A single-tube multiplex PCR assay for rapid identification and differentiation of plasmodium falciparum and plasmodium vivax in resource-limited settings.

BACKGROUND

Accurate species identification of Plasmodium falciparum and Plasmodium vivax is essential for appropriate malaria treatment, particularly in endemic regions where both species co-exist. Current molecular methods, while sensitive, often require nested PCR protocols that are time-consuming and prone to contamination.

METHODS

We developed and validated a single-tube multiplex PCR assay targeting species-specific regions of the 18S rRNA gene, incorporating a human β-globin gene as an internal control. The assay was evaluated using 162 malaria-positive samples from four geographical regions in India (Mumbai, Surat, Manipur, and Mangalore) and 80 malaria- negative controls. Performance was compared against nested PCR and microscopy-based techniques.

RESULTS

The single-tube assay demonstrated 100% sensitivity (95% CI: 97.75-100%) and 100% specificity (95% CI: 95.49-100%) compared to nested PCR. The assay successfully identified P. falciparum (n = 42, 25.93%), P. vivax (n = 12, 7.41%), and mixed infections (n = 108, 66.67%) with a detection limit of 0.2 parasites/μL. The assay required approximately 3 h total time compared to 6-8 h for nested PCR, with reduced contamination risk. Five microscopy-negative samples were PCR-positive, indicating submicroscopic parasitemia.

CONCLUSION

This single-tube multiplex PCR assay provides a rapid, sensitive, and specific method for Plasmodium species identification and differentiation suitable for clinical diagnostics and epidemiological studies in resource-limited settings.