In this project, we developed two pan-genus Tropheryma PCRs from shotgun sequencing data to detect new species of Tropheryma.

Background: Tropheryma whipplei is responsible of the classical Whipple's disease and other localized infections such as endocarditis. A new Tropheryma species was recently detected in a kidney transplant patient with recurrent pleuritis. We developed and implemented two broad-range pan-Tropheryma genus RT-PCRs aiming to detect T. whipplei, but also new Tropheryma species. Targets genes were 23S rRNA and rnpB genes. Both PCRs exhibited good analytical sensitivity, specificity and reproducibility. Here we present their clinical performances and describe a case of Tropheryma Infection due to another species of the genus. Methods: Demands for T. whipplei PCR were tested with both T. whipplei specific PCRs and Tropheryma broad-range PCRs from January 2019 to November 2022 (paused during the COVID-19 pandemics). The rnpB PCR was not performed after March 2020 due to similar performances with the 23S rRNA PCR. Results: In total, 2605 samples were tested with the broad-range 23S rRNA and 834 with both 23S rRNA and the rnpB PCRs. Sensitivity was 78.8% and 79.7% for the 23S rRNA and the rnpB PCRs, respectively. Mean CT values of the T. whipplei specific PCRs when broad-range PCRs were negative were 38.7+/-2.5. Specificity was 99.9% and 99.7% for the 23S rRNA and the rnpB PCRs, respectively. Cohen's kappa was 0.905 between the two PCRs, indicating a high level of agreement. We identified a patient where bronchoalveolar lavage and bronchial aspirate were both positive with the two broad-range PCRs with >105 copies/ml. Specific T. whipplei PCRs were all negative. Known for a panuveitis and ocular vasculitis, this 49-year-old male presented with a recurrence necessitating the introduction of oral prednisone. During work-up, the patient underwent a CT-scan showing multiple mediastino-hilar necrotic adenopathies. Transthoracic biopsies exhibited aspecific histology. Doxycyclin (1-year), hydroxychloroquin (1-year) and co-trimoxazol (1-month) treatments led to good clinical and radiological outcome. Conclusions: Real-world sensitivity of the specific T. whipplei PCR was better than the broad-range PCRs close to the limit of detection (100 to 1000 copies/ml), by targeting repeated elements. However, broad-range Tropheryma PCRs demonstrated excellent specificity and remain the only way to identify new cases of Tropheryma sp. infections, where there is a knowledge gap regarding their clinical burden.