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Latency and long-term relapses in Benign Tertian malaria
189 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 40. N o . 2. O c t o b e r , 1946.
LATENCY
A N D L O N G - T E R M RELAPSES IN B E N I G N MALARIA.
TERTIAN
BY
P. G. SHUTE, Assistant Malaria Officer, Ministry of Health, The phrase " a latent attack of malaria " is here used to denote a frank attack of fever associated with parasites in patients who were infected several months prior to the development of clinical symptoms. " A long-term relapse " denotes a clinical and parasitological attack at least 3 months after the primary attack, the latter having, been treated with a course of mepacrine or quinine, and (as far as is known), the patient having remained fit and well, without parasites in the peripheral blood between the attacks., It is my experience with therapeutic malaria that neither latency nor longterm relapses occur in malignant tertian malaria. This is based on a study of several geographical strains of both tropical and sub-tropical origin. M.T. malaria relapses, if they occur at all, do so at very short intervals, the first within a few weeks of the primary attack (and the others follow in quick succession). These, it is considered, are due to incomplete treatment of the previous attack in that not all the trophozoites were destroyed (radical cure). Latency, as seen in B.T. malaria, does not appear to occur in M.T. malaria. The incubation period in M,T. malaria varies between 6 and 18 days with an average of about 10 days. It is well known that infected persons who take small daily doses of a suppressive drug such as quinine may experience an attack of fever many weeks from the last date of infection, particularly when the drug is discontinued a week or two after infection. The ensuing attack in these cases is caused by circulating parasites which were not destroyed by suppressive treatment. Latency in B.T. malaria, as defined above, is known to occur with tropical, sub-tropical and temperate region strains, but there is some evidence to suggest that it occurs more frequently with temperate region strains than it does with tropical strains. Nothing is known about the biological factors concerned in true latency b u t we know under what circumstances it may occur. These are : - I. Under natural conditions. II. Due to drug prophylaxis.
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LATENCY IN MALARIA
III.
IV.
In the presence of mixed infections--double infection (B.T. and M.T. at the same time), the M.T. malaria developing in about 10 days and the B.T. malaria many months later. In patients who are immune to one strain and while retaining this immunity are subsequently infected with another strain. I.
LATENCY UNDER NATURAL CONDITIONS.
It is a well-known phenomenon that in Eastern Europe there is frequently a sharp malaria curve in early spring caused by B.T. malaria. Many, perhaps most, of these cases are primary attacks in persons who were infected in the previous summer and autumn. This phenomenon has been observed in Roumania, Italy, Spain, Holland and England, and according to KORTERVEG (1909.), and SWELLENGREBEL(1921), nearly all the indigenous malaria occurring in Northern Holland in the spring and summer is the result of infections contracted in the previous autumn. It may be of interest to record here some observations made at this laboratory concerning therapeutic malaria, because they bear testimony to observations made in the field. The practice at this laboratory is to prepare batches of about 200 mosquitoes about once a month in order to supply B.T. malarial material to hospitals throughout the country for malaria therapy purposes. A patient undergoing malaria therapy whose blood contains the necessary number of ripe gametocytes is selected to infect a batch of mosquitoes. After the mosquitoes have fed once or twice they are incubated until sporozoltes appear in the salivary glands, when they are ready for transmitting infection. Earlier in our work we continued to use the mosquitoes so long as we were able to find sporozoites in the salivary glands; this often exceeded a period of 2 months. It soon became apparent that the presence of sporozoites in the glands was not by itself evidence that the person bitten would develop malaria within the usual incubation period (10 to 14 days). It was seen that the majority of the patients who were bitten during the first 2 or 3 weeks following salivary gland infection of the mosquitoes, developed the disease within the normal incubation period. Many of the patients bitten later than this failed to develop fever for many months. Most of the " failures " were reinfected with a different species of parasite in order not to delay treatment. Others were reinfected by blood of the same species and a few not reinfected at all. Those patients who were infected with IV[.T. or quartan malaria developed their first attack of B.T. malaria many months after the original infective bites despite the intervention of severe attacks of fever (caused by the different species). We know that relapses do not occur in B.T. malaria induced by blood inoculations and as many of our mosquito-infected cases which failed to develop malaria within the normal incubation period developed fever several months later, these attacks were due to the original mosquito-borne infection and were therefore latent cases.
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When a batch of mosquitoes was infected on only two occasions, on alternate days, even though the infection was a particularly heavy one with the average number of o6cysts per insect being a hundred or more, cases bitten 4 to 5 weeks after gland infection failed to develop fever. It was observed that for the first few days following the invasion of the glands by sporozoites, not only were the gland cells packed with sporozoites but enormous numbers were found lying in the salivary duct. Following a number of blood meals, the gland ducts were free, or nearly free, of sporozoites, but the gland cells were still heavily infected. It is believed that this may have an important bearing on the degree of infectivity following biting. When the salivary ducts are packed with sporozoites, very large numbers would be discharged during the act of biting, but when the duct is free, though the gland cells are heavily infected, the numbers discharged would not be very great. After a heavily infected mosquito has bitten fifteen or twenty times, sporozoites can still be seen in the gland cellS, even though, in many cases, the person bitten fails to develop malaria within the usual incubation period. In the early days of our work, after observing this phenomenon, it was concluded that the finding of sporozoites in the gland cells was not°necessarily proof that the patient had been infected and we inclined to the opinion that under certain circumstances an infective mosquito may bite a person without injecting sporozoites. This may be true, especially in mosquitoes where the infection was a very light one, because the thoracic muscular p r e s s u r e exerted during the act of biting may not have squeezed out any sporozoites embedded in the gland cells. But the point of interest here is that many of the believed-to-be-failures developed fever and parasites 6 months or even a year after infection and that during this long incubation period the infected individuals remained healthy. It is worthy of note that failures seldom, if ever, occurred when the patient was bitten by even one or two mosquitoes within a week or two after the glands first became infected. One other important factor in connection with this is worth considering. Might the failures be due to the age of the sporozoites and not to numbers ? To test this a batch of infected mosquitoes which had been infected on only one occasion was fed on a rabbit over a period of 6 weeks. The actual number of times the mosquitoes bit the rabbit is not known but it is certain that they fed on at least twelve occasions. Dissections showed that the glands contained only very small numbers of sporozoites. A patient bitten by six of the batch failed to develop fever within 3 weeks. The glands-of twelve mosquitoes of the same batch were dissected and injected intravenously into another patient and fever and parasites developed within the normal incubation period. In passing it should be understood that it is not intended to imply that a relatively small number of sporozoites n e v e r give rise to fever and parasites within the usual incubation periods, but it is considered that true latency occurs only when the sporozoites injected are too few to set up an immediate attack and that there is never true latency when large numbers of sporozoites are injected.
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LATENCY IN MALARIA
That true latency occurs in nature is so well known that it is not proposed to discuss it here. It has been studied and reported upon from many countries, particularly in Europe, and especially in Holland, where the fact that no species of human malaria parasites other than that of B.T. malaria occurs in nature, has afforded opportunities for a close study of latency to be made. The subject is dealt with fully by SWELLENGREBELand DE BUCK (1938), in their book,
Malaria in the Netherlands. 1I.
LATENCY DUE TO DRUG PROPHYLAXIS.
Quinine. Patients who take quinine daily, beginning a day or two before exposure to infection and continuing for 6 to 8 days during the incubation period, usually have a slightly longer incubation period than is normally the case. Again, parasites are often difficult to find for several days even after fever has started. On the other hand, if quinine is given a day or two before infection and continued daily for 2 or 3 weeks, there is usually a single peak of fever only. If a prolonged search is made, an odd parasite may be found. In many of these cases the patient remains symptom-free for several months and then he may experience a severe attack of malaria. In such cases it is believed that the quinine has acted in the usual therapeutic manner, that the asexual parasites have been destroyed and the subsequent long-term relapse is characteristic of this species of parasite. It is significant that the interval between the primary attack and the first relapse is comparable to a prolonged incubation period where there has been no primary attack.
Pamaquin (Plasmoquine). This drug, which has very little therapeutic action on the asexual parasites of B.T. malaria, is capable of warding off a primary attack for several months, even though the drug is stopped several days before the termination of the normal incubation period. In nearly every case, however, an attack develops several months later and fever and parasites resemble in all respects a primary attack when the incubation period is normal. Following the treatment of a primary attack with a protracted incubation period, relapses may occur, in some cases only one, while in others there may be several at short intervals of a month or so. It is of interest to mention here that pamaquin is equally effective as a prophylactic against M.T. malaria, but with this species of parasite, if the drug successfully prevents the primary attack, there are no subsequent attacks either at short or long periods. Therefore, pamaquin in certain doses when given on the day of infection and for a few days afterwards, sterilizes an infection of M.T. malaria before fever or erythrocytic parasites appear. It is equally effective against the primary attacks of B.T. malaria but in this case the disease manifests itself many months later. It is believed that this difference is not
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related to the action of the drug on the erythrocytic parasites, but is due to differences in the biology of the species.
Mepacrine (Atebrin). I t is of great interest and importance to find that this drug has the same effect as pamaquin. When it is used prophylactically against certain strains of M.T. malaria, unless fever and parasites occur within about the usual incubation period, no attack develops. With B.T. malaria there is nearly always the same latent infection as when pamaquin is used. One important point in these B.T. malarial cases is that although the drugs are given for only a few days at the time of infection, the infected person remains well for several months. This is entirely different from what happens when quinine is given for the same period.
III. LATENCY IN THE PRESENCE OF MIXED INFECTIONS. Only a few cases of this series are available for study, but they are worthy of consideration. Patients may be infected with B.T. and M.T. malaria at the same time, either by mosquitoes containing sporozoites of both species of parasites or by mosquitoes, some of which are infected with B.T. malaria and others with 1V[.T. Fever occurs some 10 days later but only Nf.T, parasites are found in the blood. If, after about a week of continued fever, drugs are given which successfully abort the attack but fail to cure the disease, two or more relapses due to the same species may occur over a period of 3 or 4 months. No B.T. malarial parasites are seen and clinically and parasitologically the disease is due to one species only: that of M.T. malaria. Several months later the patient again develops fever, but this time it is due to B.T. malaria, and only parasites of this species are to be found in the blood. On the other hand, if a patient is infected with B.T. malaria 3 or 4 days before being infected with M.T. malaria, the parasites o f both species appear in the blood together, and continue to do so until treatment is given. (It was observed that in mixed infections gametocytes of both species are usually very numerous.) IV. LATENCY IN PATIENTS WHO ARE IMMUNE TO ONE STRAIN, AND WHILE RETAINING THIS IMMUNITY ARE SUBSEQUENTLYINFECTED WITH ANOTHER STRAIN. Some patients who had received a full course of malaria-therapy with our Madagascar strain failed to develop fever or parasites when they were several times reinfected with the same strain of the same species, either by blood or mosquitoes; they were immune. But if they were infected with a different strain of the same species, some developed fever and parasites but with a protracted incubation period, usually of several months duration. If, however, they were infected with a different species of parasite, fever and parasites
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LATENCY IN MALARIA
developed within the usual incubation period and the attacks were just as severe as is normal for the species. I n one series of experiments, a patient, a primary case, was i n f e c t e d b y blood inoculation with a strain of B.T. malaria from the F r e n c h Cameroons, and f r o m him a batch of mosquitoes was infected. T h e mosquito infection was a light one and in none of those dissected were there more than eight oScysts per stomach. Six patients who were i m m u n e to the Madagascar strain were bitten by mosquitoes of this batch, and although the infection seemed to have failed all the patients developed fever and parasites several m o n t h s later. T h e following s u m m a r y gives the history of the six cases in which two strains of B.T. malarial parasites were u s e d - - M a d a g a s c a r and F r e n c h Cameroon strains. Case 1.
1927.
Primary infection with Madagascar strain. Ten peaks of fever treated with quinine. 1931. Second infection with same strain. Parasites without fever. Third exposure to infection 3 months later--failed. "1932. Fourth exposure to infection 9 months later--failed. Five months later infected by mosquito bites, French Cameroon strain. Fever and parasites developed 296 days later. Treated with quinine. There were two relapses, first 1 month after end of treatment, second 2 months after end of first relapse. Case 2.
1928.
Primary attack with Madagascar strain. Ten attacks, then treated with quinine. Relapsed 5 months later. 1931. Second infection with same strain. Fever and parasites followed by spontaneous recovery. Third exposure to infection 1 month later. Bitten by over 300 infected mosquitoes-- failed. Fourth exposure to infection 1 month later. Bitten by over 100 mosquitoes-failed. 1932. Fifth exposure to infection 9 months later--failed. Two months later bitten by mosquitoes infected with French Cameroon strain. Fever and parasites developed 263 days later. Treated by quinine. There were no relapses. Case 3.
1926.
Primary attack with Madagascar strain. Ten attacks then treated with quinine. Relapsed 8 months later. 1928. Second exposure to infection--failed after being bitten by 140 infected mosquitoes. Third exposure to infection, bitten by 150 mosquitoes--failed. August, fourth exposure to infection, bitten by fifteen mosquitoes--failed. 1929. Fifth exposure to infection, bitten by thirty mosquitoes--failed. Sixth exposure to infection, bitten by forty mosquitoes--failed. Seventh exposure to infection, bitten by twenty-five mosquitoes--failed. December, exposed to infection with M.T. malaria (successful). 1930. Eighth exposure to infection, again bitten by mosquitoes infected with Madagascar strain--failed. 1931. Ninth exposure to infection, bitten by one hundred mosquitoes--failed.
P. G. SHUTE
1932.
195
Tenth exposure to infection, bitten by fifty mosquitoes--failed. In December, 1932, bitten by ten mosquitoes infected with French Cameroon strain. Fever started 253 days later and continued for 10 days when treatment was given. There were no relapses.
Case 4. 1929. First infection--with Madagascar strain. 1932. Second exposure to infection--failed. In December, 1932, bitten by eleven mosquitoes infected with French Cameroon strain. Fever started 287 days later and continued for 10 days. There were no relapses. Case 5. 1926. First infection--with Madagascar strain. 1927. Second exposure to infection--failed. Third exposure to infection--failed. 1931. Fourth exposure to i n f e c t i o n - attack started but spontaneous recovery occurred after 4 days of parasites. Fifth exposure to infection--failed. 1932. Sixth exposure to infection--failed. Eight months later, infected with French Cameroon strain. Incubation period 304 days. Fever continued for 10 days and patient was treated with quinine. There were no relapses. Case 6. 1926. First infection--with Madagascar strain. Second exposure to infection 4 months later--successful. 1932. Third exposure to infection--failed. Bitten by twelve mosquitoes infected with French Cameroon strain. Incubation period 311 days. Fever and parasites for 12 days. Treated with quinine. There were no relapses.
I n this small series of cases of B.T. malaria all the patients were i m m u n e to the M a d a g a s c a r strain. As the n u m b e r of o6cysts in the stomachs of the m o s q u i t o e s infected with the F r e n c h C a m e r o o n strain was small, the sporozoites injected would be relatively few. T w o p r i m a r y cases were infected with the F r e n c h C a m e r o o n strain b y direct b l o o d inoculation and b o t h developed fever and parasites within the usual incubation period. F o u r cases i m m u n e to the M a d a g a s c a r strain were infected by direct b l o o d inoculation with the F r e n c h C a m e r o o n s t r a i n and all developed fever and parasites within the normal incubation period. T h e two p r i m a r y cases continued to have fever until t r e a t m e n t was given, w h e r e a s the i m m u n e cases all had spontaneous recoveries after a few days. TIME FACTORS True L a t e n c y . I n nine patients infected with B . T . malaria (observed b y the writer) who failed to develop fever and parasites for several months, b u t who developed fever and parasites subsequently without reinfection, the average n u m b e r of days 1.
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LATENCY IN MALARIA
between infection and the attack was 282. On examining the findings of other workers in Europe, similar results are seen. SWELLENGREBELand BE BUCK (1938) record (page 150), an experiment where eight volunteers were infected by one or two mosquitoes. All eight developed true latent malaria, one with an incubation period of 231 days, a second with 270 days, and the remainder with incubation periods varying between 239 and 276 days.
2. Latency brought about by Drug Prophylaxis. In latency brought about by drug prophylaxis, e.g., mepacrine or pamaqu~n, the interval is about the same. In nineteen carefully selected cases Of B.T. injection the incubation period averaged 263 days.
3. Long-term Relapses. One of the greatest problems of B.T. malaria is the prevention of relapses. It is generally believed that they are due to insufficient treatment of the primary attack or previous relapse. Such is undoubtedly the case when, as the result of insufficient treatment, a primary attack is followed by a shortterm relapse (recrudescence) some few weeks later. This can be demonstrated by giving a single dose of 10 grains of quinine to a patient in the throes of a primary attack with certain strains of B.T. malaria. Fever subsides after about 3 days and parasites can seldom be found in thick smears by the 5th day. But this fever-free interval is of short duration and by the 14th day fever begins again and parasites are numerous in the peripheral blood. The single dose of the drug has aborted the attack and reduced the number of parasites below the pyrogenic threshold. Some parasites survive and in the absence of further treatment they soon increase in number sufficiently to cause clinical symptoms. There is, however, some reason for believing that long-term relapses are not due to the survival of a few asexual parasites. It is of significance that the time factor in long-term relapses is closely related to that of true latency and of latency brought about by drug prophylaxis (mepacrine and pamaquin but not quinine). In fifty-two carefully selected long-term relapses the average period of time from the last attack was 263 days. Two examples may be quoted : (1) I n August, 1943, two indigenous cases of B.T. malaria occurred in male patients in a mental hospital in Surrey. The patients shared the same ward, neither had ever been abroad nor subjected to therapeutic malaria. One patient developed B.T. malaria on 22rid August and the other on the following day. Parasites were present in the blood of both cases in moderate numbers. Type of fever.--In one patient the fever was quotidian and in the other it was tertian. Treatment of the attacks.--Both patients were treated with mepacrine 0"3 gramme daily for 7 days and fever and parasites quickly disappeared.
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In April of the following year both patients relapsed, clinically and parasitologically. In one, the interval between the primary attack and the first relapse was 238 days and in the other 229 days. AlthoUgh there were more than twenty patients sleeping in the ward, no further cases occurred and it is conjectured that a single mosquito infected both patients within a few days of each other." (2) In September, 1935, a case of indigenous B.T. malaria occurred in a young woman aged 36, living in Essex. Three weeks later a second case occurred in another house, this time in a boy aged 12; who shared a bed with his grandmother at week-ends. Eleven months later the grandmother developed malaria. The two houses were separated from each other by about 100 yards. It is believed that the three cases were infected by the same mosquito within a few days Of each other, that the first case was infected about the middle of August, and that the mosquito left the house, probably to lay eggs, and later returned to the house where the other two cases occurred. The boy was probably the next to be infected and he, too, developed the disease within the normal incubation period. The third case was probably t'he last to be infected. It is considered that the infected mosquito discharged a sufficiently large dose of sporozoites in the first two cases to produce symptoms within the usual incubation period, but that the third case received a minimum number of sporozoites, and that this is the reason for the protracted incubation period, i.e., 9 to 11 months. As a result of some recent experiments where the number of sporozoites injected is known, there is some evidence which suggests that about 2,000 sporozoites are necessary to ensure a normal incubation period. In a recent experiment the glands of five mosquitoes were dissected in Locke's fluid and an emulsion prepared. Two patients were infected, one receiving 371,300 sporozoites and the other 1,805. The patient receiving the large dose developed fever in 10 days while the patient receiving the smaller dose failed to develop either fever or Parasites and was subsequently infected by direct blood inoculation with the same strain and started fever within a week. If the number of B.T, malarial sporozoites necessary to produce clinical symptoms is in the region of 2,000 it is not difficult to understand why so many cases are latent but it is difficult to understand why so many of these latent cases, even the majority, have incubation periods of about 38 weeks, at least with certain strains of tropical and sub-tropical origin. DRUGS ON THE ~/[ORPHOLOGY OF PASASlTES. While some workers claim that they are able to distinguish quinine-affected parasites from the normal, others have failed to do so. Some years ago this was tested. I selected two patients who showed a heavy infection of B.T. malarial parasites and each was given 90 grains of quinine orally, one dose of 45 grains at 10 a.m. and a second dose 4 hours later. A series of thin films was taken at 2-hourly intervals over a period of 4 hours and stained witll Leishman. Films of patients who had not been given quinine were prepared at tile same time EFFECT OF
1~
LATENCY IN MALARIA
intervals and were stained together. The two sets of films were then examined by workers who were authorities on parasitology but all failed to distinguish which of the films contained quininized parasites. No such problem presents itself with atebrinized parasites. Following the administration of a single dose of 0.6 gramme of atebrin (mepacrine), changes in the parasite can be seen within 20 minutes. Normally, a full-grown asexual B.T. malarial parasite contains about 50 granules of pigment. Following a single large dose of atebrin, the first change which can be seen in the parasite is clumping of the pigment. In the first half-hour, instead of there being about 50 granules of pigment, there are about twenty clumps. An hour or two later the number is further reduced and usually consists of two or three lumps. After about 12 hours many of the parasites are devoid of pigment and some early disintegration of both chromatin and cytoplasm can be seen. (I have failed to observe where the pigment goes ; pigmented mononuclear cells in the peripheral circulation are not increased.) At about 24 hours, disintegration is well marked and often within 48 hours no parasites can be found in thin films. As far as my observations go, I have never seen a parasite which, after a dose of 0"6 gramme of atebrin, conforms to the normal. It is, therefore, possible that atebrin given over a period of several days may succeed in killing all the asexual parasites. Yet it is the case that many patients treated with this drug continue to relapse at relatively short intervals over a period of several montns. There is considerable evidence that plasmoquine (pamaquin), combined with quinine, reduces the relapse rate in B.T. malaria and many workers believe that this form of treatment is superior to either quinine or atebrin. When it is remembered that plasmoquine has very little action on B.T. malarial trophozoites, at least when compared with quinine or atebrin, it is difficult to understand why plasmoquinine in very small doses may reduce the relapse rate. Plasmoquine, when given a day or two before infection and continued for a few days afterwards, prevents the onset of a primary attack of B.T. malaria for several months, sometimes for a year. In M.T. malaria plasmoquine is evefi more successful and seems to be a true causal prophylactic. If this is so, then the indication is that against M.T. malaria, plasmoquine is either a sporozoiticidal drug or that it acts on the hypothetical stage of the parasite between the sporozoite and the erythrocytic parasite. What, then, is the difference between M.T. and B.T. malaria in this connection ? In B.T. malaria, as has already been noted, an attack is often delayed for nearly a year, even when the drug is given for only a few days following exposure to infection. A possible explanation is that plasmoquine kills all the sporozoites or x bodies in a M.T. infection, but not all in a B.T. infection. Even if this is so it would still not explain the very long interval between infection and symptoms in B.T. malaria. If, however, the sporozoites, after escaping from the peripheral circulation, succeeded in establishing themselves in reticulo-endothelial cells and were able to outlive the life of the host cell, they might, on being released, give rise to that
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stage of parasitism which brings about infection of the erythrocytes. Whatever the explanation of latency, it would appear that it is caused by a resting parasite and not one which is active, such as a trophozoite. In advancing the theory that it may be the sporozoite itself which is lying dormant all these months, it is of interest to note that in the insect host B.T. malarial sporozoites are able to lie dormant for several months without, apparently taking nourishment or changing their morphological character even within a wide range of temperature. The discovery of exoerythrocytic parasites in certain avian malarias, especially P. gallinaceum in the domestic fowl and P. lophurae in turkeys, has stimulated malaria research workers to attempt to demonstrate a similar cycle in mammalian malaria. From the literature it appears that these exoerythrocytic parasites are pathogenic and that in some cases they cause death to the host even before the later stages (erythrocytic parasites) have had time to develop. It would therefore seem unlikely that these forms of the parasite, even if they occur in B.T. malaria, are responsible for relapses. Furthermore, in B.T. malaria, relapses do not occur in blood-inoculated cases whereas exoerythrocytic parasites do occur in blood-inoculated cases ofP. gallinaceum or P. lophurae. Some workers have shown that a single dose of 1 gramme of quinine given orally will sterilize a primary infection of B.T. malaria induced by blood inoculation whereas the same quantity of quinine given daily for 30 days will not prevent a long-term relapse in mosquito-infected cases with the same strain. THE EVALUATION OF DRUGS I N
ANTI-RELAPSE TREATMENT.
It is a fundamental characteristic of many strains of B.T. malaria that between the primary attack and the first relapse there is usually an interval o f several months, but, as far as is known, the interval never exceeds 1 year. Following the first relapse, there is frequently a series of relapses which occur at about 1-monthly intervals and these may continue over a period of from 1 to 2 years. Are these short relapses caused by erythrocytic parasites (trophozoites), which escaped destruction during treatment of the previous relapse ? If sporozoites of B.T. malaria in human tissue cells are able to survive for many months as they are able to do in the tissue cells of a mosquito, it seems possible that they may be the direct cause of relapses. This could conceivably be the reason why one or many relapses recur at short intervals following an interval of many months up to 1 year between the primary attack and the first relapse. This phenomenon in B.T. malaria must have a very important bearing on t h e value of anti-relapse drugs. A patient who has had his first relapse 6 months to 1 year following his primary attack, followed by a number of short-term relapses, for about a year after the first relapse, will be semi-immune and such_ cases are not, at this stage of their disease, suitable for testing anti-relapse drugs. It is also the case that because so many patients infected with certain strains o f B.T. malaria do not experience a relapse for from 6 months to 1 year following
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the primary attack, the value of a drug as an anti-relapse treatment cannot be properly assessed for at least 1 year after the primary attack. I f relapses in B.T. malaria are caused by sporozoites or by an intermediate stage--between the sporozoite and the erythrocytic stages of the parasite-a drug which will prevent relapses would also conceivably be equally successful as a causal prophylactic and vice-versa. SUMMARY. The first relapse in B.T. malarial infections usually occurs several months after the treatment of the primary attack, and may be as long delayed as 1 year. A case is presented for considering that relapses are due to sporozoites which have been held up in tissue cells, a not improbable hypothesis when it is remembered that sporozoites are able to survive for many months in the tissue cells of the insect carrier. True latency is seen in those cases in which the primary attack occurs many months after infection, frequently after a delay of about 38 weeks. (The delay varies between 2 months and 1 year.) First relapses frequently occur at about the same period of time. Latency as defined in this paper occurs: (1) in nature; (2) following certain drug prophylaxis ; (3) when mixed infections of B.T. and M.T. malaria occur; (4)when patients who are immune to one strain of B.T. malaria are :infected by a different strain. The significance of the drug pamaquin (plasmoquine) is that it prevents the onset of an attack for many months,whereas, compared with quinine and mepacrine in an acute primary attack its action on erythrocytic parasites is slight. It is suggested that relapses in B.T. malaria are due to a resting phase of the parasite and that a drug which successfully prevents relapses would also act as a successful causal prophylactic and vice-versa. Sporozoites are known to survive for many months in the tissue cells of the insect host and it is suggested that they also may be able to do so in the human host. The word " prophylaxis " has been used in this paper deliberately in preference to the word " suppressive." When quinine is used as a prophylactic :against B.T. malarial infections, if the drug is discontinued a few days after exposure to infection, the onset of fever is delayed for at most a few days. When mepacrine or pamaquin is used, in one strain at least (Madagascar strain) fever seldom occurs within 2 months of infection ; and usually the period is between 34 and 38 weeks. It is, therefore, considered that while the word suppressive is applicable to quinine it is less appropriate to mepacrine and pamaquin. REFERENCES, :KoRTEWEG (1902) Klinische observaties over malaria in den winter van 1901-02. Herinneringsbundel voor Prof. ]~osenstein. Leiden : E. Ijdo, pp 263-281. SWELLENGItEBEL (1921~ Wanneer heeft ten onzent malariabesmetting plaats ? (2nd communication): Nederl. Tijdschr. v. Geneesk, 65, ii. 1486-88. ;SWELLENGREBEL• DE B U C K , (1938), Malaria in the Netherlands. Amsterdam : Scheltema & Holkema.
LATENCY
A N D L O N G - T E R M RELAPSES IN B E N I G N MALARIA.
TERTIAN
BY
P. G. SHUTE, Assistant Malaria Officer, Ministry of Health, The phrase " a latent attack of malaria " is here used to denote a frank attack of fever associated with parasites in patients who were infected several months prior to the development of clinical symptoms. " A long-term relapse " denotes a clinical and parasitological attack at least 3 months after the primary attack, the latter having, been treated with a course of mepacrine or quinine, and (as far as is known), the patient having remained fit and well, without parasites in the peripheral blood between the attacks., It is my experience with therapeutic malaria that neither latency nor longterm relapses occur in malignant tertian malaria. This is based on a study of several geographical strains of both tropical and sub-tropical origin. M.T. malaria relapses, if they occur at all, do so at very short intervals, the first within a few weeks of the primary attack (and the others follow in quick succession). These, it is considered, are due to incomplete treatment of the previous attack in that not all the trophozoites were destroyed (radical cure). Latency, as seen in B.T. malaria, does not appear to occur in M.T. malaria. The incubation period in M,T. malaria varies between 6 and 18 days with an average of about 10 days. It is well known that infected persons who take small daily doses of a suppressive drug such as quinine may experience an attack of fever many weeks from the last date of infection, particularly when the drug is discontinued a week or two after infection. The ensuing attack in these cases is caused by circulating parasites which were not destroyed by suppressive treatment. Latency in B.T. malaria, as defined above, is known to occur with tropical, sub-tropical and temperate region strains, but there is some evidence to suggest that it occurs more frequently with temperate region strains than it does with tropical strains. Nothing is known about the biological factors concerned in true latency b u t we know under what circumstances it may occur. These are : - I. Under natural conditions. II. Due to drug prophylaxis.
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III.
IV.
In the presence of mixed infections--double infection (B.T. and M.T. at the same time), the M.T. malaria developing in about 10 days and the B.T. malaria many months later. In patients who are immune to one strain and while retaining this immunity are subsequently infected with another strain. I.
LATENCY UNDER NATURAL CONDITIONS.
It is a well-known phenomenon that in Eastern Europe there is frequently a sharp malaria curve in early spring caused by B.T. malaria. Many, perhaps most, of these cases are primary attacks in persons who were infected in the previous summer and autumn. This phenomenon has been observed in Roumania, Italy, Spain, Holland and England, and according to KORTERVEG (1909.), and SWELLENGREBEL(1921), nearly all the indigenous malaria occurring in Northern Holland in the spring and summer is the result of infections contracted in the previous autumn. It may be of interest to record here some observations made at this laboratory concerning therapeutic malaria, because they bear testimony to observations made in the field. The practice at this laboratory is to prepare batches of about 200 mosquitoes about once a month in order to supply B.T. malarial material to hospitals throughout the country for malaria therapy purposes. A patient undergoing malaria therapy whose blood contains the necessary number of ripe gametocytes is selected to infect a batch of mosquitoes. After the mosquitoes have fed once or twice they are incubated until sporozoltes appear in the salivary glands, when they are ready for transmitting infection. Earlier in our work we continued to use the mosquitoes so long as we were able to find sporozoites in the salivary glands; this often exceeded a period of 2 months. It soon became apparent that the presence of sporozoites in the glands was not by itself evidence that the person bitten would develop malaria within the usual incubation period (10 to 14 days). It was seen that the majority of the patients who were bitten during the first 2 or 3 weeks following salivary gland infection of the mosquitoes, developed the disease within the normal incubation period. Many of the patients bitten later than this failed to develop fever for many months. Most of the " failures " were reinfected with a different species of parasite in order not to delay treatment. Others were reinfected by blood of the same species and a few not reinfected at all. Those patients who were infected with IV[.T. or quartan malaria developed their first attack of B.T. malaria many months after the original infective bites despite the intervention of severe attacks of fever (caused by the different species). We know that relapses do not occur in B.T. malaria induced by blood inoculations and as many of our mosquito-infected cases which failed to develop malaria within the normal incubation period developed fever several months later, these attacks were due to the original mosquito-borne infection and were therefore latent cases.
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When a batch of mosquitoes was infected on only two occasions, on alternate days, even though the infection was a particularly heavy one with the average number of o6cysts per insect being a hundred or more, cases bitten 4 to 5 weeks after gland infection failed to develop fever. It was observed that for the first few days following the invasion of the glands by sporozoites, not only were the gland cells packed with sporozoites but enormous numbers were found lying in the salivary duct. Following a number of blood meals, the gland ducts were free, or nearly free, of sporozoites, but the gland cells were still heavily infected. It is believed that this may have an important bearing on the degree of infectivity following biting. When the salivary ducts are packed with sporozoites, very large numbers would be discharged during the act of biting, but when the duct is free, though the gland cells are heavily infected, the numbers discharged would not be very great. After a heavily infected mosquito has bitten fifteen or twenty times, sporozoites can still be seen in the gland cellS, even though, in many cases, the person bitten fails to develop malaria within the usual incubation period. In the early days of our work, after observing this phenomenon, it was concluded that the finding of sporozoites in the gland cells was not°necessarily proof that the patient had been infected and we inclined to the opinion that under certain circumstances an infective mosquito may bite a person without injecting sporozoites. This may be true, especially in mosquitoes where the infection was a very light one, because the thoracic muscular p r e s s u r e exerted during the act of biting may not have squeezed out any sporozoites embedded in the gland cells. But the point of interest here is that many of the believed-to-be-failures developed fever and parasites 6 months or even a year after infection and that during this long incubation period the infected individuals remained healthy. It is worthy of note that failures seldom, if ever, occurred when the patient was bitten by even one or two mosquitoes within a week or two after the glands first became infected. One other important factor in connection with this is worth considering. Might the failures be due to the age of the sporozoites and not to numbers ? To test this a batch of infected mosquitoes which had been infected on only one occasion was fed on a rabbit over a period of 6 weeks. The actual number of times the mosquitoes bit the rabbit is not known but it is certain that they fed on at least twelve occasions. Dissections showed that the glands contained only very small numbers of sporozoites. A patient bitten by six of the batch failed to develop fever within 3 weeks. The glands-of twelve mosquitoes of the same batch were dissected and injected intravenously into another patient and fever and parasites developed within the normal incubation period. In passing it should be understood that it is not intended to imply that a relatively small number of sporozoites n e v e r give rise to fever and parasites within the usual incubation periods, but it is considered that true latency occurs only when the sporozoites injected are too few to set up an immediate attack and that there is never true latency when large numbers of sporozoites are injected.
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That true latency occurs in nature is so well known that it is not proposed to discuss it here. It has been studied and reported upon from many countries, particularly in Europe, and especially in Holland, where the fact that no species of human malaria parasites other than that of B.T. malaria occurs in nature, has afforded opportunities for a close study of latency to be made. The subject is dealt with fully by SWELLENGREBELand DE BUCK (1938), in their book,
Malaria in the Netherlands. 1I.
LATENCY DUE TO DRUG PROPHYLAXIS.
Quinine. Patients who take quinine daily, beginning a day or two before exposure to infection and continuing for 6 to 8 days during the incubation period, usually have a slightly longer incubation period than is normally the case. Again, parasites are often difficult to find for several days even after fever has started. On the other hand, if quinine is given a day or two before infection and continued daily for 2 or 3 weeks, there is usually a single peak of fever only. If a prolonged search is made, an odd parasite may be found. In many of these cases the patient remains symptom-free for several months and then he may experience a severe attack of malaria. In such cases it is believed that the quinine has acted in the usual therapeutic manner, that the asexual parasites have been destroyed and the subsequent long-term relapse is characteristic of this species of parasite. It is significant that the interval between the primary attack and the first relapse is comparable to a prolonged incubation period where there has been no primary attack.
Pamaquin (Plasmoquine). This drug, which has very little therapeutic action on the asexual parasites of B.T. malaria, is capable of warding off a primary attack for several months, even though the drug is stopped several days before the termination of the normal incubation period. In nearly every case, however, an attack develops several months later and fever and parasites resemble in all respects a primary attack when the incubation period is normal. Following the treatment of a primary attack with a protracted incubation period, relapses may occur, in some cases only one, while in others there may be several at short intervals of a month or so. It is of interest to mention here that pamaquin is equally effective as a prophylactic against M.T. malaria, but with this species of parasite, if the drug successfully prevents the primary attack, there are no subsequent attacks either at short or long periods. Therefore, pamaquin in certain doses when given on the day of infection and for a few days afterwards, sterilizes an infection of M.T. malaria before fever or erythrocytic parasites appear. It is equally effective against the primary attacks of B.T. malaria but in this case the disease manifests itself many months later. It is believed that this difference is not
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related to the action of the drug on the erythrocytic parasites, but is due to differences in the biology of the species.
Mepacrine (Atebrin). I t is of great interest and importance to find that this drug has the same effect as pamaquin. When it is used prophylactically against certain strains of M.T. malaria, unless fever and parasites occur within about the usual incubation period, no attack develops. With B.T. malaria there is nearly always the same latent infection as when pamaquin is used. One important point in these B.T. malarial cases is that although the drugs are given for only a few days at the time of infection, the infected person remains well for several months. This is entirely different from what happens when quinine is given for the same period.
III. LATENCY IN THE PRESENCE OF MIXED INFECTIONS. Only a few cases of this series are available for study, but they are worthy of consideration. Patients may be infected with B.T. and M.T. malaria at the same time, either by mosquitoes containing sporozoites of both species of parasites or by mosquitoes, some of which are infected with B.T. malaria and others with 1V[.T. Fever occurs some 10 days later but only Nf.T, parasites are found in the blood. If, after about a week of continued fever, drugs are given which successfully abort the attack but fail to cure the disease, two or more relapses due to the same species may occur over a period of 3 or 4 months. No B.T. malarial parasites are seen and clinically and parasitologically the disease is due to one species only: that of M.T. malaria. Several months later the patient again develops fever, but this time it is due to B.T. malaria, and only parasites of this species are to be found in the blood. On the other hand, if a patient is infected with B.T. malaria 3 or 4 days before being infected with M.T. malaria, the parasites o f both species appear in the blood together, and continue to do so until treatment is given. (It was observed that in mixed infections gametocytes of both species are usually very numerous.) IV. LATENCY IN PATIENTS WHO ARE IMMUNE TO ONE STRAIN, AND WHILE RETAINING THIS IMMUNITY ARE SUBSEQUENTLYINFECTED WITH ANOTHER STRAIN. Some patients who had received a full course of malaria-therapy with our Madagascar strain failed to develop fever or parasites when they were several times reinfected with the same strain of the same species, either by blood or mosquitoes; they were immune. But if they were infected with a different strain of the same species, some developed fever and parasites but with a protracted incubation period, usually of several months duration. If, however, they were infected with a different species of parasite, fever and parasites
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developed within the usual incubation period and the attacks were just as severe as is normal for the species. I n one series of experiments, a patient, a primary case, was i n f e c t e d b y blood inoculation with a strain of B.T. malaria from the F r e n c h Cameroons, and f r o m him a batch of mosquitoes was infected. T h e mosquito infection was a light one and in none of those dissected were there more than eight oScysts per stomach. Six patients who were i m m u n e to the Madagascar strain were bitten by mosquitoes of this batch, and although the infection seemed to have failed all the patients developed fever and parasites several m o n t h s later. T h e following s u m m a r y gives the history of the six cases in which two strains of B.T. malarial parasites were u s e d - - M a d a g a s c a r and F r e n c h Cameroon strains. Case 1.
1927.
Primary infection with Madagascar strain. Ten peaks of fever treated with quinine. 1931. Second infection with same strain. Parasites without fever. Third exposure to infection 3 months later--failed. "1932. Fourth exposure to infection 9 months later--failed. Five months later infected by mosquito bites, French Cameroon strain. Fever and parasites developed 296 days later. Treated with quinine. There were two relapses, first 1 month after end of treatment, second 2 months after end of first relapse. Case 2.
1928.
Primary attack with Madagascar strain. Ten attacks, then treated with quinine. Relapsed 5 months later. 1931. Second infection with same strain. Fever and parasites followed by spontaneous recovery. Third exposure to infection 1 month later. Bitten by over 300 infected mosquitoes-- failed. Fourth exposure to infection 1 month later. Bitten by over 100 mosquitoes-failed. 1932. Fifth exposure to infection 9 months later--failed. Two months later bitten by mosquitoes infected with French Cameroon strain. Fever and parasites developed 263 days later. Treated by quinine. There were no relapses. Case 3.
1926.
Primary attack with Madagascar strain. Ten attacks then treated with quinine. Relapsed 8 months later. 1928. Second exposure to infection--failed after being bitten by 140 infected mosquitoes. Third exposure to infection, bitten by 150 mosquitoes--failed. August, fourth exposure to infection, bitten by fifteen mosquitoes--failed. 1929. Fifth exposure to infection, bitten by thirty mosquitoes--failed. Sixth exposure to infection, bitten by forty mosquitoes--failed. Seventh exposure to infection, bitten by twenty-five mosquitoes--failed. December, exposed to infection with M.T. malaria (successful). 1930. Eighth exposure to infection, again bitten by mosquitoes infected with Madagascar strain--failed. 1931. Ninth exposure to infection, bitten by one hundred mosquitoes--failed.
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Tenth exposure to infection, bitten by fifty mosquitoes--failed. In December, 1932, bitten by ten mosquitoes infected with French Cameroon strain. Fever started 253 days later and continued for 10 days when treatment was given. There were no relapses.
Case 4. 1929. First infection--with Madagascar strain. 1932. Second exposure to infection--failed. In December, 1932, bitten by eleven mosquitoes infected with French Cameroon strain. Fever started 287 days later and continued for 10 days. There were no relapses. Case 5. 1926. First infection--with Madagascar strain. 1927. Second exposure to infection--failed. Third exposure to infection--failed. 1931. Fourth exposure to i n f e c t i o n - attack started but spontaneous recovery occurred after 4 days of parasites. Fifth exposure to infection--failed. 1932. Sixth exposure to infection--failed. Eight months later, infected with French Cameroon strain. Incubation period 304 days. Fever continued for 10 days and patient was treated with quinine. There were no relapses. Case 6. 1926. First infection--with Madagascar strain. Second exposure to infection 4 months later--successful. 1932. Third exposure to infection--failed. Bitten by twelve mosquitoes infected with French Cameroon strain. Incubation period 311 days. Fever and parasites for 12 days. Treated with quinine. There were no relapses.
I n this small series of cases of B.T. malaria all the patients were i m m u n e to the M a d a g a s c a r strain. As the n u m b e r of o6cysts in the stomachs of the m o s q u i t o e s infected with the F r e n c h C a m e r o o n strain was small, the sporozoites injected would be relatively few. T w o p r i m a r y cases were infected with the F r e n c h C a m e r o o n strain b y direct b l o o d inoculation and b o t h developed fever and parasites within the usual incubation period. F o u r cases i m m u n e to the M a d a g a s c a r strain were infected by direct b l o o d inoculation with the F r e n c h C a m e r o o n s t r a i n and all developed fever and parasites within the normal incubation period. T h e two p r i m a r y cases continued to have fever until t r e a t m e n t was given, w h e r e a s the i m m u n e cases all had spontaneous recoveries after a few days. TIME FACTORS True L a t e n c y . I n nine patients infected with B . T . malaria (observed b y the writer) who failed to develop fever and parasites for several months, b u t who developed fever and parasites subsequently without reinfection, the average n u m b e r of days 1.
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LATENCY IN MALARIA
between infection and the attack was 282. On examining the findings of other workers in Europe, similar results are seen. SWELLENGREBELand BE BUCK (1938) record (page 150), an experiment where eight volunteers were infected by one or two mosquitoes. All eight developed true latent malaria, one with an incubation period of 231 days, a second with 270 days, and the remainder with incubation periods varying between 239 and 276 days.
2. Latency brought about by Drug Prophylaxis. In latency brought about by drug prophylaxis, e.g., mepacrine or pamaqu~n, the interval is about the same. In nineteen carefully selected cases Of B.T. injection the incubation period averaged 263 days.
3. Long-term Relapses. One of the greatest problems of B.T. malaria is the prevention of relapses. It is generally believed that they are due to insufficient treatment of the primary attack or previous relapse. Such is undoubtedly the case when, as the result of insufficient treatment, a primary attack is followed by a shortterm relapse (recrudescence) some few weeks later. This can be demonstrated by giving a single dose of 10 grains of quinine to a patient in the throes of a primary attack with certain strains of B.T. malaria. Fever subsides after about 3 days and parasites can seldom be found in thick smears by the 5th day. But this fever-free interval is of short duration and by the 14th day fever begins again and parasites are numerous in the peripheral blood. The single dose of the drug has aborted the attack and reduced the number of parasites below the pyrogenic threshold. Some parasites survive and in the absence of further treatment they soon increase in number sufficiently to cause clinical symptoms. There is, however, some reason for believing that long-term relapses are not due to the survival of a few asexual parasites. It is of significance that the time factor in long-term relapses is closely related to that of true latency and of latency brought about by drug prophylaxis (mepacrine and pamaquin but not quinine). In fifty-two carefully selected long-term relapses the average period of time from the last attack was 263 days. Two examples may be quoted : (1) I n August, 1943, two indigenous cases of B.T. malaria occurred in male patients in a mental hospital in Surrey. The patients shared the same ward, neither had ever been abroad nor subjected to therapeutic malaria. One patient developed B.T. malaria on 22rid August and the other on the following day. Parasites were present in the blood of both cases in moderate numbers. Type of fever.--In one patient the fever was quotidian and in the other it was tertian. Treatment of the attacks.--Both patients were treated with mepacrine 0"3 gramme daily for 7 days and fever and parasites quickly disappeared.
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In April of the following year both patients relapsed, clinically and parasitologically. In one, the interval between the primary attack and the first relapse was 238 days and in the other 229 days. AlthoUgh there were more than twenty patients sleeping in the ward, no further cases occurred and it is conjectured that a single mosquito infected both patients within a few days of each other." (2) In September, 1935, a case of indigenous B.T. malaria occurred in a young woman aged 36, living in Essex. Three weeks later a second case occurred in another house, this time in a boy aged 12; who shared a bed with his grandmother at week-ends. Eleven months later the grandmother developed malaria. The two houses were separated from each other by about 100 yards. It is believed that the three cases were infected by the same mosquito within a few days Of each other, that the first case was infected about the middle of August, and that the mosquito left the house, probably to lay eggs, and later returned to the house where the other two cases occurred. The boy was probably the next to be infected and he, too, developed the disease within the normal incubation period. The third case was probably t'he last to be infected. It is considered that the infected mosquito discharged a sufficiently large dose of sporozoites in the first two cases to produce symptoms within the usual incubation period, but that the third case received a minimum number of sporozoites, and that this is the reason for the protracted incubation period, i.e., 9 to 11 months. As a result of some recent experiments where the number of sporozoites injected is known, there is some evidence which suggests that about 2,000 sporozoites are necessary to ensure a normal incubation period. In a recent experiment the glands of five mosquitoes were dissected in Locke's fluid and an emulsion prepared. Two patients were infected, one receiving 371,300 sporozoites and the other 1,805. The patient receiving the large dose developed fever in 10 days while the patient receiving the smaller dose failed to develop either fever or Parasites and was subsequently infected by direct blood inoculation with the same strain and started fever within a week. If the number of B.T, malarial sporozoites necessary to produce clinical symptoms is in the region of 2,000 it is not difficult to understand why so many cases are latent but it is difficult to understand why so many of these latent cases, even the majority, have incubation periods of about 38 weeks, at least with certain strains of tropical and sub-tropical origin. DRUGS ON THE ~/[ORPHOLOGY OF PASASlTES. While some workers claim that they are able to distinguish quinine-affected parasites from the normal, others have failed to do so. Some years ago this was tested. I selected two patients who showed a heavy infection of B.T. malarial parasites and each was given 90 grains of quinine orally, one dose of 45 grains at 10 a.m. and a second dose 4 hours later. A series of thin films was taken at 2-hourly intervals over a period of 4 hours and stained witll Leishman. Films of patients who had not been given quinine were prepared at tile same time EFFECT OF
1~
LATENCY IN MALARIA
intervals and were stained together. The two sets of films were then examined by workers who were authorities on parasitology but all failed to distinguish which of the films contained quininized parasites. No such problem presents itself with atebrinized parasites. Following the administration of a single dose of 0.6 gramme of atebrin (mepacrine), changes in the parasite can be seen within 20 minutes. Normally, a full-grown asexual B.T. malarial parasite contains about 50 granules of pigment. Following a single large dose of atebrin, the first change which can be seen in the parasite is clumping of the pigment. In the first half-hour, instead of there being about 50 granules of pigment, there are about twenty clumps. An hour or two later the number is further reduced and usually consists of two or three lumps. After about 12 hours many of the parasites are devoid of pigment and some early disintegration of both chromatin and cytoplasm can be seen. (I have failed to observe where the pigment goes ; pigmented mononuclear cells in the peripheral circulation are not increased.) At about 24 hours, disintegration is well marked and often within 48 hours no parasites can be found in thin films. As far as my observations go, I have never seen a parasite which, after a dose of 0"6 gramme of atebrin, conforms to the normal. It is, therefore, possible that atebrin given over a period of several days may succeed in killing all the asexual parasites. Yet it is the case that many patients treated with this drug continue to relapse at relatively short intervals over a period of several montns. There is considerable evidence that plasmoquine (pamaquin), combined with quinine, reduces the relapse rate in B.T. malaria and many workers believe that this form of treatment is superior to either quinine or atebrin. When it is remembered that plasmoquine has very little action on B.T. malarial trophozoites, at least when compared with quinine or atebrin, it is difficult to understand why plasmoquinine in very small doses may reduce the relapse rate. Plasmoquine, when given a day or two before infection and continued for a few days afterwards, prevents the onset of a primary attack of B.T. malaria for several months, sometimes for a year. In M.T. malaria plasmoquine is evefi more successful and seems to be a true causal prophylactic. If this is so, then the indication is that against M.T. malaria, plasmoquine is either a sporozoiticidal drug or that it acts on the hypothetical stage of the parasite between the sporozoite and the erythrocytic parasite. What, then, is the difference between M.T. and B.T. malaria in this connection ? In B.T. malaria, as has already been noted, an attack is often delayed for nearly a year, even when the drug is given for only a few days following exposure to infection. A possible explanation is that plasmoquine kills all the sporozoites or x bodies in a M.T. infection, but not all in a B.T. infection. Even if this is so it would still not explain the very long interval between infection and symptoms in B.T. malaria. If, however, the sporozoites, after escaping from the peripheral circulation, succeeded in establishing themselves in reticulo-endothelial cells and were able to outlive the life of the host cell, they might, on being released, give rise to that
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stage of parasitism which brings about infection of the erythrocytes. Whatever the explanation of latency, it would appear that it is caused by a resting parasite and not one which is active, such as a trophozoite. In advancing the theory that it may be the sporozoite itself which is lying dormant all these months, it is of interest to note that in the insect host B.T. malarial sporozoites are able to lie dormant for several months without, apparently taking nourishment or changing their morphological character even within a wide range of temperature. The discovery of exoerythrocytic parasites in certain avian malarias, especially P. gallinaceum in the domestic fowl and P. lophurae in turkeys, has stimulated malaria research workers to attempt to demonstrate a similar cycle in mammalian malaria. From the literature it appears that these exoerythrocytic parasites are pathogenic and that in some cases they cause death to the host even before the later stages (erythrocytic parasites) have had time to develop. It would therefore seem unlikely that these forms of the parasite, even if they occur in B.T. malaria, are responsible for relapses. Furthermore, in B.T. malaria, relapses do not occur in blood-inoculated cases whereas exoerythrocytic parasites do occur in blood-inoculated cases ofP. gallinaceum or P. lophurae. Some workers have shown that a single dose of 1 gramme of quinine given orally will sterilize a primary infection of B.T. malaria induced by blood inoculation whereas the same quantity of quinine given daily for 30 days will not prevent a long-term relapse in mosquito-infected cases with the same strain. THE EVALUATION OF DRUGS I N
ANTI-RELAPSE TREATMENT.
It is a fundamental characteristic of many strains of B.T. malaria that between the primary attack and the first relapse there is usually an interval o f several months, but, as far as is known, the interval never exceeds 1 year. Following the first relapse, there is frequently a series of relapses which occur at about 1-monthly intervals and these may continue over a period of from 1 to 2 years. Are these short relapses caused by erythrocytic parasites (trophozoites), which escaped destruction during treatment of the previous relapse ? If sporozoites of B.T. malaria in human tissue cells are able to survive for many months as they are able to do in the tissue cells of a mosquito, it seems possible that they may be the direct cause of relapses. This could conceivably be the reason why one or many relapses recur at short intervals following an interval of many months up to 1 year between the primary attack and the first relapse. This phenomenon in B.T. malaria must have a very important bearing on t h e value of anti-relapse drugs. A patient who has had his first relapse 6 months to 1 year following his primary attack, followed by a number of short-term relapses, for about a year after the first relapse, will be semi-immune and such_ cases are not, at this stage of their disease, suitable for testing anti-relapse drugs. It is also the case that because so many patients infected with certain strains o f B.T. malaria do not experience a relapse for from 6 months to 1 year following
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the primary attack, the value of a drug as an anti-relapse treatment cannot be properly assessed for at least 1 year after the primary attack. I f relapses in B.T. malaria are caused by sporozoites or by an intermediate stage--between the sporozoite and the erythrocytic stages of the parasite-a drug which will prevent relapses would also conceivably be equally successful as a causal prophylactic and vice-versa. SUMMARY. The first relapse in B.T. malarial infections usually occurs several months after the treatment of the primary attack, and may be as long delayed as 1 year. A case is presented for considering that relapses are due to sporozoites which have been held up in tissue cells, a not improbable hypothesis when it is remembered that sporozoites are able to survive for many months in the tissue cells of the insect carrier. True latency is seen in those cases in which the primary attack occurs many months after infection, frequently after a delay of about 38 weeks. (The delay varies between 2 months and 1 year.) First relapses frequently occur at about the same period of time. Latency as defined in this paper occurs: (1) in nature; (2) following certain drug prophylaxis ; (3) when mixed infections of B.T. and M.T. malaria occur; (4)when patients who are immune to one strain of B.T. malaria are :infected by a different strain. The significance of the drug pamaquin (plasmoquine) is that it prevents the onset of an attack for many months,whereas, compared with quinine and mepacrine in an acute primary attack its action on erythrocytic parasites is slight. It is suggested that relapses in B.T. malaria are due to a resting phase of the parasite and that a drug which successfully prevents relapses would also act as a successful causal prophylactic and vice-versa. Sporozoites are known to survive for many months in the tissue cells of the insect host and it is suggested that they also may be able to do so in the human host. The word " prophylaxis " has been used in this paper deliberately in preference to the word " suppressive." When quinine is used as a prophylactic :against B.T. malarial infections, if the drug is discontinued a few days after exposure to infection, the onset of fever is delayed for at most a few days. When mepacrine or pamaquin is used, in one strain at least (Madagascar strain) fever seldom occurs within 2 months of infection ; and usually the period is between 34 and 38 weeks. It is, therefore, considered that while the word suppressive is applicable to quinine it is less appropriate to mepacrine and pamaquin. REFERENCES, :KoRTEWEG (1902) Klinische observaties over malaria in den winter van 1901-02. Herinneringsbundel voor Prof. ]~osenstein. Leiden : E. Ijdo, pp 263-281. SWELLENGItEBEL (1921~ Wanneer heeft ten onzent malariabesmetting plaats ? (2nd communication): Nederl. Tijdschr. v. Geneesk, 65, ii. 1486-88. ;SWELLENGREBEL• DE B U C K , (1938), Malaria in the Netherlands. Amsterdam : Scheltema & Holkema.