Prevalence of Chloroquine Resistance Alleles in Plasmodium Falciparum After Two Decades of Withdrawal of Chloroquine Usagefor Treatment of Falciparum Malariain Ethiopia
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Date
2017-09-02
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Addis Ababa University
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2017
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ABSTRACT
In response to the emergence of P. falciparum (Pf) drug resistance, Ethiopia had changed anti-malaria treatment guidelines, from chloroquine (CQ) to sulfadoxin-pyrimethamine (SP) combination in 1998as first line drugs for the treatment of Pf malaria. The current status of chloroquine resistance (CQR) alleles in Pf is not known. The available few previous studies focused on clinical isolates. This study was conducted to investigate the prevalence of CQR alleles in Pf in asymptomatic and symptomatic cases. A total of 140 dried blood spot samples (DBS) from microscopy positive clinical patients (n=41) in Adama malaria center and asymptomatic individuals from Gambella (n=72) and Benishangul-Gumuz (n=27) with rapid diagnostic tests (RDT) confirmed Pf mono-infections were collected. The genomic DNA was extracted using saponin-chelex extraction method. It was then screened for the confirmation of Pf with18S nested PCR. Only 80 PCR confirmed Pf mono-infection samples were amplified using nested PCR for the three target genes which are incriminated in determining CQ resistance. These included P. falciparum chloroquine resistance transporter gene codon 76 (Pfcrt 76), P. falciparum multidrug resistance gene-1 codon 86 and 1034 (Pfmdr-1 86 and Pfmdr-1 1034). Single nucleotide polymorphisms (SNPs) of the respective targets were analyzed in Polymerase chain reaction- Restriction fragment length polymorphisms (PCR-RFLP) analysis. The overall prevalence of combined mutations of CQR alleles (Pfcrt K76T and Pfmdr-1 N86Y) was detected in 61.5% (48/78) of successfully amplified and analyzed samples. The Pfcrt K76T (CQR) mutation was detected in 61.3 % (46/75), and the Pfmdr-1 N86Y mutation was found in 2.7 % (2/75). The Pfcrt K76T mutation was found fixed in the majority of 90.9 % (30/33) of the clinical isolates from Adama and its surroundings. A relatively lower Pfcrt K76T allele was found in asymptomatic samples from Gambella and Benishangul-Gumuz with 40.7 % (11/27) and 33.3 % (5/15) respectively. The Pfmdr-1 N86Y mutation was detected in only 7.1% (2/28) of samples from Gambella and has exhibited higher reversal rate in all study sites, with 97.0 % (32/33) in clinical samples from Adama and its surroundings. The reversal rate in the asymptomatic samples was 89.3 % (25/28) from Gambella and 71.4% (10/14) from Benishangul-Gumuz. The Pfmdr-1 S1034C mutation was not detected in all analyzed samples (0/65). The major CQR determinant, Pfcrt K76T and the complementaryPfmdr-1 N86Y mutations showed statistically significant association (X2(4) =19.4; P=0.001. Although the overall CQR mutation in the analyzed targets showed a higher reversal rate; the major CQR determinant Pfcrt K76T mutation remained fixed among the parasite population in and around Adama. This most probably is because CQ is still provided for the treatment of P. vivax which sympartically occurs with Pf, resulting in co-infections and could be misdiagnosed as P. vivax and hence treated with CQ. As a result, the Pf parasite population will face the CQ pressure favoring the CQR mutations. Therefore, if CQ resistance of Pf has to be reversed for a more effective and affordable
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treatment, CQ must be withdrawn from all co-endemic localities. On the other hand, the higher reversal rate of CQR mutations documented in the present study might offer the opportunity to re-introduce CQ in Gambella and Benishangul-Gumuz regions. However; there is a need for continual assessment on the status of CQR alleles in Pf.
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Chloroquine, Drug Resistance, Chloroquine Resistance Reversal, P. Falciparum Chloroquine Resistance Transporter, P. Falciparum Multidrug Resistance Gene-1, P. Falciparum, Restriction Digestion, Single Nucleotide Polymorphism