Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of <i>Plasmodium falciparum</i> and <i>Plasmodium vivax</i> and Its Comparison with Loopamp™ Malaria

oleh: Mudsser Azam, Kirti Upmanyu, Ratan Gupta, Karugatharayil Sasi Sruthy, Monika Matlani, Deepali Savargaonkar, Ruchi Singh

Format: Article
Diterbitkan: MDPI AG 2021-09-01

Deskripsi

To strengthen malaria surveillance, field-appropriate diagnostics requiring limited technical resources are of critical significance. Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are potential point-of-care tests with high sensitivity and specificity and have been used in low-resource settings. <i>Plasmodium vivax</i>–specific consensus repeat sequence (CRS)-based and <i>Plasmodium falciparum</i>–specific 18S rRNA primers were designed, and a two-tube LAMP assay was developed. The diagnostic performance of a closed-tube LAMP assay and Loopamp™ Malaria Detection (Pan/Pf, Pv) kit was investigated using nested PCR confirmed mono- and co-infections of <i>P. vivax</i> and <i>P. falciparum</i> positive (<i>n</i> = 149) and negative (<i>n</i> = 67) samples. The closed-tube Pv LAMP assay showed positive amplification in 40 min (limit of detection, LOD 0.7 parasites/µL) and Pf LAMP assay in 30 min (LOD 2 parasites/µL). Pv LAMP and Pf LAMP demonstrated a sensitivity and specificity of 100% (95% CI, 95.96–100% and 89.85–100%, respectively). The Loopamp<sup>TM</sup> Pan/Pf Malaria Detection kit demonstrated a sensitivity and specificity of 100%, whereas Loopamp<sup>TM</sup> Pv showed a sensitivity of 98.36% (95% CI, 91.28–99.71%) and specificity of 100% (95% CI, 87.54–100%). The developed two-tube LAMP assay is highly sensitive (LOD ≤ 2 parasite/µL), demonstrating comparable results with the commercial Loopamp™ Malaria Detection (Pf/pan) kit, and was superior in detecting the <i>P. vivax</i> co-infection that remained undetected by the Loopamp™ Pv kit. The developed indigenous two-tube Pf/Pv malaria detection can reliably be used for mass screening in resource-limited areas endemic for both <i>P. falciparum</i> and <i>P. vivax</i> malaria.