TY - JOUR
T1 - Laboratory evaluation of the miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA), a simplified molecular diagnostic test for Plasmodium
AU - van Dijk, Norbert J.
AU - Menting, Sandra
AU - Wentink-Bonnema, Ellen M. S.
AU - Broekhuizen-van Haaften, Patricia E.
AU - Withycombe, Elen
AU - Schallig, Henk D. F. H.
AU - Mens, Petra F.
N1 - Funding Information: This work was supported by the EU FP7-Health-2013.0-1 project “Translation of the direct-on-blood PCR-NALFIA system into an innovative near point-of-care diagnostic for malaria” (DIAGMAL) [Grant Number 601714]. Funding Information: We would like to thank Dr. Tom van Gool of the Amsterdam University Medical Centres for kindly providing the Plasmodium -positive and -negative blood samples from returned travellers. Our thanks go to the Sanquin Amsterdam Blood Bank for the provision of blood samples from healthy donors. We also thank Zoë Piets (Amsterdam University Medical Centres) for assisting in the sensitivity and specificity assessment, and Lieke Deckers, Merel Schrooten (both Amsterdam University Medical Centres), Japhet Kabalu Tshiongo and Flory Luzolo Khote (both University of Kinshasa, Democratic Republic of the Congo) for participating in the concordance evaluation. Publisher Copyright: © 2023, The Author(s).
PY - 2023/12/1
Y1 - 2023/12/1
N2 - Background: Point-of-care diagnosis of malaria is currently based on microscopy and rapid diagnostic tests. However, both techniques have their constraints, including poor sensitivity for low parasitaemias. Hence, more accurate diagnostic tests for field use and routine clinical settings are warranted. The miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative, easy-to-use molecular assay for diagnosis of malaria in resource-limited settings. Unlike traditional molecular methods, mini-dbPCR-NALFIA does not require DNA extraction and makes use of a handheld, portable thermal cycler that can run on a solar-charged power pack. Result read-out is done using a rapid lateral flow strip enabling differentiation of Plasmodium falciparum and non-falciparum malaria infections. A laboratory evaluation was performed to assess the performance of the mini-dbPCR-NALFIA for diagnosis of pan-Plasmodium and P. falciparum infections in whole blood. Methods: Diagnostic accuracy of the mini-dbPCR-NALFIA was determined by testing a set of Plasmodium-positive blood samples from returned travellers (n = 29), and Plasmodium-negative blood samples from travellers with suspected malaria (n = 23), the Dutch Blood Bank (n = 19) and intensive care patients at the Amsterdam University Medical Centers (n = 16). Alethia Malaria (LAMP) with microscopy for species differentiation were used as reference. Limit of detection for P. falciparum was determined by 23 measurements of a dilution series of a P. falciparum culture. A fixed sample set was tested three times by the same operator to evaluate the repeatability, and once by five different operators to assess the reproducibility. Results: Overall sensitivity and specificity of the mini-dbPCR-NALFIA were 96.6% (95% CI, 82.2%–99.9%) and 98.3% (95% CI, 90.8%–100%). Limit of detection for P. falciparum was 10 parasites per microlitre of blood. The repeatability of the assay was 93.7% (95% CI, 89.5%–97.8%) and reproducibility was 84.6% (95% CI, 79.5%–89.6%). Conclusions: Mini-dbPCR-NALFIA is a sensitive, specific and robust method for molecular diagnosis of Plasmodium infections in whole blood and differentiation of P. falciparum. Incorporation of a miniature thermal cycler makes the assay well-adapted to resource-limited settings. A phase-3 field trial is currently being conducted to evaluate the potential implementation of this tool in different malaria transmission areas.
AB - Background: Point-of-care diagnosis of malaria is currently based on microscopy and rapid diagnostic tests. However, both techniques have their constraints, including poor sensitivity for low parasitaemias. Hence, more accurate diagnostic tests for field use and routine clinical settings are warranted. The miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative, easy-to-use molecular assay for diagnosis of malaria in resource-limited settings. Unlike traditional molecular methods, mini-dbPCR-NALFIA does not require DNA extraction and makes use of a handheld, portable thermal cycler that can run on a solar-charged power pack. Result read-out is done using a rapid lateral flow strip enabling differentiation of Plasmodium falciparum and non-falciparum malaria infections. A laboratory evaluation was performed to assess the performance of the mini-dbPCR-NALFIA for diagnosis of pan-Plasmodium and P. falciparum infections in whole blood. Methods: Diagnostic accuracy of the mini-dbPCR-NALFIA was determined by testing a set of Plasmodium-positive blood samples from returned travellers (n = 29), and Plasmodium-negative blood samples from travellers with suspected malaria (n = 23), the Dutch Blood Bank (n = 19) and intensive care patients at the Amsterdam University Medical Centers (n = 16). Alethia Malaria (LAMP) with microscopy for species differentiation were used as reference. Limit of detection for P. falciparum was determined by 23 measurements of a dilution series of a P. falciparum culture. A fixed sample set was tested three times by the same operator to evaluate the repeatability, and once by five different operators to assess the reproducibility. Results: Overall sensitivity and specificity of the mini-dbPCR-NALFIA were 96.6% (95% CI, 82.2%–99.9%) and 98.3% (95% CI, 90.8%–100%). Limit of detection for P. falciparum was 10 parasites per microlitre of blood. The repeatability of the assay was 93.7% (95% CI, 89.5%–97.8%) and reproducibility was 84.6% (95% CI, 79.5%–89.6%). Conclusions: Mini-dbPCR-NALFIA is a sensitive, specific and robust method for molecular diagnosis of Plasmodium infections in whole blood and differentiation of P. falciparum. Incorporation of a miniature thermal cycler makes the assay well-adapted to resource-limited settings. A phase-3 field trial is currently being conducted to evaluate the potential implementation of this tool in different malaria transmission areas.
KW - Laboratory evaluation
KW - Malaria
KW - Nucleic Acid Lateral Flow Immunoassay
KW - PCR
KW - Plasmodium
KW - Simplified molecular diagnostics
KW - miniPCR
UR - http://www.scopus.com/inward/record.url?scp=85150454780&partnerID=8YFLogxK
U2 - https://doi.org/10.1186/s12936-023-04496-4
DO - https://doi.org/10.1186/s12936-023-04496-4
M3 - Article
C2 - 36932372
SN - 1475-2875
VL - 22
JO - Malaria journal
JF - Malaria journal
IS - 1
M1 - 98
ER -