The total population of the Republic of Congo was 3,697,490 inhabitants in 2007, and 1,373,382 of these persons (37%) reside in Brazzaville, the capital city . Brazzaville is divided into seven districts: Bacongo, Makelekele, Poto-Poto, Moungali, Ouenze, Talangaï, and Mfilou. The present study was conducted in Makelekele district where 298,292 inhabitants live in urban and suburban areas. Patients were enrolled in Terinkyo urban health centre, located in the urban area of Makelekele district and Madibou health centre, located in the suburban area of the district. Previous malaria surveillance from 2003 to 2007 showed that 22.3% of febrile patients consulting Tenrikyo health centre and 44.7% of febrile patients seen at the Madibou health centre were infected with malaria parasites . In the urban area of Makelekele district, malaria is hypo- to meso-endemic, while in its suburban area, malaria is hyperendemic [24, 25].
All febrile patients were referred to the health centres’ laboratory for malaria parasite screening. Giemsa-stained thick blood films were prepared from finger-prick capillary blood and examined under the microscope. Symptomatic patients with at least 2,000 asexual parasites/μL were examined by a physician. Patients with the following criteria were included in the study: (i) mono-infection with P. falciparum ≥2,000 asexual parasites/μL, (ii) axillary temperature >38°C, (iii) absence of danger signs in young children (unable to drink or eat, vomiting more than twice in the previous 24 hours, recent history of convulsion, unconscious state or inability to sit or stand) or signs of severe and complicated malaria in older children and adults, (iv) a pack cell volume (PCV) >15%, (v) absence of other febrile illnesses, (vi) written informed consent signed by the patient (for adults) or the parents or legal guardian (for minors), and, (vii) easy accessibility of their residence for home visits . Before treatment, finger-prick capillary blood was collected on Whatmann 3MM filter paper for molecular analysis and in a capillary tube for PCV determination.
The co-blistered pack of AS + AQ (Arsucam®, Sanofi Aventis, Paris, France) containing 50 mg of artesunate and 153 mg of amodiaquine base per tablet was administered to patients under supervision. Patients received the standard daily dose of 10 mg base/kg body weight of amodiaquine and 4 mg/kg body weight of artesunate for three days. Based on the calculation using the body weight of patients, quarter, half, or three-quarter tablet was administered. For young children, the tablet was crushed and suspended in sugar-containing syrup. The patient was observed for 30 min. If vomiting occurred within 30 min following the initial drug administration, the treatment was repeated. The patient was excluded from the study if he or she vomited twice during the observation period.
The patients were followed on days 1, 2, 3, 7, 14, 21 and 28. Clinical examination and measurement of axillary temperature were performed during each visit, and any adverse events or unauthorized concomitant therapies were recorded. As recommended in the 2003 WHO protocol , blood films were examined on days 2, 3, 7, 14, 21, 28, and during any unscheduled visit if the patient became febrile. If parasites reappeared on or after day 7, finger-prick capillary blood was collected on Whatman 3MM filter paper for polymerase chain reaction (PCR) analysis.
The present study was reviewed and approved by the Congolese Ministry of Health. The WHO Secretariat Committee on Research Involving Human Subjects (SCRIHS) reviewed the study protocol and consent forms in French, English and local languages.
Thick blood films were stained with 10% Giemsa for 15 min. Asexual parasites were counted against 200 white blood cells (WBCs) and expressed as the number of asexual parasites/μL of blood, assuming a WBC count of 8,000/μL of blood. In case of hyperparasitaemia, the parasite count was determined when 500 asexual parasites were counted even if 200 WBCs were not reached. Parasite density was determined by two independent technicians. Plasmodium falciparum gametocytes were counted against 1,000 WBC. PCV was obtained by micro haematocrit centrifugation.
DNA extraction and Plasmodium falciparum genotyping
Genomic DNA was extracted from blood samples collected on filter paper using QiaAmp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. DNA was recovered in 100 μL of elution buffer. All extracted samples of parasite DNA were stored at −20°C until use.
For patients with treatment failure, the isolates collected at inclusion and recrudescent isolates were genotyped in parallel using nested PCR technique. The highly polymorphic loci, block 2 of merozoite surface protein-1 (msp-1) and the central region of merozoite surface protein-2 (msp-2), were used as markers for genotyping, as described previously . The initial amplifications were followed by individual nested PCR using specific primers for K1, MAD20, and RO33 allelic families of msp-1 and specific primers for FC27 and 3D7 allelic families of msp-2. Allelic specific positive controls and DNA-free negative controls were included in each set of reactions. Five microlitres of PCR products were loaded on 2% agarose gel, stained with SYBR Green, separated by electrophoresis, and visualized under ultraviolet transillumination.
Individual alleles were identified by the fragment length and the corresponding allele-specific primers used, and the size of the PCR products was estimated using a 100 base pair (bp) DNA ladder marker (Invitrogen, Karlsruhe, Germany). The size polymorphism in each allelic family was analysed, assuming that one band represents one amplified PCR fragment derived from a single copy of P. falciparum msp-1 or msp-2 genes. Alleles in each family were considered to be the same if the fragment size were within 20-bp interval. The minimum number of genotypes per isolate was estimated to be the highest number of fragments identified for either msp-1 or msp-2.
It was assumed that after a patient was initially treated for malaria, a subsequent episode was caused by either P. falciparum isolates present before treatment (day 0) or by P. falciparum infections that occurred after treatment. First, paired pre-treatment and post-treatment samples were genotyped using msp-2 locus. If different band profile was found, it was concluded that the re-appearance of parasites on or after day 7 was due to a new infection. If similar profile of bands was observed, these samples were further analysed for a second locus, ie, msp-1. The outcome was defined as recrudescence if the paired samples (day 0 and recrudescent sample) displayed identical alleles. The outcome was defined as new infection if the recrudescent sample had only newly identified alleles. If the recrudescent sample showed new alleles and alleles identified on day 0, this sample was recorded as mixed infections or unclassified.
The responses were classified before PCR correction and with PCR correction . The PCR-uncorrected outcomes were classified into three categories: early treatment failure (ETF), late treatment failure (LTF), which consists of two subcategories, late clinical failure (LCF) and late parasitological failure (LPF), and adequate clinical and parasitological response (ACPR). PCR-corrected outcomes were classified into three categories: recrudescence, including ETF (on or before day 3) and recrudescence after PCR analysis, adequate clinical and parasitological response (ACPR), and new infections after PCR analysis.
Signs and symptoms that developed after drug intake were observed and reported during clinical examination and questioning the patients or, in case of children, their parents or guardians, during each visit. Adverse events can also be an exacerbation of disease symptoms.
All patients consulting one of the two health centres and satisfying the inclusion criteria were enrolled after written informed consent, regardless of their age. Two age groups were constituted: ≤5 years old and >5 years old. Moreover, the residence of the patients was classified as urban (patients consulting Tenrikyo health centre) or suburban (patients consulting Madibou health centre) area.
Clinical and parasitological data were entered into pre-programmed Excel spreadsheet provided by the Department of Global Malaria Programme, WHO (Geneva, Switzerland) with the possibility to analyse PCR-uncorrected and PCR-corrected responses. Proportions and 95% confidence interval (95% CI) were calculated using Epi-info 6.04 (Centers for Disease Control and Prevention, Atlanta, USA).