Madurella real-time PCR, a novel approach for eumycetoma diagnosis

doi:10.1371/journal.pntd.0007845

. 2020 Jan 15;14(1):e0007845.

doi: 10.1371/journal.pntd.0007845. eCollection 2020 Jan.

Madurella real-time PCR, a novel approach for eumycetoma diagnosis

Amir Arastehfar ¹, Wilson Lim ², Farnaz Daneshnia ¹, Wendy W J van de Sande ², Ahmed H Fahal ³, Marie Desnos-Ollivier ⁴, Gerrit S de Hoog ^{1

5}, Teun Boekhout ^{1

6}, Sarah A Ahmed ^{1

7}

Affiliations

¹ Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands.
² Erasmus MC, University Medical Center Rotterdam, Department of Microbiology and Infectious Diseases, Rotterdam, The Netherlands.
³ Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan.
⁴ Institut Pasteur, Molecular Mycology Unit, National Reference Center for Invasive Mycoses and Antifungals, CNRS UMR2000, Paris, France.
⁵ Center of Expertise in Mycology of Radboudumc / Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.
⁶ Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands.
⁷ Faculty of Medical Laboratory Sciences, University of Khartoum, Khartoum, Sudan.

PMID: 31940343
PMCID: PMC6986762
DOI: 10.1371/journal.pntd.0007845

Madurella real-time PCR, a novel approach for eumycetoma diagnosis

Amir Arastehfar et al. PLoS Negl Trop Dis. 2020.

. 2020 Jan 15;14(1):e0007845.

doi: 10.1371/journal.pntd.0007845. eCollection 2020 Jan.

Authors

Amir Arastehfar ¹, Wilson Lim ², Farnaz Daneshnia ¹, Wendy W J van de Sande ², Ahmed H Fahal ³, Marie Desnos-Ollivier ⁴, Gerrit S de Hoog ^{1

5}, Teun Boekhout ^{1

6}, Sarah A Ahmed ^{1

7}

Affiliations

¹ Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands.
² Erasmus MC, University Medical Center Rotterdam, Department of Microbiology and Infectious Diseases, Rotterdam, The Netherlands.
³ Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan.
⁴ Institut Pasteur, Molecular Mycology Unit, National Reference Center for Invasive Mycoses and Antifungals, CNRS UMR2000, Paris, France.
⁵ Center of Expertise in Mycology of Radboudumc / Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.
⁶ Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands.
⁷ Faculty of Medical Laboratory Sciences, University of Khartoum, Khartoum, Sudan.

PMID: 31940343
PMCID: PMC6986762
DOI: 10.1371/journal.pntd.0007845

Abstract

The genus Madurella comprising four species, M. fahalii, M. mycetomatis, M. pseudomycetomatis, and M. tropicana, represents the prevalent cause of eumycetoma worldwide. The four species are phenotypically similar and cause an invariable clinical picture, but differ markedly in their susceptibility to antifungal drugs, and epidemiological pattern. Therefore, specific identification is required for optimal management of Madurella infection and to reveal proper epidemiology of the species. In this study, a novel multiplex real-time PCR targeting the four Madurella species was developed and standardized. Evaluation of the assay using reference strains of the target and non-target species resulted in 100% specificity, high analytical reproducibility (R2 values >0.99) and a lowest detection limit of 3 pg target DNA. The accuracy of the real-time PCR was further assessed using biopsies from eumycetoma suspected patients. Unlike culture and DNA sequencing as gold standard diagnostic methods, the real-time PCR yielded accurate diagnosis with specific identification of the causative species in three hours compared to one or two weeks required for culture. The novel method reduces turnaround time as well as labor intensity and high costs associated with current reference methods.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Identification of Madurella species using qPCR assay.

(A) Identification of M. fahalii and M. mycetomatis using duplex qPCR assay. (B) Identification of all target species using a tetraplex qPCR assay. (C) Identification of M. mycetomatis using a M. mycetomatis-specific qPCR assay.

Fig 2

Fig 2. Specificity of duplex qPCR assay of M. fahalii and M. mycetomatis.

(A) Tm values obtained from duplex qPCR assay for M. fahalii and M. mycetomatis. (B) Duplex qPCR assay with a blinded test set and DNA samples obtained from patients yielded 100% specificity and sensitivity if the threshold cycles were set at<20 (red line), and <30 (blue line), respectively.

Fig 3

Fig 3. Specificity and sensitivity of singleplex and tetraplex qPCR assays of Madurella species.

(A) Tm values obtained from tetraplex and M. mycetomatis-specific qPCR assays for M. fahalii, M. mycetomatis, M. pseudomycetomatis, and M. tropicana. (B) Tetraplex and MM-specific qPCR with a blinded test set and DNA samples obtained from patients yielded 100% specificity and sensitivity if the threshold cycles <30 (red line).

Fig 4

Fig 4. Receiver operation characteristic curve (ROC) obtained for the three qPCR assays revealed 100% specificity when subjected to blinded test sets.

See this image and copyright information in PMC

References

1. McGinnis MR. Mycetoma. Dermatol Clin. 1996;14:97–104. 10.1016/s0733-8635(05)70329-6 - DOI - PubMed
1. Zijlstra EE, van de Sande WWJ, Welsh O, Mahgoub ES, Goodfellow M, Fahal AH. Mycetoma: a unique neglected tropical disease. Lancet Infect Dis. 2016;16:100–112. 10.1016/S1473-3099(15)00359-X - DOI - PubMed
1. Fahal A, Mahgoub el S, El Hassan AM, Abdel-Rahman ME. Mycetoma in the Sudan: an update from the Mycetoma Research Centre, University of Khartoum, Sudan. PLoS Negl Trop Dis. 2015;9:e0003679 10.1371/journal.pntd.0003679 - DOI - PMC - PubMed
1. van de Sande WW, Fahal AH, Goodfellow M, Mahgoub el S, Welsh O, Zijlstra EE. Merits and pitfalls of currently used diagnostic tools in mycetoma. PLoS Negl Trop Dis. 2014;8:e2918 10.1371/journal.pntd.0002918 - DOI - PMC - PubMed
1. Emmanuel P, Dumre SP, John S, Karbwang J, Hirayama K. Mycetoma: a clinical dilemma in resource limited settings. Ann Clin Microbiol Antimicrob. 2018;17:35 10.1186/s12941-018-0287-4 - DOI - PMC - PubMed

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[1] McGinnis MR. Mycetoma. Dermatol Clin. 1996;14:97–104. 10.1016/s0733-8635(05)70329-6 - DOI - PubMed

[2] McGinnis MR. Mycetoma. Dermatol Clin. 1996;14:97–104. 10.1016/s0733-8635(05)70329-6 - DOI - PubMed

[3] Zijlstra EE, van de Sande WWJ, Welsh O, Mahgoub ES, Goodfellow M, Fahal AH. Mycetoma: a unique neglected tropical disease. Lancet Infect Dis. 2016;16:100–112. 10.1016/S1473-3099(15)00359-X - DOI - PubMed

[4] Zijlstra EE, van de Sande WWJ, Welsh O, Mahgoub ES, Goodfellow M, Fahal AH. Mycetoma: a unique neglected tropical disease. Lancet Infect Dis. 2016;16:100–112. 10.1016/S1473-3099(15)00359-X - DOI - PubMed

[5] Fahal A, Mahgoub el S, El Hassan AM, Abdel-Rahman ME. Mycetoma in the Sudan: an update from the Mycetoma Research Centre, University of Khartoum, Sudan. PLoS Negl Trop Dis. 2015;9:e0003679 10.1371/journal.pntd.0003679 - DOI - PMC - PubMed

[6] Fahal A, Mahgoub el S, El Hassan AM, Abdel-Rahman ME. Mycetoma in the Sudan: an update from the Mycetoma Research Centre, University of Khartoum, Sudan. PLoS Negl Trop Dis. 2015;9:e0003679 10.1371/journal.pntd.0003679 - DOI - PMC - PubMed

[7] van de Sande WW, Fahal AH, Goodfellow M, Mahgoub el S, Welsh O, Zijlstra EE. Merits and pitfalls of currently used diagnostic tools in mycetoma. PLoS Negl Trop Dis. 2014;8:e2918 10.1371/journal.pntd.0002918 - DOI - PMC - PubMed

[8] van de Sande WW, Fahal AH, Goodfellow M, Mahgoub el S, Welsh O, Zijlstra EE. Merits and pitfalls of currently used diagnostic tools in mycetoma. PLoS Negl Trop Dis. 2014;8:e2918 10.1371/journal.pntd.0002918 - DOI - PMC - PubMed

[9] Emmanuel P, Dumre SP, John S, Karbwang J, Hirayama K. Mycetoma: a clinical dilemma in resource limited settings. Ann Clin Microbiol Antimicrob. 2018;17:35 10.1186/s12941-018-0287-4 - DOI - PMC - PubMed

[10] Emmanuel P, Dumre SP, John S, Karbwang J, Hirayama K. Mycetoma: a clinical dilemma in resource limited settings. Ann Clin Microbiol Antimicrob. 2018;17:35 10.1186/s12941-018-0287-4 - DOI - PMC - PubMed

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Madurella real-time PCR, a novel approach for eumycetoma diagnosis

Affiliations

Madurella real-time PCR, a novel approach for eumycetoma diagnosis

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Substances

LinkOut - more resources

Full Text Sources