The primers’ design specific to <i>M</i>. <i>mycetomatis</i> (primer PM) or <i>M</i>. <i>fahalii</i> (primer PF).

The rDNA sequence of M. mycetomatis (A) or M. fahalii (B) was aligned with that of M. pseudomycetomatis (accession no. EU815933.1), and arrows represented the primer positions. The matched nucleotide position was represented using dot signs, while the gap and mismatch positions using corresponding substitution ones. The species name and the number of first nucleotide were represented at each line using the following abbreviations: MM, M. mycetomatis; MP, M. pseudomycetomatis; MF, M. fahalii. The nucleotide alignment and illustration were performed using Genetyx Ver. 14 (Genetyx Co., Tokyo, Japan) with its default parameters.</p

Fig 4 -

The detection limits of designed LAMP primers, (A) PV, (B) PM and (C) PFThe genomic DNAs of M. mycetomatis IFM 46458 (for PV and PM) and M. fahalii CBS 126778 (for PF) were serially diluted by 10-fold, and 10 ng—10 fg of DNAs were used as a template. LAMP reaction was performed at 63°C for 60 min, and the turbidity of the reaction solution was monitored as an indicator of amplification. The sample legends of the plots were represented on the bottom right panel, and a plot labelled with B corresponds to the data of a blank sample with a buffer used for DNA dilution.</p

Fungal and bacterial strains used in this study.

BackgroundFilamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of Madurella spp. (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii), the common causative organisms of eumycetoma.Principal findingsWe successfully designed a primer pair targeting the rDNAs of three Madurella spp. excluding M. fahalii, and detected up to 100 fg of genomic DNA extracted from isolates of M. mycetomatis and 1 pg of M. pseudomycetomatis and M. tropicana, within one hour. Second, a primer pair specific to M. mycetomatis, the most common causative species, or M. fahalii, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus Madurella from various fungal species known to cause mycetomas.ConclusionIn summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies Madurella isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.</div

Photograph showing recurrence at the right below-knee stump.

Photograph showing recurrence at the right below-knee stump.</p

Chest CT showing segmental/lobar pneumonia with parapneumonic pleural effusion.

Chest CT showing segmental/lobar pneumonia with parapneumonic pleural effusion.</p

Fig 3 -

The specificity of designed LAMP primers, (A) PV, (B) PM and (C) PF. 10 ng of the extracted genomic DNAs derived from Madurella spp. were used as templates. LAMP reaction was performed at 63°C for 60 min, and the turbidity of the reaction solution was monitored as an indicator of amplification. As for primer PV, the genomic DNA of Chaetomium rectangulare was also used. The sample legends of the plots were represented on the bottom right panel. Abbreviations: MM, M. mycetomatis IFM 46458; MP, M. pseudomycetomatis IFM 46460; MT, M. tropicana CBS201.38; MF, M. fahalii CBS129176; CR, Chaetomium rectangluare CBS 126778; B, Blank sample with a buffer used for DNA dilution.</p

Optimization of LAMP reaction temperature using primer PF.

LAMP reaction was performed using genomic DNA of Madurella fahalii CBS 129176 at the reaction temperature between 62°C and 65°C. The solid line and dotted line represent the data of the genomic DNA sample and blank using a buffer used for DNA dilution, respectively. (TIF)</p

The primers’ design targeting all four Madurella spp. (primer PV2).

(A) The alignment of rDNA sequences among Madurella strains and Chaeromium rectangluare (negative control) is shown. Arrows illustrate the primers’ positions. (B) The specificity of primer PV2. LAMP reaction was performed using genomic DNAs derived from Madurella spp. and C. rectangulare at 65°C. Abbreviations: MM, M. mycetomatis; MP, M. pseudomycetomatis; MT, M. tropicana; MF, M. fahalii; CR, Chaetomium rectangluare; B, Blank sample with a buffer used for DNA dilution. (TIF)</p

Optimization of LAMP reaction temperature using primer PM.

LAMP reaction was performed using genomic DNA of Madurella myceromatis IFM 46458 at the reaction temperature between 62°C and 65°C. The solid line and dotted line represent the data of the genomic DNA sample and blank using a buffer used for DNA dilution, respectively. (TIF)</p

LAMP amplification for clinical isolates and pathogenic fungi and bacteria, which are known as causative agents of mycetoma.

LAMP amplification for clinical isolates and pathogenic fungi and bacteria, which are known as causative agents of mycetoma.</p