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

TL;DR

A new Plasmodium malariae gene variant in Africa may cause missed detection by standard PCR tests, highlighting the need for improved diagnostic methods.

Contribution

The study reports a novel SSU rRNA gene variant in African P. malariae linked to reduced PCR amplification efficiency.

Findings

A four-base deletion in the SSU rRNA gene of P. malariae was found to reduce amplification in nested PCR.

The deletion is located in the primer-binding site of species-specific reverse primers used in secondary PCR.

Cytochrome b gene sequencing confirmed the sample was P. malariae despite the PCR anomaly.

Abstract

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.

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.

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.

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