Reduction of multiplicity of infections but no change in msp2 genetic diversity in Plasmodium falciparum isolates from Congolese children after introduction of artemisinin-combination therapy.

BACKGROUND: In this first study conducted after the introduction of artemisinin-combination therapy (ACT), the major objective was to evaluate Plasmodium falciparum genetic diversity and multiplicity of infection in isolates from Congolese children between one and nine years of age enrolled and followed up for one year. The secondary objective was to characterize the msp2 profiles of P. falciparum isolates collected from successive malaria episodes in ten children who had four or more clinical episodes during the follow up. METHODS: Three-hundred and thirteen children residing in southern part of Brazzaville participated in this study. Blood samples were obtained from all children at enrollment and checked for P. falciparum infection. Based on the one year follow-up data, two clinical groups were considered according to the number of malaria episodes presented over the follow up period: "protected"(children who did not experience any episode) and "unprotected" (those who experienced more that two episodes). Therefore, the msp2 genetic diversity of P. falciparum isolates collected at enrollment in the two groups was characterized by allele-specific nested PCR and compared. The msp2 profiles of P. falciparum isolates collected from successive malaria episodes was also characterized by allele-specific nested PCR. RESULTS: Forty-three percent of FC27 and fifty-seven percent of 3D7 in protected vs fifty-six percent of FC27 and forty-four percent of 3D7 in isolates from unprotected children were detected. Seven and two alleles belonging to the FC27, and six and three alleles belonging to 3D7 families were distinguished in isolates from protected and unprotected children respectively. The mean multiplicity of infection (MOI) values at inclusion for the msp2 locus was 1.29 and 1.43 for protected and unprotected children respectively. 43 isolates were obtained from the ten children who had four or more clinical episodes during the follow up. A total of 63 alleles or fragments corresponding to 57% (36/63) FC27 and 43% (27/63) 3D7 were detected. The variant 400bp of FC27 was the most prevalent. 46% (20/43), 42% (18/43), 2% (1/43) and 2% (1/43) of isolates were found to have 1, 2, 3 and 4 parasite genotypes respectively and the mean MOI was 1.78. CONCLUSION: This study shows that the introduction of ACT in the Republic of Congo has reduced the MOI but not the genetic diversity of P. falciparum isolates from children living in Southern districts of Brazzaville.

Genetic polymorphism of merozoite surface protein 2 and prevalence of K76T pfcrt mutation in Plasmodium falciparum field isolates from Congolese children with asymptomatic infections.

BACKGROUND: In order to prepare the field site for future interventions, the prevalence of asymptomatic Plasmodium falciparum infection was evaluated in a cohort of children living in Brazzaville. Plasmodium falciparum merozoite surface protein 2 gene (msp2) was used to characterize the genetic diversity and the multiplicity of infection. The prevalence of mutant P. falciparum chloroquine resistance transporter (pfcrt) allele in isolates was also determined. METHODS: Between April and June 2010, 313 children below 10 years of age enrolled in the cohort for malaria surveillance were screened for P. falciparum infection using microscopy and polymerase chain reaction (PCR). The children were selected on the basis of being asymptomatic. Plasmodium falciparum msp2 gene was genotyped by allele-specific nested PCR and the pfcrt K76T mutation was detected using nested PCR followed by restriction endonuclease digestion. RESULTS: The prevalence of asymptomatic P. falciparum infections was 8.6% and 16% by microscopy and by PCR respectively. Allele typing of the msp2 gene detected 55% and 45% of 3D7 and FC27 allelic families respectively. The overall multiplicity of infections (MOI) was 1.3. A positive correlation between parasite density and multiplicity of infection was found. The prevalence of the mutant pfcrt allele (T76) in the isolates was 92%. CONCLUSION: This is the first molecular characterization of P. falciparum field isolates in Congolese children, four years after changing the malaria treatment policy from chloroquine (CQ) to artemisinin-based combination therapy (ACT). The low prevalence of asymptomatic infections and MOI is discussed in the light of similar studies conducted in Central Africa.