Abstract
BackgroundMolecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method.Methodology and Significant FindingsPublished genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples.ConclusionThis RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.
Highlights
2 billion people are exposed to malaria with morbidity surpassing 250 million cases and close to 1 million deaths per year [1]
Most non-histidine rich protein (HRP)-2 based tests (LDH and aldolase) are commonly pan-species test that allow for the speciation of P. falciparum and/or non-falciparum species when used in conjunction with HRP-2 based tests
We were able to amplify any of the four species of human malaria parasites (P. falciparum, P. vivax, P. malariae and P. ovale) within 20 minutes (Figure 2)
Summary
2 billion people are exposed to malaria with morbidity surpassing 250 million cases and close to 1 million deaths per year [1]. The existing tools for the diagnosis of malaria include microscopy, parasite antigen/enzyme detection kits (commonly referred to as rapid diagnostic tests (RDTs)) and molecular tools (reviewed in [3]). Each of these diagnostic tools has its own advantages and limitations. Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria They require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. We attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform We refer to this as the RealAmp method
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
