A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.
Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar's test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar's test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in malaria diagnosis in low to high parasite density settings.
Malaria Research Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.
Sylvie
S
Kemleu
PATH, Seattle, Washington, United States of America.
Dylan
D
Guelig
Malaria Research Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.|Faculty of Medicine and Pharmaceutical Sciences, University of Douala, Douala, Cameroon.
Carole
C
Eboumbou Moukoko
Malaria Research Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.|Faculty of Sciences, University of Douala, Douala, Cameroon.
Estelle
E
Essangui
PATH, Seattle, Washington, United States of America.
Steven
S
Diesburg
Hematology Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.
Abas
A
Mouliom
Hematology Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.
Bernard
B
Melingui
Hematology Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.
Jeanne
J
Manga
Malaria Research Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.
Christiane
C
Donkeu
Hematology Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.
Annie
A
Epote
Public Health & Epidemiology Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.
Gaëtan
G
Texier
PATH, Seattle, Washington, United States of America.
Paul
P
LaBarre
PATH, Seattle, Washington, United States of America.
Robert
R
Burton
Malaria Research Unit, Centre Pasteur du Cameroun, Yaoundé, Cameroon.
Lawrence
http://orcid.org/0000-0001-7717-4832
L
Ayong
D001483:Base Sequence; D006801:Humans; D057230:Limit of Detection; D016778:Malaria, Falciparum / Q000175:diagnosis* / Q000469:parasitology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010963:Plasmodium falciparum / Q000235:genetics*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D016415:Sequence Alignment