In situ loop-mediated isothermal amplification (LAMP) for identification of Plasmodium species in wide-range thin blood smears

• Published in: Malaria Journal
• Main Authors: Muneaki Hashimoto, Hirokazu Sakamoto, Yusuke Ido, Masato Tanaka, Shouki Yatsushiro, Kazuaki Kajimoto, Masatoshi Kataoka
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2018
• Subjects: Malaria; Plasmodium; Diagnosis; Parasite species; In situ LAMP; Thin blood smears; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic; Triton
• Online Access: https://doi.org/10.1186/s12936-018-2381-7; https://doaj.org/article/e88d6409c86e45d7bb8510c90118d8fa

Abstract Background Five species of Plasmodium are known to infect humans. For proper treatment of malaria, accurate identification of the parasite species is crucial. The current gold standard for malaria diagnosis is microscopic examination of Giemsa-stained blood smears. Since the parasite species are identified by microscopists who manually search for the parasite-infected red blood cells (RBCs), misdiagnosis due to human error tends to occur in case of low parasitaemia or mixed infection. Then, molecular methods, such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are required for conclusive identification of the parasite species. However, since molecular methods are highly sensitive, false-positive results tend to occur due to contamination (carry over) or the target gene products may be detected even after clearance of the parasites from the patient’s blood. Therefore, accurate detection of parasites themselves by microscopic examination is essential for the definitive diagnosis. Thus, the method of in situ LAMP for the parasites was developed. Results Red blood cell suspensions, including cultured Plasmodium falciparum, strain 3D7, infected-RBCs, were dispersed on cyclic olefin copolymer (COC) plate surfaces rendered hydrophilic by reactive ion-etching treatment using a SAMCO RIE system (hydrophilic-treated), followed by standing for 10 min to allow the RBCs to settle down on the plate surface. By rinsing the plate with RPMI 1640 medium, monolayers of RBCs formed on almost the entire plate surface. The plate was then dried with a hair drier. The RBCs were fixed with formalin, followed by permeabilization with Triton X-100. Then, amplification of the P. falciparum 18S rRNA gene by the LAMP reaction with digoxigenin (DIG)-labelled dUTP and a specific primer set was performed. Infected RBCs as fluorescence-positive cells with anti-DIG antibodies conjugated with fluorescein using fluorescent microscopy could be detected. Conclusions The present work shows that the potential of ...