Multiplexed Isothermal Amplification Assay for the Detection of Plasmodium falciparum and Trichomonas vaginalis in Urine.

Trichomoniasis and malaria, caused by Trichomonas vaginalis (T. vaginalis) and Plasmodium falciparum (P. falciparum), respectively, remain major public health threats, primarily in resource-limited regions like sub-Saharan Africa. In particular, malaria in pregnancy (MiP) poses serious diagnostic challenges due to the placental sequestration of P. falciparum, often leading to underdetection by conventional tools like microscopy or rapid diagnostic tests (RDTs). Meanwhile, T. vaginalis, the most prevalent nonviral sexually transmitted infection, exacerbates poor maternal and fetal outcomes when co-infections occur. In this study, we present a multiplexed isothermal nucleic acid amplification assay (iso-IMRS) for simultaneous detection of P. falciparum and T. vaginalis. We built on prior work validating iso-IMRS for P. falciparum and PCR-IMRS for T. vaginalis using a modified workflow better suited to near point-of-care (POC) settings. The assay was tested in simulated and human urine matrices, aiming for a low-cost, non-invasive, and field-ready diagnostic solution. The assay detected P. falciparum and T. vaginalis at 100 copies/μL in processed human urine, demonstrating clinically relevant sensitivity for noninvasive, multiplexed detection. However, use with a neat urine sample requires a processing step that may limit POC use. In our clinics in Kenya, we process samples nearby, and a fast molecular test that does not require thermocycling, with a turnaround time within the span of a typical prenatal visit, will positively impact our patients.