A qPCR identification scheme to detect the most common causative agents of actinomycetoma in Africa
8 affiliations
- 1Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
- 2ErasmusMC, Department of Medical Microbiology and Infectious Diseases, Rotterdam, The Netherlands
- 3Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan
- 4The Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan
- 5Faculty of Medical laboratory Sciences, University of Khartoum, Khartoum, Sudan
- 6Centre de Recherche sur les maladies Tropicales Négligées, UFR Sciences de la Santé, Université Gaston Berger, Saint-Louis, Senegal
- 7Department of medical microbiology and parasitology, faculty of Medical Laboratory sciences, University of Dongola, Dongola, Sudan
- 8Sydney Institute for Infectious Diseases, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia
Abstract
Mycetoma is a neglected tropical disease characterized by mutilating tumorous lesions in the subcutaneous tissue. The causative agents are found embedded in granules called grains. Mycetoma is either caused by bacteria (actinomycetoma) or fungi (eumycetoma). To initiate the appropriate treatment, it is important to identify the causative agent rapidly and molecular identification for eumycetoma revolutionized the time to identification. For actinomycetoma this was not possible yet. Here we developed a multiplex qPCR identification scheme for the most common causative agents of actinomycetoma in Africa. Whole genome sequencing was used to identify species-specific gene families for Actinomadura madurae, Actinomadura pelletieri, Streptomyces somaliensis and Streptomyces sudanensis. qPCR primers and probes were developed on these species and validated against DNA isolated from mycetoma strains and grains. Each probe was unique with no cross-reactivity with other tested species. The limit of detection ranged from 0.000013 to 0.00067 ng bacterial DNA. When the qPCRs were validated against 28 grain samples, all fungal grains remained negative and 11 out of 12 Actinomadura grains were correctly identified. This resulted in a sensitivity of 85.7% for the A. pelletieri probe and a specificity of 100%. For the A. madurae probe, a sensitivity and specificity of 100% was obtained. The actinomycetoma qPCR developed in this study can be used to identify the most common causative agents of actinomycetoma in Africa. Author summary: Mycetoma is a devastating disease with large tumorous lesions. It can be caused by fungi and bacteria. Most laboratories use either histology and/or culturing to identify the causative agents, but these procedures can take up to six weeks till a positive identification is obtained. Recently we demonstrated that qPCR can be used directly on mycetoma grains and can be used to discriminate actinomycetoma from eumycetoma within 2 hours. Furthermore, for actinomycetoma identification to the genus level can be achieved with this qPCR. However, if a species identification was needed there were no qPCR assays available to achieve this. Therefore, in this project we aimed to develop qPCR methods to identify the most common causative agents of actinomycetoma in Africa. Instead of using classical barcode genes, we searched the genomes of Actinomadura madurae, Actinomadura pelletieri, Streptomyces somaliensis and Streptomyces sudanensis to find conserved unique genes for each lineage. qPCRs were designed on these genes and validated against a large cohort of fungal and bacterial mycetoma causative agents. No cross reaction was observed. Next, we also validated these qPCRs against the mycetoma grains. In these clinical samples a sensitivity of 85.7% and a specificity of 100% was achieved. The next step is to validate this qPCR identification scheme throughout different mycetoma endemic regions in Africa and beyond.
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BibTeX
@article{watson2026qpcr,
title = {{A qPCR identification scheme to detect the most common causative agents of actinomycetoma in Africa}},
author = {Andrew K. Watson and Sahar Bakhiet and Emmanuel E. Siddig and Carole Pab Minlekib and Rihab Mohammed and Willemien Zandijk and Mickey Konings and Ahmed H. Fahal and Maguette Faye and Katarzyna Mickiewicz and Fatehmeh Mazraati Tajabadi and Doudou Sow and Jeffrey Errington and Wendy W. J. van de Sande},
journal = {PLOS Neglected Tropical Diseases},
year = {2026},
month = sep,
doi = {10.1371/journal.pntd.0014710},
url = {https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014710},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A qPCR identification scheme to detect the most common causative agents of actinomycetoma in Africa
AU - Andrew K. Watson
AU - Sahar Bakhiet
AU - Emmanuel E. Siddig
AU - Carole Pab Minlekib
AU - Rihab Mohammed
AU - Willemien Zandijk
AU - Mickey Konings
AU - Ahmed H. Fahal
AU - Maguette Faye
AU - Katarzyna Mickiewicz
AU - Fatehmeh Mazraati Tajabadi
AU - Doudou Sow
AU - Jeffrey Errington
AU - Wendy W. J. van de Sande
PY - 2026
DA - 2026/09/08
JO - PLOS Neglected Tropical Diseases
DO - 10.1371/journal.pntd.0014710
UR - https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014710
AB - Mycetoma is a neglected tropical disease characterized by mutilating tumorous lesions in the subcutaneous tissue. The causative agents are found embedded in granules called grains. Mycetoma is either caused by bacteria (actinomycetoma) or fungi (eumycetoma). To initiate the appropriate treatment, it is important to identify the causative agent rapidly and molecular identification for eumycetoma revolutionized the time to identification. For actinomycetoma this was not possible yet. Here we developed a multiplex qPCR identification scheme for the most common causative agents of actinomycetoma in Africa. Whole genome sequencing was used to identify species-specific gene families for Actinomadura madurae, Actinomadura pelletieri, Streptomyces somaliensis and Streptomyces sudanensis. qPCR primers and probes were developed on these species and validated against DNA isolated from mycetoma strains and grains. Each probe was unique with no cross-reactivity with other tested species. The limit of detection ranged from 0.000013 to 0.00067 ng bacterial DNA. When the qPCRs were validated against 28 grain samples, all fungal grains remained negative and 11 out of 12 Actinomadura grains were correctly identified. This resulted in a sensitivity of 85.7% for the A. pelletieri probe and a specificity of 100%. For the A. madurae probe, a sensitivity and specificity of 100% was obtained. The actinomycetoma qPCR developed in this study can be used to identify the most common causative agents of actinomycetoma in Africa. Author summary: Mycetoma is a devastating disease with large tumorous lesions. It can be caused by fungi and bacteria. Most laboratories use either histology and/or culturing to identify the causative agents, but these procedures can take up to six weeks till a positive identification is obtained. Recently we demonstrated that qPCR can be used directly on mycetoma grains and can be used to discriminate actinomycetoma from eumycetoma within 2 hours. Furthermore, for actinomycetoma identification to the genus level can be achieved with this qPCR. However, if a species identification was needed there were no qPCR assays available to achieve this. Therefore, in this project we aimed to develop qPCR methods to identify the most common causative agents of actinomycetoma in Africa. Instead of using classical barcode genes, we searched the genomes of Actinomadura madurae, Actinomadura pelletieri, Streptomyces somaliensis and Streptomyces sudanensis to find conserved unique genes for each lineage. qPCRs were designed on these genes and validated against a large cohort of fungal and bacterial mycetoma causative agents. No cross reaction was observed. Next, we also validated these qPCRs against the mycetoma grains. In these clinical samples a sensitivity of 85.7% and a specificity of 100% was achieved. The next step is to validate this qPCR identification scheme throughout different mycetoma endemic regions in Africa and beyond.
SN - 1935-2735
ER -