberculosis in any organ. The failure to induce the disease in guinea-pigs by injecting them with the blood (ram tuberculous individuals is supposed by Rosen­berger to depend upon the attenuation or death of the organisms in the blood-stream. The experiments of Liebermeister are, however, convincingly opposed to this view.

Sources of Error.-That Rosenberger should report constant positive results in a large series of cases, and that Ravenel and Smith, Cotton and Schroeder, and Mohler, together with others, uniformly negative ones, and that negative results should practically result in the fifty cases herein reported IS, to say the least, extremely perplexing. To say that organisms are present in the blood only at certain stages of the disease is not tenable, as Rosen­berger asserts to have found them in all stages. How, then, can such discrepancies be accounted for? All sources of contamination seemed to have been excluded by Rosenberger in his investigation, but Burnham and Lyons, and more recently Brem, have demonstrated at least one source through which error can arise. Brem, intent upon confirming or refuting Rosenberger's work and finding acid-fast organisms fairly constantly, though his inoculation experiments proved negative, was led to suspect the water that he made use of for his solutions and also his chemicals and stains. All were critically examined for the presence of the acid· fast organism, but with no result, until at last, afler fixing the sediment from the dislilled water upon a slide by means of albumin, he was able to demonstrate acid-fast organisms in greater or less numbers in every specimen examined . It is true that Rosenberger examined his solutions with negative results, but, as Brem points out, the failure to find the water bacilli may have been due to the fact that no albuminous material was used as a fixative. Having demonstrated acid fast

ABSTRACTS AND REPORT.

bacilli in all the cases examined at Bryn Mawr, and three of those cases otlter than tuberculosis, also aroused suspicions that an error existed somewhere. All materials used were examined, but with negative results, until, albumin being used, innumerable acid-fast organisms were found in the distilled water. This water was distilled from a Jewell apparatus some time previous to its use. The large number of acid-fast organisms which it contained can be the better appreciated when it is understood that the demijohn which contained fully two gallons of the water was shaken up, and only approximately 90 cc. of it centrifuged and the sediment from it examined. Every field showed large numbers of these organisms. The greater number of them were typical for the tubercle bacillus, and were identical with those found in the blood specimens. However, organisms typical of the tubercle bacilli were demon­strable on the slide. Tinctorially, they accord with tubercle bacilli in many respects. They will resist 25 per cent. aqueous solution of nitric acid and also Pappenheim's solution, but, unlike the tubercle bacillus, they are promptly decolorised by a 25 per cent. solution of nitric acid in alcohol. Two guinea-pigs were inoculated with the sediment from the water, but, un­fortunately, they died prematurely.

At the Phipps Institute the distilled water was also examined, but with negative results. As the bottle in which the distilled water was kept was a small one, it was necessary to replenish it repeatedly, so that the water originally used for the blood examinations was not examined. It is readily conceivable, however, that the bottle still contained them, but in such small numbers that the examination of many slides would have been necessary to demonstrate them, as 124 blood slides had been carefully examined before one was met with which revealed the acid-fast organisms.

In this series of cases there is no doubt that the source of error was the distilled water, and, with this experience, coupled with that of Brem, the author is led to suspect strongly that· the same factor is responsible for the finding of alleged tubercle bacilli in the blood. (Joseph McFarland, Sixth Annual Report of the Henry Phipps Institute, 1912, p. 127.)

THE DETECTION OF ANTHRAX SPORES IN INDUSTRIAL MATERIAL.

As indicated by the title, the object of the investigations carried out by the authors was to discover the most satisfactory means of detecting anthrax contamination in industrial material. In the preliminary portion of the paper tables are given showing the number of cases of anthrax in man and in animals in Great Britain and Ireland during the years 1906 to 191I. There is also a brief consideration of the" causes of the dissemination of the disease among domestic animals."

The first point considered in the body of the paper is the comparative value of the methods usually employed for the netection of anthrax con­tamination, and the authors state that one of the objects of their paper is to demonstrate the superiority of the inoculation method over the method of plate cultivation.

Having given a brief summary of the investigations carried out by other investigators, the authors pass on to the description of the technique of their methods of plate cultivation and animal inoculation.