Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum.

Mutations in the chloroquine resistance (CQR) transporter gene of Plasmodium falciparum (Pfcrt; chromosome 7) play a key role in CQR, while mutations in the multidrug resistance gene (Pfmdr1; chromosome 5) play a significant role in the parasite's resistance to a variety of antimalarials and also modulate CQR. To compare patterns of genetic variation at Pfcrt and Pfmdr1 loci, we investigated 460 blood samples from P. falciparum-infected patients from four Asian, three African, and three South American countries, analyzing microsatellite (MS) loci flanking Pfcrt (five loci [approximately 40 kb]) and Pfmdr1 (either two loci [approximately 5 kb] or four loci [approximately 10 kb]). CQR Pfmdr1 allele-associated MS haplotypes showed considerably higher genetic diversity and higher levels of subdivision than CQR Pfcrt allele-associated MS haplotypes in both Asian and African parasite populations. However, both Pfcrt and Pfmdr1 MS haplotypes showed similar levels of low diversity in South American parasite populations. Median-joining network analyses showed that the Pfcrt MS haplotypes correlated well with geography and CQR Pfcrt alleles, whereas there was no distinct Pfmdr1 MS haplotype that correlated with geography and/or CQR Pfmdr1 alleles. Furthermore, multiple independent origins of CQR Pfmdr1 alleles in Asia and Africa were inferred. These results suggest that variation at Pfcrt and Pfmdr1 loci in both Asian and African parasite populations is generated and/or maintained via substantially different mechanisms. Since Pfmdr1 mutations may be associated with resistance to artemisinin combination therapies that are replacing CQ, particularly in Africa, it is important to determine if, and how, the genetic characteristics of this locus change over time.

Hepatitis E virus infection in work horses in Egypt.

Hepatitis E virus (HEV) is an important cause of hepatitis among young Egyptian adults with high seroprevalence rates seen in both rural areas of the Nile Delta and in suburban Cairo. Because natural antibodies to HEV have been detected in animals and zoonotic transmission is postulated, we surveyed work horses in Cairo for evidence of HEV exposure and viremia. Sera from 200 Cairo work horses were tested by ELISA for the presence of IgG anti-HEV antibody revealed a seropositivity of 13%. Among 100 samples processed for detection of viral genome by means of nested polymerase chain reaction (N-PCR), 4% were positive and indicative of viremia. Viremic animals were less than 1 year old. Relative to PCR-negative horses, PCR-positive animals demonstrated significant elevation of AST (p=0.03). Phylogenetic analysis of a 253-bp fragment, in the ORF-1,2,3 overlap region of the HEV genome from the viremic animals showed that three of these viral strains to be identical, and closely related (97-100% nucleotide identity) to two human isolates from Egypt, and distant (78-96%) from 16 other HEV isolates from human and animals and shared 99.6% NI with the fourth strain. The consensus sequence of the four strains was origin obtained elsewhere. These data indicated that horses acquire HEV infection and suggest that cross-species transmission may occur. Whether horses play a role in the transmission of HEV needs further investigation.

Mefloquine treatment for uncomplicated falciparum malaria in young children 6-24 months of age in northern Ghana.

Mefloquine (MQ) single dose 20 mg/kg treatment of falciparum malaria was evaluated in 186 children of 6-24 months of age in northern Ghana. There were 15 RII/RIII-type parasitologic failures, all with Day 2 MQ blood levels significantly lower than children whose parasitemias cleared before Day 7 and remained clear through 28 days. Predictors of RII/RIII parasitologic response were vomiting after MQ dosing, Day 2 MQ levels < 500 ng/mL, and undetectable Day 2 levels of the carboxymefloquine metabolite. There were 50 cases of delayed RI parasitologic failure, but 71% of these cases had undetectable Day 28 blood levels of MQ and drug levels in the remaining 29% ranged below the 620 ng/mL level that suppresses MQ sensitive strains of P. falciparum. Drug levels among infants that tolerated MQ well were not associated with age, weight, hemoglobin, parasitemia, and pre-existing symptoms of vomiting or diarrhea. An observed recurrent parasitemia of 34,400 trophozoites/microL against a MQ blood concentration of 550 ng/mL was taken as indication of tolerance to suppressive levels of the drug at this location.

Bionomics of phlebotomine sandflies at a peacekeeping duty site in the north of Sinai, Egypt.

A longitudinal entomological survey for sandflies was conducted from 1989 to 1991 at a focus of enzootic cutaneous leishmaniasis in Northeast Sinai, Egypt, within the border region monitored by multinational peacekeepers. Standardized sampling with CDC light traps, oiled paper "sticky traps", and human landing collection was employed to determine monthly trends in species composition, density, sex ratio, and reproductive status of vector sandflies. Each collection method independently defined sandfly seasonality as the period May-November in 1990, and March-October in 1991. Plebotomus papatasi was the only anthropophagic species found and comprised more than 94% of the sandfly population. Two population peaks (May, July) were observed for this species in both survey years. Density of P. papatasi in underground bunkers was higher than outside but inflated by a greater proportion of male flies. During 1990, the proportion of gravid P. papatasi increased progressively during the 5 months period from May to September and averaged 29.5% and 29.7% for interior and exterior collections, respectively. Density of P. papatasi was greater during 1991, but proportions of gravid flies were significantly lower in each survey month and averaged 14.9% and 12.3% for interior and exterior collections, respectively. Seasonal rates of Leishmania-infected P. papatasi averaged 0.8% and 0.9% in 1989 and 1990, but fell to zero in 1991, suggesting an unstable focus of Leishmania major transmission. Proportions of gravid flies may be a valid indicator of the physiological age and epidemiologic importance of the vector sandfly population at this focus. The strong correlation of sticky trap indices to human-landing/biting rates shows that this is an accurate, inexpensive, and no-risk alternative to human bait collections.

Mefloquine is highly efficacious against chloroquine-resistant Plasmodium vivax malaria and Plasmodium falciparum malaria in Papua, Indonesia.

BACKGROUND: During the period of 1996-1999, we prospectively monitored 243 Javanese adults and children after arriving in Papua, Indonesia, and microscopically documented each new case of malaria by active surveillance. METHODS: In a randomized, open-label, comparative malaria treatment trial, 72 adults and 50 children received chloroquine for each incident case of malaria, and 74 adults and 47 children received mefloquine. RESULTS: Among 975 primary treatment courses, the cumulative 28-day curative efficacies were 26% and 82% for chloroquine against Plasmodium falciparum malaria and Plasmodium vivax malaria, respectively. Mefloquine cure rates were far superior (96% against P. falciparum malaria and 99.6% against P. vivax malaria). CONCLUSIONS: Mefloquine is a useful alternative treatment for P. vivax malaria and P. falciparum malaria in areas such as Papua, where chloroquine is still recommended as the first-line therapeutic agent.

Use of bioassay and microplate assay to detect and measure insecticide resistance in field populations of Culex pipiens from filariasis endemic areas of Egypt.

Insecticide and resistance bioassays and microplate assays were performed on Culex pipiens mosquitoes to determine the level and mechanisms of resistance. Culex pipiens larvae were collected from three filariasis-endemic areas of Egypt and reared to adults for subsequent production and testing of F1 generation larvae and adults. Bioassays were performed using World Health Organization (WHO) methods with the diagnostic doses of 6 organophosphate insecticides for larvae and 1 organochlorine (OC), 4 pyrethroid, 2 organophosphate, and 2 carbamate insecticides for adults. Microplate assays were performed to measure levels of beta esterase, acetylcholinesterase, insensitive acetylcholinesterase, oxidases, and glutathione-S-transferase enzymes. Larval bioassay results showed clear indications of resistance to organophosphate insecticides. Adult bioassays also showed widespread, significant resistance to many insecticides from all four classes, including the OC, DDT. The Qalubiya larval population was susceptible only to malathion, whereas Sharkiya larvae were susceptible to malathion, temephos, and chlorpyrifos. On the other hand, larval specimens from Assiut were resistant to all insecticides tested. Larval bioassay results were supported by those of microplate assays in showing elevated levels of glutathione S-transferase in populations from all three areas. In general, microplate results confirmed patterns of resistance observed using bioassays, and mechanisms of resistance were evident for all three areas sampled. Mechanisms of resistance are discussed in relation to microplate and bioassay results for the areas sampled and pesticides used.

Primaquine for prevention of malaria in travelers.

An expanding risk and range of endemic malaria threatens travelers. Primaquine is an old drug recently demonstrated to offer effective prophylaxis. Clinical trials conducted in Indonesia, Kenya, and Colombia showed that a primaquine base (30 mg per day) had protective efficacy against Plasmodium falciparum and Plasmodium vivax of 85%-93%. Among 339 children (age, >8 years) and adults taking this regimen for 12-52 weeks, there was no greater risk of adverse symptomatic events among primaquine users than among recipients of placebo in double-blind studies. Among 151 subjects evaluated after 20 or 52 weeks of daily primaquine therapy, methemoglobinemia was found to be mild (<13%; typically <6%) and transient (duration, <2 weeks). We consider primaquine base (0.5 mg/kg per day consumed with food) to be safe, well-tolerated, and effective prophylaxis against malaria for nonpregnant persons and those with normal glucose-6-phosphate dehydrogenase levels. Primaquine's major advantage over most drugs for chemoprophylaxis is that it does not have to be taken before entering or beyond 3 days after leaving a malarious area.

A randomized, double-blind, placebo-controlled, dose-ranging trial of tafenoquine for weekly prophylaxis against Plasmodium falciparum.

Tafenoquine is a promising new 8-aminoquinoline drug that may be useful for malaria prophylaxis in nonpregnant persons with normal glucose-6-phosphate dehydrogenase (G6PD) function. A randomized, double-blind, placebo-controlled chemoprophylaxis trial was conducted with adult residents of northern Ghana to determine the minimum effective weekly dose of tafenoquine for the prevention of infection by Plasmodium falciparum. The primary end point was a positive malaria blood smear result during the 13 weeks of study drug coverage. Relative to the placebo, all 4 tafenoquine dosages demonstrated significant protection against P. falciparum infection: for 25 mg/week, protective efficacy was 32% (95% confidence interval [CI], 20%-43%); for 50 mg/week, 84% (95% CI, 75%-91%); for 100 mg/week, 87% (95% CI, 78%-93%); and for 200 mg/week, 86% (95% CI, 76%-92%). The mefloquine dosage of 250 mg/week also demonstrated significant protection against P. falciparum infection (protective efficacy, 86%; 95% CI, 72%-93%). There was little difference between study groups in the adverse events reported, and there was no evidence of a relationship between tafenoquine dosage and reports of physical complaints or the occurrence of abnormal laboratory parameters. Tafenoquine dosages of 50, 100, and 200 mg/week were safe, well tolerated, and effective against P. falciparum infection in this study population.

Atovaquone/proguanil therapy for Plasmodium falciparum and Plasmodium vivax malaria in Indonesians who lack clinical immunity.

Thirty-eight of 295 subjects participating in a randomized, double-blind, placebo-controlled trial of the efficacy of daily administration of atovaquone/proguanil for malaria prevention developed malaria at some time during the 20-week prophylaxis period. These subjects (3 atovaquone/proguanil recipients and 35 placebo recipients) were treated with 4 tablets of atovaquone/proguanil per day for 3 days. Atovaquone/proguanil provided safe, well-tolerated, and effective therapy for uncomplicated malaria in nonimmune Indonesians.

Randomized, placebo-controlled trial of atovaquone/proguanil for the prevention of Plasmodium falciparum or Plasmodium vivax malaria among migrants to Papua, Indonesia.

The increasing prevalence of resistance to antimalarial drugs reduces options for malaria prophylaxis. Atovaquone/proguanil (Malarone; GlaxoSmithKline) has been >95% effective in preventing Plasmodium falciparum malaria in lifelong residents of areas of holoendemicity, but data from persons without clinical immunity or who are at risk for Plasmodium vivax malaria have not been described. We conducted a randomized, double-blinded study involving 297 people from areas of nonendemicity in Indonesia who migrated to Papua (where malaria is endemic) < or =26 months before the study period. Subjects received prophylaxis with 1 Malarone tablet (250 mg of atovaquone and 100 mg of proguanil hydrochloride; n=148) or placebo (n=149) per day for 20 weeks. Hematologic and clinical chemistry values did not change significantly. The protective efficacy of atovaquone/proguanil was 84% (95% confidence interval [CI], 44%-95%) for P. vivax malaria, 96% (95% CI, 72%-99%) for P. falciparum malaria, and 93% (95% CI, 77%-98%) overall. Atovaquone/proguanil was well tolerated, safe, and effective for the prevention of drug-resistant P. vivax and P. falciparum malaria in individuals without prior malaria exposure who migrated to Papua, Indonesia.

Very high risk of therapeutic failure with chloroquine for uncomplicated Plasmodium falciparum and P. vivax malaria in Indonesian Papua.

Chloroquine remains the first-line therapy for uncomplicated malaria in Indonesia. Among a series of trials of chloroquine for malaria on this archipelago conducted since 1990, we now report the highest risk of therapeutic failure yet observed. A clinical trial of standard chloroquine therapy for uncomplicated malaria at Arso PIR V in northeastern Indonesian Papua was conducted during 1995. We enrolled 104 non-immune subjects infected with Plasmodium falciparum (n = 55), P. vivax (n = 29), or P. falciparum plus P. vivax (n = 20) and administered supervised standard chloroquine therapy (10 + 10 + 5 mg/kg at 24-hour intervals). The 28-day cumulative incidence of therapeutic failure was 95% for P. falciparum, 84% for P. vivax, and 100% for mixed infections. Only one subject each for P. falciparum and P. vivax remained free of parasites at day 28. All recurrent parasitemias occurred with whole blood levels of chloroquine plus desethylchloroquine exceeding 100 ng/ml. These findings document almost complete failure of chloroquine against P. falciparum or P. vivax near the northeastern coast of Indonesian Papua.

Alterations in Plasmodium falciparum genotypes during sequential infections suggest the presence of strain specific immunity.

Many of the asexual stage Plasmodium falciparum proteins that are the targets of host protective responses are markedly polymorphic. The full repertoire of diversity is not defined for any antigen. Most studies have focused on the genes encoding merozoite surface proteins 1 and 2 (MSP1, MSP2). We explored the extent of diversity of some of the less studied merozoite surface antigens and analyzed the degree of complexity of malaria field isolates by deriving nucleotide sequences of several antigens. We have determined the genotype of apical membrane antigen 1 (AMA1) in a group of 30 field samples, collected over 29 months, from individuals living in an area of intense malaria transmission in Irian Jaya, identifying 14 different alleles. AMA1 genotyping was combined with previously determined MSP2 typing. AMA1 had the greatest power in distinguishing between isolates but methodological problems, especially when mixed infections are present, suggest it is not an ideal typing target. MSP1, MSP3, and glutamate-rich protein genotypes were also determined from a smaller group of samples, and all results were combined to derive an extended antigenic haplotype. Within this subset of 10 patients, nine different genotypes could be discerned; however, five patients were all infected with the same strain. This strain was present in individuals from two separate villages and was still present 12 months later. This strain was predominant at the first time point but had disappeared at the fourth time point. This significant change in malaria genotypes could be due to strain-specific immunity developing in this population.

New haplotypes of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene among chloroquine-resistant parasite isolates.

Mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene were examined to assess their associations with chloroquine resistance in clinical samples from Armopa (Papua) and Papua New Guinea. In Papua, two of the five pfcrt haplotypes found were new: SVIET from Armopa and CVIKT from an isolate in Timika. There was also a strong association (P < 0.0001) between the pfcrt 76T allele and chloroquine resistance in 50 samples. In Papua New Guinea, mutations in the pfcrt gene were observed in 15 isolates with chloroquine minimum inhibitory concentrations (MICs) of 16-64 pmol, while the remaining six isolates, which had a wild-type pfcrt gene at codon 76, had MICs of 2-8 pmol. These observations confirm that mutations at codon 76 in the pfcrt gene are present in both in vivo and in vitro cases of chloroquine resistance, and that detection of the pfcrt 76T allele could predict potential chloroquine treatment failures.

Serologic evidence of infection with ehrlichiae and spotted fever group rickettsiae among residents of Gag Island, Indonesia.

The causative agents of scrub and murine typhus are considered endemic to Indonesia. However, the presence of spotted fever group rickettsiae and ehrlichiae have not been previously described in this country. During an investigation of arthropod-borne diseases on Gag Island, located northwest of the island of New Guinea in eastern Indonesia, the prevalence of antibody to the etiologic agents of monocytic ehrlichiosis, spotted fever rickettsiosis, and scrub and murine typhus were determined. Analysis of 55 blood samples from residents of Gag Island showed seroreactivity to antigen preparations of Ehrlichia chaffeensis (7 of 48, 14.6%), two spotted fever group rickettsiae: Rickettsia rickettsii (5 of 48, 10.4%) and R. conorii (10 of 49, 20.4%), Orientia tsutsugamushi (5 of 53, 9.4%), and R. typhi (1 of 48, 2.1% [by an indirect immunofluorescence assay] and 1 of 50, 2.0% [by an enzyme-linked immunosorbent assay]). These results show serologic evidence of infection with ehrlichiae and spotted fever group rickettsiae for the first time in Indonesia in a location where the prevalence of antibody to O. tsutsugamushi and R. typhi was lower.

Assessing the Parasight-F test in northeastern Papua, Indonesia, an area of mixed Plasmodium falciparum and Plasmodium vivax transmission.

User-friendly, reliable, and inexpensive methods for diagnosing malaria are needed at the primary health care level. During a randomized treatment trial, the Parasight-F test was assessed on days 0, 3, 7, and 28 against standard light microscopy of Giemsa-stained thick blood smears for diagnosing Plasmodium falciparum parasitemia in patients with P. falciparum (n = 84) or P. vivax (n = 59) malaria. The median P. falciparum parasite count on day 0 was 2,373/microL (range = 20-74,432/microL). At the start of treatment, the Parasight-F test had a sensitivity of 95.2% (80 of 84; 95% confidence interval [CI] = 88.2-98.7), and a specificity of 94.9% (56 of 59; 95% CI = 85.8-98.9). On day 7, this test showed false-positive results in 17 (16.3%) of 104 patients (95% CI = 9.8-24.9). The Parasight-F test performed well when compared with light microscopy in detecting P. falciparum parasitemia in patients presenting with clinical malaria. However, the high false-positive rate on day 7 limits its use for patient follow-up.

Short report: therapeutic efficacy of chloroquine combined with primaquine against Plasmodium falciparum in northeastern Papua, Indonesia.

Chloroquine combined with primaquine was evaluated for therapy of uncomplicated malaria caused by Plasmodium falciparum in nonimmune Javanese migrants to northeastern Papua, Indonesia. Subjects were randomized to treatment with standard chloroquine therapy (25 mg/kg in 3 doses over the course of 48 hours) with 30 mg primaquine administered daily for 28 days (n = 25) or a placebo of primaquine (n = 28). The 14-day cumulative incidence of therapeutic failure was 56% with primaquine and 79% with placebo (odds ratio [OR], 0.35; 95% confidence interval [CI], 0.1-1.3; P = 0.08). Primaquine administered daily created a marginally significant improvement in therapeutic efficacy at day 14, but not at day 7 (20% versus 36%; OR, 0.2; 95% CI, 0.1-1.8; P = 0.2) or day 28 (82% versus 93%; OR, 0.31; 95% Cl, 0.04-2.1; P = 0.23). This report corroborates studies suggesting that therapeutic doses of primaquine exert no discernible effect on parasitemia by P. falciparum.

Characteristics of severe anemia and its association with malaria in young children of the Kassena-Nankana District of northern Ghana.

Severe anemia is thought to be the principal underlying cause of malaria death in areas of intense seasonal malaria transmission such as the Kassena-Nankana District of northern Ghana. Factors associated with severe anemia in young children, 6-24 months old, were elucidated by analyzing results of 2 malaria-associated anemia surveys (1996, 2000), separated by 4 years, but conducted in the same community and at the same seasonal time point. Age-adjusted comparison confirmed that the proportion of severely anemic children and overall mean hemoglobin (Hb) levels in the November 2000 sample were significantly improved over those of the 1996 sample (17.5 versus 26.4%, P = 0.03; Hb 7.5 versus 6.9 g/dL, P = 0.002). Weight-for-age Z-scores also indicated a significant improvement in the 2000 sample (-1.93 versus -2.20, P < 0.05). Independently, each survey identified statistically significant associations between severe anemia and age, parasite rate, fever, and sex. Relative to children with Hb > or = 6.0 g/dL, those with severe anemia (Hb < 6.0 g/dL) were older, more frequently parasitemic (odds ratio [OR], 1.60; 95% confidence interval [CI], 1.08-2.35), more often febrile (OR, 2.44; 95% CI, 1.71-3.48), and predominantly male (OR, 1.50; 95% CI, 1.05-2.13). An association was identified in both surveys between severe anemia and residence in the northern part of the district, but no clear link was observed in relation to irrigation. Blood transfusions, a likely surrogate index of severe anemia in young children, followed a distinct seasonal pattern. Evidence suggests that dramatic peaks and troughs of severe anemia are regular and possibly predictable events that may be used to gauge the health and survival of young children in this area.

Short report: in vivo sensitivity of Plasmodium falciparum to halofantrine in southern central Vietnam.

Drug-resistant Plasmodium falciparum is present in Vietnam. We assessed the in vivo sensitivity of P. falciparum to halofantrine in two villages in the southern part of central Vietnam. Halofantrine (8 mg/kg x 3 doses) was administered to 37 patients with either P. falciparum (n = 32) or mixed P. falciparum/P. vivax malaria (n = 5). End points were parasite sensitivity or resistance (RI/RII/RIII) determined by parasite clearance, persistence, or recurrence during 28 days of follow-up. By day 28, 31 (93.9%) of 33 (95% confidence interval = 79.8-99.2%) patients were sensitive. Two patients had recurrent P. falciparum parasitemia on days 14 and 21. Halofantrine effectively treated uncomplicated P. falciparum malaria in these Vietnamese patients.

Age-dependent impairment of IgG responses to glycosylphosphatidylinositol with equal exposure to Plasmodium falciparum among Javanese migrants to Papua, Indonesia.

Immune responses directed at glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum may offer protection against symptomatic malaria. To independently explore the effect of age on generation of the anti-GPI IgG response, we measured serum anti-GPI IgGs in a longitudinal cohort of migrant Javanese children (6-12 years old) and adults (> or = 20 years old) with equivalent numbers of exposures to P. falciparum in Papua, Indonesia. While the peak response in adults was achieved after a single infection, comparable responses in children required > or = 3-4 infections. Significantly fewer children (16%) than adults (41%) showed a high (optical density > 0.44) anti-GPI IgG response (odds ratio [OR] = 3.8, 95% confidence interval [CI] = 2.3-6.3, P < 0.0001), and adults were more likely to show a persistently high response (OR = 5.5, 95% CI = 1.0-56.8, P = 0.03). However, the minority of children showing a strong response were significantly less likely to experience symptoms with subsequent parasitemia compared with those with a weak response (OR = 4.0, 95% CI = 1.1-13.8, P = 0.02). This effect was not seen among high- and low-responding adults (OR = 1.2, 95% CI = 0.5-2.8, P = 0.60). Host age, independent of cumulative exposure, apparently represents a key determinant of the quantitative and qualitative nature of the IgG response to P. falciparum GPI.

Seasonal malaria attack rates in infants and young children in northern Ghana.

The incidence density of infection and disease caused by Plasmodium falciparum in children aged six to 24 months living in the holoendemic Sahel of northern Ghana was measured during the wet and dry seasons of 1996 and 1997. At the beginning of each season, a cohort composed of 259 and 277 randomly selected children received supervised curative therapy with quinine and Fansidar and primaquine for those with normal glucose-6-phosphate dehydrogenase activity. The 20 weeks of post-therapy follow-up consisted of three home visits weekly and examination of Giemsa-stained blood films once every two weeks. Blood films were also taken from children brought to clinic with illness. The incidence density of parasitemia after radical cure was 4.7 infections/person-year during the dry season and 7.1 during the wet season (relative risk = 1.51, 95% confidence interval [CI] = 1.25-1.81; P = 0.00001). Although the mean parasitemia count at time of reinfection in the dry season (3,310/microl) roughly equaled that in the wet season (3,056/microl; P = 0.737), the risk ratio for parasitemia > 20,000/microl during the wet season was 1.71 (95% CI = 1.2-2.4; P = 0.0025). The risk ratio for parasitemia > 20,000/microl with fever during the wet season was 2.45 (95% CI = 1.5-4.1; P = 0.0002). The risk ratio for anemia (hemoglobin < 8 g/dl) at first post-radical cure parasitemia showed no difference between seasons (1.0; 95% CI = 0.73-1.4; P = 0.9915). We did not see seasonal differences in anemia known to exist in this region, probably because the longitudinal cohort design using first parasitemia as an end point prevented the subjects from developing the repeated or chronic infections required for anemia induction. These findings bear upon the design of malaria drug and vaccine trials in holoendemic areas.