First field and laboratory evaluation of LAMP assay for malaria diagnosis in Cubal, Angola

Abstract

Background Malaria is a globally distributed infectious disease. According to the World Health Organization, Angola is one of the six countries that account for over half the global malaria burden in terms of both malaria cases and deaths. Diagnosis of malaria still depends on microscopic examination of thin and thick blood smears and rapid diagnostic tests (RDTs), which often lack analytical and clinical sensitivity. Molecular methods could overcome these disadvantages. The aim of this study was to evaluate, for the first time to our knowledge, the performance of a loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria in an endemic area in Cubal, Angola, and to assess the reproducibility at a reference laboratory. Methods A total of 200 blood samples from patients attended at Hospital Nossa Senhora da Paz, Cubal, Angola, were analysed for Plasmodium spp. detection by microscopy, RDTs, and LAMP. LAMP assay was easily performed in a portable heating block, and the results were visualized by a simple colour change. Subsequently, the samples were sent to a reference laboratory in Spain to be reanalysed by the same colorimetric LAMP assay and also in real-time LAMP format. Results In field tests, a total of 67/200 (33.5%) blood samples were microscopy-positive for Plasmodium spp., 98/200 RDT positive, and 112/200 (56%) LAMP positive. Using microscopy as reference standard, field LAMP detected more microscopy-positive samples than RDTs (66/67; 98% vs. 62/67; 92.5%). When samples were reanalysed at a reference laboratory in Spain using both colorimetric and real-time assays, the overall reproducibility achieved 84.5%. Conclusions This is the first study to our knowledge in which LAMP has been clinically evaluated on blood samples in a resource-poor malaria-endemic area. The colorimetric LAMP proved to be more sensitive than microscopy and RDTs for malaria diagnosis in field conditions. Furthermore, LAMP showed an acceptable level of reproducibility in a reference laboratory. The possibility to use LAMP in a real-time format in a portable device reinforces the reliability of the assay for molecular diagnosis of malaria in resource-poor laboratories in endemic areas. Graphical Abstract

The Laboratory Diagnosis of Malaria: A Focus on the Diagnostic Assays in Non-Endemic Areas

“…It is a simple method based on the isothermal amplification, not requiring special equipment, and producing results that can be read visually or with a real-time turbidimetry. LAMP allows to reduce the time for the result within the recommended 2 h for the diagnosis of malaria and showed to have high efficiency, allowing DNA amplified 10 9 -10 10 times in 15-60 min [87][88][89]. Advantages of LAMP based assays also include the use of small amounts of blood samples on filter papers and the tolerance of inhibitory substances present in blood samples (hemoglobin/immunoglobulin), but it currently lacks sufficient accuracy [21].…”

The Laboratory Diagnosis of Malaria: A Focus on the Diagnostic Assays in Non‐Endemic Areas

“…It is a simple method based on the isothermal amplification, not requiring special equipment, and producing results that can be read visually or with a Real-time turbidimetry. LAMP allows to reduce the time for the result within the recommended 2 hours for the diagnosis of malaria and showed to have high efficiency, allowing DNA amplified 10 9 -10 10 times in 15-60 min [87][88][89]. Advantages of LAMP based assays also include the use of small amounts of blood sample on filters papers and the tolerance to inhibitory substances present in blood samples (hemoglobin/immunoglobulin), but it currently lacks sufficient accuracy [21].…”