═══════════════════════════╤═══════════╤═══════════╤═══════════
DILUTION OF SERUM │ + │ +– │ –
───────────────┬───────────┼───────────┼───────────┼───────────
Convalescents │1-1 │ 3│ 0│ 2
│1-10 │ 5│ 2│ 7
│1-40 │ 2│ 3│ 9
│1-80 │ 0│ 1│ 13
│1-160 │ 0│ 0│ 14
───────────────┼───────────┼───────────┼───────────┼───────────
Normal Controls│1-10 │ 1│ 2│ 0
│1-40 │ 0│ 1│ 2
───────────────┴───────────┴───────────┴───────────┴───────────
The complete agglutination as would be indicated by +++ or ++ was
not seen.
Agglutination tests were carried out with the sera of 14 convalescents
and 3 normal individuals. A polyvalent emulsion of strains of the
influenza bacillus isolated from our cases was used. The results are
shown in Table III. Tubes were incubated at 37.5° C. The results did not
indicate anything in the nature of a specific reaction. Dr. Frost
carried out this work during the height of the epidemic, but we were
unable to continue it further. A short review of the work of others will
be found near the end of this paper. Miss Thompson and Mr. Mock studied
complement fixation, using the sera of 15 convalescents against an
antigen of B. influenzæ. Their results were negative. The antigen
appeared to be slightly more anti-complementary than were emulsions of
staphylococcus or B. coli. Huntoon also noted this anti-complementary
character of emulsions of B. influenzæ.
Attempts were made to estimate the amount of complement present in the
fresh blood serum of influenza patients. The technique was to use a 1-4
dilution of the patient’s serum, adding measured amounts of this to a 1
per cent. blood emulsion, with 1 unit of amboceptor and determine the
smallest amount necessary to bring about complete hemolysis. This test
was carried out on eight patients ill for only a few days. The average
amount of the dilute serum was 0.181 c.cm. Fifteen patients,
convalescent after a moderate illness, gave an average of 0.276 c.cm.
Two patients seriously ill with temperatures of 104.3° F. and 105° F.
required 0.4 c.cm. to bring about complete hemolysis. We would not like
to draw any very definite conclusions where we are dealing with such
small fractional differences. This lessening of complement has been
noted in other infectious diseases and may be important in the questions
of immunity in influenza. Dr. Frost carried out a number of cutaneous
tests after the method of Von Pirquet, using a polyvalent, weakly
alkaline emulsion of influenza bacilli in 25 per cent. glycerin. Eleven
convalescents were tested and none of them showed any local or general
reaction. The suggestion that these results may indicate an increase in
resistance is discussed in another place. A number of strains of
pneumococci which we had isolated from our autopsy cases were
differentiated by the agglutination method. Type I was found 3 times;
type II, 10 times; type IV, 9 times. Four showed agglutination with both
type I and type II sera. Type IV pneumococcus was isolated in one case
from the right and left bronchus as well as the lung. In another case
the same type pneumococcus was recovered from the lung and pleural
fluid. These results are similar to those found by numerous workers.
_The Hemophilic Bacteria_
The discovery by Pfeiffer of the hemophilic character of the bacillus
found by him in cases of influenza opened up a new group of
micro-organisms known as the hemophilic bacteria. Davis (1915) has laid
particular stress on the group character of these bacilli, and the more
they are studied the more clear does it become that there are several
distinct members. The B. influenzæ is by far the most important as well
as the most frequently found of the group and is considered as the type
organism.
All these bacteria require for their growth the presence of some form of
hemoglobin. The actual amount necessary may be very small, and Davis
suggested that it may have a catalytic action. A great deal of work has
been done in attempts to discover just what portions of the hemoglobin
are necessary to bring about this phenomenon. In our discussion on media
for the influenza bacillus we will briefly describe some of the various
hemoglobin preparations that have been used successfully. It must at
this point be emphasized that blood is very useful in many media to
stimulate the growth of a great variety of bacteria, and the transfers
made from such luxuriantly growing cultures may grow very poorly or not
at all on ordinary media, and this might easily lead to erroneous
conclusions on the hemophilic character of the organisms studied. There
are certain bacteria which grow so much better on media containing blood
that such media are sometimes necessary for their isolation, although
after a few transfers they will grow on ordinary media. This is true for
bacillus pertussis, and throughout the literature a good deal of
confusion has arisen in not recognizing this temporary hemophilic
character of certain bacteria. The true hemophilic bacteria do not grow
except in the presence of hemoglobin in some form or other. The problem
becomes almost academic when we consider the small amounts of hemoglobin
that are necessary. Davis has shown that a dilution of 1 in 180,000 is
sufficient, and in the interesting discussion between Cantani and Ghon
and Preyss it was demonstrated that hematin or other hemoglobin product
was necessary in the agar before B. influenzæ would grow in the presence
of other bacteria, and that this hematin could be derived from the blood
in the meat which was used in making the basic infusion.
_Symbiosis._—The fact that other bacteria can bring to growth the
influenza bacillus on media otherwise unsuited to its needs brings up
the interesting problem of symbiosis, which is one of the most important
characters of the influenza bacillus. Not only do other bacteria make
possible the growth of B. influenzæ on media on which the influenza
bacillus will not grow, but they stimulate a better growth on blood agar
and other more or less favorable media. Grassberger first noted this
stimulating character of other bacteria and described and illustrated
the very large colonies of B. influenzæ which develop in the
neighborhood of colonies of staphylococcus and other bacteria.
Staphylococci killed by heat were found to have a similar effect.
Meunier nicely described this phenomenon by using the term satellites
for the circles of B. influenzæ colonies which develop about the
colonies of other bacteria. A great number of workers have since noted
this characteristic relationship between B. influenzæ and other
bacteria, and occasionally have laid stress on its importance in the
problems of the infections by the influenza bacillus. Allen particularly
emphasized the probable importance of this in discussing the problem of
carriers of B. influenzæ as sources of danger. There seems no doubt that
this symbiotic relationship depends on so altering the hemoglobin
products as to render them more readily available for the influenza
bacillus. This is indicated by the fact that on various media containing
hemoglobin, altered so that it encourages the growth of B. influenzæ, no
such symbiotic stimulation can be demonstrated. This phenomenon is quite
peculiar to this bacillus, distinguishes it from most of the other
members of the group, and should be always determined before an organism
is classed as B. influenzæ.
_Other Hemophilic Bacteria._—The question of a pseudo-influenza bacillus
was first raised by Pfeiffer and has been studied by many workers after
him. Grassberger, who carefully investigated this problem, worked more
particularly with two strains showing the extreme of variation between
the small characteristic morphology of the B. influenzæ and the thread
forms supposed to be characteristic of the so-called pseudo-influenza
bacillus. The great majority of workers have agreed with him in
concluding that this morphological variation is not sufficient nor
constant enough to justify separating two such groups. Nevertheless many
reports indicate peculiar tendencies of certain strains toward thread
formation. There seems to be suggestive evidence that the organism
described by Cohen in 1909 under the name B. meningitidis
cerebrospinales septicemicus is different from true B. influenzæ.
Although the cultural characters were apparently identical, this
organism was definitely pathogenic for guinea pigs and rabbits. The
involvement of joints in the cases reported by Longo and others would
suggest a greater pathogenic power for these strains. Prasek and Zatelli
reported a similar bacillus from meningitis, and Davis found that his
meningitis strains were more pathogenic for rabbits than were others.
Wollstein has studied this question very carefully and found a marked
difference between the strains from the meninges and those from the
respiratory tract in their pathogenicity for rabbits. The strains with a
tendency to thread formation were usually also those grown from the
meninges, but she concluded from the results of serological tests that
all strains of B. influenzæ are of one race, irrespective of their
origin or virulence. The question is still an open one, as Batten and
others described strains from the meninges which are non-pathogenic, and
Ritchie found his strains from meningitis pathogenic for guinea pigs but
not for rabbits. The irregularity and wide divergence in the results of
blood cultures may have a definite relationship to these differences in
the pathogenicity of strains.
Other hemophilic bacteria include the bacillus described by Friedberger
under the name of B. hemoglobinophilus canis. This organism is to be
found in the preputial secretion of dogs. It does not show the
phenomenon of symbiosis, and I have found that it grows rather more
freely and is more resistant to drying than is the influenza bacillus.
Krage has confirmed Friedberger’s findings growing this bacillus from 60
per cent. of his dogs, and believed it a pyogenic organism just as B.
influenzæ may be.
The hemophilic and hemolytic organisms described by Davis, which he
isolated from pathological urine, were non-symbiotic and non-pathogenic.
Koch has described a similar organism from puerperal infection. Whether
the hemophilic organism described by Thalhimer from the uterus in a case
of puerperal infection, those found by Cohen in urethral discharge in
one case and the pelvic exudate of another, and the findings of Kretz in
pyelitis, Wright in pyelonephrosis and Klieneberger in cystitis cases,
possibly refer to this same bacillus is, of course, uncertain. Pritchett
and Stillman found a somewhat similar bacillus, which they called
Bacillus X, from the mouths of 24 persons. It was hemophilic and
hemolytic, stouter than B. influenzæ and showed long tangled threads in
blood broth. It was non-pathogenic and is probably the same as Davis’
organism.
Davis described another hemophilic bacillus from a patient with purulent
foci which was non-hemolytic and non-symbiotic. It was grown from an
abscess of the shoulder joint, the blood and the bronchial secretion of
an infant. Cyanosis was a marked feature of this case. Paranhos
described a hemophilic bacillus from meningitis, which, however, was
Gram positive, and Moon reported an anærobic hemophilic bacillus from an
infection of the ethmoid sinus. The work of Jordan would suggest that
there may be two groups of B. influenzæ based on the indol production.
_Morphology._—The morphology of B. influenzæ has received more than
usual attention. In what we consider its characteristic form, it is an
extremely small bacillus, usually single but sometimes in pairs, and not
infrequently exhibiting polar staining. In direct smears, where there
are many bacteria present, they are frequently arranged in the schools
so frequently described. The development of thread forms is today
considered quite characteristic for B. influenzæ. The organisms vary
from moderately long bacillary forms to very long twisted or curled
threads suggesting leptothrix. In such cultures chains of tiny bacilli
are also quite often noted. At the other extreme we have exceedingly
tiny coccoid forms, resembling in size the B. bronchisepticus, which, as
Ferry has shown, are small enough to pass through many grades of
filters.
It is the thread forms, as discussed above, that have received most
attention in relation to the so-called pseudo-influenza bacillus. The
observations of Wollstein, Lacy and many others showed these forms to be
common in meningeal infections and that, as a rule, they are more
pathogenic for animals than other strains. Another interesting and
important observation is that emphasized by Dick and Murray of the
possible confusion of these forms with Gram negative leptothrix. That
this confusion is liable to occur is illustrated by reports such as
Macdonald finding leptothrix in a meningeal infection, now looked upon
as an example of influenzal meningitis, and the probable B. influenzæ
reported by Dick, and, as quoted by Dick and Murray, the finding of a
Gram negative leptothrix as the cause of broncho-pneumonia by Kato. The
2 per cent. leptothrix reported by Nuzum and his co-workers from the
recent epidemic may be still another example. Equally important is the
recognition of the great frequency of this thread development in the
majority of B. influenzæ cultures on ordinary blood agar media, or even
in the water of condensation of fresh blood agar tubes. The delayed
growth of this bacillus on ordinary blood agar would lead to its being
frequently overlooked unless smears are made, and the irregular thread
forms are recognized as being the B. influenzæ. This development of
thread forms was particularly noted in my work before pickings were made
to the Voges heated blood agar, but because I had been forewarned by
discussing these morphological variations with Lacy, I was able to
recognize them as forms of B. influenzæ. Most of my early isolations
showed these predominating, and they were also noticed in cultures sent
from the Public Health Laboratory at Washington. These cultures on
further transfer, however, showed in 24 hours the typical small form on
ordinary blood agar as well as on the Voges medium. On the latter the
development of thread forms was greatly delayed and frequently did not
appear at all, although after long periods other abnormal, swollen and
irregular shapes sometimes developed.
_Media in Growth of B. Influenzæ_
The discovery of the hemophilic character of B. influenzæ has been
confirmed by a long list of investigators. The agar smeared with pigeon
blood as used by Pfeiffer has not, however, been found fully
satisfactory and many modifications have been made. The fact that
hemoglobin in some form is necessary for the growth of these bacteria
has led to a great deal of study in attempts to discover the chemical
part, or parts, essential for this purpose. Hemoglobin in very small
amount, as shown by Davis and others, is sufficient to make media
suitable for growing B. influenzæ. This fact has led to much confusion,
owing to the difficulty of eliminating all possible sources from which
some form of hemoglobin might enter the media. Kitasato used a glycerin
agar and succeeded in growing the influenza bacillus for 10 transfers.
Pielicke, however, did not consider that Kitasato was actually dealing
with the influenza bacillus, but that he as well as Babes, Bruschettini
and Markel had most probably streptococci in their cultures. Besson held
the same view of Kitasato’s organism. It would further appear from the
illustrations of Klein that he also grew streptococci and not the B.
influenzæ. The first culture of the influenza bacillus was probably
obtained by Bujiwid in February, 1890. He grew on agar smeared with the
spleen pulp of an influenzal patient a tiny bacillus which he was unable
to grow on blood free medium, but he did not appreciate its importance
until Pfeiffer’s article appeared. Teissier in his book on “L’ Influenza
en Russie” mentioned this culture.
The hemophilic character of these bacteria indicates that they are
rather strict parasites, and despite the researches of Nastjukoff with
various egg media, and Cantani with a number of supposedly
non-hemoglobin additions to the agar, as well as the studies on
symbiosis, with other bacteria, by Cantani, Neisser, Luerssen and many
others, it still remains true that some form of hemoglobin is necessary
for their growth. Fresh blood either incorporated in the medium or
smeared on the surface is not the best medium for these bacteria.
Altered hemoglobin is much more favorable, and a variety of methods have
been devised to bring about those alterations which stimulate the growth
of B. influenzæ. One of the earliest, as well as one of the very best,
of these is the method of Voges, who added blood to melted agar at a
temperature of about 100° C. I have found this medium exceptionally
suited to growing B. influenzæ, and I consider it excellent for the
primary culture from the original material, for pickings from plates and
to obtain a heavy growth of B. influenzæ for any purpose. The medium was
used by Delius and Kolle (1897), Grassberger (1898), who spoke very
highly of it, and Paltauf (1899), who said that the use of this medium
made the demonstration of B. influenzæ possible when only a very few
were present. A great many other workers have used it with success, and
during the recent epidemic it has gradually found its place. Levinthal’s
medium (1918) is practically the same, although he boiled and filtered
the agar after the addition of the blood. The growth of B. influenzæ on
the Voges agar can properly be described as luxuriant, and to anyone
only accustomed to the use of ordinary blood agar it is an agreeable
surprise to see this supposedly delicate bacillus growing so remarkably
well.
Various other methods have been used to bring about this beneficial
change in hemoglobin. Gioelli (1896) used a medium made up of 1.1 per
cent. hemoglobin and 21.5 per cent. malt extract. This is reddish brown
in color, becomes clear when neutralized with potassium hydrate and
remains so on heating. This added to agar is reported as very favorable
in growing this bacillus. Ghon and Preyss described a medium made up of
meat, peptone, salt and agar prepared in the ordinary way, but not
filtered for at least a week, and then only roughly. This medium is
favorable for symbiotic growths. He further used beef blood heated in a
soda solution and blood heated in water as hemoglobin preparations to be
added to agar. Thalhimer found an amorphous hemoglobin medium to be more
favorable than when a purer hemoglobin was used. W. F. Robertson found a
hemoglobin agar, prepared by allowing sheep’s blood to clot, decanting
off most of the serum, freezing and then thawing what remains and adding
1 c.c. of this to an agar tube at about 60° C., to be very favorable for
the growth of B. influenzæ. Cantani used a blood treated with pepsin and
hydrochloric acid, digested some days in the incubator, filtered and
made weakly alkaline. This mixture was heated for a few minutes,
refiltered and added to the medium. He speaks of it as extraordinarily
good for B. influenzæ. Blood treated with trypsin has been used by
Matthews, Averill, Young and Griffiths, Harris, A. Fleming and others.
Fleming further found that this alteration in hemoglobin can be brought
about in a number of other ways. Blood boiled in agar (suggesting the
Voges agar) and the tubes slanted while hot, blood boiled in water, the
clotted blood precipitated and the clear fluid added to agar, or more
rapidly by adding equal quantities of sulphuric acid to the blood and a
similar amount of potassium hydrate he obtained altered blood suitable
for media. He reported that by any of these methods he could obtain a
medium very stimulating to the growth of B. influenzæ. By the addition
of brilliant green (1 in 500,000) he inhibited the growth of
staphylococcus, streptococcus and pneumococcus. For storing cultures of
B. influenzæ Fleming found a minced meat medium with the addition of
blood to be the best. I have found this medium without the blood to be
an excellent one for keeping a great variety of cultures. Bernstein and
Loewe have reported the use of gentian violet (1 in 5,000) for the same
purpose as the brilliant green used by Fleming. Avery’s oleate blood
agar medium he reported to be largely selective. It checked the growth
of pneumococci and streptococci, but gave luxuriant growths of B.
influenzæ. Pritchett and Stillman have used it with excellent results
recovering B. influenzæ from a very high percentage of the cases
studied.
The use of symbiotic bacteria has been extensively studied in
investigations of the biology of B. influenzæ, and it has been shown, as
noted elsewhere, that such accessory bacteria will bring to growth B.
influenzæ on media otherwise quite unsuited to its needs. It has been
further found that on various preparations of hematin agar, on which B.
influenzæ refused to grow, such media could be rendered favorable for
their growth by the addition of living or freshly killed cultures of
staphylococcus and many other bacteria. And although the method is well
known, it has not been extensively used for the purposes of isolation.
Many of the workers, however, have pointed out the importance of looking
for growth of the influenza bacillus in the neighborhood of the more
easily grown bacteria which almost always develop in cultures from the
respiratory tract. Grassberger has particularly studied this problem and
has made practical application of the method. Accidental contamination
of plates with air bacteria have made possible, in some instances, the
isolation of B. influenzæ—as, for example, in the finding of Heyrovsky
from a case of empyema of the gall bladder—while other workers have
pointed out the difficulty of demonstrating growth where B. influenzæ is
pure in the material cultured, and the comparative ease and relative
luxuriance of growth where other bacteria are present. To just what this
stimulating effect is due has been much discussed, and it is generally
agreed that the hemoglobin is markedly changed and rendered more
available by the action of these germs. It is to be noted that on a
medium containing blood altered by heating or by the various methods as
described by Fleming the foreign bacteria no longer show any symbiotic
action on B. influenzæ. Grassberger considered the effect of the
bacteria on the blood to be the same as that of heating. Allen laid
particular stress on this symbiotic character. He used a staphylococcus,
either living or killed, in making his cultures and noted the difficulty
of growing B. influenzæ from material in which it occurred pure. W. F.
Robertson made use of these facts of symbiosis for both isolation and
stimulation of growth. He employed alternate drills of M. catarrhalis or
pneumococcus with the B. influenzæ, and Brown and Orcutt used strains of
hemolytic streptococci for the same purpose. The latter authors
considered that the beneficial effect of the streptococci was merely due
to the setting free of the hemoglobin. The fact that similar results are
to be obtained by the use of non-hemolytic bacteria as well as forms
giving green color changes to the blood makes this explanation
untenable. In my own studies I have confirmed the results of several
previous workers. I have found that B. influenzæ is stimulated in its
growth by the presence near it of colonies of staphylococcus pyogenes
aureus and albus, pneumococci, streptococcus viridans and hemolyticus
and other bacteria. The largest colonies of the bacillus I have obtained
were those growing near the periphery of a colony of an air nocardia. I
have also noted that emulsions of a staphylococcus killed by boiling for
five minutes, when added to ordinary blood agar, had a marked
stimulating effect, although no evidence of hemolysis was present. This
effect was practically absent if the emulsion was boiled for 15 minutes,
or after being killed was left at room temperature for several days.
There was no evidence of these stimulating effects by any of these
methods when heated blood agar was used, the colonies on this medium
growing equally large by themselves. Comparative studies of the effect
of different bacteria can be simply carried out as follows: Smear evenly
the surface of an ordinary blood agar plate with an emulsion of B.
influenzæ. Seed this plate at various points with minimal amounts of the
various bacteria. After various periods of incubation the size of the B.
influenzæ colonies about the other bacterial growths can be estimated,
and impression preparations on cover glasses will give very interesting
pictures.
The growth of B. influenzæ in primary cultures from sputa and similar
sources is to be explained by the probable presence of traces of blood
or altered hemoglobin as well as the symbiotic relationship with other
bacteria. Fichtner used fresh heated sputum (60 to 65° C.) in place of
blood, and Richter a medium made with sterilized pus. Parker, in her
study of a filterable poison produced by the B. influenzæ, found veal
infusion broth with 10 per cent. defibrinated blood heated to 75° C.
until the blood coagulated and settled on standing to be the best for
the purpose. Jordan in his study of indol production by these bacteria
used a meat infusion broth with 5 per cent. sheep’s blood added at 90°
C. or over and filtered through cotton or paper. Wittingham and Sims
noted that in using blood from influenza cases the bacteria frequently
did not grow, more especially B. influenzæ; and Rivers found human blood
poorer than cat or rabbit blood for growing this organism, as did
Minaker and Irvine. It would seem clear from this review of some of the
suggestive work on the methods of growing B. influenzæ that little
attention should be given to the results of many workers, where ordinary
media were used, particularly when the difficulties of isolation were
not appreciated.
_B. Influenzæ as a Pathogenic Bacterium_
If B. influenzæ is the causative agent in clinical influenza, there is
certainly ample evidence that it is pathogenic to man. The symptoms of
toxemia, which are so manifest in the pandemic disease as well as in the
sporadic cases, would indicate that the etiological agent is markedly
toxicogenic. Animal experiments by Pfeiffer, and a long list of
investigators following him, would seem to show that the majority of
cultures of B. influenzæ do not have any power of establishing
themselves in the animal tissue. Killed cultures showed equally as high
toxic effects as the living, and so it was generally concluded that many
of the general effects in influenzal infections were of a toxic nature.
There are many exceptions to the above-mentioned failures to produce
infections in animals. Cantani obtained very constant positive results
by subdural injections. He first clearly showed that killed cultures
were markedly toxic and that virulence could be raised very definitely
by animal passage. By injecting brain emulsion with a culture he
obtained a subcutaneous abscess in a rabbit which after eight days still
contained the living organism. Nastjukoff found that animals with a
lowered resistance, or definitely ill from, for example, an artificial
tuberculosis, became infected while others did not. Jacobson showed that
B. influenzæ injected with streptococci caused a definite mixed
infection, and that after six passages the influenza bacillus alone
could produce a fatal infection. Saathoff (1907) confirmed Jacobson’s
findings and found pneumococci equally effective. Davis (1915) also
confirmed the principle established by Jacobson of the symbiotic
relation of other bacteria to infection with B. influenzæ. He used a
culture of a non-virulent staphylococcus pyogenes aureus, and was able
to produce death invariably in guinea pigs after intraperitoneal
injection. From the heart’s blood, as a rule, only the hemophilic
bacillus was recovered. He also found animal passage increased the
virulence, and further that M. catarrhalis and an avirulent
streptococcus had the same effect as the staphylococcus. Slatineanu
(1901) found that he could infect animals with B. influenzæ if the
cultures were injected along with weak solutions of lactic acid, and
that after animal passage by this method the bacillus became more
virulent and would eventually kill by itself. It must not be forgotten
in this connection that strains of B. influenzæ from meningitis cases
are frequently definitely pathogenic for animals. The importance of
considering these various factors in a discussion of infection by this
organism is, of course, very evident. Ecker found his strains pathogenic
for mice after subcutaneous injection, and the bacilli were readily
obtained from the heart’s blood. Spooner and his co-workers from their
results of more than a hundred intraperitoneal injections concluded that
the organism is not pathogenic for mice.
In all animal experiments it is of the greatest importance that the
bacteria be known which may interfere in the experiments through
spontaneous infection (often liable to be induced by the injection) from
the animal’s own flora, as well as the greater susceptibility of
previously diseased animals (Nastjukoff). It would appear from the
results of Bruschettini and Cornil and Chantemesse in the early days of
the influenza bacillus, and those of Lamb and Brannin in their recent
study, that these authors did not seriously consider the spontaneous
infection of guinea pigs and rabbits with B. bronchisepticus or the
bacillus of rabbit septicæmia, both morphologically, very similar to B.
influenzæ. Rosenow in his experiments with streptococci from cases of
influenza has also apparently failed to realize the importance of the
lung lesions produced by the B. bronchisepticus in guinea pigs as
reported by Theobald Smith, myself and many others.
Parker has found a filterable poison from the influenza bacillus which
developed rapidly (6 to 8 hours) in a special heated blood broth medium,
deteriorated rapidly even in the cold, and killed rabbits in quantities
of 2 c.c. in from 1 to 3 hours. Rabbits could further be immunized
against this poison, and their sera protected other rabbits against
fatal doses. This is the first time that a true powerful toxine has been
obtained. Couret and Herbert obtained toxine from B. influenzæ in
Avery’s oleate broth. Huntoon and Ross also clearly demonstrated toxine
production by this organism so that it would appear, with this
confirmation, that the B. influenzæ can be definitely classed among the
toxine producers. Toxemia being the most striking clinical
characteristic of influenza, we have in these findings very strong
evidence of the etiological importance of this hemophilic bacillus to
the disease. A very interesting observation was made by Latapie that the
serum of a goat immunized against influenza bacillus is toxic if it is
used shortly after the injection of the microbes, but that this toxicity
is absent three weeks after the last injection. It would appear to me
that the evidence of a filterable virus from the secretions of the
respiratory tract does not eliminate the very probable toxine from such
materials. The production of toxine by this organism probably depends,
as is the case with very many of our toxine formers, on the most
favorable combinations of conditions. That it is not readily formed in
artificial cultures, or that it is very unstable if formed, is evidenced
by the frequent failures of a great many workers. It has been suggested
that different symbiotic conditions in the respiratory tract determine
the amount of toxine produced. Huntoon found a high toxine production in
mixed cultures with streptococci. This, however, does not appear to be
necessary, as there is ample evidence of severe toxemia from pure
infections with B. influenzæ in various parts, such as the accessory
sinuses of the head, the meninges, the lungs and other parts of the
respiratory tract.
It is not fundamentally necessary that a toxine producing organism be
present in overwhelming numbers before it can be accepted as the cause
of the toxemia. Nor, on the other hand, must we have toxemia every time
the organism is found. The prevalent idea among bacteriologists would
appear to be the reverse of what I have just stated. It would, indeed,
be extremely difficult to make bacteriological diagnoses of a great many
of our diseases, where the etiological factor is well established, if
these conditions were required. We do not do so, for example, in
diphtheria, examinations of stools for typhoid, nor in infections with
the tetanus bacillus. We recognize carrier cases of meningococcus, B.
typhosus, hemolytic streptococci and many others, without detracting
seriously from their importance in definite types of infection. Formerly
the specificity of the different bacteria for definite disease processes
was very rigid, but today we interpret more broadly the finding of
gonococcus in endocarditis, the meningococcus in bacteremia, B. typhosus
in osteomyelitis, streptococci and pneumococci in all manner of
infections and many other bacteriological results. True it is that the
various bacteria show predilections for attacking certain tissues, but
the varying susceptibilities bring about the greatest variations in the
manifestations of these infections.
The B. influenzæ is not confined to the causation of severe pandemic or
epidemic influenza, but includes in its field purulent bronchitis,
meningitis, sinusitis, conjunctivitis and many other pathological
processes. It further should be recognized as a relatively frequent
cause of complications in measles and other diseases.
_Infections of the Respiratory Tract_
The disease influenza is primarily an infection of the respiratory
tract. It varies from one of the most acute and fatal diseases we know
of through all grades of severity—from chronic infections lasting over
years to the familiar three or five day fever. This graduation is to be
found more or less marked in all our bacterial infections, but would
seem to be not generally recognized or appreciated as occurring in
infections with the influenza bacillus. That Pfeiffer was dealing with
one phase of the disease when the influenza bacillus was discovered does
not invalidate the results of numerous workers which have been added
since then.
Probably the greatest confusion in attempts to get a clear picture of
this protean disease has been and is a non-recognition of influenza as a
frequent complication of other diseases, such as measles (Jochmann,
Susswein, Tedesko and very many others). The second cause for this
confusion has been the misinterpretation of the facts demonstrating the
rather frequent occurrence of carriers. During an epidemic the vast
majority of patients show the disease as an upper respiratory infection
of varying degrees of intensity, but which usually subsides after
periods of from three to five days of fever. Along with this we have
other graded manifestations of further involvement of the tract with
laryngitis, bronchitis, bronchiolitis and all degrees of
broncho-pneumonia. To prevent the severe lung involvement prompt
treatment must be carried out, under which rest in bed is by long odds
the most important. This will be discussed in another paper of this
series, and was particularly well demonstrated in the results at the
Naval Hospital as verbally reported to me by D. G. Richey. The
interesting point is that the infection can be controlled, but this does
not indicate the etiological factor as different from that acting in the
more severe cases.
The epidemiological evidence would seem to show very clearly that the
incubation period is approximately two days, and that a period of six
weeks is the usual limit for the severe wave of the epidemic in
different localities. In my opinion, during this period every exposed
individual in a community has received the influenza bacillus in the
respiratory tract, and that all the susceptible individuals are attacked
and show more or less evidence of the infection. As a consequence of
this general distribution we have great numbers of individuals carrying
the organism, and the aftermath is to be noted in other and later
manifestations of the same infection.
Sporadic cases of influenza appear during inter-epidemic periods and
more or less healthy carriers are frequent. Scheller’s study in
Königsberg showed, if we can rely on his figures, that the carriers were
very numerous during an epidemic year (winter 1906-1907), being 24 to 33
per cent.; that as the epidemic became less widespread (winter
1907-1908) it fell to 10 to 13 per cent.; as it was disappearing (summer
1908) he found only 1.5 to 3.3 per cent.; while when the epidemic was
completely over (winter 1908-1909) there were no carriers of B.
influenzæ found. These results are taken from studies of sputa and
throat smears of 138, 218, 155 and 185 cases, respectively, for the
periods mentioned. The monumental work of Tedesko, who reported the
results of 1,479 cultures, covering 11 years (1896-1906), would indicate
that B. influenzæ is continually present in the population. However, in
carefully analyzing his results, it is very clear that in the great
majority of his cases it was of definite etiological significance.
Lobular pneumonia, acute, purulent and chronic bronchitis, and most
frequently clinical influenza, are the prominent diagnoses in all his
tables. He was able to grow B. influenzæ repeatedly from individual
patients for many months.
Lord in similar studies (1902, 1905, 1908) brought out somewhat similar
facts. He laid particular stress on the cases of chronic bronchitis with
numerous B. influenzæ in the sputum and a probable confusion of these
with pulmonary tuberculosis. He was able to follow a number of his
patients for several years. B. influenzæ was grown in culture from the
sputum of one of these in 1902; in November, 1903; in February, 1904,
and in February, 1905. In other cases the organism was shown to be
present by culture practically continuously for months and even years.
Lord, with Scott and Nye, in a recently published article (1919)
reviewed his former results and showed a relatively high incidence of B.
influenzæ in the respiratory tract of apparently healthy people. Davis
studied 534 cases, further indicating the prevalence of this organism in
the community.
The B. influenzæ has been recovered from the respiratory tract during
the clinically pure influenza, from the sputum and lung in influenzal
pneumonia, and from the purulent sputum in all grades of bronchitis.
These should all be looked upon as true infections by the influenza
bacillus, the varying manifestations merely differing with the
resistance of the individual. In the epidemic in the fall of 1918
pneumonia was the outstanding feature. Preceding this in the English
publications we have reports of outbreaks of purulent bronchitis.
Macdonald and his co-workers, finding the B. influenzæ frequently
present, considered the condition as one indication of a virulent
infection by this organism. Hammond, Rolland and Shore reported similar
cases, and Abrahams and his co-workers looked upon the cases of purulent
bronchitis as occupying a position, without any definite line of
demarcation, between those with definite broncho-pneumonia on the one
side and those with simple bronchial catarrh on the other. H. E.
Robertson emphasized the serious nature of influenzal purulent
bronchitis and the almost epidemic character and rather high mortality
of the outbreak in the winter and spring of 1917-1918. There were also
numerous mild outbreaks of influenza before the overwhelming culmination
of the last three months of 1918, as reported by Orticoni and many
others and noted by Johnston in this series of papers. Greenwood in an
epidemiological study emphasized the point, previously made evident by
Parsons for the pandemic of 1889-1892, that the mass attack is preceded
by numbers of individual cases. In this country it was noted during the
winter of 1917-1918 and the following spring that the B. influenzæ was
rather frequently found in the respiratory infection in our army camps
(Soper, Cole and MacCallum and others).
It is well recognized that when the actual epidemic struck there were
comparatively few bacteriologists familiar with the B. influenzæ. The
real difficulties of isolation, the more favorable media, the facts of
symbiosis, the importance of carriers, the varying manifestations of the
infection and many of the other vitally important points, although more
or less fully reported in the literature, were nevertheless practically
unknown. It was my own experience, and that of many others. This must be
seriously considered in analyzing many of the reports on bacteriological
findings throughout the period of the severe wave and even after.
_Results of Others During the Recent Pandemic_
It will be impossible to review the numerous reports on the recent
epidemic that have appeared. Many of these can be discounted, as far as
the finding of B. influenzæ is concerned, for the reasons mentioned
above. The often quoted report of Little, Garofalo and Williams, who did
not even use a hemoglobin medium, will serve as an example. Little
attention should be given to others where the large numbers of cases
precluded the requisite time and media necessary for such a difficult
problem. Friedlander and his co-workers in their report from Camp
Sherman made no mention of the number of sputa, throat swabs or
autopsies which they examined bacteriologically. The incidence of
influenza showed a total of 10,979 cases, 2,001 of pulmonary œdema or
pneumonia and 842 deaths. They recorded one culture from the sputum with
pneumococcus predominating which gave two colonies of B. influenzæ, and
this bacillus was grown from the lung exudate at one autopsy. Their
conclusions that “B. influenzæ (Pfeiffer) has not been demonstrated as
the causative organism” is certainly true from their results, but that
“the frequency of its detection has not exceeded the frequency of its
existence under normal conditions” can hardly be considered as
established, if we accept the many results mentioned above as indicating
its presence during inter-epidemic times, unless they mean by normal
conditions practically complete freedom from this organism.
The prevalence of B. influenzæ in various sections of this country may
be indicated by the following reports chosen from many available ones.
Keegan, from the First Naval District Hospital, found B. influenzæ 19
times from 23 in cultures grown from the lungs. In 6 cases these
cultures were pure. Medalia reported from Camp McArthur the following.
Out of 2,279 sputa of influenza suspects, 76.8 per cent. showed “B.
influenzæ” in smears, and 445 sputa from cases of broncho-pneumonia
showed it in 54 per cent. It was found in culture in only 10.6 per cent.
of these last cases. He considered sputum smears of practical diagnostic
help. He further grew B. influenzæ twice from the blood during life,
once with a pneumococcus and once alone. Necropsy cultures gave B.
influenzæ in 2 of 3 cultures from the brain, 19 of 34 from the heart, 19
of 36 from the spleen, 54 of 65 from both lungs, 50 of 62 from the right
pleura and 47 of 62 from the left pleura. The percentage of positive
results ranged from 53 in the spleen to 83 in the lungs. Nuzum and his
associates only found B. influenzæ in 4 of 100 cases from the bronchial
secretions, but it is interesting to note that he grew it in practically
pure culture from both lungs of one case at autopsy. Synnott and Clark
in Camp Dix found streptococci and pneumococci predominating, and,
although making no particular effort to study the B. influenzæ or
determine its frequency, they found it in the majority of cases when it
was looked for. Blanton and Irons reported as follows from Camp Custer.
From cultures of the nose and throat of 357 examined before the epidemic
struck, B. influenzæ was found in 5.1 per cent.; in 366 throat cultures
of influenza cases without physical signs of pneumonia the same organism
was grown in 44, or 8 per cent.; sputa typed for pneumococci 740 times
from influenza cases with pneumonia gave isolations of B. influenzæ 38
times, or 5 per cent.—8 times alone, but here it should be remarked that
these latter isolations were only attempted after the organism was
suspected from the morphological picture of the smears; from 280
autopsies B. influenzæ was recovered 8 times from the lung and 3 times
from the heart’s blood. This report covered the period from the outbreak
of the epidemic, October 5 (or as given by Soper, September 30) to
October 22, at the outside a period of 22 days. During this time 366
throat cultures, 510 blood cultures, 740 sputa typed for pneumococci,
280 autopsies with cultures from both lung and heart’s blood, made a
total of primary cultures of well over 2,000. The technical difficulties
would make it almost impossible to handle such a mass of material and
get reliable results for the incidence of B. influenzæ.
Brem, Bolling and Casper in Camp Fremont found B. influenzæ in 259 from
537 selected cases in swabs from the nasopharynx. It was also noted in a
fair number of other examinations. Opie and his co-workers found B.
influenzæ to be very frequent at Camp Pike. Spooner, Scott and Heath
isolated B. influenzæ at Camp Devens from the sputa of 104 cases, from
nasopharyngeal swabs in 11 out of 18 attempts and from the pleural fluid
8 times out of 45, twice pure. From 37 autopsies they found B. influenzæ
in 23 and in pure culture in at least 1 lobe of the lung in 16. From 82
blood cultures at autopsy B. influenzæ was recovered twice. Nichols and
Stimmel studied lung punctures during life and grew the B. influenzæ
from 7 out of 10 attempts, 5 times in pure culture. Stone and Swift at
Fort Riley found B. influenzæ in 18.7 per cent. of 928 sputa and in 5.2
per cent. of 77 sputa from fatal cases. He recovered it from autopsy
material; 21 times from 51 lungs, once alone; twice from 26 pleural
fluids; twice from 30 heart bloods; 19 times from the sinuses of 40, and
9 times from the ear and mastoid of 17 cases.
Lamb and Brannin at Camp Cody examined 80 typical cases early in the
epidemic. They found B. influenzæ predominated in 46 per cent. being
present with pneumococci on 41 per cent. of the plates. They also grew
the influenza bacillus from a fair number of other cases.
Wollstein and Goldbloom in the Babies Hospital of the City of New York
found the B. influenzæ in 13 of 17 sputa during life and in both lungs
of all 18 autopsies as well as in the heart’s blood of one. Kotz found
it in half of his 30 cases. Pritchett and Stillman grew the influenza
bacillus from 41 of 49 cases of influenza, from 40 of 43 cases of
influenza with broncho-pneumonia, from all of six other
broncho-pneumonia cases and from 11 of 20 cases of lobar pneumonia,
making a total of 98 positive findings from 118 or 82 per cent. They
further found 25 positives from 54 convalescent and 74 from 177 normal
sputa. Wolbach found this organism in pure culture in one or more lobes
of the lungs of 9 from 23 cultured cases. It was demonstrated in 23 of
28 either by culture or in section.
Similar results are to be found in reports from Great Britain. Martin
noted a great increase in the numbers present as the sputum became more
purulent. Hicks and Gray found B. influenzæ by culture in 75 per cent.
of their cases. They were seen in direct smears in only 70 per cent.
Gotch and Wittingham considered M. catarrhalis to be the etiological
factor as it was found in all of their 50 cases. B. influenzæ was grown
in 8 per cent., although B. influenzæ-like bacilli, were seen in 62 per
cent. of their smears. Averill, Young and Griffiths studied the sputum
from 41 cases and found B. influenzæ in 32. It is interesting that
Macdonald and Lyth determined the incubation period to be 41 hours as a
minimum in their own experience and that from the posterior nares of one
of them B. influenzæ was obtained.
Schofield and Cynn found the B. influenzæ in Korea. Kraus in Brazil
found it in the sputum in 62 per cent. of his cases of influenza. It was
also found in the organs of 27 who had died, being in pure culture in
five. It has further been found in France, Italy and practically all
parts of the world where investigations have been made. The German
literature is at present only available in the report of the British
Medical Research Committee which is written in a more or less popular
manner with a rather strong tendency against the importance of B.
influenzæ. Dietrich, Simmonds, Bergmann and others, however, found B.
influenzæ rather frequently. Such quotations as “Uhlenhuth, a diehard of
bacteriologic orthodoxy, has clearly shown signs of uneasiness” and “one
empyema and one throat swab yielded the looked for growth” will indicate
why this review is of little use. It is certainly necessary to “look
for” the B. influenzæ to get results of any worth.
Secondary, ancillary or symbiotic bacteria are of cardinal importance in
these infections. It has been considered by some writers as
characteristic for the influenza bacillus to be followed so frequently
with such a variety of secondary invaders. Sahli looked upon the complex
of B. influenzæ, pneumococcus and streptococcus as the true etiological
cause of influenza. Abrahams and his associates discussed the symbiotic
effect of the B. influenzæ in raising the virulence of pneumococci
previously present in the patient and many other investigators lay
stress on these symbiotic relationships.
Pneumococci appear to be the commonest of these secondary
micro-organisms judging from the various published reports, but the fact
must not be overlooked that, particularly in America, the typing of
pneumococci has drawn a disproportionate attention to this group.
Hemolytic streptococci have received much attention (Ely and his
co-workers and several others). M. catarrhalis (Gotch and Wittingham and
several of the British writers), members of the B. mucosus capsulatus
group (Nichols and Stimmel, Rucker and Wenner), staphylococcus aureus
(Patrick), various ill-defined streptococci (Rosenow and several British
writers), capsulated cocci apparently different from pneumococci, B.
pestislike forms and many others have been given more or less attention,
often as clearly recognized secondary infections, but not infrequently
as of primary significance.
B. influenzæ, however, is the organism most regularly found in this
pandemic where carefully looked for, and the evidence of its lowering
the general resistance to bacterial invasion is very strong. The
experiments of Ghedini and Fedeli showing the effect of the toxine on
muscular tone and those of Ghedini and Breccia who found a similar
effect on blood vessels are worthy of note.
The fact that the flora differs so widely in various regions is what one
might expect and many investigators have emphasized the significance of
this. Bacteria in the mouth and throat are readily transmitted from
individual to individual and under the conditions in the training camps
and our modern life, the development of local flora is not surprising.
That it is of very great importance is recognized by all and it is often
a determining factor in the severity of the infection. Nevertheless,
influenza in this pandemic has been almost equally severe whatever the
secondary organism may have been.
I have discussed in another place the suggestion of the stimulating
effect of various bacteria on the growth and toxine production of B.
influenzæ. Huntoon showed the effect of hemolytic streptococci in
cultures to be helpful in toxine production. An important point,
however, is that no one bacterium has been shown to be exclusive in thus
affecting the growth on media of the influenza bacillus, and in the
animal experiments in raising the invasive and pathogenic power of this
organism the same appears to be true. The infection in influenza, in the
vast majority of cases, rapidly becomes a mixed one. The secondary
organisms at times completely dominating the field, at least as far as
numbers go, most frequently invade the blood stream and it would appear
often play the important role in many of the secondary conditions.
_Chronic Infections_
B. influenzæ is a frequent finding in the sputum of patients with
chronic bronchitis, pulmonary tuberculosis and other chronic conditions
in the respiratory tract. Boggs recovered this bacillus from two cases
of bronchiectasis, Richards and Gurd had a similar case and Tedesko
reported several. The literature is filled with references to the
finding of B. influenzæ in cases of chronic bronchitis. Those reported
by Lord, Madison and Tedesko quoted above will serve as examples. The
frequent positive cultures in cases of pulmonary tuberculosis so often
referred to in reviews of the literature and the significance of these
findings, as pointed out by Scheller, are important as bearing on the
much debated subject of the effect of influenza on this disease. These
types of chronic infection by the influenza bacillus should be more
generally recognized as they undoubtedly will become more numerous
following this last epidemic if we can judge from the experience of the
past.
_Infections of the Pleura_
The recovery of B. influenzæ from the pleural cavity is not uncommon as
is shown in the above review. The findings of MacCallum, Cole and others
during the spring of 1918 are particularly interesting. Beall in 1906
reported a case of empyema with large quantities of green pus in which
B. influenzæ was found in pure culture.
_Sinuses of the Head_
Infection of the accessory sinuses of the head has long been recognized
as occurring in influenza. Frankel found B. influenzæ in 4 from 40
infected antra. Lindenthal, who was particularly interested in the
question of sporadic influenza, found the bacillus in one or more of the
head sinuses in six of eight carefully studied cases. He considered that
the B. influenzæ remained in these areas during inter-epidemic times and
from hence caused the sporadic outbreaks of influenza. Howard and
Ingersoll reviewed the literature up to 1898 and grew B. influenzæ from
one of three acute antral diseases. They did not find it, however, in 12
chronic cases. Clemens believed the influenza bacillus to be present in
the sinuses rather frequently in cases where it was overgrown or
difficult to culture from the lower respiratory secretions. Moszkowski
grew it in one case from the pus of the antrum. Tedesko recorded several
positive results and many others are reported in the literature.
The two cases reported by Lacy (1918), the findings during the present
epidemic by Stone and Swift of B. influenzæ in 13 of 28 sphenoidal and 6
of 12 ethmoidal sinuses cultured at necropsy, those by Spooner, Scott
and Heath, of B. influenzæ in four frontal sinuses and in eight
sphenoidal, and the recovery by Wolbach of B. influenzæ in cultures from
the sinuses in certain cases where the lung cultures were negative,
emphasize the importance and frequency of the infection by this organism
in these cavities. Keegan, who laid particular stress on lung punctures
and autopsy examinations, pointed out that in throat cultures the
probability that the influenza focus is often not in the pharynx but in
some recess of the nasal cavity.
H. E. Robertson in the spring of 1918 reported the infection of the
sinuses in seven cases of tracheo-bronchitis with patches of
broncho-pneumonia and the growth of B. influenzæ from sphenoid, ethmoid
or frontal sinuses of all these cases. He also found this organism in
the sphenoid of six cases dying with various diseases as well as in two
accident cases with death under 24 hours. The importance of these
results was laid stress on by the author, not only on account of the
probable toxic absorption and the general menace of spread, but, more
particularly, because such individuals, acting as carriers, could
furnish foci for the spread of epidemics.
_Eye and Ear_
Infections of the eye by the influenza bacillus are quite common. This
subject is fully discussed by Axenfeld (text-book, “The Bacteriology of
the Eye”). Giani and Picchi found it in the eye in 66 per cent. of
influenza cases, in 90 per cent. of epidemic conjunctivitis, and in the
normal eye of 5.8 per cent. Wynekoop, in 1903, reported having found
this organism in cases of conjunctivitis in 1899. Guiral, in the recent
epidemic, found influenza bacillus constantly present in the secretions
in cases of what seemed to be Week’s conjunctivitis. Ulceration of the
cornea was rather common. One such case is mentioned in which there was
no pain in the eyes, but general symptoms of influenza. The middle ear
is also sometimes infected. Between the report of Kossel in 1893 and
that of Stone and Swift in 1918, who found the middle ear and mastoid to
contain B. influenzæ in 8 of 17 cases, there have been many references
in the literature to this complication by the influenza bacillus. The
evidence indicates, however, that in the middle ear, as in the pleural
cavity, the secondary bacteria are far more often the important ones.
_Meninges_
Influenzal meningitis seems to stand by itself as a manifestation of the
pathogenic effects of B. influenzæ. The literature is too voluminous to
review in this place, but the evidence would seem to point to a more
invasive and pathogenic type of this organism, if not to a separate
member of the group.
_Invasion of the Blood Stream_
The evidence in clinical influenza would suggest at times a bacteremia
in addition to the severe toxemia, which is such a constant feature of
the disease. Simultaneously with the discovery of B. influenzæ, Canon
reported finding bacilli of similar morphology in blood smears, but was
unable to grow them, and it would appear at least doubtful that he was
dealing with the influenza bacillus. Meunier is probably the first who
grew this organism from the blood. He recovered it from 8 blood cultures
out of 10 in cases of broncho-pneumonia following measles, and in one
other case of broncho-pneumonia. A very full discussion of this question
is to be found in Canon’s book on “The Bacteriology of the Blood in
Infectious Diseases.” Of particular interest are the results of Ghedini,
who made a careful study of 28 influenza patients. B. influenzæ was
grown from the blood in 18 of these at the height of the fever, while in
the 10 negative cases the disease was milder or the blood was taken only
after the temperature had fallen. The amount of blood used was 20-30
c.c., and it was cultured in lecithin broth. In practically all of his
cases several cultures were taken, and in a number of the positive cases
negative results were obtained both before and after the acme of the
fever. He also grew the bacillus from 8 of 14 spleen punctures of these
patients. Madison (1910) reported the recovery of this bacillus from the
blood of a patient with a primary broncho-pneumonia who recovered. This
author also used about 30 c.c. of blood. Thursfield, in 1910, also
reported two cases of B. influenzæ bacteremia in which the organisms
were recovered at the height of the temperature. One had influenza, the
other phlebitis, and both recovered. Tedesko and several others have
found it in the heart’s blood in many cases, more especially in
broncho-pneumonia after measles.
During the present epidemic the positive cultures of this bacillus from
the blood have been rather infrequent. J. S. Fleming had 2; 2 are quoted
in the report of the Influenza Committee of the Advisory Board to the D.
G. M. S. (Peters and Cookson); Medalia had 2 during life and 19 of 34 at
autopsy; Orticoni, Barbie and Leclerc in 5 of 10 blood cultures in one
series, and 7 of 19 in another; Stone and Swift 2 at autopsy; McKeekin,
in Australia, influenza-like bacilli in 4; Blanton and Irons three times
in the heart’s blood, one of these pure; Spooner, Scott and Heath twice
in the heart’s blood at autopsy, and Wollstein and Goldbloom from the
heart’s blood in one child. In the majority of these findings the
bacillus was not found in pure culture. Abrahams and his associates
found the B. influenzæ along with a pneumococcus and M. catarrhalis from
the heart’s blood in one case. In our positive blood culture there was
evidence of the same mixture being present.
Before drawing sweeping conclusions against the invasion of the blood by
B. influenzæ it must be remembered that the quantity of blood used has
been generally only about 10 c.c., and often much less, the difficulty
of observing growth if the culture is pure has been largely overlooked,
the use of more favorable media than blood agar and the possible
inhibitory action of influenzal blood, as suggested by Wittingham and
Sims, Rivers and others, has not been considered, and further that
sufficient care has not been exercised to obtain blood at the most
favorable period in the disease. It may be recalled that the problem is
quite similar to that of demonstrating the organisms in the blood in
patients with streptococcus viridans bacteremia.
All the available evidence, however, points to the invasion of the blood
in influenzal infections as being a very fleeting one. Unless this is
true, it would be surprising in the many hundreds of blood cultures
which have been taken in the concentrated study of patients during the
recent pandemic, if more successful cultures had not been obtained.
General infections with localization of B. influenzæ in different parts
of the body are here of interest—such as that reported by Slawyk and
others. Whether the strains causing meningitis, and which apparently
more frequently invade the blood, are really different members of the
hemophilic group or only forms with a higher invasive power is still, I
believe, an open question.
_Endocarditis_
In endocarditis the B. influenzæ is probably, after streptococci, the
organism most frequently isolated from the blood. Rosenthal from heart’s
blood at autopsy, Schlangenhaufer, Jehle two cases, Horder (1907) six
cases, and who believed he was the first to isolate B. influenzæ from
the blood, Tedesko in a number at autopsy, Spat, F. J. Smith, Saathoff,
Libman four cases, Sacquepee, McPhedran, Mann, Rainaford and Warren
three cultures from two patients, and a number of others all bear
witness to its frequency.
Other organs of the body are sometimes found to contain B. influenzæ.
Adrian, Schultes, Basile and Tedesko have all recovered this organism
from the diseased appendix. Several years ago a bacillus, considered, to
be B. influenzæ, was grown from the pus of an appendix abscess in our
laboratories. Wright found it in pyelonephrosis. Klieneberger found
influenza-like bacilli in cases of cystitis. Menko reported the bacillus
from orchitis, and Cohn found numerous influenza-like bacilli in the
discharge from urethritis. Meunier found it in pure culture in a case of
osteoperiostitis. Huyghe, Besancon and Griffon recovered it from
infected joints, as did Pacchioni in a general infection. Weil found it
in the pus about the hip joint one month after an attack of influenza.
This short review serves to illustrate that the influenza bacillus,
although generally limited to infections in the respiratory tract, is,
nevertheless, capable of infecting other parts.
_Immunity—Phagocytosis_
Phagocytosis of the B. influenzæ has been very frequently noted in the
study of sputum smears. It has been observed, moreover, that this
phenomenon occurs most frequently when the patient is on the road to
recovery (Pfeiffer, Martin, and others), and it may indicate an
important reaction on the part of the body to this organism. Tunnicliff
in a recent report, however, did not find the opsonic index to be raised
above the normal in her patients, and Tunnicliff and Davis had
difficulty with a spontaneous phagocytosis of this bacillus. This
difficulty was to a large extent absent in her later study.
_Agglutination_
Agglutination tests have been used by many investigators in attempts to
determine a specific reaction in the sera of persons suffering from
influenza. Such reactions develop, as we know, against secondary
infecting bacteria, so that unqualified conclusions cannot be drawn that
agglutinins in the sera of patients against B. influenzæ indicate the
etiological importance of this organism. Vagedes using a dilution of
1-50 found 8 positives among 27 patients tested. Lord found the test
most inconstant. Ghedini obtained useful results by using serum in
dilutions 1-20 to 1-30, and had 17 positives from 28 influenza cases. He
found agglutinins present three to four days after the height of the
infection, and noted that the sera became practically normal after three
to four weeks. Fichtner, although he obtained agglutination with sera of
influenza patients in high dilutions (1-100 and 1-750), found his
controls were often agglutinated, and consequently drew no conclusions.
Wollstein (1906) did a series of agglutination tests, using various
strains of B. influenzæ. The sera of patients she found very
unsatisfactory, but by immunizing rabbits with this organism she
obtained sera with titres up to 1 in 400. She could find no differences
among the various strains studied. Somewhat similar results were
obtained by her in 1915 working with strains from the meninges and the
respiratory tract. Odaira carried out a rather extensive series of
tests, using immunized rabbit sera and a special method of making his
bacterial emulsions. He was able to distinguish B. influenzæ from both
B. pertussis and the so-called Cohen’s bacillus of meningitis.
Friedberger’s dog bacillus, however, could not be differentiated from B.
influenzæ by this means. A. Fleming during the recent epidemic had good
results with the sera of 21 patients. He incubated at 50° C. for two
hours. He also used sera of immunized rabbits and got marked
agglutination against the homologous strain, but varying results with
other strains. He noted some strains agglutinated readily, while others
did not. Eyre and Lowe noted an increase in agglutinins in the sera of
people vaccinated against the influenza bacillus. Couret and Herbert
could distinguish two types and a possible third among their strains.
Park and his co-workers found numerous types by means of agglutination.
Absorption of agglutinins was found helpful by these last two workers.
There are so many factors capable of altering the sensitiveness of
bacteria to agglutination, as in the well-known experiments of Neufeld,
that we must recognize that much work is still to be done before we can
properly interpret the results of these agglutination tests.
_Binding of Complement_
Complement fixation tests were carried out by Odaira but his results
were much less satisfactory than those he obtained by means of
agglutination. Rapaport made an extensive study of this test, using the
sera of patients in various stages of convalescence. Three hundred and
fifteen convalescents showed 54.5 per cent. positive while 300 controls
only gave 9.5 per cent. positive results. Most of the positive cases
were in patients three to five days after their illness, but the
reaction was found in convalescents after from 1 to 45 days. Sera from
acutely ill patients at times showed negative or slightly positive
reactions but these same sera after keeping for some days and retesting
often gave strongly positive results. This would appear to be a
promising field for investigation.
_Anaphylaxis_
Hypersensitiveness was noted by W. F. Robertson in chronic infections
with B. influenzæ. Wollacott in a letter to the British Medical Journal
suggested that the severity of the recent outbreak of influenza may
possibly be due to the development of a state of anaphylaxis. There
would seem to be at least some evidence in favor of such a view in the
fact that the severe outbreak was preceded by epidemics of a milder form
of influenza and that the influenza bacillus was probably widely spread
during this time. Greenwood, as quoted above, noted that primary cases
always precede the mass attack. Of course, the term anaphylaxis has been
used to explain almost everything. Nevertheless, the theory is
interesting. The skin tests which we did for hypersensitiveness were, as
I have noted above, negative but there is a possibility that the failure
of the reaction may indicate a higher resistance or even an antitoxin,
now that the bacillus can be classed as a toxicogenic one.
Anti-influenza sera have been produced by a few investigators (Latapie,
Wollstein) but have not found any practical application during this
pandemic. Vaccination is discussed elsewhere in these studies.
_Experiments on the Human_
There has never been in the history of medicine so many experiments on
human beings as have been carried out in the attempts to discover the
etiological factor in the recent pandemic of influenza. Davis has called
attention to a successful human inoculation with pure cultures of B.
influenzæ which he performed in 1906. During the present investigation
at least 200 men have volunteered as experimental subjects, and the
results of many different methods of attempting to transmit the disease,
have been disappointing and inconclusive. I will not attempt to review
the reports at present available, as a great deal of the work done has
not yet appeared in print. The important point is that the results do
not affect the various views held as to the causative agent in pandemic
influenza nor the massive evidence for transmission of the disease under
natural epidemic conditions.
It is my opinion, as expressed above, that practically all of the
population are rapidly infected during such a pandemic as we have had.
The resistant have escaped, and it would appear to be very difficult to
break down this resistance. The human experiment carried out by
Pettenkofer on himself and his assistant with vibrion choleræ is an
example, but we have numerous others demonstrating the same kind of
phenomena in most of our diseases of established bacterial origin. In
diphtheria we have an explanation in the varying antitoxic content of
the sera, but we really know very little of what are the actual factors
in preventing or determining infection among exposed individuals in the
natural history of most diseases. The reports of Leonard Hill and Gregor
are well worth reading in this connection, as well as the editorial in
the same number of the British Medical Journal. We are not in a position
to be very dogmatic on the causes of epidemics. The mere presence of the
bacteria or any other living virus is not in itself sufficient to
explain the phenomenon, and one of the chief objects of this paper is to
indicate from the collected facts, that in the words of Flexner, “the
case against the influenza bacillus is not proved.”
_Conclusions_
1. B. influenzæ is one of a group of hemophilic bacteria and there are
probably strains of this organism which may be differentiated which will
lead to further subdivisions of the group.
2. B. influenzæ as we understand it today, is distinguished by its
morphological and staining characters; its requiring hemoglobin in some
form for its development; its showing symbiotic reactions with other
bacteria which stimulate its growth; the production of a toxine and its
usual low pathogenicity for animals.
3. The media found most favorable for its growth are those containing
blood with the hemoglobin content altered in certain ways, (1) by
heating, (2) the addition of various chemicals, (3) by the action of
other bacteria or their products. The heated blood agar I have found to
be a most efficient and readily prepared medium.
4. Since B. influenzæ is so difficult to isolate, it is necessary to be
very cautious in interpreting results unless the greatest effort has
been made to demonstrate the presence of this organism.
5. B. influenzæ should be considered, from the evidence at hand, as the
bacterial causative agent in epidemic influenza, and it should be
recognized that secondary infections following the primary attack by
this organism are both frequent and important. This view I believe the
logical one, unless much more convincing evidence than we have today may
demonstrate another more probable living virus as the cause.
6. B. influenzæ is a frequent etiological factor in purulent and chronic
bronchitis, broncho-pneumonia and other acute and chronic respiratory
infections, in meningitis, endocarditis, sinusitis, conjunctivitis and
other conditions, as well as in complications of many other diseases.
7. There are many carriers of the bacillus among our population, both in
apparently normal individuals and in those suffering from chronic
infections of bronchi, sinuses or other parts.
8. The problem of what constitutes resistance or susceptibility to this
infection are as far from solution as they are in most other respiratory
diseases, and the attempts to explain the reasons for epidemics have
been as futile as they are for meningitis and many other respiratory
epidemics.
9. It would not appear that the immunological reaction against this
infection has been discovered, but the possibility of its being of an
antitoxic nature opens an interesting field for investigation.