Development and Application of Two Multiplex Real-Time PCR Assays for the Detection of
            <i>Mycobacterium ulcerans</i>
            in Clinical and Environmental Samples

Fyfe, Janet; Lavender, Caroline J.; Johnson, Paul; Globan, Maria; Sievers, Aina; Azuolas, Joseph; Stinear, Timothy P.

doi:10.1128/aem.02971-06

Cited by 210 publications

(340 citation statements)

References 31 publications

Supporting

Mentioning

330

Contrasting

Unclassified

Order By: Relevance

“…hominissuis IS901 and IS1245 are used most commonly . For direct detection of M. ulcerans and M. marinum, PCR and real time PCR system specific for the insertion sequences IS2404 and IS2606 has been described (Fyfe et al 2007). For total mycobacterial DNA analysis in soil, different attempts have been made at DNA isolation.…”

Section: Identification Of Mycobacteriamentioning

confidence: 99%

Mycobacteria in water, soil, plants and air: a review

Hruška¹,

Kaevska²

2012

Vet. Med.

View full text Add to dashboard Cite

Amazingly, despite the 24 143 papers on mycobacteria, indexed in the Web of Science database during the last six years, published by 67 008 authors from 13 128 organizations located in 166 countries or territories, internationally accepted legal directives on how to control the public health risk associated with environmental mycobacteria have yet to be developed. Mycobacteria are human and animal pathogens, causing not only tuberculosis and leprosy, but mycobacterioses of skin, soft tissues and lung. Due to their cell wall composition and their adaptability mycobacteria can survive in different habitats for years. Their immunomodulatory ability has been recognised for more than 50 years and hundreds of papers published during the last two decades have demonstrated that small chemical products derived from mycobacterial cells participate in inflammatory pathways involved the pathogenesis of important human diseases like Crohn's disease, asthma, type 1 diabetes mellitus, psoriasis, arthrosis, Blau syndrom, sarcoidosis, autism etc. Mycobacteria can influence inflammatory pathways not only as live organisms, but also by means of components derived from dead cells. Pasteurisation or cooking does not affect this ability. Hence, how many mycobacterial cells are ingested, what factors play a role concurrently, and how long the harmful effect persists become important questions. This paper presents only a short review based on selected papers about mycobacteria in water, soil, plants and air with the aim of attracting attention to this significant global problem and of making the first steps towards protection of people. Selected bibliographic references of published data from 2007 to 2012 are presented in easy-to-navigate tables.Keywords: Mycobacterium; water; soil; plant; vegetables; air; biofilm; sediment; determination; zoonoses; food safety List of abbreviations CFU = colony forming units; DGGE = denaturation gradient gel electrophoresis; IMS = immuno-magnetic separation; IS = insertion sequence; ITS = intergenic transcribed spacer; MAC = Mycobacterium avium complex; MAP/Map = Mycobacterium avium subsp. paratuberculosis; MTC = Mycobacterium tuberculosis complex; MWF = metal working fluids; nPCR = nested PCR; NTM = non-tuberculous mycobacteria; PCR = polymerase chain reaction; PPM = potentially pathogenic mycobacteria; qPCR = quantitative real time PCR; RFLP = restriction fragment length polymorphism

show abstract

Section: Identification Of Mycobacteriamentioning

confidence: 99%

Mycobacteria in water, soil, plants and air: a review

Hruška¹,

Kaevska²

2012

Vet. Med.

View full text Add to dashboard Cite

show abstract

“…15 A previously reported M. ulcerans mouse model was used with modifications. 16 The animal model protocol no.…”

mentioning

confidence: 99%

Experimental Survival of Mycobacterium ulcerans in Watery Soil, a Potential Source of Buruli Ulcer

Tian¹,

Lépidi²,

Nappez³

et al. 2016

The American Society of Tropical Medicine and Hygiene

View full text Add to dashboard Cite

Abstract. The reservoir of Mycobacterium ulcerans causing Buruli ulcer (BU) remains unknown. Here, sterilized watery soil was mixed with 2 × 10 6 colony-forming units (CFU)/g of M. ulcerans Agy99 or M. ulcerans ATCC 33728 and incubated in a microaerophilic atmosphere in the presence of negative controls. Both M. ulcerans strains survived in soil for 4 months with a final inoculum of 300-440 CFU/g. Further, three groups of five mice with and without footpad scarification were exposed to control soil or M. ulcerans-inoculated soil. Although no specific clinical and histopathological lesions were observed in control animals, red spots observed on 8/20 scarified feet in 8/10 challenged mice yielded inflammatory infiltrates and positive real-time polymerase chain reaction detection of M. ulcerans DNA in five mice. BU can be acquired as an inoculation infection with watery soil as a transient source of infection. These experimental observations warrant additional field observations. Moreover, the only environmental M. ulcerans isolate was recovered from a water strider in Benin. 7 These observations suggest a link between BU and stagnant and slow-flowing aquatic environments in West Africa. However, freshwater itself may not be the ultimate source of M. ulcerans. In particular, fishermen who are presumably in frequent contact with water have a lower prevalence of BU than other groups in endemic regions in Africa. 8,9 In addition, the M. ulcerans inoculum has been estimated to be 100-1,000 mycobacteria/mL in water, based on real-time polymerase chain reaction (RT-PCR) targeting IS2404.3 This estimated inoculum level is three to four times lower than the levels reported for major waterborne human pathogens such as Vibrio. 10We have recently shown that Mycobacterium tuberculosis, a Mycobacterium that is phylogenetically closely related to M. ulcerans, retains its infectivity after an extended stay in soil. 11 We therefore hypothesized that watery soil could be a source of M. ulcerans.To test the hypothesis, M. ulcerans ATCC 19423 TMT (strain Agy99) and M. ulcerans ATCC 33728 (both obtained from the American Type Culture Collection, Masassas, VA) were cultured in an incubator at 32°C on Middlebrook 7H10 Agar (Becton Dickinson, Le Pont de Claix, France), supplemented with OADC (oleic acid, bovine albumin, dextrose, and catalase) (Becton Dickinson). Natural soil was steam sterilized at 134°C for 15 minutes, and the sterility was then assessed by inoculating 2 g of steam-sterilized soil onto Middlebrook 7H10 Agar incubated at 32°C for 4 weeks. A 20-g sample of sterilized soil was inoculated and mixed with 2 × 10 6 colonyforming units (CFU)/g of each of the two M. ulcerans isolates, placed into 25-cm 2 cell culture flasks (Corning, Corning, NY) and thoroughly mixed. Three flasks were prepared for each M. ulcerans strain. The flasks were placed inside a transparent sterile plastic container where they were exposed to a microaerophilic atmosphere created using a CampyGen atmosphere generation system (Oxoid Ltd., Basingstoke, Un...

show abstract

“…Of the nondecontaminated suspensions, the single-run PCR had higher positivity rates than the nested PCR and rates very similar to those of the real-time quantitative PCR, known to be very sensitive (7). One would expect a nested PCR assay to be more sensitive than a single-step PCR assay.…”

Section: Discussionmentioning

confidence: 94%

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

et al. 2012

View full text Add to dashboard Cite

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...

show abstract

Development and Application of Two Multiplex Real-Time PCR Assays for the Detection of Mycobacterium ulcerans in Clinical and Environmental Samples

Cited by 210 publications

References 31 publications

Mycobacteria in water, soil, plants and air: a review

Mycobacteria in water, soil, plants and air: a review

Experimental Survival of Mycobacterium ulcerans in Watery Soil, a Potential Source of Buruli Ulcer

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

Contact Info

Product

Resources

About