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Correlation of molecular markers, Pfmdr1-N86Y and Pfcrt-K76T, with in vitro chloroquine resistant Plasmodium falciparum, isolated in the malaria endemic states of Assam and Arunachal Pradesh, Northeast India.

Shrivastava SK, Gupta RK, Mahanta J, Dubey ML - PLoS ONE (2014)

Bottom Line: However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1.The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC50 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC50 0.32 µM/L blood).The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Post Graduate Institute of Medical Education and Research, Chandigarh, India; Basic and Clinical Immunology of Parasitic Diseases, Centre for Infection and Immunity of Lille, Institut Pasteur de Lille, France.

ABSTRACT
The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum is not clearly understood. However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1. These genes encode for digestive vacuole transmembrane proteins Pfcrt and Pgh1, respectively. The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC50 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC50 0.32 µM/L blood). All the CQ resistant isolates showed the presence of Pfmdr1 and Pfcrt mutations. CQ sensitive isolates were negative for these mutations. Strong linkage disequilibrium was observed between the alleles at these two loci (Pfmdr1-N86Y and Pfcrt-K76T). The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum. The result necessitates the evaluation of CQ in vivo therapeutic efficacy in endemic areas for more effective malaria control strategies.

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Representative photomicrograph showing the results of RFLP after digestion of PCR amplified product with endonucleases, Apo1 and AfI III for Pfmdr1 gene (86Y) in chloroquine resistant Plasmodium falciparum isolates.In photograph, (M) is 100 bp DNA ladder; (C) is undigested product as control; (P) is Apo1; (F) is Afl III and (R1- R4) are chloroquine resistant P. falciparum isolates.
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pone-0103848-g001: Representative photomicrograph showing the results of RFLP after digestion of PCR amplified product with endonucleases, Apo1 and AfI III for Pfmdr1 gene (86Y) in chloroquine resistant Plasmodium falciparum isolates.In photograph, (M) is 100 bp DNA ladder; (C) is undigested product as control; (P) is Apo1; (F) is Afl III and (R1- R4) are chloroquine resistant P. falciparum isolates.

Mentions: To ascertain the genetic polymorphism leading to the CQ resistance, nested PCR for Pfmdr1 and Pfcrt genes was performed. To confirm the status of the amplicon, the PCR product was digested with Apo1 and Afl III. On nested PCR for Pfmdr1, all the isolates showed the Pfmdr1-codon 86 region amplicon with the product size of 501 bp. On digestion with the ApoI in the case of CQ sensitive isolates, ApoI digested the amplicon at codon 86, and generated three fragments of 250 bp, 226 bp and 25 bp, respectively. Afl III was unable to digest the amplicon of the sensitive isolates (Table 1; Figure 1). However, Afl III generated two fragments of 279 bp and 222 bp, respectively from the amplicon of all the CQ resistant isolates indicating a mutant allele at codon 86 (86T). ApoI did not digest the amplicon of the CQ resistant strains at this site, and generated fragments of 476 bp and 25 bp (Table 1; Figure 2). The nested PCR of Pfcrt gene showed an amplicon of 145 bp. In the case of CQ sensitive isolates, the ApoI digestion of amplicon resulted in two fragments of 111 bp and 34 bp respectively, suggesting the presence of K76 allele at codon 76. On the other hand, mutant allele 76T was observed in all the CQ resistant isolates with an undigested fragment of 145 bp (Table 1; Figure 3).


Correlation of molecular markers, Pfmdr1-N86Y and Pfcrt-K76T, with in vitro chloroquine resistant Plasmodium falciparum, isolated in the malaria endemic states of Assam and Arunachal Pradesh, Northeast India.

Shrivastava SK, Gupta RK, Mahanta J, Dubey ML - PLoS ONE (2014)

Representative photomicrograph showing the results of RFLP after digestion of PCR amplified product with endonucleases, Apo1 and AfI III for Pfmdr1 gene (86Y) in chloroquine resistant Plasmodium falciparum isolates.In photograph, (M) is 100 bp DNA ladder; (C) is undigested product as control; (P) is Apo1; (F) is Afl III and (R1- R4) are chloroquine resistant P. falciparum isolates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126653&req=5

pone-0103848-g001: Representative photomicrograph showing the results of RFLP after digestion of PCR amplified product with endonucleases, Apo1 and AfI III for Pfmdr1 gene (86Y) in chloroquine resistant Plasmodium falciparum isolates.In photograph, (M) is 100 bp DNA ladder; (C) is undigested product as control; (P) is Apo1; (F) is Afl III and (R1- R4) are chloroquine resistant P. falciparum isolates.
Mentions: To ascertain the genetic polymorphism leading to the CQ resistance, nested PCR for Pfmdr1 and Pfcrt genes was performed. To confirm the status of the amplicon, the PCR product was digested with Apo1 and Afl III. On nested PCR for Pfmdr1, all the isolates showed the Pfmdr1-codon 86 region amplicon with the product size of 501 bp. On digestion with the ApoI in the case of CQ sensitive isolates, ApoI digested the amplicon at codon 86, and generated three fragments of 250 bp, 226 bp and 25 bp, respectively. Afl III was unable to digest the amplicon of the sensitive isolates (Table 1; Figure 1). However, Afl III generated two fragments of 279 bp and 222 bp, respectively from the amplicon of all the CQ resistant isolates indicating a mutant allele at codon 86 (86T). ApoI did not digest the amplicon of the CQ resistant strains at this site, and generated fragments of 476 bp and 25 bp (Table 1; Figure 2). The nested PCR of Pfcrt gene showed an amplicon of 145 bp. In the case of CQ sensitive isolates, the ApoI digestion of amplicon resulted in two fragments of 111 bp and 34 bp respectively, suggesting the presence of K76 allele at codon 76. On the other hand, mutant allele 76T was observed in all the CQ resistant isolates with an undigested fragment of 145 bp (Table 1; Figure 3).

Bottom Line: However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1.The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC50 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC50 0.32 µM/L blood).The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Post Graduate Institute of Medical Education and Research, Chandigarh, India; Basic and Clinical Immunology of Parasitic Diseases, Centre for Infection and Immunity of Lille, Institut Pasteur de Lille, France.

ABSTRACT
The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum is not clearly understood. However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1. These genes encode for digestive vacuole transmembrane proteins Pfcrt and Pgh1, respectively. The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC50 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC50 0.32 µM/L blood). All the CQ resistant isolates showed the presence of Pfmdr1 and Pfcrt mutations. CQ sensitive isolates were negative for these mutations. Strong linkage disequilibrium was observed between the alleles at these two loci (Pfmdr1-N86Y and Pfcrt-K76T). The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum. The result necessitates the evaluation of CQ in vivo therapeutic efficacy in endemic areas for more effective malaria control strategies.

Show MeSH
Related in: MedlinePlus