Interactions between oral contraceptives and malaria infections in rhesus monkeys*

Bulletin ofthe World Health Organization, 62 (6): 931 - 939 (1984) © World Health Organization 1984 Interactions between oral contraceptives and malaria infections in rhesus monkeys* G. P. DUTTA,' S. K. PURI,2 K. K. KAMBOJ,3 S. K. SRIVASTAVA,4 & V. P. KAMBOJ5 The interaction oforal contraceptives with malaria infection (Plasmodium cynomolgi B and P. coatneyi) in adultfemale rhesus monkeys (Macaca mulatta) was studied. The oral contraceptives (Norinyl and Ovral-28) were administered for 12 consecutive menstrual cycles, from day 5 to 25 of each cycle, at either '13 of the human dose of Norinyl (norethisterone 0.33 mg + ethinylestradiol 0.012 mg) or 116 of the human dose of Ovral-28 (norgestrel 0.083 mg + ethinylestradiol 0.008 mg). The animals were divided into three groups for each infection (control, Norinyl and Ovral-28 treated) with 10 monkeys in each group for P. cynomolgi B infection and 12 in each group for P. coatneyi infection. The animals were infected after 6 cycles of oral contraceptive administration, and the course ofinfection was studied during the 7th and 8th cycles. This wasfollowed by radical cure during the 9th and 10th cycles and rechallenge in the 11th and 12th cycles. The present study showed that (1) the contraceptive-treated animals maintained a slightly increased cumulative parasite load; (2) the contraceptives did not interfere with the radical curative action of chloroquine; (3) the contraceptive-treated animals showed no significant change in the course ofparasitaemia on rechallenge or in the malaria indirect fluorescent antibody levels; (4) the liver function tests were not altered significantly by the administration of contraceptives and subsequent infection; and (5) the haematological changes observed in the contraceptive-treated animals were similar to those observed in the control group. Malaria continues to be a major health problem and a cause of morbidity and mortality in many tropical and subtropical countries of the world. In addition, many of these countries are facing the problem of an unprecedented population explosion. Global efforts are, therefore, under way to control this population problem through family planning including the large-scale use of hormonal contra- ceptives. Currently more than 50 million women, primarily in the developed countries, are taking combined oral contraceptive pills, and the use of such * From the Central Drug Research Institute, Post Box No. 173, Lucknow 226001, India. This paper and the one by Collins et al. (Bulletin of the World health Organization, 62 (4): 627-637(1984)) on the same WHO-sponsored project were meant to be published in the same issue of the Bulletin. The two teams carried out this work independently in Atlanta, GA, USA, and in Lucknow, India, with equal priority. Publication of the present paper took longer during processing and interested readers should study the two papers together. ' Assistant Director, Division of Microbiology, Central Drug Research Institute, Lucknow, India. Requests for reprints should be addressed to this author. 2 Research Associate, Division of Microbiology. 3 Senior Research Fellow, Division of Microbiology. 4 Scientist, Division of Microbiology. 5 Deputy Director, Division of Endocrinology. contraceptives for fertility regulation is expected to increase in the developing cpuntries in the future. The interactions between tropical diseases like malaria and oral contraceptives have not been adequately studied so fariA preliminary report from the Gambia indicated that women taking Ovral-28 were exposed to a greater risk of malaria infection, as shown by the prevalence rate and higher levels of parasitaemia (1). In view of the lack of any controlled study, the WHO Special Programme of Research, Development and Research Training in Human Reproduction proposed that two centres (Centers for Disease Control, Atlanta, GA, USA, and the Central Drug Research Institute, Lucknow, India) should investigate the interaction of oral contraceptives (Norinyl and Ovral-28) with malaria infections (Plasmodium cynomolgi B and P. coatneyl) using adult female rhesus monkeys (Macaca mulatta) as host. The major objective of this study was to evaluate the following: the effect of the adminis- tration of oral contraceptives on the course of malaria infection and on the development of malaria immunity and malaria antibody titres, the changes in haematological parameters and liver function tests, 4493 931- 932 G. P. DUTTA ET AL. the efficacy of the antimalarial drug chloroquine during oral contraceptive administration, and the basal progesterone levels during oral contraceptive therapy with concurrent malaria infection. MATERIALS AND METHODS Maintenance and acclimatization of animals Female rhesus monkeys (Macaca mulatta), weighing 4-7 kg, were kept under quarantine for one month in the primate house of the Central Drug Research Institute, Lucknow, and were fed on a standard animal diet. They were certified to be free from tuberculosis by the tuberculin ocular test and chest X-ray. Blood smears were checked to exclude malaria, trypanosomiasis or other protozoan infections. Animals showing a negative indirect fluorescent antibody test for malaria were included in the study. The animals were caged individually and subjected to 12 hours of daily exposure to artificial light (fluorescent lighting) from 07h 00 to 19h 00. Female monkeys with evidence of menstrual bleeding and parity (as judged by breast size and state of genital region sex skin) were included in the study. Study groups The acclimatized female rhesus monkeys were randomly assigned to one of the following study groups: (1) P. cynomolgi B control group (10 monkeys) (2) P. cynomolgi B + Norinyl (10 monkeys) (3) P. cynomolgi B + Ovral-28 (10 monkeys) (4) P. coatneyi control group (12 monkeys) (5) P. coatneyi+ Norinyl (12 monkeys) (6) P. coatneyi + Ovral-28 (12 monkeys) The oral contraceptives were administered to the designated groups from day 5 to 25 of each menstrual cycle. Induction of menstrual cycle The menstrual cycle in each monkey was induced initially by intramuscular injection of 0.25 ml Menstrogen forte (1 ml containing 5 mg estradiol benzoate and 50 mg progesterone) daily for 3 days: 95% of the animals menstruated after 6-14 days. They were then observed for three consecutive cycles by using macroscopic and microscopic techniques and those showing regular cycles were identified. Selection of ovulating monkeys The occurrence of ovulation in the selected animals was monitored by their plasma progesterone levels at four different times in each menstrual cycle - on days 10, 14, 18 and 22 (the first day of menstrual bleeding was taken as day 1 of the cycle), in accord with WHO recommended procedures.' The criterion adopted to indicate positive ovulation was a progesterone level of 0.5-3.5 nmol/l on day 10 and above 10 nmol/l on days 14 or 18 of the menstrual cycle. The progesterone levels of each animal were followed for three consecutive cycles. In order to ensure regular ovulation .in each cycle, the female monkeys were kept in proximity of a dominant male. Animals with normal 'functioning of the pituitary- ovarian-uterine axis, as shown by the hormonal levels, were selected for further study involving oral contraceptive administration for twelve cycles. Oral contraceptives Two types of contraceptive pills (Norinyl and Ovral-28) were supplied by WHO for use in the study: Norinyl contains norethisterone 1 mg + ethinyl- estradiol 0.035 mg (human dose). Ovral-28 contains norgestrel 0.5 mg+ ethinyl- estradiol 0.05 mg (human dose). The pills were macerated in gum acacia, suspended in distilled water, and administered orally from day 5 to 25 in each cycle. Determination of the ovulation-inhibiting dose of oral contraceptives: treatment cycles 1-3 Norinyl administered daily at 1/6 of the human dose (norethisterone 0.167 mg + ethinylestradiol 0.006 mg) resulted in anovulatory cycles as judged by endogenous progesterone levels, but the menses did not occur after drug withdrawal in over half the animals. The dose of this pill was therefore increased to 1/3 of the human dose in the subsequent study. Ovral-28 at 1/6 of the human dose (norgestrel 0.083 mg + ethinylestradiol 0.008 mg) administered daily to monkeys for the first three cycles produced complete inhibition of ovulation in all the animals and the menses were regular after drug withdrawal. So this dose was continued throughout the study. Synchronization ofthe menstrual cycle and ovulation inhibition studies: treatment cycles 4-6 Norinyl at 1/3 (norethisterone 0.33 mg+ ethinyl- estradiol 0.012 mg) and Ovral-28 at 1/6 of the human dose administered daily were found to inhibit ovulation during cycles 4-6 in all the monkeys, and the menses after drug withdrawal were normal. These a Programme for the Provision of Matched Assay Reagents for the Radioimmunoassay of Hormones in Reproductive Physiology. Method manual. 5th edition, 1981. Geneva, World Health Organization (unpublished document). ORAL CONTRACEPTIVES AND MALARIA IN MONKEYS 933 doses of oral contraceptives were therefore continued during the period of malaria infection, cure, and rechallenge (treatment cycles 7-12). Malaria infections: treatment cycles 7 and 8 Blood-induced P. cynomolgi B or P. coatneyi infections in both the test groups (animals treated with oral contraceptive) and in the control groups were used in this study. Infection was introduced on day 5 of the 7th cycle using 5 x 104 parasitized RBC for intravenous inoculation. The course of malaria infection was studied by recording daily the parasitaemia per 104 RBC in thin blood smears stained with Giemsa. No drug intervention was required during the course of P. cynomolgi B infection. Since untreated infections with P. coatneyi occasionally resulted in severe anaemia followed by death of some animals, it was considered safe in this study to administer quinine sulfate (300 mg for 1-2 doses) when the primary parasitaemia reached approximately 200 parasites/104 RBC. The course of parasitaemia (with both infections) was recorded for a period of 8 weeks (from day 6 of the 7th cycle to day 5 of the 9th cycle). Oral contraceptives were continued during this period. Radical cure with chloroquine: treatment cycles 9 and 10 Chloroquine phosphate was administered for 3 days at a dose of 20 mg (chloroquine base) per kg body weight per day in 10 ml of distilled water by orogastric tube. This treatment was given on days 6-8 of the 9th treatment cycle. Blood smears were examined twice weekly up to day 5 of the 11th cycle. Oral contraceptives were continued during this period. Rechallenge with malaria infections: treatment cycles 11 and 12 All the animals were rechallenged with their respective malaria infections on about day 6 of the 11th cycle. The inoculum comprised 5 x l04 parasitized RBC (intravenous route) per animal, and the course of blood parasitaemia per 104 RBC was monitored daily for 8 weeks (up to day 5 of the 13th cycle). Oral contraceptives were continued during this period. Assay of hormonal levels Plasma progesterone levels were measured throughout the study on days 10, 14, 18 and 22 of each cycle in order to monitor the ovulation-inhibitory action of the oral contraceptives. b b See footnote a. Assay of malaria antibody levels The indirect fluorescent antibody (IFA) test was used to monitor weekly the serum malaria antibody levels of the experimental monkeys using anti- monkey IgG labelled with fluorescein isothiocyanate (2). Observations were recorded with an AO (American Opticals) fluorescence microscope using a BG-12 filter. Assay ofserum protein fractions Serum protein fractions (a,, Ci2, and -y globulins) and the albumin/globulin (A/G) ratio were assayed using cellulose acetate membrane electrophoresis on a Beckman microzone electrophoresis cell, and the membranes after staining with Ponceau-S were scanned under a Beckman densitometer (3). Liverfunction and haematological tests The serum levels of aspartate aminotransferase (formerly glutamic-oxaloacetic transaminase, GOT), alanine aminotransferase (formerly glutamic-pyruvic transaminase, GPT), and lactate dehydrogenase (LDH) (4), and bilirubin (5) were monitored at weekly intervals. The effect of the malaria infections and concurrent oral contraceptive administration on different haematological parameters, namely, haemoglobin (Hb), RBC count, packed cell volume (PCV), erythrocyte sedimentation rate (ESR after 1 hour), platelet count, total leukocyte count and differential leukocyte counts, and the total absolute polymorph (TAP) and total absolute lymphocyte (TAL) counts, was studied (6, 7). In order to understand the type of anaemia, other parameters were calculated including mean cell (corpuscular) volume (MCV), mean cell (corpuscular) haemoglobin (MCH), and the mean cell (corpuscular) haemoglobin concentration (MCHC). RESULTS Course of P. cynomolgi B infections (Fig. 1) The Norinyl group, compared with the controls, maintained a higher level of parasitaemia throughout the phase of primary infection, although the increase was significant (P < 0.05) only during the 8th treatment cycle. Compared with the controls, the Ovral-28 group also maintained a high level of parasitaemia throughout the phase of primary infection, but the increase was significant only in the 8th treatment cycle. After rechallenge (11th treatment cycle), the Ovral-28 group maintained a slightly higher level of parasitaemia, compared with the control and Norinyl groups, although the differences G. P. DUTTA ET AL. Im 1I0 S In Uz oi. Sth Cytb number Fig. 1. Geometric mean daily parasitaemia of rhesus monkeys infected with P. cynomolgi B (7th to 9th treatment cycle) and after rechallenge (1 1 th treatment cycle), in control/Norinyl/Ovral-28 groups. (Parasitaemia plotted on semi-log graph). CH = Chloroquine 20 mg/kg x 3 day treatment; INOC = Inoculation with 5 x 104 parasitized RBC. between the groups were not significant. During the 12th cycle, the parasitaemia remained at a very low level in all three groups. A comparison of the cumulative parasitaemia during the 8 weeks of primary infection (7th-9th cycles) in the control and both contraceptive groups (Table 1) showed that the Norinyl group maintained a 1.46 times higher cumulative parasitaemia while the Ovral-28 group maintained a 1.84 times higher level, compared with the control group. On rechallenge also (1 I th cycle), the parasite load in the two contraceptive groups was slightly higher in the Ovral-28 group. During the 7th treatment cycle, the peak parasitaemia levels were significantly higher in both contraceptive groups, compared with the controls (P < 0.05) (Table 2). A comparison of the course of Table 1. Cumulative parasitaemiaa in P. cynomolgi B infected rhesus monkeys (control, Norinyl and Ovral-28 groups) during the 7th, 8th, 9th and 11th treatment cycles Total (7th-9th Group 7th cycle 8th cycle 9th cycle cycle) 11th cycle Control 2115.70 92.08 2.86 2210.64 293.51 Norinyl 3030.70 188.04 3.57 3222.31 363.31 Ovral-28 3454.55 605.19 4.31 4064.05 420.74 e Based on daily parasite counts/104 RBC. Table 2. Peak parasitaemia levels in P. cynomolgi B infected rhesus monkeys compared with a control group (parasites/104 RBC) treated with oral contraceptives, Peak parasitaemia levels 7th cycle 8th cycle 11 th cycle Serial number Control Norinyl Ovral-28 Control Norinyl Ovral-28 Control Norinyl Ovral-28 1 684 1828 1254 2.4 520.0 482.0 126 Died 95 2 656 822 523 82.0 7.8 2.4 130 284 92 3 274 526 1042 1.6 2.7 42.0 114 134 136 4 261 522 484 1.5 481.0 2.5 95 112 151 5 765 854 893 7.0 8.0 2.5 91 116 124 6 764 725 425 50.0 30.0 30.0 160 85 298 7 281 887 825 44.0 6.5 4.5 146 425 250 8 442 846 655 2.4 2.5 80.0 71 342 170 9 484 876 957 2.1 69.0 504.0 210 60 270 10 791 657 984 1.7 15.0 32.0 75 128 91 Mean 540.20± 854.30:± 804.20± 19.47± 114.25± 118.19± 121.80± 187.33± 167.70± ±S.D. 217.67 368.87 272.95 28.76 204.75 199.10 42.70 129.21 77.69 S.E. 68.83 116.65 86.31 9.09 64.75 62.96 13.50 43.07 24.57 Significance P < 0.05 P < 0.05 NS NS NS NS 934 ORAL CONTRACEPTIVES AND MALARIA IN MONKEYS infection and peak parasitaemia levels in individual monkeys during the primary infection also showed that 50%o of the control animals developed primary peak levels above 5%, while 80%o of the Ovral-28 group and 1000/o of the Norinyl group of animals developed peak levels above 5 0o. Course of P. coatneyi infections (Fig. 2) During the 7th treatment cycle, the control group maintained a significantly higher level of parasitaemia compared with both contraceptive groups (P < 0.05). However, during the 8th treatment cycle both contraceptive groups maintained a significant,ly higher parasitaemia level compared with the control group (P < 0.05). During the rechallenge phase ( 11th and 12th treatment cycles) there was no significant difference in the parasitaemia levels among the three groups. Cumulative parasitaemia during the 8 weeks of primary infection (7th-9th cycles) in the control and both contraceptive groups (Table 3) showed that the Norinyl group maintained a 1.25 times higher cumulative parasitaemia while the Ovral-28 group maintained a 1.14 times higher level, compared with the control group. During rechallenge (11th cycle), ' *CoZrol e oinyl 0-. Owel 'U t INOC INOC 7dh Ith 9th 11 th Cceb number Fig. 2. Geometric mean daily parasitaemia of rhesus monkeys infected with P. coatneyi (7th to 9th treatment cycle) and after rechallenge (1 1 th treatment cycle), in control/Norinyl/Ovral-28 groups. (Parasitaemia plotted on semi-log graph). CH e Chloroquine 20 mg/kg x 3 days treatment; INOC= Inoculation with 5 x 104 parasitized RBC. Table 3. Cumulative parasitaemiaa in P. coatneyi infected rhesus monkeys (control, Norinyl and Ovral-28 groups) during the 7th, 8th, 9th and 11th treatment cycles Total (7th-9th Group 7th cycle 8th cycle 9th cycle cycle) 11th cycle Control 1987.42 1371.23 135.27 3493.92 560.50 Norinyl 1738.00 2493.75 145.58 4377.33 769.17 Ovral-28 1827.75 2041.58 118.40 3987.73 707.20 a Based on daily parasite counts/104 RBC. the parasite load in both contraceptive groups was slightly higher. The primary peak parasitaemia in the different groups during the first two weeks after infection (7th treatment cycle) could not be considered reliable for statistical analysis because the parasitaemia had been suppressed by 1-2 doses of quinine (300 mg each time) which was administered to all the P. coatneyi- infected monkeys to prevent severe anaemia. Analysis of the peak parasitaemia levels during the 8th cycle showed a significantly higher peak level in the Norinyl group compared with the controls (P < 0.05). Similarly, the Norinyl-treated animals showed significantly higher peak levels of parasitaemia during rechallenge (11th cycle) (P < 0.005) (Table 4). The Ovral-28 group showed exceptionally high parasitaemia in two animals (No. 1 and 5), reaching levels up to 12% and 9.8%. Two monkeys in this group succumbed to the infection owing to anaemia (No. 5 and 12). Serological responses IFA antibody titres. Primary IFA titres after P. cynomolgi infection (Fig. 3) in the control and both contraceptive (Norinyl and Ovral-28) groups were similar and the peak levels of the geometric mean of reciprocal titres attained by the 3rd week of infection were 2.95, 2.83 and 3.16, respectively. After cure with chloroquine, the titres declined to 1.84, 1.87 and 1.96, respectively, in the 3rd week of radical cure. Upon rechallenge, the titres again increased to 2.98, 2.94 and 3.04, respectively, by the 4th week of rechallenge. In the case of P. coatneyi infection also (Fig. 4), peak antibody titres in the control, Norinyl and Ovral-28 groups were attained in the 3rd week of the 8th cycle and the geometric mean values were 2.94, 2.83 and 3.07, respectively. After radical cure, these values declined to 1.86, 1.88 and 1.84 and, on subsequent rechallenge, the titres increased to 3.09, 2.96 and 2.89, respectively. 935 936 G. P. DUTTA ET AL. Table 4. Peak parasitaemia levels in P. coatneyi infected rhesus monkeys treated with oral contraceptives, compared with a control group (parasites /104 RBC) Peak parasitaemia levels 7th cycle 8th cycle 11th cycle Serial number Control Norinyl Ovral-28 Control Norinyl Ovral-28 Control Norinyl Ovral-28 1 324 380 623 376 466 1202 132 170 137 2 245 350 280 120 379 286 215 225 164 3 456 456 374 158 371 328 172 235 218 4 260 474 470 330 480 124 90 195 117 5 481 646 980 152 244 Died 104 248 Died 6 422 242 256 388 376 91 93 148 178 7 551 462 421 554 137 226 121 262 175 8 546 466 582 84 587 210 217 145 93 9 384 431 478 450 652 574 157 244 178 10 452 444 210 148 380 155 114 225 122 11 324 558 437 86 456 412 170 176 237 12 236 484 321 188 180 557 142 160 Died Mean 390.08±t 449.42±: 452.67 252.83+ 392.33± 378.64± 143.92± 202.75± 161.90± ± S.D. 112.07 100.37 208.22 158.78 152.50 317.72 43.37 41.89 45.29 S.E. 32.35 28.97 60.11 45.84 44.02 95.79 12.52 12.09 14.32 Significance NS NS P < 0.05 NS P < 0.005 NS IUW I. 1 000 - U_ .I .c Cycle number 7th 8th 9th 10th 11th 12thCycle number Fig. 3. Geometric mean of reciprocal IFA titres of P. Fig. 4. Geometric mean of reciprocal IFA titres of cynomolgi B infected rhesua monkeys (control/Norinyl/ P. coatneyl Infected rheaus monkeys (control/Norinyl/ Ovral-28 groups) from 7th to 12th treatment cycle. Ovral-28 groups) from 7th to 12th treatment cycle. (Titres plotted on semi-log graph.) (Titres plotted on seml-log graph.) -^ .Control e* -e Norinyl o-. Ovrel E a I ~~~~~~~.a I ORAL CONTRACEPTIVES AND MALARIA IN MONKEYS Albumin/globulin ratio. The A/G ratios of control, Norinyl and Ovral-28 groups before P.cynomolgi infection (5th treatment cycle) were 1.02±0.15, 1.03±0.20, and 0.92±0.15 and after malaria infection (8th cycle) the values declined to 0.72 ± 0.09, 0.68 ± 0.14 and 0.73 ± 0.04, respectively. Similarly, after P. coatneyi infection the A/G ratios declined to 0.88±0.11, 0.80±0.11 and 0.78±0.12, respectively. The A/G ratios in all the study groups reverted to the normal range after radical cure. On rechallenge, there was only marginal decrease in the A/G ratios. The percentage of globulin fractions (ca, 0Y2, ) did not show significant alteration though there was increase in the gammaglobulins after infection in various study groups. Liverfunction tests The serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and total bilirubin were determined at weekly intervals throughout the study (treatment cycles 1-12) in the control and both contraceptive groups infected with P. cynomolgi B and with P. coatneyi. No significant alterations in the enzyme levels were observed during oral contraceptive treatment with malaria infection and their values were within normal limits. The normal bilirubin values ranged between 1.7 and 10.3 imol/l and there was no change after P. cynomolgi infection in any of the groups. However, in the animals with P. coatneyi infection, there was a significant increase in total bilirubin during the 3rd and 4th weeks after infection and the peak values recorded in the control, Norinyl and Ovral-28 groups were 13.2± 8.7, 16.8±13.2, and 19.2± 18.2 smol/l, respectively. Haematological studies Preinfection phase (6th treatment cycle). The oral contraceptive administration for six cycles did not produce significant changes in any of the haemato- logical test results. All the values recorded were within the normal limits as observed in healthy monkeys. Malaria infection phase/radical cure/rechallenge. In the control group, some of the haematological parameters were significantly altered during the primary malaria infection. The most significant change was the onset of anaemia, indicated by a decline of Hb, RBC count and PCV (P< 0.001) during the 8th cycle. The anaemia observed was normocytic and normochromic. Following radical cure, there was recovery from the anaemia and during rechallenge no further change occurred. The changes in ESR and platelets were within normal limits. The total leukocyte count was slightly elevated during the malaria infection. The differential leukocyte count as well as the total absolute counts showed a decrease in polymorphs and a corresponding elevation in lymphocytes. In the two contraceptive groups, the degree of anaemia observed after malaria infection was comparable to that in the control group. After chloroquine therapy, there was recovery from the anaemia and no significant change was recorded after rechallenge. The changes in ESR and platelets were within normal limits. The changes in the total and differential leukocyte counts and in the TAP and TAL counts in both contraceptive groups were almost the same as those observed in the control group after malaria infection, radical cure and rechallenge. Progesterone levels Normal female monkeys showed plasma progesterone levels of 0.5-3.5 nmol/l on day 10, above 10 nmol/l on either day 14 or 18, and then declined to normal levels again on day 22 of the menstrual cycles. Treatment cycles 1-3: Both Norinyl and Ovral-28 at 1/6 of the human dose effectively suppressed ovulation as shown by progesterone levels which remained below 2.5 nmol/l on all the four days of sampling. The menstrual cycle after drug withdrawal was normal in the Ovral-28 group but erratic in the Norinyl group. In a few animals, higher levels of progesterone indicative of ovulation were observed. Treatment cycles 4-6: Ovral-28 at 1/6 dose and Norinyl at 1/3 dose suppressed ovulation, and regular menses returned after drug withdrawal in all the animals. Progesterone levels remained below 2.5 nmol/l and the same contraceptive doses were used during the malaria infection. Treatment cycles 7-8, 9-10 and 11-12: There was no interference with the malaria infection or with radical cure using chloroquine or with rechallenge, the progesterone levels remaining low in the animals in both contraceptive groups, and their ovulation continued to be suppressed. DISCUSSION The use of steroid hormones in family planning in countries with a high prevalence of tropical diseases like malaria has been the cause of some concern after the preliminary observation by Bray (1) of a slightly higher malaria parasite density among women using an oral contraceptive (Ovral-28). The present study has shown that oral contra- ceptive administration for 6 cycles in female rhesus 937 G. P. DUTTA ET AL. monkeys infected with malaria (P. cynomolgi B and P. coarneyl) produced a slightly increased cumulative parasite load during the phase of primary infection over a period of 8 weeks. The results based on mean daily parasitaemia measurements also showed a significantly higher level of parasitaemia during the 8th treatment cycle with both contraceptives, and the peak levels were also elevated in some animals. These findings suggest that the administration of oral contraceptives (Norinyl or Ovral-28) lowers the host animal's resistance to malaria infections. However, the nature of this impaired immune response is not known. The present study also shows that oral contra- ceptive administration for 8 cycles did not interfere with the efficacy of the radical curative action of chloroquine (dose of 20 mg per kg body weight daily for 3 days) against malaria infections. The results of the rechallenge experiments after 8 weeks of radical cure show that the acquired immunity of the monkeys treated with oral contra- ceptives for 10 cycles was not significantly impaired because the course of parasitaemia in all the study groups, upon rechallenge, was more or less similar. Further, the course of antibody production as measured by the IFA test, during primary infection or rechallenge, was not significantly different in the contraceptive groups compared with the control group. Moreover, the decline of IFA titres after radical cure was similar in all the groups. Further rechallenge experiments revealed a rapid increase of IFA titres in all the groups to the level observed after primary infection, which suggests that the continued administration of oral contraceptives did not interfere with the ability of the host to mount a fresh antibody response to reinfection. Comparative analysis of several serum biochemical parameters for liver function did not reveal signi- ficant alterations in the control or contraceptive groups of monkeys following malaria infection with P. cynomolgiB or P. coatneyi. During the early phase of only P. coatneyi infection, a significant increase in serum bilirubin was observed which might have been due to administration of quinine (1-2 doses of 300 mg) during primary peak parasitaemia. In an earlier study, no increase of bilirubin was observed in animals with untreated P. coatneyi infection (8). In the case of P. cynomolgi B infection, there was no change in serum bilirubin levels in either the control or the contraceptive groups. A significant fall in the haemoglobin, RBC, and PCV levels in animals with P. cynomolgi B and P. coatneyi infections is linked with the onset of anaemia during the primary phase of infection. The anaemia, observed in both the control and contra- ceptive groups, was normocytic and normochromic, and there was complete recovery after radical cure of the malaria infection with chloroquine. Anaemia was not observed after rechallenge. There was no significant change in the plasma progesterone levels of the monkeys as a result of the malaria infection or the administration of a curative regimen of chloroquine (20 mg per kg body weight for 3 days) to the control or the contraceptive-treated groups. These results show that the chemotherapeutic use of chloroquine did not interfere with the efficacy of the two oral contraceptives. We therefore conclude that the institution of early radical curative malaria therapy is warranted among oral contraceptive users, because this would protect them against any possible complications arising out of steroid interaction with malaria. ACKNOWLEDGEMENTS We are grateful to the WHO Special Programme of Research, Development and Research Training in Human Reproduction for supporting this work (Project No. 78149B) and for the supply of oral contraceptives and reagents for progesterone assay under the WHO Matched Assay Reagents Programme. We should also like to thank Dr Nitya Anand, DIreetor of the Central Drug Research Institute, Lucknow, for continued support and guidance during the course of the presernt study, and Mr K.L. Gulati and Mr G.P. Dikshit for technical help. The strains of malaria used in this study were kindly supplied by Dr W.E. Collins, Centers for Disease Control, Atlanta, GA, USA. RESUMt iNTERACTIONS ENTRE LES CONTRACEPTIFS ORAUX ET L'INFECTION PALUDIQUE CHEZ LE SINGE RHESUS Les interactions entre les contraceptifs oraux et l'infection paludique (A Plasmodium cynomolgi B et P. coatneyi) ont et etudiees chez des femelles adultes de singe rhesus (Macaca mulatta). Les contraceptifs oraux (Norinyl et Ovral-28) ont et administres pendant 12 cycles menstruels consecutifs, du 5' au 25' jour du cycle, a une dose trois fois plus faible que chez la femme dans le cas du Norinyl (0,33 mg de norethisterone + 0,012 mg d'ethinylestradiol) 938 ORAL CONTRACEPTIVES AND MALARIA IN MONKEYS 939 et six fois plus faible dans celui de l'Ovral-28 (0,083 mg de norgestrel + 0,008 mg d'ethinylestradiol). Pour chaque infection, les animaux etaient r6partis en trois groupes (un groupe t6moin et deux groupes trait6s, l'un par le Norinyl et l'autre par l'Ovral-28) de 10 animaux chacun dans le cas de l'infection a P. cynomolgi B et 12 animaux dans le cas de l'infection a P. coatneyi. L'infection exp6rimentale a eu lieu au bout de 6 cycles d'administration de contraceptifs oraux et son evolution a ete etudiee pendant les deux cycles suivants, le 7' et le 8'. Apres une cure radicale au cours des 9' et IO' cycles, les animaux ont de nouveau ete exposes au parasite pendant les 1 I et 12' cycle. Cette etude a donne les resultats suivants: 1) les animaux sous contra- ceptifs ont presente une charge parasitaire cumulative l6gerement accrue; 2) les contraceptifs n'ont pas entrave l'action curative radicale de la chloroquine; 3) chez les animaux sous contraceptifs, 1'6volution de la parasitemie apres la seconde exposition n'a pas 6te sensiblement diff6rente, pas plus que les titres d'anticorps antiplasmo- cliques, mesures par immunofluorescence indirecte; 4) les epreuves de la fonction h6patique n'ont pas Wte sensiblement modifi6es par l'administration des contraceptifs ni par l'infection ult6rieure; et 5) les modifications h6matologiques constatees ont 6t6 identiques chez les animaux sous contra- ceptifs et dans les groupes temoins. REFERENCES 1. BRAY, R.S. Contraception, 14: 417-425 (1976). 2. COLLINS, W.E. & SKINNER, J.C. American journal of tropical medicine and hygiene, 21: 690-695 (1972). 3. PECK, J.M., ed. Beckman microzone, electrophoresis manual. Fullerton, California, Beckman Instruments Inc., 1979. 4. WOOrrON, I.D.P. Microanalysis in medical bio- chemistry. London, Churchill, 1964, pp. 102-118. 5. MALLOY, H.T. & EVELYN, K.A. Journal of biological chemistry, 118: 481 (1937). 6. DACIE, J.V. & LEWIS, S.M. Practical haematology. London, Churchill, 1968. 7. EASTHAM, R.D. Clinical haematology, Fifth Indian edition. Bombay, Varghese, 1977. 8. DESOWITZ, R.S. ET AL. Annals of tropical medicine and parasitology, 61: 365-374 (1967).