A Genotypic Approach for Detection, Identification, and Characterization of Drug Resistance in Mycobacterium ulcerans in Clinical Samples and Isolates from Ghana

Abstract

Abstract. Standardized antimycobacterial therapy is considered the treatment of choice for Buruli ulcer disease. To assess the prevalence of drug resistance among clinical Mycobacterium ulcerans isolates in Ghana, we conducted a sequence-based approach to detect mutations associated with drug resistance. We subjected clinical samples to direct DNA sequencing of rpoB and rpsL genes and compared culture and whole-genome extracts regarding the efficiency of sequence analysis; 99.1% ( rpoB ) and 100% ( rpsL ) of the patients harbored M. ulcerans wild type. In one isolate (0.9%), a point mutation of the rpoB gene at codon Ser522 leading to an amino acid change was detected. Culture extracts yielded a significantly higher sequencing efficiency than whole-genome extracts. Our data suggest a low level of drug resistance in Ghana. However, mutations associated with drug resistance do occur and require monitoring. Improved techniques are necessary to enhance the efficiency of sequence analysis of whole-genome extracts.

Detection of Viable Mycobacterium ulcerans in Clinical Samples by a Novel Combined 16S rRNA Reverse Transcriptase/IS2404 Real-Time qPCR Assay

“…Sequence analysis of 16S rRNA genes from the listed strains revealed 100% nucleotide concordance of the corresponding genomic regions amplified by the 16S rRNA RT-qPCR; no SNPs or mutations were detected, suggesting a high selectivity of the assay. Sequencing primers are described in Table 3 [11].a M. ulcerans cultures were available from previous studies from Kamerun ( n  = 21) and Ghana ( n  = 4) at DITM [8] or were available at KCCR ( n  = 4) from the present study. All strains were of human origin (BUD patients) and confirmed by conventional IS 2404 PCR and sequencing of rpoB - and rpsL -genes that revealed the M. ulcerans Agy99 wild-type sequences (GenBank accession no.…”

“…All strains were of human origin (BUD patients) and confirmed by conventional IS 2404 PCR and sequencing of rpoB - and rpsL -genes that revealed the M. ulcerans Agy99 wild-type sequences (GenBank accession no. CP000325.1) [11], [12].bResults of the 16S rRNA RT-qPCR of mycobacterial RNA extracts.cResults of the IS 2404 qPCR of mycobacterial DNA extracts.dResults of the IS 2404 qPCR of genomic DNA (gDNA) wipeout controls (see Protocols S2 and S3); a positive result indicates gDNA contamination of RNA extracts following DNAse digestions, and a negative result indicates RNA extracts free of gDNA.…”

Comparison of Two Assays for Molecular Determination of Rifampin Resistance in Clinical Samples from Patients with Buruli Ulcer Disease

“…A pilot study on molecular drug resistance testing conducted by our group from 2004 through 2007 in Ghana revealed a low level (0.9%) of RMP resistance. However, the overall test efficiency of the assay applied in the pilot study (referred to here as assay A) was low (35%) (10). Therefore, the aim of this study was to develop an improved sequencing assay (referred to here as assay B).…”

Susceptibility Profiles ofMycobacterium ulceransIsolates to Streptomycin and Rifampicin in Two Districts of the Eastern Region of Ghana

“…Mycobacteria therapy has had its fair share of multidrug resistance (MDR), with a lot of cases being reported particularly for Mycobacterium tuberculosis (MTB) [23, 29–31]. To date, however, there had been few reports of emergent resistant M. ulcerans isolates [8, 32]. This necessitates more studies into resistant M. ulcerans isolates.…”

“…This monitoring activity is very necessary, especially with the current use of antibiotics for BU treatment. Generally, the prevalence of drug resistance among clinical Mycobacterium ulcerans isolates in Ghana has been reported to be low (0.9%) [32]. Meanwhile, there is no guarantee that the rate would continue to remain low.…”