Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Method for Simultaneous Detection of Spotted Fever Group Rickettsiae and Malaria Parasites by Dipstick DNA Chromatography.

Lavel Chinyama Moonga,Ryo Nakao,Kyoko Hayashida,Chihiro Sugimoto,Naoko Kawai,Boniface Namangala,Junya Yamagishi

doi:10.3390/diagnostics10110897

Lavel Chinyama Moonga, Ryo Nakao + Show 5 more

Open Access

https://doi.org/10.3390/diagnostics10110897

Copy DOI

Abstract

Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis’s clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.

Highlights

Undifferentiated febrile illnesses (UFIs) are the most common clinical presentation at healthcare centers in low- and middle-income countries [1,2]
Rickettsioses and malaria are among the common cause of acute UFI, with the Diagnostics 2020, 10, 897; doi:10.3390/diagnostics10110897
Rickettsiosis caused by Rickettsia africae causes a significant proportion of acute febrile illnesses among returning travelers from sub-Saharan Africa [9]

Summary

Introduction

Undifferentiated febrile illnesses (UFIs) are the most common clinical presentation at healthcare centers in low- and middle-income countries [1,2]. The acute undifferentiated febrile manifestation leads to challenges in the differential diagnosis of the definitive cause of acute UFI, mostly in incapacitated diagnostic settings. Most clinical diagnostic approaches tend to target more common diseases, such as malaria, missing out on other pathogens, including rickettsiae [4,6]. Spotted fever group (SFG) rickettsioses are neglected diseases and represent a large proportion of emerging and re-emerging infectious diseases with a worldwide distribution. Rickettsiosis caused by Rickettsia africae causes a significant proportion of acute febrile illnesses among returning travelers from sub-Saharan Africa [9]. Rickettsia felis is a common cause of febrile illness in sub-Saharan

Methods

Results

Discussion

Conclusion