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

Abstract

We compared two DNA extraction methods (a semiautomated method using a Maxwell kit and a modified Boom method) and three amplification procedures (a single-step PCR, a nested PCR, and a real-time quantitative PCR) on 74 surgical tissue specimens from patients with clinically suspected Buruli ulcer. All of these procedures were compared before and after decontamination. We observed that, among the procedures tested, real-time PCR after the modified Boom extraction method or a single-run PCR assay after the Maxwell 16 extraction method, performed on nondecontaminated suspensions, are the best for the molecular diagnosis of Mycobacterium ulcerans disease. Mycobacterium ulcerans disease, commonly called Buruli ulcer (BU), is a skin disease mainly endemic to certain riverine areas of West and Central Africa (4,12). The disease may present with a diverse range of clinical symptoms, and, due to a possible confusion with other tropical skin diseases, a diagnosis based strictly on clinical observation is not always accurate, leading to the necessity of confirmatory tests such as microscopy, culture, histopathology, and PCR (3, 16).Microscopy is comparatively straightforward to perform, and the materials and skills required are available in regions of endemicity; but diagnosis based on microscopy lacks sensitivity (16). Therefore, even when samples are negative when tested by microscopy, another test should be carried out to confirm the diagnosis. 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. However, only a few studies have evaluated these methods (5). Nevertheless, such an evaluation would be essential to identify the best method for the detection of M. ulcerans by PCR.Since several laboratories which perform PCR also routinely perform culture, PCR is sometimes carried out on suspensions of specimens that have been subjected to a decontamination protocol in preparation for culture. To our knowledge, the effects of this decontamination step on PCR results have not, to date, been investigated.In this study, we compared in two separate laboratories two extraction methods (a semiautomated method using the Maxwell 16 kit [Promega, Leiden, The Netherlands] and a modification of the method of Boom [11]) and three amplification procedures (a single-step PCR, a nested PCR, and a real-time quantitative PCR) routinely performed on surgical tissue specimens from patients with suspected BU. All of these procedures were applied o...

Implementation of a National Reference Laboratory for Buruli Ulcer Disease in Togo

“…Whereas until the early 2000s laboratory diagnostic services for endemic countries were mainly provided by external reference laboratories, until 2011 six African countries (Ivory Coast, Ghana, Benin, Cameroon, Central African Republic, Democratic Republic of Congo) installed their own reference laboratories upon increasing demand for local diagnostic capacity [6], [10], [11], [18], [20]–[26], [29], [30], [32], [37], [44]–[46]. Due to the absence of laboratory facilities a number of countries still require support from external reference laboratories; in general however, the role of external reference laboratories has shifted to development of improved laboratory techniques for application in endemic countries, technical support and training of local laboratory staff, as well as external quality assurance for newly established reference laboratories [6], [11], [21], [23]–[32], [37]–[40], [43].…”

“…In the case of Togo, extensive preparatory work conducted in the context of previous research projects by DAHWT and DITM [13], vast expertise gained from a longstanding cooperation with partners in Ghana [21], [23], [25], [26], [29], [40], as well as continuous exchange of information with other “BuruliVac” partners [6], [32] facilitated the implementation of a national reference laboratory considerably.…”

“…Whereas microscopy is an appropriate and cost-effective first-line test for peripheral laboratories, PCR is considered the method of choice and WHO recommends PCR confirmation of at least 50% of suspected BUD cases [3], [7]–[13]. Microscopy and various PCR assays have been successfully implemented in other endemic countries and case confirmation rates of 29–78% (microscopy) and 54–83% (PCR) were reported [10], [12]–[32].…”

Transdisciplinary Research and Action to Stop Buruli Ulcer: A case Study from Philanthropy

“…An illustrative and open access video-guide of how-to-do-it and WHO supported teaching and training activities improved the accuracy of diagnosis undertaken by qualified health staff in BU affected countries. In addition, optimization of PCR-based diagnosis by ITM improved laboratory confirmation of clinical cases [6,7].…”

Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities

“…The two automated extraction platforms tested were the semi-automated QuickGene-Mini80™ (Autogen/FujiFilm, Holliston, MA) and the automated Maxwell®16 (Promega, Madison, WI) platforms. Both systems were designed to extract nucleic acids from various tissues and cell types in clinical labs (Affolabi et al 2012) and both systems have proven successful for a wider variety of additional sample types including spores (Shipley et al 2012), plant leaves, seeds, and fungi (Affolabi et al 2012; Foley et al 2011; Khokhar et al 2011; Schagat et al 2007). The manual kit, PowerBiofilm DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA), was designed to extract DNA from biofilm material and is representative of the kits widely used by environmental microbiologists (Ferrando and Tarlera 2009; McBeth et al 2010).…”