Deep learning approach to describe and classify fungi microscopic images

Preliminary diagnosis of fungal infections can rely on microscopic examination. However, in many cases, it does not allow unambiguous identification of the species by microbiologist due to their visual similarity. Therefore, it is usually necessary to use additional biochemical tests. That involves additional costs and extends the identification process up to 10 days. Such a delay in the implementation of targeted therapy may be grave in consequence as the mortality rate for immunosuppressed patients is high. In this paper, we apply a machine learning approach based on deep neural networks and Fisher Vector (advanced bag-of-words method) to classify microscopic images of various fungi species. Our approach has the potential to make the last stage of biochemical identification redundant, shortening the identification process by 2-3 days, and reducing the cost of the diagnosis

Impact of biological treatment on intestinal microbiom in children with Crohn's disease

Crohn’s disease (CD) is a chronic, inflammatory illness of the digestive tract, characterized by alternating periods of remission and recurrence. The pathogenesis of CD is still unclear but probably is a result of a complex interaction between immunological, genetic and microbiological disorders. In recent years, there has been an increasing extent of evidence that gut microbiota plays a very important role in the pathogenesis of CD. Currently, the most effective treatment is biological therapy using anti-TNF monoclonal antibodies. It is interesting whether biological drugs resulting in fast remission, contributes to the normalization of the gut microbiota. Due to the fact that the children’s population is a significant percentage of all patients with CD, it is important to pay close attention to the problem of microbiological disorders in this age group. The aim of this study was to investigate whether there are quantitative changes of chosen bacteria species and fungi of the genus Candida in children with Crohn's disease relative to healthy children and assesment of quantitative changes in patients after biological treatment. In the group of children with Crohn’s disease, the numbers in Candida were significantly higher (9.74×1017 CFU/g) than in the control group (9.35×1010 CFU/g, p = 0.011). Biological therapy led to a significant reduction in the amount Candida (5.91×1011) and was comparable with the number in the control group. In the case of bacteria, we observed an increase in S. marcescens (3,4×108) in the patients group compared to the controls (1,85×108) and an increase in L. fermentum (2,34×1010) in relation to healthy children (3,31×108, p = 0,048) Biological treatment had an impact on the decrease in L. fermentum (4,76×109, p = 0.05)

Evaluation of the activity of thermostable DNA polymerases in the presence of heme, as a key inhibitor in the real time PCR method in diagnostics of sepsis

The study aim was evaluation of the usefulness of several thermostable DNA polymerases in real time PCR conducted in the presence of the heme. Our study had the advantage of testing several different polymerases, one of which proved to be the least sensitive to heme activity. We also found that there is no need of supplementing the reaction mixture with protective substances like BSA. Selection of the appropriate polymerase can increase the efficiency of the PCR reaction which is very important for diagnosis of sepsis and for other analyses performed on DNA template isolated from the blood

A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood

BACKGROUND: The study describes the application of the PCR method for the simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may cause sepsis. Material for the study was sterile blood inoculated with four species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed method is based on nested-multiplex real-time PCR . RESULTS: Analysis of the obtained data shows that sensitivity of nested-multiplex real-time PCR remained at the level of 10(1) CFU/ml for each of the four studied species of microorganisms and the percentage of positive results of the examined blood samples from the patients was 70% and 19% for the microbiological culture method. The designed primers correctly typed the studied species as belonging to the groups of Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi. CONCLUSIONS: Results obtained by us indicated that the designed PCR methods: (1) allow to detect bacteria in whole blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the main groups of microorganisms within a few hours

Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis

BACKGROUND: Microbiological diagnosis of sepsis relies primarily on blood culture data. This study compares four diagnostic methods, i.e. those developed by us: nested, multiplex, qPCR (qPCR) and FISH with commercial methods: SeptiFast (Roche) (SF) and BacT/ALERT® 3D blood culture system (bioMérieux). Blood samples were derived from adult patients with clinical symptoms of sepsis, according to SIRS criteria, hospitalized in the Intensive Care Unit. RESULTS: Using qPCR, FISH, SF, and culture, microbial presence was found in 71.8%, 29.6%, 25.3%, and 36.6% of samples, respectively. It was demonstrated that qPCR was significantly more likely to detect microorganisms than the remaining methods; qPCR confirmed the results obtained with the SF kit in all cases wherein bacteria were detected with simultaneous confirmation of Gram-typing. All data collected through the FISH method were corroborated by qPCR. CONCLUSIONS: The qPCR and FISH methods described in this study may constitute alternatives to blood culture and to the few existing commercial molecular assays since they enable the detection of the majority of microbial species, and the qPCR method allows their identification in a higher number of samples than the SF test. FISH made it possible to show the presence of microbes in a blood sample even before its culture

Antimicrobial properties of selected copper alloys on Staphylococcus aureus and Escherichia coli in different simulations of environmental conditions : with vs. without organic contamination

Background: Hospital equipment made from copper alloys can play an important role in complementing traditional methods of disinfection. Aims of the study: The aim of this study was to assess the dynamics of the antimicrobial properties of selected copper alloys in different simulations of environmental conditions (with organic contamination vs. without organic contamination), and to test alternatives to the currently used testing methods. Materials and Methods: A modification of Japanese standard JIS Z 2801 as well as Staphylococcus aureus (SA) and Escherichia coli (EC) suspended in NaCl vs. tryptic soy broth (TSB) were used in tests performed on seven commonly used copper alloys, copper, and stainless steel. Results: A much faster reduction of the bacterial suspension was observed for the inoculum prepared in NaCl than in TSB. A faster reduction for EC than for SA was observed in the inoculum prepared in NaCl. The opposite results were found for the inoculum based on TSB. A significant correlation between the copper concentration in the copper alloys and the time and degree of bacterial suspension reduction was only observed in the case of EC. Conclusions: This study confirmed the antimicrobial properties of copper alloys, and additionally showed that Staphylococcus aureus was more resistant than Escherichia coli in the variant of the experiment without organic contamination. However, even for SA, a total reduction of the bacterial inoculum’s density took no longer than 2 h. Under conditions simulating organic contamination, all of the tested alloys were shown to have bactericidal or bacteriostatic properties, which was contrary to the results from stainless steel

Changes in the intestinal microbiota are seen following treatment with infliximab in children with crohn's disease

The aim of the study was to determine the impact of biological treatment with tumor necrosis factor α antibodies (anti-TNF-α) on the intestinal microbiome of children with severe Crohn’s disease (CD) and to evaluate the differences in the intestinal microbiome between patients treated with biological therapy and healthy children. Microbiota composition was analyzed by 16S next-generation sequencing (NGS) and microbial profiles were compared between studied groups. Fifty-four samples (from 18 patients before and after anti-TNF-α induction therapy and 18 healthy children) were used in the sequencing analysis. Shannon’s diversity index (p = 0.003, adj. p = 0.010) and observed operational taxonomic units (OTUs) (p = 0.007, adj. p = 0.015) were different between controls and patients with prior therapy for CD. Statistically significant dissimilarities between beta diversity metrics, indicating distinct community composition across groups, were observed in patients with CD before and after therapy. We did not observe any differences between controls and patients with CD after therapy. Core microbiome analysis at species level showed that 32 species were present only in patients with CD but not in controls. The results show that biological treatment is associated with changes in the intestinal microbiome of patients with CD: these changes result in an intestinal microbiome pattern similar to that seen in healthy children. Long-term observation is necessary to determine whether treatment can lead to full restoration of a healthy-like microbiome

The effect of linagliptin treatment on gut microbiota in patients with HNF1A-MODY or type 2 diabetes : a preliminary cohort study

Wstęp: W wielu dotychczas przeprowadzonych badaniach oceniano związek między cukrzycą a mikroflorą jelitową. Obserwowano zmianę mikroflory jelitowej pod wpływem inhibitorów dipeptydylopeptydazy-4 w modelach zwierzęcych. Celem niniejszego badania była ocena wpływu linagliptyny na florę bakteryjną okrężnicy u ludzi. Materiał i metody: W prospektywnym badaniu kohortowym wzięło udział 24 pacjentów: 5 chorych na cukrzycę monogenową, związaną z mutacją HNF1A, i 19 chorych na cukrzycę typu 2. Próbki kału pobrano przed i 4 tygodnie po intensyfikacji aktualnego leczenia za pomocą linagliptyny lub pochodnej sulfonylomocznika. Z próbek kału wyizolowano rRNA, a następnie wykonano sekwencjonowanie 16S nowej generacji. Wyniki: U 9 pacjentów do leczenia dołączono linagliptynę, a u 15 pacjentów dołączono sulfonylomocznik lub zwiększono jego dawkę. Po leczeniu linagliptyną nie zaobserwowano zmian na poziomach taksonomicznych L2–L7 na podstawie analizy składu mikrobiomów (ANCOM). To samo odnosiło się do różnorodności alfa [wskaźnik różnorodności Shannona, p = 0,59; wskaźnik równomierności Pielou, p = 0,68; obserwowane operacyjne jednostki taksonomiczne (OTU), p = 0,77] i różnorodności beta (nieważony wskaźnik UniFrac, p = 0,99; ważony wskaźnik UniFrac, p = 0,93; wskaźnik Braya–Curtisa, p = 0,98; wskaźnik Jaccarda, p = 0,99). Również po intensyfikacji leczenia pochodną sulfonylomocznika nie zaobserwowano zmian na poziomach taksonomicznych L2–L7 w ANCOM ani zmian w różnorodności alfa (wskaźnik różnorodności Shannona, p = 0,19; wskaźnik równomierności Pielou, p = 0,21; obserwowane OTU, p = 0,42) i różnorodności beta (nieważony wskaźnik UniFrac, p = 0,99; ważony wskaźnik UniFrac, p = 0,99; wskaźnik Braya–Curtisa, p = 1; wskaźnik Jaccarda, p = 0,99). Wnioski: Po 4 tygodniach od dołączenia linagliptyny do aktualnego leczenia cukrzycy nie zaobserwowano zmian w mikroflorze okrężnicy. Konieczne są dalsze badania w celu ustalenia, czy linagliptyna wpływa na mikroflorę okrężnicy u ludzi.Introduction. Many studies have evaluated the relationship between diabetes and microbiota. In animal models, the dipeptidyl peptidase-4 inhibitors altered the gut microbiota. We investigated whether linagliptin alters the gastrointestinal flora in humans. Materials and methods. This prospective cohort study enrolled 24 patients: 5 patients with maturity onset diabetes of the young associated with HNF1A mutation and 19 patients with type 2 diabetes mellitus. Stool samples were collected at baseline and 4 weeks after treatment intensification with either linagliptin or a sulphonylurea alongside current treatment. Faecal 16S rRNA was analysed by next-generation sequencing. Results. Nine patients initiated linagliptin whereas 15 patients initiated or increased the dose of a sulphonylurea. After linagliptin treatment, we did not observe changes in taxa in L2–L7 based on analysis of composition of microbiomes (ANCOM). The same held true for pairwise alpha diversity (Shannon diversity, p = 0.59; Pielou’s measure of evenness, p = 0.68; and observed operational taxonomic units [OTUs], p = 0.77) and beta diversity distances (unweighted UniFrac, p = 0.99; weighted UniFrac, p = 0.93; Bray-Curtis, p = 0.98; and Jaccard, p = 0.99). Similarly, after sulphonylurea intensification, we did not observe changes in taxa in L2–L7 in ANCOM, nor were there changes in alpha diversity (Shannon diversity, p = 0.19; Pielou’s measure of evenness, p = 0.21; and observed OTUs, p = 0.42) or beta diversity distances (unweighted UniFrac, p = 0.99; weighted UniFrac, p = 0.99; Bray-Curtis, p = 1; and Jaccard, p = 0.99). Conclusion. We did not observe changes in colonic microbiota 4 weeks after addition of linagliptin to current diabetes treatment. Further studies are required to determine whether linagliptin influences the colonic microbiota in human