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

The risk of non-human primate (NHP) malaria transmission to humans is increasing, with <i>Plasmodium knowlesi</i> and <i>Plasmodium cynomolgi</i> emerging as significant zoonotic threats, particularly in Malaysia. While <i>P. knowlesi</i> is well-documented, <i>P. cynomolgi</i> infections in humans remain underreported, largely due to diagnostic challenges. Routine microscopy and standard molecular diagnostic tools often misdiagnose <i>P. cynomolgi</i> infections as <i>P. vivax</i> due to morphological similarities and genetic homology. We report a new case of a human <i>P. cynomolgi</i> infection misdiagnosed as <i>Plasmodium vivax</i> 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 <i>P. vivax</i>. 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 <i>P. vivax</i> and <i>P. cynomolgi</i>. This case highlights the diagnostic limitations in detecting <i>P. cynomolgi</i>, which shares high genetic similarity with <i>P. vivax</i>, leading to potential cross-reactivity and diagnostic inaccuracies. As <i>P. cynomolgi</i> emerges as the second zoonotic malaria species after <i>P. knowlesi</i> 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.