A novel method for extracting nucleic acids from dried blood spots for ultrasensitive detection of low-density Plasmodium falciparum and Plasmodium vivax infections

Malaria Journal

Research

Si Thura¹  Zay Yar Han²  Hnin Wai Lwin²  Kay Thwe Han²  Kayvan Zainabadi³  Christopher V. Plowe³  Myaing M. Nyunt³  Amed Ouattara³  Matthew Adams³ 

[1] Community Partners International, Yangon, Myanmar;Department of Medical Research, Ministry of Health and Sports, Yangon, Myanmar;Division of Malaria Research, Institute for Global Health, University of Maryland School of Medicine, Baltimore, USA;

关键词: Malaria;    Malaria elimination;    Plasmodium falciparum;    Plasmodium vivax;    Ultrasensitive PCR;    Limits of detection;    Molecular surveillance;    Asymptomatic infection;    Diagnostics;    Myanmar;    Southeast Asia;    Dried blood spot;    DBS;    Low transmission;   

DOI  :  10.1186/s12936-017-2025-3

 received in 2017-04-07, accepted in 2017-09-07,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundGreater Mekong Subregion countries are committed to eliminating Plasmodium falciparum malaria by 2025. Current elimination interventions target infections at parasite densities that can be detected by standard microscopy or rapid diagnostic tests (RDTs). More sensitive detection methods have been developed to detect lower density “asymptomatic” infections that may represent an important transmission reservoir. These ultrasensitive polymerase chain reaction (usPCR) tests have been used to identify target populations for mass drug administration (MDA). To date, malaria usPCR tests have used either venous or capillary blood sampling, which entails complex sample collection, processing and shipping requirements. An ultrasensitive method performed on standard dried blood spots (DBS) would greatly facilitate the molecular surveillance studies needed for targeting elimination interventions.MethodsA highly sensitive method for detecting Plasmodium falciparum and P. vivax 18S ribosomal RNA from DBS was developed by empirically optimizing nucleic acid extraction conditions. The limit of detection (LoD) was determined using spiked DBS samples that were dried and stored under simulated field conditions. Further, to assess its utility for routine molecular surveillance, two cross-sectional surveys were performed in Myanmar during the wet and dry seasons.ResultsThe lower LoD of the DBS-based ultrasensitive assay was 20 parasites/mL for DBS collected on Whatman 3MM filter paper and 23 parasites/mL for Whatman 903 Protein Saver cards—equivalent to 1 parasite per 50 µL DBS. This is about 5000-fold more sensitive than standard RDTs and similar to the LoD of ≤16–22 parasites/mL reported for other ultrasensitive methods based on whole blood. In two cross-sectional surveys in Myanmar, nearly identical prevalence estimates were obtained from contemporaneous DBS samples and capillary blood samples collected during the wet and dry season.ConclusionsThe DBS-based ultrasensitive method described in this study shows equal sensitivity as previously described methods based on whole blood, both in its limit of detection and prevalence estimates in two field surveys. The reduced cost and complexity of this method will allow for the scale-up of surveillance studies to target MDA and other malaria elimination interventions, and help lead to a better understanding of the epidemiology of low-density malaria infections.

【 授权许可】

CC BY   
© The Author(s) 2017