Plasmodium falciparum initially infects the liver, and, after approximately 7 days, parasites begin to emerge from the liver to parasitize red cells. A prophylactic agent against the liver stage of infection ("causal" prophylactic agent) needs to be administered for only approximately 7 days or less after infection, whereas an agent effective solely against the blood stage of the infection ("suppressive" prophylactic agent) needs to be administered for several weeks after infection. Mefloquine and doxycycline are insufficiently effective against liver-stage parasites, and patients require dosing for 4 weeks after leaving the endemic area.

Azithromycin is an azalide analog of erythromycin that was recently licensed for the treatment of bacterial and chlamydial diseases. In a rodent model, we found that azithromycin has a causal prophylactic efficacy superior to that of doxycycline [5]. In addition, we did a study in which volunteers were exposed to the bites of P. falciparum-infected mosquitoes and given azithromycin for as long as 5 days after exposure to the bites. Azithromycin, 500 mg administered 2 days before exposure to the bites followed by 250 mg/d administered for 5 days after exposure to the bites, was successful prophylaxis for three of four persons [6].

Because the previous clinical study involved only 4 drug recipients, we did a new study of causal prophylaxis using 10 drug recipients. Because we obtained results showing unexpectedly poor efficacy, we subsequently entered another cohort of 10 drug recipients to determine the combined causal efficacy as well as the suppressive efficacy of azithromycin against P. falciparum malaria in the challenge model.

Methods

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Study Design

We did an open-label, controlled phase II study in which two cohorts were entered sequentially.

In cohort 1, we evaluated the causal prophylactic efficacy of azithromycin. Ten persons received 500 mg of the drug on day 14 before the challenge, followed by 250 mg/d from day 13 before the challenge through day 7 after the challenge. Dosing from day 14 before the challenge to day 0 was intended to allow serum concentrations to reach a steady state; dosing from days 1 to 7 after the challenge was intended to provide causal prophylaxis. In the 12 preceding experiments at our clinical research center, all of approximately 40 control volunteers inoculated with this strain of P. falciparum became infected. In our present study, two controls did not receive azithromycin and were concomitantly infected to verify that the parasitologic challenge was infectious.

In cohort 2, we evaluated the combined causal and suppressive prophylactic efficacy of azithromycin. Ten persons received 500 mg of the agent on day 14 before the challenge, followed by 250 mg/d from day 13 before the challenge through day 28 after the challenge. Dosing from days 8 through 28 was intended to provide suppressive prophylaxis. Two controls were concomitantly challenged.

Inclusion and Exclusion Criteria

Men and women aged 18 through 46 years were eligible if they were healthy on the basis of history, physical examination, and results of laboratory tests (complete blood count; serum levels of sodium, potassium, chloride, urea nitrogen, creatinine, calcium phosphate, total protein, albumin, aspartate aminotransferase, alkaline phosphatase, total bilirubin, lactate dehydrogenase, and uric acid) and had no history of exposure to malaria in the previous 2 years. Persons who did not object to the high likelihood of becoming parasitemic were permitted to be controls.

Study Procedures

For each cohort of 12 volunteers, all persons were infected [7] by exposure to the bites of five female Anopheles stephensi mosquitoes infected with the chloroquine-sensitive, mefloquine-resistant NF54 strain of P. falciparum.

During drug administration, adverse reactions and symptoms of malaria were assessed by means of daily interviews for subjective symptoms (chills, headache, photophobia, back pain, muscle ache, stomach ache, anorexia, nausea, vomiting, diarrhea, vaginal itching), by daily recording of body temperature, and by repetition of the entrance laboratory tests on day 4 before the challenge and day 5 after the challenge.

To make the definitive diagnosis of malaria, on days 5 through 28 (for cohort 1) or on days 5 through 35 (for cohort 2), thick blood smears were obtained, stained with Giemsa, and examined for P. falciparum by an investigator blinded to whether or not the study participant had received azithromycin. Positive results were confirmed by two other investigators. In addition, all persons not already diagnosed as having malaria were instructed to contact study personnel until day 70 if they had symptoms that suggested malaria. In each thick smear, 200 fields were examined. Patent malaria was defined as the presence of at least two parasites per smear. If the volunteer had patent parasitemia, he or she was treated with chloroquine (1500-mg base given over 3 days).

Treatment Agents

Azithromycin in the form of 250-mg tablets was provided by Pfizer Central Research. Groton, Connecticut. Chloroquine in the form of Aralen Phosphate tablets was purchased from Winthrop Pharmaceuticals. New York, New York.

Protocol Approval

Our protocol was approved by the Office of the Surgeon General of the United States Army and filed under IND with the U.S. Food and Drug Administration. All volunteers gave written informed consent.

Statistical Methods

Computation of exact CIs of a proportion was done using the binomial distribution.

Results

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Prophylactic Efficacy of Azithromycin

Cohort 1 consisted of 10 volunteers who received azithromycin until 7 days after challenge with P. falciparum sporozoites and 2 volunteers who were challenged with sporozoites but received no treatment. Both of the controls who received no treatment and 6 of the 10 volunteers who received treatment with the drug developed parasitemia. One participant was removed from the study on day 25 because of noncompliance with follow-up. He was not patent (parasitemic) on that day, and no other participant became patent between day 23 and the end of the study on day 70. We consider this participant to be a prophylactic success. All but one of the persons who received treatment with the drug and in whom prophylaxis failed had delayed patency (mean day of patency, 19 days; range, 11 to 23 days) compared with the controls (mean day of patency, 10 days; range, 9 to 11 days). In cohort 1, the efficacy of treatment was 40% (95% CI, 12% to 74%).

Cohort 2 consisted of 10 persons who received azithromycin until 28 days after exposure to sporozoites and 2 controls who received no treatment. Both controls became patent (on days 11 and 13); none of the participants who received drug treatment became patent. In cohort 2, the efficacy of azithromycin was 100% (lower 95% CI, 70%).

Of the 10 participants in both cohorts who became parasitemic, only 3 had temperatures greater than 37.7 °C, and 7 had symptoms characterized by chills, headache, myalgia, and anorexia. No volunteer required hospitalization.

Adverse Effects of Azithromycin

Although each day during the period of drug administration, study participants were interviewed for subjective side effects, we found only four instances of headache, four instances of stomach ache, and one instance of diarrhea not temporally linked to parasitemia. Each of these events was mild and lasted at most 1 day. In addition, one patient developed flulike symptoms 2 days after receiving the last dose of the drug. Neither the one woman in cohort 1 nor the three women in cohort 2 reported symptoms of vaginitis.

The only abnormality in laboratory variables was total bilirubin values somewhat higher than the upper limit of normal (24 µmol/L) in 3 participants. In 1 volunteer, the value on day 4 before the challenge (37.6 µmol/L) had returned to normal by day 5 after the challenge (15.4 µmol/L) while the participant was still receiving the drug. In 1 participant, the values were relatively constant during therapy (27.4 µmol/L on day 4 before the challenge and 34.2 µmol/L on day 5 after the challenge) and then returned to normal after therapy. In the third participant, a value of 30.8 µmol/L was first recorded on day 5 and levels returned to normal after therapy.

Discussion

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A causal and suppressive prophylactic regimen of azithromycin, 250 mg/d for 28 days after a single challenge with P. falciparum sporozoites, was successful in 10 of 10 persons studied. In contrast, a causal prophylactic regimen of azithromycin, 250 mg/d for 7 days after challenge, was successful in only 4 of 10 persons.

We had hoped that azithromycin would be sufficiently concentrated in the liver to be completely causally prophylactic, because it has been reported that azithromycin liver concentrations are approximately 10 times that of serum concentrations [8]. The results of treatment for 7 days after the challenge show that, as with doxycycline [9], azithromycin has only partial causal prophylactic activity in the human challenge model. The mean percent efficacy determined here (40% [CI, 12% to 74%]) is much less than that recently achieved by Kuschner and colleagues [6] in a trial with a similar but not identical design; in their study, prophylaxis was successful in three of four volunteers (75% [CI, 30% to 95%]). Kuschner and colleagues used a different strain of P. falciparum and used azithromycin capsules rather than the new azithromycin formulation (tablets) used in our study. Although chance or these differences in study conditions might account for the difference in efficacy, we believe that the most likely explanation is that our study, with its greater numbers of participants, is more representative of the causal prophylactic activity of azithromycin in the challenge model.

Extrapolation of the results from the challenge model to the field is uncertain. Mosquito bites may be more frequent in the field, but the percentage of mosquitoes that are infected would be lower than in this model. In addition, the larger number of patients available in the field would provide greater statistical confidence. Nevertheless, the 40% efficacy of azithromycin in this study and the total efficacy of 50% (7 of 14 volunteers) in both causal prophylaxis studies suggest that the regimen of 250 mg/d for 7 days after exposure is unlikely to be highly effective in the field. In contrast, the 100% efficacy for the regimen of 250 mg/d for 28 days after challenge indicates that, in the field, the combined causal and suppressive clinical efficacy of azithromycin may be high.

Few adverse effects, all of which were mild, were seen in this nonblinded study, even though 250-mg tablets were administered daily for 6 weeks in cohort 2. The drug regimens approved in the United States are 1000 mg as a single dose and a 5-day regimen of 500 mg on day 1 followed by 250 mg on days 2 through 5. Our study therefore considerably extends the period over which azithromycin is administered to immunocompetent persons and provides data on the lack of side effects associated with this regimen.

Our study shows that azithromycin has the potential to be at least as effective as mefloquine and doxycycline as a prophylactic agent for malaria. Compared with mefloquine (pregnancy class C: teratogenic in laboratory animals but no evidence of teratogenicity in humans) and doxycycline (pregnancy class D: positive evidence of human fetal risk), azithromycin is in pregnancy class B (no evidence of teratogenicity in laboratory animals but no well-controlled clinical studies) and may be used during pregnancy if clearly needed. A liquid formulation for young children is now being developed. If effective, azithromycin may have a therapeutic index for some clinical populations that is superior to that of mefloquine and doxycycline.

Drs. Berman, Wesche, and Schuster: Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, DC 20307-5100.

Drs. Kuschner, Magill, and Wellde: Division of Communicable Disease and Immunology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.

Dr. Schneider: Department of Entomology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.

Dr. Dunne: Pfizer Central Research, Eastern Point Road, Groton, CT 06340.