DNA-Chip-basierte Diagnose der Onychomykose und Tinea pedis.

HINTERGRUND UND ZIELE: Onychomykose (OM) und Tinea pedis (TP) sind häufige Pilzinfektionen der Haut. Aktuell basiert die Diagnose vornehmlich auf mikroskopischem Direktnachweis und/oder Kultur. Beide Methoden haben jedoch eine geringe bis mäßige Sensitivität und benötigen teilweise mehrere Wochen, bis endgültige Laborergebnisse vorliegen. Um die Diagnose kutaner Pilzinfektionen zu verbessern, wurden PCR-basierte Methoden entwickelt. Hier haben wir hier die Sensitivität und Spezifität einer Chip-basierten Multiplex-PCR mit mikroskopischen Direktnachweis und verglichen. PATIENTEN UND METHODIK: In einer monozentrischen, prospektiven Studie wurden bei Patienten mit Verdacht auf OM (n  =  67) oder TP (n  =  73) Schuppenpräparate entnommen und mittels mikroskopischem Direktnachweis, Kultur und DNA-Chip-Technologie der Erregernachweis durchgeführt. In einem weiteren Ansatz wurde überprüft, ob Abstriche als Alternative zur Entnahme eines Schuppenpräparates verwendet werden können. Hierfür wurden 24 weitere OM/TP-Patienten rekrutiert und die Ergebnisse der DNA-Chip-Technologie aus Abstrichen mit denen aus den Schuppenpräparaten verglichen. ERGEBNISSE: Im Vergleich aller Methoden hatte die DNA-Chip-Technologie die höchste Sensitivität, eine Kombination von DNA-Chip-Technologie mit mikroskopischem Direktnachweis erhöhte dies weiter. Ergebnisse dieser kombinierten Labordiagnostik sind innerhalb von 24 Stunden verfügbar. Der Vergleich der Probenentnahmetechniken (Abstrich beziehungsweise Schuppenpräparat) zeigte vergleichbare Ergebnisse. SCHLUSSFOLGERUNGEN: Die molekulare Diagnostik (mittels DNA-Chip-Technologie) hat eine hohe Sensitivität für die OM- und TP-Diagnostik, insbesondere in Kombination mit dem mikroskopischen Direktnachweis.

DNA chip-based diagnosis of onychomycosis and tinea pedis.

BACKGROUND AND OBJECTIVES: Onychomycosis (OM) and tinea pedis (TP) are common fungal infections. Currently, diagnosis is based on direct microscopy and culture that have a low to moderate sensitivity and/or require up to 3-4 weeks until results are obtained. PCR techniques have emerged for the diagnosis of fungal infections, but little is known about their sensitivity and specificity in diagnosing. Here, we compared the diagnostic value of a DNA-chip technology, that detects 56 fungal pathogens, in a single-center prospective diagnostic study with microscopy and culture in suspected OM/TP. PATIENTS AND METHODS: Microscopy, culture and DNA microarray assays were performed on scraping material from patients with suspected OM (n = 67) or TP (n = 73). To test whether swabs can be used as an alternative for scraping, PCR yields were compared in a further 13 patients with OM and 11 patients with TP. RESULTS: DNA microarrays had the highest sensitivity. Combination of DNA-chip technology with microscopy further increased the sensitivity, and results from this combined laboratory diagnosis can be obtained within 24 hours. Comparison of sampling techniques (scraping, dry or wet swab) for DNA-chip assays showed similar results in suspected OM or TP. CONCLUSIONS: DNA-chip technology shows high sensitivity for OM and TP diagnosis, especially when combined with microscopy.

Expression of epidermal antimicrobial peptides is increased in tinea pedis.

BACKGROUND: Tinea pedis is often chronic or recurrent, but not all individuals are equally susceptible to this infection. Dermatophytes are able to induce the expression of antimicrobial peptides and proteins (AMPs) in human keratinocytes and certain AMPs can inhibit the growth of dermatophytes. OBJECTIVE: The focus of this study was to analyse the secretion of relevant AMPs, especially RNase 7, human beta-defensin-2 (hBD-2) and the S-100 protein psoriasin (S100A7), in patients with confirmed tinea pedis. METHODS: To verify the diagnosis, skin scales were obtained from all patients (n = 13) and the dermatophytes were identified by potassium hydroxide mount, culture and molecular analysis. To determine the AMP concentrations, the lesional skin area of the foot was rinsed with a buffer that was subsequently analysed by ELISA. The corresponding area of the other unaffected foot as well as defined healthy skin areas of the forearm and forehead and samples from age and gender-matched healthy volunteers served as controls. RESULTS: In tinea pedis patients the AMP concentrations were higher in lesional skin than in non-lesional skin and in healthy skin of controls. In particular, concentrations of hBD-2 and psoriasin were significantly elevated. CONCLUSIONS: The induction of AMPs in tinea pedis might be triggered directly by the dermatophytes; furthermore, attendant inflammation and/or differentiation processes may play a role. Our results indicate that there is no defect in the constitutive expression and induction of the analysed AMPs by dermatophytes in the epidermis of affected patients. However, it is not known why the elevated AMP concentrations fail to efficiently combat dermatophyte growth.

[Arthroderma crocatum on human skin]. / Arthroderma crocatum auf menschlicher Haut.

We report on three cases in which Arthroderma (A.) crocatum was isolated from human skin in Germany. The characteristics and epidemiology of this rare geophilic and probably mostly apathogenic dermatophyte are described paying special attention to its gymnothecia. The combination of KOH mount, culture and genetic analysis is the foundation for clinically meaningful conclusions. It is likely that the prevalence of A. crocatum is currently underestimated.

Prediction of people's origin from degraded DNA--presentation of SNP assays and calculation of probability.

The characterization of externally visible traits by DNA analysis is already an important tool when investigating ancient skeletal remains and may gain similar importance in future forensic DNA analysis. This, however, depends on the different legal regulations in the different countries. Besides eye or hair color, the population origin can provide crucial information in criminal prosecution. In this study, we present the analysis of 16 single-nucleotide polymorphisms (SNPs) combined to two robust SNaPshot assays with a detection threshold of 25-pg DNA. This assay was applied to 891 people from seven different populations (West Africa, North Africa, Turkey, Near East, Balkan states, North Europe, and Japan) with a thorough statistical evaluation. The prediction model was validated by an additional 125 individuals predominantly with an ancestry from those same regions. The specificity of these SNPs for the prediction of all population origins is very high (>90 %), but the sensitivity varied greatly (more than 90 % for West Africa, but only 25 % for the Near East). We could identify West Africans with a certainty of 100 %, and people from North Africa, the Balkan states, or North Europe nearly with the same reliability while determination of Turks or people from the Near East was rather difficult. In conclusion, the two SNaPshot assays are a powerful and reliable tool for the identification of people with an ancestry in one of the above listed populations, even from degraded DNA.

Emerging genetic patterns of the European Neolithic: perspectives from a late Neolithic Bell Beaker burial site in Germany.

The transition from hunting and gathering to agriculture in Europe is associated with demographic changes that may have shifted the human gene pool of the region as a result of an influx of Neolithic farmers from the Near East. However, the genetic composition of populations after the earliest Neolithic, when a diverse mosaic of societies that had been fully engaged in agriculture for some time appeared in central Europe, is poorly known. At this period during the Late Neolithic (ca. 2,800-2,000 BC), regionally distinctive burial patterns associated with two different cultural groups emerge, Bell Beaker and Corded Ware, and may reflect differences in how these societies were organized. Ancient DNA analyses of human remains from the Late Neolithic Bell Beaker site of Kromsdorf, Germany showed distinct mitochondrial haplotypes for six individuals, which were classified under the haplogroups I1, K1, T1, U2, U5, and W5, and two males were identified as belonging to the Y haplogroup R1b. In contrast to other Late Neolithic societies in Europe emphasizing maintenance of biological relatedness in mortuary contexts, the diversity of maternal haplotypes evident at Kromsdorf suggests that burial practices of Bell Beaker communities operated outside of social norms based on shared maternal lineages. Furthermore, our data, along with those from previous studies, indicate that modern U5-lineages may have received little, if any, contribution from the Mesolithic or Neolithic mitochondrial gene pool.

STR-typing of ancient skeletal remains: which multiplex-PCR kit is the best?

AIM: To comparatively test nine co mmercially available short tandem repeat (STR)-multiplex kits (PowerPlex 16, 16HS, ES, ESI17, ESX17, S5 [all Promega]; AmpFiSTR Identifiler, NGM and SEfiler [all Applied Biosystems]) for their efficiency and applicability to analyze ancient and thus highly degraded DNA samples. METHODS: Fifteen human skeletal remains from the late medieval age were obtained and analyzed using the nine polymerase chain reaction assays with slightly modified protocols. Data were systematically compared to find the most meaningful and sensitive assay. RESULTS: The ESI, ESX, and NGM kits showed the best overall results regarding amplification success, detection rate, identification of heterozygous alleles, sex determination, and reproducibility of the obtained data. CONCLUSION: Since application of these three kits enables the employment of different primer sequences for all the investigated amplicons, a combined application is recommended for best possible and--most importantly--reliable genetic analysis of ancient skeletal material or otherwise highly degraded samples, e.g., from forensic cases.

The SNPforID Assay as a Supplementary Method in Kinship and Trace Analysis.

OBJECTIVE: Short tandem repeat (STR) analysis using commercial multiplex PCR kits is the method of choice for kinship testing and trace analysis. However, under certain circumstances (deficiency testing, mutations, minute DNA amounts), STRs alone may not suffice. METHODS: We present a 50-plex single nucleotide polymorphism (SNP) assay based on the SNPs chosen by the SNPforID consortium as an additional method for paternity and for trace analysis. The new assay was applied to selected routine paternity and trace cases from our laboratory. RESULTS AND CONCLUSIONS: Our investigation shows that the new SNP multiplex assay is a valuable method to supplement STR analysis, and is a powerful means to solve complicated genetic analyses.

Biodiversity of mycobial communities in health and onychomycosis.

Onychomycosis (OM) is a common fungal nail infection. Based on the rich mycobial diversity in healthy toenails, we speculated that this is lost in OM due to the predominance of a single pathogen. We used next generation sequencing to obtain insights into the biodiversity of fungal communities in both healthy individuals and OM patients. By sequencing, a total of 338 operational-taxonomic units were found in OM patients and healthy controls. Interestingly, a classifier distinguished three distinct subsets: healthy controls and two groups within OM patients with either a low or high abundance of Trichophyton. Diversity per sample was decreased in controls compared to cases with low Trichophyton abundance (LTA), while cases with a high Trichophyton abundance (HTA) showed a lower diversity. Variation of mycobial communities between the samples showed shifts in the community structure between cases and controls-mainly driven by HTA cases. Indeed, LTA cases had a fungal ß-diversity undistinguishable from that of healthy controls. Collectively, our data provides an in-depth characterization of fungal diversity in health and OM. Our findings also suggest that onychomycosis develops either through pathogen-driven mechanisms, i.e., in HTA cases, or through host and/or environmental factors, i.e., in cases with a low Trichophyton abundance.

The more the merrier? How a few SNPs predict pigmentation phenotypes in the Northern German population.

Human pigmentation traits are of great interest to many research areas, from ancient DNA analysis to forensic science. We developed a gene-based predictive model for pigmentation phenotypes in a realistic target population for forensic case work from Northern Germany and compared our model with those brought forth by previous studies of genetically more heterogeneous populations. In doing so, we aimed at answering the following research questions: (1) do existing models allow good prediction of high-quality phenotypes in a genetically similar albeit more homogeneous population? (2) Would a model specifically set up for the more homogeneous population perform notably better than existing models? (3) Can the number of markers included in existing models be reduced without compromising their predictive capability in the more homogenous population? We investigated the association between eye, hair and skin colour and 12 candidate single-nucleotide polymorphisms (SNPs) from six genes. Our study comprised two samples of 300 and 100 individuals from Northern Germany. SNP rs12913832 in HERC2 was found to be strongly associated with blue eye colour (odds ratio=40.0, P<1.2 × 10(-4)) and to yield moderate predictive power (AUC: 77%; sensitivity: 90%, specificity: 63%, both at a 0.5 threshold for blue eye colour probability). SNP associations with hair and skin colour were weaker and genotypes less predictive. A comparison with two recently published sets of markers to predict eye and hair colour revealed that the consideration of additional SNPs with weak-to-moderate effect increased the predictive power for eye colour, but not for hair colour.

Determination of population origin: a comparison of autosomal SNPs, Y-chromosomal and mtDNA haplogroups using a Malagasy population as example.

Y-chromosomal and mitochondrial DNA (mtDNA) polymorphisms have been used for population studies for a long time. However, there is another possibility to define the origin of a population: autosomal single-nucleotide polymorphisms (SNPs) whose allele frequencies differ considerably in different populations. In an attempt to compare the usefulness of these approaches we studied a population from Madagascar using all the three mentioned approaches. Former investigations of Malagasy maternal (mtDNA) and paternal (Y chromosome) lineages have led to the assumption that the Malagasy are an admixed population with an African and Asian-Indonesian heritage. Our additional study demonstrated that more than two-third of the Malagasy investigated showed clearly a West African genotype regarding only the autosomal SNPs despite the fact that 64% had an Asian mtDNA and more than 70% demonstrated an Asian-Indonesian heritage in either mtDNA or Y-chromosomal haplogroup or both. Nonetheless, the admixture of the Malagasy could be confirmed. A clear African or Asian-Indonesian heritage according to all the three DNA approaches investigated was only found in 14% and 1% of male samples, respectively. Not even the European or Northern African influences, detected in 9% of males (Y-chromosomal analysis) and 11% of samples (autosomal SNPs) were consistent. No Malagasy in our samples showed a European or Northern African origin in both categories. So, the analysis of autosomal SNPs could confirm the admixed character of the Malagasy population, even if it pointed to a greater African influence as detectable by Y-chromosomal or mtDNA analysis.

Highly reliable quantification of proteins such as members of the HSP70 superfamily based on the grey scale index via immune detection stained bands on a Western blot.

The HSP70 superfamily is a reliable biomarker for hyperthermia, hypothermia and hypoxia. The Enzyme-linked Immunosorbent Assay (ELISA) respectively immunohistochemically staining methods are the typically used techniques for the quantification of those proteins. As the costs for reagents and devices as well as the work schedule of these methods are immense it was the goal of our study to develop an easy and reliable method to quantify the concentration of specific proteins. We established a procedure to measure the relative concentration of proteins fixed on ROTI(®) PVDF membranes via Western blot, calculating the relative protein concentration in dependency to the grey scale index of the normalized and digitalized pictures of the bands on the blots.