title: 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.
authors.affiliation: Johns Hopkins University School of Medicine, Broadway Research Building,733 N. Broadway, Suite 147, Baltimore, MD 21205, USA.
authors.forename: Tyler
authors.initials: T
authors.lastname: Brown
authors.forename: Linda S
authors.initials: LS
authors.lastname: Smith
authors.forename: Eh Kalu Shwe
authors.initials: EK
authors.lastname: Oo
authors.forename: Kum
authors.initials: K
authors.lastname: Shawng
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Lee
authors.forename: David
authors.initials: D
authors.lastname: Sullivan
authors.forename: Chris
authors.initials: C
authors.lastname: Beyrer
authors.forename: Adam K
authors.initials: AK
authors.lastname: Richards
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000962:Antimalarials / Q000494:pharmacology*; D058070:Asymptomatic Diseases; D002648:Child; D002675:Child, Preschool; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D016778:Malaria, Falciparum / Q000469:parasitology*; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics; D008875:Middle Aged; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics; D002052:Myanmar / Q000453:epidemiology; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics; D055815:Young Adult
chemical_list: D000962:Antimalarials; D016054:DNA, Protozoan; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; C513498:Mdr1 protein, Plasmodium falciparum; C416369:PfCRT protein, Plasmodium falciparum