Primary malarial thrombocytopenia in the rhesus monkey

70 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.

Vol. 60.

No.

1.

1966.

P R I M A R Y M A L A R I A L T H R O M B O C Y T O P E N I A IN T H E R H E S U S M O N K E Y BY

WILLIAM A. SODEMAN, JR. AND GEOFFREY M. JEFFERY

Laboratory of Parasite Chemotherapy, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda 14, Maryland The association of thrombocytopenia with malaria in man has been reported during the last 40 years by PAISSEAUand LEMAII~ (1916), Mast OVA (1924), SrmAGERand I~AN (1946), MALDONADO-AI~LmqI)Eet al. (1960), Kvvm et al. (1962), and HILL et al. (1964). Kuvm et al. (1962) and HILL et al. (1964) described the presence of thrombocytopenia during attacks of sporozoite-induced and blood-induced malaria in non-immune volunteers. Thrombocytopenia was stated to occur regularly in the early phase of Plasmodium vivax and P. cynomolgi bastianellii infections; we subsequently observed similar thrombocytopenia in sporozoite-induced P. cynomologi (M strain) infections in non-immunes. With the hope of establishing a suitable model system we have carried out an investigation of platelet alterations in the rhesus monkey during P. cynomologi (M strain) infection. Methods

5 rhesus monkeys were used for these studies; 3 were intact, and in 2 the spleen had been removed 36 days before infection. All monkeys were infected by intravenous inoculation of salivary glands, or by bites of Anopheles quadrimaculatus infected with P. cynomologi (M strain) or both. Daily platelet counts and white blood counts were made, and thick and thin films were stained with Giemsa for parasite counts. In 4 of the monkeys, daily microhaematocrit determinations were made and sera were frequently tested for malarial antibody by the indirect fluorescent antibody (FA) test. Platelets were counted by phase microscopy according to the method of BRECHER et al. (1953). Results

The 5 monkeys showed a significant fall in the platelet count after the onset of patent parasitaemia (Fig.). A consistent but mild leucopenia was noted before or concurrently with maximum parasitaemia. The severity of infection was enhanced in the splenectomized monkeys, as shown by prolongation of high parasite density. This increase in the severity of the infection did not affect the thrombocytopenia with regard to time of onset, duration or depth of fall of platelet count. Details of the thrombocytopenic response are summarized in the Table. Minimum platelet counts ranged from 23,750 to 60,000 per c.mm. of blood, or about 12% of the normal level. All platelet counts returned to normal levels in the face of continuing, though less severe, parasitaemia. No antimalarial agents were administered during the periodof

WILLIAM A. SODEMAN~JR. AND GEOFFREY M. JEFFERY

71

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i~ ~oc

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i i 1'5 20~ 25~ ~0 IO TIME (days) FROM INFECTION

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72

PRIMARYMALARIALTHROMBOCYTOPENIAIN THE RHESUSMONKEY

TABLE. Thrombocytopenic response in P. eynomolgi infection in rhesus monkeys. Prepatent period (days)

Day of maxamum parasitaemia

Day of minimum platelet count

Mean platelet count (prepatent period) _ SD

Minimum platelet count

WJ1

12

22

22

393500 ± 96750

41560

173

10

19

18

434500 ± 29250

23750

174

8

16

16

520938 ± 41750

49500

167

10

18

19

388500 ± 58000

60000

168

8

14

19

323500 -- 52250

43500

Monkey

study. Progressive anaemia was noted, with decrease of the haemotocrit from 40-45% to 21-26°//o. Reticulocyte counts as such were not performed; however, it was observed that reticulum, regarded as cell remains in thick Giemsa smears (WALKER, 1952), was scanty until after platelet densities had begun to recover. At that time increased reticuhim became evident. Considerable difference in the ability to produce malarial antibody measurable by the indirect FA test was noted between intact and splenectomized monkeys (FIG.). A measurable antibody titre had occurred by the 14th day in the intact monkeys but was not apparent until the 23rd and 25th days in the splenectomized monkeys. Discussion

Whenever specifically sought, thrombocytopenia has been noted as a regularly occurring pathological phenomenon in human and simian P. vivax-like malaria. Review of the literature suggests several mechanisms for thrombocytopenic syndromes observed in malarious patients, which may be grouped into 4 general categories: (1) coincident idiopathic thrombocytopenia; (2) hypersplenism induced by malaria; (3) drug-induced purpuras; and (4) primary malarial th~ombocytopenia. Idiopathic thrombocytopenia unrelated to the infection occasionally occurs in malarious individuals. Such an occurrence is suggested by a case reported by OOSTERHUIS (1960) where exacerbations ofpurpura and haemorrhage were not clearly related to exacerbations of parasitaemia, or improved by antimalarial chemotherapy. Malarial infection with accompanying splenomegaly, causing hypersplenism with platelet depression, represents a second general category of malarial thrombocytopenia, the characteristic clinical picture of which is discussed by ~vIALDONADO-ALLEbIDEet al. (1960). A confirmatory platelet response can be expected to follow splenectomy. The drug-induced purpuras form the third general association between malaria and thrombocytopenia, with quinine the most common offender. SHRAGERand KEAN (1946) reported 9 such occurrences in a series of 6,000 cases of quinine-treated malaria at Gorga.s Hospital in the Canal Zone. Primary malarial thrombocytopenia is the name we have given to the thrombocytopenic syndrome directly related to malaria infection. Our results permit us to outline the platelet response in rhesus monkeys with sporozoite-induced P. cynomologi. A fall in the platelet count is noted in the non-immune monkey 6-10 days after the

WILLIAM A. SODEMAN, JR. AND GEOFFREY M. JEFFERY

73

occurrence of patent parasitaemia. The fall is progressive over a period of 2-4 days. Then spontaneously, i.e., without specific or non-specific treatment, the platelet count rises and returns to normal levels in 2-6 days. A positive tourniquet test and impaired clot retraction may be noted at the time of lowest platelet count. A similar pattern of thrombocytopenic response has been noted during P. vivax and P. cynomolgi infection in non-immune human volunteers. The platelet depression is noted earlier in man, beginning 1-3 days after patency, with lowest counts noted 4-12 days after patency. The general pattern of thrombocytopenia and recovery remains the same whether the infection is blood-induced or sporozoite-induced. Case reports suggestive of primary malarial thrombocytopenia have been made by PAISSEAUand LEMAIRE(1916) and SHRAGER and KEAN(1946). MASLOVA(1924) has also observefl thrombocytopenia which appeared to be directly related to the acute attack of malaria in patients from an endemic area. Several explanations for this phenomenon have been postulated by HILL et al. (1946), including (1) platelet destruction, (2) sequestration, (3) an auto-immune mechanism (i.e., platelet agglutinins), or (4) decreased production or inhibited release, or both. To these we would add a fifth possible explanation: removal by a non-specific immune mechanism such as immune-adherence. Increased platelet destruction is an implied rather than an observed phenomenon. It has been suggested that platelets may adhere to erythrocyte stroma or may be lysed by circulating toxic agents. Recovery of platelet counts in the face of continuing parasitaemia suggests that such a mode of platelet destruction is not active in the rhesus monkey. Platelet sequestration by an acutely enlarged spleen is well documented, as is acute splenomegaly early in malaria infection. It would not seem unreasonable to expect that the acute malarial splenomegaly contributes to the development ofthrombocytopenia. However, the absence of any demonstrated effect of splenectomy on the primary malarial thrombocytopenic response in two of our monkeys suggests that a splenic contribution to platelet depression is not essential to the primary thrombocytopenic response. Platelet agglutinins have not been investigated in either monkeys or human subjects infected with malaria. Immune adherence, i.e., the clumping of platelets by antigenantibody complexes, has been well described in general terms but has not been investigated in malarial infection. Malaria antibody (as measured by the indirect FA test) and antigen (the parasite) were present in intact monkeys before the appearance of thrombocytopenia. In the splenectomized monkeys, however, appearance of antibody was delayed until platelet counts had begun to return to normal (Fig.). In the absence of measurable antibody, immune adherence would not seem a valid explanation for platelet depression. The remaining suggestion of impaired production or release of platelets best coordinates the available information on this syndrome, though it should be recognized that more than one mechanism may contribute to the platelet fall. Although the severity of the leucopenia was variable, in all cases a fall in white blood count was present. Evidence of reticulocyte production was notably absent in spite of demonstrable anaemia until platelet and leucocyte densities had begun to return to normal. The coincidence of decrease in peripheral leucocytes and thrombocytes, plus absence of peripheral reticulocytes, suggests a primary dysfunction in production or release of formed elements, i.e. a marrow failure. It is interesting to note in this regard that lymphocytes were not affected. Lymphocytes claim their origin from lymphoid follicles outside the marrow spaces and a relative lymphocytosis is not infrequently reported in malaria. The small number of reported investigations of marrow performance in human or

74

PRIMARY MALARIAL THROMBOCYTOPENIA IN THE RHESUS MONKEY

simian malaria have been limited in scope. The most comprehensive studies were those of KNOTTGEN (1949, 1961) in which both P. vivax and P. falciparum malaria was studied in Europeans returning from tropical areas. Marrow was obtained at variable times in the course of the malarial attack, usually once per patient. Megakaryocytes were not mentioned but abnormalities were noted in the progression of both red and white cell series. A dyspoietic anaemia with atypical red cell precursors was described. Nuclear abnormalities in erythrocyte precursors were common. White cell abnormalities were also noted, with nuclear abnormalities and giant forms. Cell counts showed a relative increase in metamyelocytes and a "reactive marrow" which in the face of leucopenia suggests arrest of maturation, although this interpretation was not made by Knfittgen. It is apparent that the marrow studies reported in human malaria demonstrate abnormal cellular development consistent with the impression that malaria infection may be accompanied by altered hematopoiesis producing, among other things, peripheral thrombocytopenia.

Summary In 3 intact and 2 splenectomized monkeys infected with Plasmodium cynomolgi a significant thrombocytopenia was observed. Platelet counts were at their lowest point at about the time of maximum parasitaemia but returned to normal in the face of continuing parasitaemia. This syndrome appears to have direct relation to the malaria infection and has been termed primary malarial thrombocytopenia. REFERENCES BRECHER, G., SCHEIDERMAN, M. & CRONKITE, E. P. (1953). Amer. J. clin. Path., 23, 15. HILL, G. J., KNIGHT,V. • JEFFERY,G. M. (1964). Lancet, 1, 240. KNOTTGEN,H. (1949). Z. Tropenmed. Parasit., 1, 178. (1963). Ibid., 14, 423. KtrVlN, S. F., BEYE, H. K., STOHLMAN,F., CONTACOS,P. G. & COATNEY, G. R. (1962). Trans. R. Soc. trop. Med. Hyg., 56, 371. MALDONADo-ALLENDE,I., GARCIACASTELLANOS, J. A., NELLI, E., MARISTANY,G., RYSSER, O. & CASTAGNO, R. (1960). Prensa med. Argent., 47, 1475. MASLOVA, A. N. (1924). Russk. Zh. trop. Med., 3, 7. OOSTERHUIS,G. J. (1960). Doc. Med. geogr, trop., 4, 85. PAISSEAU, G. & LEMAIRI~,H. (1916). Bull. Mdm. Soc. mgd. H6p. Paris, 32, 1672. SHRAGER,J. & KEAN, B. H. (1946). Amer. J. reed. Sci., 212, 54.