Evaluation of the fat-tailed gerbil, Pachyuromys duprasi (Rodentia: Gerbillidae), as a new animal model for studies of Leishmania major infection and transmission.

The fat-tailed gerbil Pachyuromys duprasi is a common burrowing rodent found across the northern Sahara Desert from Morocco to Egypt. There is overlap in the geographical distribution and ecological habitats of P. duprasi, several Old World Leishmania species, and numerous sand fly vectors of Leishmania, but there are no records that document the natural occurrence of this gerbil with any species of Leishmania or phlebotomine sand fly. Experiments were conducted to determine its potential as a natural host and laboratory animal model for Leishmania major. Captive-born P. duprasi were inoculated subcutaneously (s.c.) in the tail with promastigotes or amastigotes of an Egyptian strain of L. major and monitored for signs of infection. Local swelling and erythema was visible 10-12 days after amastigote inoculation, and within 3-4 weeks swelling had increased tail widths by up to 78%. Infections progressed more slowly and less conspicuously following inoculation with promastigotes. Tissue density of amastigotes in the gerbil's tail lesions after inoculating with either stage of L. major was significantly lower than that produced in the footpads of BALB/c mice by the same parasite and incubation period. Laboratory transmission of L. major to P. duprasi by sand fly bite was demonstrated and acquisition of L. major, by bite, from tail lesions of infected P. duprasi to laboratory-reared Phlebotomus papatasi was also achieved with 10% of biting flies developing promastigote infections. The acquisition and development of L. major infections in P. papatasi after biting an infected P. duprasi and the susceptibility of P. duprasi to L. major delivered at low densities by sand fly bites indicate that fat-tailed gerbils could serve as a natural host and reservoir of L. major.

Experimental acquisition, development, and transmission of Leishmania tropica by Phlebotomus duboscqi.

We report experimental infection and transmission of Leishmania tropica (Wright), by the blood-feeding sand fly Phlebotomus duboscqi (Neveu-Lemaire). Groups of laboratory-reared female sand flies that fed "naturally" on L. tropica-infected hamsters, or artificially, via membrane feeding device, on a suspension of L. tropica amastigotes, were dissected at progressive time points post-feeding. Acquisition, retention and development of L. tropica through procyclic, nectomonad, and leptomonad stages to the infective metacyclic promastigote stage, and anterior progression of the parasites from abdominal midgut bloodmeal to the thoracic midgut were demonstrated in both groups. Membrane feeding on the concentrated amastigote suspension led to metacyclic promastigote infections in 60% of sand flies, whereas only 3% of P. duboscqi that fed naturally on an infected hamster developed metacyclics. Sand flies from both groups re-fed on naïve hamsters, but despite infections in 25-50% of membrane-fed and 2-3.5% of naturally fed flies, no skin lesions developed in the hamsters. After four months of observation these animals were euthanized and necropsied. Screening of the organs and tissue by polymerase chain reaction (PCR) that targeted the small subunit RNA gene, amplified generic Leishmania DNA from liver, spleen, bone marrow, and blood, but only from hamsters bitten by membrane-infected P. duboscqi. These results are notable in demonstrating the ability of P. duboscqi, originating from Kenya, to acquire, retain, develop, and transmit a Turkish strain of L. tropica originally isolated from a human case of cutaneous leishmaniasis. This marks the first demonstration of complete development and transmission of L. tropica by a member of the Phlebotomus subgenus of sand flies.

Failure of Supervised Chloroquine and Primaquine Regimen for the Treatment of Plasmodium vivax in the Peruvian Amazon.

The widespread use of primaquine (PQ) and chloroquine (CQ), together, may be responsible for the relatively few, isolated cases of chloroquine-resistant P. vivax (CQRPV) that have been reported from South America. We report here a case of P. vivax from the Amazon Basin of Peru that recurred against normally therapeutic blood levels of CQ. Four out of 540 patients treated with combination CQ and PQ had a symptomatic recurrence of P. vivax parasitemia within 35 days of treatment initiation, possibly indicating CQ failure. Whole blood total CQ level for one of these four subjects was 95 ng/ml on the day of recurrence. Based on published criteria that delineate CQRPV as a P. vivax parasitemia, either recrudescence or relapse, that appears against CQ blood levels >100 ng/mL, we document the occurrence of a P. vivax strain in Peru that had unusually high tolerance to the synergistic combination therapy of CQ + PQ that normally works quite well.

Virus isolations and high population density implicate Culex antennatus (Becker) (Diptera: Culicidae) as a vector of Rift Valley Fever virus during an outbreak in the Nile Delta of Egypt.

In June, 2003, Egypt's hospital-based electronic disease surveillance system began to record increased cases of acute febrile illness from governorates in the Nile Delta. In response to a request for assistance from the Egyptian Ministry of Health and the World Health Organization (WHO), the U.S. Naval Medical Research Unit No. 3 (NAMRU-3) provided assistance in identifying the cause and extent of this outbreak. Testing of human clinical samples (n=375) from nine governorates in Egypt identified 29 cases of RVF viremia that spanned the period of June to October, and a particular focus of disease in Kafr el Sheikh governorate (7.7% RVF infection rate). Veterinary samples (n=101) collected during this time in Kafr el Sheikh and screened by immunoassay for RVFV-specific IgM identified probable recent infections in cattle (10.4%) and sheep (5%). Entomologic investigations that focused in rural, rice growing villages in the Sidi Salim District of Kafr el Sheikh during August-September, 2003, collected, identified, and tested host-seeking female mosquitoes for the presence of pathogenic viruses. Three isolates of RVF virus (RVFV) were obtained from 297 tested pools of female mosquitoes and all three RVFV isolates came from Cx. antennatus (Becker). While Cx. pipiens has been considered the primary vector of RVF virus in Egypt and is often the most common man-biting species found, Cx. antennatus was the dominant species captured at the 2003 outbreak location in Kafr el Sheikh governorate. This is the first time that Cx. antennatus has been found naturally infected with RVFV in Egypt.

Effects of ivermectin on blood-feeding Phlebotomus papatasi, and the promastigote stage of Leishmania major.

Ivermectin (IVM) is a chemically modified macrocyclic lactone of Streptomyces avermitilis that acts as a potent neurotoxin against many nematodes and arthropods. Little is known of IVM's effect against either blood-feeding Phlebotomus sand flies, or the infective promastigote stage of Leishmania transmitted by these flies. We injected hamsters subcutaneously with two standard IVM treatments (200 and 400 µg/kg body weight) and allowed cohorts of Leishmania major-infected Phlebotomus papatasi to blood-feed on these animals at various posttreatment time points (4 h, 1, 2, 6, and 10 days). Infected and uninfected sand flies that bit treated and untreated hamsters served as controls. Serum levels of IVM in low- and high-dose-treated hamsters were determined at the five time points. Sand fly mortality following blood feeding was recorded at 24-h intervals and, in relation to IVM treatment, was time and dose dependent. Mortality was most rapid and greatest among infected flies that fed nearest to time of dosing. Mean survival of infected sand flies after feeding on untreated hamsters was 11.5 days, whereas that of infected sand flies that fed 4 h, 1 day, or 2 days posttreatment on high-dose-treated hamsters (400 µg/kg) was 1.6, 2.1, and 2.7 days, respectively. Infected and uninfected sand flies that blood fed 6 days following low-dose IVM treatment (200 µg/kg) still experienced significantly greater mortality (p < 0.02) than controls. Promastigotes dissected out of surviving flies that fed on IVM-treated hamsters showed typical motility and survival. Moreover, 21.7% of IVM-treated hamsters developed lesions after being fed upon by infected sand flies. L. major promastigotes appeared to be tolerant to ng/mL blood levels of IVM that caused significant mortality for up to 10 days posttreatment in blood-feeding P. papatasi.

Sulfadoxine-pyrimethamine-based intermittent preventive treatment, bed net use, and antenatal care during pregnancy: demographic trends and impact on the health of newborns in the Kassena Nankana District, northeastern Ghana.

Demographics and health practices of 2,232 pregnant women in rural northeastern Ghana and characteristics of their 2,279 newborns were analyzed to determine benefits associated with intermittent preventive treatment (IPTp), antenatal care, and/or bed net use during pregnancy. More than half reported bed net use, 90% reported at least two antenatal care visits, and > 82% took at least one IPTp dose of sulfadoxine-pyrimethamine. Most used a bed net and IPTp (45%) or IPTp alone (38%). Low birth weight (< 2,500 grams) characterized 18.3% of the newborns and was significantly associated with female sex, Nankam ethnicity, first-born status, and multiple births. Among newborns of primigravidae, IPTp was associated with a significantly greater birth weight, significantly fewer low birth weight newborns, improved hemoglobin levels, and less anemia. Babies of multigravidae derived no benefit to birth weight or hemoglobin level from single or multiple doses of sulfadoxine-pyrimethamine during pregnancy. No differences or benefits were seen when a bed net was the only protective factor.

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.

Laboratory demonstration of the acquisition and development of leishmania major in the sand fly Phlebotomus kazeruni (Diptera: Psychodidae).

Phlebotumus kazeruni, a blood-feeding, xerophilic sand fly species found broadly throughout North Africa and Western Asia, is a suspected vector of cutaneous leishmaniasis (CL). Following successful laboratory colonization of this species, we employed the murine (BALB/c) infection model to determine whether our Sinai strain of P. kazeruni was able to successfully acquire, develop, and transmit a Sinai strain of Leishmania major. Groups of female sand flies were fed 1) by membrane, hamster blood containing culture-produced L. major promastigotes, 2) by membrane, hamster blood containing a suspension of L. major tissue amastigotes, and 3) directly upon L. major lesions in BALB/c mice. Samples of blood-fed sand flies from each group were dissected on selected days post-feeding and examined by light microscope for acquired and developing Leishmania infections. Female P. kazeruni acquired viable parasites by the three feeding methods. Development of ingested parasites to infective-stage metacyclic forms was observed and seen to progress from midgut to the mouthparts. Promastigote infection rates were 20% in flies fed directly upon infected mice, 35% in those fed amastigotes via membrane, and 100% in flies fed culture promastigotes via membrane. Direct blood fee-ding upon BALB/c mice was more avid (P < 0.001) among previously blood-fed flies, possibly indicative of selection and colony adaptation to murine blood-feeding. Although we failed to demonstrate clear transmission of infective-stage L. major promastigotes by feeding infected flies upon a susceptible murine host, and producing lesions in the animal, the progressive development of L. major from amastigote to metacyclic-stage promastigotes, and movement of the parasites from sand fly midgut to its mouthparts, provides evidence that P. kazeruni could serve as a vector for this parasite.

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.

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.

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.

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.

Malaria transmission dynamics at a site in northern Ghana proposed for testing malaria vaccines.

We studied the malaria transmission dynamics in Kassena Nankana district (KND), a site in northern Ghana proposed for testing malaria vaccines. Intensive mosquito sampling for 1 year using human landing catches in three micro-ecological sites (irrigated, lowland and rocky highland) yielded 18 228 mosquitoes. Anopheles gambiae s.l. and Anopheles funestus constituted 94.3% of the total collection with 76.8% captured from the irrigated communities. Other species collected but in relatively few numbers were Anopheles pharoensis (5.4%) and Anopheles rufipes (0.3%). Molecular analysis of 728 An. gambiae.s.l. identified Anopheles gambiae s.s. as the most dominant sibling species (97.7%) of the An. gambiae complex from the three ecological sites. Biting rates of the vectors (36.7 bites per man per night) were significantly higher (P<0.05) in the irrigated area than in the non-irrigated lowland (5.2) and rocky highlands (5.9). Plasmodium falciparum sporozoite rates of 7.2% (295/4075) and 7.1% (269/3773) were estimated for An. gambiae s.s. and An. funestus, respectively. Transmission was highly seasonal, and the heaviest transmission occurred from June to October. The intensity of transmission was higher for people in the irrigated communities than the non-irrigated ones. An overall annual entomological inoculation rate (EIR) of 418 infective bites was estimated in KND. There were micro-ecological variations in the EIRs, with values of 228 infective bites in the rocky highlands, 360 in the lowlands and 630 in the irrigated area. Approximately 60% of malaria transmission in KND occurred indoors during the second half of the night, peaking at daybreak between 04.00 and 06.00 hours. Vaccine trials could be conducted in this district, with timing dependent on the seasonal patterns and intensity of transmission taking into consideration the micro-geographical differences and vaccine trial objectives.

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.

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.

Detection of malaria sporozoites by standard ELISA and VecTestTM dipstick assay in field-collected anopheline mosquitoes from a malaria endemic site in Ghana.

We compared the VecTestTM dipstick assay for detection of Plasmodium sporozoites in Anopheles vectors of malaria with standard circumsporozoite (CS) microplate ELISA for detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in Anopheles mosquitoes. Mosquitoes were collected from a malaria endemic site (Kassena Nankana district) in northern Ghana. Of 2620 randomly sampled mosquitoes tested, the standard CS-ELISA gave a sporozoite rate of 10.8% compared with 11.2% by VecTestTM, which was not statistically different (P = 0.66). Visual reading of the CS-ELISA results gave a sporozoite rate of 13.4%, which was higher than the other tests (P > 0.05). To allow a more objective evaluation of the sensitivity of the dipstick, an additional 136 known CS-ELISA-positive specimens were analysed. The prevalence of the test (including the additional samples) was 14.6% and 14.7% for CS-ELISA and dipstick, respectively (P > 0.05). The estimated prevalence by visual assessment of the CS-ELISA results was 17.5%. The relative specificity and sensitivity of the VecTestTM dipstick and visually read ELISA were estimated based on the CS-ELISA as a gold standard. The specificities of the dipstick and visual ELISA were high, 98.0% and 96.6%, respectively. However, the sensitivities of the two assays were 88.8% for VecTest and 100% for visual ELISA (P < 0.01). Concordance between VecTest and CS-ELISA was good (kappa = 0.86). Similarly, there was a good concordance between the dipstick and the visually read ELISA (kappa = 0.88). Extrapolating from PfCSP controls (titrated quantities of P. falciparum sporozoites), mean sporozoite loads of CS-ELISA-positive An. gambiae (286 +/- 28.05) and An. funestus (236 +/- 19.32) were determined (P = 0.146). The visual dipstick grades showed high correlation with sporozoite load. The more intense the dipstick colour, the higher the mean sporozoite load (+ = 108, ++ = 207, +++ = 290, r = 0.99, r2 = 1). The VecTest dipstick offers practical advantages for field workers needing rapid and accurate means of detection of sporozoites in mosquitoes.

Seasonal profiles of malaria infection, anaemia, and bednet use among age groups and communities in northern Ghana.

We conducted all-age point prevalence surveys to profile the severity and seasonality of malaria and anaemia in Kassena-Nankana District of northern Ghana. Random cross-sectional surveys were timed to coincide with the end of low (May 2001) and high (November 2001) malaria transmission seasons and to yield information as to the potential value of haemoglobin (Hb) levels and parasitaemia as markers of malaria morbidity and/or malaria vaccine effect. Parasitaemia was found in 22% (515 of 2286) screened in May (dry-low transmission), and in 61% of the general population (1026 of 1676) screened in November (wet-high transmission). Malaria prevalence in May ranged from 4% (infants <6 months and adults 50-60 years) to 54% (children 5-10 years). Age-specific malaria prevalence in November ranged from 38% (adults 50-60 years) to 82% (children 5-10 years). Differences between low- and high-transmission periods in the prevalence of severe anaemia (SA) among young children (6-24 months) were unexpectedly comparable (low, 3.9%vs. high, 5.4%; P = 0.52) and greatly reduced from levels measured in this same community and age group in November 2000 (12.5%) and November 1996 (22.0%). Despite the lower frequency of anaemia/SA in young children surveyed in 2001, it was still clear that this condition was strongly associated with parasitaemia and that children under 5 years of age experienced a significant drop in their mean Hb levels by the end of the high transmission season. Prevalence of parasitaemia was significantly lower (P < 0.01) among infants and young children (<2 years) whose parents reported the use of bednets. There was a significantly lower risk of parasitaemia among infants [odds ratio (OR) 6-8] and young children (OR 3-4) living in the central, more urbanized sector of the study area.