Study participants, clinical information, and diagnostic results.

Table 6 shows suspected BUD cases with ulcerative lesions enrolled in the pre-treatment cohort (Figure 1), clinical information, and diagnostic results. Swab samples from 24 suspected BUD cases were subjected to 16S rRNA RT/IS2404 qPCR viability assay (swab 1 in PANTA), microscopic examination and enumeration of acid fast bacilli (AFB) following Ziehl-Neelsen staining (swab 2, direct smear), and conventional IS2404 dry-reagent-based (DRB) PCR (swab 3 in Cell Lysis Solution [Qiagen]). 18 patients were laboratory confirmed by IS2404 qPCR and 15 out of those were RNA positive; the quantification by IS2404 qPCR revealed a bacillary load (1–2 bacilli per sample) below the lower limit of detection of the RNA assay for samples from three RNA negative patients. All samples from six IS2404 qPCR negative study participants were also RNA negative. Direct correlation of AFB enumeration with IS2404 qPCR quantification is not feasible due to inhomogeneous distribution of M. ulcerans in different clinical samples. NA, not applicable; Neg, negative test result; Pos, positive test result.aResults of the 16S rRNA RT/IS2404 qPCR viability assay. Clinical swab samples in PANTA were directly processed at KCCR, and M. ulcerans DNA and cDNA were transported to DITM and subjected to qPCR.bRoutine diagnostics were conducted following standardized procedures at KCCR [3].cNo., consecutive number of study participants.dYes, IS2404 qPCR confirmed BUD patients; No, IS2404 negative study participants.eDuration of disease before presentation of study participants in weeks.fCategory of lesion according to the World Health Organization's clinical criteria [1].gResults of the IS2404 qPCR with corresponding cycle threshold (Ct)-values.hThe bacillary load in the respective swab samples (No. 2) was estimated on the basis of IS2404 quantification given an IS2404 copy number of 209 copies per M. ulcerans genome [9]. For bacterial numbers iResults of the 16S rRNA RT-qPCR.kMIC, microscopic detection and enumeration of AFB was conducted at KCCR including external quality assurance by DITM. The following scale was applied: 0 = negative, +1 = 10–99 AFB/100 fields, +2 = 1–10 AFB/1 field, +3 = more than 10 AFB/1 field.lPCR, conventional, single target gel-based IS2404 DRB PCR.