A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections

Abstract

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.

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

“…Accurate diagnosis of malaria infection is critical. 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] .…”

“…Meantime it shows limited sensitivity when detecting low-density infection [16,22] , and less sensitivity to non-P.f P.v as 66.0-88.0%, P.o as 5.5-86.7%, P.m as 21.4-45.2% [19] . PCR is one of the most sensitive and reliable methods for detecting DNA of malaria, with a high sensitivity and specificity limit to 1-10 malaria parasites/μl [10,11] . However, it requires technical expertise and infrastructure which limit its wide range of applications.…”

“…The patients with asymptomatic Plasmodium falciparum infection may be malaria-infected reservoir [7,8] . Therefore, a high sensitivity diagnostic methods able to detect low-density malaria parasite emias for the elimination of malaria is critical [6,9] .There is urgent need for extremely sensitive and field-based diagnostic system [10] .…”

Sensitivity of Loop-Mediated Isothermal Amplification Increases 100-Fold after TritonTM X-100 Treatment of Plasmodium-Infected Erythrocytes

“…Although the IRBC membrane is broken by the increase in osmotic pressure caused by the dilution of the RBCs in water, the pressure might be insufficient to break the PV membrane and/or parasite plasma membrane. Therefore, we postulated that treatment of IRBC with a detergent would increase the sensitivity of LAMP assay [13]. This potential effect of a detergent has not been investigated yet.…”

“…Therefore, it is very important to establish a LAMP method that does not require any instruments and power supply for centrifugation and/ or boiling. Previously, Kemleu, et al reported the until-date most sensitive method for a reverse transcriptase (RT)-LAMP in a field setting [13]. However, since that method requires reverse transcriptase, RT-LAMP is thus more expensive than LAMP.…”