Plasmodium mono and mixed-infections in India: a tale of see-saw in species misidentification.

Introduction: Malaria is still a public health challenge across many regions of the world. With significant efforts, it has become a target for elimination in near future. However, elimination requires early and accurate diagnosis and despite the fact that malaria elimination is nearing in several countries, specific and sensitive diagnosis of malaria and its causative species remains inadequate in many near-elimination settings, including India. With the advent and increasing usage of nucleic acid-based detection of Plasmodium infection, the diagnostic limitations of microscopy, including errors in species identification, have become more prominent. Methods: The purpose of this study was to determine the magnitude of Plasmodium species misidentification by microscopy in India based on previously published reports that performed microscopy and PCR to the same samples. A total of 2706 microscopy-PCR pairs were extracted from 16 different locations across 11 Indian states. Region-specific and species-wise misidentification rates were also estimated. Results: The analyses revealed 15% misidentification rate (408/2706). Surprisingly, microscopy misidentified >98% of mixed-infections (400/405) as mono-infections (almost all as P. falciparum mono-infections). The study identifies Jharkhand and Madhya Pradesh as major contributors (>20%) to Plasmodium species misidentification by microscopy. Discussion: These findings suggest that we are overestimating P. falciparum burden, potentially wasting elimination resources, and underestimating non-falciparum species. The study also addresses an important issue concerning analysis of misidentification and sub-microscopic infection data. It proposes an analysis approach that is expected to help in deciphering misidentification and sub-microscopic infections in a more granular manner, generating actionable data for countries targeting malaria elimination.