A qPCR identification scheme to differentiate actinomycetoma from eumycetoma

BACKGROUND: Mycetoma is a Neglected Tropical Disease of the skin. It is characterized by large tumorous lesions in the subcutaneous tissue which hinder the patients in their daily activities. The disease can be caused by either bacteria (actinomycetoma) or fungi (eumycetoma), and treatment depends on the entity. Therefore, in 2022, a Target Product Profile was published by the World Health Organisation in which it was stated that there is an urgent need to develop a diagnostic tool that can differentiate the two reliably within a day. OBJECTIVES: To develop a qPCR that can not only differentiate actinomycetoma from eumycetoma but can also identify the actinomycetoma causative agents to the genus level. METHODS: We developed singleplex, quadruplex and triplex qPCRs that can be used, depending on the endemic region and validated this against a large strain collection and grains. RESULTS: The limit of detection (LOD) of each of the probes ranged from 0.000002 ng for the fungal probe to 0.002 ng for the Nocardia genus probe in a singleplex reaction. Higher LODs were obtained in the multiplexes. The sensitivity and specificity were both 100% for most combinations, although some cross-reactivity with the Actinomadura and Streptomyces genus probes was noted, mainly with non-mycetoma causative agents. CONCLUSION: The developed qPCR meets the requirements set in the Target Product Profile from the World Health Organisation. IMPORTANCE: In this study a qPCR is developed which can differentiate actinomycetoma from eumycetoma and identify actinomycetoma causative agents to the genus level. This qPCR adheres to the minimal requirements set in the Target Product Profile for a mycetoma diagnostic test by the World Health Organisation. When the qPCR is used on mycetoma grain it reduces the time to identification to only a few hours. With hand-held qPCRs this can also be performed in the field.