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

Abstract

BackgroundFive 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.ResultsRed 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.ConclusionsThe present work shows that the potential of in situ LAMP for the identification of Plasmodium species at the single cell level on hydrophilic-treated COC palates, allowing highly sensitive and accurate malaria diagnosis. The findings will improve the efficacy of the gold standard method for malaria diagnosis.

Poor diagnostic performance of a species-specific loop-mediated isothermal amplification (LAMP) platform for malaria

“…The easy application of LAMP facilitates the further development of malaria-LAMP point-of-care applications, like the combination of LAMP with lateral flow platforms [34–36] or the in situ application on microscopic slides [37], as well as LAMP from blood samples on filter papers [38, 39]. Even from body fluids other than blood like saliva and urine, LAMP-based detection of Plasmodium spp.…”

Evaluation of Visual LAMP detection of Plasmodium falciparum and non-Plasmodium falciparum

“…Microscope examination of blood smears is still considered the gold standard for the diagnosis of malaria, which was labor-intensive, time-consuming [7,10,16] , with detection limit of 100 malaria parasites/µl [10,16] . It was usually unable to detect low-density and mixed infections, the sensitivity and specificity of which depends on the skill of the technician, quality and staining quality of blood smear, and so on [3,17] . Compared with molecular diagnostic methods, in malaria endemic areas, only half of malaria patients were correctly diagnosed.…”

“…At the same time, due to increasing travel and population movements to malaria-endemic areas, imported malaria is still widespread [21] . Particularly, asymptomatic malaria infection and lowdensity malaria parasite emias are hard to be detected by microscope examination and RDT due to below the detection threshold [11,24] , causing misdiagnosis or delayed diagnosis [3,21] . But they could maintaining transmission of malaria as infected intermediate host [24,26] .…”

“…There are 4 kinds of malaria parasites, of which Plasmodium falciparum is the most harmful, toxic, and with the highest fatality rate [2,3] . According to The World Malaria Report 2018, $3.1 billion was invested in control and eradication of malaria in 2017, with a total of 219 million malaria cases worldwide and 435,000 deaths.…”

“…The common detection methods of malaria parasites are mainly optical microscope examination and RDT. In the chronic infection stage, due to the malaria parasite emias always maintain low density, optical microscope examination and RDT leak detection [3,6] . The RDT is insensitive to Plasmodium vivax and Plasmodium ovale.…”