Molecular surveillance for drug-resistant Plasmodium falciparum in clinical and subclinical populations from three border regions of Burma/Myanmar: cross-sectional data and a systematic review of resistance studies

Abstract

Background: Confirmation of artemisinin-delayed parasite clearance in Plasmodium falciparum along the Thai-Myanmar border has inspired a global response to contain and monitor drug resistance to avert the disastrous consequences of a potential spread to Africa. However, resistance data from Myanmar are sparse, particularly from high-risk areas where limited health services and decades of displacement create conditions for resistance to spread. Subclinical infections may represent an important reservoir for resistance genes that confer a fitness disadvantage relative to wild-type alleles. This study estimates the prevalence of resistance genotypes in three previously unstudied remote populations in Myanmar and tests the a priori hypothesis that resistance gene prevalence would be higher among isolates collected from subclinical infections than isolates collected from febrile clinical patients. A systematic review of resistance studies is provided for context.

Methods: Community health workers in Karen and Kachin States and an area spanning the Indo-Myanmar border collected dried blood spots from 988 febrile clinical patients and 4,591 villagers with subclinical infection participating in routine prevalence surveys. Samples positive for P. falciparum 18 s ribosomal RNA by real-time PCR were genotyped for P. falciparum multidrug resistance protein (pfmdr1) copy number and the pfcrt K76T polymorphism using multiplex real-time PCR.

Results: Pfmdr1 copy number increase and the pfcrt K76 polymorphism were determined for 173 and 269 isolates, respectively. Mean pfmdr1 copy number was 1.2 (range: 0.7 to 3.7). Pfmdr1 copy number increase was present in 17.5%, 9.6% and 11.1% of isolates from Karen and Kachin States and the Indo-Myanmar border, respectively. Pfmdr1 amplification was more prevalent in subclinical isolates (20.3%) than clinical isolates (6.4%, odds ratio 3.7, 95% confidence interval 1.1 - 12.5). Pfcrt K76T prevalence ranged from 90-100%.

Conclusions: Community health workers can contribute to molecular surveillance of drug resistance in remote areas of Myanmar. Marginal and displaced populations under-represented among previous resistance investigations can and should be included in resistance surveillance efforts, particularly once genetic markers of artemisinin-delayed parasite clearance are identified. Subclinical infections may contribute to the epidemiology of drug resistance, but determination of gene amplification from desiccated filter samples requires further validation when DNA concentration is low.

Figure 1

Anti-malarial, drug-resistance studies in Myanmar and neighbouring countries, by region and year. Blue squares represent the locations of in vivo, in vitro and molecular studies conducted between 1996 and 2009; locations of the current study (2008–2009) appear as red circles (n = 13 sites). Studies reporting prevalence of pfmdr1 copy number amplification or pfcrt K76T haplotypes are numbered on the map and on the x-axis of the bar figures, in order of data collection year. Previous molecular studies from Myanmar appear in insets; molecular studies from neighbouring countries appear on the country-wide map. Locations of villages forcibly displaced between 2008–2011 are indicated by red shading. See manuscript text for additional information on displaced villages, and Additional file 1 for a complete list of molecular studies and abstracted prevalence estimates.

Figure 2

Scatterplots and boxplots of pfmdr1 copy number estimate precision and Plasmodium falciparum beta-tubulin PCR cycle thresholds (Ct) for febrile clinical patients (grey circles and boxplots) and active screening participants (empty circles and boxplots) A: The scatterplots demonstrate precision of pfmdr1 copy number (CN) estimate (y-axis) diminishes as DNA concentration (x-axis) decreases among active screening participants (bottom panel) and febrile clinical patients (top panel). The mean cycle threshold and mean precision of pfmdr1 CN is indicated by the black point on each scatterplot. A three-cycle increase in the Ct corresponds to a 10-fold decrease in relative concentration of Pf DNA. Pfmdr1 precision is calculated as the range/mean for each isolate, with higher values indicating lower precision. The standard deviation of pfmdr1 CN estimates was 0.200 for clinic patients and .516 for screening participants (not shown) B: Boxplots display the distribution of DNA concentration (top panel) and pfmdr1 CN precision. Boxes represent inter-quartile ranges (IQR); whiskers represent the value of [upper/lower quartile +/− (IQR*1.5)]. Screening participants had lower DNA concentration and less precise estimation of pfmdr1 CN.

Figure 3

Systematic review of in vivo, in vitro, and molecular resistance studies in Myanmar and neighbouring countries, 1996–2009[[1,7,9,10,13,39,40,60-82]].