Clinical Specimens Of Patients Research Articles

Associated Microorganisms in Clinical Specimens from Neurology Intensive Care Unit of A Tertiary Care Hospital In Adana, Turkey

Objective: The aim of this study was to determine our microorganisms breeding in culture cause of nosocomial infections in neurology intensive care unit (NICU) of a tertiary care hospital. Methods: This study was performed at the neurology intensive care unit, Adana City Research and Training Hospital, Turkey. A retrospective review of medical records was conducted for all patients admitted to the NICU, and clinical specimens of patients suspected of having infection were received, from September 2017 through July 2018. Data was collected using a neurology intensive care unit electronic database. MedCalc 15.8 software program (MedCalc Belgium) was used for statistical evaluation. Results: Out of the 84 patients evaluated, there was no microbial growth in the clinical specimens of 24 (28.2%) patients. Microbial growths were observed in blood, urine or tracheal aspirate cultures of 60 (71.8%) of the patients, while there were microbial growths detected in blood cultures of 46 (54.1%) patients; urine cultures of 47 (55.27%) patients, and tracheal aspirate cultures of 15 (17.6%) patients. The highest percentage of bacterial recovery was Escherichia coli (23.6%), which were followed by Staphylococcus epidermidis (10.7%), Acinetobacter baumannii (9.5%), and Staphylococcus aureus (9.5%) respectively. Conclusion: Clinical specimens from neurology intensive care unit of Adana City Research and Training Hospital, Turkey, yielded Gram-negative (Acinetobacter baumannii and Escherichia coli), as well as Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) bacterial species.

Designing luminescent ruthenium prodrug for precise cancer therapy and rapid clinical diagnosis

The effective design of a targeted drug delivery system could improve the therapeutic efficacy of anticancer drugs by reducing their undesirable adsorption and toxic side effects. Here, an RGD-peptide functionalized and bioresponsive ruthenium prodrug (Ru-RGD) was designed for both cancer therapy and clinical diagnosis. This prodrug can be selectively delivered to cervical tumor sites to enhance theranostic efficacy. The benzimidazole-based ligand of the complex is susceptible to acidic conditions so, after reaching the tumor microenvironment, ligand substitution occurs and the therapeutic drug is released. The deep-red emissions produced by both one-photon and two-photon excitation increases the potential of Ru-RGD for use in the deep tissue imaging of 3D tumor spheroids. The specific accumulation of the Ru prodrug in tumor sites allows for precise tumor diagnosis and therapy in vivo. Luminescence staining of 38 clinical patient specimens shows that Ru-RGD exhibits differences in binding capability between cervical cancer and normal tissue, with a sensitivity of 95% and a specificity of 100%. This study thus provides an approach for the effective design and application of targeted metal complexes in cancer therapy and clinical diagnosis.

Detection of Efflux MexAB-associated Multidrug Resistant (MDR) Pseudomonas aeruginosa Isolated from Patients in Torbat Heydarie, Northeast Iran

Aims of the study : Pseudomonas aeruginosa ( P. aeruginosa ) is one of the leading causes of hospital acquired infections. Infections with P. aeruginosa are often hard to treat because of existence of different mechanisms of antibiotic resistance changes in permeability of drugs and activity multidrug efflux pumps . The aim of current study was to determine the antibiotic resistance pattern of P. aeruginosa and existence of efflux pump MexAB genes using PCR technique. Materials and Methods: 506 isolates cultured from different clinical specimens of patients hospitalized at Nohom Dey and Razi hospitals of Torbat Heydarie (northeast Iran) were collected and used in this study. Isolates were identified using conventional bacteriology and their susceptibility to different antibiotics were assessed using agar disk diffusion method. The PCR assay was used to detect efflux pump MexAB genes. Results: From 506 isolates, 50 were identified as P. aeruginosa and these were isolated from isolated from blood, tracheal, burn, and wound. Incidence of P. aeruginosa was greater in males than females , wound infections had the highest number of occurrence and patients between 30-50 years were the most infected age group. In total, 60.86% of strains were multidrug resistant (MDR). The PCR technique revealed that most of the P. aeruginosa isolates and all the MDR strains contained MexA and MexB genes. Conclusions: The emergence of MDR microorganisms poses serious therapeutic problems for patients. Determining bacterial resistance mechanisms is complex. In this way, efflux systems were responsible for antibiotic resistance and played an important role in the MDR phenotype among P. aeruginosa isolates.

Overexpression of satellite alpha transcripts leads to chromosomal instability via segregation errors at specific chromosomes.

The impairment of the stability of the chromosomal structure facilitates the abnormal segregation of chromosomes, thus increasing the risk of carcinogenesis. Chromosomal stability during segregation is managed by appropriate methylation at the centromere of chromosomes. Insufficient methylation, or hypomethylation, results in chromosomal instability. The centromere consists of satellite alpha repetitive sequences, which are ideal targets for DNA hypomethylation, resulting in the overexpression of satellite alpha transcript(SAT). The overexpression of SAT has been reported to induce the abnormal segregation of chromosomes. In this study, we verified the oncogenic pathway via chromosomal instability involving DNA hypomethylation and the overexpression of SAT. For this purpose, we constructed lentiviral vectors expressing SAT and control viruses and then infected human mammary epithelial cells with these vectors. The copy number alterations and segregation errors of chromosomes were evaluated by microarray-based comparative genomic hybridization (array CGH) and immunocytochemistry, respectively. The levels of hypomethylation of satellite alpha sequences were determined by MethyLight polymerase chain reaction. Clinical specimens from 45patients with breast cancer were recruited to verify the data invitro. The results of immunocytochemistry revealed that the incidence of segregation errors was significantly higher in the cells overexpressing SAT than in the controls. An array CGH identified the specific chromosomes of 8q and 20q as frequent sites of copy number alterations in cells with SAT overexpression, although no such sites were noted in the controls, which was consistent with the data from clinical specimens. A regression analysis revealed that the expression of SAT was significantly associated with the levels of hypomethylation of satellite alpha sequences. On the whole, the overexpression of SAT led to chromosomal instability via segregation errors at specific chromosomes in connection with DNA hypomethylation, which was also recognized in clinical specimens of patients with breast cancer. Thus, this oncogenic pathway may be involved in the development of breast cancer.

Prevalence and risk factors for CTX-M gram-negative bacteria in hospitalized patients at a tertiary care hospital in Kilimanjaro, Tanzania

Emergence and spread of extended spectrum beta-lactamase (ESBL)-producing gram-negative bacteria, mainly due to CTX-M, is a major global public health problem. Patients infected with ESBL-producing gram-negative bacteria have an increased risk of treatment failure and death. We investigated the prevalence and risk factors for CTX-M gram-negative bacteria isolated from clinical specimens of patients hospitalized at a tertiary care hospital in Kilimanjaro, Tanzania. Isolated gram-negative bacteria from inpatients admitted at Kilimanjaro Christian Medical Centre (KCMC) between August 2013 and August 2015 were fully genome sequenced. The prevalence of ESBL-producing gram-negative bacteria was determined based on the presence of blaCTX-M. The odds ratio (OR) and risk factors for ESBL-producing gram-negative bacteria due to CTX-M were assessed using logistic regression models. The overall CTX-M prevalence (95% CI) was 13.6% (10.1–18.1). Adjusted for other factors, the OR of CTX-M gram-negative bacteria for patients previously hospitalized was 0.26 (0.08–0.88), p = 0.031; the OR for patients currently on antibiotics was 4.02 (1.29–12.58), p = 0.017; the OR for patients currently on ceftriaxone was 0.14 (0.04–0.46), p = 0.001; and the OR for patients with wound infections was 0.24 (0.09–0.61), p = 0.003. The prevalence of ESBL-producing gram-negative bacteria due to CTX-M in this setting is relatively low compared to other previous reports in similar settings. However, to properly stop further spread in the hospital, we recommend setting up a hospital surveillance system that takes full advantage of the available next-generation sequencing facility to routinely screen for all types of bacterial resistance genes.

Microchamber Cultures of Bladder Cancer: A Platform for Characterizing Drug Responsiveness and Resistance in PDX and Primary Cancer Cells

Precision cancer medicine seeks to target the underlying genetic alterations of cancer; however, it has been challenging to use genetic profiles of individual patients in identifying the most appropriate anti-cancer drugs. This spurred the development of patient avatars; for example, patient-derived xenografts (PDXs) established in mice and used for drug exposure studies. However, PDXs are associated with high cost, long development time and low efficiency of engraftment. Herein we explored the use of microfluidic devices or microchambers as simple and low-cost means of maintaining bladder cancer cells over extended periods of times in order to study patterns of drug responsiveness and resistance. When placed into 75 µm tall microfluidic chambers, cancer cells grew as ellipsoids reaching millimeter-scale dimeters over the course of 30 days in culture. We cultured three PDX and three clinical patient specimens with 100% success rate. The turn-around time for a typical efficacy study using microchambers was less than 10 days. Importantly, PDX-derived ellipsoids in microchambers retained patterns of drug responsiveness and resistance observed in PDX mice and also exhibited in vivo-like heterogeneity of tumor responses. Overall, this study establishes microfluidic cultures of difficult-to-maintain primary cancer cells as a useful tool for precision cancer medicine.

Chronic lower extremity wound infection due to Kerstersia gyiorum in a patient with Buerger\u2019s disease: a case report

BackgroundKerstersia gyiorum is an extremely rare pathogen of human infection. It can cause chronic infection in patients with underlying conditions. It can easily be misdiagnosed if proper diagnostic methods are not used.Case presentationA 47-year-old male patient with a history of Buerger’s Disease for 28 years presented to our hospital with an infected chronic wound on foot. The wound was debrided, and the specimen was sent to Microbiology laboratory. Gram staining of the specimen showed abundant polymorphonuclear leukocytes and gram-negative bacilli. Four types of colonies were isolated on blood agar. These were identified as Kerstersia gyiorum, Proteus vulgaris, Enterobacter cloacae, Morganella morganii by Maldi Biotyper (Bruker Daltonics, Germany). The identification of K. gyiorum was confirmed by 16S ribosomal RNA gene sequencing. The patient was successfully recovered with antimicrobial therapy, surgical debridement, and skin grafting.ConclusionsThis is the first case of wound infection due to K. gyiorum in a patient with Buerger’s Disease. We made a brief review of K. gyiorum cases up to date. Also, this case is presented to draw attention to the use of new and advanced methods like MALDI-TOF MS and 16S rRNA gene sequencing for identification of rarely isolated species from clinical specimens of patients with chronic infections and with chronic underlying conditions.

Phenotypic and genotypic characterization of biofilm formation among Staphylococcus aureus isolates from clinical specimens, an Atomic Force Microscopic (AFM) study.

Staphylococcus aureus is a major cause of nosocomial infections. Biofilm formation is an important factor for bacterial pathogenesis. Its mechanisms are complex and include of many genes depends on expression of icaADBC operon involved in the synthesis of a polysaccharide intercellular adhesion. The aim of study was to investigate biofilm forming ability of Staphylococcus aureus strains by phenotypic and genotypic methods. Also Atomic Force microscope (AFM) was used to visualize biofilm formation. 140 Isolates were collected from clinical specimens of patients in Milad Hospital, Tehran and diagnosed by biochemical tests. The ability of strains to produce slime was evaluated by CRA method. For diagnosing of bacterial EPS, Indian ink staining were used and finally biofilm surface of 3 isolates observed by AFM. The prevalence of icaA and icaD genes was determined by PCR. By CRA method 15% of samples considered as positive slime producers, 44.28% as intermediate and 40.71% indicative as negative slime producers. 118 staphylococcus aureus strains showed a distinct halo transparent zone but 22 strains showed no halo zone. AFM analysis of Slime positive isolates showed a distinct and complete biofilm formation. In slime negative strain, there was not observed biofilm. The prevalence of icaA, icaD genes was 44.2% and 10% of the isolates had both genes simultaneously. There is a relationship between exopolysaccharide layer and biofilm formation of Staphylococcus aureus isolates. The presence of icaAD genes among isolates is not associated with invitro formation of biofilm. AFM is a useful tool for observation of bacterial biofilm formation.

USE OF PROBIOTIC ACID BACTERIA FOR THE CONTROL OF MULTIDRUG RESISTANT BACTERIA ISOLATED FROM CLINICAL INFECTIONS

This study was carried out to evaluate the microbial infections in natural way and reduce the antibiotic resistance. One hundred bacterial isolates were collected from clinical specimens of patients suffering from bacterial infection (55 females and 45 males). The bacterial isolates were obtained from 8 different specimens with the following percentage representations: urine (30.58 %), blood (20%), abscess (20%), wound (9.41%), throat and tube swab (5.88%), ear discharges (5.88%), vaginal swabs (5.88%) and eye swab (2.35%).The results showed that (29.41%) Escherichia coli, (23.53%) Klebsiella pneumoniae, (17.65%) Staphylococcus aureus, (15.29%) Pseudomonas aeruginosa, (8.24%) Proteus vulgaris, (5.88%) Acinetobacter baumannii. Among 17 antibiotics tested, meropenem, a carbapenem antibiotic, was the most effective drug against most of the gram-negative and gram-positive bacteria. Detection of biofilm by two different methods show that most of isolates were multidrug resistant. Three species of lactic acid bacteria namely L.acidophilus (DSM20079), L.plantarum (DSM20174) and L.salivarus (DSM20555) were used for reducing the microbial infections. Bacteriocins were isolated from MRS broth culture of these lactic acid bacteria through the precipitation method using 1N HCL and were tested against different pathogenic bacteria. Antibacterial activity of bacteriocins extracted from L. acidophilus (DSM20079) showed maximum activity against most isolated bacteria as compare to others. Also, L.salivarus (DSM20555) have the highest activity against Methicillin-resistant Staph aureus. The present study has showed the antibacterial role of bacteriocin isolated from lactic acid bacteria can be used for treatment variety of human diseases.

Assessment of <i>Pseudomonas aeruginosa</i> resistance profiles in intensive care units: Five-year outcomes

Aim: Pseudomonas aeruginosa is a non-fermentative Gram-negative bacilli which infections in hospitals have high morbidity and mortality rates. The aim of this study is to determine the antimicrobial susceptibility profiles of P.aeruginosa strains isolated in Izmir Katip Celebi University Ataturk Training and Research Hospital between January 2011 and October 2015. Materials and Methods: In our hospital, between January 2011- October 2015, 1056 P.aeruginosa strains isolated from various clinical specimens of patients who have been in intensive care units were included in the study. The strains were isolated and identified by the BD Phoenix™ Automated Microbiology System (Becton-Dickinson, USA) system. Antibiotic susceptibilities were established by disk diffusion and were evaluated according to the Clinical Laboratory Standards Institute (CLSI) criteria in 2011-2014 and EUCAST criteria in 2015. Results: The highest antibiotic resistance rates were found for cefoperazone/sulbactam, aztreonam, ceftazidime, piperacillin/ tazobactam and cefepime. Conclusion: In our hospital, resistance rates of P. aeruginosa strains isolated from intensive care unit were higher consistent with data reported in our country. The results of this study will help the establishment of more appropriate treatment protocols against P.aeruginosa infections and will aid in preventing the development of a high number of resistant strains.

Utility of 16S rRNA PCR performed on clinical specimens in patient management

Broad-range 16S rRNA PCR can be used for the detection and identification of bacteria from clinical specimens in patients for whom there is a high suspicion of infection and cultures are negative. The aims of this study were (1) to compare 16S rRNA PCR results with microbiological culture results, (2) to assess the utility of 16S rRNA PCR with regard to antimicrobial therapy, and (3) to compare the yield of 16S rRNA PCR for different types of clinical specimen and to perform a cost analysis of the test. A retrospective study was performed on different clinical specimens which had 16S performed over 3 years (2012-2015). Standard microbiological cultures were performed on appropriate media, as per the laboratory protocol. Patient clinical and microbiological data were obtained from the electronic medical records and laboratory information system, respectively. 16S rRNA PCR was performed in a reference laboratory using a validated method for amplification and sequencing. The outcomes assessed were the performance of 16S rRNA PCR, change of antimicrobials (rationalization, cessation, or addition), and duration of therapy. Concordance of 16S rRNA PCR with bacterial cultures was also determined for tissue specimens. Thirty-two patients were included in the study, for whom an equal number of specimens (n=32) were sent for 16S rRNA PCR. 16S rRNA PCR could identify an organism in 10 of 32 cases (31.2%), of which seven were culture-positive and three were culture-negative. The sensitivity was 58% (confidence interval (CI) 28.59-83.5%) and specificity was 85% (CI 61.13-96%), with a positive predictive value of 70% (CI 35.3-91.9%) and negative predictive value of 77.2% (CI 54.17-91.3%). Antimicrobial therapy was rationalized after 16S rRNA PCR results in five patients (15.6%) and was ceased in four based on negative results (12.5%). Overall the 16S rRNA PCR result had an impact on antimicrobial therapy in 28% of patients (9/32). The highest concordance of 16S rRNA PCR with bacterial culture was found for heart valve tissue (80%), followed by joint fluid/tissue (50%). Despite the low diagnostic yield, results of 16S rRNA PCR can still have a significant impact on patient management due to rationalization or cessation of the antimicrobial therapy. The yield of 16S rRNA PCR was highest for heart valves.

Heterogeneity of the Tumor Microenvirontment in Diffuse Large B-Cell Lymphoma Revealed By Deep Phenotyping Using Mass Cytometry (CyTOF)

BACKGROUNDDiffuse large B-cell lymphoma (DLBCL) is a high-grade malignancy and the most common subtype of lymphoma worldwide. Standard therapy with anthracycline-based chemotherapy and rituximab is able to cure about two thirds of patients but a significant proportion will develop relapsed/refractory disease. Salvage treatment options in this setting are limited and only effective in a minority of patients. Most of these patients will eventually die from progressive disease, underscoring the pressing clinical need for the development of new treatment strategies.Checkpoint blockade therapy has demonstrated efficacy in a variety of cancers and involves modulating the host's immune response towards an effective anti-tumoral attack. Trials using programmed cell death protein 1 (PD-1) blockers in advanced hematological malignancies demonstrated remarkable activity in patients with Hodgkin lymphoma. Unfortunately, results in DLBCL were marginal and short lived. The mechanisms underlying the failure of immune checkpoint blockade in DLBCL are not known. To better understand mechanisms of immune evasion we utilized mass cytometry to the study of immune subsets in the tumor microenvironment (TME) of DLBCL.METHODSViably cryopreserved cell suspensions created from clinical specimens of patients with DLBCL were obtained from our biorepository. Specimens were stained using a cocktail of metal-tagged antibodies recognizing over 32 surface proteins and acquired on the mass cytometer (CyTOF2™, Fluidigm). Control tissue consisted of reactive lymph node (LN) tissue, tonsil and spleen, obtained from patients without diagnosis of cancer. Analysis was performed using Cytobank (www.cytobank.com). Institutional Review Board approved the conduct of this study.RESULTSThirteen patient samples were available for analysis (7 DLBCL, 6 controls). DLBCL samples originated from LN (4), spleen (2), and adrenal gland (1). Control tissue consisted of tonsil (3), reactive LN (2), and normal spleen (1).Global analysis of CD45+ cells showed that T-cells were more commonly found in the TME of DLBCL than controls (DLBCL 47.2% vs. 24.5% in controls; Figure 1). The difference persisted when the analysis was restricted to LN specimens (50.2% in DLBCL vs. 25.1% in normal LN). On both DLBCL and control samples the majority of T-cells were CD4+ (61.1% in DLBCL; 75.3% in control) compared to CD8+ T-cells (33.3% in DLBCL; 20% in control). DLBCL samples had increased proportion of regulatory T-cells (Treg) compared to controls (16.9% vs. 6.5%), even after stratifying for tissue of origin (Figure 2). Multiparametric analysis of cytotoxic (CD8+) T-cell subsets revealed that majority of CD8+ T-cells in DLBCL has an effector memory (Tem) phenotype (81.9%) compared to tissue-matched control (43%). PD-1 expression was present in the majority of CD8+ T-cells and mapped to the Effector Memory and Effector subsets. The majority of PD-1 positive cells co-expressed CD27 (>80%). One of the samples (DLBCL arising in the spleen from CLL background) also demonstrated bright PD-1 expression in B-cells (Figure 3).CONCLUSIONSMass cytometry reveals that the tumor microenvironment of DLBCL is enriched with CD4+ T-cells including a higher proportion of Treg compared to tissue-matched control. We also demonstrated that most cytotoxic T-cells in the TME of DLBCL have an effector memory phenotype and express other markers of activation and prior antigenic exposure. Co-expression of PD-1 and CD27 was common in CD8+ T-cells, providing rationale for studies of combination immunotherapy. PD-1 expression in B-cells of DLBCL arising from CLL background deserves further investigation. [Display omitted] [Display omitted] [Display omitted] DisclosuresAnsell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding.

Detection of extended-spectrum β-lactamase (ESBL) and plasmid-borne blaCTX-M and blaTEM genes among clinical strains of Escherichia coli isolated from patients in the north of Iran.

Escherichia coli is an important cause of hospital-acquired infections worldwide. Antimicrobial resistance leads to treatment failure of hospital infections caused by E. coli. Production of extended-spectrum β-lactamases (ESBLs) is one of the major causes of antibiotic resistance in these bacteria. This study aimed to investigate the frequency of blaTEM and blaCTX-M genes in ESBL-producing E. coli strains isolated from clinical specimens of patients admitted to six hospitals in the north of Iran. A total of 160 E. coli strains were isolated from various clinical samples of hospitalised patients. Antibiotic resistance patterns were determined by the Kirby-Bauer disk diffusion method. The double-disk phenotypic confirmatory test was carried out amongst β-lactam-resistant isolates to detect ESBL-producing strains. Plasmid DNA of ESBL-producing strains was extracted and subjected to PCR for detection of the blaTEM and blaCTX-M genes, and isolates were extensively verified by sequencing. The highest resistance rate was to amoxicillin; all E. coli isolates (100%) were susceptible to imipenem. Amongst the 160 clinical E. coli isolates, 83 (51.9%) were ESBL-positive, of which 27 (32.5%) and 72 (86.7%) were positive for blaTEM and blaCTX-M, respectively. This study is the first report of an ESBL phenotype disseminated in hospitals in the north of Iran. These findings showed that there was a direct relationship between the development of resistance to β-lactam antibiotics and production of TEM and CTX-M enzymes.

Characterisation and Isolation of Candida Species from ICU Patients in Nobel Medical College Teaching Hospital, Biratnagar

Background Fungi have emerged as major causes of human diseases. Intensive Care Units (ICU), harbor almost all the risk factors for opportunistic fungal infections. Among these, Candida infections are very common with recent trends being rise in the non-Candida albicans (NCA) species along with an increase in resistance of these species to antifungal drugs. Increament in invasive Candidasis during last three decades have been reported in several studies, among all Candida species the Candida albicans is considered as the most common infectious agent the other non-albicans like C. Tropicalis, C. glabrata, C. parapsilosis C. krusei, and C. dubliniensis were raised as infectious agents. The aim of current study is to characterize the candida species from the clinical specimens of patients admitted in the ICU of Tertiary Care hospital in Eastren Nepal and to perform their antifungal susceptibility.Material and Methods The study was carried out in the tertiary care hospital Nobel Medical College and Teaching Hospital Biratnagar Nepal over a period of 6 monthsbetween September 2015 to February 2016. The following techniques were employed to characterize the isolates in the study – Gram’s stain, culture on Sabouraud’s Dextrose Agar, Germ Tube test, morphology in Cornmeal Agar and chromogenic agar media, sugar fermentation and sugar assimilation tests, and the results were interpreted by using standard protocols.Results Out of 50 candida isolates from different clinical samples including 37 blood samples, 9 urines and 4 Endo Tracheal (ET) tube, the most common species was C.albicans (44%) followed by C.tropicalis (26%), C.Glabrata (18%), C.Parapsilosis (08%), C.Krusei (02%), and C.Dubliniensis(02%).Conclusion The purpose of the study is to show the value of species isolation, identification and antimicrobial sensitivity testing of the ICU & NICUs patients.Journal of Nobel Medical CollegeVolume 5, Number 1, Issue 8, January-July 2016, 61-65

Spa Typing of Staphylococcus aureus Strains Isolated From Clinical Specimens of Patients With Nosocomial Infections in Tehran, Iran.

BackgroundThe incidence of nosocomial Staphylococcus aureus infection is increasing annually and becoming a true global challenge. The pattern of Staphylococcus aureus protein A (spa) types in different geographic regions is diverse.ObjectivesThis study determined the prevalence of methicillin-resistant S. aureus and different spa types in S. aureus clinical isolates.Materials and MethodsDuring a six-month period, 90 S. aureus isolates were recovered from 320 clinical specimens. The in vitro susceptibility of various S. aureus isolates to 16 antibiotic discs was assessed using the Kirby-Bauer disk diffusion method. Molecular typing was carried out with S. aureus protein A typing via polymerase chain reaction.ResultsThe frequency of methicillin-resistant S. aureus in our study was 88.9%. Twenty-three (25.5%) isolates were positive for panton-valentine leukocidin encoding genes. S. aureus presented a high resistance rate to ampicillin (100%) and penicillin (100%). No resistance was observed to vancomycin, teicoplanin, or linezolid. The rates of resistance to the majority of antibiotics tested varied between 23.3% and 82.2%. The rate of multidrug resistance among these clinical isolates was 93.3%. The 90 S. aureus isolates were classified into five S. aureus protein A types: t037 (33.3%), t030 (22.2%), t790 (16.7%), t969 (11.1%), and t044 (7.7%). Eight (8.9%) isolates were not typable using the S. aureus protein A typing method.ConclusionsWe report a high methicillin-resistant S. aureus rate in our hospital. Additionally, t030 and t037 were the predominant spa-types among hospital-associated S. aureus. Our findings emphasize the need for continuous surveillance to prevent the dissemination of multidrug resistance among different S. aureus protein A types in Iran.

Distribution and antifungal susceptibility of Candida species in patients with increased risk for fungal infections

Candida species are opportunistic yeasts that can be a serious threat for immunocompromised and critically ill patients, and a cause for increased morbidity and mortality in hospitalized patients. The aim of this study was to determine the frequency and distribution of different Candida species in clinical specimens in patients with increased risk for fungal infections, and to determine the antifungal susceptibility profile of invasive Candida species to antifungal agents. During a two year period, clinical specimens from 120 patients divided into 4 groups were analysed at the Institute of microbiology and parasitology, Faculty of Medicine, Skopje, Republic of Macedonia. Each of these 4 groups consisted of specimens from 30 patients, with primary immune deficiency, critically ill patients treated in the intensive care units (ICU), patients with mucosal candidiasis only, and patients with cystic fibrosis. All specimens were investigated with conventional mycological methods. Identification of Candida species was performed with VITEK-2 system (bioMérieux, France). E-test strips of fluconazole, voriconazole, amphotericin B and caspofungin (AB bioMerieux, France) were used for determination of the antifungal susceptibility profile. In this study, a total of 115 isolates of Candida species were confirmed in different clinical specimens (91 isolates from mucosal surfaces and 24 isolates from blood culture). Colonisation of mucosal membranes of gastrointestinal, respiratory and/or urinary tracts was registered in 56.67% (17/30), 56.67% (17/30), 90% (27/30) and 100% (30/30) of the specimens in the first, second, third and fourth group respectively. In all four groups of patients, the following Candida species were confirmed: C. albicans - 55%, C. glabrata - 17.6%, C. parapsilosis - 7.7%, C. tropicalis - 6.6%, unidentified Candida species - 4.4%, C. dubliniensis - 3.3%, C. kefyr - 2.2%, and one isolate of C. rugosa, C. pelliculosa and C. krusei each. Positive blood culture was registered in 23.33% specimens from the first group, 43.33% in the second group, 23.08% of the third group, and in one specimen of the fourth group. The most frequent isolates from blood culture were C. tropicalis and C. krusei, followed by C. albicans, C. parapsilosis and C. tropicalis, and in the second group C. albicans and C. pelliculosa were equally distributed, followed by C. parapsilosis and C. glabrata. All invasive isolates of Candida species were susceptible to amphotericin B, voriconazole and caspofungin. Resistance to fluconazole was registered in 8.3% (2/24) of all confirmed Candida species. Dose-dependent susceptibility to fluconazole was confirmed in 46% (11/24) of the isolates. Our study confirms high prevalence of colonisation and candidemia with non-albicans Candida species. Resistance to antifungal agents was registered only in two isolates of C. krusei. An epidemiological study is necessary for surveillance of dynamics of candidemia and antifungal susceptibility profile of invasive isolates of Candida species in our patients.

Antimicrobial Susceptibility Pattern of Enterococci Isolated From Patients in Tehran

Background: Enterococci are one of the most common nosocomial pathogens and the emergence of multidrug-resistant strains has been increasing. Objectives: We studied the antimicrobial susceptibility of enterococci isolated from different clinical specimens of patients in Tehran. Materials and Methods: From the beginning of April 2013 to the end of June 2013, a total of 146 enterococci were isolated from the Pars General Hospital in Tehran. The antimicrobial susceptibility pattern of the isolates against ampicillin, clindamaycin, ciprofloxacin, erythromycin, levofloxacin, linezolid, nitrofurantoin, tetracycline, and vancomycin was determined using the disk diffusion method according to the guidelines of clinical laboratory standards institute (CLSI). Results: The rates of resistance were high to clindamycin, tetracycline, and erythromycin (97.2%, 89%, and 74.5%, respectively); moderate to ciprofloxacilin and levofloxacilin (40.6% and 36.4%, respectively); and low to ampicillin and nitrofurantoin (13.8% and 3.5%, respectively). All isolates were linezolid sensitive. Vancomycin-resistant enterococci (VRE) accounted for 9.6% of the isolates. Conclusions: VRE and a high rate of resistance to some of antimicrobial agents were found among the enterococci isolated from patients in Tehran. These findings highlight the importance of regular supervision of antimicrobial susceptibilities.

Dissemination of fusidic acid resistance among Staphylococcus aureus clinical isolates.

BackgroundA significant trend towards increased fusidic acid (FA) resistance among Staphylococcus aureus with increased duration of use is of concern. The aim of the present study is to investigate the dissemination of fusidic acid resistance among Staphylococcus aureus clinical isolates.MethodsThe susceptibility of S. aureus isolates to antimicrobial agents was determined by disc-diffusion method. The minimal inhibitory concertrations(MICs) of fusidic acid and vacomycin for fusidic acid-resisitant isolates were determined by ager dillution method. FA resistance determinants were determined by PCR and DNA sequencing. SCCmec typing, spa typing and multi-locus sequence typing were used for the determination of molecular characteristics for S. aureus isolates.ResultsA total of 392 non-duplicate S. aureus isolates including 181 methicillin-resistant S. aureus (MRSA) isolates, which were isolated from the clinical specimens of patients at a Chinese tertiary hospital from January, 2012 to September, 2013, were collected for investigating FA resistance. Among 392 S. aureus clinical isolates tested, 56 (14.3 %) with FA MIC values ranging from 2 μg/ml to ≥128 μg/ml were resistant to FA. The proportions of FA resistance among MRSA and MSSA isolates were 27.1 % (49/181) and 3.3 % (7/211). There was a trend of rapidly increased FA resistance among S. aureus and MRSA isolates from 5.2 % and 8.9 % in 2012 to 24.9 % and 45.1 % in 2013. Acquired FA resistance gene, fusB, was present in 73.2 % (41/56) of FA-resistant S. aureus isolates. fusC and fusA mutation were not found in any of tested isolates. A total of 9 sequence types (STs) and 12 spa types were identified among the 56 FA-resistant S. aureus isolates. ST5 accounting for 66.1 % (37/56) was the most prevalent ST. The majority (92.9 %, 52/56) of the isolates tested belonged to clonal complex 5(CC5). t2460 was the most prevalent spa type, accounting for 67.9 % (38/56) . ST5-MRSA- II-t2460 was predominant clone, accounting for 75.5 % (37/49) of FA-resistant MRSA isolates and 66.1 % (37/56) of FA-resistant S. aureus isolates. Five of 7 FA-resistant MSSA isolates belonged to ST630-MSSA.ConclusionIncreased FA resistance among S. aureus isolates was found in China. fusB was predominant FA resistance determinant. The spread of CC5 clone, especially novel ST5-MRSA- II-t2460 clone with high-level resistance to FA, was responsible for the increase of FA resistance.

Avaliação da suscetibilidade da Klebsiella pneumoniae aos beta-lactâmicos

Background and Objective: Klebsiella pneumoniae is a Gram negative bacilli causing infections related to health care. In recent years, it has become a multiressistente microorganism, because of the production spectrum beta-lactamase enzymes extended and carbapenemases. Therefore, the aim of this study was to evaluate the susceptibility profile of K. pneumoniae in relation to carbapenems; identifying carbapenemase production and to determine the susceptibility profile of the isolates compared to other β -lactams. Methods: This is research on naexperimental basis with quantitative and qualitative approach, which used 27 isolates from different clinical specimens ofpatients whit infection, assigned by reference laboratories. Prior to susceptibility testing, the saples were re-isolated on MacConckey Agar culture meddium and the plates were incubated with semeios at 37° C for 24 hours. Susceptibility testing to carbapenems and other beta-lactams were performed according to the method of Kirby-Bauer. The antibiotic disks used in the tests were amoxicillin - clavulanate, cefoxitin, cefotaxime, ceftazidime, cefepime, imipenem, meropenem and ertapenem. The test for the detection of the enzyme carbapenemase was performed by the use of antibiotics: meropenem, and ertapenem, by Modified Hodge test. Results:All isolates were susceptible to beta-lactam group of carbapenems and no production of carbapenemases, but 18.5 % produced extend-spectrum beta- lactamase extended spectrum and 40.7 % of the strains were resistant to other beta-lactams. Conclusion: The production of beta- lactamase extended spectrum was one of the main factors involved in resistance to antibiotics tested , but other resistance mechanisms were not identified possibly involved. KEY WORDS: Klebsiella pneumoniae. Disease Susceptibility. Beta-lactams.

The JCPYV DNA load inversely correlates with the viral microrna expression in blood and cerebrospinal fluid of patients at risk of PML

BackgroundIn light of their regulatory role, changes in the expression of Polyomavirus JC (JCPyV) microRNAs may be relevant for virus reactivation and the development of progressive multifocal leukoencephalopathy (PML). ObjectivesTo investigate the presence of JCPyV-DNA and JCPyV microRNA expression in clinical specimens of patients at risk for PML. Study designThe JCPyV-DNA and microRNA status was assessed in peripheral blood mononuclear cells (PBMCs) and plasma from 100 HIV patients, in serum and cerebrospinal fluid (CSF) from 14 HIV PML patients and in PBMCs and plasma from 50 healthy controls using Multiplex real-time PCR and JCPyV miRNA-J1-3p and −5p stem-loop RT-PCR. The JCPyV-DNA microRNA-expressing region was also sequenced. ResultsA positive JCPyV-DNA status was more prevalent in HIV patients (67%, 67/100) compared to healthy controls (18%, 9/50). Among these, 46% and 42% of the HIV patients and 18% and 0% of the healthy controls were positive based on PBMC and plasma determinations, respectively. PBMC JCPyV microRNA positivity was observed in 22 out of 46 (48%) JCPyV+ HIV patients and in 3 out of 9 (33%) JCPyV+ healthy controls. Moreover, JCPyV microRNAs in exosomes were found in 6 out of 100 (6%) HIV plasma samples, in 12 out of 50 (24%) healthy samples, in 6 out of 14 (43%) serum samples, and in 3 out of 5 (60%) HIV PML CSF samples. Of note, the JCPyV-DNA load was inversely correlated with expression of the viral microRNA. The JCPyV microRNA genomic expression region showed a different combination of three mutations. ConclusionsThe low levels of JCPyV microRNA expression in HIV patients with high JCPyV-DNA prevalence observed in this study highlight the potential clinical relevance of JCPyV microRNAs in PML risk assessment.