# Development and Validation of In‐House Conventional and Multiplex PCR Methods for the Detection and Identification of Lophomonas spp.: An Innovative Approach

## TL;DR

This study developed and validated new PCR methods to detect and identify Lophomonas species, showing that multiplex PCR is more sensitive and accurate than traditional microscopy.

## Contribution

The development and validation of in-house conventional and multiplex PCR methods for Lophomonas detection and species identification.

## Key findings

- Multiplex-PCR showed 100% sensitivity in detecting Lophomonas infections.
- Multiplex-PCR had strong agreement with conventional PCR (κ = 0.96).
- All positive samples were identified as L. blattarum using multiplex-PCR.

## Abstract

Pulmonary lophomoniasis is an emerging disease caused by the protozoan pathogen Lophomonas spp. Recently, a conventional polymerase chain reaction (PCR) method has been developed. However, its sensitivity and specificity remain to be fully established. Therefore, this study aimed to develop in‐house conventional and multiplex PCR for the detection and identification of Lophomonas infections. Additionally, we attempted to compare the diagnostic performance of these novel PCR tests with the current microscopic examination method using BAL samples.

We studied 120 bronchoalveolar lavage (BAL) specimens of the patients clinically suspected of having lophomoniasis. The specimens were examined using three methods: microscopic examination (Giemsa staining), in‐house conventional PCR, and multiplex‐PCR. Moreover, multiplex‐PCR was used for the simultaneous identification of two species of Lophomonas.

Out of the 120 BAL specimens tested, 30 (25%) tested positive through microscopic wet mount examination. Among the three techniques, multiplex‐PCR was the most sensitive (100%, 95% CI, 88.3–100), while Giemsa staining had the lowest sensitivity (86.2%, 95% CI, 69.4–94.5). The data reveal a strong agreement between multiplex‐PCR and conventional PCR (κ = 0.96), while the lowest agreement was found between multiplex‐PCR and microscopy methods (κ = 0.16). The study also confirmed the presence of L. blattarum species in all samples using multiplex‐PCR.

This study demonstrates that the in‐house multiplex‐PCR is a robust and accurate diagnostic test for the detection and identification of Lophomonas species. Therefore, our findings suggest that this method may be a powerful tool to overcome some diagnostic pitfalls for lophomoniasis.

Sequence alignment of the SSU rRNA of the two Lophomonas species, L. blattarum and
L. striata
. The red rectangle highlights the position of the reverse universal primer; the yellow, the L. blattarum‐specific forward primer; and the green, the
L. striata
‐specific forward primer used in multiplex PCR. The 3′ end of each primer was designed based on targeting nucleotides distinguishing the two species.