# Molecular and Microscopic Challenges in Detecting Plasmodium cynomolgi Co-Infections with Plasmodium vivax: A Case Report

## TL;DR

A human case of Plasmodium cynomolgi malaria was misdiagnosed as P. vivax, highlighting the need for better diagnostic tools to detect this emerging zoonotic malaria.

## Contribution

A new case of misdiagnosed P. cynomolgi infection in a human is reported, revealing diagnostic limitations due to genetic and morphological similarities with P. vivax.

## Key findings

- P. cynomolgi was misdiagnosed as P. vivax using routine microscopy and qPCR.
- Real-time PCR and sequencing confirmed a co-infection with P. vivax and P. cynomolgi.
- P. cynomolgi is an emerging zoonotic malaria species in Southeast Asia requiring improved diagnostics.

## Abstract

The risk of non-human primate (NHP) malaria transmission to humans is increasing, with Plasmodium knowlesi and Plasmodium cynomolgi emerging as significant zoonotic threats, particularly in Malaysia. While P. knowlesi is well-documented, P. cynomolgi infections in humans remain underreported, largely due to diagnostic challenges. Routine microscopy and standard molecular diagnostic tools often misdiagnose P. cynomolgi infections as P. vivax due to morphological similarities and genetic homology. We report a new case of a human P. cynomolgi infection misdiagnosed as Plasmodium vivax in a 32-year-old male with no prior malaria history or travel to endemic countries. The initial diagnoses made by the microscopy and qPCR conducted by the Kota Bharu Public Health Laboratory in Kelantan identified the infection as P. vivax. However, cross-examination by the Institute for Medical Research (IMR) revealed the presence of mixed-species infection, prompting further analysis. The real-time PCR and sequencing performed at MAPELAB, Spain, confirmed the co-infection of P. vivax and P. cynomolgi. This case highlights the diagnostic limitations in detecting P. cynomolgi, which shares high genetic similarity with P. vivax, leading to potential cross-reactivity and diagnostic inaccuracies. As P. cynomolgi emerges as the second zoonotic malaria species after P. knowlesi capable of infecting humans in Southeast Asia, improved diagnostic methods are urgently needed. Enhanced molecular diagnostics and comprehensive epidemiological studies are essential to elucidate transmission dynamics, assess public health implications, and inform effective malaria control strategies.