Elsevier

Acta Tropica

Volume 113, Issue 2, February 2010, Pages 190-194
Acta Tropica

Monitoring of in vitro susceptibilities and molecular markers of resistance of Plasmodium falciparum isolates from Thai-Myanmar border to chloroquine, quinine, mefloquine and artesunate

https://doi.org/10.1016/j.actatropica.2009.10.016Get rights and content

Abstract

Malaria is one of the major causes of morbidity and mortality worldwide. The major factor which has aggravated the situation is the emergence of multidrug resistant Plasmodium falciparum malaria. To successfully deal with the problem, thorough understanding of the molecular bases for reduced parasite sensitivity to existing antimalarial drugs is of considerable importance. The objective of this work was to broaden the insight into the molecular mechanisms of resistance of P. falciparum to quinoline-containing antimalarials and artemisinin derivatives. Polymorphisms of the candidate genes pfmdr1 and pfcrt were investigated in relation to the susceptibility (in vitro sensitivity) of P. falciparum isolates to chloroquine (CQ), mefloquine (MQ), quinine (QN) and the artemisinin derivative – artesunate (AS). A total of 26 P. falciparum isolates were successful cultured. In vitro sensitivity results indicate the increase in susceptibility of P. falciparum strains in Thailand to CQ, while the susceptibility to MQ and QN was markedly declined. The pattern of cross-resistance was observed between MQ vs QN vs AS. Only one point mutation in the pfmdr1 gene, i.e., N86Y was observed with low prevalence of 7.7% (2/26). In contrast, the mutations at positions 76T, 220S, 271E, 326S, 356T and 371I in the pfcrt gene were identified in almost all isolates (25 isolates, 96.2%). The association between polymorphisms of the pfmdr1 and susceptibility of the parasite to MQ and QN was observed (increased susceptibilities to MQ and QN in isolates with mutations). Moreover, the correlation between pfmdr1 gene amplification and susceptibility of the parasite to MQ, QN and AS was observed (decreased susceptibilities to MQ, QN and AS in isolates with increased pfmdr1 copy number).

Introduction

Multidrug resistant Plasmodium falciparum including resistance to structurally related antimalarial aminoquinolines such as chloroquine, quinine and mefloquine, is still problematic along the border areas of Thailand, especially Thai-Myanmar border (Wongsrichanalai et al., 2002). The policy of antimalarial treatment for uncomplicated falciparum malaria in Thailand has been revised several times during the past 30 years in order to counter the rapid emergence and spread of drug resistance. The artemisinin combination therapy (ACT), i.e., a 3-day artesunate–mefloquine combination is currently being used as the first-line treatment of uncomplicated falciparum malaria throughout the country (Na-Bangchang and Congpuong, 2007). Recently however, there has been a report of modest increase in resistance of this combination in areas along the Thai-Cambodian and Thai-Myanmar borders (Carrara et al., 2009, Lim et al., 2009, Noedl et al., 2008, Rogers et al., 2009, Vijaykadga et al., 2006). The extensive deployment of antimalarial drugs in the past years, has provided a remarkable selection pressure on malaria parasites to evolve resistance mechanisms. Due to this alarming emergence of antimalarial resistance, surveillance is a key element to a successful malaria control program. Although in vivo test is a gold standard for assessing antimalarial efficacy and monitoring of drug resistance, it is resource intensive, requiring controlled clinical trials with bona fide clinical outcomes and monitoring of therapeutic drug level (Guerin et al., 2009). In vitro sensitivity test together with molecular surveillance system can help target in vivo studies to where they are needed the most. Molecular surveillance is high-throughput and can be performed in a central laboratory on dried blood spots, which are easily collected in the field. Expanded molecular surveillance could also accurately help mapping antimalarial resistance and enable sub-national treatment policies in countries with marked geographic variation in drug susceptibility.

Several possible factors have contributed to antimalarial drug resistance. Among these factors, polymorphisms of genes involved in vial process of malaria parasite are proposed as the key factor. The parasitic genes involed in transportation or efflux of antimalarial drugs are attractive candidates as molecular markers of antimalarial drug resistance. The most recognized genes are pfcrt which encodes the transmembrane protein PfCRT and pfmdr1 which encodes a P-glycoprotien homologue 1 (Pgh1). The associations between gene mutation and/or mutation and/or amplification and resistance of P. falciparum to antimalarials however have been controversial and debatable during the past decades, depending on geographical areas reported (Wilson et al., 1993, Price et al., 2004). In recent studies, increasing copy number of pfmdr1 gene has been proposed to be a predictor of ACT treatment failure (Alker et al., 2007, Nelson et al., 2005, Price et al., 2004). In consistence with the in vitro results, increasing copy number of pfmdr1 results in increasing resistance to mefloquine, lumefantrine and artesunate (Lim et al., 2009).

In the present study, we investigated the association between in vitro susceptibility and the polymorphisms of pfcrt, pfmdr1 including amplification of pfmdr1 gene in P. falciparum isolates collected from an area along the Thai-Myanmar border during 2007–2008. In addition, cross-resistance patterns among the tested drugs (CQ, QN, MQ and AS) were also investigated.

Section snippets

Culture system for parasite maintenance

A total of 50 P. falciparum field isolates were collected from malaria endemic area of Thailand along the Thai-Myanmar border. All isolates were preserved with freezing solution (7.56 g sorbital, 180 ml of 0.9% NaCl and 70 ml of 99% glycerol) and stored in a liquid nitrogen tank until transportation to Pharmacology and Toxicology Unit, Faculty of Allied Health Sciences, Thammasat University. All were adapted to continuous culture according to the methods of Trager and Jensen (1976) with

In vitro drug susceptibilities of P. falciparum isolates to antimalarial drugs

A total of 26 isolates were successfully adapted and investigated for their susceptibilities to CQ, QN, MQ and AS. The criteria for discrimination between the resistant and sensitive response of the parasite isolates to CQ, QN, and MQ followed the criteria described by Ceruttij et al. (1999) and Pickard et al. (2003). CQ susceptibility was categorized into three levels, i.e., sensitive (S: IC50 < 25 nM), moderately resistant (MR: 25  IC50 < 100 nM), and highly resistant (HR: IC50  100 nM). QN

Discussion

P. falciparum drug resistance has been rapidly developed along Thailand borders since the past decade. The present study investigated susceptibility profiles of P. falciparum isolates collected during 2007–2008 from the Thai-Myanmar border to CQ, MQ, QN and AS (Fig. 2) (Na-Bangchang and Congpuong, 2007). It is remarkable that 3 isolates were resistant to QN. Furthermore, results from this study confirm cross-resistance patterns among MQ, QN and AS. The strong correlation between the activities

Acknowledgements

The study was supported by Thammasat University and Commission for Higher Education, Ministry of Education, Thailand. WC, RW and KN are supported by Commission for Higher Education, Ministry of Education. We are grateful to Malaria Research Unit, Chulalongkorn University, Thailand for parasites isolates.

References (42)

  • C. Wongsrichanalai et al.

    Epidemiology of drug-resistant malaria

    Lancet Infect. Dis.

    (2002)
  • J. Wooden et al.

    PCR and strain identification in Plasmodium falciparum

    Parasitol. Today

    (1993)
  • A.P. Alker et al.

    Pfmdr1 and in vivo resistance to artesunate–mefloquine in falciparum malaria on the Cambodian-Thai border

    Am. J. Trop. Med. Hyg.

    (2007)
  • T.N. Bennett et al.

    Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy

    Antimicrob. Agents Chemother.

    (2004)
  • V.I. Carrara et al.

    Changes in the treatment responses to artesunate–mefloquine on the northwestern border of Thailand during 13 years of continuous deployment

    PLoS One

    (2009)
  • Ceruttij et al.

    Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method

    Mem. Inst. Oswaldo. Cruz.

    (1999)
  • W. Chaijaroenkul et al.

    In vitro antimalarial drug susceptibility in Thai border areas from 1998–2003

    Malar. J.

    (2005)
  • S.C. Chaiyaroj et al.

    Analysis of mefloquine resistance and amplification of pfmdr1 in multidrug-resistant Plasmodium falciparum isolates from Thailand

    Am. J. Trop. Med. Hyg.

    (1999)
  • N. Chen et al.

    Pfcrt Allelic types with two novel amino acid mutations in chloroquine-resistant Plasmodium falciparum isolates from the Philippines

    Antimicrob. Agents Chemother.

    (2003)
  • N. Chen et al.

    Sequence polymorphisms in pfcrt are strongly associated with chloroquine resistance in Plasmodium falciparum

    J. Infect. Dis.

    (2001)
  • K. Congpuong et al.

    Molecular epidemiology of drug resistance markers of Plasmodium falciparum malaria in Thailand

    Trop. Med. Int. Health

    (2005)
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