Utilization of 18s ribosomal RNA LAMP for detecting Plasmodium falciparum in microscopy and rapid diagnostic test negative patients

Abstract

In this study, Plasmodium falciparum was detected in patients that were declared negative for malaria microscopy and rapid diagnostic test kit (mRDT), using Plasmodium 18s rRNA loop-mediated isothermal amplification (LAMP) technique. The main aim of this study was to assess the usefulness of LAMP assay for detecting pre-clinical malaria, when microscopy and mRDT were less sensitive. DNA was obtained from 100 μL of whole blood using the boil and spin method. Subsequently, the Plasmodium 18s rRNA LAMP assay was performed to amplify the specific Plasmodium 18s rRNA gene. Microscopy and mRDT negative samples [697/2223 (31.2%)] were used for this study. Compared to frequencies obtained for the other demographic variables, most of the patients were < 6 years (37.7%), females (59.0%), peri-urban dwellers (39.0%) and patients that sought outpatient department services (39.3%). Overall, the prevalence of Plasmodium 18s rRNA was 17.5%. when stratified by study variables, Plasmodium 18s rRNA LAMP positivity was higher in patients over 30 years [58/122 (54.2%)], males [69/122 (56.5%)], rural dwellers [69/122 (56.5%)] and patients that sought OPD services [68/122 (55.7%)]. The risk of being infected with Plasmodium when routine tests were negative was higher in 15-30-year group (OR = 3.03, 95% CI: 1.6-5.8, p = 0.0007), patients > 30 years (OR = 15.2, 95% CI: 8.3-27.7, p<0.001), males (OR = 2.1, 95% CI: 1.4-3.2, p = 0.0002) and rural dwellers (OR = 2.2, 95% CI:1.4-3.6, p = 0.0009). However, risk was lower in post-natal children (OR = 0.3, 95% CI: 0.18-0.51, p<0.001). Majority (81.5%) of the infected patients presented with headache, herpes labialis, diarrhea and vomiting. We demonstrated the lack of sensitivities of microscopy and mRDT for one-time diagnosis of malaria. Therefore, it is essential to utilize a sensitive technique such as Plasmodium 18s rRNA LAMP to increase the detection rate of Plasmodium infection.

Fig 1. Quality control of LAMP assay.

EC–extraction control, RC1 –reaction control (master mix), RC2—reaction control: master mix with nuclease free water, PC 1 –positive control (24,087 parasites/μL), PC2—positive control (812 parasites/μL), PC3—positive control (6 parasites/μL), NC–negative control, NR1/2 –patient negative reactions, PR1/2 –patient positive reactions.

Fig 2. Monthly trends in suspected and Plasmodium 18s rRNA positive cases.

Major rainfall–April–June, minor rainfall–March, September and October, no or very low rains—July, August, November–February.