Accuracy of the Polymerase Chain Reaction of Ulnar Perineural Subcutaneous Aspirate Guided by Ultrasound for the Diagnosis and Monitoring of Leprosy Cure

Overview

\*\*Brief Summary\*\* Leprosy is a chronic granulomatous infectious disease caused by \*Mycobacterium leprae\* or \*Mycobacterium lepromatosis\*, characterized by peripheral nerve involvement that may lead to progressive neurological damage, disability, and deformities if not diagnosed and treated early. The diagnosis of leprosy is primarily clinical and epidemiological, supported by laboratory methods such as bacilloscopy and biopsy; however, these tests have limited sensitivity, particularly due to the bacillus' tropism for peripheral nerve structures. Ultrasonography has emerged as a non-invasive imaging method capable of detecting morphological changes in peripheral nerves, including nerve enlargement, fascicular abnormalities, and inflammatory hypervascularization. Despite its diagnostic value, ultrasonography alone cannot detect the presence of the bacillus. This prospective cohort study aims to evaluate the diagnostic and prognostic accuracy of combining clinical evaluation, peripheral nerve ultrasonography, and molecular detection techniques using subcutaneous perineural aspirate. Patients with suspected leprosy attending the Leprosy Outpatient Clinic at the University Hospital of Brasília will undergo clinical evaluation, ultrasound examination of the ulnar nerves, and ultrasound-guided subcutaneous perineural aspirate for molecular detection of \*Mycobacterium leprae\* DNA and RNA using real-time PCR and RT-PCR. Participants will be followed for one year, with assessments performed at diagnosis and after one year of treatment. The study will compare clinical, imaging, and molecular findings to determine whether perineural subcutaneous aspirate combined with ultrasonography improves early detection and diagnostic accuracy compared with conventional methods such as bacilloscopy and biopsy. The study aims to contribute to improved diagnostic strategies for leprosy, enabling earlier detection of neural involvement and potentially reducing disease transmission and long-term disability.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae and is characterized by skin and peripheral nerve involvement. Neural impairment is the main determinant of disability and long-term morbidity. Early diagnosis remains challenging because current diagnostic methods have important limitations. Slit-skin smear microscopy and skin biopsy have low sensitivity in many clinical settings, especially in paucibacillary and primary neural forms. Nerve palpation is widely used but lacks reproducibility and does not distinguish among etiologies of neuropathy. Nerve biopsy, although more precise in selected cases, is invasive and may cause permanent neurological sequelae. High-resolution ultrasonography has emerged as a promising tool for evaluation of leprosy neuropathy because it allows objective assessment of peripheral nerve cross-sectional area, asymmetry, fascicular abnormalities, and intraneural or epineural hypervascularity using Doppler. However, ultrasound cannot directly detect the bacillus. Molecular methods have expanded the diagnostic possibilities in leprosy. Real-time PCR targeting the repetitive element RLEP can detect M. leprae DNA with high sensitivity. However, DNA detection alone does not distinguish viable from non-viable bacilli. Detection of RNA by RT-PCR, including targets such as 16S rRNA and sodA, may provide evidence of bacillary viability because RNA degrades rapidly after cell death. This approach may improve treatment monitoring and refine the concept of cure. The present study proposes a minimally invasive strategy based on ultrasound-guided ulnar perineural subcutaneous aspirate. Biological material will be collected adjacent to the affected nerve, avoiding direct nerve biopsy and potentially increasing the sensitivity of molecular detection in the neural microenvironment. Participants with suspected leprosy will be assessed clinically, by high-resolution ultrasound of the ulnar nerves, and by molecular detection of M. leprae DNA and RNA in aspirated material. They will be evaluated at baseline, near the time of diagnosis, and after one year of treatment. The main objective is to determine the diagnostic accuracy of ultrasound-guided perineural aspirate PCR/RT-PCR compared with conventional methods. Secondary objectives include correlation of ultrasound abnormalities with molecular positivity, assessment of bacillary viability over time, and development of workflows potentially applicable to the public health system, including referral centers and settings using portable ultrasound.