title: An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples.
abstract: BACKGROUND
  Highly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions. While traditional microscopy and rapid diagnostic tests (RDTs) are suitable for the diagnosis of symptomatic malaria infection, more sensitive tests are needed to screen for low-density, asymptomatic infections that are targeted by interventions aiming to eliminate the entire reservoir of malaria infection in humans.
  
  
  METHODS
  A reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA of Plasmodium falciparum and Plasmodium vivax. Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR.
  
  
  RESULTS
  Limits of detection (LoD) were determined under simulated field conditions. When 0.3 mL blood samples were stored for 14 days at 28 °C and 80% humidity, the LoD was less than 16 parasites/mL for P. falciparum and 19.7 copies/µL for P. vivax (using a plasmid surrogate), about 10,000-fold lower than RDTs. Of the 1739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive for P. falciparum, 108 were positive for P. vivax, and 127 were positive for either P. vivax and/or P. falciparum using ultrasensitive RT-PCR.
  
  
  CONCLUSIONS
  This ultrasensitive RT-PCR method is a robust, field-tested screening method that is vastly more sensitive than RDTs. Further optimization may result in a truly scalable tool suitable for widespread surveillance of low-level asymptomatic P. falciparum and P. vivax parasitaemia.
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. madams@medicine.umaryland.edu.
authors.forename: Matthew
authors.initials: M
authors.lastname: Adams
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. sjoshi@medicine.umaryland.edu.
authors.forename: Sudhaunshu N
authors.initials: SN
authors.lastname: Joshi
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. gmbambo@medicine.umaryland.edu.
authors.forename: Gillian
authors.initials: G
authors.lastname: Mbambo
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. awang@medicine.umaryland.edu.
authors.forename: Amy Z
authors.initials: AZ
authors.lastname: Mu
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. shampton@medicine.umaryland.edu.
authors.forename: Shay M
authors.initials: SM
authors.lastname: Roemmich
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. bshrestha@medicine.umaryland.edu.
authors.forename: Biraj
authors.initials: B
authors.lastname: Shrestha
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. kstrauss@medicine.umaryland.edu.
authors.forename: Kathy A
authors.initials: KA
authors.lastname: Strauss
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. neddingt@medicine.umaryland.edu.
authors.forename: Nicole Eddington
authors.initials: NE
authors.lastname: Johnson
authors.affiliation: Defence Services Medical Research Centre, Ministry of Defence, Nay Pyi Taw, Myanmar. drkhine@gmail.com.
authors.forename: Khine Zaw
authors.initials: KZ
authors.lastname: Oo
authors.affiliation: Defence Services Medical Research Centre, Ministry of Defence, Nay Pyi Taw, Myanmar. hlaingtm@gmail.com.
authors.forename: Tin Maung
authors.initials: TM
authors.lastname: Hlaing
authors.affiliation: Department of Medical Research, Ministry of Health, Yangon, Myanmar. drzayarhan@gmail.com.
authors.forename: Zay Yar
authors.initials: ZY
authors.lastname: Han
authors.affiliation: Department of Medical Research, Ministry of Health, Yangon, Myanmar. drkaythwehan@yahoo.com.
authors.forename: Kay Thwe
authors.initials: KT
authors.lastname: Han
authors.affiliation: Community Partners International, Yangon, Myanmar. sithura@cpintl.org.
authors.forename: Si
authors.initials: S
authors.lastname: Thura
authors.affiliation: UCLA Division of General Internal Medicine and Health Services Research, Los Angeles, USA. adamrichards@mednet.ucla.edu.
authors.forename: Adam K
authors.initials: AK
authors.lastname: Richards
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. fhuang@medicine.umaryland.edu.|National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China. fhuang@medicine.umaryland.edu.
authors.forename: Fang
authors.initials: F
authors.lastname: Huang
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. mnyunt@medicine.umaryland.edu.
authors.forename: Myaing M
authors.initials: MM
authors.lastname: Nyunt
authors.affiliation: Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA. cplowe@medicine.umaryland.edu.
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Plowe
mesh_terms: D058345:Asymptomatic Infections; D001769:Blood / Q000469:parasitology*; D016054:DNA, Protozoan / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D006801:Humans; D016778:Malaria, Falciparum / Q000175:diagnosis*; D016780:Malaria, Vivax / Q000175:diagnosis*; D002052:Myanmar; D010963:Plasmodium falciparum / Q000235:genetics / Q000302:isolation & purification*; D010966:Plasmodium vivax / Q000235:genetics / Q000302:isolation & purification*; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S