Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

doi:10.1128/JCM.01544-15

Comparative Study

. 2015 Oct;53(10):3280-5.

doi: 10.1128/JCM.01544-15. Epub 2015 Aug 5.

Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

Sarah A Ahmed ¹, Wendy W J van de Sande ², Marie Desnos-Ollivier ³, Ahmed H Fahal ⁴, Najwa A Mhmoud ⁴, G S de Hoog ⁵

Affiliations

¹ Faculty of Medical Laboratory Sciences, University of Khartoum, Khartoum, Sudan CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Netherlands.
² Erasmus MC, Department of Medical Microbiology and Infectious Diseases, Rotterdam, Netherlands.
³ Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycologie et Antifongiques, Paris, France.
⁴ Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan.
⁵ Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China Basic Pathology Department, Federal University of Paraná State, Curitiba, Paraná, Brazil King Abdulaziz University, Jeddah, Saudi Arabia s.hoog@cbs.knaw.nl.

PMID: 26246484
PMCID: PMC4572522
DOI: 10.1128/JCM.01544-15

Comparative Study

Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

Sarah A Ahmed et al. J Clin Microbiol. 2015 Oct.

. 2015 Oct;53(10):3280-5.

doi: 10.1128/JCM.01544-15. Epub 2015 Aug 5.

Authors

Sarah A Ahmed ¹, Wendy W J van de Sande ², Marie Desnos-Ollivier ³, Ahmed H Fahal ⁴, Najwa A Mhmoud ⁴, G S de Hoog ⁵

Affiliations

¹ Faculty of Medical Laboratory Sciences, University of Khartoum, Khartoum, Sudan CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Netherlands.
² Erasmus MC, Department of Medical Microbiology and Infectious Diseases, Rotterdam, Netherlands.
³ Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycologie et Antifongiques, Paris, France.
⁴ Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan.
⁵ Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China Basic Pathology Department, Federal University of Paraná State, Curitiba, Paraná, Brazil King Abdulaziz University, Jeddah, Saudi Arabia s.hoog@cbs.knaw.nl.

PMID: 26246484
PMCID: PMC4572522
DOI: 10.1128/JCM.01544-15

Abstract

Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide is Madurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of M. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection of M. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples.

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Figures

FIG 1

FIG 1

Sensitivity of Madurella mycetomatis LAMP using serial dilution of CBS 109801^T DNA seen in 2% agarose gel electrophoresis. Lane M, 200-bp DNA marker; lanes 1 to 8, 15, 7.5, 3.75, 1.88, 0.94, 0.47, 0.23, and 0.12 ng.

FIG 2

FIG 2

Specificity of Madurella mycetomatis LAMP detected in 2% agarose gel electrophoresis. Lane M, DNA ladder; lane 1, M. mycetomatis CBS 109801^T; lane 2 M. tropicana CBS 201.38^T; lane 3, M. pseudomycetomatis CBS 129177^T; lane 4, M. fahalii CBS 129176^T; lane 5, Trematosphaeria grisea CBS 332.50^T; lane 6, Falciformispora senegalensis CBS 196.79^T; lane 7, F. tompkinsii CBS 200.70; lane 8, Medicopsis romeroi CBS 252.60^T.

FIG 3

FIG 3

Gel representation of the sensitivity of Madurella mycetomatis-specific RPA using primer pair P2MaduF and P5MAduR. Serial dilutions of CBS 109801^T DNA were used. Lane M, 200-bp DNA marker; lanes 1 to 8, 15, 7.5, 3.75, 1.88, 0.94, 0.47, 0.23, and 0.12 ng.

FIG 4

FIG 4

Specificity of Madurella mycetomatis RPA using primer pair P2MaduF and P5MAduR and seen in 2% agarose gel electrophoresis. Lane M, 200-bp DNA marker; lane 1, M. mycetomatis CBS 109801^T; lane 2, M. tropicana CBS 201.38^T lane 3, M. pseudomycetomatis CBS 129177^T; lane 4, M. fahalii CBS 129176^T; lane 5, Trematosphaeria grisea CBS 332.50^T; lane 6, Falciformispora senegalensis CBS 196.79^T; lane 7, F. tompkinsii CBS 200.70; lane 8, Medicopsis romeroi CBS 252.60^T.

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References

1. van de Sande WWJ. 2013. Global burden of human mycetoma: a systematic review and meta-analysis. PLoS Negl Trop Dis 7:e2550. doi:10.1371/journal.pntd.0002550. - DOI - PMC - PubMed
1. Mahgoub ES, Murray IG. 1973. Mycetoma. William Heinemann, Medical Books, London, United Kingdom.
1. Ahmed AO, van Leeuwen W, Fahal A, van de Sande W, Verbrugh H, van Belkum A. 2004. Mycetoma caused by Madurella mycetomatis: a neglected infectious burden. Lancet Infect Dis 4:566–574. doi:10.1016/S1473-3099(04)01131-4. - DOI - PubMed
1. van de Sande WW, Mahgoub el S, Fahal AH, Goodfellow M, Welsh O, Zijlstra E. 2014. The mycetoma knowledge gap: identification of research priorities. PLoS Negl Trop Dis 8:e2667. doi:10.1371/journal.pntd.0002667. - DOI - PMC - PubMed
1. Fahal A, Mahgoub el S, El Hassan AM, Abdel-Rahman ME, Alshambaty Y, Hashim A, Hago A, Zijlstra EE. 2014. A new model for management of mycetoma in the Sudan. PLoS Negl Trop Dis 8:e3271. doi:10.1371/journal.pntd.0003271. - DOI - PMC - PubMed

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[1] van de Sande WWJ. 2013. Global burden of human mycetoma: a systematic review and meta-analysis. PLoS Negl Trop Dis 7:e2550. doi:10.1371/journal.pntd.0002550. - DOI - PMC - PubMed

[2] van de Sande WWJ. 2013. Global burden of human mycetoma: a systematic review and meta-analysis. PLoS Negl Trop Dis 7:e2550. doi:10.1371/journal.pntd.0002550. - DOI - PMC - PubMed

[3] Mahgoub ES, Murray IG. 1973. Mycetoma. William Heinemann, Medical Books, London, United Kingdom.

[4] Mahgoub ES, Murray IG. 1973. Mycetoma. William Heinemann, Medical Books, London, United Kingdom.

[5] Ahmed AO, van Leeuwen W, Fahal A, van de Sande W, Verbrugh H, van Belkum A. 2004. Mycetoma caused by Madurella mycetomatis: a neglected infectious burden. Lancet Infect Dis 4:566–574. doi:10.1016/S1473-3099(04)01131-4. - DOI - PubMed

[6] Ahmed AO, van Leeuwen W, Fahal A, van de Sande W, Verbrugh H, van Belkum A. 2004. Mycetoma caused by Madurella mycetomatis: a neglected infectious burden. Lancet Infect Dis 4:566–574. doi:10.1016/S1473-3099(04)01131-4. - DOI - PubMed

[7] van de Sande WW, Mahgoub el S, Fahal AH, Goodfellow M, Welsh O, Zijlstra E. 2014. The mycetoma knowledge gap: identification of research priorities. PLoS Negl Trop Dis 8:e2667. doi:10.1371/journal.pntd.0002667. - DOI - PMC - PubMed

[8] van de Sande WW, Mahgoub el S, Fahal AH, Goodfellow M, Welsh O, Zijlstra E. 2014. The mycetoma knowledge gap: identification of research priorities. PLoS Negl Trop Dis 8:e2667. doi:10.1371/journal.pntd.0002667. - DOI - PMC - PubMed

[9] Fahal A, Mahgoub el S, El Hassan AM, Abdel-Rahman ME, Alshambaty Y, Hashim A, Hago A, Zijlstra EE. 2014. A new model for management of mycetoma in the Sudan. PLoS Negl Trop Dis 8:e3271. doi:10.1371/journal.pntd.0003271. - DOI - PMC - PubMed

[10] Fahal A, Mahgoub el S, El Hassan AM, Abdel-Rahman ME, Alshambaty Y, Hashim A, Hago A, Zijlstra EE. 2014. A new model for management of mycetoma in the Sudan. PLoS Negl Trop Dis 8:e3271. doi:10.1371/journal.pntd.0003271. - DOI - PMC - PubMed

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Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

Affiliations

Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

Authors

Affiliations

Abstract

Figures

References

Publication types

MeSH terms

Substances

LinkOut - more resources

Full Text Sources