Detection of Pan-Dermatophytes and Trichophyton rubrum Using Recombinase Polymerase Amplification-Lateral Flow Dipstick Assay.
Mycopathologia
BACKGROUND:Traditional methods for diagnosing onychomycosis are characterized by limited sensitivity and prolonged processing times, and heavily rely on the skill level of laboratory personnel. OBJECTIVES:To develop a fast, simple, user-friendly, and reliable molecular assay that offers high sensitivity and specificity for the detection of common dermatophytes in nail specimens. METHODS:We developed a technique that integrates recombinase polymerase isothermal amplification with lateral flow dipstick (RPA-LFD) for the detection of pan-dermatophytes and Trichophyton rubrum, and evaluated its analytical sensitivity and specificity. This method was applied to analyze 190 nail specimens, with the results compared with traditional microscopy and fungal culture. RESULTS:The RPA-LFD assay demonstrated an analytical sensitivity of 10 pg/reaction for pan-dermatophytes and 1 pg/reaction for T. rubrum. In clinical evaluations for tinea unguium, the sensitivity of the RPA-LFD, fungal culture, and microscopy methods, as determined through latent class analysis, was estimated to be 91.0%, 70.8%, and 93.9%, respectively. Correspondingly, the specificity of these methods-RPA-LFD, fungal culture, and microscopy-was assessed at approximately 95.7%, 98.0%, and 94.3%. CONCLUSIONS:Our RPA-LFD assay exhibited high sensitivity and specificity in the detection of dermatophytes. Due to its technical simplicity, enhanced sensitivity, and reduced processing times, it represents a promising alternative to conventional fungal culture methods for the mycological detection and identification of dermatophytes.
10.1007/s11046-024-00921-7
Molecular characterization of a subgroup IE intron with wide distribution in the large subunit rRNA genes of dermatophyte fungi.
Jackson Colin J,Barton Richard C,Clark C Graham,Kelly Steven L
Medical mycology
Group I introns have the ability to catalyse their own excision (self-splice) from pre-RNA, and are found in a wide range of eukaryotic organisms. In fungal nuclear genomes, they have been identified in the small subunit (SSU) and large subunit (LSU) of the ribosomal RNA gene. Sequencing of the 3' region of the LSU rRNA gene of the dermatophyte Trichophyton interdigitale revealed a 393 bp group I intron, Tin.2563, containing the four characteristic conserved motifs (P,Q,R and S) essential for self-splicing. The predicted secondary structure revealed nine sets of conserved paired regions (P1-P9), with most similarity to a subgroup IE intron of the entomopathogenic hyphomycete Beauveria bassiana. Tin.2563 was inserted at a site in the LSU rDNA corresponding to position 2563 of the Escherichia coli 23S rRNA. PCR and sequence analysis showed an intron to be present at an identical location in the LSU rDNA of many dermatophytes, although its distribution was erratic. In contrast, an intron was present at the same location in multiple isolates (n = 20) of the clinically important anthrophilic species Trichophyton rubrum and T. interdigitale. Conservation of intron insertion site, subgroup and P helix sequences showed intron genotyping to be unsuitable for strain identification in dermatophytes. Phylogenetic analysis of intron sequences from different dermatophyte species indicated that lateral transfer of the element was likely to be a rare event.
10.1080/13693780802385445
Variation in restriction fragment length polymorphisms among serial isolates from patients with Trichophyton rubrum infection.
Gupta A K,Kohli Y,Summerbell R C
Journal of clinical microbiology
Molecular genotyping of strains of Trichophyton rubrum and T. mentagrophytes from patients with onychomycosis of the toes was performed to ascertain whether the fungal genotype changes over the course of time as sequential samples were obtained from patients receiving antifungal therapy and during follow-up. Sixty-six serial strains of T. rubrum and 11 strains of T. mentagrophytes were obtained from 20 patients (16 patients with T. rubrum, 4 with T. mentagrophytes) who were treated with oral antifungal therapy and observed over periods of up to 36 months. These strains were screened for genetic variation by hybridization of EcoRI-digested genomic DNAs with a probe amplified from the small-subunit (18S) ribosomal DNA and adjacent internal transcribed spacer regions. A total of five restriction fragment length polymorphism (RFLP) types were observed among 66 strains of T. rubrum. Two major RFLP types, differentiated by one band shift, represented 68% of the samples. None of the patients had a unique genotype. More than one RFLP type was often observed from a single patient (same nail) over a period of 1, 2, or 3 years, even in cases that did not appear cured at any time. Samples taken from different nails of the same patient had either the same or a different genotype. The genotypic variation did not correspond to any detectable phenotypic variation. Furthermore, no correlation was observed between the efficacy of the treatment administered and the genotype observed. While the DNA region studied distinguished among T. rubrum, T. mentagrophytes, and T. tonsurans, intraspecific RFLP variation was observed for T. rubrum and T. mentagrophytes strains. While independent multiple infection and coinhabitation of multiple strains may explain the presence of different genotypes in a nail, microevolutionary events such as rapid substrain shuffling, as seen in studies of repetitive regions in Candida species, may also produce the same result. The recovery of multiple strains during the course of sequential sampling of uncured patients further suggests that the typing system is not able to distinguish between relapse or reinfection, ongoing infection, and de novo infection.
10.1128/JCM.39.9.3260-3266.2001
Stability of tandemly repetitive subelement PCR patterns in Trichophyton rubrum over serial passaging and with respect to drug pressure.
Hryncewicz-Gwóźdź Anita,Jagielski Tomasz,Kalinowska Katarzyna,Baczyńska Dagmara,Plomer-Niezgoda Ewa,Bielecki Jacek
Mycopathologia
Trichophyton rubrum is the most significant agent of dermatomycoses worldwide, primarily causing tinea pedis and tinea unguium. PCR analysis of tandemly repetitive subelements (TRS) within the rDNA nontranscribed spacer region is a major tool for molecular typing of T. rubrum. The aim of this study was to investigate the stability of TRS PCR patterns by analyzing isogenic strains of T. rubrum. Twenty-seven groups of isogenic T. rubrum strains were examined, each composed of an original clinical isolate and its 3 subcultures, maintained on a drug-free medium, a medium containing fluconazole and itraconazole. TRS typing was performed for the original strains and their subcultures grown after 12 passages, at 4-week intervals, on respective media. To add more objectivity to the results, TRS typing for each of the isogenic strain was performed three times, using DNA isolated from three different colonies. Among 27 groups of isogenic strains, all but one were exclusively composed of strains with identical TRS-1 and TRS-2 PCR patterns. In one group, 3 isolates from the last, twelfth passage had identical TRS-1 PCR profiles (type 1), yet different TRS-2 PCR profiles, as compared with the original strain (type I vs. type II). The mechanism underlying the genotype switch was a deletion of a single repeat unit in the TRS-2 locus, as evidenced by sequence analysis. In the interpretation of TRS typing results, microevolutionary events need to be taken into account, urging drawing epidemiological conclusions with caution and in conjunction with other genotyping data and traditional contact tracing information.
10.1007/s11046-012-9565-4
Fast and sensitive detection of Trichophyton rubrum DNA from the nail samples of patients with onychomycosis by a double-round polymerase chain reaction-based assay.
Gupta A K,Zaman M,Singh J
The British journal of dermatology
BACKGROUND:Trichophyton rubrum is one of the most frequently isolated pathogens in onychomycosis. Isolation of T. rubrum from nail samples by traditional methods is time-consuming and has a high false-negative rate of detection. OBJECTIVES:To investigate the detection of T. rubrum in nail samples using DNA detection methods. METHODS:A total of 62 nail samples from onychomycosis patients with T. rubrum infection were evaluated by culture on Sabouraud's dextrose agar plus chloramphenicol, cycloheximide and gentamicin and compared with genotyping methods utilizing DNA extracted directly from nails. Trichophyton rubrum DNA isolated directly from nails was amplified using two different conserved regions [actin gene and internal transcribed spacer 1 (ITS)] in double-round polymerase chain reaction (PCR) assays. RESULTS:Forty-eight of 62 (77.4%) samples were potassium hydroxide (KOH) positive, but T. rubrum culture was positive in only 14 of 62 (22.6%) samples. By contrast, direct T. rubrum DNA detection rate was 59.7% (37/62) by actin gene and 45.2% (28/62) by ITS1 region PCR assays corresponding to higher detection frequencies compared with culture with P < 0.001 and < 0.008, respectively. The combined detection of actin and ITS1 was 69.4% (43/62). Interestingly, T. rubrum DNA was detected in 9 out of 14 (64.3%) of KOH- and culture-negative samples. Importantly, 15 culture-negative samples collected from patients undergoing antifungal treatment tested PCR positive using the actin region. CONCLUSIONS:These results suggest that a direct DNA detection protocol is more sensitive, accurate and faster than traditional culture-based methods. It can be useful to detect T. rubrum in patients undergoing antifungal therapy and who have been reported mycologically cured on the basis of a culture-based method.
10.1111/j.1365-2133.2007.08110.x
Evaluation of a PCR melting profile method for intraspecies differentiation of Trichophyton rubrum and Trichophyton interdigitale.
Leibner-Ciszak Justyna,Dobrowolska Anita,Krawczyk Beata,Kaszuba Aleksandra,Stączek Paweł
Journal of medical microbiology
In order to identify the source of infections caused by dermatophytes, as well as the pathogen transmission pathway, there is a need to determine methods that allow detailed genetic differentiation of the strains within the dermatophyte genera. In this work, a PCR melting profile (PCR-MP) technique based on the ligation of adaptors and the difference in melting temperatures of DNA restriction fragments was used for the first time for intraspecies genotyping of dermatophytes. Clinical isolates and reference strains of dermatophytes isolated from skin, scalp, toenails and fingernails were used for this study. PCR-MP and random amplification of polymorphic DNA (RAPD) were used to type 11 isolates of Trichophyton rubrum, 40 isolates of Trichophyton interdigitale and 14 isolates of Microsporum canis. The results distinguished five types (containing one subtype) characteristic for T. rubrum and seven types characteristic for T. interdigitale using the PCR-MP technique. Analysis conducted using RAPD revealed five types for T. rubrum and four types for T. interdigitale isolates. No differentiation was observed for the M. canis isolates with either method. These results demonstrate that PCR-MP is a reliable method for the differentiation of T. rubrum and T. interdigitale strains and yields a discriminatory power that is at least equal to that of RAPD.
10.1099/jmm.0.013458-0
Molecular typing of Trichophyton rubrum clinical isolates from Poland.
Hryncewicz-Gwóźdź Anita,Jagielski Tomasz,Sadakierska-Chudy Anna,Dyląg Mariusz,Pawlik Krzysztof,Baran Eugeniusz,Szepietowski Jacek C
Mycoses
The aim of this study was to investigate the intraspecific diversity of Trichophyton rubrum clinical isolates. Thirty clinical isolates of T. rubrum were selected for molecular typing by PCR amplification of two tandemly repetitive elements (TRS-1 and TRS-2) of the rDNA and randomly amplified polymorphic DNA (RAPD) analysis with primers designated 1 and 6. The assignment to the species T. rubrum was achieved by nested PCR of ITS1. Five PCR types were produced from the TRS-1 and three from the TRS-2 locus. Thirteen and 23 individual profiles were obtained by RAPD, with primer 1 and 6 respectively. At the phylogenetic level, 26 (87%) isolates were allocated into four clusters, with each cluster comprising isolates of over 80% similarity. The reproducibility of TRS typing was 100%, whereas that of RAPD was 40% and 30%, when using primer 1 and 6 respectively. Neither correlation between the morphological characteristics and the TRS-1-TRS-2 or RAPD genotype nor between TRS-1-TRS-2 and RAPD genotyping was observed. Although both the TRS amplification and RAPD analysis possess the ability to discriminate between T. rubrum strains, the TRS typing method is particularly valuable as its results are much more reproducible, more easily interpreted and recorded than those generated by RAPD.
10.1111/j.1439-0507.2010.02007.x
Identification and differentiation of Trichophyton rubrum clinical isolates using PCR-RFLP and RAPD methods.
Hryncewicz-Gwóźdź A,Jagielski T,Dobrowolska A,Szepietowski J C,Baran E
European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology
Trichophyton rubrum represents the most frequently isolated causative agent of superficial dermatophyte infections. Several genotyping methods have recently been introduced to improve the delineation between pathogenic fungi at both the species and the strain levels. The purpose of this study was to apply selected DNA fingerprinting methods to the identification and strain discrimination of T. rubrum clinical isolates. Fifty-seven isolates from as many tinea patients were subjected to species identification by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and strain differentiation using a randomly amplified polymorphic DNA (RAPD) method, with two primers designated 1 and 6. Using PCR-RFLP, 55 of the isolates studied were confirmed to be T. rubrum. Among those, a total of 40 and five distinct profiles were obtained by RAPD with primers 1 and 6, respectively. The combination of profiles from both RAPD assays resulted in 47 genotypes and an overall genotypic diversity rate of 85.4%. A dendrogram analysis performed on the profiles generated by RAPD with primer 1 showed most of the isolates (87.3%) to be genetically related. PCR-RFLP serves as a rapid and reliable method for the identification of T. rubrum species, while the RAPD analysis is rather a disadvantageous tool for T. rubrum strain typing.
10.1007/s10096-010-1144-3
Genotyping of Trichophyton rubrum by analysis of ribosomal-DNA intergenic spacer regions.
Yang Guoling,An Lijia,Li Qiao,Lin Jingrong,Liu Weida,Jin Liji,Lin Xiran
Mycopathologia
An attempt was made to explore the genotyping of Trichophyton rubrum (T. rubrum) and the relationship between genotype and geographical origin using ribosomal restriction endonuclease polymorphic analysis. The total DNA was extracted by cetyltrimethyl ammonium bromide (CTAB). The probe was amplified from part of the 18S, ITSI, 5.8S, and ITSII region of T. rubrum standard strain with the universal fungal primers NS5 [5'-AACTT AAAGG AATTG ACGGA AG-3'] and ITS4 [5'-TCCTC CGCTT ATTGA TATGC-3']. The genomic DNA of 49 clinical T. rubrum isolates digested by EcoR1 were hybridized with this probe, and the hybridization patterns were used as the basis of genotyping. Of the data from 49 strains of T. rubrum studied (21 from Nanjing, 26 from Dalian, and two from Beijing), 20 individual patterns (DNA Type A-T) were identified, among which Type A-C accounted for 48.98% of all the strains. The DNA patterns of Nanjing strains were represented by three bands, those of Dalian strains were represented by four bands. The DNA typing of T. rubrum by Southern blotting was highly sensitive and highly distinguishable. The DNA patterns of Nanjing strains were obviously different from those of Dalian strains.
10.1007/s11046-007-9021-z