Dry-Reagent-Based PCR as a Novel Tool for Laboratory Confirmation of Clinically Diagnosed Mycobacterium ulcerans -Associated Disease in Areas in the Tropics Where M. ulcerans Is Endemic

Abstract

After tuberculosis and leprosy, Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial disease in immunocompetent humans. The disease occurs in tropical countries, with foci in West Africa, Central Africa, and the western Pacific. BU is defined as an infectious disease involving the skin and the subcutaneous adipose tissue characterized by a painless nodule, papule, plaque, or edema, evolving into a painless ulcer with undermined edges and often leading to invalidating sequelae. Due to the fundamental lack of understanding of modes of transmission, disease control in endemic countries is limited to early case detection through improved active surveillance and surgical treatment. The laboratory confirmation of BU is complicated by the absence of a diagnostic "gold standard." Therefore, misclassification and delayed diagnosis of BU may occur frequently, causing a considerable socioeconomic impact in terms of treatment costs due to prolonged hospitalization. In order to respond to the urgent need to develop reliable tools for early case detection and to overcome technical difficulties accompanying the implementation of diagnostic PCR procedures in tropical countries, a dry-reagent-based PCR formulation for the detection of M. ulcerans in diagnostic specimens has been developed at the Bernhard Nocht Institute for Tropical Medicine. Following technical and clinical validation, the assay has been successfully installed and field tested at the Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi, Ghana. Preliminary results show an excellent diagnostic sensitivity of >95%.

Differential Diagnosis of Ulcerative Cutaneous Lesions: the Emergence of Buruli Ulcer in Western European Countries

“…Despite the inconclusive PCR results for M. ulcerans and the absence of isolation in culture, positive acid-fast stain in the lesions biopsies with negative PCR for other mycobacteria and the compatible histopathology (with a sensitivity of up to 90%), reinforced the diagnostic hypothesis. In fact, it is known that transporting the sample in suboptimal conditions may lead to inconclusive results in the PCR (in the present case, the sample was transported for processing in other, distant, units, and no specific reagents or transportation buffer solutions were used) [10]. Additionally, the growth in culture is very difficult, with a sensitivity of 20-60% [11].…”

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

“…Briefly, DNA was prepared using the Puregene DNA isolation kit (Gentra Systems) as described elsewhere ( Table 1 ). 19 Amplification of rpoB and rpsL genes. A partial sequence of the mycobacterial rpoB gene (342 bp) encompassing the RRDR was amplified by Mycobacterium genus-specific primers as described by Kim and others.…”

“…Mycobacterial cultures were subjected to a confirmatory IS2404 PCR. 3,5,[18][19][20][21] Samples subjected to sequence analysis. Suspensions of IS2404 PCR confirmed M. ulcerans cultures ( N = 87) dissolved in 700 μL Cell Lysis Solution (Qiagen, Hilden, Germany ) followed by inactivation at 80°C for 20 minutes, and IS2404 PCR-positive whole-genome extracts (total of 156 genome extracts) derived from swab ( N = 68) and tissue samples ( N = 88) were subjected to sequence analysis of rpoB and rpsL genes at the Department of Infectious Diseases and Tropical Medicine, University of Munich (DITM).…”

Effects of Decontamination, DNA Extraction, and Amplification Procedures on the Molecular Diagnosis of Mycobacterium ulcerans Disease (Buruli Ulcer)

“…Cultivation of M. ulcerans also lacks sensitivity and takes time to give results (6), rendering it less useful for the routine management of patients. Histopathology is sensitive, specific for BU, and helpful for differential diagnosis but is rarely available in countries where BU is endemic due to a lack of specialists trained and experienced in this technique (16).PCR has been shown to be sensitive as well as specific and is increasingly used for BU diagnosis in countries of endemicity (10,14). Various commercial and in-house DNA extraction and amplification procedures are used, mainly targeting the insertion sequence IS2404 of the M. ulcerans genome.…”

“…PCR has been shown to be sensitive as well as specific and is increasingly used for BU diagnosis in countries of endemicity (10,14). Various commercial and in-house DNA extraction and amplification procedures are used, mainly targeting the insertion sequence IS2404 of the M. ulcerans genome.…”