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CHAPTER I.
THE ETIOLOGY OF INFLUENZA
ОглавлениеFrancis G. Blake, M.D.; Thomas M. Rivers, M.D.; James C. Small, M.D.
The bacteriologic investigation which will be described was made at Camp Pike, Arkansas, during the period of the influenza epidemic from September 6 to December 5, 1918. The data presented are limited to observations made during life in uncomplicated cases of influenza and to control studies in normal individuals, and in cases of measles. Bacteriologic studies made at autopsy will be described in a subsequent part of this report.
Because of the wide variations in opinion concerning the relationship of various bacteria to influenza that have arisen during the progress of the recent pandemic, a brief review of the salient features of the earlier literature seems advisable. In 1892 Pfeiffer8 found a small, Gram-negative, hemophilic bacillus in all cases of influenza, often in almost pure culture, both during life and at autopsy. He stated that the organism was found only in cases of influenza or in those convalescent from the disease. Similar bacilli occasionally found in other conditions he classified as pseudoinfluenza bacilli. He furthermore showed that freshly isolated cultures were pathogenic for monkeys, producing a disease not unlike influenza, though lacking in what he considered the characteristic lung lesions. He therefore felt justified in claiming that this bacillus, which he designated B. influenzæ, was the cause of epidemic influenza. Pfeiffer’s work, though hailed by many as unassailable, has failed to stand the test of time in two respects. It has been definitely shown, by Wollstein9 in particular, that there is no justification for recognizing a group of pseudoinfluenza bacilli, organisms so classified by Pfeiffer being indistinguishable from B. influenzæ. Furthermore, numerous investigations have demonstrated that B. influenzæ may frequently be found in a variety of diseases affecting the respiratory tract and in a small proportion of normal individuals. Kretz10 found it 47 times in 950 examinations, usually associated with disease of the respiratory tract. Süsswein,11 Liebscher,12 Jehle,13 Wollstein,9 Davis14 and many others have demonstrated its presence in cases of measles. Lord15 isolated B. influenzæ in 30 per cent of 186 sputums from patients with acute and chronic infection of the respiratory tract. Boggs16 found it in frequent association with chronic bronchiectasis. Wollstein9 17 showed that it was often present in the respiratory diseases of infants, and was not an infrequent cause of meningitis. Rosenthal18 found that one in six of normal individuals harbors influenza bacilli and therefore considered it purely a saprophyte, a position, of course, thoroughly untenable in the face of indisputable evidence that it may be highly pathogenic. The widely accepted statement that B. influenzæ is nonpathogenic for animals has apparently served in considerable degree to shake belief in its etiologic relationship to epidemic influenza. It would appear, however, that this opinion is not founded upon fact. Reference is again made to the work of Wollstein19, who has shown that virulent strains of B. influenzæ, when freshly isolated from the human host, are highly pathogenic for rabbits and monkeys and that nearly all strains are more or less pathogenic for mice and guinea-pigs.
None of these modifications of Pfeiffer’s original work, however, would seem to constitute any valid reason for abandoning the conception of the etiologic importance of B. influenzæ. On the contrary, they are quite in harmony with well-established facts concerning other bacteria which cause infections of the respiratory tract. Such bacteria are frequently found in normal individuals leading a saprophytic existence, are often associated with other disease conditions, and tend to show marked variations in virulence.
Since the outbreak of scattered epidemics of influenza beginning in 1915–16, which finally culminated in the pandemic of 1918–19, a vast amount of literature on the subject has appeared. No attempt has been made thoroughly to analyze this, because much of it is not available, much of it abounds in contradictions which it is difficult to harmonize at the present time, and much of it has been written on the basis of insufficient data gathered under the handicap of war conditions by men without sufficient time to undertake special investigation, or it is feared, in many instances, not sufficiently qualified by previous bacteriologic training.
The sum and substance of opinion in 1918 would seem to be best summarized by quoting from the published report compiled by the British Medical Research Commission:20 “Although Pfeiffer may yet furnish reasons why the verdict should not be pronounced, there is already sufficient material to shake the orthodox conception out of its high altar. Two facts stand out prominently: the generally acknowledged, or by some reluctantly admitted, absence of B. influenzæ from organs on postmortem examinations, and the universally recorded findings of diplostreptococci, singly or in association with the Pfeiffer bacillus.” Comment on this opinion will be made in the general discussion at the end of this paper.
In undertaking a study of the bacteriology of influenza, it seemed essential to bear in mind certain clinical features of the disease which will be discussed in greater detail in a subsequent paper. It suffices to say for our present purpose that it is felt that influenza in itself should be regarded as a self-limited disease of short duration (two to five days in most instances), the most prominent local manifestation of which is a rapidly progressing attack upon the mucous membranes of the respiratory tract. Among the cases observed during the epidemic at Camp Pike uncomplicated influenza never proved fatal and death invariably was associated with a complicating pneumonia. In a large majority of cases pneumococci, S. hemolyticus, or less frequently other bacteria in addition to B. influenzæ were associated with the pneumonia. It is felt, therefore, that in any attempt to determine the primary cause of influenza bacteriologic studies made during life in early uncomplicated cases of the disease are of primary importance and that the bacteriology of the sputum of patients with complicating pneumonia and the bacteriology of autopsies can only properly be used as valuable supplements to data so obtained.
Since cultures from the respiratory tract must often of necessity contain many bacteria which play no part in the production of influenza, it is essential to have a working knowledge of the bacteria that may be encountered by the methods employed. It is also important that such knowledge as may have been gained in interepidemic periods be amplified by study of the bacterial flora present at various periods throughout the course of an epidemic, both in normal individuals and in other disease conditions. These points have been borne in mind throughout the present study and such observations have formed an essential part of the work.
Methods.—In an investigation of this nature the culture methods employed should be suitably directed to determine primarily what bacteria are present and in what relative proportion they exist. The use of culture or animal inoculation methods that are highly selective in character, enhancing the growth of certain bacteria and retarding or inhibiting the growth of others, are of great additional value, but can only properly be used secondarily in order to augment the results obtained by nonselective culture methods. As the most suitable medium for the purpose in hand plain meat infusion agar, titrating 0.1+ to 0.3+ to phenolphthalein, to which 5 per cent of sterile defibrinated horse blood was added, was used. Since growth on freshly poured plates is greatly superior to that on plates that have been stored, the agar was melted as needed, the blood being added when the medium had cooled to approximately 45° C. Cultures from the nose and throat were made by swabbing the mucous membranes with a sterile applicator, touching the applicator to a small area on the surface of a blood agar plate, and spreading the inoculum over the surface of the medium with a platinum needle, insuring as wide a separation as possible. Direct cultures of selected and washed specimens of sputum were made when possible. In many instances, of course, it was impossible to get sufficiently satisfactory specimens to permit of washing, especially when cultures were made very early in the disease. To supplement direct culture of the sputum the mouse inoculation method as employed for the determination of pneumococcus types was used. This is, of course, a highly selective method, of particular value in the detection of pneumococcus and B. influenzæ when they are present in relatively small numbers as compared with other bacteria. Plates were examined after twenty to twenty-four hours’ incubation and again at the end of thirty-six to forty-eight hours when necessary.
In the present study, attention has been centered upon B. influenzæ, S. hemolyticus, and the various immunologic types of pneumococci, other organisms encountered having played no significant part in the cases studied except in rare instances. B. influenzæ was identified by its morphologic, staining and cultural characteristics and conformed to the classical description given by Pfeiffer. S. hemolyticus was identified by its morphologic, staining, and cultural characteristics on blood agar, supplemented by a confirmatory hemolytic test with washed sheep corpuscles, and bile solubility test. Pneumococci were identified by morphologic, staining and cultural characteristics, bile solubility test, and agglutination with specific antipneumococcus immune sera. Note was made in most instances of the presence of other organisms, such as members of the Gram-negative diplococcus, staphylococcus, diphtheroid and streptococcus viridans groups, but no attempt was made further to isolate or identify them.
Bacillus Influenzæ in Cases of Influenza.—On October 10, 1918, at the height of the epidemic at Camp Pike, search for B. influenzæ was made in a group of 23 consecutive cases of uncomplicated influenza from one to six days after the onset of the disease. From each individual simultaneous cultures on blood agar plates were made (a) from the nose, (b) from the throat, and (c) from the sputum, and the sputum from each case was injected into the peritoneal cavity of a white mouse. A similar study of 5 consecutive cases was made on November 19. The results are presented in Table II.
By means of multiple cultures taken simultaneously from different portions of the respiratory tract no difficulty was encountered in demonstrating B. influenzæ in all these cases of uncomplicated influenza. Not only was B. influenzæ found in all cases, but often in very large numbers predominating over all other bacteria on at least one of the plates from each patient, and in occasional instances occurring in nearly pure culture. One culture made about two hours after onset of the initial coryza is of interest. There was at the time a profuse serous nasal discharge. One drop of this allowed to fall on the surface of a blood agar plate gave a practically pure culture of B. influenzæ.
| Table II | |||||
|---|---|---|---|---|---|
| Presence of B. Influenzæ in 28 Cases of Influenza | |||||
| NO. | DAY OF DISEASE | NOSE | THROAT | SPUTUM CULTURE | SPUTUM PASSED THROUGH MOUSE |
| 1 | 1 | + | + | + | + |
| 2 | 4 | − | + | + | + |
| 3 | 5 | − | − | + | − |
| 4 | 4 | − | − | + | + |
| 5 | 3 | − | − | + | + |
| 6 | 4 | − | + | + | c |
| 7 | 2 | − | + | − | c |
| 8 | 4 | + | + | + | − |
| 9 | 5 | − | + | + | + |
| 10 | 2 | + | − | − | − |
| 11 | 2 | − | + | c | + |
| 12 | 3 | c | + | + | + |
| 13 | 3 | − | − | − | + |
| 14 | 2 | − | − | + | + |
| 15 | 3 | c | − | − | + |
| 16 | 1 | − | + | + | + |
| 17 | 3 | − | + | − | + |
| 18 | 4 | + | + | c | + |
| 19 | 6 | − | − | + | + |
| 20 | 1 | − | + | + | + |
| 21 | 2 | − | + | − | + |
| 22 | 4 | + | − | + | + |
| 23 | 3 | c | − | − | + |
| 24 | 2 | + | − | − | − |
| 25 | 1 | − | − | + | + |
| 26 | 5 | − | − | + | + |
| 27 | ? | − | + | − | + |
| 28 | 1 | − | − | + | + |
| 6 | 14 | 17 | 22 | ||
| c indicates that the plate was contaminated. |
During the latter part of November and in early December a small secondary wave of influenza occurred at Camp Pike. In a series of 48 consecutive cases, B. influenzæ was readily found in all by means of combined throat cultures and mouse inoculation of the sputum, 33 times (68.7 per cent) in the throat cultures, 39 times (81.3 per cent) in the sputum. These cases were cultured on admission to the receiving ward of the hospital within twenty-four to forty-eight hours after onset and were all early cases of influenza without complications at the time the cultures were made. In 90 more consecutive cases in this series 62 or 68.9 per cent showed B. influenzæ in a single throat culture taken on admission.
A summary of all cultures made in cases of uncomplicated influenza is presented in Table III.
| Table III | |||
|---|---|---|---|
| Presence of B. Influenzæ in Cases of Influenza | |||
| METHOD | NUMBER OF CASES CULTURED | B. INFLUENZÆ FOUND | |
| NUMBER | PER CENT | ||
| Nose culture | 28 | 6 | 21.4 |
| Throat culture | 166 | 109 | 65.7 |
| Sputum culture | 28 | 17 | 60.7 |
| Sputum (mouse passage) | 76 | 61 | 80.3 |
| Combined nose, throat and sputum cultures and sputum inoculation | 28 | 28 | 100 |
| Combined throat cultures and sputum inoculation | 48 | 48 | 100 |
Of any single method used the intraperitoneal inoculation of a white mouse with a specimen of the patient’s sputum proved the most efficient in demonstrating the presence of B. influenzæ. No single method served to demonstrate B. influenzæ in all cases, but by simultaneous cultures from the nose, throat, and deeper air passages no difficulty was met in showing that B. influenzæ was invariably present, usually in abundance somewhere in the respiratory tract during the acute stage of the disease. This result is not out of harmony with the rapidly progressive character of the attack upon the mucous membranes of the respiratory tract in influenza.
Of interest in this connection are certain observations which suggest that the presence of B. influenzæ in predominant numbers at least is in many cases coincident with the acute stage of influenza and that the organisms show a tendency rapidly to diminish in abundance with the progress of the disease to recovery. In 82 cases of influenza cultured on the day of admission to the hospital, B. influenzæ was present in 52 (63.4 per cent) of the throat cultures. Repeated throat cultures in this group of cases from the fourth to the eighth day after admission when the temperature had fallen to normal, showed that B. influenzæ was still present in demonstrable numbers in the throat of only 25 cases or 30.5 per cent. Not only was there a material reduction in the number of patients in whom B. influenzæ could be demonstrated by the throat culture method, but the contrast in the predominance of B. influenzæ on the plates made early in the disease with those made during convalescence was often very striking. It is only fair to say, however, that some cases continued to carry B. influenzæ in their throats in large numbers throughout the period of observation.
Presence of Pneumococcus in Cases of Influenza.—It seemed of some importance to determine the prevalence of pneumococcus in cases of influenza, not because of any possibility that pneumococci might bear an etiologic relationship to the disease, but more by way of comparison with the prevalence of B. influenzæ, since both organisms are found in the mouths of normal individuals and are also frequently found together in the pneumonias that complicate influenza. The results obtained in cases of influenza early in the disease before the development of either a purulent bronchitis or of pneumonia are presented. The presence of pneumococcus was determined by the intraperitoneal inoculation of white mice with the saliva or sputum.
Twenty-four cases examined on September 27 and 28 gave the results shown in Table IV. These patients had been in the hospital from two to five days at the time the determinations were made.
| Table IV | ||
|---|---|---|
| Pneumococcus in Cases of Influenza | ||
| NUMBER | PER CENT | |
| Pneumococcus, Type I | 0 | 0 |
| Pneumococcus, Type II | 0 | 0 |
| Pneumococcus, Atypical II | 0 | 0 |
| Pneumococcus, Type III | 2 | 8.3 |
| Pneumococcus, Group IV | 15 | 62.5 |
| No pneumococci found | 7 | 29.2 |
From November 27 to December 1, the pneumococci present in 47 consecutive cases of influenza were determined. In this group specimens of sputum were collected shortly after admission of the patients to the receiving ward of the hospital. The results are shown in Table V.
| Table V | ||
|---|---|---|
| Pneumococci in Cases of Influenza | ||
| NUMBER | PER CENT | |
| Pneumococcus, Type I | 0 | 0 |
| Pneumococcus, Type II | 0 | 0 |
| Pneumococcus, Atypical II | 2 | 4.3 |
| Pneumococcus, Type III | 0 | 0 |
| Pneumococcus, Group IV | 25 | 53.2 |
| No pneumococci found | 20 | 42.5 |
The results obtained show that pneumococci found in early uncomplicated cases of influenza, both early and late in the course of the epidemic, differ in no respect from those found in the mouths of normal individuals at any time.
Similar studies of the prevalence of S. hemolyticus as determined by throat cultures in early cases of influenza are shown in Table VI.
The only point of interest in these observations is the increased prevalence of S. hemolyticus in cases examined late in the epidemic of influenza as compared with that found early in the epidemic. The significance of this will be discussed in other parts of this report.
| Table VI | ||||
|---|---|---|---|---|
| S. Hemolyticus in Cases of Influenza | ||||
| DATE | NUMBER OF CASES CULTURED | S. HEMOLYTICUS FOUND | S. HEMOLYTICUS NOT FOUND | PER CENT POSITIVE FOR S. HEMOLYTICUS |
| Sept. 25–26 | 100 | 6 | 94 | 6 |
| Nov. 27–Dec. 5 | 138 | 39 | 99 | 28.3 |
Presence of Bacillus Influenzæ in Normal Men.—For comparison with the results obtained in cases of influenza a fairly extensive study of the prevalence of B. influenzæ in normal individuals has been made at various times prior to and throughout the course of the epidemic. This was deemed of special importance, since it was obvious that the results obtained by previous workers during interepidemic periods would not in all probability coincide with those obtained in the presence of a widespread epidemic of influenza where the opportunity for the dissemination of B. influenzæ was almost unlimited.
From the results obtained in the multiple cultures in cases of influenza it is obvious that only like methods can be compared. The results obtained in normal individuals have, therefore, been tabulated in groups dependent upon the culture method employed. These groups have been subdivided according to the time and the place of the study, such explanatory notes as seem necessary being added. (See Tables VII-IX.)
The most striking feature of the figures presented in Table VII is the wide variation in the incidence of B. influenzæ in different groups varying all the way from 11.1 to 68 per cent. Analysis of these differences brings out certain points of great interest. It is apparent that the percentage of cases carrying B. influenzæ depended in large part upon the prevalence of respiratory diseases in the group from which the data were obtained. In the studies made at Camp Funston prior to the fall outbreak of influenza in epidemic proportions, it is noteworthy that “bronchitis” and pneumonia were prevalent throughout the summer in those groups showing a relatively high incidence of B. influenzæ. At the time these studies were made the presence of influenza in these organizations was not recognized, but in view of knowledge gained throughout the course of the epidemic at Camp Pike, it seems not improbable that influenza in mild form was present throughout the summer in certain organizations at Camp Funston. This would seem more likely in view of the fact that this commission has clearly demonstrated that a considerable epidemic of influenza swept through Camp Funston in March, 1918, and was followed by recurring smaller epidemics in April and May.21 In contrast with these groups showing a high incidence of B. influenzæ is that of the 210th Engineers, an organization entirely free from respiratory diseases during the period of our study.
| Table VII | ||||||
|---|---|---|---|---|---|---|
| Incidence of B. Influenzæ in Normal Men as Determined by Intraperitoneal Inoculation of White Mice With Saliva or Sputum | ||||||
| DATE | PLACE | ORGANIZATION | NUMBER EXAMINED | B. INFLUENZÆ PRESENT | PER CENT POSITIVE FOR B. INFLUENZÆ | REMARKS |
| 1918 | ||||||
| Aug. 13 | Camp Funston, Kans. Detention Camp, No. 2 | 22 Prov. Colored Co. 164th Depot Brigade | 25 | 6 | 24 | Bronchitis and pneumonia were prevalent in this organization of recently drafted negroes during July and August, 1918 |
| Aug. 18 | Camp Funston, Kans. Detention Camp, No. 2 | Co. D. 3rd Dev. Bn. | 25 | 11 | 44 | Recently drafted southern negroes not fit for full military duty. Bronchitis and pneumonia were prevalent in this organization during July and August, 1918 |
| Aug. 20 | Camp Funston, Kan. | 70th Inf. | 25 | 11 | 44 | 25 men presenting themselves at sick call for various complaints; not strictly normal; respiratory diseases not prevalent |
| Aug. 22 | Ft. Riley, Kan. | Quarters 4 M M.O.T.C. | 32 | 16 | 50 | Recently drafted white men of 4 to 8 weeks’ service. Pneumonia fairly prevalent in this organization |
| Aug. 26 | Camp Funston, Kan. | 210th Eng. | 27 | 3 | 11.1 | About one mile distant from Camp Funston proper. No sickness in this organization |
| Nov. 12 | Hot Springs, Ark. | Drafted men assembled to entrain for camp | 50 | 11 | 22 | 50 men selected from isolated farm communities; 12 gave a history of “influenza” within the preceding 8 weeks |
| Nov. 25 | Camp Pike, Ark. | Miscellaneous | 26 | 13 | 50 | 12 of this group had influenza during the epidemic |
| Dec. 10 | Camp Pike, Ark. | Miscellaneous | 25 | 17 | 68 | 12 of this group had influenza during the epidemic |
| Summary: | Normals | 235 | 88 | 37.4 | ||
| Cases of influenza (for comparison) | 76 | 61 | 80.3 |
On November 12 search was made for B. influenzæ in 50 normal drafted men who had assembled at Hot Springs, Ark., on that date preparatory to entraining for Camp Pike. These men were all from isolated farming communities where influenza was only moderately prevalent and where there was little opportunity for the wide dissemination of B. influenzæ such as occurs when large bodies of men are assembled in camps. Twelve of the 50 gave a history of influenza within the preceding eight weeks. The cultures were made by the same methods as those used at Camp Pike, the laboratory car “Lister” being taken to Hot Springs for that purpose. The incidence of B. influenzæ was only 22 per cent. In striking contrast with this figure are the figures of 50 and 68 per cent obtained in the last two groups studied at Camp Pike after the epidemic had swept through the camp: 24 of the 51 men in these groups had influenza during the epidemic.
It is of interest to record that the incidence of pneumococcus in these cases was approximately the same in all groups and bore no relation to the prevalence of influenza, bronchitis, or pneumonia.
| Table VIII | ||||||
|---|---|---|---|---|---|---|
| Incidence of B. Influenzæ in Normal Men as Determined by Throat Cultures on Blood Agar Plates | ||||||
| DATE | PLACE | ORGANIZATION | NUMBER EXAMINED | B. INFLUENZÆ PRESENT | PER CENT POSITIVE FOR B. INFLUENZÆ | REMARKS |
| Sept. 14–Oct. 5 | Camp Pike, Ark. | Med. Detachment, Base Hos.; personnel on measles wards | 82 | 14 | 17.1 | 82 throat cultures in 42 individuals |
| Nov. 5–9 | Camp Pike, Ark. | Miscellaneous | 296 | 71 | 23.9 | Number among this group who had had influenza not recorded |
| Nov. 12 | Hot Springs, Ark. | Drafted men assembled to entrain for camp | 64 | 0 | 0 | Men, in large part from isolated farm communities; 13 gave a history of “influenza” within the preceding 8 weeks |
| Nov. 25 | Camp Pike | Miscellaneous | 26 | 13 | 50 | 12 of this group had influenza during the epidemic |
| Dec. 10 | Camp Pike | Miscellaneous | 25 | 13 | 52 | 12 of this group had influenza during the epidemic |
| Summary | Normals | 493 | 111 | 22.5 | ||
| Cases of influenza (for comparison) | 166 | 109 | 65.7 |
The results obtained by throat culture are quite similar to those obtained by the mouse inoculation method. The entire absence of B. influenzæ in the group of 64 throat cultures made in the draft men assembled at Hot Springs as compared with the relatively high incidence in the last two groups examined at Camp Pike is very striking.
In consideration of the figures presented in Table IX it is important to remember that the group of 50 men from Hot Springs were all from isolated farm communities, had not previously been assembled and had not been in continuous contact with a widespread epidemic of influenza. On the other hand, the two groups of normal men at Camp Pike were studied immediately after the epidemic had swept through the camp and had been constantly in contact with epidemic influenza for a period of three months, 24 of the 51 actually having had the disease during this period. The fact that in the group of men from Hot Springs, B. influenzæ was found only by the mouse inoculation method is noteworthy, since it indicates that the organism was present in relatively small numbers and could be detected only by a highly selective method.
