A qPCR identification scheme to differentiate actinomycetoma from eumycetoma

Area of Science:

• Medical Diagnostics
• Infectious Diseases
• Molecular Biology

Background:

• Mycetoma is a Neglected Tropical Disease causing debilitating skin lesions.
• Distinguishing between fungal (eumycetoma) and bacterial (actinomycetoma) causes is crucial for effective treatment.
• Current diagnostic methods lack the speed required for timely intervention.

Purpose of the Study:

• To develop a rapid quantitative polymerase chain reaction (qPCR) assay.
• To differentiate between actinomycetoma and eumycetoma.
• To identify the causative agents of actinomycetoma to the genus level.

Main Methods:

• Development of singleplex, triplex, and quadruplex qPCR assays.
• Validation of assays using a comprehensive collection of mycetoma strains and clinical samples (grains).
• Assessment of limit of detection, sensitivity, and specificity for each assay component.

Main Results:

• The developed qPCR assays demonstrated high sensitivity and specificity (100% for most combinations).
• The limit of detection varied by probe, with fungal probes at 0.000002 ng and Nocardia genus probes at 0.002 ng in singleplex reactions.
• Minor cross-reactivity was observed with Actinomadura and Streptomyces probes, primarily with non-mycetoma agents.

Conclusions:

• The developed qPCR test effectively differentiates mycetoma types and identifies causative agents.
• The assay meets the World Health Organization's Target Product Profile requirements for mycetoma diagnostics.
• This rapid diagnostic tool can significantly reduce identification time to a few hours, enabling field use with portable qPCR devices.