NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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The kidneys, lungs, and heart presented no marked changes. Some
punctiform hæmorrhages were present on the parietal peritoneum
and on the under surface of the arachnoid.
Autopsy No. 2.—A female patient. Age about 15. Death from bron-
cho pneumonia following cancrum oris.
Spleen enlarged ; length six inches ; consistence firm ; not pigmented.
Liver not enlarged but showed lobular changes.
Small intestine apparently normal. A few anchylostomes were pres-
ent, also some round worms and a few whip-worms (Tricho-
cephalus).
Large intestine.—Mucous membrane for the most part normal. In the
sigmoid flexure and rectum numerous ulcers about the size of a
split-pea were present. Many of these presented thickened and
raised edges, so that they had an almost pustular appearance.
In a few a central mass of altered blood pigment was present
reaching as deep as the muscular coats.
The lungs showed patches of congestion and consolidation.
Autopsy No. 3.—A male patient. Age about 20. Death from purulent
peritonitis. The abdomen contained purulent fluid.
Spleen much enlarged ; length 8 inches ; consistence firm ; not pig-
mented.
Liver not enlarged but lobular changes visible. A small abscess cavity
about the size of a marble was found in the left lobe ; it con-
tained soft caseous material.
Small intestine.—Patches of congested mucous membrane were present,
and on the edges of the valvulæ conniventes small red dots
resembling grains of Cayenne pepper (capillaries of villi) were
seen. Anchylostomes were not found.
Large intestine.—The mucous membrane was greatly thickened
throughout and dark pink in colour from congestion. Small
erosions were present throughout the gut. Patches, several
square inches in extent, showed projecting small red granul-
ations and were covered by tenacious pink mucus. Small ulcers,
often hidden by the overlapping mucous membrane, were numer-
ous, and large ulcers extending over several square inches were
situated at intervals along the gut. Perforation of the base of
one of these had taken place.
J
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The lymphatic glands in the retroperitoneal tissue were not apparent-
ly enlarged.
There were some punctiform hæmorrhages on the parietal peritoneum.
The red marrow in the sternum seemed increased in amount.
3. The importance of the disease.
Cases exhibiting the clinical picture and post mortem appearances described
above are by no means rare, nor are they confined to Madras. Ross and others
have long ago noted the presence in Bengal of cases of fever accompanied by
great enlargement of the spleen in which malarial parasites could not be found.
In 1901, in conjunction with Stephens and James, I examined in the hospitals
of Calcutta more than 6o cases which had been diagnosed as malarial cachexia.
All showed greatly enlarged spleens and an irregular, often high, temperature.
The operation of spleen puncture was not performed in any of the cases, but no
malarial parasites were found in the peripheral blood, and the cases in which
leucocyte counts were made exhibited no increase in the large mononuclear
leucocytes. The post mortem lesions in some of these cases were identical with
those found in fatal cases of infection with the new parasites. At the time we
considered that the diagnosis of malaria in such cases was made on very slender
grounds. I now recognise the identity of such cases with those I have seen
in Madras in which the new parasite is found. Exactly similar cases were seen
by us in many parts of India, and I have no hesitation in saying that cases of
infection with these parasites are clinically identical with cases diagnosed as
" malarial cachexia with enlarged spleen," so well recognised throughout India.
The question naturally arises as to the relation of the cases in Madras to
the disease known as kala azar in Assam. In the descriptions of kala azar
stress is laid on the pigmentation of the skin, on the extreme weakness of the
patients, and on the more rapidly fatal termination than in the cases of enlarged
spleen seen in and about Calcutta. During my work with the Royal Society's
Malaria Commission in India we did not study kala azar, but in the Madras
cases I can detect no increase of pigmentation, and the weakness of the patients
and the duration of the disease do not seem different to that in ordinary cases of
so-called " malarial cachexia with enlarged spleen." I have, however, seen a
specimen from the spleen of a case of kala azar which was sent by Dr.
Bentley to Captain Donovan in which the bodies were very numerous.* If they
are the cause of this disease, then the disease of Assam called kala azar and the
common disease of India called " malarial cachexia with enlarged spleen "
must be essentially of the same nature, though the one may possibly be a more
severe form of the disease than the other.
* Blood films obtained from the spleens of five cases of Kale azar which were sent to Simla by Dr. Bentley
have been examined and the new parasite has been found to be present in large numbers in all. Malarial para-
sites were not found in any of these films.—Ed.
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4. A description of the parasites.
When the bodies are present at all, they are usually to be found in large
numbers. They are seen with difficulty in fresh preparations, but can be detected
and distinguished from blood platelets. They are rather more refractile than
platelets and often have a faint greenish tinge. One or two more refractile
masses can be made out in the body of the parasite and in many cases an appear-
ance like a grain of pigment is seen. On careful focussing this is seen to be a
minute refractile spot like " Manson's spot " in lymphocytes. The bodies are
quite motionless.
In films stained by Romanowsky's method the bodies are seen with great
clearness. They exhibit a remarkable uniformity in size and appearance. The
majority are about 2.5 micromillimetres in diameter, but forms may be found
which are a little larger, 3 to 3.5 micromillimetres, or a little smaller, 2 micro-
millimetres. Very occasionally still smaller bodies, 1.5 micromillimetres in diameter,
may be encountered. Most of the bodies are approximately circular in outline,
but very many, and especially the larger forms, are irregularly oval and very much
resemble a cockle-shell in shape (Figures 7 and 8). This, indeed, appears to be
the most typical shape and even in the apparently round forms an approach to
this shape can be made out. Occasionally specimens are found more elongated
and distinctly pear-shaped.
The bodies are very clearly outlined, and appear to possess a distinct and
comparatively resistant cuticle. They retain their proper shape and are very
rarely seen distorted. I have seen a body which had been crushed and from
which the two chromatin masses had been squeezed out. The larger mass still
lay partially within the body which had an appearance exactly resembling that of
a ruptured cuticle.
The bodies are very resistant to hypotonic solutions and may be recovered
by centrifugalisation from blood that has been laked by such a solution.
Except in very rare small forms which show only a single chromatin mass,
the bodies invariably possess two chromatin masses—a large one staining lightly
and a small one staining intensely with the red colouring matter of Romanowsky's
stain.
The two chromatin masses are usually situated opposite to each other in the
shorter axis of the parasite.
The small chromatin mass is usually rod-shaped, but may appear as a
dot only. It is usually contained in what appears to be protoplasm displaced to
the periphery of the body by the vacuole or vacuoles. It may appear close to
the periphery or centrally situated. In the latter case it is probably in reality
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on the periphery also, since it seems to be generally situated in some part of an
arc lying immediately beneath the cuticle and in a plane at right angles to the .
length of the body when this can be made out. There is often a " tail " joining
it to the larger mass. As a rule, this appears to be formed only of a strand of
protoplasm. On one or two occasions a minute chromatin particle appeared to
be situated in it.
The large chromatin mass stains much less darkly than the small one and is
obviously of a quite different nature. It frequently stains so as to show a darker
and lighter portion. It is very commonly bilobed, and often heart-shaped, or
formed of two oval masses lying in contact. In some cases bodies are seen in
which the large chromatin mass stains very faintly although other bodies in the
same field stain normally. The large chromatin mass is situated near what
would be the hinge in the cockle shell shaped forms, and in the thicker end of
the pear-shaped forms.
The body substance usually stains pink by the method employed (fixa-
tion in alcohol, and staining by watery solutions of eosin and Romanowsky
blue). In some specimens the protoplasm stains faintly blue. In a few forms
a uniform pink staining hyaline substance is seen filling the body of the parasite.
In the majority of bodies one or two vacuoles are present which in many cases
have pushed the pink staining substance to the periphery. A ridge of proto-
plasm left between the vacuoles forms the " tail," sometimes seen joining the
two chromatin masses. The protoplasm is not as a rule uniformly disposed around
the periphery, but is collected in greater amount on one or other side of the small
chromatin mass.
The bodies have a very regular external form and arrangement of parts. A
cockle shell will best represent the shape of the parasite. In the thicker por-
tion, where the body of the cockle is placed, lies the large chromatin mass,
often divided into two lobes or even into two separate bodies—one in each valve.
Near the centre of the convex free edge of the valves and lying partly
in each is the small chromatin rod. The forms seen lying flat appear as in
figs. 7 and 8. The pear shaped forms are these seen edgeways. In many aspects
the bodies appear nearly circular. Various views of our imaginary body will rep-
resent almost all the arrangements of the chromatin masses and the shapes
assumed by the parasite in films (see descriptive diagram).
Developmental forms.—Forms may be seen which appear to show division
of the bodies into two. Appearances showing both longitudinal and transverse
fission are seen. In the former, two pear shaped bodies are found lying side by
side. The large chromatin masses are sittuated in the thick end of the pear
and the small rod bodies at the thinner ends. Division occurs first at the thick
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end, and the small chromatin mass may often be seen undivided when the larger
masses are widely separated.
In the forms which apparently show transverse division the two large chro-
matin masses are at opposite poles separated by the two (or the as yet undivided)
small masses. Both these appearances represent the same process from different
points of view (see accompanying diagram). Division takes place in the line of
separation of what would be the two valves of the shell form. Seen from one
direction (A) the double pear-shaped form is seen. Seen from B. apparent
transverse division. The bilobed, heart-shaped and double forms of the chromatin
mass appear to be early stages of the same process.
In addition to the above, forms are seen showing division into three or more
bodies. These forms are more or less circular, of larger size than the largest of the.
single forms, and may be as large as a red blood corpuscle. In typical speci-
mens the large chromatin masses are arranged peripherally, whilst the small rod
shaped masses are nearer the centre. The formation of from 3 to 6 bodies is
most common.
Asexual and sexual forms.—No appearances have been seen by me which
seem to point to the presence of two forms of the parasite. Variations in the bodies
appear to be insignificant and to depend mostly upon the point of view from
which they are seen. The two most distinct forms are those with and those
without vacuoles. The forms showing uniform pink body substance are often
quite large. Another form is that with a very faintly staining chromatin mass.
Relation of the parasites to the red cell.—In ordinary preparations taken by
splenic puncture or in smears taken. post mortem, many of the bodies are. free
and found scattered over the slide. Other forms are seen included in leucocytes
and macrophages. Others again are found surrounded by some substance which
stains faintly blue and appears granular or reticular in structure. Bodies lying in
such material may occur singly or in numbers up to 10 or more. According to
Laveran this blue staining substance is the red cell rapidly disorganised by the
presence of the parasite. Ross mentions it only as a matrix.
Most of the apparently free bodies seen in films are, I believe, derived from
the leucocytes, and especially from enormous cells which are crowded with the
bodies and which readily become broken up. In many cases the bodies may be
seen to have been carried in streaks from such cells by the action of the slide or
needle used in spreading the film. Free bodies are generally scanty when blood
flows readily into the syringe during puncture. When this is the case very few
splenic cells are seen in the preparations and at the same time very few parasites.
They are more abundant when actual splenic pulp is used than when the films
are made from blood taken from the spleen. In sections enormous numbers of
bodies are seen included in cells, but I have searched in vain for free bodies, and if
present at all they must be in very small number only.
The blue staining substance in which some of the bodies are seen is of great
interest, and upon the interpretation of its nature the zoological position assigned
to the parasite depends. The appearance of this substance varies slightly in differ-
ent preparations. It stains faintly or more darkly blue and shows a hyaline, finely
granular or reticular structure. It may be, and often is, about the size of a red
cell but it may be very much smaller and it may be larger. If small, it usually con-
tains only a single body. When five or more bodies are present the size exceeds
that of a red cell. The outline is often very distinct, but it is sometimes blurred
or quite irregular. In some cases the resemblance to a red cell altered by the
simple tertian parasite is very marked. Distinct stippling is not, however, seen.
Bodies resembling these, but not containing parasites, are generally numerous in
preparations showing included forms. If blood containing the bodies is placed in
a hypotonic solution of ammonium oxalate and the centrifugalised sediment
examined, the blue staining material around the bodies becomes more clearly
defined and stains more darkly blue. At the same time the protoplasm of the
mononuclear leucocytes shews a marked tendency to budding and separation from
the cell and stains more clearly. But the red blood corpuscles are seen only as
ghosts.
In one of the cases examined by us during life and post mortem, bodies resem-
bling altered blood cells contained undoubted malarial pigment. In the large
macrophages similar pigment was seen included in the same cell as the
parasites.
I think there can be no doubt, therefore, that very many of the blue staining
bodies in which included parasites are seen, are pieces of detached cell protoplasm
derived from the large mononuclear cells and especially from the macrophages.
In many preparations the process of detachment may be observed, and buds of
protoplasm containing parasites are seen projecting from the cell mass. The
process appears to be assisted by the addition of hypotonic solutions and may
be largely due to the fluid always present in the syringe when used for spleen
puncture.
Whether the blue substance is always of leucocytic nature, whether some of
the bodies seen are really red cells (Fig. 19), I hesitate to say ; but however
striking the resemblance to the red cell may have been, we have always found the
protoplasm of the leucocytes to be similarly stained.
Relation of the parasites to leucocytes.—A most striking feature in connection
with the bodies is their presence in great numbers in an unchanged state in the
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leucocytes. They are found especially in the large mononuclear cells. Such
cells contain from one to twenty bodies. The polymorphonuclear cells also
often contain parasites, but rarely more than two or three. I have seen single
bodies in eosinophil cells and in finely eosinophil myelocytes ; also in the
nucleus of some cells, but I am not sure whether these latter were not superimposed
bodies. In addition to the leucocytes noted above, large cells with spherical
nuclei, quite distinct from the large mononuclear forms, are seen in preparations.
They are usually packed with parasites and have a most characteristic appearance.
They usually contain from 20 to 100 bodies, but may contain as many as 250.
Some of these cells are of gigantic size, but become broken up in films and are
seen to advantage only in sections.
Bodies included in the leucocytes and in the large cells described above
show no signs of intracellular digestion. They stain very clearly, and when
liberated from the cell show a clear outline. They do not appear to lie in
vacuoles in the cell protoplasm.
5. The distribution of the parasite in the body.
a. Peripheral blood.—I have been unable to find the bodies in the peri-
pheral blood even of cases in which they occurred abundantly in
the spleen. The examination of peripheral blood taken at 6 P. M.
and at midnight as well as of blood taken during high fever
(104° F.) has been negative. I have not so far been able to detect
the parasites in the peripheral blood even after centrifugalisation.
b. Blood from the deeper vessels.—The parasites were not found in blood
taken post mortem from the deeper vessels of the abdominal wall.
c. Blood from the spleen.—When blood from the spleen flows freely into
the syringe the parasites are found in it, but only, in small num-
bers.
d. Blood from the liver and portal vein.— In blood from the liver the
parasites may be abundant. In blood taken post mortem from the
portal vein a considerable number of the bodies were found, es-
pecially in the large mononuclear cells.
e. Petechiae from the arachnoid.— In pe t e c hæ f r om t he a r a c hnoi d a f a i r
number of the bodies were seen, all included in poymorpho-
nuclear cells.
f.
I n t h e t i s s u e s . —
I n
s t u d y i n g t h e o r g a n i s m i n t h e t i s s u e s I h a v e m a d e
use of a modification of Romanowsky's method of staining. It is
founded upon the observation of Stephens that the characteristic
staining with Romanowsky's stain could be as well obtained by
placing the films first in the eosin solution and then in the methy-
lene blue solution as by mixing the two solutions together before
pouring them on the slide.
So far as I am aware, it has not yet been found possible to stain organisms
in sections by Romanowsky's method. Treated in the ordinary way, sections
stain merely with the methylene blue, and the nuclei show no trace of the charac--
teristic red body of the Romanowsky stain. Films treated first by alcohol, xylol,
and the paraffin bath, still stain quite readily, so that the reason why sections do
not stain appears to be a physical one. Stephens found that Romanowsky staining
was developed when films were placed first in eosin and then in the methylene
blue solution, and we were accustomed to use this method when dealing with large
numbers of films. By employing this method of saturating sections first with eosin
and then placing them in the methylene blue solution I have been able to stain the
nuclei and included parasites with the characteristic red of Romanowsky's stain.
The following is an account of the method.
Fix the tissues in absolute alcohol or saturated aqueous solution of perchl--
oride of mercury. Use small slabs I to 2 millimetres thick placed upon paper so
that they may harden flat. Remove carefully all adherent paper fibre and embed
in paraffin, using, after complete dehydration (1 to 4 hours in absolute alcohol)
only 15 minutes in xylol and at most 20 minutes in hard paraffin.
Cut thin sections. Flatten by floating out on water not quite hot enough
to melt paraffin. Float on to a slide. Remove the excess of water and press firmly
with filter paper. No fixative is necessary if this be done. Dry a few seconds
at a height above the flame just sufficient not to melt the paraffin. Melt the
paraffin and treat as usual with xylol, absolute alcohol, and water.
Stain for 10 to 15 minutes in 1—1,000 eosin (Höchst, B. A. Grübler).
Pour off the excess of eosin and press the section with filter paper. Add
an excess of the methylene blue solution prepared according to the following
formula and allow to stain for 15-20 minutes.
1 0 0 c . c . o f m e t h y l e n e b l u e ( p u r e m e d i c i n a l , G r ü b l e r ) s o l u t i o n i n d i s t i l l e d
water.
5 c. c. of a 10 per cent. solution of chemically pure sodium carbonate.
Leave in tropical sun until a strong red colour is seen on shaking.
Dilute 25 times for use.
Pour off the excess of stain and wash rapidly in 70 per cent. alcohol. The
washing in alcohol should be quite momentary. If the washing is too prolonged
too much red colour will be removed, and if too short some precipitate will be left,
Transfer as rapidly as possible to water.
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If, after washing, the section is dark purple or blue, wash in 1 to 4 0 0
a c e t i c
acid in distilled water, and at once plunge into water. The acetic acid removes
the excess of methylene blue staining and makes the tissue much clearer. If too
long washing is given, the red colour will also be removed, If the section is
light reddish or light purple after washing with alcohol the acetic acid bath is
unnecessary.
Allow the section to dry upon the slide. It is on account of the necessity
for this procedure that thin sections must be used. In the case of very thin
sections shrinkage is negligable, but in thick sections it is considerable.
Mount in Canada balsam or keep as a film and examine merely by placing a
drop of cedar oil on the section.
Quite unshrunken tissues may be examined after staining by washing and
mounting in glycerine. The result is useless as a permanent preparation since
the glycerine dissolves out the red stain, but it is useful for temporarily displaying
the relation of parasites to the tissues.
Several kinds of tissues may be examined in this way, and the parasites
displayed by spreading portions of the tissues upon a slide, drying, fixing in
alcohol, and staining as above. Omentum, teased muscle, connective and other
tissues give good results. Micro-organisms are very well shown when present.
The changes in the organs and the distribution of the parasite in the tissues
were similar in all three fatal cases.
Films made from the liver substance showed many bodies free and in
leucocytes. The most striking feature was the presence of large leucocytic cells
crowded with the bodies. The liver cells were free from parasites.
Sections of the liver showed very clearly the relation of the parasites to the
tissues. The lobular capillaries were dilated and the liver cells, especially in
certain areas, were much atrophied. In the capillaries were seen numerous large
cells crowded with parasites. The bodies of these cells were usually situated in
the lumen of the capillary, but attached by several processes to the capillary wall.
In many cases they were applied closely and flattened against the capillary wall.
The protoplasm of these cells was often retracted so as to form a globular mass,
but more usually was stretched out along the capillary in a very characteristic
way. The appearance of these cells irresistibly suggests large amœbæ crawling
about the capillaries. In some parts of the sections these cells were extremely
numerous, forming the bulk of the tissue. In one of the cases the cells were
so crowded with bodies that their nuclei were often hidden. The exact nature
of these large cells is doubtful, but they appear to be identical with the large