AN INVESTIGATION CONCERNING DOHLES LEUCO-

CYTIC '' INCLUSION BODIES IN SCARLET ))

FEVER AND OTHER D1SEASES.l

By WILLIAMMACEWEN, M.B., Ch.B. (alas.), D.P.H. (Cantab.),
Assistant Physician, Ruchill Fe?jei.Hospital, Glasgow.
" INCLUSION BODIES,"occurring in the polymorpho-nuclear leucocytes
of the blood of patients suffering from scarlet fever, were first described
by Dohle (191lS1) in November 1911, and were later confirmed by
Kretschmer (1912 ') in March 1 9 1 2 . Since then much work has
been done by many workers, not only in confirming the observations
of Dohle and Kretschmer, but in carrying the investigations further,
so as to include Inany other diseases.
The so-called inclusion bodies are found in the cytoplasni of the
polymorpho-nuclear cells, generally situated near the periphery of the
cell. Their shape varies-bacillary, coccoid, diplococcal, streptococcal,
pyriform, annular, and amorphous fornis being found. I n size also
they vary greatly,-sometimes being little larger than the neutrophil
granules, and a t others filling a considerable portion of the cell. The
number of inclusions found in one cell likewise is very variable,
depending upon the severity of the case, but from one to four is a
usual number. They are not present in all the polymorphs, but in
scarlet fever about 70 per cent. of these cells contain them, depending
upon the type of infection and severity of the case.
Extra-cellular forms of inclusion bodies can occasionally be seen,
although some writers have denied their presence. These extra-
cellular forms are sometimes to be found lying quite free, and a t others
attached to the periphery of the polymorphs. Bodies resembling
inclusions are also found in specimens of pus taken from scarlet
fever patients.
I n addition to the various forms of inclusions already mentioned,
Dohle describes a spirochztal form, which he has named the Spiyochceta
scarlatina?. Curiously, little attention has been paid to these spiro-
chaAal forms by writers on this subject. They appear to be quite
distinct from the inclusions already described, and occur as thin thread-
like structures, sometimes coiled, sonietimes straight, which are only to
Received September 1, 1913.
 DOHLB’S ( L ~ USION
~ c zBODIES ,’ INSCAHLETEE VXR. 457

be seen in good specimens, and are very delicate and difficult to find.
While the writer can confirm their presence in the blood of some
cases suffering from scarlet fever, they are not confined to that disease,
as he has also found them in several diseases, such as measles, typhus,
and erysipelas. The presence of these threads, therefore, cannot be
regarded as peculiar to scarlet fever, and so many spirochstes have of
late years been described as occurring in the blood that one hesitates
to acknowledge these threads as such. This point, however, would
require further investigation.
I n scarlet, fever, as a rule, inclusions are found present during the
first week of illness, but after this time they tend to disappear.
Kolmer (1912 4), however, found them present in one case on the
forty-fourth day of the illness, just before dismissal. If the temper-
ature remains elevated, as in septic scarlet fever, the inclusions will be
found as long as the temperature continues to swing. They are said
to appear also in cases of relapse accompanied by a rise of temperature.
I n septic and hsmorrhagic scarlet fever, conditions in which there is a
marked leucocytosis, the inclusion bodies occur in great numbers and
are often of large size.
I n malignant scarlet fever, Nicoll and Williams (19 1 2 3, found that
inclusions are absent. The explanation of this is probably to be found
i n t h e fact, that in this condition the onset of the disease and death of
the patient occur within a very short period, and the tissues have no
time to react.
Inclusion bodies have now been observed in many other conditions
besides scarlet fever. They have been described as occurring in
erysipelas, typhus, septic conditions (especially of streptococcal origin),
measles, diphtheria, pneumonia, broncho-pneumonia, whooping-cough,
osteomyelitis, cancer, etc. Their presence has not, however, been
demonstrated in the blood of normal persons.
Kolmer found inclusion bodies absent in thirty cases of serum
sickness which occurred on an average of ten days after admission to
hospital. Their absence, he says, in this condition excludes scarlet
fever, while their presence may be due to the primary attack of
diphtheria.
I have, however, found inclusion bodies occurring in cases in which
the serum rash appeared ten days after the acute diphtheritic
symptoms had disappeared.
My attention was first drawn to the subject of inclusion bodies
about a year after the publication of Dohle’s original paper, by Dr.
Brownlee, the superintendent of Ruchill Fever Hospital, and I there-
after spent a considerable time in testing the various staining reagents
and observing their reactions, and in examining large numbers of cases
with a view to satisfying myself as ti0 the existence of these bodies.
Having satisfied myself as to the presence of these bodies, and having
become familiar with the best methods of preparation of the specimens.
 458 WILLIAM M A Ci? W E N .

I next examined many cases of various diseases, and tabulated the

results, which I now present.
SCARLET FEvER.-oUt of a series of a hundred cases examined, ninety-six
were found to be positive, and only four proved negative. Of these four cases
one was desquamating on admission, the date of rash being unknown, and
another was in the twenty-second day of illness. As inclusions tend to dis-
appear after the first week, these two cases may be discounted. The third
and fourth cases are particularly interesting and instructive. Case No. 3 had
a very indefinite rash on admission, when the negative examination of the
blood was made, but later developed a typical scarlet rash. Case No. 4 had
a definite rash on admission, and in every respect resembIed a case of scarlet
fever, but later a second rash developed, and thereafter inclusion bodies were
found in abundance.
These cases were taken practically as t,hey were admitted to hospital-
acute, mild, and doubtful cases being included. With a few exceptions the
blood was taken during the first week of illness, and usually before the
temperature had settled.
Thus it may be said that of this series of cases every case of scarlet fever in
the first week of illness yielded a positive result, while in two doubtful cases,
in which a negative blood result was obtained, the subsequent development of
a typical scarlet fever rash proved the condition on admission to have been
other than scarlet fever. The presence of inclusion bodies, taken in con-
junction with the general blood picture, is therefore a point of considerable
importance in deciding whether a doubtful case is or is not one of scarlet fever.
A further series of forty-five convalescent cases (four to six weeks) recover-
ing from scarlet fever was examined. Out of this number only one case
could be said without doubt to still retain inclusions in the polymorphe, the
others being very doubtful or negative.
~IPETHERIA.-FOUrteell cases were examined, of which ten were positive
and four negative. In all the above cases the blood was examined on the
second day after admission t o hospital, while the symptoms were still acute.
SERUM srcKNEss.-Four cases suffering from anti-diphtheritic seruni rashes
mere examined. Three were found t o contain inclusion bodies, while the
fourth was negative. The rashes in three of the cases occurred ten days after
injection of serum and fourteen days in the case of the fourth.
&lEASLES.-A series of thirty-five cases was examined during the acute
stage of the illness. Twelve were positive and the remainder negative. I n
this fever oral sepsis is a common complication, and may account for the
presence of inclusions in some of the cases.
GERMANMEAsr,Es.-Forty cases were examined, and all but one proved
negative. The exception was a child just recovering from whooping-cough
and broncho-pneumonia. As inclusions are found in these conditions, this
case may be discounted. The absence of inclusions from the blood of patients
suffering from German measles is of considerable importance clinically, as the
differential diagnosis between mild cases of scarlet fever and German measles
is often a matter of some difficulty.
TypHus.-seven cases were examined, and all were found to be positive.
The inclusions were often large and tended to be bacillary in shape;
spirochaetal forms were occasionally found, and indefinitely staining ovoid or
circular areas were also occasionally found in the polyniorph cells., In the
cases examined a marked leucocytosis was found present, the whole blood
picture resembling that of scarlet fever.
ERysrpmAs.-twenty-five cases were examined, and all were positive with
the exception of two, which were mild cases. The inclueions were well
marked and plentiful, giving a very definite positive result, and a marked
leucocytosis was generally present.
 D6HLE’S “INCLUSIONBODIES” INSCARLETFE VER. 459

PaTarsrs.-Twenty cases were examined, of which sixteen were positire

and four negative. The sixteen positive cases were advanced in the disease,
the temperature being elevated and the sputum purulent. The secondary infec-
tion, so common in the lung, is probably sufficient explanation of the presence
of inclusion bodies. The inclusions are small and not very numerous, nor is
there a marked leucocytosis.
PNEuMoNIA.-Three cases were examined and proved positive. The
following septic conditions all proved markedly positive, and were accompanied
by a pronounced leucocytosis :-abscess of the lung, enipyema, tonsillar
abscess, adenitis, osteomyelitis.

TECHNZQ
UE.

Blood films were made on either slides or cover-glasses. Thin

films are desirable. Methyl-alcohol was used throughout as a fixative,
except when Leishman’s stain was used, when no fixing is required.
STAlNING.-of all the stains experimented with, Manson’s (borax
methylene-blue) was found by far the most reliable. I n fact, a negative
finding could not be definitely given with any of the other stains tried,
With this stain the red blood cells are stained a greenish-blue; the nucleus of
the polymorphs a deep blue, the inclusion bodies slightly lighter than the
nucleus, while the cytoplasm is stained a very light blue-green. The stain is
made up with methylene-blue, 2 grms.; borax, 5 grms.; water, 100 C.C.
8s stock. A few drops of the stock solution are diluted before use with
water until the solution is just translucent. The films are stained for from ten to
thirty seconds, then washed in water, dried, and mounted. Giemsa’s stain is
not very suitable for demonstrating the presence of inclusion bodies, as the
neutrophil granules of the polymorph cells are greatly enlarged and so tend to
obscure the inclusions. Slightly understained specimens are more suitable for
this purpose. The inclusions with this stain are pale blue in tint.
Leishman’s stain is better than Giemsa’s for inclusions, which are stained
a light blue tint, while the nucleus of the polymorphs is stained a deep purple.
In good preparations a very pretty differentiation is obtained.
Pappenheim’s stain (methyl-green and pyronin) stains the inclusions a
deep red, the nucleus of the polymorphs a dark green or blue, while the
cytoplasm is of a light red tint. Various other stains have been tried, but
none have proved nearly so suecessful as Manson’s, which has the further
advantage of great simplicity in use-a point of home importance when large
numbers of slides have to be prepared. Before leaving the subject of stains, a
few points regarding the nature of the inclusions may be considered in regard
to their staining reaction.

Some writers (Glomset, 1 9 1 2 5, have suggested that inclusion

bodies are merely fragments broken off from the nucleus. When we
consider the staining reactions above mentioned, we see that with
Leishman’s stain the nucleus has a deep purple colour, while the
inclusions are stained a light blue; with Pappenheim the nucleus
stains a deep blue-green and the inclusions a deep red (Pappenheim
stains bacteria a deep red). Further, if blood filnis are stained with
hzmatoxylin, as in Israel Pappenheim’s stain, the inclusions are not
stained a t all, while the nucleus of the polymorphs is stained a deep
blue. It does not appear probable that the inclusions are of nuclear
origin if they remain unstained with haematoxylin; and in all the
460 WILLIAM MACE WEN:

above staining methods the inclusion bodies stain differently from

the nucleus, in some cases being of a lighter tint, while in others the
staining reaction is quite different.
Kolmer regards the inclusions as being of streptococcal origin, and
this theory is supported by the above staining reactions. There is,
however, one point which may be worthy of notice, namely, that the
inclusion bodies in scarlet fever are Gram-negative. This staining
reaction would disprove the theory of streptococcal origin, unless the
staining reaction of the streptococci was changed after ingestion by
the polymorphs. I n this connection it would also be worthy of
consideration whether a degenerative change in a portion of the
nucleus, or of the cytoplasm, say of toxic origin, could alter the
staining reaction of the affected portion.
I n the hope of producing inclusions in the polymorph cells of the
lower animals, the following experiments were tried.
1. A culture of streptococci, emulsified in saline, was injected
into the loose areolar tissue of the abdominal wall of a rabbit. A
local abscess formed, but no inclusion bodies could be discovered.
Daily examinations of the blood were made u p to the twelfth day,
without success.
2. A similar experiment tried with pneumococci had a similar
negative result.
3. Blood, taken from the arm of a patient suffering from acute
scarlet fever, was injected into the abdominal cavity of a guinea-pig,
in the hope of producing inclusions. The result, however, was again
negative.

CONCLUSIONS.

1. That the inclusion bodies are to be found in practically every

case of scarlet fever during the first week of the fever. After that
time they tend to disappear.
2. That the inclusions are not of etiological importance in scarlet
fever.
3. That in scarlet fever the inclusions are large and plentiful, and
are accompanied by a marked leucocytosis.
4. That the so-called Spirochmta scarlatinae is not peculiar to
scarlet fever, being found in other diseases, and that therefore it has
no claim to its title. It is indeed doubtful if the thin thread-like
structures are spirocha-Aes. These thread-like bodies are separate and
distinct from inclusion bodies.
5. Inclusions are to be found occasionally in measles, but are
usually small in size and few in number. Further, the blood picture
differs markedly from that of scarlet fever, there being a leucopmia
and not a leucocytosis.
6 . I n diphtheria, inclusions may be present during the acute stage
 DGHLE’S “INCLUSIONBODIES” INSCARLET FE VZR. 461

of the disease, but tend to disappear as the throat condition clears up

and the temperature falls. I n serum rashes, inclusion bodies may be
present, although small in size and few in number.
7. Inclusions were found to be absent in all the cases of German
measles examined. This fact, coupled with the absence of leucocytosis
in this condition, is a valuable aid in differentiating between German
measles and scarlet fever.
8. I n typhus, erysipelas, and septic conditions generally, inclusions
are large in size and plentiful in number. In these conditions a,
marked leucocytosis was found, the general blood picture most closely
resemloiing that of scarlet fever.
9. Inclusion bodies can be found in phthisis, but when present are
small in size and few in number.
10. The presence of sepsis, especially of streptococcal origin, plays
an important part in the origin of inclusions.
11. Inclusion bodies, taken along with the general blood picture,
are an aid to differential diagnosis if judiciously used, always re-
membering that definite sepsis will produce inclusions in the
polymorph cells.

My thanks are due to Dr. Brownlee, superintendent of Ruchill

Fever Hospital, for having drawn my attention to the work of Dohle
and other writers, and to the Ruchill staff for affording me every
facility for obtaining specimens of blood.

REFERENCES.

1. DOHLE . . . . . . . Centralbl. f . Rakteriol. u. Parasitenk., Jena,

1912, Abth. 1, Orig. Bd. lxv. S. 13.
2. KRETSCHMER. . . . . Berl. klin. Wchnschr., 1912, No. 11, S. 49.
3. NICOLLAND WILLIAMS . , Arch. Pediat., N. Y., 1912, vol. xxiv. p. 350.
4. KOLMER.. . . . . . Anaer. Journ. Dis. of Children, 1912, vol.
iv. p. 91.
5. GLOMSET . . . . . . Journ. Infect. Diseases, Chicago, 1912, vol.
xi. p. 468.

3 1 - ~ ~ .OF PATH.-VOL. XVIII.