Related to Horton Hospital Malaria Therapy Unit
Description:

‘In the earlier work on malaria-therapy, the fever was induced by the injection of infected blood. It was recognised, however, that in certain cases there might be serious objections to the method of inducing malarial attacks by the direct inoculation of blood from patient to patient. This would be the case especially if the donor were suffering from general pareisis, under which conditions the risk of transmitting a syphilitic virus, possibly one with neurotropic characters, to a non-infected patient should never be taken.’ (4)

 

‘The laboratory portion of this unit was under the control of Colonel James, assisted by Mr. P.G. Shute and one laboratory assistant (later two) supplied by the Ministry of Health, while Dr. W.D. Nicol, a medical officer at the London County Council’s Mental Health Service was in charge of the clinical side of the work.

The origianal ward consisted of mosquito-proofed wards holding 12 beds, and two small rooms fitted up for laboratory work in connection with the examination of blood and with the infection of mosquitoes. Since that time there has been considerable expansion, necessitated by a steadily increasing demand for the treatment of cases. The special wards now provide for 23 patients (11 males and 12 females), and it is hoped to extend this accommodation in the near future. These wards are used only for the care of patients suffering from the acute stages of malaria, or who are infective to mosquitoes. Patients who have received treatment, who do not require special observation for malaria, and who are no longer infective to mosquitoes, are accommodated in other parts of the hospital. The laboratory unit now consists of five rooms for routine and research work, a special insectary for breeding mosquitoes, and a room (the temperature of which is thermostatically controlled) for the incubation and preservation of infected mosquitoes.’ (5)

 

‘The Ministry of Health’s Malaria Laboratory at Horton is the “official” source of supply of pure strains of malaria parasites for the purpose of malaria-therapy at mental hospitals, general hospitals, nursing homes, etc., in this country generally. Originally it was intended that the only “official” material supplied for this purpose should be infective mosquitoes, but requests for such treatment soon exceeded expectations. [note: ‘The number of hospitals in England and Wales which have been supplied frequently with infected material is 284 at present...’]

The experimental transmission of malaria by mosquito bites necessitates the carriage of teh infected insects and their application to the patient by a trained assistant. In practice this has not been found possible in all cases treated in institutions situated at long distances from the laboratory. [note: ‘In some of the large provincial hospitals... the infection is often transmitted by direct blood inoculation from patient to patient. Every few months, however, the series of blood inoculations is started afresh by means of mosquito inoculation supplied from Horton.’]. (5)

 

‘In all early work, reliance was placed entirely upon infections with benign tertian malaria (Plasmodium vivax). Such infections are relatively harmless provided that the physical state of the patient is fairly good before the inoculation is made, and careful watch is kept on his condition... By the timely administration of appropriate treatment and by careful nursing to keep the temperature within bounds, it is usually possible to hold the infection in check and damp down its severity. Where these precautions are not taken, the case mortality has sometimes been as high as 10-14 per cent. (Meagher, 1929). However, where specialist experience in the care and management of cases of malaria is assisted by organised arrangements for expert laboratory control and experienced trained nursing, the actual death rate directly attributable to benign tertian malaria is seldom more than about 3.4.per cent.

Under the special controlled laboratory and hospital conditions at Horton, it has been possible to investigate with safety the therapeutic possibilities not only of P. vivax, but also of P. falciparum, P. malariae, P. ovale and P. knowelsi. Infection with these other species of Plasmodium have not been found to be useful for routine distribution as has been that of P. vivax... Infections with P. falciparum are usually much more severe than those with P. vivax, and, if not very carefully watched and controlled, may be very dangerous and may quickly lead to fatal results (James, Nicol and Shute, 1932). [note: Infections with the Roumanian strain of P. falciparum, at present in use at Horton, are rapidly controlled by atebrin.’] In the case of P. ovale, the infections produced are usually less severe than those of P. vivax, and are therefore less suitable as a rule for obtaining beneficial results in general paresis. This species is also less easy to transmit by the technique of mosquito infection. While P. malariae may give good and prolonged bouts of fever, it has not been found possible to transmit the infection by mosquito bite... and the disease is liable to become chronic with [6-7] possible deleterious effects to the patient (anaemia, albuminaria). The results obtained by infections with P. knowelsi were too irregular and uncertain to make this parasite suitable for routine use in malaria therapy.

Infections with these other species of Plasmodium have, however, been found very useful in special circumstances. They are suitable for certain patients who have resisted infection with P. vivax... and also for patients who have... developed... some degree of tolerance to infection with P. vivax. P. malariae has proved especially useful under such conditions.’ (6-7)

 

‘Several different strains of P. vivax derived from countries as far apart as India, Roumania and Madagascar, have been tried at Horton in the practice of malaria-therapy. The one selected as the most satisfactory from all points of view is the so-called “Madagascar” strain at present in use. This strain was isolated originally in 1925 from the blood of a lascar working on a ship coming from Madagascar. [note: ‘It is uncertain whether the original infection was contracted in Madagascar or in some other country abroad, but for the sake of convenience it is known as the “Madagascar” strain.’]... [7-8]

This strain has proved itself... to be a reliable and certain therapeutic agent for malaria-therapy, [note: ‘the “Madagascar” strain of P. vivax has been given to workers in many other countries, and has been used in most of the malaria laboratories of Europe for the treatment of general paresis and for experimental purposes. The countries include Austria, France, Germany, Holland, Italy, Roumania and Spain.’] and one which is easy to transmit both by blood and mosquito inoculation. Since it was established, 132 batches of mosquitoes making a total of over 27,000 insects, have been infected with it at Horton.’ (7-8)

 

‘All requests for infective material are received at the Ministry of Health, whence they are passed on to the Malaria Laboratory at Horton, where appropriate steps are taken to fulfil the demand. For patients in the vicinity of London, infected mosquitoes are usually provided, unless the medical officer asks especially for blood. For patients further afield either blood or mosquito infections are used according to the special circumstances.’ (8)

 

‘From the mass of data which has accumulated in the office of the Ministry of Health and in the laboratory at Horton, much very valuable information has been obtained, not only with regard to malaria-therapy generally, but also with regard to malarial immunity and epidemiology, the action of anti-malarial drugs, and many other facts of the utmost importance to malariologists’ (9)

 

‘Since the last detailed descriptions of the methods used at the Horton Laboratory were published about 10 years ago (James and Shute, 1926; James, 1927a), many changes have been made as the result of experience...’ (11)

 

‘Anopheles maculipennis var. atroparvus, an indigenous species of mosquito which breeds freely in captivity, is the insect of choice for the work of malaria transmission in England. The specimens used for routine work belong to an English strain obtained from an area on the Thames Estuary...

In the early work all the insects had to be caught in nature. As the collecting area was about 60 miles distant from Horton, the mosquitoes available had to be used sparingly as was consistent with efficiency. To overcome this difficulty... attempts were made to breed A. maculipennis var. atroparvus on a large scale at Horton. In 1934 a special insectary was constructed and a colony of this mosquito was successfully established. [Note: ‘Details of the construction of this insectary and of the breeding technique have been given by Shute (1936).'] This was derived originally from a single female and has flourished so well that an ample supply of insects is available at all seasons of the year. If, during winter, extra numbers are needed for some special experiments, those available are supplemented with those caught in nature.

When mosquitoes are needed, the usual procedure is to collect 100-200 pupae from the breeding pens in the insectary. These are put into clean water in a glass vessel, which is placed in a breeding cage in the hot room. The insects which have emerged are collected from the cage as required.

It has been found that freshly-hatched females of the species do not feed so greedily as do older ones, and that their first blood meal is commonly a small one. For this reason, as a rule the laboratory-bred insects are not allowed to feed upon an [11-12] infective patient until they have already had one or more blood meals, usually on a rabbit. In our work such specimens were, therefore, about one week old, and possibly in a few instances two, at the time of the first infective feed.

Most of the mosquitoes collected in nature show the remains of a blood meal when captured. Those not required for immediate use are stored in cages in an outhouse, and are allowed to feed on a rabbit at appropriate intervals. When required for transmission work, the insects are placed in the hot room for 48 hours so that any previous blood meal is completely digested, after which they will usually feed readily, especially if they have been deprived of water for 24 hours.’ (11-12)

 

‘When a suitable case is found, a batch of about 100-200 mosquitoes are allowed to feed on it. The jars used for feeding purposes are all cylindrical, of thick glass with a heavily flanged mouth, and measure about 3 ½ inches (9 cms.) in diameter and height. The bottom of each jar is fitted with a disc of dry filter paper to absorb any of the moisture which is liable to accumulate during the process of feeding, especially if the patient has fever or is sweating at the time. Over the mouth of each jar is tied a piece of mosquito netting having an opening in the middle about the size of the diameter of a test-tube. The insects are captured in test-tubes and transferred through the opening into the jar. When about 30 have been inserted, the end is covered with another layer of netting which is tied in position.

[13-14]

The mouths of the netting-covered jars are applied to the outer surface of the patient’s thigh and pressed firmly against the skin. [note: ‘One assistant cannot deal with more than four jars at the same time in a satisfactory manner. If a greater number of jars is being used another assistant is required, and the remainder are applied to the external surface of the opposite thigh.’] If the patient’s skin be cold or the room temperature low, the insects may be stimulated to feed more readily if a hot-water bottle be applied to the skin for about 15 minutes before the insects are allowed to feed. When the insects have been starved for about a couple of days the majority will usually take a blood meal within about 20 minutes.

When most of the mosquitoes seem to have fed, the jars are removed. The insects are then released into a small gauze cage and those which have taken an obvious blood meal are separated from the rest. [note: ‘This is quickly determined by catching each insect separately in a test tube, and holding it close to a strong light, when the bright red blood of the newly ingested meal is easily detected.’] The former insects are released into a storage cage while the latter are either given another opportunity of feeding un the next day or are destroyed. [note: ‘No mosquito which has had an opportunity of making an infective feed is ever placed among the normal insects, because there is always a risk that it may have imbibed a minute, but undetected, blood meal sufficient to infect it. Such insects mixed with normal ones might transmit infection when the latter are being nourished upon a non-infected patient, or might produce a mixed infection as the result of a later feed upon a patient infected with another species of Plasmodium.’]

...[fed again to ensure high infection rates of mosquitoes, large number of infective agents]... [note: ‘James (1926, 1931b) has discussed in detail the factors that may influence the acquisition of transmissible malarial infection in a suitable anopheline host. By the improved laboratory technique which has now been gradually evolved at Horton, it is now possible to avoid most of those difficulties.’]’ (13-14)

 

‘The Conservation of Infected Mosquitoes.

After the insects have received an infecting feed, they are stored in gauze cages at temperatures ranging from 70˚ to 80˚F. (21˚-27˚C.) to allow of the development of the parasite in those that have acquired a plasmodial infection. For experimental purposes, temperatures outside this range have been tried, but for routine work the average now employed is about 75˚F. (24˚F). To ensure that conditions are favourable for the prolonged survival of the insects at these temperatures, a relative humidity of about 80 per cent. or over is maintained. [14-15]

In the earlier work the insects were conserved in ordinary bacteriological incubators kept at a suitable temperature and humnidity. In 1932 a special incubation room was constructed. This has greatly facilitated the routine work and produced much more satisfactory results – a much larger stock of infected insects can now be maintained and the duration of life among them is considerably larger.

The methods that have been employed in this laboratory may be divided, therefore, into two groups – (i) that in which the mosquitoes were stored in ordinary incubators, and (ii) that in which they were kept in the specially heated room. The survival rates are very different in these two groups, that in the former only being about half that in the latter (vide Sinton and Shute, 1938).

A description of these two methods is given below:-

(i) Incubator Group of Insects. – The incubator was heated electrically. The humidity was kept at a high level by inserting a bowl of water and by hanging wet cloths in it. No attempt was made to maintain a constant degree of humidity; the object aimed at was merely to keep it at a high level but below saturation point.

The insects were confined in cages with an end dimension of 8 inches by 8 inches, and a length of 11 inches. The top and bottom of each cage was of wood, while the slides were covered with mosquito-netting, and at one end was a cotton sleeve to allow of the insertion of the insects. A watchglass full of water was placed in each cage to provide facilities for egg-laying (James 1927a).

(ii) Heated-Room Group of Insects. – The room used has a floor area about 11 feet square and a height of about 8 feet. The north side [note: ‘This prevents the marked variations in temperature which may be caused by sunlight beating into the room during the summer months.’] of the room is occupied almost exclusively by a large glass window, which is made in two layers with an air-space between them to minimise the loss of heat.

The room is heated by four electrical heaters, one placed low down on each wall. [note: ‘It is important that a bright reflecting surface be placed above and behind each heater. These reflections should be inclined downwards and outwards so that the heat is thrown towards the middle of the room, otherwise the distribution of the heat may be very unequal.’] These heaters are controlled thermostatically so that the temperature can be regulated at the required level. For ordinary routine work this is between 75˚F and 80˚F. (24-26˚C)

The relative humidity of the room is maintained at about 75-80 per cent. by large pans of water placed on the floor. [15-16] When necessary the humidity is increased by means of damp cloths wetted by a flow of hot water from a tap at a sink in the corner of the room.

The insects are kept in large cubical cages (Figs. 1 and 2 [images of cages with 3 segments – one image includes rabbit in one of the segments. Caption: ‘Figs. 1 and 2 – Cage with mosquito gauze removed to show the animal compartment surrounded by wire netting (A). In fig. 1 the door (B) is closed, while in fig. 2 it is open to show the rabbit, with an area for feeding on its back denuded of hair, resting in the metal tray (D) which is partly withdrawn. The wire (C) on which a damp cloth is hung during the feeding period, is also shown.’]) each side of which measures about 18 inches (45 cms.). The sides and top are covered with mosquito-gauze. The bottom is made of wood, while a thin layer of wood is fixed over the top to protect the gauze, and is in contact with it. The gauze should be tightly stretched to prevent injury by the rabbit or other animal inserted for feeding purposes.

One section of the cage, equal to one-fourth the space, is shut off from the rest by wire netting of ½-1 inch mesh. This provides a compartment for housing a rabbit or other animal. It is closed with a wooden door, [note: ‘Where several species of Plasmodium are being used to infect insects at the same period, the woodwork of the cages is painted a distinctive colour for each species (red for P. vivax; blue for P. falciparum; green for P. ovale; black for P. malariae, and white for uninfected insects). No cage is ever used for any other type of infection except that appropriate to its colour. This ensures that there is no risk of the accidental transmission of a mixed infection because a mosquito has been overlooked in a cage considered to be empty, and so used for the storage of insects infected with another species of Plasmodium.’] and is provided with a metal tray having edges about 3 inches high (Fig. 2). The latter facilities the rapid introduction and withdrawal of the animal, and thus diminishes the risk of insects escaping at this time. At the same end of the cage near the door, a cotton sleeve attached to the mosquito-gauze, opens into the cage and is used for the introduction of mosquitoes. A small earthenware basin filled with water is also placed inside the cage to provide a place for oviposition by gravid insects.

Parallel with and about 1 inch outside the top bar on each side of the cage, is fixed a metal wire. A damp cloth is hung over each of these bars whenever a rabbit is placed in the cage. The increased humidity so produced appears to stimulate the feeding activities of the insects. The cloth should not be in contact with the netting as this would allow the mosquitoes to suck up moisture, an act which is undesirable as it makes the disinclined to take a blood meal for some time afterwards. Contact with the moist cloth also tends to cause premature rotting of the netting.

The cages are arranged on shelves around the sides of the hot chamber.’ (14-16)

 

‘Feeding of Infected Insects.

In the earlier experiments with the incubator, raisins, dates, bananas, etc., were provided for the insects to feed upon [16-17] in the intervals between meals. It was soon found, as noted by numerous other workers, that many of the mosquitoes so fed developed an infection with bacteria and moulds in their gut and tissues. This usually resulted in a heavy death rate. [note: ‘Although no comparative experiments have been made, we formed the impression that such accidents were more frequent among insects stored in incubators than among those kept in large rooms, i.e. where there was a free circulation of air. De Buck and Swellengrebel (1935) have reported very good results with insects fed entirely on sugar-water and stored in a warm room.’]

It was also found that insects which had been allowed to feed on such a diet showed less inclination to take a blood meal subsequently than did insects which had been fed on blood only (Darling, 1910; James and Shute, 1926). This is a distinct disadvantage in the practice of malaria-therapy by mosquito bites, although there was no evidence to show that such a diet had any effect in retarding or preventing the development of the plasmodial infection in insects so fed (James, 1931b). For these reasons, in all the later work blood meals were provided either from man or from rabbits. [note: ‘Mosquitoes usually bite rabbits almost entirely in the region of the nose, so that if many insects are placed with a rabbit, this area may be bitten so frequently that a cankerous sore soon develops. If the hair be removed from the back of a rabbit an admirable feeding place can be exposed (Fig. 2). This is best done by the use of a depilatory paste... The loose hair is cut away roughly with scissors from a large area on the back of the rabbit, and the paste is applied with a wooden spatula. After about 2 minutes the paste is removed and the area thoroughly washed with water and dried.’]

When A. maculipennis var. atroparvus is kept at a temperature of about 79˚F. (26˚C), a large blood meal is digested in about 48 hours, and, unless the insects are fed at least every second day, a high death rate occurs at this temperature.

In the case of mosquitoes kept in the incubator, this meant that they had to be collected into test-tubes, transferred to a feeding jar, and applied to a man or rabbit every time a blood meal was necessary. Many of the insects which only took a small meal, or showed no inclination to bite during the period of application of the feeding jar, were liable to die in the intervals between opportunities for feeding, unless these were given at least once daily. In many instances two daily applications were made, each lasting about one hour. Apart from deaths due to starvation, the very frequent handling of these insects, necessitated by the feeding technique, must have been a factor in diminishing their expectation of life.

The conditions were very different in the case of the insects kept in a warm room. If it were not considered necessary to [17-18] give the insects another infecting meal, a prepared rabbit was placed in the small compartment of their cage every evening, and left there over-night. As mentioned previously, a damp cloth was hung on either side of the cage to stimulate feeding while the rabbit was present in the cage. These insects were only handled when given their original infecting feeds, and not again until they were removed to transmit the infection. Their opportunities for feeding lasted about 16 hours daily as compared with the maximum of 2 hours in the case of the insects stored under incubator conditions.

Every few days one or more mosquitoes devoid of blood were removed and dissected to determine the presence, the rate of development and the intensity of any plasmodial infection acquired.

In the earlier work with the incubators, the mortality among the infected insects was so high that it was necessary to conserve as many of them as possible to meet the demands for such mosquitoes. For this reason when sporozoites had been detected in their salivary glands, the batch of infects insects was stored in a refrigerator at about 5˚C., and removed only when taken out to infect a patient, or for necessary blood meals at long intervals (vide Second General Report of the Malaria Commission, 1927). In this way a single batch of infected mosquitoes could be used for a long period, sometimes for 3 months (James and Shute, 1926).

Because of the survival rates of the infected insects have been nearly doubled since the introduction of the hot room for storage, the necessity of keeping batches of infected mosquitoes for long periods has almost disappeared. This result has been aided by improvements in technique, by the larger numbers of insects available from the insectary, and by the increase in the patients being treated at Horton, which has resulted in the presence of a greater number of suitable cases upon whom to infect insects. At present the mosquitoes are stored continuously in the hot room, and usually remain highly infective for at least one month. At the end of which time, the transmission of infection may become less certain, so a new and more recently infected batch is then employed. Infective insects are only stored in the refrigerator when a very large batch, say 500, have been infected at one time, or for experimental investigations.’ (16-18)

 

‘Transmission of Infection by Mosquitoes.

When a request is received for the transmission of malarial infection by mosquitoes, about 15-20 insects, starved for 48 hours and free from blood, are placed in a feeding jar and taken by a trained assistant to the hospital where the patient is. During transit the feeding jars are packed in a thick leather case, to prevent breakage and thus the escape of infective insects.

The mosquitoes are given an opportunity of biting by applying the gauze-covered end of the feeding jar to the external surface of the patient’s thigh (vide supra). If the patient is residing a distance of several hours’ journey from the laboratory, two batches of insects are often taken, and these are given an opportunity of feeding immediately on arrival, and again a few hours later. To be absolutely certain that the transmission will be effected, an attempt is made to get at least 10 mosquitoes to bite, but with the heavily infected insects commonly used, a much smaller number would be sufficient. [note: ‘On some occasions when the insects have shown little inclination to bite, the infected salivary glands have been dissected out, crushed, and suspended in Locke’s fluid, as described by James, Nicol and Shute (1927). The resultant suspension was then injected intravenously. This procedure is also very useful for experimental purposes, and makes it possible to inoculate a known number of sporozoites (Shute, 1937).’]

After biting, the insects are brought back again to the laboratory. Some of those showing evidence of a blood meal are dissected to confirm the presence of sporozoites in their salivary glands, and so show that infective material had been inoculated during biting. The remainder are replaced in the cage in the incubation room.’ (19)