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