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Background Increased resistance by Plasmodium falciparum parasites led to the withdrawal of the antimalarial drugs chloroquine and sulphadoxine-pyrimethamine in Ethiopia. Since 2004 artemether-lumefantrine has served to treat uncomplicated P. falciparum malaria. However, increasing reports on delayed parasite clearance to artemisinin opens up a new challenge in anti-malarial therapy. With the complete withdrawal of CQ for the treatment of Plasmodium falciparum malaria, this study assessed the evolution of CQ resistance by investigating the prevalence of mutant alleles in the pfmdr1 and pfcrt genes in P. falciparum and pvmdr1 gene in Plasmodium vivax in Southern and Eastern Ethiopia. Methods Of the 1,416 febrile patients attending primary health facilities in Southern Ethiopia, 329 febrile patients positive for P. falciparum or P. vivax were recruited. Similarly of the 1,304 febrile patients from Eastern Ethiopia, 81 febrile patients positive for P. falciparum or P. vivax were included in the study. Of the 410 finger prick blood samples collected from malaria patients, we used direct sequencing to investigate the prevalence of mutations in pfcrt and pfmdr1. This included determining the gene copy number in pfmdr1 in 195 P. falciparum clinical isolates, and mutations in the pvmdr1 locus in 215 P. vivax clinical isolates. Results The pfcrt K76 CQ-sensitive allele was observed in 84% of the investigated P.falciparum clinical isolates. The pfcrt double mutations (K76T and C72S) were observed less than 3%. The pfcrt SVMNT haplotype was also found to be present in clinical isolates from Ethiopia. The pfcrt CVMNK-sensitive haplotypes were frequently observed (96%). The pfmdr1 mutation N86Y was observed only in 14.9% compared to 85.1% of the clinical isolates that carried sensitive alleles. Also, the sensitive pfmdr1 Y184 allele was more common, in 96% of clinical isolates. None of the investigated P. falciparum clinical isolates carried S1034C, N1042D and D1246Y pfmdr1 polymorphisms. All investigated P. falciparum clinical isolates from Southern and Eastern Ethiopia carried only a single copy of the mutant pfmdr1 gene. Conclusion The study reports for the first time the return of chloroquine sensitive P. falciparum in Ethiopia. These findings support the rationale for the use of CQ-based combination drugs as a possible future alternative. [ABSTRACT FROM AUTHOR]

Background With 75% of the Ethiopian population at risk of malaria, accurate diagnosis is crucial for malaria treatment in endemic areas where Plasmodium falciparum and Plasmodium vivax coexist. The present study evaluated the performance of regular microscopy in accurate identification of Plasmodium spp. in febrile patients visiting health facilities in southern Ethiopia. Methods A cross-sectional study design was employed to recruit study subjects who were microscopically positive for malaria parasites and attending health facilities in southern Ethiopia between August and December 2011. Of the 1,416 febrile patients attending primary health facilities, 314 febrile patients, whose slides were positive for P. falciparum, P. vivax or mixed infections using microscopy, were re-evaluated for their infection status by PCR. Finger-prick blood samples were used for parasite genomic DNA extraction. Phylogenetic analyses were performed to reconstruct the distribution of different Plasmodium spp. across the three geographical areas. Results Of the 314 patients with a positive thick blood smear, seven patients (2%) were negative for any of the Plasmodium spp. by nested PCR. Among 180 microscopically diagnosed P. falciparum cases, 111 (61.7%) were confirmed by PCR, 44 (24.4%) were confirmed as P. vivax, 18 (10%) had mixed infections with P. falciparum and P. vivax and two (1.1%) were mixed infections with P. falciparum and P. malariae and five (2.8%) were negative for any of the Plasmodium spp. Of 131 microscopically diagnosed P. vivax cases, 110 (84%) were confirmed as P. vivax, 14 (10.7%) were confirmed as P. falciparum, two (1.5%) were P. malariae, three (2.3%) with mixed infections with P. falciparum and P. vivax and two (1.5%) were negative for any of the Plasmodium spp. Plasmodium falciparum and P. vivax mixed infections were observed. Plasmodium malariae was detected as mono and mixed infections in four individuals. Conclusion False positivity, under-reporting of mixed infections and a significant number of species mismatch needs attention and should be improved for appropriate diagnosis. The detection of substantial number of false positive results by molecular methodologies may provide the accurate incidence of circulating Plasmodium species in the geographical region and has important repercussions in understanding malaria epidemiology and subsequent control. [ABSTRACT FROM AUTHOR]

Background: Increased resistance by Plasmodium falciparum parasites led to the withdrawal of the antimalarial drugs chloroquine and sulphadoxine-pyrimethamine in Ethiopia. Since 2004 artemether-lumefantrine has served to treat uncomplicated P. falciparum malaria. However, increasing reports on delayed parasite clearance to artemisinin opens up a new challenge in anti-malarial therapy. With the complete withdrawal of CQ for the treatment of Plasmodium falciparum malaria, this study assessed the evolution of CQ resistance by investigating the prevalence of mutant alleles in the pfmdr1 and pfcrt genes in P. falciparum and pvmdr1 gene in Plasmodium vivax in Southern and Eastern Ethiopia., Methods: Of the 1,416 febrile patients attending primary health facilities in Southern Ethiopia, 329 febrile patients positive for P. falciparum or P. vivax were recruited. Similarly of the 1,304 febrile patients from Eastern Ethiopia, 81 febrile patients positive for P. falciparum or P. vivax were included in the study. Of the 410 finger prick blood samples collected from malaria patients, we used direct sequencing to investigate the prevalence of mutations in pfcrt and pfmdr1. This included determining the gene copy number in pfmdr1 in 195 P. falciparum clinical isolates, and mutations in the pvmdr1 locus in 215 P. vivax clinical isolates., Results: The pfcrt K76 CQ-sensitive allele was observed in 84.1% of the investigated P.falciparum clinical isolates. The pfcrt double mutations (K76T and C72S) were observed less than 3%. The pfcrt SVMNT haplotype was also found to be present in clinical isolates from Ethiopia. The pfcrt CVMNK-sensitive haplotypes were frequently observed (95.9%). The pfmdr1 mutation N86Y was observed only in 14.9% compared to 85.1% of the clinical isolates that carried sensitive alleles. Also, the sensitive pfmdr1 Y184 allele was more common, in 94.9% of clinical isolates. None of the investigated P. falciparum clinical isolates carried S1034C, N1042D and D1246Y pfmdr1 polymorphisms. All investigated P. falciparum clinical isolates from Southern and Eastern Ethiopia carried only a single copy of the mutant pfmdr1 gene., Conclusion: The study reports for the first time the return of chloroquine sensitive P. falciparum in Ethiopia. These findings support the rationale for the use of CQ-based combination drugs as a possible future alternative.

Background: With 75% of the Ethiopian population at risk of malaria, accurate diagnosis is crucial for malaria treatment in endemic areas where Plasmodium falciparum and Plasmodium vivax co-exist. The present study evaluated the performance of regular microscopy in accurate identification of Plasmodium spp. in febrile patients visiting health facilities in southern Ethiopia., Methods: A cross-sectional study design was employed to recruit study subjects who were microscopically positive for malaria parasites and attending health facilities in southern Ethiopia between August and December 2011. Of the 1,416 febrile patients attending primary health facilities, 314 febrile patients, whose slides were positive for P. falciparum, P. vivax or mixed infections using microscopy, were re-evaluated for their infection status by PCR. Finger-prick blood samples were used for parasite genomic DNA extraction. Phylogenetic analyses were performed to reconstruct the distribution of different Plasmodium spp. across the three geographical areas., Results: Of the 314 patients with a positive thick blood smear, seven patients (2%) were negative for any of the Plasmodium spp. by nested PCR. Among 180 microscopically diagnosed P. falciparum cases, 111 (61.7%) were confirmed by PCR, 44 (24.4%) were confirmed as P. vivax, 18 (10%) had mixed infections with P. falciparum and P. vivax and two (1.1%) were mixed infections with P. falciparum and P. malariae and five (2.8%) were negative for any of the Plasmodium spp. Of 131 microscopically diagnosed P. vivax cases, 110 (84%) were confirmed as P. vivax, 14 (10.7%) were confirmed as P. falciparum, two (1.5%) were P. malariae, three (2.3%) with mixed infections with P. falciparum and P. vivax and two (1.5%) were negative for any of the Plasmodium spp. Plasmodium falciparum and P. vivax mixed infections were observed. Plasmodium malariae was detected as mono and mixed infections in four individuals., Conclusion: False positivity, under-reporting of mixed infections and a significant number of species mismatch needs attention and should be improved for appropriate diagnosis. The detection of substantial number of false positive results by molecular methodologies may provide the accurate incidence of circulating Plasmodium species in the geographical region and has important repercussions in understanding malaria epidemiology and subsequent control.