One-step PCR: A novel protocol for determination of pfhrp2 deletion status in Plasmodium falciparum

• DOI: 10.1371/journal.pone.0236369
• Publication Date: 2020-07-23T17:56:21Z
• Authors: Sophie Jones; Gireesh Subramaniam; Mateusz M. Plucinski; Dhruviben Patel; Jasmine Padilla; Michael Aidoo; Eldin Talundzic
• Keywords: Dried blood

Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis remote locations. However, emergence of pfhrp2 deleted parasites is threatening the efficacy RDTs, and World Health Organization (WHO) has highlighted surveillance these deletions as a priority. Nested PCR used to confirm deletion but costly laborious. Due spurious amplification paralogue pfhrp3, identity nested exon 1 product must be confirmed by sequencing. Here we describe new one-step method for detection pfhrp2. To determine sensitivity specificity, all PCRs were performed triplicate. Using photo-induced electron transfer (PET) 18srRNA true positive, had comparable 95.0% (88.7–98.4%) 1, 99.0% (94.6–99.9%) 2, 98.0% (93.0–99.8%), specificity 93.8% (69.8–99.8%) 100.0% (79.4–100.0%) (74.4–100.0%). Sequencing revealed that one step does not amplify pfhrp3. Logistic regression models applied measure 95% level clinical isolates provided estimates 133p/μL (95% confidence interval (CI): 3-793p/μL) whole blood (WB) samples 385p/μL CI: 31–2133 p/μL) dried spots (DBSs). When considering protocol attributes, less expensive, faster more suitable high throughput. In summary, developed accurate may ideal application WHO investigating symptomatic individuals presenting health care facilities.