NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
the large mononuclears in malarial films, are distinguished from these by the
presence of a deeply notched nucleus and by the presence of scanty granula-
tions in the protoplasm.
In suspected cases of malaria, slides for making differential counts should
be taken, if possible, during an apyretic interval, as it is at such times that the
difference in the proportions of the different varieties of leucocytes is most
marked.
When all possible care to place each variety of leucocyte met with under
its proper heading in the count has been taken, a result which gives the number
of large mononuclear elements as over 15 per cent of all the leucocytes may be
regarded as a proof of recent malarial infection.
II.—Methods for the examination of malaria parasites in mosquitoes.
The forms of the malaria parasite which it is important to be able to
recognise in the tissues of anopheles mosquitoes are zygotes, in the stomach wall,
and sporozoites, in the salivary glands.
To dissect out the salivary glands for the demonstration of sporozoites.—
The salivary glands consist of six lobes, three on each side, and the ducts of the
three lobes of each side unite to form a single duct which, after joining with its
fellow of the opposite side, passes forwards and ends in the first portion of the
alimentary canal close to the base of the proboscis.
The object in a dissection of the salivary glands is to remove the six lobes
cleanly and entirely from the tissues which surround them. This requires care-
ful dissection and cannot be done,—except occasionally by chance,—by fixing the
mosquito's head with one needle and the thorax with another and pulling the
needles apart, as is usually recommended. The position of the salivary glands
in an anopheles is shown in the figure (Fig. 4).
A g r a p h i c a p p e a r s h e r e – s e e i m a g e F i g . 4 . o f p a g e 8
8
9
The mosquitoes to be dissected are killed with chloroform and the species
of each carefully identified. They are then placed in a normal saline solution
in watch glasses—one watch glass for the members of each species. This is
necessary because it is of course impossible to identify an anopheles after it has
been some little time in the salt solution.
The importance of exactly identifying each anopheles before dissection is
very great, for the finding of sporozoites in any particular mosquito is of little
value unless we know the exact species.
For dissection, a mosquito is taken out of the watch glass, its legs and wings
are removed, and it is laid on its side in a drop of salt solution on a glass
slide.
With a sharp triangular needle or a fine entomological knife, the thorax is
cut across just behind the glands in the direction shown in the figure (AB,
Fig. 4), and the hind part of the thorax with the abdomen attached is discarded.
The top of the fore-part of the thorax is then removed by a clean cut in the
direction (CD, Fig. 4).
If now the head of the mosquito is held, as near the neck as possible
with the point of one needle, and the remaining tissues surrounding the gland
with the other, and the needles are gently and carefully separated, the glands
will remain attached to the head and almost clear of all surrounding tissues.
The preparation is now examined with a low power of the microscope
( 1 / 4 t h i n c h o b j e c t i v e ) t o s e e w h e t h e r t h e g l a n d i s r e a l l y a t t a c h e d t o t h e h e a d a s i t
should be.
The dissection is completed by a clean cut across the neck at its point of
junction with the head (EF, Fig. 4), and by this cut, and perhaps one or two
final touches with the needles, the six lobes will be freed of all outside tissues
— a m o s t i m p o r t a n t p o i n t i f s p o r o z o i t e s a r e t o b e d e t e c t e d . A c o v e r - g l a s s i s
now placed on the glands and firm pressure applied to it with a needle. By
this operation the sporozoites will be squeezed out and will be seen with greater
ease.
T h e 1 / 6 t h i n c h o b j e c t i v e i s n o w u s e d , a n d i f s p o r o z o i t e s a r e p r e s e n t , t h e y w i l l
be seen in large numbers as transparent sickle-shaped bodies floating about in the
salt solution. The oil-immersion lens is not necessary for the detection of
sporozoites, but if in doubt, it may be used and each part of the crushed gland
thoroughly searched.
It is a mistake to think that sporozoites in infected mosquitoes are present
only in the so-called middle lobe of the glands : as a rule they are equally
numerous in all the lobes.
To stain sporozoites.— The sporozoites that have been demonstrated by the
c
O
10
above method may be easily stained. The excess of salt solution is removed
with blotting-paper and the cover-glass slipped off the slide in the same
manner as is done when making a cover slip preparation of blood. The slide
and cover-slip are then dried in the air, fixed by immersion in absolute alcohol
for half an hour, and stained by Romanowsky's method like an ordinary
blood film.
Examination with the oil-immersion lens will show the sporozoites singly
and in groups as sickle-shaped bodies, pointed towards the ends, and with one
or more brilliant red chromatin dots at or near the centre of each.
It would be of little or no advantage to enumerate the appearances and
structures that may be mistaken for sporozoites. The best way to avoid error
is to ensure that no other structures except the glands are included under the
cover-glass. If in doubt, the specimen may be fixed and stained as above
described.
In making dissections of a large number of mosquitoes caught in a village,
several other parasites besides sporozoites will almost certainly be found.
The most interesting of these are the bodies which for want of a better name
we have called the "sausage bodies." They are small oval or crescent-shaped
bodies, which are occasionally found in great numbers in the tissues of the
head surrounding the salivary glands, but not as a rule in the glands them-
selves. When stained with Romanowsky's stain they present a red capsule,
blue body substance, and two round chromatin patches in the centre surrounded
by a clear area. Their nature is unknown.
To dissect out the stomach to demonstrate zygotes.— In order to demons-
trate zygotes successfully, the stomach of the mosquito must be quite empty
of blood. The method of dissection is as follows :—
The mosquito is divided into two by a cut across the thorax as for the
dissection of the salivary glands, and the head and fore-part of the thorax
discarded.
The abdomen is laid in a drop of salt solution on a slide, and while the
point of one needle holds the thorax to the slide, the pointed end of the other
needle is laid across the abdomen near the seventh or eighth segment. The
needles are then gently separated, and the stomach with the malpighian
tubes attached comes away with the hind segments. The malpighian tubes,
which will probably be lying over the stomach after it has been pulled out,
are carefully drawn away, and a cover-glass applied. The weight of the cover-
glass is usually sufficient to flatten out the stomach, and it is inadvisable to use
any pressure on it. The preparation is now examined with a ¼th inch objective,
and if the zygotes are fairly large they can easily be seen under this power.
It will be found as a rule that most of the zygotes are situated within the
11
posterior third of the stomach (nearest the origin of the malpighian tubes)
although in some cases they are more uniformly spread over the whole stomach.
The number of zygotes present varies greatly ; sometimes only ten or fifteen can
be counted, at other times as many as four or five hundred, so that the whole
surface of the organ is studded with them.
Very young zygotes can only be made sure of by examining the whole of
the specimen with the oil-immersion lens : they can then be recognised by
the presence of grains of malarial pigment the appearance of which is very
characteristic. These grains of pigment may be arranged in little groups or in a
line across the zygote.
There are so many cells in and around the stomach and malpighian tubes
that bear a superficial resemblance to zygotes that their detection presents more
difficulty to those who are unfamiliar with their appearance than does the
detection of sporozoites. In this case,—as indeed in the detection of malaria
parasites in the blood,—the difficulty lies not so much in recognising them
when they are really present, but in not mistaking other structures for them
when they are absent.
Those, however, who are unfamiliar with the appearance of zygotes
can study them easily by feeding a few of the common Indian culex ( C. Fati-
gans) on a sparrow with proteosoma parasites in its blood. In a few days
almost all the culex will have zygotes in large numbers, developing in the
stomach wall.
To make permanent preparations of zygotes.—Fairly good preparations
of zygotes may be made by simply mounting the fresh stomach in a 5 per
cent. solution of formalin and ringing the cover-glass thickly with balsam.
Such specimens keep good for some months at any rate.
The staining of zygotes in order to show any definite structure in them
is a difficult matter and requires special methods. Moderately good specimens
may, however, be obtained by allowing the stomach to dry as flatly as possible on
the slide, fixing it first in formalin and then in alcohol, and staining very lightly
with methylene blue. The specimen can then be permanently mounted in
xylol balsam.
To carry out feeding experiments.—The chief difficulties in carrying out
feeding experiments arise from two causes : first, the difficulty in getting a
suitable case on which to feed the mosquitoes, and second, the difficulty in
making the anopheles feed and in keeping them alive sufficiently long for
the parasites to develop.
The first of these difficulties is the more important, and is the one which
has probably been responsible for the many negative results in feeding experi-
ments that have been recorded. Thus Dr. Daniels, working with A. Fuliginosus
C 2
12
(Giles) and A. Rossii (Giles), states that neither of these species carry the
malignant Tertian (crescent) variety of the malaria parasite.
Positive results have, however, been obtained by us with both those species
of anopheles for simple Tertian, malignant Tertian and Quartan parasites. We
have also shown that zygotes and sporozoites will readily develop in six
other species of Indian anopheles with which we have experimented (viz.,
A. Culicifacies, A. Fluviatilis, A. Stephensii, A. Theobaldi, A. Turkhudi, A.
Barbirostris), so that we may infer that almost certainly all the species of
Indian anopheles are capable of harbouring the malaria parasite under experi-
mental conditions, and negative results in feeding experiments with anopheles
are more likely to be due to failure in carrying out the details of the experiment
than to the fact that any species of anopheles is hostile to the parasite.
The most important thing is, as we have said, to obtain a suitable case
for the anopheles to feed upon. By this is meant a case in which a large
number of gametes are ripe for flagellation at the time of the experiment. It
is practically useless to carry out a feeding experiment on a case in which,
by the examination of fresh specimens of blood, a fair number of flagellating
bodies have not been seen shortly before the experiment.
Having procured a good case on which to carry out the experiment, the
best method of feeding the mosquitoes and keeping them alive can be easily
devised after a few trials. We have found that anopheles do not, however,
live so well in the mosquito netting boxes that are usually recommended for
feeding experiments as they do in glass bottles, but if the latter are used a
rough piece of cardboard or stiff paper must be placed in the bottles for the
anopheles to rest comfortably upon. This detail, which is due to the ingenuity
of Dr. Christophers, is very important.
The following is the plan we have found most successful :—
A number of large wide-mouthed glass jars are obtained—glass prune jars
or crystalized fruit bottles do well—and thoroughly cleaned and dried. Into each
of these bottles is fixed lengthwise a piece of rough cardboard on which the
anopheles can rest easily. The mouth of the jar is closed by a removable cover of
mosquito netting. Ten or twelve female anopheles (preferably including several
species) are placed in each bottle. The patient upon whom it is intended to
feed the mosquitoes should sit on the ground, and the bottles (closed by their
mosquito netting covers) be arranged on a low stool or box in front of him, so
that he may be able with comfort to rest both his forearms on the mouths of the
bottles placed upright on the box. In this manner he must remain until the
anopheles have fed. When using mosquitoes bred from larvæ it is often
difficult to get them to feed on the first night, but once having fed there will
be no difficulty on subsequent nights.
13
After the, mosquitoes have fed, the bottles should be inverted-the cover
having been removed—over a small tin of water in which a piece of paper
is floating for the mosquitoes to rest upon when laying their eggs. With care
this manipulation can be easily performed without letting any escape.
The bottles should be labelled and kept in a dark cupboard until the next
night when the mosquitoes must be again allowed to feed.
Each morning the water in the tin should be renewed and any mosquitoes
that have died removed from it. If they are fed each night the mosquitoes will
keep alive for from twelve to twenty days or more, and no other food than blood
is necessary.
When it is desired to examine any of the mosquitoes, they should be kept
without being fed until they have got rid of all the blood they have ingested.
This usually takes about two days, but the longer they can be kept alive without
food the more satisfactory will be the examination of the stomach.
If the weather is cool the bottles may with advantage be kept in an
incubator at a temperature of between 70°—80ºF., by which means the parasites
will develop more readily.
It has been proved by Dr. Stephens that fertilization is not essential for
14
the development of zygotes, so that it is not absolutely necessary to place any
males in the bottles with the females, although in order to imitate natural condi
tions more closely it is possibly of advantage to do so.
Either mosquitoes bred from larvæ, or those caught in a village may be
used; the advantage of employing the latter is that they will usually feed more
readily on the first night.
For infecting a healthy person, all that is necessary is to allow the anopheles:
which have previously fed on the case of malaria to bite the healthy man in the
same manner as they did the malaria case. It is of course useless to attempt to
infect a healthy person with malaria unless sporozoites have been detected in the
glands of some of the anopheles of the same batch as those which are being
used in the infecting experiment, and experiments which simply consist in feeding
a number of anopheles for some days on a case of malaria, and then on a healthy
man, without simultaneous dissection of the mosquitoes, are of little if any value;
and when negative results are obtained, such experiments are liable to throw
discredit on the fact that malaria is conveyed only by mosquitoes.
III.—The investigation of malaria in hospitals and jails.
There are many opportunities for the study of individual. cases of malaria in
the hospitals of India, but as a rule it is difficult to take advantage of them owing
to the fact that the great majority of the cases have been treated with quinine
before admission. For this reason it is quite exceptional to be able to find
malarial parasites, or even to see a typical Tertian or Quartan chart in hospital
cases; for although irregular fever may continue for some time afterwards, yet
the administration of even small doses of quinine almost invariably causes the
disappearance of parasites from the peripheral circulation.
On one occasion we visited the out-patient department of one of the large
hospitals and examined the blood of all the patients who presented themselves
complaining of "fever" On that day we did not find parasites or other indica
tion of malaria in any of the cases, but in spite of this they were all diagnosed
as "ague" by the native assistant physician in charge and given ten grains of
quinine twice a day.
Possibly some of these cases were in reality cases of malaria who would
therefore have benefited by the quinine, but at any rate it would appear to be
preferable in all cases to wait a few days before giving a drug which, once given,
lessens the chances of an accurate diagnosis of the case being afterwards made
by an examination of the blood, or, in the absence of this, by an inspection of the
temperature chart.
In all ordinary cases of malaria it is quite unnecessary to begin quinine
treatment immediately, and the advantage which accrues in the treatment of a
5
patient from the knowledge of whether his case is one of simple Tertian, of Quar-
tan, or of malignant Tertian fever far outweighs any remote danger there may
be in withholding quinine for a few days until the diagnosis can be accurately
made. I say "for a few days" advisedly, because in a first attack of malaria it
sometimes happens that during the first or even during the second day of fever,
parasites may be absent from the peripheral circulation. During the first day
or two also the temperature may be continuous and give us no clue as to the
nature of the fever. If we give quinine during this time we do so empirically
and without the least knowledge of the nature of the case, and in doing so we
do away at once with almost all chance of afterwards finding out (should the
fever not yield to quinine) whether the case is one of malaria or not. In very
severe cases of malaria, as, for instance, where patients are brought into hospital
in a semicomatose condition, parasites can usually be found by a few minutes
examination, and we are then in a position to treat the case with appropriate
vigour.
This question of the routine administration of quinine during the first few
days in nearly all cases of fever has an important bearing on the question of the
differentiation of Indian fevers. Unless we can absolutely exclude malaria by
repeated examinations of the blood before quinine has been taken, it is impos-
sible to say definitely that a case of fever is not malaria.
I t i s q u i t e p o s s i b l e t h a t i n d e f i n i t e c a s e s o f f e v e r w h i c h c a n n o t b e c l a s s e d a s
enteric fever, Malta fever, relapsing fever, or malarial f ever occur in India, but
if in such cases quinine has been given early in the disease it is impossible
to definitely exclude malaria, because a malarial fever may continue in spite of
the administration of quinine in ordinary doses, and in spite of the absence
of parasites from the peripheral circulation. If, however, in such a case we
have refrained from giving quinine, we are able, by repeated examinations
of the blood, to definitely exclude malaria and can therefore, with confidence,
search for some other cause of the fever.
It appears, therefore, that until the plan of not beginning quinine treatment
until an accurate diagnosis of every case of fever has been made, is adopted
systematically for a year or so in one of the large hospitals of India, our
knowledge of the non-malarial fevers will remain no further advanced than it
has been for many years past. This, together with a close study of individual
cases of malaria, is the kind of work which can be best carried out in hospitals
and jails.
The large class of cases which are characterised by the presence of greatly
enlarged spleens and much anæmia, accompanied frequently by long continued
irregular fever, are also very common in Indian hospitals and offer excellent
material for study, but although it is generally considered that these cases are
16
hardly, if at all, amenable to quinine, it is quite exceptional to find in any hospital
a case of this nature which is not being treated more or less vigorously with
this drug, so that to prove definitely the connection between such cases and
malaria becomes a difficult matter.
IV.—The investigation of malaria among the general population.
It is certain that an examination of the statistics of admissions into hos-
pitals for malaria gives us no real indication of the amount of malaria prevailing
in a district at the time, and from a study of hospital cases we can learn nothing
regarding the conditions under which the disease prevails. The method of inves-
tigation by which, above all others, the most valuable advances in our knowledge
of malaria are likely to be made, is that of the investigation of the disease as it
occurs among the general population of a place or district. The study of cases
in hospitals and a detailed study of the structure of parasites is of little or no
importance compared to a comparative study of the conditions under which
malaria prevails in different places, and as this is a method of investigation
which has received little attention in India up to the present, special importance
should be attached to it.
An outline of the principles upon which such investigations have been
carried out by us is as follows
:—
Having selected a village or series of villages in any district for in-
vestigation, our first object is to find out, as accurately as possible, the
amount of malaria and the liability to infection existing at the time of the
investigation.
We have found in India, as was previously found in Africa by Koch and by
Stephens and Christophers, that in any place which is more or less malarious, a
certain number of the young children will have malaria parasites in their blood;
and the percentage of young children so affected affords the most accurate test
of the amount of malaria and the liability to infection existing in the place
under examination.
The term "Endemic Index" has been given to this percentage of infected
children, and the first thing to do on arriving at a village is to determine this
endemic index.
For this purpose films of blood must be taken from at least thirty or forty
children, none of whom should be over ten years of age. As may be imagined,
this is often a difficult matter in Indian towns and villages, but with tact and
patience it can be done, and except in one or two villages in Bengal we have
never failed to get the required number.
The blood films should be taken by the method previously described, and
any other method is practically impossible under the circumstances. At the
same time as the films are being taken, the spleen of each child may be