Molecular Detection Challenges of Human Plasmodium knowlesi infection by Polymerase Chain Reaction

Abstract

Plasmodium knowlesi is the fifth Plasmodium sp. causing malaria in humans. There were 545 P. knowlesi malaria cases reported in Indonesia in 2010-2021 period. The first case was reported from South Kalimantan, and more cases were reported in Sumatra and Kalimantan Island. The morphology of P. knowlesi is difficult to distinguish from other Plasmodium species, especially with P. falciparum and P. malariae. Therefore, molecular identification is still the most promising method for diagnosing P. knowlesi infection. Objective: This study aimed to analyze the molecular detection method of human P. knowlesi infection using Polymerase Chain Reaction (PCR) and sequencing techniques. Methods: DNA was isolated from malaria blood samples. P. knowlesi detection was conducted by nested PCR using primer rPLU1 and rPLU5 for nested 1 and Kn1f and Kn3r for nested 2. The PCR products were directly sequenced. The sequences were analysed using Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI). Results: Blood samples from ten malaria patients from Maluku province were collected after informed consent. The P. knowlesi-specific PCR amplification resulted in a band of approximately 420 bp in all samples. Sequence analysis showed the highest similarity (89-92 %) with many global P. falciparum strains. However, BLAST analysis for part of sequences also showed high similarities with several P. knowlesi H strains 18s rRNA from Peninsular Malaysia. Primer analysis using BLAST demonstrated the specificity of Kn3r-nested 2 primer, however, Kn1f primer showed a cross-reactive with other Plasmodium sp, including P. falciparum and P. vivax. Conclusion: Molecular detection of P. knowlesi infection is challenging. A new target gene for primer design and detection method with higher specificity for human P. knowlesi examination is needed to develop.