Research Article: Identification of the Mycobacterium ulcerans Protein MUL_3720 as a Promising Target for the Development of a Diagnostic Test for Buruli Ulcer

Date Published: February 10, 2015

Publisher: Public Library of Science

Author(s): Anita Dreyer, Katharina Röltgen, Jean Pierre Dangy, Marie Thérèse Ruf, Nicole Scherr, Miriam Bolz, Nicholas Jay Tobias, Charles Moes, Andrea Vettiger, Timothy Paul Stinear, Gerd Pluschke, Pamela L. C. Small.

Abstract: Buruli ulcer (BU) caused by Mycobacterium ulcerans is a devastating skin disease, occurring mainly in remote West African communities with poor access to health care. Early case detection and subsequent antibiotic treatment are essential to counteract the progression of the characteristic chronic ulcerative lesions. Since the accuracy of clinical BU diagnosis is limited, laboratory reconfirmation is crucial. However, currently available diagnostic techniques with sufficient sensitivity and specificity require infrastructure and resources only accessible at a few reference centres in the African endemic countries. Hence, the development of a simple, rapid, sensitive and specific point-of-care diagnostic tool is one of the major research priorities for BU. In this study, we have identified a previously unknown M. ulcerans protein, MUL_3720, as a promising target for antigen capture-based detection assays. We show that MUL_3720 is highly expressed by M. ulcerans and has no orthologs in other prevalent pathogenic mycobacteria. We generated a panel of anti-MUL_3720 antibodies and used them to confirm a cell wall location for MUL_3720. These antibodies could also specifically detect M. ulcerans in infected human tissue samples as well as in lysates of infected mouse footpads. A bacterial 2-hybrid screen suggested a potential role for MUL_3720 in cell wall biosynthesis pathways. Finally, we demonstrate that a combination of MUL_3720 specific antibody reagents in a sandwich-ELISA format has sufficient sensitivity to make them suitable for the development of antigen capture-based diagnostic tests for BU.

Partial Text: Buruli ulcer (BU) is a neglected mycobacterial skin disease, reported from tropical and subtropical countries world-wide with highest incidence rates in Western Africa [1]. Populations in rural areas with limited access to health facilities are most affected and often seek medical advice at late disease stages [2]. Advances in the clinical management of BU have shifted options for treatment from surgical resection to combination antibiotic therapy [1]. While PCR analysis targeting the insertion sequence IS2404 has evolved into the gold standard for laboratory diagnosis of BU, this test is only available at a few reference centres. Therefore, the diagnosis of BU is currently often based on clinical findings and antibiotic therapy is started before laboratory diagnostic results can be obtained. BU has a wide range of clinical manifestations including non-ulcerative forms such as subcutaneous nodules or papules, plaques and oedema, which may progress to chronic ulcerative lesions. Due to this diversity of disease presentations the accuracy of clinical diagnosis is limited [1, 3–5] and thus a significant proportion of patients reporting with skin lesions may not receive adequate treatment. This includes cases of cutaneous tuberculosis which may be misdiagnosed as BU and thus receive the recommended eight week course of Streptomycin/Rifampicin combination chemotherapy for BU [5], which is much too short for the treatment of tuberculosis. As for IS2404 PCR, two of the other three currently applied methods for laboratory reconfirmation of BU—histopathology and cultivation of the extremely slow-growing mycobacteria—equally require expensive equipment and expertise [4, 6–8] not accessible at peripheral health facilities. The only available point-of-care diagnostic test, direct-smear examination by microscopy for the detection of acid fast bacilli (AFB), has limited sensitivity and specificity [6]. Hence, one of the major research priorities for BU is the development of a fast, low-tech, sensitive and specific point-of-care diagnostic test, which can be directly implemented at peripheral health centres.

Attempts to develop a diagnostic tool based on serological approaches have been equivocal [9–11], so we decided to focus on direct detection of M. ulcerans antigens in BU patient specimens. In the present study, we identified the MUL_3720 protein as a promising target in antigen capture-based diagnostic tests for M. ulcerans. Based on 2D gel electrophoretic analyses, MUL_3720 is one of the most highly expressed proteins in vitro. The high expression of MUL_3720 is considered an advantage with respect to developing a sensitive antigen detection test for the diagnosis of BU.



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