An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples

Malaria Journal

Methodology

Si Thura¹  Khine Zaw Oo²  Tin Maung Hlaing²  Kay Thwe Han³  Zay Yar Han³  Gillian Mbambo⁴  Kathy A. Strauss⁴  Amy Z. Mu⁴  Nicole Eddington Johnson⁴  Myaing M. Nyunt⁴  Sudhaunshu N. Joshi⁴  Christopher V. Plowe⁴  Shay M. Roemmich⁴  Biraj Shrestha⁴  Matthew Adams⁴  Fang Huang⁵  Adam K. Richards⁶ 

[1] Community Partners International, Yangon, Myanmar;Defence Services Medical Research Centre, Ministry of Defence, Nay Pyi Taw, Myanmar;Department of Medical Research, Ministry of Health, Yangon, Myanmar;Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA;Howard Hughes Medical Institute/Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA;National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People’s Republic of China;UCLA Division of General Internal Medicine and Health Services Research, Los Angeles, USA;

关键词: Malaria;    Malaria elimination;    Plasmodium falciparum;    Plasmodium vivax;    RT-PCR;    Limits of detection;    Nucleic acid;   

DOI  :  10.1186/s12936-015-1038-z

 received in 2015-08-08, accepted in 2015-12-08,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundHighly 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.MethodsA 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.ResultsLimits 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.ConclusionsThis 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.

【 授权许可】

CC BY   
© Adams et al. 2015