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Multiple invasion of erythrocyte by malaria parasites
268 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE. Vol. 64. No. 2. 1970.
MULTIPLE INVASION OF ERYTHROCYTE BY MALARIA PARASITES C H U N G C. WANG
Department of Health, Washington Heights Health District, New York In malaria, invasion of the erythrocyte by two or more merozoites is most commonly observed in Plasmodium falciparum infections, but may occur with P. vivax and P. ovale and very rarely with P . malariae. The significance of multiple invasion has been a moot question. GRASSI (1920) recorded a single erythrocyte which contained a gametocyte and a schizont of P. vivax and postulated the theory of parthenogenesis, assuming that a single malaria merozoite~had divided into sexual and asexual forms. TI~ONSON (1917) and WENYON (1926) refuted this theory by demonstrating that this was merely a multiple invasion. They showed that the so-called parthenogenetic body consisted of two parasites in a single erythrocyte with ill defined membranes separating the gametocyte and the schizont. SCIJOFFNER and DEGRAAF (1937) suggested 3 theories for multiple invasion: 1. The merozoites adhere to each other and fail to part when they invade a single erythr0cyte. 2. The immature reficulocytes are especially prone to invasion by several merozoites at the same time. 3. The ring forms undergo binary fission. It would be difficult to explain by the first and second theories the existence of the ring forms of different sizes if they entered the erythrocyte simultaneously. The ring forms should be of the same size if they have the same stage of development. Certainly the mixed invasion of a gametocyte and a schizont in one erythrocyte contradicts the theory of binary fission. At the Tropical Disease Diagnostic Service of New York City multiple invasions have been observed in all the four species. This observation prompted a study of the relationship between the frequency of multiple invasion and the intensity of malaria infection.
Materials and methods According to BOYD (1944), in P. malariae infections the parasitized erythrocytes seldom exceed 10,000 per c.mm., i.e., less than 0.2% of the erythrocytes; with P. vivax the figure is 50,000 per c.mm., i.e., less than 1% of the erythrocytes. Of the P. vivax patients reported here, in none did the parasites reach the ceiling set by Boyd. Therefore, unless the parasitaemia is so high that almost 10% of the erythrocytes are infected, as in heavy P. falciparum infection, it is difficult to express the malaria parasites as a simple percentage of the erythrocytes present. In animal experiments where the malaria parasitaemia is high, counting up to 10,000 erythrocytes is sufficient to get a workable ratio of the infected erythrocytes for comparison. We find that in the cases presented The author wishes to thank Dr. Harold W. Brown and Dr. Louis H. Miller, Columbia University; Dr. Eileen H. Pike, New York Medical College; Dr. Howard B. Shookhoff, Department of Health, New York City and Columbia University; Dr. Vincent F. Guinee, Director, Bureau of Preventable Diseases, Department of Health, New York City; for advice and reading the manuscript.
CHUNG C. WANG
269
here, including some with low parasitaemia, a count up to 50,000 erythrocytes is necessary to get a workable ratio of the infected erythrocytes. Our study is based upon 4 cases each of P. vivax, P. falciparum and P. ovale infection, and one of P. malariae, seen during 1967 and 1968. The number of parasites per 50,000 erythrocytes were counted with the help of a calibrated ocular reticule after the method described by PETERS (1965). A blood cell counter was used to facilitate the tabulation of the normal erythrocyte, the single invasion, double invasion, triple invasion and quadruple invasion. All the counts were done on thin blood films stained with Giemsa. All the patients were from the United States except one who was a student from Liberia. 7 of them acquired malaria in West Africa, 3 in Vietnam, 2 in Pakistan and one by transfusion in New York City. Two were females and 11 males; ages ranged from 12 to 45 years. Results
TABLE I.
Patient
No. of parasitized cells per 50,000 erythrocytes
Multiple invasion of erythrocytes
Cells with single invasion
Cdlswith double invasion
Cells with triple invasion
Cells with quadruple invasion
A--P. vivax infections
4
39
37
64
61
94
88
0
B--P. f alciparum infections 1
2
2
9
3
108
102
4
10,000
8,621
1,259
99
21
C--P. ova~ infections 1
5
5
0
0
0
2
15
15
0
0
0
3
16
16
0
0
0
4
238
234
3
1
0
152
2
0
0
D--P. malariaeinfecfions 1
154
270
MULTIPLE INVASION OF ERYTHROCYTE BY MALARIA PARASITES
It seems that when the parasitized erythrocytes increased in numbers, the multiple invasions also increased in all the species, but the rate of multiple invasions with respect to the height of parasitaemia varies slightly from species to species. One triple invasion of a single erythrocyte was seen in the blood films from patients No. 3 and No. 4 of the P. vivax infections. These triple invasions were not included in the count because they were found only after extensive search.
Comment THOMSON (1917) found multiple invasion associated with P. vivax infections when parasitaemia was high; we also found that where multiple invasions were present the parasitaemia was high--our tables suggest a positive correlation between multiple invasion and the height of parasitaemia. It has been often stated that multiple invasion of erythrocytes is pathognomonic of P. falciparum infections, but the exceedingly high intensity of parasitaemia in these infections is probably a major factor in the frequent Occurrence of multiple invasions. When the parasitaemia of the other species becomes unusually high, multiple invasions may also be found. It should be noted that delay in diagnosis and a high intensity of parasitaemia often go hand in hand. Thus in Thomson's group, soldiers with malaria were thought at first to be febrile because of wound infections. There may be other unknown species factors which may deter the development of multiple invasions, such as the species preference for immature erythrocytes, or the inability of the erythrocyte to accommodate more than one parasite without rupture. Anaemia may enhance the development of multiple invasion because the numbers of available erythrocytes per unit volume are reduced. However, the occurrence of multiple invasion may be mainly due to the presence of a greater number of parasitized erythrocytes, which results in a greater number of maturing schizonts, presenting the opportunity for more multiple invasions. Statistical analysis on these cases is not practicable. The relationship between multiple invasion and the intensity of parasitaemia should be confirmed statistically in controlled animal experiments.
Summary 13 cases of light and heavy infections with P. vivax, P. falciparum, P. ovale and P. malariae were studied. The results suggest a positive correlation between multiple invasion of erythrocytes and the intensity of parasitaemia. REFERENCES BOYD, M. F. (1944). ]. Am. med. Ass., 124, 1179. GRASSI, B. (1920). Studi di un Zoologo sulla Malaria, Rome. PETERS, W. (1965). Exp. Parasit., 17, 80. SCHUFFNER, W. • DE GRAAF,W. (1937). Besondere Eigenschaften der Malariaplasmodien, Nocht Festschrift, Hamburg. THOMSON, J. D. (1917). ft. R. Army med. Cps., 29, 379. WENYON, C. M. (1926). Protozoology, 2. London: Bailliere, Tindall & Cox.
MULTIPLE INVASION OF ERYTHROCYTE BY MALARIA PARASITES C H U N G C. WANG
Department of Health, Washington Heights Health District, New York In malaria, invasion of the erythrocyte by two or more merozoites is most commonly observed in Plasmodium falciparum infections, but may occur with P. vivax and P. ovale and very rarely with P . malariae. The significance of multiple invasion has been a moot question. GRASSI (1920) recorded a single erythrocyte which contained a gametocyte and a schizont of P. vivax and postulated the theory of parthenogenesis, assuming that a single malaria merozoite~had divided into sexual and asexual forms. TI~ONSON (1917) and WENYON (1926) refuted this theory by demonstrating that this was merely a multiple invasion. They showed that the so-called parthenogenetic body consisted of two parasites in a single erythrocyte with ill defined membranes separating the gametocyte and the schizont. SCIJOFFNER and DEGRAAF (1937) suggested 3 theories for multiple invasion: 1. The merozoites adhere to each other and fail to part when they invade a single erythr0cyte. 2. The immature reficulocytes are especially prone to invasion by several merozoites at the same time. 3. The ring forms undergo binary fission. It would be difficult to explain by the first and second theories the existence of the ring forms of different sizes if they entered the erythrocyte simultaneously. The ring forms should be of the same size if they have the same stage of development. Certainly the mixed invasion of a gametocyte and a schizont in one erythrocyte contradicts the theory of binary fission. At the Tropical Disease Diagnostic Service of New York City multiple invasions have been observed in all the four species. This observation prompted a study of the relationship between the frequency of multiple invasion and the intensity of malaria infection.
Materials and methods According to BOYD (1944), in P. malariae infections the parasitized erythrocytes seldom exceed 10,000 per c.mm., i.e., less than 0.2% of the erythrocytes; with P. vivax the figure is 50,000 per c.mm., i.e., less than 1% of the erythrocytes. Of the P. vivax patients reported here, in none did the parasites reach the ceiling set by Boyd. Therefore, unless the parasitaemia is so high that almost 10% of the erythrocytes are infected, as in heavy P. falciparum infection, it is difficult to express the malaria parasites as a simple percentage of the erythrocytes present. In animal experiments where the malaria parasitaemia is high, counting up to 10,000 erythrocytes is sufficient to get a workable ratio of the infected erythrocytes for comparison. We find that in the cases presented The author wishes to thank Dr. Harold W. Brown and Dr. Louis H. Miller, Columbia University; Dr. Eileen H. Pike, New York Medical College; Dr. Howard B. Shookhoff, Department of Health, New York City and Columbia University; Dr. Vincent F. Guinee, Director, Bureau of Preventable Diseases, Department of Health, New York City; for advice and reading the manuscript.
CHUNG C. WANG
269
here, including some with low parasitaemia, a count up to 50,000 erythrocytes is necessary to get a workable ratio of the infected erythrocytes. Our study is based upon 4 cases each of P. vivax, P. falciparum and P. ovale infection, and one of P. malariae, seen during 1967 and 1968. The number of parasites per 50,000 erythrocytes were counted with the help of a calibrated ocular reticule after the method described by PETERS (1965). A blood cell counter was used to facilitate the tabulation of the normal erythrocyte, the single invasion, double invasion, triple invasion and quadruple invasion. All the counts were done on thin blood films stained with Giemsa. All the patients were from the United States except one who was a student from Liberia. 7 of them acquired malaria in West Africa, 3 in Vietnam, 2 in Pakistan and one by transfusion in New York City. Two were females and 11 males; ages ranged from 12 to 45 years. Results
TABLE I.
Patient
No. of parasitized cells per 50,000 erythrocytes
Multiple invasion of erythrocytes
Cells with single invasion
Cdlswith double invasion
Cells with triple invasion
Cells with quadruple invasion
A--P. vivax infections
4
39
37
64
61
94
88
0
B--P. f alciparum infections 1
2
2
9
3
108
102
4
10,000
8,621
1,259
99
21
C--P. ova~ infections 1
5
5
0
0
0
2
15
15
0
0
0
3
16
16
0
0
0
4
238
234
3
1
0
152
2
0
0
D--P. malariaeinfecfions 1
154
270
MULTIPLE INVASION OF ERYTHROCYTE BY MALARIA PARASITES
It seems that when the parasitized erythrocytes increased in numbers, the multiple invasions also increased in all the species, but the rate of multiple invasions with respect to the height of parasitaemia varies slightly from species to species. One triple invasion of a single erythrocyte was seen in the blood films from patients No. 3 and No. 4 of the P. vivax infections. These triple invasions were not included in the count because they were found only after extensive search.
Comment THOMSON (1917) found multiple invasion associated with P. vivax infections when parasitaemia was high; we also found that where multiple invasions were present the parasitaemia was high--our tables suggest a positive correlation between multiple invasion and the height of parasitaemia. It has been often stated that multiple invasion of erythrocytes is pathognomonic of P. falciparum infections, but the exceedingly high intensity of parasitaemia in these infections is probably a major factor in the frequent Occurrence of multiple invasions. When the parasitaemia of the other species becomes unusually high, multiple invasions may also be found. It should be noted that delay in diagnosis and a high intensity of parasitaemia often go hand in hand. Thus in Thomson's group, soldiers with malaria were thought at first to be febrile because of wound infections. There may be other unknown species factors which may deter the development of multiple invasions, such as the species preference for immature erythrocytes, or the inability of the erythrocyte to accommodate more than one parasite without rupture. Anaemia may enhance the development of multiple invasion because the numbers of available erythrocytes per unit volume are reduced. However, the occurrence of multiple invasion may be mainly due to the presence of a greater number of parasitized erythrocytes, which results in a greater number of maturing schizonts, presenting the opportunity for more multiple invasions. Statistical analysis on these cases is not practicable. The relationship between multiple invasion and the intensity of parasitaemia should be confirmed statistically in controlled animal experiments.
Summary 13 cases of light and heavy infections with P. vivax, P. falciparum, P. ovale and P. malariae were studied. The results suggest a positive correlation between multiple invasion of erythrocytes and the intensity of parasitaemia. REFERENCES BOYD, M. F. (1944). ]. Am. med. Ass., 124, 1179. GRASSI, B. (1920). Studi di un Zoologo sulla Malaria, Rome. PETERS, W. (1965). Exp. Parasit., 17, 80. SCHUFFNER, W. • DE GRAAF,W. (1937). Besondere Eigenschaften der Malariaplasmodien, Nocht Festschrift, Hamburg. THOMSON, J. D. (1917). ft. R. Army med. Cps., 29, 379. WENYON, C. M. (1926). Protozoology, 2. London: Bailliere, Tindall & Cox.