Malaria in Vietnam is exhibiting increasingly complex epidemiological patterns, characterized by a significant shift in the species composition, with the proportion of Plasmodium malariae rising noticeably from 2% to 52.8% during the 2023 – 2024 period. Meanwhile, malaria diagnosis remains challenging due to the typically low parasitemia levels of P. malariae and its morphological resemblance to other Plasmodium species under light microscopy, often leading to misidentification - particularly in asymtomatic cases. Objective: This study aimed to standardize a species-specific diagnostic approach for P. malariae based on Nested PCR. Method: The assay focused on amplifying the 18S rRNA gene of P. malariae using two rounds of PCR. The initial amplification utilized the primer pair PLU5/PLU6, followed by a second round using MAL1/MAL2 primers. Various parameters were optimized, including the number of cycles in the primary PCR (25, 30, and 35 cycles) and the secondary amplification step (25 and 30 cycles), and annealing temperatures for the primer pair MAL1/MAL2 (54°C, 56°C, 58°C, and 60°C). Amplified products were resolved via electrophoresis on 2% agarose gels with 0.5X TAE buffer, electrophoresed at 110 volts for 30 minutes, and visualized using the ChemiDoc-It² Imaging System. Result: The optimized Nested PCR protocol consist of 35 cycles for the primary PCR, 25 cycles for the second round, with an annealing temperature of 54°C in 1 minute for the MAL1/MAL2 primers. The optimized protocol demonstrated a limit of detection as low as 0.5 parasites/µL, and the total time required was 3 hours and 50 minutes. Conclusion: The Nested PCR protocol for the specific detection of P. malariae has been successfully standardized and is applicable in clinical practice, offering enhanced diagnostic efficacy and reduce turnaround time, thereby contributing to improved cost-effectiveness in diagnostic workflows.

Malaria, Plasmodium Malariae, Nested-PCR, Protocol Standardization