Longitudinal cohort study of the epidemiology of malaria infections in an area of intense malaria transmission I. Description of study site, general methodology, and study population.

A large-scale longitudinal cohort project was initiated in western Kenya in June 1992. The primary purpose of the project was to study Plasmodium falciparum malaria in a highly endemic area using a comprehensive and multidisciplinary approach, which included epidemiology, entomology, and immunology. Between June 1992 and July 1994, pregnant women living in 15 rural villages were identified during a monthly census and 1,164 were enrolled. The women were followed-up throughout their pregnancy and they, along with their newborn infants and direct siblings of the infants' less than 15 years of age, were monitored over time. As of May 1995, 1,017 infants had been born to these women. This paper presents the design and general methodology used in this study and describes the initial experience with intense monitoring of a large population over a prolonged period.

Longitudinal cohort study of the epidemiology of malaria infections in an area of intense malaria transmission II. Descriptive epidemiology of malaria infection and disease among children.

A large-scale longitudinal cohort project was initiated in western Kenya in June 1992. Between June 1992 and July 1994, 1,848 children less than 15 years of age were monitored prospectively for a mean of 236 days. During this period, 12,035 blood smears were examined for malaria and only 34% were found to be negative. Parasite prevalence (all species) decreased with age (from a high of 83% among children 1-4 years old to 60% among children 10-14 years old). Even more dramatic decreases were noted in the prevalence of high density falciparum infection (from 37% among children 12-23 months old to < 1% among 10-14-year-old children) and in clinical malaria (20% to 0.3% in the same age groups). Children < 1 year of age accounted for 55% of all cases of anemia detected. Anemia was consistently associated with high density infection in children < 10 years of age (20% to 210% increased risk relative to aparasitemic children). These results demonstrate the relationship between high-density malaria infection and two clinical manifestations of malarial illness.

Field evaluation of a polymerase chain reaction-based nonisotopic liquid hybridization assay for malaria diagnosis.

In a blind field evaluation of a nonisotopic liquid hybridization assay for detection of malaria parasites, 100 blood samples were tested from an area in which malaria is endemic; light microscopy was used as the reference test. Sensitivity, specificity, and positive and negative predictive values of the hybridization assay were 100%. One sample that was microscopy-negative and hybridization-positive was positive when reexamined. Another sample that was microscopy-positive and hybridization-negative was negative at reexamination. The detection limit of the test was > or = 0.0005% parasitemia. Four samples with mixed infections were misdiagnosed by microscopy as single-species infections. Four samples diagnosed as mixed infections by microscopy and single infection by the hybridization test had no evidence of a second Plasmodium species upon reexamination. The polymerase chain-reaction-based nonisotopic liquid hybridization assay was better than conventional light microscopy in detecting low-grade parasite infection and offers an exceptional advantage for detecting mixed infections.

Evaluation of the QBC method to detect malaria infections in field surveys.

The conventional peripheral blood film method used to diagnose malaria is characterized by low sensitivity in scanty parasitaemia and can be time consuming when required to rule out infection. The Quantitative Buffy Coat (QBC) method has been proposed to be quicker and more sensitive. We conducted a malaria survey in April 1992 among school-children in Kisumu (holoendemic) and Webuye (hypoendemic) areas of Western Kenya. Peripheral blood samples were examined by thick blood smear (TBS) stained with Giemsa solution, and by the QBC method. A total of 360 paired samples were analyzed. There were 175 (49%) positive TBS and 201 (56%) positive QBC. Of the 185 TBS classified as negative, 30 (16%) were positive by QBC. When parasite density by TBS was > or = 100/300 WBCs, the sensitivity of QBC was 100%. Overall sensitivity for QBC was 98%, with a specificity of 84%. Negative predictive value for the QBC was 98%, and had a calculated accuracy of 92%. It took an average of 44 minutes to process a TBS and a further average of 2.6 minutes to examine a negative TBS. For the QBC the mean time to process and to examine was 7.09 and 1.04 minutes respectively. We conclude that the QBC is quicker, with high sensitivity, and will prove useful in clinical and epidemiological screening, especially when parasitaemia is low.

Sensitivity of falciparum malaria to chloroquine and amodiaquine in four districts of western Kenya (1985-1987).

In-vivo and in-vitro studies to determine the sensitivity of Plasmodium falciparum malaria to chloroquine and amodiaquine were conducted in 4 districts of Western Kenya over a 2-year-period. Patients aged 5-60 years, were treated with chloroquine or amodiaquine base 25 mg/kg over 3 days. Recurrence of parasitaemia within 7 days (R1 resistance) or failure to clear parasites (R11 resistance) was observed in 27% of infections in West Pokot district, 51% in Busia, 45% in Bungoma and 19% in Rusinga Island. R111 resistance (failure to decrease parasitaemia by at least 75%) was documented in Rusinga Island. The proportions of parasites with minimum inhibitory concentrations (MICs) for chloroquine greater than 114 nM in in-vitro tests ranged from 37% in Busia to 68% in Bungoma. For amodiaquine, 20% of 30 isolates tested had MICs greater than 80 nM. We conclude that resistance to chloroquine is now established in the area and amodiaquine may be useful in uncomplicated chloroquine resistant falciparum infections in the region.

PIP: Between 1985-1987, researchers recruited 419 5-60 year old persons with moderate falciparum malaria to take part in a series of studies in West Pokot, Rusinga Island, Busia, and Bungoma districts in western Kenya to determine malaria parasite sensitivity to chloroquine and amodiaquine. The subject received 25mg/kg of the antimalarial for 3 days. Chloroquine resistance was highest in Busia in May 1986 (51%) followed by Bungoma (45%), Busia in July 1986 (38%), West Pokot (27%), and Rusinga Island (19%). No RIII resistance (failure to decrease parasitemia by at least 75%) occurred in any of the 4 studies. In July 1986, researchers compared the different sensitivity levels of chloroquine and amodiaquine among 119 school children in Busia. None of the children exhibited RIII resistance. None of the falciparum malaria parasites in the 58 children receiving amodiaquine showed RII resistance (failure to clear parasites) compared to 3.3% of those receiving chloroquine. Yet 15.5% of students receiving amodiaquine harbored parasites with RI resistance (recurrence of parasitemia within 7 days), especially delayed RI resistance. Still this percentage was much lower than RI resistance among the students receiving chloroquine (34.4%). Total resistance was significantly much higher in the chloroquine group than the amodiaquine group (p.01). Moreover in vitro studies revealed that the percentages of parasites with minimum inhibitory concentrations (MICs) for chloroquine 114 nM (indicating resistance) varied from 37% in Busia to 68% in Bungoma. Further 20% had MICs for amodiaquine 80 mM (indicating resistance). In conclusion, chloroquine resistant falciparum malaria parasites have established themselves in western Kenya. Despite growing resistance, amodiaquine should still be used in uncomplicated infections, if the health practitioner is able to follow up on the patients.

Chlorproguanil/dapsone for the treatment of non-severe Plasmodium falciparum malaria in Kenya: a pilot study.

Chlorocycloguanil, the active metabolite of chlorproguanil, was synergistic in vitro with dapsone against 2 culture-adapted Plasmodium falciparum isolates from Kenya; maximal synergy occurred at lower concentrations that it did with pyrimethamine and sulfadoxine. 48 children with asymptomatic P. falciparum infections were treated with chlorproguanil (at a target dose of 1.2 mg/kg) and dapsone (target dose of 1.2 or 2.4 mg/kg); all were free of parasitaemia by day 7. The following numbers had recurrences on days 14, 21, and 28, respectively: 1 of 48, 7 of 47, and 7 of 40. All 39 children treated with pyrimethamine (target dose 1.2 mg/kg) and sulfadoxine (target dose 24 mg/kg) were cleared of infection, while the following had recurrences on days 14, 21, and 28: 1 of 39, 2 of 38, and 2 of 36. The rate of decrease in parasitaemia was the same in the 2 groups, and there was no change in haematocrit or haemoglobin during the follow-up. The rate of recurrence in the children receiving chlorporguanil/dapsone was higher, probably because these drugs have a much shorter clearance time than pyrimethamine/sulfadoxine. Chlorproguanil/dapsone is an effective combination for treating P. falciparum malaria and deserves further study.

Chloroquine treatment of falciparum malaria in an area of Kenya of intermediate chloroquine resistance.

106 children aged 1-10 years who had pure Plasmodium falciparum infections and temperatures greater than or equal to 38 degrees C were treated with chloroquine base, 25 mg/kg body weight. 29% of the infections were sensitive in vivo, 41% recurred within 4 weeks (RI), 26% were RII resistant, and 4% were RII resistant. Rieckmann micro in vitro tests were successful in 64% of isolates obtained from these children; 63% were resistant to chloroquine. In 58 paired isolates obtained before and after treatment, the level of chloroquine sensitivity was lower in the parasites persisting or recurring after treatment. All children except 2 of the 4 with RIII resistance became afebrile an average of 1.4 d after starting treatment and their other symptoms resolved in an average of 1.8 d. By day 28, 57% of the children with RI resistance and 78% of those with RII resistance had recurrence of fever and other symptoms, compared with 19% of children with sensitive infections. No relationship was observed between the clinical or parasitological response and age, nutritional status, haematocrit, splenomegaly, presence of sickle-cell trait, or seropositivity to malaria by enzyme-linked immunosorbent assay. The study demonstrates that, in most children with malaria in an area of intermediate chloroquine resistance, fever and other symptoms resolve at least temporarily when treated with chloroquine.

Effectiveness of amodiaquine as treatment for chloroquine-resistant Plasmodium falciparum infections in Kenya.

Studies were conducted in Malindi, Kenya, to assess the response of Plasmodium falciparum to chloroquine and amodiaquine in vivo (by an extended 14-day test) and in vitro (with the Rieckmann micro test). In-vivo resistance was demonstrated in 19 of 69 (28%) infections treated with chloroquine, but in only 2 of 60 (3.3%) of those treated with amodiaquine (p less than 0.001). In-vitro resistance to chloroquine was demonstrated in 15 of 23 (65%) tests. In contrast, 22 of the same 23 isolates were sensitive to amodiaquine in vitro. Effective concentrations by probit analysis for 50% and 99% (EC50 and EC99) inhibition, respectively, were 180.7 and 4319.6 nmol/l for chloroquine and 12.2 and 147.0 nmol/l for amodiaquine. The results suggest that amodiaquine is effective for the treatment of chloroquine-resistant falciparum malaria in Kenya.