INTRODUCTION PATIENTS, MATERIALS, AND METHODS

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1 Am. J. Trop. Med. Hyg., 66(5), 2002, pp Copyright 2002 by The American Society of Tropical Medicine and Hygiene A RETROSPECTIVE EXAMINATION OF SPOROZOITE-INDUCED AND TROPHOZOITE-INDUCED INFECTIONS WITH PLASMODIUM OVALE: DEVELOPMENT OF PARASITOLOGIC AND CLINICAL IMMUNITY DURING PRIMARY INFECTION WILLIAM E. COLLINS AND GEOFFREY M. JEFFERY Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta Georgia Abstract. A retrospective analysis was made of clinical and parasitologic parameters in patients with induced Plasmodium ovale infection to document the initial clinical and parasitologic response and their subsequent development of clinical and parasitologic immunity, and to determine the effect of previous homologous and heterologous malaria on subsequent infection with this parasite. The prepatent periods were relatively uniform. Eight patients injected with sporozoites that had been stored frozen had a median prepatent period of 14 days (range days). Thirty-five patients infected via the bites of infected mosquitoes had a median prepatent period of 15 days (range days). In eight patients previously infected with P. vivax, the median prepatent period was 16 days. High-intensity fever ( 104 F) was frequently seen, with instances of fever 106 F recorded on many occasions. Fever > 101 F and > 104 F occurred for much shorter periods of time than had been observed in patients infected with P. falciparum. Parasite counts > 10,000/ L were infrequent; in most patients, such parasite counts rarely lasted more than two or three days. Gametocytemia was generally of low density and lasted only a few days. The overall length of the clinical and parasitologic period was much shorter compared with that seen in patients infected with P. falciparum. Previous infection with P. ovale did not prevent reinfection, but resulted in reduced levels of parasitemia and fever. Previous infection with heterologous species of Plasmodium did not prevent infection; some reduction in the frequency and intensity of fever and parasite counts was evident. Previous infection with homologous or heterologous parasites failed to eliminate the production of infective gametocytes. A total of 462 lots of mosquitoes were fed on 67 patients with no previous history of infection. Of these feedings, 168 (36.4%) resulted in infection as determined by the presence of oocysts on the midguts of dissected mosquitoes. As shown, the infection rate increased with the density of gametocytes even though 48 (23.4%) of 205 lots of mosquitoes fed when no gametocytes were detected were infected. INTRODUCTION Progress on the development of antimalarial vaccines against Plasmodium falciparum prompted us to review archival records collected at a time when the induction of plasmodial infection was a preferred treatment for patients with neurosyphilis. 1 4 In previous reports, we examined the parasitologic and clinical response to infection and the development of immunity in patients with primary and secondary infections with homologous P. falciparum 1,2 and the heterologous parasites P. malariae, P. ovale, and P. vivax. 3,4 Here we report a similar retrospective examination of records of patients with primary and secondary infections with P. ovale. This parasite is found mainly in Africa and its endemicity, along with that of P. malariae, coincides with that of P. falciparum. Thus, in areas where vaccines against P. falciparum may be introduced, knowledge on the heterologous parasites may prove vital in the accurate assessment of the efficacy of these vaccines. This information also should be useful when candidate vaccines for P. ovale are developed. Reports on many of these induced infections with P. ovale have been previously presented. 5 9 Infections were characterized by short primary clinical attack (8.1 paroxysms per attack) and a mean maximum parasitemia of 8,314/ L. The tendency to produce multiple broods resulted in frequent quotidian periodicity. 5 Clinical relapses generally terminated spontaneously after only a few febrile episodes. It was indicated that previous infections with P. vivax, P. falciparum, or P. malariae did not significantly affect subsequent infection with P. ovale. 5 Plasmodium ovale is a relapsing malaria in that parasitemia arising from resting stages in the liver (hypnozoites) results in the reappearance of parasitemia following 492 treatment with schizonticidal drugs such as chloroquine. Preerythrocytic schizonts in the liver have been described. 10 Here, we present a summary examination of the data from all the patients for whom records were available. A database was extracted from parasitologic and fever records acquired between 1949 and 1963 at National Institutes of Health installations then located at the South Carolina State Hospital in Columbia, South Carolina, and the Georgia State Hospital in Milledgeville, Georgia. An historical perspective on the use of malariatherapy for the treatment of patients with neurosyphilis has been previously reported. 1 PATIENTS, MATERIALS, AND METHODS management. Consent for whatever treatments the hospital staff determined necessary was granted by the families of the patients or the courts when patients were admitted to the hospitals. The decision to treat a neurosyphilitic patient with malaria was made as part of standard patient care by the medical staff of either the South Carolina State Hospital or the Georgia State Hospital. care and evaluation of clinical endpoints (e.g., fever) were the responsibility of the medical staff. During infection, the temperature, pulse, and respiration were checked every four hours and every hour during paroxysms (fevers) by hospital personnel. During paroxysms, patients were treated symptomatically. Infections were terminated at the direction of the attending physician. The U.S. Public Health Service (PHS) personnel provided the parasites for inoculation and monitored the daily parasite counts to determine the course of infection. Mosquito colonies were maintained by the PHS staff for mosquito infection and subsequent transmissions to other patients as assigned by

2 P. OVALE IN HUMANS 493 the hospital staff. All patients undergoing malariatherapy lived in screened wards of the hospital to prevent possible infection of local anophelines. Treatment. Treatment with noncurative doses of antimalarial drugs (primarily 5 10 grains [325 or 650 mg] of quinine sulfate) was occasionally given. Infections were terminated by the use of drugs appropriate for the different parasites. Mosquito feeding. Mosquitoes of the species Anopheles quadrimaculatus and An. albimanus were allowed to feed on patients during periods of overt or presumed gametocytemia to produce mosquito infections for transmission to additional patients. Samples of fed mosquitoes were dissected after appropriate incubation periods to determine oocyst and/or sporozoite rates and intensity. Whenever possible, salivary gland dissections were made on all mosquitoes fed on recipient patients to determine rates and intensity of sporozoites in the donor mosquitoes. Methods of mosquito rearing, maintenance, and use in malaria transmission have been described in detail in previous publications. 10,11 Strains of P. ovale. The Donaldson strain was isolated in June 1949 from a serviceman who had returned to the United States from the Pacific area some 3.5 years earlier. The infection apparently originated either in the Philippines or the Admiralty Islands; the best chance of acquiring the infection was on Luzon in the Philippines. 5 The Liberian strain was isolated in England by Dr. A. R. D. Adams (Liverpool School of Tropical Medicine) from a patient returning from Liberia and made available to the PHS laboratory by Professor P. C. C. Garnham of the London School of Hygiene and Tropical Medicine. 9 The first patient in the United States was inoculated with parasitized blood after four human passages in England. 12 s were infected either by the intravenous inoculation of parasitized blood or via sporozoites. In some cases, blood TABLE 1 Prepatent periods for 43 sporozoite-induced infections with the Donaldson (35) and Liberian (8) strains of Plasmodium ovale Previous malaria* Species of Anopheles Route of inoculation No. of mosquitoes No. positive Prepatent period (days) G-346 quadrimaculatus + albimanus Bites G-480 quadrimaculatus Bites 52? 13 G-455 Po quadrimaculatus + albimanus Bites 45? 13 S-812 Pm, Pf quadrimaculatus Bites G-460 quadrimaculatus + albimanus Bites G-449 quadrimaculatus Bites G-484 quadrimaculatus Inoc G-487 quadrimaculatus Inoc G-344 quadrimaculatus Bites G-418 quadrimaculatus + albimanus Bites G-329 quadrimaculatus Bites G-306 quadrimaculatus Bites G-354 quadrimaculatus Bites G-357 quadrimaculatus Bites G-356 quadrimaculatus Bites G-488 quadrimaculatus Inoc G-472 quadrimaculatus Bites G-450 Pv quadrimaculatus Inoc G-459 Pv quadrimaculatus Inoc S-1135 quadrimaculatus Bites G-467 quadrimaculatus Bites G-331 quadrimaculatus + albimanus Bites G-386 quadrimaculatus Bites G-377 quadrimaculatus Bites G-190 Pv quadrimaculatus Bites G-266 Pv, Pf, Po, Pm quadrimaculatus Bites S-1074 quadrimaculatus Bites?? 16 S-1089 quadrimaculatus Bites G-402 quadrimaculatus Bites G-340 quadrimaculatus + albimanus Inoc G-355 quadrimaculatus Bites G-490 albimanus Inoc S-1080 quadrimaculatus Bites 4? 16 G-373 Pv quadrimaculatus + albimanus Bites G-332 Pv quadrimaculatus Bites G-223 Pv quadrimaculatus Bites G-267 Pv quadrimaculatus Bites G-91 Pv, Pm quadrimaculatus + albimanus Bites G-409 quadrimaculatus Bites S-1129 Pf quadrimaculatus Bites G-335 quadrimaculatus Bites S-1017 Pv quadrimaculatus Bites 15? 18 G-481 quadrimaculatus + albimanus Inoc *Po P. ovale; Pm P. malariae; Pf P. falciparum; Pv P. vivax. Inoculated with the Liberian strain; all others infected with the Donaldson strain. Inoculated with dissected salivary glands that had been stored frozen.

3 494 COLLINS AND JEFFERY FIGURE 1. Prepatent periods for 43 sporozoite-induced infections with the Donaldson and Liberian strains of Plasmodium ovale. and/or sporozoites were preserved frozen and were thawed immediately before inoculation into the patient. 13 Parasitemia. Thick and thin peripheral blood films were made daily by the method of Earle and Perez, 14 stained with Giemsa, and examined microscopically for the presence of parasites. The threshold of detection was approximately 10 parasites/ L. and sexual parasites were recorded per microliter of blood. During the later stages of the infection, when parasites were infrequently seen and at low densities, and no symptoms of malaria were evident, blood films were usually made two or three times a week. Data presentation. The parasite counts and fever records from the two hospitals have been combined. Reported here are data recorded for 177 patients. These patients were infected with P. ovale for the treatment of paresis and other mental disorders associated with tertiary syphilis. The following measurements were considered for each patient: 1) the presence of fever 101 F and 104 F, 2) parasite counts 1,000/ L and 10,000/ L, and 3) the presence of 100 gametocytes/ L for each patient. These parasitologic and fever records were examined to determine the development of immunity following primary and secondary malariatherapy. Evidence of developing immunity was the following: 1) reduction of all fever episodes ( 101 F), 2) reduction in high intensity fever episodes ( 104 F), and 3) reduction in days with parasite densities 1,000/ L, and 4) reduction in days with parasite densities 10,000/ L. Two different types of infections were considered: 1) sporozoite-induced infections and 2) trophozoite-induced infections. Presented graphically are 1) frequency of fever 101 F and 104 F (number of patients with fever/number of patients remaining) for each of the first 60 days of patent par- TABLE 2 parasitemia, days of fever, and length of parasitemia for 30 sporozoite-induced infections with the Donaldson (28) and Liberian (2) strains of Plasmodium ovale in patients with no history of malaria s of fever s of parasitemia G C,Q S , Q G-357 3, S , G , C G-377 7, C G-409 8, G-355 3, C,Q,R S , G-346 2, C G-329 8, C G-402 2, G , C G-480 4, Q G , G-331 5, S , T G-460 7, G , Q G , G-335 3, G , T,Q G-490 7, C,R G-472 9, C G , G-467 6, G , G , G , G , C,R *C chloroquine; Q quinine; R primaquine; T bismuth thioglycolate. Liberian strain of P. ovale; all others are the Donaldson strain. No fever chart available.

4 P. OVALE IN HUMANS 495 FIGURE 2. and sexual parasitemia, fever, and mosquito infection for patients with primary infections with Plasmodium ovale. A, patient G-467 injected with sporozoites of the Donaldson strain; fever 104 Fon10days.B, patient G-475 infected with trophozoites of the Liberian strain; fever 104 F on 7 days. * days when mosquitoes were infected. asitemia, 2) frequency of parasite counts 1,000/ L and 10,000/ L during the first 60 days of patent parasitemia, and 3) the frequency of gametocytemia 100/ L during this same period. Prepatent periods for the sporozoite-induced infections are also plotted. The days of maximum parasitemia, number of days with fever 101 F and 104 F, the number of days of asexual parasitemia 10/ L and 10,000/ L, and gametocytemia 100/ L are presented in tabular form for each of the 177 patients. For sporozoite-induced infections, the species of mosquito, and the route of inoculation (bites or injection by syringe), the number of mosquito bites, the intensity of the salivary gland infections, and the prepatent periods are given. The intensity of the salivary glands was determined by rating the number of sporozoites present at dissection as 1+ (1 10 sporozoites), 2+ ( sporozoites), 3+ (101 1,000 sporozoites), or 4+ (> 1,000 sporozoites). The total number of + ratings was then determined. RESULTS s were divided into three groups. Group I consisted of 30 patients who received their primary infections via sporozoites, Group II consisted of 70 patients who received their primary infections via trophozoite inoculation, and Group III consisted of 77 patients who were infected with P. ovale following infection with P. falciparum, P. malariae, P. vivax, and/or P. ovale. FIGURE 3. Frequency of fever, asexual parasitemia, and gametocytemia during the first 60 days in patients with sporozoite-induced infections with the Donaldson and Liberian strains of Plasmodium ovale. A, frequency of fever ( 101 F and 104 F). B, frequency of asexual parasitemia ( 1,000/ L and 10,000/ L). C, frequency of gametocytemia ( 10/ L and 100/ L). Prepatent periods for the 43 sporozoite-induced infections ranged from 12 to 20 days (Table 1 and Figure 1). The median prepatent period was 15 days. There were 35 transmissions via mosquito bite using An. quadrimaculatus and/or An. albimanus. Eight infections were induced by the injection of sporozoites from salivary glands that had been stored frozen for extended periods.

5 TABLE 3 parasitemia, days of fever, and days of parasitemia for 70 trophozoite-induced infections with the Donaldson (66) and Liberian (4) strains of Plasmodium ovale in patients with no history of malaria s of fever s of parasitemia G , C G-455 3, G-358 7, C S , G-391 7, C G , Q,C S , S , S , S , G , T,C G , C G-443 4, G-361 8, C G-468 7, C G , C G , C G , C G-317 5, S , S , G-405 6, C G-410 8, C G-470 7, C S , S Q S , Q G , S , C S , S , G , S , G-448 9, G-442 9, G , S , S , S , Q S , G-434 9, G-309 4, G-421 9, S , G , P G , G-447 6, G , S , G , Q G , G-374 8, S , S , G-478 1, G , Q S , G ,408 9 # # S , G , G-479 2, G-399 7, S , S , G-417 7, G-485 8, G , G , S , G , *C chloroquine; Q quinine; T bismuth thioglycolate; P chlorguanide. No fever chart available. Liberian strain of P. ovale; all others are the Donaldson strain.

6 P. OVALE IN HUMANS 497 Group I. This group was composed of 30 patients with sporozoite-induced infections who had no record of previous infection with Plasmodium. Prepatent periods ranged from 12 to 20 days (mean 14.9 days, median 14 days) (Table 1 and Figure 1). The maximum parasitemia, days of fever, and days of parasitemia are shown in Table 2. Treatments with chloroquine and primaquine terminated the parasitemia, whereas treatments with quinine and bismuth thioglycolate were given to temporarily reduce parasitemia. A chart of patient G-467, who was infected with sporozoites of the Donaldson strain (Figure 2A), is illustrative of the data available for these patients. Fever. There were 306 episodes of fever 101 F in the 29 infections for whom fever records were available; of these, 144 (47.1%) were 104 F (Table 2). The frequency of fever 101 F and 104 F for these patients (day of fever divided by patients still infected) is shown in Figure 3A. Peak incidence of fever occurred on days 8, 9, and 10. Parasitemia. Periods of parasitemia ranged from seven to 76 days; 11 of the 30 patients had maximum parasite counts 10,000/ L (Table 2). Only 22 days of parasite counts 10,000/ L were recorded. The mean maximum parasite count was 9,711/ L (range ,600/ L). The daily frequency of parasitemia > 1,000/ L and > 10,000/ L (Figure 3B) indicated a peak on days 9 and 10. Twenty-two of the 30 patients produced detectable gametocytemia; only seven of these had gametocyte densities > 100/ L. The daily frequency of gametocytemia (Figure 3C) indicated a peak on day 10. Group II. This group was composed of 70 patients with primary trophozoite-induced infections with P. ovale (Table 3). A chart of patient G-475, infected with trophozoites of the Liberian strain (Figure 2B) is illustrative of the data available for these patients. There were 644 episodes of fever 101 F in the 67 patient infections for whom fever records were available; of these, 288 (44.7%) were 104 F (Table 2). The frequency of fever 101 F and 104 F for these patients is shown in Figure 4A. Peak fever occurred most frequently on day 11. TABLE 4 Infection of mosquitoes with Plasmodium ovale by feeding on patients with no history of infection* / L FIGURE 4. Frequency of fever, asexual parasitemia, and gametocytemia during the first 60 days in patients with trophozoite-induced infections with the Donaldson and Liberian strains of Plasmodium ovale. A, frequency of fever ( 101 F and 104 F). B, frequency of asexual parasitemia ( 1,000/ L and 10,000/ L). C, frequency of gametocytemia ( 10/ L and 100/ L). 0 Pos./exam Pos./exam. 100 Pos./exam. Totals Percent 3 1/2 1/ /6 1/1 4/ /12 4/4 1/1 11/ /9 6/6 2/2 10/ /12 7/8 2/3 13/ /9 6/13 9/10 18/ /5 5/15 5/7 12/ /8 4/16 4/7 10/ /10 1/15 3/4 5/ /10 3/22 1/3 4/ /11 4/20 1/4 6/ /11 4/17 1/3 5/ /16 1/10 1/1 4/ /8 2/7 0/3 2/ /8 1/5 0/1 3/ /9 3/4 1/2 6/ /6 7/9 1/1 11/ /8 6/6 2/3 9/ /6 6/7 7/ /6 6/7 7/ /4 1/1 2/ /2 2/3 1/ /27 5/5 1/1 16/ Totals 48/205 85/201 35/56 168/462 Percent * Pos./exam positive/ examined.

7 498 COLLINS AND JEFFERY TABLE 5 parasitemia, days of fever, and length of parasitemia for 1 sporozoite-induced and 8 trophozoite-induced infections with the Donaldson (3) and Liberian (6) strains of Plasmodium ovale in patients with a history of infection with Plasmodium ovale parasitemia/ L s of fever s of parasitemia G-455* G G G-451 9, G G-449 1, G-435 1, G-473 4, G * sporozoite-induced infection. Infected with the Liberian strain of P. ovale; previous infection with the Donaldson strain. G-447 was rechallenged twice with the Donaldson strain. Parasitemia. Periods of parasitemia ranged from 10 to 112 days; 26 of 70 patients had maximum parasite counts 10,000/ L (Table 3). Twenty-two days of parasite counts >10,000/ L were recorded. The mean maximum parasite count was 7,257/ L (range ,200/ L). The frequency of parasitemia > 1,000/ L and > 10,000/ L (Figure 4B) indicated a peak on days 9 and 10. Fifty-eight of the 69 patients produced detectable gametocytemia; in only seven of these were gametocyte densities 100/ L. The frequency of gametocytemia (Figure 4C) indicated a peak on day 10. Mosquito infection. Mosquitoes were fed on patients to obtain sporozoite infections for transfer to other patients. Gametocyte counts were routinely low when compared with infections with P. vivax. Thus mosquitoes were frequently fed when no gametocytes were detected on the blood film (Table 4). A total of 462 lots of mosquitoes were fed on 67 of the 100 patients with no previous history of infection. Of these feedings, 168 (36.4%) resulted in infection as determined by the presence of oocysts on the midguts of dissected mosquitoes. As shown, the infection rate increased with the presence of gametocytes, even though 48 (23.4%) of 205 lots of mosquitoes, fed when no gametocytes were detected, were infected. Group III. This group was composed of 77 patients. Nine patients had been previously infected with P. ovale, 18 had been previously infected with P. falciparum, 27 had been previously infected with P. vivax, nine had been previously infected with P. malariae, and 14 had been previously infected with two or more species of Plasmodium before being infected with P. ovale. Previous infection with P. ovale. The nine patients infected previously with P. ovale (Table 5) had maximum parasite counts from 70 to 9,360/ L (mean 954/ L). There were 12 episodes of fever and only two episodes of fever 104 F. Only two patients produced detectable gametocytemia. Mos- TABLE 6 parasitemia, days of fever, and length of parasitemia for 1 sporozoite-induced and 17 trophozoite-induced infections with the Donaldson strain of Plasmodium ovale in patients with a history of infection with Plasmodium falciparum s of fever s of parasitemia S-830 1, G-268 4, S , S , S , C G-387 4, G-289 1, S , S S , C S , S , C S , S , S , S , C S , C G-308 1, *C chloroquine. No fever chart available. Sporozoite-induced infection.

8 P. OVALE IN HUMANS 499 quitoes were fed on four of the patients; infection occurred on three of 12 feeding days. Previous infection with P. falciparum. The 18 patients previously infected with P. falciparum (Table 6) had maximum parasite counts from 930 to 14,653/ L (mean 3,620/ L). Fever 101 F occurred on 113 occasions (7.2 episodes per patient); 58 of these (51.3%) measured 104 F. There were only four occasions when the parasite count was > 10,000/ L. The peak day for frequency of parasitemia 1,000/ L (Figure 5A) was day 7. were observed in 15 of the 18 patients. Mosquitoes were fed on 10 patients; mosquitoes were infected on five of 20 occasions (20%). Previous infection with P. vivax. The 27 patients previously infected with P. vivax (Table 7) had maximum parasite counts from 464 to 35,520/ L (mean 4,078/ L). Fever 101 F occurred on 185 occasions (7.1 episodes per patient) for the 26 patients in which fever charts were available; 82 of these fevers (44.3%) were 104 F. There were only seven occasions in which the parasite count was 10,000/ L. The peak day for frequency of parasitemia 1,000/ L (Figure 5B) was day 8. were observed in 19 of the 27 patients. Mosquitoes were fed on 14 patients; mosquitoes were infected on 57 of 79 occasions (72.2%). Previous infection with P. malariae. The nine patients previously infected with P. malariae (Table 8) had maximum parasite counts from 820 to 7,920/ L (mean 3,241/ L). Fever 101 F occurred on 33 occasions (4.1 episodes per patient) for the eight patients in which fever charts were available; 14 of these (42.4%) were 104 F. There were no occasions when the parasite count was 10,000/ L. The peak days for frequency of parasitemia 1,000/ L (Figure 5C) were days 7, 9, and 10. were observed in four of the nine patients. Mosquitoes were fed on only one patient; mosquitoes were infected. Previous infection with multiple species of Plasmodium. The 14 patients previously infected with P. vivax, P. malariae, P. falciparum, and/or P. ovale (Table 9) had maximum parasite counts from 1,250 to 12,000/ L (mean 3,424/ L). Fever 101 F occurred on 66 occasions (5.1 episodes per patient) for the 13 patients in which fever charts were available; 29 of these (43.9%) were 104 F. There were only three occasions in which the parasite count was > 10,000/ L. The peak day for frequency of parasitemia 1,000/ L was day 7. were observed in eight of the 14 patients. Mosquitoes were fed on seven patients; mosquitoes were infected on six of 13 occasions (46.2%). Mean daily parasite counts. All of the high-density parasitemia occurred during the first 20 days of patent parasitemia. Comparisons were made between the mean daily parasite counts for the different groups of patients (Table 10). There were no significant differences on any days between the geometric mean parasite counts for sporozoite-induced and trophozoite-induced primary infections. For those patients previously infected with P. falciparum, there was a significant difference between the primary sporozoite-induced infections on day 9 and the trophozoite-induced infections on day 10. For those patients previously infected with P. vivax, there was a significant difference between both groups of the primarily infected patients on days 10 through 14 and days 19 and 20; there was a significant difference from the trophozoite-induced primary infections and those previously infected with P. vivax on days 15 through 18. With patients FIGURE 5. Frequency of asexual parasitemia during the first 60 days of patent parasitemia of Plasmodium ovale. A, frequency of asexual parasitemia ( 1,000/ L and 10,000/ L) in 18 patients previously infected with P. falciparum. B, frequency of asexual parasitemia ( 1,000/ L and 10,000/ L) in 27 patients previously infected with P. vivax. C, frequency of asexual parasitemia ( 1,000/ L and 10,000/ L) in nine patients previously infected with P. malariae. previously infected with P. malariae, there was a significant difference between the trophozoite-induced primary infections on day 16. For those patients previously infected with P. ovale, there was a significant difference between both the sporozoiteinduced and trophozoite-induced patients with primary infections on days 6 through 20. For patients with multiple spe-

9 500 COLLINS AND JEFFERY TABLE 7 parasitemia, days of fever, and length of parasitemia for 8 sporozoite-induced and 19 trophozoite-induced infections with the Donaldson (26) and Liberian (1) strains of Plasmodium ovale in patients with a history of infection with Plasmodium vivax s of fever s of parasitemia G-373 4, Q,C S , C G-332 1, C G , C G-190 5, C G-450 7, G , C G-267 7, C S , C S , G-21 4, C S , C G-432 4, C G-171 5, C S , C S , C S-533 1, G-440 5, T S , C S , S-768 2, C G , S-670 4, C S G , C G-454 6, C S *Q quinine; C Chloroquine; T bismuth thioglycolate. Sporozoite-induced infection. No fever chart available. Liberian strain of P. ovale; all others are the Donaldson strain. cies infections, there were significant differences between both of the primary infection groups and this group on days 9 through 14. An examination of the curves of the geometric mean parasite counts for the different groups of patients (Figure 6) shows the marked effect of prior infection with P. ovale and the somewhat reduced curves for those previously infected with other species of Plasmodium. Relapse. Sporozoite-induced infections with P. ovale frequently relapse after treatment with effective schizonticidal drugs such as chloroquine (1,500 mg over a three-day period). G-335 spontaneously controlled the primary attack; parasites reappeared in the peripheral blood films on day 240 of patent parasitemia. The patient was treated with chloroquine (as above) on day 245; parasites reappeared on day 397. The patient was again treated with chloroquine (day 397). Parasites reappeared on day 517 and the patient was again treated on day 519. No additional parasites were seen through day 1,067. There was no way to determine if the reappearance of parasites at day 240 was a recrudescence or a relapse; it is TABLE 8 parasitemia, days of fever, and length of parasitemia for 9 trophozoite-induced infections with the Donaldson strain of Plasmodium ovale in patients with a history of infection with Plasmodium malariae s of fever s of parasitemia S S , S # S , C S , S , S , C S , S , *C chloroquine. No fever chart available.

10 P. OVALE IN HUMANS 501 TABLE 9 parasitemia, days of fever, and length of parasitemia for 14 patients infected with the Donaldson strain of Plasmodium ovale following previous infection with P. vivax (Pv), P. malariae (Pm), P. ovale (Po), and/or P. falciparum (Pf) Previous malaria s of fever s of parasitemia G-432 Pv, Po, Pf 1, G-266 Pv, Pf, Po, Pm 6, S-812 Pm, Pf 3, G-91 Pv, Pm 12, S-1053 Pm, Pf 1, S-911 Pv, Pm 5, C S-1270 Pf, Pv 1, C S-1109 Pm, Pf 1, S-1322 Pf, Pm 3, S-1102 Pf, Pm 1, C S-1121 Pm, Pf 6, S-1246 Pf, Pm 2, S-1325 Pv, Pf 4, G-266 Pv, Pf 8, T *C chloroquine; T bismuth thioglycolate. Sporozoite-induced infection. No fever chart available. obvious that the reappearance of parasitemia on days 397 and 517 were relapses. s G-346, G-329, G-472, and G-190 were treated with 1,500 mg of chloroquine following their primary attack (days 30, 35, 57, and 45, respectively); relapses occurred at days 100, 142, 224, and 186, respectively. s G-354, G-332, and G-267 were similarly treated with chloroquine (days 5, 27, and 44, respectively), and failed to relapse through 240, 273, and 409 days of observation, respectively. Eleven sporozoite-induced infections (in patients G-418, G-331, G-386, G-340, G-449, G-467, G-306, G-481, G-344, G-148, and G-91) were not treated after their primary attack; after periods of undetected parasitemia following the primary attack, parasites reappeared on days 155, 159, 122, 140, 152, 210, 100, 107, 187, 148, and 126, respectively. It is impossible to say with certainty whether the reappearance was due to recrudescence or relapse; however, the interval suggests that these also may be relapses from persistent liver-stage parasites. Three additional patients with sporozoite-induced infections (T-402, T-460, and T-484), were not treated; following the primary attack, parasites failed to reappear after 168, 473, and 128 days of observation. DISCUSSION As in previous reports, 1 4 there were two major objectives in conducting the retrospective analysis of clinical and parasitologic parameters in patients with induced P. ovale infec- TABLE 10 Geometric mean parasite counts for the first 20 days of patent parasitemia for patients infected with Plasmodium ovale No previous infection Previously infected with s of patent parasitemia Sporozoite-induced (Group I) Trophozoite-induced (Group II) P. falciparum P. vivax P. malariae P. ovale , , * 1, , , , , , * 1, , , , , , * 1, , , , , , * 901.8* 10 3, , , * 1, * 443.7* 11 2, , * * 330.1* 12 2, , * * 134.6* 13 1, , * * 130.4* , * * 114.4* * * * * * * * * 43.5 * Significantly different from Groups I and II. Significantly different from Group I. Significantly different from Group II. Multiple species

11 502 COLLINS AND JEFFERY secondary treatment with chloroquine. In the absence of treatment with chloroquine, some patients experienced reappearance of parasitemia suggestive of relapses, based on the long interval between the primary attack and the reappearance of parasitemia. s treated with primaquine (15 mg/ day for 14 days) failed to relapse. Acknowledgments: We gratefully acknowledge Dr. Martin D. Young, who directed the U.S. Public Health Service installations in Columbia, South Carolina and Milledgeville, Georgia during the time these data were collected. We thank many members of the technical staff who assisted in the compilation and analysis of the data. Authors addresses: William E. Collins, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Mailstop F-12, 4770 Buford Highway, Atlanta, GA Geoffrey M. Jeffery (Public Health Service, retired), 1093 Blackshear Drive, Decatur, GA FIGURE 6. Geometric mean parasite counts during the first 20 days of patent parasitemia with Plasmodium ovale for groups of patients with and without previous infection with homologous and heterologous species of Plasmodium. tion: 1) to document the initial clinical and parasitologic response and the subsequent development of clinical and parasitologic immunity in humans infected with P. ovale, and 2) to determine the effect of previous homologous and heterologous malaria on subsequent infection with this parasite. The prepatent periods were relative uniform. The eight patients injected with sporozoites that had been stored frozen had median prepatent periods of 14 days (range days). The patients infected via the bites of infected mosquitoes had median prepatent periods of 15 days (range days). In the eight patients previously infected with P. vivax, the median prepatent period was 16 days. Fever was the primary indicator of clinical malaria. The frequency of high-intensity fever ( 104 F) was highly noticeable, with instances of fever 106 F being recorded on many occasions. However, the occurrence of fever 101 F and 104 F covered much shorter periods than had been observed in patients infected with P. falciparum. 1 3 Parasite counts 10,000/ L were infrequent; in most patients, parasite counts at this level rarely lasted more than two or three days. Gametocytemia was generally of low density and lasted only a few days. The overall lengths of the high-intensity clinical and high-density parasitologic periods were markedly short compared with that seen in patients infected with P. falciparum. Previous infection with P. ovale did not prevent reinfection, but resulted in reduced levels of parasitemia and fever. Previous infection with heterologous species of Plasmodium did not prevent infection; some reduction in the frequency and intensity of fever and parasite counts was evident. Previous infection with homologous or heterologous parasites failed to eliminate the production of infective gametocytes. Some of the sporozoite-induced infections were observed long enough to determine periods of relapse. The interval was highly variable. Some patients had several relapses following REFERENCES 1. Collins WE, Jeffery GM, A retrospective examination of sporozoite- and trophozoite-induced infections with Plasmodium falciparum: development of parasitologic and clinical immunity during primary infection. Am J Trop Med Hyg 61(suppl): Collins WE, Jeffery GM, A retrospective examination of secondary sporozoite- and trophozoite-induced infections with Plasmodium falciparum: development of parasitologic and clinical immunity following secondary infection. AmJTrop Med Hyg 61(suppl): Collins WE, Jeffery GM, A retrospective examination of sporozoite- and tophozoite-induced infections with Plasmodium falciparum in patients previously infected with heterologous species of Plasmodium: effect on development of parasitologic and clinical immunity. Am J Trop Med Hyg 61(suppl): Collins WE, Jeffery GM, A retrospective examination of the patterns of recrudescence in patients infected with Plasmodium falciparum. Am J Trop Med Hyg 61(suppl): Jeffery GM, Young MD, Wilcox A, The Donaldson strain of malaria. 1. History and characteristics of the infection in man. AmJTropMedHyg3: Wilcox A, Jeffery GM, Young MD, The Donaldson strain of malaria. 2. Morphology of the erythrocytic parasites. Am J Trop Med Hyg 3: Jeffery GM, The Donaldson strain of malaria. 3. The infection in the mosquito. AmJTropMedHyg3: Jeffery GM, Young MD, The Donaldson strain of malaria. 4. An evaluation and status. Am J Trop Med Hyg 3: Jeffery GM, Wilcox A, Young MD, A comparison of West African and West Pacific strains of Plasmodium ovale. Trans R Soc Trop Med Hyg 49: Mayne B, Young MD, The technique of induced malaria as used in the South Carolina State Hospital. Venereal Dis Information 22: Collins WE, Jeffery GM, Methods and Techniques for the Handling of Mosquitoes in Human and Animal Malaria Studies. Proceedings of the 49th Annual Meeting of the New Jersey Mosquito Extermination Association, Garnham PCC, Bray RS, Cooper W, Lainson R, Qwad FI, Williamson J, The pre-erythrocytic stage of Plasmodium ovale. Trans R Soc Trop Med Hyg 49: Jeffery GM, Rendtorff RC, Preservation of viable human malaria sporozoites by low-temperature freezing. Exp Parasitol 4: Earle WC, Perez M, Enumeration of parasites in the blood of malarial patients. J Lab Clin Med 17: