Is gut microbiota of patients with ALS different from that of healthy individuals?

Background/aim: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Several studies have shown that alterations of microbiota increase the risk of neurodegenerative disorders. We aimed to reveal whether there is a difference in the gut microbiota of patients with ALS. Materials and methods: The participants are divided into three groups. Group 1 comprised patients with ALS. Healthy family members living in the same house of the patients formed Group 2. Lastly, sex- and age-matched healthy people were included in Group 3. Fecal samples were collected in 15-mL falcon tubes and stored at -80 °C. Genomic DNA isolation was performed on samples. Bacterial primers selected from the 16S rRNA region for the bacterial genome and ITS1 and ITS4 (internal transcribed spacer) were used for the identification of DNA. Next generation sequence analysis (NGS) and taxonomic analyses were performed at the level of bacterial phylum, class, order, family, genus, and species. Alpha and beta diversity indexes were used. The linear discriminant analysis (LDA) effect size method (LEfSe) was applied to identify a microbial taxon specific to ALS disease. Results: The relative abundances of the Succinivibrionaceae and Lachnospiraceae families were significantly lower in patients. The dominant families among patients were Streptococcaceae and Ruminococcaceae, while the dominant families among healthy controls were Bacteroidaceae and Succinivibrionaceae. The LEfSe analysis revealed that four families (Atopobiaceae, Actinomycetaceae, Erysipelatoclostridiaceae, Peptococcacceae) differed significantly between the patients and healthy controls (LDA values> 2.5, p < 0.05). Conclusion: Comparison with family members living in the same house is the strength of this study. We found that there were changes in the microbiota of the patients, consistent with the literature. Studies that analyze the composition of the gut microbiota in the predisease period may be needed to understand whether dysbiosis is caused by the mechanisms inherent in the disease or whether it is dysbiosis that initiates the disease.

MicroRNA expression profile of alveolar epithelial cells infected with Aspergillus fumigatus.

There are limited number of studies investigating the role of microRNAs (miRNAs) in Aspergillus infections. In this study, we designed an in vitro aspergillosis model to identify differentially expressed Aspergillus-related miRNAs. For this purpose, carcinoma cell lines "A549" and "Calu-3" were infected with Aspergillus fumigatus. Total miRNA was isolated at 0, 1, 6, and 24 h post-infection. Quantitative real-time PCR assay was conducted to screen 31 human miRNAs that were possibly related to aspergillosis. Up- and downregulated miRNAs were detected in the infected cells. Highest level of miRNA expression was detected at 6 h post-infection. miR-21, hsa-miR-186-5p, hsa-miR-490-5p, miR-26a-5p, miR-26b-5p, hsa-miR-424-5p, hsa-miR-548d-3p, hsa-miR-196a-5p, miR-150-5p, miR-17-5p, and hsa-miR-99b-5p were found to be significantly upregulated (p < 0.001) at 6 h after A. fumigatus infection compared with the controls. Among the screened miRNAs, hsa-miR-145-5p (p < 0.001); hsa-miR-583 and hsa-miR-3978 (p < 0.01); and miR-21-5p, hsa-miR-4488, and hsa-miR-4454 (p < 0.05) were found to be downregulated compared with the controls. In conclusion, screening the identified miRNAs may reveal the personal predisposition to aspergillosis, which might be valuable from the perspective of personalized medicine.

Bacterial and fungal communities in chronic rhinosinusitis with nasal polyps.

OBJECTIVE: Multiple inflammatory mechanisms dynamically interact in the development of chronic rhinosinusitis with nasal polyps (CRSwNP). Disruption of the relationship between host and environmental factors on the mucosal surface leads to the development of inflammation. Microorganisms constitute the most important part of environmental factors. METHODS: 28 volunteers (18 CRSwNP patients and 10 healthy individuals) were included in the study. Eight patients were recurrent nasal polyposis cases, and the remaining were primary cases. Swab samples were taken from the middle meatus under endoscopic examination from all participants. After DNA extraction, a library was created with the Swift Amplicon 16S + ITS kit and sequenced with Illumina Miseq. Sequence analysis was performed using QIIME, UNITE v8.2 database for ITS and Silva v138 for 16S rRNA. RESULTS: The predominant bacteria in all groups were Firmicutes, Proteobacteria, Actinobacteria as phyla and Staphylococcus, Corynebacterium, Sphingomonas as genera. Comparison of bacterial communities of CRSwNP patients and control group highlighted Corynebacterium, as the differentiating taxa for control group and Streptococcus, Moraxella, Rothia, Micrococcus, Gemella, and Prevotella for CRSwNP patients. The predominant fungal genus in all groups was Malassezia. Staphylococcus; showed a statistically significant negative correlation with Dolosigranulum. Corynebacterium had a positive correlation with Anaerococcus, and a negative correlation with Neisseria, Prevotella, Fusobacterium and Peptostreptococcus. CONCLUSION: Nasal microbiome of CRSwNP patients shows greater inter-individual variation than the control group. Corynebacterium is less abundant in patients with CRSwNP compared to the control group. Malassezia is the predominant fungus in the nasal cavity and paranasal sinuses and correlates positively with the abundance of Corynebacterium.

Inhaler Steroid Use Changes Oral and Airway Bacterial and Fungal Microbiome Profile in Asthma Patients.

INTRODUCTION: The full spectrum of bacterial and fungal species in adult asthma and the effect of inhaled corticosteroid use is not well described. The aim was to collect mouthwash and induced sputum samples from newly diagnosed asthma patients in the pretreatment period and in chronic asthma patients while undergoing regular maintenance inhaled corticosteroid therapy, in order to demonstrate the bacterial and fungal microbiome profile. METHODS: The study included 28 asthmatic patients on inhaler steroid therapy, 25 steroid-naive asthmatics, and 24 healthy controls. Genomic DNA was isolated from induced sputum and mouthwash samples. Analyses were performed using bacterial primers selected from the 16S rRNA region for the bacterial genome and "panfungal" primers selected from the 5.8S rRNA region for the fungal genome. RESULTS: Dominant genera in mouthwash samples of steroid-naive asthmatics were Neisseria, Haemophilus, and Rothia. The oral microbiota of asthmatic patients on inhaler steroid treatment included Neisseria, Rothia, and Veillonella species. Abundant genera in induced sputum samples of steroid-naive asthma patients were Actinomyces, Granulicatella, Fusobacterium, Peptostreptococcus, and Atopobium. Sputum microbiota of asthma patients taking inhaler steroids were dominated by Prevotella and Porphyromonas. Mucor plumbeus and Malassezia restricta species were abundant in the airways of steroid-naive asthma patients. Choanephora infundibulifera and Malassezia restricta became dominant in asthma patients taking inhaled steroids. CONCLUSION: The oral and airway microbiota consist of different bacterial and fungal communities in healthy and asthmatic patients. Inhaler steroid use may influence the composition of the oral and airway microbiota.

[Comparison of the Results of Galactomannan and Beta Glucan Antigen Tests in the Suspicion of Invasive Pulmonary Aspergillosis]. / Invaziv Pulmoner Aspergilloz Süpheli Hastalarda Galaktomannan ve Beta Glukan Antijen Testlerinin Karsilastirilmasi.

Opportunistic fungal infections are an important cause of morbidity and mortality in immunocompromised patients. Invasive aspergillosis (IA) has an important place among these infections with ~ 250.000 cases annually. Reducing the mortality rate due to invasive aspergillosis is possible with early diagnosis and treatment of the disease. Because of the low sensitivity in microscopic examination, the time consuming of culture growth, and the difficulties in distinguishing colonization/infection, serological methods are frequently used in the diagnosis of invasive aspergillosis. The aim of this study was to determine the diagnostic performance of galactomannan and beta glucan tests for the diagnosis of invasive pulmonary aspergillosis (IPA). Sixty patients, followed up with the suspicion of invasive pulmonary aspergillosis in Gazi University Hospital were included in the study. The clinical classification of the patients was made according to the revised European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) criteria. A total of 10 patients were classified as probable invasive aspergillosis and 20 patients were classified as possible invasive fungal disease. Demographic data of the patients and various risk factors were recorded. One hundred and thirty serum and nine bronchoalveolar lavage (BAL) fluid samples were studied with Plateliaᵀᴹ Aspergillus Ag (Bio-Rad, France), Dynamiker Aspergillus Galactomannan and Dynamiker Fungus (1-3)-beta-D-Glucan (Dynamiker, China) kits. Sensitivity and specificity values were calculated according to U.S. Food and Drug Administration (FDA) approved Plateliaᵀᴹ Aspergillus Ag test. According to this study, the most important risk factors in the development of IPA were the use of steroids and immunomodulatory drugs. The sensitivity of the galactomannan test in the probable group was 77.8%, the specificity was 96.7%, the sensitivity of the beta glucan test was 61.1%, and the specificity was 92.6%. When these two tests were evaluated together, it was observed that the sensitivity in the probable group increased to 83.3% and the specificity decreased to 89.3%. The combined use of galactomannan and beta glucan tests increases the diagnostic sensitivity. Although the presence of prolonged neutropenia is an important risk factor for IA, the use of steroids and immunomodulatory drugs should be kept in mind in non-neutropenic patients.

Frequency of azole resistance in clinical and environmental strains of Aspergillus fumigatus in Turkey: a multicentre study.

OBJECTIVES: Aspergillus fumigatus causes several diseases in humans and azole resistance in A. fumigatus strains is an important issue. The aim of this multicentre epidemiological study was to investigate the prevalence of azole resistance in clinical and environmental A. fumigatus isolates in Turkey. METHODS: Twenty-one centres participated in this study from 1 May 2018 to 1 October 2019. One participant from each centre was asked to collect environmental and clinical A. fumigatus isolates. Azole resistance was screened for using EUCAST agar screening methodology (EUCAST E.DEF 10.1) and was confirmed by the EUCAST E.DEF 9.3 reference microdilution method. Isolates with a phenotypic resistance pattern were sequenced for the cyp51A gene and microsatellite genotyping was used to determine the genetic relationships between the resistant strains. RESULTS: In total, resistance was found in 1.3% of the strains that were isolated from environmental samples and 3.3% of the strains that were isolated from clinical samples. Mutations in the cyp51A gene were detected in 9 (47.4%) of the 19 azole-resistant isolates, all of which were found to be TR34/L98H mutations. Microsatellite genotyping clearly differentiated the strains with the TR34/L98H mutation in the cyp51A gene from the strains with no mutation in this gene. CONCLUSIONS: The rate of observed azole resistance of A. fumigatus isolates was low in this study, but the fact that more than half of the examined strains had the wild-type cyp51A gene supports the idea that other mechanisms of resistance are gradually increasing.

Epidemiology and Risk Factors of Candidemia Among Hospitalized Patients in a Turkish Tertiary Care Hospital.

BACKGROUND: Candidemia, which constitutes 50 - 70% of invasive Candida infections, is an important clinical condition with high mortality and difficulty in diagnosis and treatment. Our objective was to determine the epidemiology, risk factors of candidemia, the distribution, and antifungal susceptibilities of Candida spp. responsible for candidemia among hospitalized patients in Gazi University Medical Faculty Hospital. METHODS: This was a laboratory-based, prospective observational study conducted between 2009 and 2010. The definition of nosocomial candidemia was based on CDC criteria. All relevant demographic and clinical data were collected from patient files. Candida spp. were identified by API ID32C system. Antifungal susceptibility testing was performed using broth microdilution method according to CLSI. RESULTS: Seventy-one candidemia episodes were identified with the incidence of 0.94 cases/1,000 hospital admissions. C. albicans was isolated in 47.9% of the admissions and in 52.1% of non-albicans Candida admissions. Among the latter, C. parapsilosis and C. tropicalis were the most frequent species. The most common risk factors were use of antibiotics (94.4%), hospitalization in the last 1 month (93%), history of hospitalization in ICU (74.6%), and CVC use (70.4%). Abdominal surgery, urethral catheter insertion, and use of piperacillin/tazobactam was found to increase the risk of C. albicans. A history of hospitalization within the last 3 months increased the risk of developing candidemia with non-albicans Candida spp. In total, fluconazole resistance was 20% (24.2% for C. albicans and 16.2% for non-albicans Candida strains) and voriconazole resistance was 5.7% (12.1% for C. albicans and 0% for non- albicans Candida). CONCLUSIONS: This study provided a relevant source of information for the prediction of high-risk patients and the implementation of prevention strategies for nosocomial candidemia.

[A case of Peritonitis Caused by Wickerhamomyces anomalus (Candida pelliculosa) Related to Peritoneal Dialysis]. / Wickerhamomyces anomalus (Candida pelliculosa)'un Etken Oldugu Periton Diyalizi Iliskili Bir Peritonit Olgusu.

Fungal peritonitis is less commonly seen than bacterial peritonitis in patients undergoing peritoneal dialysis (PD), but it is a serious complication with high morbidity and mortality. It often results in catheter loss and modifying therapy from PD to hemodialysis. The causative organisms are often Candida species. In this report, a PD-associated peritonitis caused by Wickerhamomyces anomalus (Candida pelliculosa), a rare fungal infection agent with increasing clinical importance by causing different clinical pictures was presented. An outpatient peritoneal fluid culture was sent from a 48-yearold male patient, who had been undergoing continuous peritoneal dialysis (CAPD) for 9 years, due to abdominal pain and blur in peritoneal fluid during dialysis. The patient admitted to the emergency department four days later due to the persistence of his complaints. A sample of peritoneal fluid was taken in the emergency department and sent to the laboratory for microbiological analysis. In the direct microscopical examination of the peritoneal fluid; cell number was determined as 210/mm3, and no microorganisms were seen in the Gram and methylene blue staining. The patient was admitted to the nephrology service with a pre-diagnosis of PD-associated peritonitis. Enterobacter aerogenes was grown in the peritoneal fluid culture which was sent from the dialysis outpatient clinic four days ago. The peritoneal fluid sample sent from the emergency department was inoculated on 5% sheep blood , EMB and chocolate agars and no growth was detected. As the patient's complaints and peritoneal fluid leukocyte count continued to increase, peritoneal fluid cultures were repeated and recurrent growth of yeast was detected in cultures. The yeast was identified as Candida pelliculosa by matrix assisted laser desorption ionization time-of-flight mass spectrofotometry (MALDI-TOF) VITEK®MS (bioMerieux, France). The species identification was confirmed by sequencing the target ITS gene regions on the rRNA and the isolate was identified as 100% Wickerhamomyces anomalus (sexual reproduction form of Candida pelliculosa, teleomorph). The reference microdilution method was performed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) in order to test the antifungal susceptibility. After 24 hour incubation, the minimal inhibitory concentrations (MIC) were determined as 0.03 µg/ml for amphotericin B, 0.125 µg/ml for caspofungin 0.125 µg/ml for voriconazole, 0.03 µg/ ml for itraconazole and 4 µg/ml for fluconazole. Fluconazole and anidulafungin were started for the treatment of fungal peritonitis. The patient's peritoneal dialysis catheter was removed and hemodialysis was applied to the patient. Clinical and laboratory symptoms regressed with antifungal therapy and the patient's anidulafungin treatment was discontinued for 14 days after the catheter removal. In conclusion, in patients undergoing CAPD, as in our case, fungal pathogens should also be considered although it is rare, when there is no laboratory and clinical improvement, and the response to treatment is not complete in PD-associated peritonitis to prevent delays in diagnosis and treatment.

Succinivibrionaceae is dominant family in fecal microbiota of Behçet's Syndrome patients with uveitis.

Behçet's Syndrome (BS) is a multisystem vasculitis with various clinical manifestations. Pathogenesis is unclear, but studies have shown genetic factors, innate immunity and autoinflammation to have an important role in the disease course. Diversity in the microbial community of gut microbiota may significantly contribute to the activation of the innate immune system. The clinical features of BS present themselves in clusters and each cluster may be a consequence of different disease mechanisms. For this reason we aimed to investigate the gut microbiota of BS patients with uveitis. In addition to healthy controls, we have aimed to compare the gut microbiota of BS with that of Familial Mediterranean Fever (FMF) and Crohn's Disease (CD) as both diseases have innate and autoinflammatory features in their pathogenesis. Seven patients with BS, 12 patients with FMF, 9 patients with CD and 16 healthy controls (HC) were included in the study. Total genomic DNAs were isolated from fecal samples of the patients. Partial 16S rRNA gene was sequenced using the PGM Ion Torrent (Thermo Fisher Scientific, Waltham, MA, USA) for microbiota analysis. Statistical analysis showed that significant differences were detected on the microbial community of four groups. Succinivibrionaceae is dominant and the signature family, whereas Bacteroides was absent in BS patients.

[Candida hellenica Candidemia Occurred After Esophagus Surgery]. / Özofagus Cerrahisi Sonrasi Gelisen Candida hellenica Kandidemisi.

In this study, a case of candidemia caused by Candida hellenica as the first report in our country was presented. Fluconazole and liposomal amphotericin B treatment was initiated in a 20-year-old male patient in October 2018 due to the diagnosis of candidemia following esophageal surgery. The patient had a history of multiple esophageal operations. The patient was discharged during the last 24 hours due to the lack of fever, improvement in general condition and lack of growth in blood cultures. Germination tube test of the Candida isolate grown in blood culture was negative and the colony morphology in corn meal tween 80 agar was not defining. It was identified as C.hellenica according to the profile obtained from the ID32C® (bioMérieux, France) method based on carbohydrate assimilation. The target ITS regions of the rRNA genes were amplified by polymerase chain reaction and sequenced using suitable primers for the confirmation of the identification on species level. The DNA sequences obtained were searched by using the "National Center for Biotechnology Information (BLAST)" (http://www.ncbi.nlm.nih.gov/ BLAST/) database and the isolate was identified as C.hellenica with a 99% homology with GenBank sequences. MALDI-TOF (Vitek MS, bioMerieux) could not identify the yeast isolate. The reference microdilution method was performed according to the recommendations of the Clinical and Laboratory Standards Institute in order to test the antifungal susceptibility. The minimal inhibitory concentrations for the isolate, determined after 24-hour incubation were 0.25 µg/ml for amphotericin B, 8 µg/ml for fluconazole, 0.25 µg/ml for voriconazole, and 0.25 µg/ml for itraconazole. As our case had a previous history of gastrointestinal tract surgery it was thought that gastrointestinal tract was the endogenous source of candidemia by leading to mucosal disruption and this mucosal disruption might facilitate the translocation of Candida. The carbohydrate assimilation test ID32C®, was able identify the causative agent of candidaemia at the species level in this case. However, uncommon or previously unrecognized organisms may be misidentified by commercial systems. While the phenotypic definition is sufficient in routine laboratories, it is mandatory to confirm the microorganism species definition by DNA sequence analysis, as done in this case. We have presented a correctly identifed and successfully treated candidemia case. Although the candidemia was not mortal in our patient, the mortality rate of candidemia which is 50%, should be remembered. A total of two C.hellenica infections have been reported in the literature, including one candidaemia and one respiratory tract colonization. Our successfully treated case was presented to draw attention to this rare infectious agent.

Retrospective analysis of the association of the expression and single nucleotide polymorphisms (SNPs) of the TLR4, PTX3 and Dectin-1 (CLEC/A) genes with development of invasive aspergillosis among haematopoietic stem cell transplant recipients with oncohaematological disorders.

OBJECTIVES: Several studies described single nucleotide polymorphisms (SNPs) on pattern recognition receptor (PRR) such as toll-like receptors (TLRs), dendritic cell-associated C-type lectin-1 (Dectin-1/CLEC7A) genes of patients with invasive fungal infections (IFIs) caused by Candida and Aspergillus. We screened TLR4, Dectin-1 and PTX3 polymorphisms in a Turkish population with invasive aspergillosis (IA) underlying haematological malignancies. METHODS: In this case-control study, a cohort of 59 patients with haematological malignancies were included. There were 26 IA patients assigned by the EORTC-MSG criteria and 33 patients with no evidence of fungal disease. DNA and RNA were isolated from frozen bone marrow and serum samples. RNA levels and polymorphisms of TLR4 (rs4986790, rs4986791), Dectin-1 (rs16910526, rs7309123) and PTX3 (rs2305619, rs3816527) were determined. The odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated by unconditional logistic regression analysis. RESULTS AND CONCLUSIONS: TLR4, PTX3 and Dectin-1 genes were downregulated in aspergillosis cohort under similar haematological conditions. TLR4 expression was 0.0626 ± 0.032 in controls when compared to IA patients as 0.0077 ± 0.014, and the difference was significant (P = .026). There was a difference in also the PTX3 gene among IA (0.0043 ± 0.004) and control (0.5265 ± 0.0043) groups (P = .035). The Dectin-1 (CLEC/A) expression was downregulated in IA group (0.1887 ± 0.072 & 0.0655 ± 0.010) but not statistically significant (P > .05). Conditional logistic regression analyses indicated that the GT genotype of rs16910526 polymorphism in Dectin-1 gene was associated with lower risk of IA (odds ratio = 3.635, 95% confidence interval = 0.690-3.138, P = .04).

In Vitro Efficacy of Chlorhexidine and a riboflavin/UVA Combination on Fungal Agents of Keratitis.

Purpose: Mycotic keratitis is a global ophthalmological problem because it is difficult to diagnose and treat. The aim of the current study was to evaluate the efficiency of using antifungal agents amphotericin B (AMB), voriconazole (VRC), 0.02% chlorhexidine (CHX), and a combination of riboflavin and UVA treatment against two fungal genera (Aspergillus and Fusarium) responsible for keratitis.Methods: We evaluated antifungal efficiencies of riboflavin/UVA and the antifungal drugs VRC, AMB, and CHX (alone and in combination) against fungal inocula at four concentrations. We recorded colony counts of isolates for Aspergillus terreus, A. flavus, A. fumigatus, Fusarium falciforme, F. proliferatum, and F. solani on Mueller-Hinton agar plates.Results: Fungal suspensions exposed to the following treatment combinations did not allow fungal growth: riboflavin/UVA and VRC, riboflavin/UVA and AMB, riboflavin/UVA and CHX, and CHX alone. We observed a statistically significant reduction (P < .05) in the number of colonies on agar plates when fungal suspensions were treated with riboflavin/UVA, VRC, and AMB only.Conclusions: Riboflavin/UVA treatment in combination with AMB, VRC, and CHX are capable of killing keratitis-inducing fungi (P < .05). The antiseptic CHX exerted a considerable antifungal effect on all strains we examined. Therefore, we recommend CHX as additional therapy against mycotic keratitis, particularly when keratitis is caused by multi-resistant members of Fusarium.

Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit.

BACKGROUND: The prevalence of opportunistic yeast infections has increased in recent decades as the result of an increasing immunocompromised patient population. AIMS: To evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, to investigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypic method. METHODS: Both regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, were amplified using PCR and then sequenced. The sequences were compared to reference data available from the GenBank database of the National Center for Biotechnology Information using the BLASTn tool. RESULTS: Using ID32C, 88% (44/50) of all strains were identified accurately at the species level, although 6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Candida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the four isolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITS region, but the remaining two were not distinguishable from closely related species. Based on the D1/D2 region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Trichosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions. CONCLUSIONS: Identifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapid and reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest a two-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agar with Tween® 80 and API ID32C®) and sequence analysis of the ITS and D1/D2 region.

Is Induced Sputum A Useful Noninvasive Tool in the Diagnosis of Pulmonary Sarcoidosis?

OBJECTIVES: In patients with pulmonary sarcoidosis, the provocation of sputum expectoration through the inhalation of hypertonic saline has been investigated as an alternative diagnostic tool for invasive procedures. We aimed to assess the diagnostic value of induced sputum (IS) by observing its cell distribution in patients with a confirmed histopathological diagnosis of sarcoidosis. MATERIALS AND METHODS: In this prospective, cross-sectional study, we compared the IS results of 20 patients with a histopathologically confirmed pulmonary sarcoidosis diagnosis and 24 healthy volunteers. The percentages of macrophages, lymphocytes, neutrophils, and eosinophils in IS and the CD4/CD8 ratio were compared. RESULTS: The percentage of lymphocytes in IS was significantly higher in the pulmonary sarcoidosis patients compared to the control group (41.6% vs 8.9%, p<0.001). There were no significant differences in the other IS cell percentages and CD4+/CD8+ ratio between the groups. Sputum induction was well tolerated. CONCLUSION: Sputum induced by the inhalation of hypertonic saline is a safe, inexpensive, less invasive, and easily repeated method and can be a valuable alternative to other invasive methods in the diagnosis of pulmonary sarcoidosis.

Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit / Identificación de levaduras de interés médico mediante análisis de secuencia del espaciador interno transcrito y de la región D1/D2 de la subunidad ribosómica grande

Background: The prevalence of opportunistic yeast infections has increased in recent decades as the result of an increasing immunocompromised patient population. Aims: To evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, to investigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypic method. Methods: Both regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, were amplified using PCR and then sequenced. The sequences were compared to reference data available from the GenBank database of the National Center for Biotechnology Information using the BLASTn tool. Results: Using ID32C, 88% (44/50) of all strains were identified accurately at the species level, although 6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Candida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the four isolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITS region, but the remaining two were not distinguishable from closely related species. Based on the D1/D2 region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Trichosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions. Conclusions: Identifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapid and reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest a two-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agar with Tween(R) 80 and API ID32C(R)) and sequence analysis of the ITS and D1/D2 region

Antecedentes: La prevalencia de infecciones oportunistas por levaduras ha aumentado en las últimas décadas como resultado de una población de pacientes inmunocomprometidos cada vez mayor. Objetivos: Evaluar la secuencia del gen del ARN ribosomal (ARNr) para identificar especies de levaduras médicamente importantes, investigar el rendimiento del espaciador transcrito interno del gen ARNr (ITS) y las regiones D1/D2 en la identificación de levaduras clínicamente relevantes, y comparar estos resultados con los de un método fenotípico estándar. Métodos: Ambas regiones del ARNr de 50 cepas de levaduras con 45 aislamientos clínicos y 5 cepas de referencia se amplificaron mediante PCR y posteriormente se secuenciaron. Las secuencias se compararon con los datos de referencia disponibles en la base de datos GenBank(R) del Centro Nacional de Información Biotecnológica mediante la herramienta BLASTn. Resultados: Mediante el método ID32C el 88% (44/50) de todas las cepas se identificaron con precisión y el 6% se identificaron erróneamente; dos aislamientos de Candida eremophila fueron identificados como Candida glabrata y Candida tropicalis, y un aislamiento de Saprochaete clavata fue identificado como Saprochaete capitata. Dos de los cuatro aislamientos identificados por métodos fenotípicos como Trichosporon asahii se catalogaron así al analizar la región ITS, pero las dos restantes no se distinguían de las especies estrechamente relacionadas. En base a la secuencia de la región D1/D2, estos cuatro aislamientos se identificaron, con un 100% de similitud, como T. asahii, Trichosporon japonicum y Trichosporon asteroides. El aislamiento identificado como Trichosporon inkin mediante ID32C no se pudo distinguir de Trichosporon ovoides al analizar las regiones ITS y D1/D2. Conclusiones: La identificación de levaduras de interés médico mediante la secuenciación de las regiones ITS y D1/D2 es una alternativa rápida y confiable a los métodos de identificación convencionales. Para un algoritmo de diagnóstico sugerimos un procedimiento de dos pasos que integre métodos convencionales (morfología microscópica en agar de harina de maíz con Tween(R) 80 y API ID32C(R)) y análisis de la secuencia de las regiones ITS y D1/D2

In vitro activity of eravacycline in combination with colistin against carbapenem-resistant A. baumannii isolates.

The synergistic activity of eravacycline in combination with colistin on carbapenem-resistant A. baumannii (CRAB) isolates was evaluated in this study. Minimum inhibitory concentrations (MICs) of eravacycline and colistin were determined by the broth microdilution method. MICs values ranged between 1 to 4 mg and 0.5 to 256 mg l-1 for eravacycline and colistin, respectively. In vitro synergy between eravacycline and colistin was evaluated by using the chequerboard methodology. Synergistic activity was found in 10% of the strains, and additive effect in 30%. No antagonism was detected. Similar activity was also observed in colistin-resistant CRAB isolates. The result of this study indicates that eravacycline and colistin combination may be a potential therapeutic option for the treatment of CRAB related infections.

First multicentre report of in vitro resistance rates in candidaemia isolates in Turkey.

OBJECTIVES: This study investigated the antifungal resistance rates of isolates from candidaemia patients in 12 tertiary-care centres in Turkey. METHODS: A total of 1991 Candida spp. isolates from 12 centres isolated from 1997-2017 were included in the study. Species/species complex (SC) identification was performed using conventional methods in all centres, occasionally accompanied by MALDI-TOF/MS. Antifungal susceptibility testing was performed for amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole and micafungin (as echinocandin class representative) using the CLSI microdilution method. Resistance rates were determined according to CLSI clinical breakpoints (CBPs). For drugs and species with undetermined CBPs, epidemiological cut-off values were used for wild-type (WT)/non-WT categorisation. RESULTS: No or low rates of resistance were detected in general for tested Candida spp. isolates. Specifically, overall resistance to fluconazole in isolates of Candida parapsilosis SC and Candida glabrata SC were 7.7% and 0.9%, respectively. Resistance rates for C. parapsilosis SC varied extensively from one center to other (0-47.1%). Importantly, no echinocandin resistance was detected. Rates of non-WT isolates were also generally low: fluconazole against Candida lusitaniae, 4.3%; posaconazole against C. parapsilosis SC, 3.5%; posaconazole against Candida krusei, 1.9%; and voriconazole against C. glabrata SC, 0.5%. CONCLUSION: This is the first multicentre report of antifungal resistance rates among candidaemia isolates in Turkey, suggesting low resistance rates in general. Due to varying rates of fluconazole resistance in C. parapsilosis SC isolates that was detected at remarkably high levels in some centres, further studies are warranted to explore the source, clonal relatedness and resistance mechanisms of the isolates.