First report of a Plasmodium malariae SSU rRNA gene variant in Africa associated with reduced amplification by nested PCR.

Background: Nested polymerase chain reaction (PCR) targeting the small subunit ribosomal ribonucleic acid (SSU rRNA, 18S rRNA) region is widely used to differentiate Plasmodium species. We identified a variant of the Plasmodium malariae SSU rRNA gene that suggests nested PCR may fail to detect P. malariae strains with unknown mutations.

Case presentation: A 56-year-old Japanese man developed a fever 2 months after returning from a 2-month stay in Sierra Leone. Quartan malaria was suspected based on blood smear findings, and nested PCR confirmed P. malariae infection. However, the secondary PCR band obtained using P. malariae-specific primers was fainter than the primary PCR band amplified with universal primers-a reversal of the typical pattern. Sequence analysis revealed a four-base deletion in the SSU rRNA gene within the primer-binding site of the species-specific reverse primer used in the secondary PCR, suggesting that mutations in this region may partially impair amplification and hinder species identification. Cytochrome b gene sequencing confirmed 100% identity with P. malariae.

Conclusions: These findings underscore the need for continued molecular surveillance and sequence-based validation to ensure accurate diagnosis of Plasmodium infections, particularly in regions where genetic variants and zoonotic strains are emerging.