New Strategy for Rapid Diagnosis and Characterization of Fungal Infections: The Example of Corneal Scrapings

PURPOSE: The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate diagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types. The objective of this work was to develop a new diagnostic strategy based on real-time polymerase-chain reaction high-resolution melting analysis (PCR-HRM) that a) detects yeasts and filamentous Fungi, b) differentiates yeasts from filamentous Fungi, and c) discriminates among relevant species of yeasts. METHODS: PCR-HRM detection limits and specificity were assessed with a) isolated strains; b) human blood samples experimentally infected with Fungi; c) blood experimentally infected with other infectious agents; d) corneal scrapings from patients with suspected fungal keratitis (culture positive and negative) and e) scrapings from patients with suspected bacterial, viral or Acanthamoeba infections. The DNAs were extracted and mixed with primers diluted in the MeltDoctor® HRM Master Mix in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn (5'TCCTCCGCTT ATTGATATGCT) and the second for filamentous Fungi, containing the forward primer FilamUn (5'TGCCTGTCCGAGCGTCAT) and FungUn. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were systematically monitored. RESULTS: PCR-HRM detected 0.1 Colony Forming Units (CFU)/µl of yeasts and filamentous Fungi, differentiated filamentous Fungi from yeasts and discriminated among relevant species of yeasts. PCR-HRM performances were higher than haemoculture and sensitivity and specificity was 100% for culture positive samples, detecting and characterizing Fungi in 7 out 10 culture negative suspected fungal keratitis. CONCLUSIONS: PCR-HRM appears as a new, sensitive, specific and inexpensive test that detects Fungi and differentiates filamentous Fungi from yeasts. It allows direct fungal detection from clinical samples and experimentally infected blood in less than 2.30 h after DNA extraction

Contribution of the Microbial Communities Detected on an Oil Painting on Canvas to Its Biodeterioration

In this study, we investigated the microbial community (bacteria and fungi) colonising an oil painting on canvas, which showed visible signs of biodeterioration. A combined strategy, comprising culture-dependent and -independent techniques, was selected. The results derived from the two techniques were disparate. Most of the isolated bacterial strains belonged to related species of the phylum Firmicutes, as Bacillus sp. and Paenisporosarcina sp., whereas the majority of the non-cultivable members of the bacterial community were shown to be related to species of the phylum Proteobacteria, as Stenotrophomonas sp. Fungal communities also showed discrepancies: the isolated fungal strains belonged to different genera of the order Eurotiales, as Penicillium and Eurotium, and the non-cultivable belonged to species of the order Pleosporales and Saccharomycetales. The cultivable microorganisms, which exhibited enzymatic activities related to the deterioration processes, were selected to evaluate their biodeteriorative potential on canvas paintings; namely Arthrobacter sp. as the representative bacterium and Penicillium sp. as the representative fungus. With this aim, a sample taken from the painting studied in this work was examined to determine the stratigraphic sequence of its cross-section. From this information, “mock paintings,” simulating the structure of the original painting, were prepared, inoculated with the selected bacterial and fungal strains, and subsequently examined by micro-Fourier Transform Infrared spectroscopy, in order to determine their potential susceptibility to microbial degradation. The FTIR-spectra revealed that neither Arthrobacter sp. nor Penicillium sp. alone, were able to induce chemical changes on the various materials used to prepare “mock paintings.” Only when inoculated together, could a synergistic effect on the FTIR-spectra be observed, in the form of a variation in band position on the spectrum.The FTIR analyses performed in this study were financed by the Junta de Andalucía (RNM-325 group). The molecular analyses performed in this study were financed by the Austrian Science Fund (FWF) project ‘Hertha-Firnberg T137’ and the Spanish Ministry of Science and Innovation (Project CTQ2008-06727-C03-03). G. Piñar also thanks the “Elise-Richter V194-B20” projects

Rapid Qualitative Urinary Tract Infection Pathogen Identification by SeptiFast® Real-Time PCR

Background Urinary tract infections (UTI) are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical. Aim To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast®) and to compare the results with dipslide and microbiological culture. Design of study Pilot study with prospectively collected urine samples. Setting University hospital. Methods 82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast®) was performed in all samples. Results 61 samples were SeptiFast® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%), 477/492 (97%) and 238/246 (97%), respectively. Sensitivity and specificity of the SeptiFast® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast® pathogen identifications were available at least 43 hours prior to culture results. Conclusion The SeptiFast® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods

Real-Time PCR Improves Helicobacter pylori Detection in Patients with Peptic Ulcer Bleeding

Background and aims: Histological and rapid urease tests to detect H. pylori in biopsy specimens obtained during peptic ulcer bleeding episodes (PUB) often produce false-negative results. We aimed to examine whether immunohistochemistry and real-time PCR can improve the sensitivity of these biopsies. Patients and Methods: We selected 52 histology-negative formalin-fixed paraffin-embedded biopsy specimens obtained during PUB episodes. Additional tests showed 10 were true negatives and 42 were false negatives. We also selected 17 histology-positive biopsy specimens obtained during PUB to use as controls. We performed immunohistochemistry staining and real-time PCR for 16S rRNA, ureA, and 23S rRNA for H. pylori genes on all specimens. Results: All controls were positive for H. pylori on all PCR assays and immunohistochemical staining. Regarding the 52 initially negative biopsies, all PCR tests were significantly more sensitive than immunohistochemical staining (p<0.01). Sensitivity and specificity were 55% and 80% for 16S rRNA PCR, 43% and 90% for ureA PCR, 41% and 80% for 23S rRNA PCR, and 7% and 100% for immunohistochemical staining, respectively. Combined analysis of PCR assays for two genes were significantly more sensitive than ureA or 23S rRNA PCR tests alone (p<0.05) and marginally better than 16S rRNA PCR alone. The best combination was 16S rRNA+ureA, with a sensitivity of 64% and a specificity of 80%. Conclusions: Real-time PCR improves the detection of H. pylori infection in histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during PUB episodes. The low reported prevalence of H. pylori in PUB may be due to the failure of conventional tests to detect infection

Molecular Tools for Monitoring the Ecological Sustainability of a Stone Bio-Consolidation Treatment at the Royal Chapel, Granada

Background: Biomineralization processes have recently been applied in situ to protect and consolidate decayed ornamental stone of the Royal Chapel in Granada (Spain). While this promising method has demonstrated its efficacy regarding strengthening of the stone, little is known about its ecological sustainability.Methodology/Principal Findings: Here, we report molecular monitoring of the stone-autochthonous microbiota before and at 5, 12 and 30 months after the bio-consolidation treatment (medium/long-term monitoring), employing the well-known molecular strategy of DGGE analyses. Before the bio-consolidation treatment, the bacterial diversity showed the exclusive dominance of Actinobacteria (100%), which decreased in the community (44.2%) after 5 months, and Gamma-proteobacteria (30.24%) and Chloroflexi (25.56%) appeared. After 12 months, Gamma-proteobacteria vanished from the community and Cyanobacteria (22.1%) appeared and remained dominant after thirty months, when the microbiota consisted of Actinobacteria (42.2%) and Cyanobacteria (57.8%) only. Fungal diversity showed that the Ascomycota phylum was dominant before treatment (100%), while, after five months, Basidiomycota (6.38%) appeared on the stone, and vanished again after twelve months. Thirty months after the treatment, the fungal population started to stabilize and Ascomycota dominated on the stone (83.33%) once again. Members of green algae (Chlorophyta, Viridiplantae) appeared on the stone at 5, 12 and 30 months after the treatment and accounted for 4.25%, 84.77% and 16.77%, respectively.Conclusions: The results clearly show that, although a temporary shift in the bacterial and fungal diversity was observed during the first five months, most probably promoted by the application of the bio-consolidation treatment, the microbiota tends to regain its initial stability in a few months. Thus, the treatment does not seem to have any negative side effects on the stone-autochthonous microbiota over that time. The molecular strategy employed here is suggested as an efficient monitoring tool to assess the impact on the stone-autochthonous microbiota of the application of biomineralization processes as a restoration/conservation procedure.This work was supported by the European Regional Development Fund (ERDF), Junta de Andalucía (Spain) and the “Fortalecimiento de la I+D+i” program from the University of Granada, co-financed by grant RNM-3493 and Research Group BIO-103 from Junta de Andalucía, as well as by the Spanish Government through “José Castillejo” program from the “Ministerio de Educación, Cultura y Deporte” (I+D+i 2008-2011), and by the Austrian Science Fund (FWF) under Grant “Elise-Richter V194-B20”

Molecular Analysis of Microbial Communities in Endotracheal Tube Biofilms

Ventilator-associated pneumonia is the most prevalent acquired infection of patients on intensive care units and is associated with considerable morbidity and mortality. Evidence suggests that an improved understanding of the composition of the biofilm communities that form on endotracheal tubes may result in the development of improved preventative strategies for ventilator-associated pneumonia. (n = 5). DGGE profiling of the endotracheal biofilms revealed complex banding patterns containing between 3 and 22 (mean 6) bands per tube, thus demonstrating the marked complexity of the constituent biofilms. Significant inter-patient diversity was evident. The number of DGGE bands detected was not related to total viable microbial counts or the duration of intubation.Molecular profiling using DGGE demonstrated considerable biofilm compositional complexity and inter-patient diversity and provides a rapid method for the further study of biofilm composition in longitudinal and interventional studies. The presence of oral microorganisms in endotracheal tube biofilms suggests that these may be important in biofilm development and may provide a therapeutic target for the prevention of ventilator-associated pneumonia

Influence of Uranium on Bacterial Communities: A Comparison of Natural Uranium-Rich Soils with Controls

This study investigated the influence of uranium on the indigenous bacterial community structure in natural soils with high uranium content. Radioactive soil samples exhibiting 0.26% - 25.5% U in mass were analyzed and compared with nearby control soils containing trace uranium. EXAFS and XRD analyses of soils revealed the presence of U(VI) and uranium-phosphate mineral phases, identified as sabugalite and meta-autunite. A comparative analysis of bacterial community fingerprints using denaturing gradient gel electrophoresis (DGGE) revealed the presence of a complex population in both control and uranium-rich samples. However, bacterial communities inhabiting uraniferous soils exhibited specific fingerprints that were remarkably stable over time, in contrast to populations from nearby control samples. Representatives of Acidobacteria, Proteobacteria, and seven others phyla were detected in DGGE bands specific to uraniferous samples. In particular, sequences related to iron-reducing bacteria such as Geobacter and Geothrix were identified concomitantly with iron-oxidizing species such as Gallionella and Sideroxydans. All together, our results demonstrate that uranium exerts a permanent high pressure on soil bacterial communities and suggest the existence of a uranium redox cycle mediated by bacteria in the soil

Metagenomic Profile of the Bacterial Communities Associated with Ixodes ricinus Ticks

Assessment of the microbial diversity residing in arthropod vectors of medical importance is crucial for monitoring endemic infections, for surveillance of newly emerging zoonotic pathogens, and for unraveling the associated bacteria within its host. The tick Ixodes ricinus is recognized as the primary European vector of disease-causing bacteria in humans. Despite I. ricinus being of great public health relevance, its microbial communities remain largely unexplored to date. Here we evaluate the pathogen-load and the microbiome in single adult I. ricinus by using 454- and Illumina-based metagenomic approaches. Genomic DNA-derived sequences were taxonomically profiled using a computational approach based on the BWA algorithm, allowing for the identification of known tick-borne pathogens at the strain level and the putative tick core microbiome. Additionally, we assessed and compared the bacterial taxonomic profile in nymphal and adult I. ricinus pools collected from two distinct geographic regions in Northern Italy by means of V6-16S rRNA amplicon pyrosequencing and community based ecological analysis. A total of 108 genera belonging to representatives of all bacterial phyla were detected and a rapid qualitative assessment for pathogenic bacteria, such as Borrelia, Rickettsia and Candidatus Neoehrlichia, and for other bacteria with mutualistic relationship or undetermined function, such as Wolbachia and Rickettsiella, was possible. Interestingly, the ecological analysis revealed that the bacterial community structure differed between the examined geographic regions and tick life stages. This finding suggests that the environmental context (abiotic and biotic factors) and host-selection behaviors affect their microbiome