The diagnostics and treatment of tropical diseases

Chapter I: Malaria (1)

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DEFINITION AND SYNONYMS

=Definition.=—Malaria is a protozoal disease caused by three species of _Plasmodium_. In the clinically benign types of malaria we have that of benign tertian, due to _P. vivax_, with a tertian periodicity and that of quartan, due to _P. malariae_ and showing a quartan or seventy-two hour periodicity. The clinically malignant type of malaria is due to _P. falciparum_, the parasite of malignant tertian or aestivo-autumnal malaria.

The benign malarial fevers are characterized by a frank chill
and well marked distinctions of cold, hot and sweating stages.
In malignant tertian there is an indefinite or dumb chill with
prolonged hot stage. Diagnostic of malaria are periodicity,
parasites and splenic enlargement. The malignant tertian parasite
is the one responsible for the so-called cerebral and algid
manifestations of perniciousness. Man is the intermediate host of
the parasite while the sexual cycle or sporogony goes on in some
species of mosquito of the anopheline subfamily, the definitive
host.

=Synonyms.=—Remittent Fever, Intermittent Fever, Ague, Marsh Fever, Paludism, Jungle Fever.

French: Paludisme. German: Wechselfieber.

HISTORY AND GEOGRAPHICAL DISTRIBUTION

=History.=—Hippocrates, who considered malaria as intimately
connected with bile, divided the disease into quotidian, tertian
and quartan, differentiating such types of fever from continuous
fevers. It is interesting to note that Celsus recognized two types
of tertian fever, the one benign and similar to quartan fever, the
other far more dangerous, with a fever occupying thirty-six of the
forty-eight hours, not entirely subsiding in the remission, but
being only mitigated.

In the time of Caesar views were expressed by Varro that swamp air
might be the cause of malaria and furthermore that animals, so
small that the eye could not follow them, might transmit diseases
by way of the mouth or nose.

In the view of our present knowledge it is remarkable that Lancisi,
in 1718, should have associated marshes with the development of
gnats, which insects he thought could not only introduce with
their proboscides the putrefying organic matter of such swamps but
animalcules as well.

In 1638 Countess del Chinchon, the wife of the Viceroy of Peru, was
cured of an intermittent fever by the employment of the bark of
certain trees which bark was introduced into Europe in 1640. The
origin of the name cinchona is thus explained.

While Morton and Sydenham in 1666 noted the specific action of
cinchona in certain fevers it remained for Torti, in 1753, by the
use of cinchona, clinically to differentiate those fevers which
were cured by cinchona from those which failed to yield to this
specific. Quinine was not introduced until after 1820. Audouard, in
1803, was the first to draw attention to the splenic enlargement of
malaria.

The views of Nott and Beauperthuis as to transmission of malaria
and yellow fever by insects are considered under the latter disease.

In 1847 Meckel announced that the dark color of malarial organs
was due to a pigment and in 1848 Virchow noted that this pigment
was contained in cells. In 1875, Kelsch observed pigmented bodies
in malarial blood and in 1880 came to the conclusion that these
pigmented cells were diagnostic of malaria.

The year 1880 is the most important one in the history of malaria
for on November 6, 1880, Laveran, at Constantine, first saw the
parasites of malaria while carrying on investigations as to the
origin of the pigmented bodies and melaniferous leucocytes. He not
only noted the findings of spherical pigmented bodies but also of
crescents and in particular the flagellation of the male gamete
which demonstrated to him that these were living bodies.

The name _Oscillaria malariae_ was proposed on account of the
movements of the flagellate body, but had to be dropped as not
valid, the generic name _Oscillaria_ having been previously applied.

When these bodies were demonstrated to various Italian authorities,
in 1882, they were thought by them to be degenerated red cells.

It may be stated that at this time the Italians, influenced by
the work of Pasteur, were convinced that an organism, _Bacillus
malariae_, reported by Klebs and Crudeli (1879) to have been
isolated from water and soil of malarious districts, was the cause
of malaria. This bacillus was said to be cultivable on ordinary
media and to be capable, when injected into man, of producing
malaria.

By 1885 the Italians were convinced that the bodies discovered by
Laveran were the cause of malaria and Marchiafava, by staining with
methylene blue, noted the ring forms and the increase in size up
to that of the sporulating parasites. To Golgi we not only owe the
discovery that the malarial paroxysm coincides with the period when
the sporulating forms (merocytes) simultaneously reach maturity
but also the exact working out of the cycle of quartan malaria.
He even showed three stages of development of the parasites in a
triple quartan. It may be stated that Golgi, Marchiafava and Celli
are the ones to whom we owe our first knowledge of the existence
of different species of parasites for different kinds of malaria.
In these investigations they showed that as a rule they could
reproduce a certain type of malaria by injecting the blood of
such a case of malaria into a well man. Gerhardt, in 1884, was
the first to produce malaria by the injection of malarial blood.
Laveran insisted all this time that there was but a single species
of malaria. About this period a great deal of research was carried
on as to the origin of malarial parasites and it was found that
many animals harbored parasites similar to the malarial parasites
of man. In 1891 the chromatin staining method of Romanowsky was
introduced which by bringing out the variations in chromatin
distribution led to more accurate study of species and cycles.

Our present exact knowledge as to the existence of 3 species of
malaria is largely due to the careful examinations made by Koch of
fresh and stained malarial blood preparations.

In 1894 Manson formulated the hypothesis of the mosquito
transmission of malaria. He based this upon the fact that the
flagellation of the male gamete does not take place for several
minutes after the removal of the blood from the peripheral
circulation. He also suggested that larvae might feed upon infected
mosquitoes dying upon the water and thus acquire the disease.

Ross for two years had mosquitoes feed upon the blood of malarial
patients which contained crescents but as he used insects of the
genera _Culex_ and _Stegomyia_ he failed to observe development
in the tissues of the mosquitoes. In 1897 he used 8 dappled-wing
mosquitoes (Anopheline) and in two of these, upon dissection, he
noted pigmentary bodies different from anything he had observed in
hundreds of dissections of other mosquitoes. At this time he was
forced to discontinue this work for about six months.

In 1886 Metschnikoff from observation of sporulating parasites in
the brain capillaries at the autopsy of a malarial case considered
them to be coccidial in nature. In 1892 Pfeiffer, studying the
Coccidia showed that there was an endogenous cycle going on in
the epithelial cells as well as the long known exogenous cycle
connected with the ingestion of oocysts passing out in the feces
of an animal infected with coccidiosis. He suggested that malaria
might similarly have an exogenous cycle as well as the well-known
endogenous one. Opie noted hyaline and granular forms of parasites
in the blood of crows and MacCallum, working with this malaria-like
disease of birds (_Halteridium_), observed the fecundation of a
granular female parasite by the flagellum-like process of the
hyaline male cell.

In 1898, in India, working with a malarial disease of sparrows
(_Proteosoma_), Ross infected 22 out of 28 healthy sparrows by
mosquitoes which had previously fed on sick sparrows. He noted in
the culicine mosquito employed for transmission the same cycle of
development as that subsequently worked out for human malaria, in
anopheline mosquitoes, by Grassi and Bignami, in Italy.

Koch’s great work in connection with malaria was to demonstrate
that the malaria-like infections of other animals had no part in
the causation of human malaria and that the malarial parasite could
only circulate between man and certain mosquitoes.

In order to demonstrate conclusively the connection between
infected mosquitoes and malaria Sambon and Low lived for three of
the most malarious months of 1900, in one of the most malarious
sections of the Roman Campagna, in a mosquito screened hut and did
not contract malaria.

Infected mosquitoes were also sent to London from Italy and allowed
to feed upon Doctor P. T. Manson and Mr. George Warren. After
a period of incubation these volunteers came down with typical
malaria with parasites in the blood.

In 1911 Bass first cultivated the parasites of malaria.

=Geographical Distribution.=—Malaria is so widely distributed
over all parts of the tropical and subtropical world that it would
require too much space to give its geographical distribution other
than as given in the accompanying chart. The malaria belt may be
said to extend from 60° N. to 40° S. Many of the islands of the
Pacific are exempt.

ETIOLOGY AND EPIDEMIOLOGY

=Etiology.=—There are at least three species of animal parasites which produce human malaria, _Plasmodium vivax_, the cause of benign tertian, _P. malariae_ of quartan and _P. falciparum_ of aestivo-autumnal. These parasites belong to the haemamoeba type of the order Haemosporidia, of the class Sporozoa and of the phylum Protozoa.

This type of Haemosporidia is characterized by invasion of red
cells, amoeboid movement, pigment production and the extrusion of
flagellum-like processes from the male sporont after the blood is
taken from the animal and allowed to cool.

Other Haemosporidia which are very important in diseases of
domesticated animals, but not for man, are those of the piroplasm
type.

These parasites of the red cells do not produce pigment and do
not “exflagellate.” It is to parasites of this type that some
authorities have ascribed the cause of blackwater fever, a
condition undoubtedly connected with malaria.

It has been thought proper by some to consider the malarial parasites as belonging to two genera, the genus _Plasmodium_, characterized by round sexual forms and including _P. vivax_ and _P. malariae_ and the genus _Laverania_, characterized by crescent-shaped sexual forms and including but one species _L. malariae_, that of aestivo-autumnal malaria.

Craig recognizes a quotidian form and a tertian form for the
aestivo-autumnal parasite. Manson formerly held the view that three
different species of crescent-bearing parasites were concerned
in malignant infections; one, of tertian periodicity, _Laverania
malariae_, and two, of quotidian periodicity, _L. praecox_, a
pigmented form, and _L. immaculata_, a form in which pigment is
only observed in the crescent formation and does not exist in the
ring form schizonts. He has abandoned this view. Stephens has noted
a parasite which has more nuclear material than _P. falciparum_
(_P. tenue_).

_Malaria of Animals_.—Other Haemosporidia of the haemamoeba type
are found in birds, monkeys, bats, squirrels and possibly in
reptiles (the parasites of reptiles, while intracorpuscular and
pigment producing, do not exflagellate). Of particular interest is
the so-called bird malaria or _Proteosoma_, a parasite very similar
to the human malarial ones.

The life cycle of this parasite was demonstrated before that of the
malarial parasites of man.

Although Koch in his work showed that these malaria-like parasites
of other animals were not infectious for man, Fermi has recently
carried out well-controlled experiments, by feeding laboratory
bred anophelines on the blood of various animals showing such
infections, and subsequently on men, with invariably negative
results.

Accumulated experience shows that man is not susceptible to any of the animal malarias and that the three human species can only exist in man as an intermediate host and in certain species of anopheline mosquitoes as definitive hosts. Culicine mosquitoes never transmit malaria.

_Malaria-Transmitting Mosquitoes_.—In the United States,
_Cellia albimana_, _C. argyrotarsis_, _Anopheles crucians_,
_A. quadrimaculatus_ and _A. pseudopunctipennis_ are efficient
transmitters of malaria. Rather remarkable is the experience of
Beyer in New Orleans that _A. crucians_ will only transmit _P.
falciparum_ while _A. quadrimaculatus_ will transmit _P. vivax_ and
_P. malariae_, but not _P. falciparum_. Further experiments have
shown that _A. crucians_ will transmit _P. vivax_ as well as _P.
falciparum_.

As showing the uncertainty attaching to the question of a certain
anopheline species being efficient hosts for malaria may be cited
the case of _A. punctipennis._ This species has been frequently
reported as incapable of transmitting malaria and quite recently
Mitzmain reported experiments on 219 females of the species which
had fed on crescent containing blood and which were dissected
from three to thirty-eight days after such feedings with negative
findings in stomach and salivary glands. Furthermore, these
mosquitoes failed to transmit malaria to healthy persons. Control
experiments with _A. quadrimaculatus_ and _A. crucians_ were
successful. In June, 1916, Dr. King reported 33% of positive
findings after dissection of _A. punctipennis_ which had fed on
malignant tertian cases and 85% of success where the man bitten
had benign tertian malaria. These results showed as high a degree
of success as that obtained with the control _A. crucians_ and _A.
quadrimaculatus_.

From the above it must be evident that there are other factors
involved besides that of the host species as both Mitzmain and King
are expert epidemiologists.

A species which may be the chief transmitter in one country may
be unimportant, though present, in another country. Thus _Cellia
albimana_ is the chief malarial transmitter of Panama although _C.
argyrotarsis_ is present. In Brazil the conditions are reversed,
probably due to _C. albimana_ thriving best where slightly brackish
pools of standing water abound, as in Panama.

In the Philipines _A. febrifer_ seems the important transmitter. It
freely enters houses and is a vicious biter.

In India the species which seem most active in transmitting malaria
are _Myzomyia culicifacies_ and _M. listoni_; while in Africa, _M.
funesta_ is very efficient.

In Europe _A. maculipennis_ and _A. bifurcatus_ are important.

The following species of anophelines selected from the different genera are important transmitters of malaria.

_Anopheles maculipennis_.—Wings with four spots located at bases
of both forked cells and of second and third longitudinal veins. No
costal spots. Palpi yellowish brown and unbanded. Legs unbanded.

_Anopheles punctipennis_.—Wings with black costa showing yellow
spots at apical third and at apex. The apical spot involves the
first long vein and upper branch of first fork cell. The larger
spot at the apical third passes through the first long vein and to
the second vein just before it branches. In _A. pseudopunctipennis_
the markings are as above but the fringe has yellow spots.

_Myzomyia funesta_.—Wings with four yellow spots on a black costa
and two black line spots on third longitudinal vein. Palps with
three white rings. Proboscis unbanded. Legs with faint apical bands.

_Pyretophorus costalis_.—Costa black with five or six small yellow
spots. Palps with two narrow white bands and white tip. Femora and
tibiae with yellow spots. Apical tarsal bands.

_Myzorhynchus pseudopictus_.—Black costa with two pale yellow
spots. Wing fringe unspotted. Black palps with four pale bands.
Apex of palps white.

_Nyssorhynchus fuliginosus_.—Black costa with three large yellow
spots. Numerous black dots on the longitudinal veins. Palpi black
with white tip and two narrow white bands. Last three hind tarsal
segments white.

_Cellia argyrotarsis_.—Black costa with two distinct and several
smaller white spots.

While anophelines are usually rural or at any rate preferring the suburbs of cities yet we can differentiate between domesticated and wild anophelines, these latter keeping away from man and consequently not playing a transmitting rôle.

Another factor in their becoming an efficient host appears to rest
in the feeding habits of such anophelines, one which is voracious
and fills and then ejects by rectum the blood taken from the
malarial patient is more apt to be a transmitter than a species
less greedy.

By an _efficient host_ is meant a species in which full development of the parasite takes place.

LIFE HISTORY OF THE MALARIAL PARASITE

Malaria can be transmitted by subcutaneous or intravenous injection of the blood of a patient with the disease into a well person, the same type being reproduced.

=Transmission of Malaria.=—Such a method of transmission is only of scientific interest and the regular method is as follows: An infected anopheline at the time of feeding on the human blood introduces through a minute channel in the hypopharynx the infecting sporozoite of the sexual cycle.

When man is first infected by sporozoites we have starting up a
nonsexual cycle (schizogony) which is completed in from forty-eight
to seventy-two hours, according to the species of the parasite.
The falciform sporozite bores into a red cell, assumes a round
shape and continues to enlarge (schizont). Approaching maturity,
it shows division into a varying number of spore-like bodies. At
this stage the parasite is termed a merocyte. When the merocyte
ruptures, these spore-like bodies or merozoites enter a fresh cell
and develop as before.

=Malarial Toxin.=—At the time that the merocyte ruptures it is supposed that a toxin is given off which causes the malarial paroxysm.

Rosenau, by injecting, intravenously, filtered blood, taken from
a patient at the time of sporulation of the parasites caused a
malarial paroxysm. No parasites developed later. Another man who
received a small amount of unfiltered blood allowed a slight
paroxysm and four days later showed parasites in his blood. Hence
the parasite will not pass through the pores of a Berkefeld filter.

=Schizogony.=—The nonsexual cycle goes on by geometric progression from the first introduction of the sporozoite, but it is usually about two weeks before a sufficient number of merocytes rupture simultaneously to produce sufficient toxin for symptoms (period of incubation). This cycle is termed _schizogony_. It is considered that there must be several hundred parasites per cubic millimeter sporulating to be capable of producing symptoms.

=Gametes.=—After a varying time, whether by reason of necessity
for renewal of vigor of the parasite by a respite from sporulation,
or whether from a standpoint of survival of the species, sexual
forms (gametes) develop. Some think that sporozoites of sexual and
nonsexual character are injected at the same time. It is usually
considered, however, that sexual forms develop from preexisting
nonsexual parasites. The developing gametes are often termed
sporonts. Strictly, the sexual parasites in the blood should be
called gametocytes. The gametes take about twice as long to reach
maturity as schizonts. The life of a crescent has been estimated
as about ten days and that of the gametes of benign tertian and
quartan about one-half this period.

=Sporogony.=—The gametes show two types the one which contains
more pigment, has less chromatin, and stains more deeply blue is
the female—a macrogametocyte; the other with more chromatin, less
pigment, and staining grayish green or light blue is the male—a
microgametocyte. When the gametes are taken into the stomach of the
Anophelinae, the male cell throws off spermatozoa-like projections,
which have an active lashing movement and break off from the now
useless cell carrier and are thereafter termed microgametes.
These fertilize the macrogametes and this body now becomes a
zygote. (Following nuclear reduction with formation of polar
bodies the macrogametocyte becomes a macrogamete). This process
of exflagellation can be observed in a wet preparation under the
microscope. There is first seen a very active movement of the
pigment of the male gamete and finally long delicate bulbous-tipped
flagellum-like processes are thrown off (exflagellated) and push
aside the red cells by their progressive motion. MacCallum saw a
female _Halteridium_ fertilized by the microgamete, after which it
was capable of a worm-like motion (vermiculus or ookinete).

By a boring-like movement the vermiculus stage of the zygote goes
through the walls of the mosquito’s stomach, stopping just under
the delicate outer layer of the stomach or mid-gut. In three or
four days after fertilization the zygote becomes encapsulated and
is then often called an oocyst. It continues to enlarge until about
the end of one week it has grown to be about 50µ in diameter and
has become packed with hundreds of delicate falciform bodies. Some
only contain a few hundred, others several thousand.

=Zygotes.=—In some of his observations Darling has noted that the
zygote of benign tertian malaria grows larger and more rapidly
than that of aestivo-autumnal and that the pigment is clumped
rather than in belts or lines as with aestivo-autumnal. Darling has
also noted that mosquitoes do not tend to become infected unless
the gamete carrying man has more than 12 gametes to the cubic
millimeter of blood. Rouband notes that the oocysts of _P. vivax_
are feebly refractile with fine granules of gray pigment in loose
chains while _P. falciparum_ ones are highly refractile with large
grains of black pigment. At a temperature of 25°C. vivax completes
its cycle in 11 days while the zygote of the crescent requires 14
days. Apparently it is possible for a mosquito to carry both types
of parasites.

The capsule of the mature zygote ruptures about the tenth day
and the sporozoites are thrown off into the body cavity. They
make their way to the salivary glands and thence, by way of the
veneno-salivary duct, in the hypopharynx, they are introduced into
the circulation of the person bitten by the mosquito, and start a
nonsexual cycle. As the sexual life takes place in the mosquito,
this insect is the definitive host and man only the intermediate
host. The sexual cycle or _sporogony_ in the mosquito takes about
ten to twelve days.

=Efficient Mosquito Hosts.=—It must be remembered that only certain genera and species of Anophelinae are known malaria transmitters; thus Stephens and Christophers, in dissecting 496 mosquitoes of the species _M. rossi_, did not find a single gland infected with sporozoites.

With _M. culicifacies_, however, 12 in 259 showed infection. A
mosquito which is capable of carrying out the complete sporogonous
cycle is an efficient host and in the case of malaria the mosquito
is the definitive host (sexual life of parasite).

=Malarial Index.=—Mosquito dissection is one method of determining the endemicity of malaria or the _malarial index_. There are two other methods: 1. by noting the prevalence of enlarged spleens, and 2. by determining the number of inhabitants showing malarial parasites in the blood. This index is best determined from children between two and ten years of age, as children under two show for a general average too high a proportion of parasites in the peripheral blood while those over ten years of age show too great an incidence of enlarged spleens.

Barber working in the Philippines with children from five to ten
years of age obtained a spleen index of 13.3 and a parasitic index
of 11.

=As Before Stated there are Three Species of Malarial Parasites:= 1. _Plasmodium vivax_, that of benign tertian—cycle, forty-eight hours; 2. _Plasmodium malariae_, that of quartan—cycle, seventy-two hours; and 3. _Plasmodium falciparum_, that of aestivo-autumnal or malignant tertian—cycle of forty-eight hours.

=Multiple Infections.=—Variations in cycles may be produced by
infected mosquitoes biting on successive nights, so that one crop
will mature and sporulate twenty-four hours before the second.
This would give a quotidian type of fever. In aestivo-autumnal
infections anticipation and retardation in the sporulation cause
a very protracted paroxysm, lasting eighteen to thirty-six hours;
this tends to give a continued or remittent fever instead of the
characteristic intermittent type.

=Plasmodium Vivax.=—In fresh, unstained preparations, taken at
the time of the paroxysm or shortly afterward, the benign tertian
schizont, or nonsexual parasite, is seen as a grayish white, round
or oval body, whose outlines cannot be distinctly differentiated
from the infected red cell. They are about one-fifth of the
diameter of the red cell and are best picked up by noting their
amoeboid activity. In about eighteen hours fine pigment particles
appear and make them more distinct. After twenty-four hours the
lively motion of the pigment and the projection of pseudopod-like
processes, in a pale and swollen red cell, make their recognition
very easy. When about thirty to thirty-six hours old the amoeboid
movement ceases. Approaching the merocyte stage the pigment tends
to clump into one or two pigment masses and one can recognize
small, oval, highly refractile bodies within the sporulating
parasite.

The gametes or sexual forms do not show amoeboid movement, but
the fully developed gamete, which is generally larger than the
red cells, has abundant pigment, which is actively motile in
the male gamete and nonmotile in the female. The male gamete
is more refractile, is rarely larger than a red cell and shows
yellow-brown, short rod-like particles of pigment. About fifteen
minutes after the making of a fresh preparation these male gametes
throw out four to eight long, slender, lashing processes, which
are about 15 to 20 microns long. These spermatozoon-like bodies
now break off from the useless parent cell and with a serpent-like
motion glide away in search of a female gamete, knocking the red
cells about in their passage through the blood plasma.

The female gamete is larger than a red cell, is rather granular and
has more abundant dark-brown pigment than the male.

=Stained Smears.=—In dried smears, stained by some Romanowsky
method, as that of Wright, Leishman or Giemsa, we note small
oval blue rings, about one-fifth of the diameter of the infected
yellowish-pink erythrocyte. One side of the ring is distinctly
broader than the rather fine opposite end, which seems to hold
a round, yellowish-brown dot, the chromatin dot, and has a
resemblance to a signet ring. These small tertian rings of the
nonsexual parasites (schizont) are seen about the time of the
commencement of the sweating stage of the paroxysm. Two chromatin
dots in the line of the ring are rare as is also true of more than
one ring in a red cell.

When the parasite is about twenty-four hours old we note that
it contains much pigment and has an amoeboid or multiple
figure-of-eight contour, is about three-fourths the size of a red
cell and that the infected red cell is about one and one-half times
as large as in the beginning and presents a washed-out appearance.
It is an anaemic-looking cell. We also note, as characteristic of
a benign tertian infection, reddish-yellow dots in the pale red
cell, which are known as Schüffner’s dots. These, practically, are
characteristic for benign tertian.

A few hours before the completion of its forty-eight-hour cycle the
contained pigment begins to clump, the chromatin to divide and,
finally, we have a sporulating parasite, in which the 16 to 20
small, round, bluish bodies, with chromatin dots, are irregularly
distributed over the area of the merocyte.

The gametes, or sexual parasites, show a thicker blue ring and have
the chromatin dot in the center of the ring. The pigmentation of
the half-grown gametes is more marked than that of schizonts of
equal size. The shape of the gametes is not amoeboid, as is that
of the twenty-four to thirty-six-hour-old schizont, but round or
oval. _The full-grown gametes have the pigment distributed and the
chromatin in a single aggregation—just the opposite of nonsexual
parasites._ The male gamete stains a light grayish blue and has
a very large amount of chromatin, usually centrally placed. The
female gamete stains a pure blue, has only about one-tenth as much
chromatin as plasma, with the chromatin often placed at one side.
The pigment of the female gamete is dark brown while that of the
male is yellowish brown.

=Plasmodium Malariae.=—In fresh preparations the young quartan
schizont has only slight amoeboid movement and, as development
proceeds, the rather dark brown, coarse pigment tends to arrange
itself peripherally about the band-shaped or oval parasite.

The infected red cell shows but little change. At the end of
seventy-two hours the rather regular daisy form of the merocyte is
more distinct than that of the benign tertian merocyte.

The distinctions between the male and female gametes are similar to
those of the benign tertian gametes. In Romanowsky-stained smears
it is difficult to distinguish the young quartan schizont from
the benign tertian one but, after twenty-four hours, the tendency
of the quartan schizont to assume equatorial band forms across a
red cell of normal size and staining characteristics and without
Schüffner’s dots makes the differentiation easy. In the fully
developed sporulating parasite or merocyte the eight merozoites
assume a regular distribution, giving it a daisy appearance.

The gametes show practically the characteristics of the benign
tertian ones but are smaller.

=Plasmodium Falciparum.=—The young schizont of malignant tertian
is extremely difficult to detect in fresh preparations, there being
noted early in the rather long continued, hot stage, as small
crater-like dots, about one-sixth of the diameter of a red cell
which, however, show an active amoeboid movement.

Malignant tertian blood tends to show rather marked vacuolation
of the red cells and these central vacuoles have a resemblance
to young ring forms. The malarial parasites are most often
peripherally placed and they do not enlarge and diminish in size on
focusing up and down as do the vacuoles.

Later on in the hot stage these ring-like dots enlarge to become
about one-third of the diameter of a red cell, most often occupying
the periphery of the infected red cell. About this time, or at the
very commencement of the pigmentation, the schizont-containing red
cells disappear from the peripheral circulation so that the further
development is rarely observed in blood specimens.

The infected cell is brassy in color and shrunken in shape—it
shows evidences of degeneration. The gametes appear as
crescent-shaped bodies, which are absolutely characteristic of
malignant tertian, the male gamete being more hyaline and delicate
while the female one is more granular and larger.

In Romanowsky-stained preparations we see, while the fever is
sustained, small hair-like rings, with geometrical outline, with
frequently two chromatin dots in one end of the ring and a single
red cell often showing two or more of these young rings. The rings
are often seen as if plastered on the periphery of the red cells
or as if having destroyed a rounded section of the rim of the
red cell. As the fever declines the rings tend to disappear from
the peripheral circulation. The infected red cells often show
polychromatophilia and distortion.

FIG. 9.—Estivo-autumnal malarial parasites, and small ring forms and crescents. (Todd.)]

In old aestivo-autumnal cases, or those with severe infection, we
may see adult rings and merocytes, which latter are smaller than
those of benign tertian, show from 10 to 12 irregularly placed
merozoites and a sharply clumped mass of pigment.

The gametes are the striking crescent-shaped bodies and these show
the distinctions of blue-staining for the female, with lighter
gray-blue to purplish staining and abundance of chromatin for
the male. The chromatin staining of crescents does not stand out
so well as that of the round form gametes of benign tertian and
quartan.

The black pigment of the female tends to be clumped toward the
center while the rather generally distributed pigment of the male
is reddish brown rather than black in a stained preparation.

This variation of pigment color may be due to the effect of
chromatin staining, as the black of the pigment is the same in male
and female gametes in fresh blood preparations.

_Stained Smear Preferred._—As regards differentiation of species and cycle the examination of stained smears is more satisfactory and definite, as well as less time consuming. Still, one obtains many points of differentiation in the fresh preparation and should study such a preparation while carrying out the staining of his dried smear.

Central vacuolation of red cells is common in malarial anaemia and
may be mistaken for nonpigmented parasites.

Malarial rings are usually peripheral and do not vary in size as
one focuses up and down as do the central vacuoles.

_Quinine-affected Parasite._—A very puzzling but well-recognized
finding in cases treated with quinine or salvarsan is the so-called
quinine-affected parasite. Such parasites lack definiteness of
outline and show poor chromatin staining. The gametes do not seem
to show these effects from the drug.

=Certain questions connected with the life history of the malarial
parasite in man which are of interest.=

1. _Extracellular location._—It is usual to consider the parasite
as developing within a red cell and in this position to destroy the
red cell. Rowley-Lawson, however, thinks that the parasites are
exclusively extracellular and that they adhere to the red cells by
loop-like pseudopodia which encircle a portion of the red cell and
digest the haemoglobin of such an area.

2. _Relapses._—There are several views as to the etiology of
relapses in malaria. These views are taken up under relapses (see
page 35).

3. _Malarial toxin._—Nature of the toxic material thrown off by
the parasite at the time of simultaneous sporulation. Rosenau’s
experiments tend to show that there is a fever-producing toxin
thrown off at this time. Other authors have thought that a
haemolysin and an endotheliolysin were thrown off at the same
time. Brown considers that the pigment produced by the parasite,
in its metabolism of the haemoglobin of the red cell, may act as a
haemolysin, he having found that intravenous injections of haematin
were capable of producing marked anaemia. It is well known that
a far greater number of red cells are destroyed in a paroxysm
than would be accounted for by the actual percentage of red cells
destroyed by parasites. The endothelial cells take up actively this
malarial pigment or haemozoin and are damaged or destroyed thereby.
Haematin injections also tend to destroy leucocytes and platelets.

Rowley-Lawson is of the opinion that the greater red cell
destruction than would be represented by percentage of cells
showing parasites is explained by parasites migrating from cell to
cell so that many red cells may be destroyed by a single parasite.

4. _Transmission to larvae._—There has been an idea that
sporozoites might enter the ovaries and ova as well as the salivary
glands so that a second generation of mosquitoes might transmit
malaria. There is no proof that such a method is ever operative.

5. _Congenital malaria._—There has been some question as to the
possibility of congenital malaria. Heiser has recently reported the
case of an infant which showed crescents in its blood by the end of
one week from birth. The mother showed the same infection and the
child must have been infected through the placental circulation.

Clark in numerous examinations of the blood of the new-born
failed to find infection even when the mother’s blood teemed with
parasites. In one case where the child showed infection shortly
after birth there had been an accident to the placenta and he
believes that instances of so-called congenital malaria are to be
explained in this way.

6. _Cultivation of parasite._—As to cultivation of malarial
parasites. Bass takes from 10 to 20 cc. of blood from the malarial
patient’s vein in a centrifuge tube which contains 1/10 cc. of
50% glucose solution. A glass rod, or a piece of tubing extending
to the bottom of the centrifuge tube is used to defibrinate the
blood. After centrifugalizing there should be at least one inch
of serum above the cell sediment. The parasites develop in the
upper cell layer, about 1/50 to 1/20 inch from the top. All of the
parasites contained in the deeper-lying red cells die. To observe
the development, red cells from this upper 1/20 inch portion are
drawn up with a capillary bulb pipette.

Should the cultivation of more than one generation be desired,
the leucocyte upper layer must be carefully pipetted off, as the
leucocytes immediately destroy the merozoites. Only the parasites
within red cells escape phagocytosis. Sexual parasites are much
more resistant. Bass thinks he observed parthenogenesis. The
temperature should be from 40° to 41°C. and strict anaerobic
conditions observed. Aestivo-autumnal organisms are more resistant
than benign tertian ones. Dextrose seems to be an essential for the
development of the parasites.

Bass considers that _P. vivax_ has a disc-like structure which
enables it to squeeze through the brain capillaries while adult
schizonts of _P. falciparum_ have a solid oval form which causes
them to be caught in the capillaries.

The Thompsons have rather simplified the method of Bass. They draw
10 cc. of blood into a test tube containing the usual amount of
glucose solution. They then defibrinate the blood by stirring with
a thick wire for about five minutes and remove the wire with the
adhering clot. They then pour this defibrinated blood into several
small sterile test tubes, which should contain at least a one-inch
column. Rubber caps are adjusted over the cotton plugs and the
tubes placed in the incubator. They note the tendency of cultures
of _P. falciparum_ to agglutinate which is not true of _P. vivax_.

They think this agglutination the great cause of the plugging of
capillaries in pernicious malaria. They note 32 merozoites as
maximum number in sporulation of _P. falciparum_ while _P. vivax_
has usually 16 or more, but never as many as 32.

This would explain the shorter incubation period of malignant
tertian. The pigment of _P. falciparum_ clumps much earlier in the
developing schizont than that of _P. vivax_ and is much coarser and
more discrete.

While Bass thought he noted parthenogenesis in cultures others have
failed to observe any evidence of it.

7. _Immunity._—As to immunity. There is no real immunity to
malaria, it is a continuance of the infection, but the parasites
are not in sufficient numbers to give rise to fever. If, however,
the patient becomes chilled or fatigued or otherwise depressed,
fever results.

This apparent immunity is also kept up by reinfection, because
if natives leave the locality for a length of time they lose it.
Patients who show this apparent immunity to one form of malaria
have no such resistance to the other types. Bass states that immune
bodies are produced in malaria and that immune processes contribute
to control of the infection, but that it is not lasting and is not
effective against new infection.

8. _Perniciousness._—Causes of perniciousness. This is taken up
under perniciousness in malaria. (See page 31.)

9. _Quinine-affected parasite._—Effect of quinine on malarial
parasites. It is usually thought that the merozoites at the time
of being thrown off from the merocyte are most vulnerable, while
the gametes are only slightly affected, if at all. Still, the young
forms from which gametes develop are destroyed. Quinine causes
parasites to disappear from the peripheral circulation and produces
degenerative changes in such parasites as may remain. Bass thinks
that quinine makes the red cell permeable to the lytic action of
serum. Anaemia may cause degenerative changes in parasites similar
to that from quinine.

10. _Anaphylaxis and the paroxysm._—Abrami has brought forward
evidence in favor of the malarial paroxysm being due to the
outpouring of merozoites into the blood plasma which act as
foreign antigen. It is noted that the dissemination of merozoites
takes place some hours before the cold stage which is one of the
manifestations of anaphylactic shock. They note a leucopenia and
lowering of the blood pressure preceding the paroxysm as evidence
of a haemoclastic crisis.

The Anopheline Mosquito

The ova of culicine mosquitoes are usually deposited in a scooped-out raft-like mass of about 250 eggs set vertically. The raft is easily seen with the eye, being about ⅕ inch long. The anopheline eggs are oval in shape with pleated air cell projections laterally. They are laid upon the surface of the water, to the number of about 100, in star, triangle or ribbon patterns. The egg stage is two to four days but shorter, however, in the tropics.

The larval stage is the most important one to be acquainted
with because in this stage one can most readily distinguish the
anopheline or possible malaria transmitter from a culicine species.
One can more readily and quickly make a survey for anophelines by
examining the collections of water for larvae than in any other
way. The anopheline larva seems to prefer the surface, on which
it lies flat and out of the water. To keep it from turning over
on its long axis, it has little rosette-like hair tufts on the
dorsal surface of the 5 or 6 middle abdominal segments (palmate
hairs). There are feathered lateral hairs projecting from thorax
and abdominal segments. The head is very small in comparison with
the thorax and can be rotated with lightning-like rapidity. There
is no projecting breathing tube or syphon from the next to the last
abdominal segment, as is characteristic of _Culex_, _Stegomyia_ or
any other culicine genus.

In addition, culicine larvae do not float parallel to the surface
of the water, but hang suspended at an angle, with only the tip
of the syphon pushed upward to the surface. The lateral hairs or
bristles are not feathered and the head is much larger than that of
the anopheline larvae. It is the fact of the surface position of
these anopheline larvae which enables them to worm their way over
film layers of water or between blades of grass, in grass or rush
studded pools or swamps.

In the pupal stage it is rather difficult to differentiate
species of mosquitoes from each other, so that, other than to
recognize that the bloated shrimp-like body is a mosquito pupa, is
unnecessary.

DIFFERENTIATION OF CULICINAE AND ANOPHELINAE

It is impossible even for an entomologist to determine the species of mosquitoes without recourse to elaborate keys and tables. It is a comparatively easy matter, however, to decide as to whether the mosquito is a probable malaria transmitter or not.

_The male anopheline._—While certain characteristics of the male
are used to separate the Aedinae from other subfamilies, yet it is
only with the female that we concern ourselves in differentiating
the Culicinae from the Anophelinae. Therefore, it is first
necessary to distinguish the male from the female. If the antennae
have not been torn off, this can be decided by the highly adorned
plumose antennae of the male, those of the female being sparsely
decorated with short hairs. The palpi of the male _Anopheles_ tend
to be clubbed, while those of the _Culex_ are straight. If the
antennae have been broken off, look for the claspers at the end of
the abdomen.

Male mosquitoes do not feed on blood but on fruits and flowers instead. The puncturing parts of the male are not sufficiently resistant to penetrate the skin.

_The female anopheline._—Having determined that the insect is
a female, we then proceed to place it either in the subfamily
Culicinae or Anophelinae by a study of the relative length of the
palpi to the proboscis. If the palpi are much shorter than the
proboscis, it belongs to the Culicinae; if about as long or longer,
to the Anophelinae. The palpi of the female Megarhininae are also
long, but the proboscis is curved.

Having settled on the subfamily, we separate the genera by considering such points as character and distribution of scales on back of head, wings, thorax, and abdomen; banding of proboscis, legs, abdomen, and thorax, shape of scales on wings, and location of cross veins.

Anophelinae show abundant upright forked scales on occiput. The
mesothorax shows sparse hairs or scales with a smooth scutellum. As
a rule, the wings are spotted (dappled) and the location of these
spots gives the best clue to the different species of the genera.
With the exception of _Bironella_ the first submarginal cell is
large. This cell is longer than the second posterior one.

In the resting position _Culex_ allows the abdomen to droop, so
that it is parallel to the wall. The angle formed by the abdomen
with head and proboscis gives a hunchback appearance.

_Anopheles_ when resting on a wall goes out in a straight line at
an angle of about 45°. It resembles a bradawl.

The scutellum of _Anopheles_ is simple, that of _Culex_ trilobed.
_Anopheles_ has but one spermatheca; _Culex_ has three.

=Anophelinae=

{ 1. Scales on wings, large and lanceolate.
1. Scales on head only; { _Anopheles._ Palpi only slightly scaled.
hairs on thorax and { 2. Wing scales small and narrow and lanceolate.
abdomen. { _Myzomyia_. Only a few scales on palpi.
{ 3. Large inflated wing scales.
{ _Cycloleppteron._

2. Scales on head and {
thorax (narrow curved { 1. Wing scales small and lanceolate.
scales). Abdomen with { _Pyretophorus._
hairs. {

{ 1. Abdominal scales only on ventral surface.
{ Thoracic scales like hairs. _Myzorhynchus._
{ Palpi rather heavily scaled.
3. Scales on head and { 2. Abdominal scales narrow, curved or
thorax and abdomen. { spindle-shaped. Abdominal scales as tufts
Palpi covered with { and dorsal patches. _Nyssorhynchus._
thick scales. { 3. Abdomen almost completely covered with
{ scales and also having lateral tufts.
{ _Cellia._
{ 4. Abdomen completely scaled. _Aldrichia._

NOTE.—Of the above genera only _Cycloleppteron_ and _Aldrichia_
are unproven malarial transmitters.

The female anopheline mosquito alone bites man, the male feeding
on fruits and flower juices. The female absolutely requires blood
for the development of her eggs after fertilization by the male
mosquito.

The anopheline mosquito bites at night or toward evening and
selects some dark place or dark colored wall to sleep against
during the day. Hence the advantage of a buff colored wall
interior. It is well to remember that the malarial incidence may be
kept down by killing the mosquitoes inside of a house by striking
them with a folded paper or piece of wire gauze on a handle (fly
swatter).

It is not a bad plan to have a dark colored surface in a room to
attract them and make their destruction easy.

Anophelines do not like wind and seek protection of underbrush.
As regards distance of flight from breeding places Metz has noted
that _A. crucians_ were not distributed generally over 7000 feet
and rarely were found between 7000 and 9000 feet beyond which
distance they were not found. Some anophelines get accustomed to
feeding exclusively on animals. Mosquitoes may hibernate through
the winter and possibly cause new infections the following spring.
Cases of malaria in the spring are however usually due to relapses.
Mitzmain’s negative experiments with hibernating mosquitoes _prove
man_ to be the _winter carrier_.

The malarial zygote will not develop in the stomach of the mosquito if the temperature is below 16°C. (60°F.). It would seem that the zygote of _P. malariae_ will develop at a lower temperature than that of the other two species, _P. falciparum_ requiring the highest temperature.

Our views as to temperature requirements for the development of zygotes in the mosquito must be changed as King has recently shown that _P. vivax_ sporonts will survive exposure to temperatures of 30°F. for two days and _P. falciparum_ temperatures of 35°F. for one day. This proves that temperatures approximating freezing ones will fail to destroy the parasites of hibernating mosquitoes.

Wenyon has found experimentally that mosquitoes which had fed on
malarial blood and kept at incubator temperatures for a week to
allow development of zygotes showed inhibition of development of
zygotes when kept at temperatures corresponding to hibernating
ones. This treatment did not kill the zygotes but complete
development took place when subsequently the mosquitoes were again
subjected to incubator temperatures. From this it would seem that
the zygotes remain viable during the winter hibernation. This is
at variance with Mitzmain’s views who regarded hibernation as
destructive to zygotes.

The mosquito does not seem to suffer from her malarial infection—quite different from the serious affection that filariasis causes in the mosquito.

=Epidemiology.=—This matter has been considered rather extensively under the historical and etiological discussions.

It may be stated however that the requirements for the spread of malaria are: (1) Men who have sexual forms of the malarial parasite in their peripheral circulation; (2) efficient anopheline hosts, and (3) an atmospheric temperature above 60°F. (16°C.).

It is a well recognized fact in the tropics that the natives
seem to have an immunity to malaria yet may carry parasites in
their circulation and serve as carriers. The native children to
a striking degree harbour parasites and to them malaria is a
prime cause of death. After repeated infections, if not fatal, a
temporary immunity is acquired. Many localities in the tropics
owe freedom from malaria to an absence of anophelines, as for
instance Barbadoes. Again malaria-bearing mosquitoes may acquire
the habit of feeding on animal blood other than that of man. It is
well recognized that rural populations are more liable to malaria
than those of towns and as the population of a country moves to
the industrial centres human blood may become difficult to obtain
and the anophelines turn to other sources of blood supply. It has
been suggested that mosquitoes may suffer from other infections
which may be inimical to the development of malarial zygotes (black
spores of Ross). Anophelines bite chiefly at sunset and at night
from which fact there would seem to be some value in shutting the
windows towards nightfall as is the custom in many malarious parts
of the world.

Pools containing a border growth of grass or rushes are often
selected by anophelines for depositing eggs. The small fish
or tadpoles, which prey on the larvae, cannot work their way
through the obstacles and, again, petroleum oil cannot be easily
distributed in a network of grass. Anophelines of different species
and of different countries seem to vary in their selection of water
for depositing their eggs. We should not generalize but go out and
search for breeding places.

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The diagnostics and treatment of tropical diseasesChapter I: Malaria (1)

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