1. Rapid DNA-probe based screening assay for differential diagnosis
of dermatomycoses
Kroschwald L.1, Weber C. 2, Augustin M. 2, Kurtzer R. 2, Bröse E. 1, Wiedenfeld A. 1, Ahrendt R. 2, Aichinger E. 2 and Bauer A. 1
1Universitätsklinikum Dresden, Dermatologie, Dresden, Germany 2Biotype Diagnostic GmbH, Dresden, Germany
Background and objectives
Worldwide, dermatomycosis and fungal infections of skin and mucosa are the most common diseases with increasing prevalence up to 26% and a high number of unreported cases 1-3. The causative agent of superficial mycoses are mainly dermatophytes, yeasts and to a lesser extend non-dermatophyte mould. In central europe most frequent for Tinea capitis is Microsporum canis 4, for onychomycoses Trichophyton rubrum, T. interdigitale 5 and Epidermophyton flocossum 6 but also Scopulariopsis brevicaulis, Aspergillus spp. or Candida spp. 7-9 (Fig.1).
An accurate and rapid detection of fungi is most important for the choice of antimycotics and the success of treatment. For a long time, identification of fungi was almost based on morphological features examined by microscopy or microbial culture. Meanwhile, molecular methods using different genetic targets and techniques are available. The aim of this study was to improve a recently evaluated multiplex-based PCR diagnostic test kit 10 to fulfill quality standards for pathogen detection based on DNA techniques by means of a DNA probe (MiQ) while maintaining already achieved performance. Here we present the clinical validation using 266 clinical samples.
Method
The pathogen amplification is based on a modular multiplex PCR approach which consists of three multiplex PCRs (Fig.2). PCR 1 is designed as a screen, determining whether the infection is caused by dermatophytes, fungi, yeast or a combination. Simultaneously, T. rubrum, the most common pathogen, is identified as well. With PCR 2 and 3 a possible dermatophytic infection is analyzed. The amplicons are detected on a lateral flow platform combining the nucleic acid lateral flow immunoassay (NALFIA) and nucleic acid lateral flow test strip (NALFT) technologies. The hapten-tagged amplicons from the multiplex PCR hybridized with labelled amplicon-specific probes and will be detected by a colour reaction on the lateral flow test strip (LFT) (Fig.3).
Prior to DNA extraction, patient samples are spiked with a control DNA sample which serves as extraction control and as an internal amplification control for the PCR itself. Given the possibility of negative clinical samples, this hapten-amplicon-antibody complex will also serve as control for the functionality of the LFT, the lyophilized and liquid reagents and a sufficient gold labeling of all PCR amplicons.
Results and discussion
In total, 266 patients treated at the Department of Dermatology (University Hospital Carl Gustav Carus, TU Dresden, Germany) were analysed. According to the clinical manifestations, 116 nail clippings, 90 skin scrapings and 60 smears from mucosa and wepping skin lesions were collected (Fig. 5). The accuracy and reproducibility were verified by comparing culture, microscopy and qPCR with the new assay. Therefrom, 135 were tested positive in microscopy, 161 in culture, 227 in culture or microscopy or both, 144 in MycodermQS Lateral Flow PCR-Assay and 213 in qPCR, respectively (Fig. 6).
The definition of a reference standard pose to a be a challange because microscopy and culture are described with high variances in sensitivity (50 – 80 % 11,12). Therefore, we set pPCR as golden standard. Based on the threshold for DNA-concentration within the clinical samples (≥ 0.2 pg/µl; ≥ 0.5 pg/µl and ≥ 1 pg/µl ), sensitivity, specificity, positive and negative predictive value of more than 83 %, 100 %, 100 % and more than 86 % respectively, were calculated (Fig. 7). These values are comparable to other published PCR tests for dermatophytes 10, In 60 % (158 samples) of all cases, less than 1 pg DNA was determined. Thereof, 128 were tested positive in microscopy, culture or both. This can be explained by amount and heterogeneity of clinical samples, their preparation and DNA extraction. Looking closer to the genus and species identification, from 22 or 7 lateral flow negative samples, for 21 samples the TME specific amplicon was detected, genus specific detection was not possible. In one case a Candida spp. positive sample from culture could not be confirmed by the assay. For the above described experiments a minimum of four subsamples were needed per sampling point. Therefore, the amount of pathogenetic DNA varied. In comparison to conventional standard methods, qPCR and the new assay were performed from the same subsample.
Keeping in mind that other PCR-based assays can identify only one or a few species, this commercial kit is dedicated to detect and differentiate all frequently observed human pathogenetic dermatophytes, yeasts and moulds in central europe in three multiplex PCRs. The test can be finished in less than one working day while sample lysis for DNA extraction should be performed overnight. Furthermore, the assay includes an internal PCR amplification control (quality sensor, QS) and has proven to be a reliable approach which clearly outperformed the conventional diagnostic by time, sensitivity and specifity.
Figure 1: Most frequent dermatophytes: Scopulariopsis brevicaulis (A), Microsporum canis (B), Trichophyton rubrum (C); depicted as culture macroscopic (left), culture microscopic (middle) and clinical picture (right).
Figure 2: Dermatophytes, yeasts and non- dermatophyte moulds detectable with the multiplex PCR Mentype® MycoDermQS NALFT.
* QS = DNA extraction and PCR amplification control according to RiliBÄK and MiQ
** TME (Trichophytum, Microsprorum, Epidermophyton) also detects the following types:
Trichophyton terrestre (geophilic): Arthroderma insingulare; Arthroderma quadrifidum; Arthroderma lenticulare; Microsporum ferrugineum
*** Candida spp. types genus specifically detected :
C. albicans; C. tropicalis; C. glabrata; C. krusei (Pichia kudriavzevii, Issatchenkia orientalis); C. guilliermondii
(Meyerozyma guilliermondii); C. parapsilosis
PCR 1: Screening Dermatophytes / Yeasts / Mould
QS*
T. rubrum
Dermatophyton spp. (TME)**
Candida spp.***
S. brevicaulis
T. interdigitale/ A. vanbreuseghemii
T. mentagrophytes
T. tonsurans
T. violaceum
T. erinacei/ A. benhamiae/
T. verrucosum
PCR 2: Trichophyton spp.
M. audouinii
M. gypseum
M. canis
E. floccosum
PCR 3: Microsporum spp./ Epidermophyton
Figure 3: NALFT with hybridization haptene + antibody (Ab) (compliant with MiQ 2011 )
(3) Lateral Flow Detection
of hapten-antibody-complex
(1) Multiplex PCR
Haptene:
Sample
Pad
Reagent
(Gold conj. Ab)
Detection
Zone
Wick
Lateral Flow Teststripe
Anti-
Hapten-Ab
(2) Hybridization
with labeled probes:
probes
Figure 4: Workflow in pictures: multiplex-PCR (a), centrifugation (b), hybridization with DIG-labelled probes (c) in a thermo cycler (d) and detection via lateral flow test strips (LFT) (f,g)
Figure 5: Distribution of the clinical samples according to their sampling location investigated for this study from april to june 2014.
Figure 6: Results of positive tested samples from the clinical analyses by using microscopy, culture, Lateral flow and qPCR in percent.
Figure 7: Sensitivity, specifity, positive predictive value (PPV) and negative predictive value (NPV) based on the threshold set for DNA-concentration (≥0.2 pg; ≥0.5 pg and ≥1 pg) with pPCR as diagnostic reference standard.
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