The diagnostic ambiguity for Polymerase Chain Reaction in the detection of Lophomonas blattarum

Objective: Since the first reported case of Lophomonas blattarum (L. blattarum) infection in humans of 1993, subsequent cases had primarily been identified through microscopic examination until 2019. That year marked a turning point, as the introduction of polymerase chain reaction (PCR) primers specifically designed for L. blattarum enabled molecular-level identification and led to a growing number of reported studies. However, several concerns persist regarding potential misdiagnoses, as some researchers suggest the parasite may be confused with ciliated bronchial epithelial cells. Although PCR initially appeared to offer a promising solution to these diagnostic challenges, subsequent analyses revealed that the primers used lack specificity for Lophomonas and amplify commensal trichomonads such as Pentatrichomonas hominis and Tetratrichomonas spp. In this study, we aimed to investigate the presence of L. blattarum in bronchoalveolar lavage fluid (BAL) samples using molecular methods. Method: We examined BAL samples submitted to the Microbiology Laboratory of Gazi University Hospital for routine microbiological analysis between September 2022 and May 2023. We performed initial microscopic examination using saline and native-Lugol staining methods. Samples displaying structures morphologically compatible with L. blattarum were further evaluated with trichrome and Giemsa staining to allow for more detailed structural characterization under the microscope. Samples compatible with L. blattarum trophozoites were subjected to DNA isolation and amplification for PCR. The PCR products were electrophoresed on a 1.5% agarose gel and a band of 214 base pairs was visualized. Bidirectional sequencing of the amplified products was carried out using the Sanger method, and we compared the resulting sequences against the BLASTn database for confirmation. Results: In this study, out of 685 BAL samples (n=685), 14 showed L. blattarum-like morphology under microscopy, and PCR targeting the parasite’s specific gene region was performed on these 14 samples. Electrophoresis revealed bands in only two of these samples. Sequencing of the amplified regions demonstrated homology with Trichomonas tenax sequences in the GenBank database. Conclusion: Given the current lack of conclusive data on the life cycle, transmission routes, pathogenicity, and clinical manifestations of Lophomonas spp., there is no available standardized diagnostic protocol, which highlights the critical need for future research to employ rigorously validated, specific diagnostic methods to ensure patient safety and strengthen the reliability of the existing knowledge.