Noninvasive Approach of Plasmodium falciparum Molecular Detection for Malaria Surveillance in Malaria Endemic Areas in Cameroon

Abstract

Background: Accurate, cost-effective, and noninvasive alternative molecular methods are needed for detecting low malaria parasitemia. The currently-used nested polymerase chain reaction (nPCR) requires blood as well as skilled personnel in order to minimise the risk of bloodborne disease transmission. Therefore, this study is aimed at assessing the accuracy of a noninvasive and more affordable malaria diagnosis with saliva using the loop-mediated isothermal amplification (LAMP) technique.

Methods: A cross-sectional study was conducted in the Centre and Southwest regions of Cameroon. Matched blood and saliva samples collected from symptomatic and asymptomatic participants were tested for malaria using rapid diagnostic tests, microscopy, PCR, and LAMP. Statistics were performed using R studio software at 95% confidence interval.

Results: A total of 100 participants (65% symptomatic and 35% asymptomatic) aged between 1 and 74 years with a balanced gender distribution ratio of 1.08 were included in our study. The prevalence of malaria was 61%, 57%, 59%, 42%, 35%, 17%, and 16% for blood-RDT, blood-PCR, blood-LAMP, blood-RT-LAMP, saliva-PCR, saliva-RT-LAMP, and saliva-LAMP, respectively. Both saliva and blood showed a sensitivity of 43.90% and respective specificities of 68.75% and 57.62%. When using RT-LAMP, sensitivities of 49.38% and 48.21% and specificities of 94.11% and 66.67% were recorded for saliva and blood, respectively. Sensitivities of 70.23% and 73.49% and specificities of 62.5% and 76.47% were recorded, respectively, for saliva-LAMP and saliva-RT-LAMP when compared to saliva-PCR as the gold standard. Saliva-LAMP and saliva-RT-LAMP had a fair agreement (к = 0.221 and 0.352, respectively) with saliva-PCR. Homemade LAMP and RT-LAMP technologies match the WHO recommendations and after proper validation in a larger sample size, could serve for malaria diagnosis in developing countries.

Figure 1

Malaria prevalence by gender and age group. (a). Malaria prevalence in male and female participants by PCR and LAMP. (b). Malaria prevalence in each age group by PCR and LAMP. (c). Malaria prevalence of different techniques using blood and saliva.

Figure 2

Electropherograms of P. falciparum in blood and saliva using PCR and LAMP. (a). Electropherogram of P. falciparum in blood and saliva using PCR. (b). Electropherograms of P. falciparum in blood and saliva using LAMP and the image of tubes showing the changing of color. (c). P. falciparum amplification curves by the real-time LAMP Genie II system. PC: positive control; B1: blood sample 1; B2: blood sample 2; NC: negative control; S1: saliva sample 1; S2: saliva sample 2; NSS: negative saliva sample; MWM: 100 base pair molecular weight maker represented on the gels by band size numbers only.