Candidemia Cases Caused by Candida parapsilosis Complex Species: Epidemiology and Antifungal Susceptibility of Strains.

BACKGROUND: Candida parapsilosis is a common non-albicans Candida species isolated from blood cultures. The increase in fluconazole-resistant C. parapsilosis complex isolates is worrying, especially in strains with Y132F changes in the ERG11 gene since this ultimately leads to outbreaks. This study aimed to investigate the distribution and antifungal susceptibility of C. parapsilosis complex species isolated from bloodstream, clinical characteristics of patients, prevalence of risk factors, and to determine ERG11 gene region mutations in strains that were not susceptible to fluconazole. METHODS: Between 2014 and 2018, 96 patients with C. parapsilosis candidemia were evaluated. Thermo Scientific SensititerTM YeastOneTM YO10 was used for antifungal susceptibility testing. The ERG11 gene region sequence analysis was performed for fluconazole non-susceptible isolates. RESULTS: All the strains were defined as C. parapsilosis sensu stricto. The rate of fluconazole resistance was 6.3%, and that of susceptibility to fluconazole at an increased dose was 2.1%. Two isolates showed Y132F or G458S ERG11 changes associated with azole resistance, with the most common change being identified as R398I, which was shown not to encode azole resistance. No resistance to echinocandins and amphotericin B was observed. The use of broad-spectrum antibiotics (83.3%) was the most common risk factor. CONCLUSIONS: This study highlights the importance of susceptibility testing when making a decision to use fluconazole in the treatment of C. parapsilosis candidemia. The presence of resistance associated with ERG11 Y132F changes indicated that azole resistance should be closely monitored. Increasing awareness of fluconazole-resistant C. parapsilosis candidemia will help identify strategies to overcome these infections.

[Investigation of Ganciclovir Resistance in Cytomegalovirus Isolates by Phenotypic and Genotypic Methods]. / Sitomegalovirüs Izolatlarinda Gansiklovir Direncinin Fenotipik ve Genotipik Yöntemlerle Arastirilmasi.

Ganciclovir-resistant cytomegalovirus (CMV) strains are reported following long-term antiviral agent use, especially for immune-suppressive patients. In this study, it was aimed to investigate the mutations in the UL97 gene of CMV, which causes ganciclovir (GCV) resistance by genotypic and phenotypic methods in patients who developed CMV infection following hematopoietic cell (HCT) or solid organ transplantation (SOT). Thirty patients who had HCT or SOT in Mediterranean University Hospital and developed CMV infection during routine follow-up with a viral load of CMV over 1000 copies/mL were included in the study. CMV DNA was analyzed by an automated system (Cobas Ampliprep/COBAS TaqMan CMV Test, Roche Diagnostics, Germany) quantitatively. DNA sequence analysis of the regions including codons 420-664 in the UL97 gene region was done by the Sanger sequencing method to detect mutations causing antiviral resistance and compared with defined mutations. In order to investigate antiviral resistance by phenotypic methods, heparinized blood samples of the patients were collected, 'buffy coat (leukocyte layer)' was inoculated into MRC-5 cells by centrifugation method and CMV growth in these cells was controlled with monoclonal antibodies when growth was detected, virus titer was determined and plaque reduction test was applied as recommended. It was determined that 22 of the 30 patients were HCT recipients and eight were SOT (five kidney, three liver) recipients. When the CMV serology pattern of the patients was evaluated before transplantation, 29 (96.7%) patients were found to be seropositive and one (3.3%) patient was found to be seronegative. Totally, nine CMV UL97 mutations were detected in seven (23.3%) pediatric patients who had HCT, including six seropositive and one seronegative case. In addition, one mutation (D605E) not known to cause GCV resistance was detected in a seronegative recipient and three previously unidentified mutations were detected (1474T, F499S, V559A) in a seronegative recipient. Five of the mutations defined were UL97 mutations with a defined clinical resistance against GCV in each of the five recipients (C603W, C592G, H520Q, M460V, A594T). In the plaque reduction test using 3 µM, 12 µM, 48 µM and 96 µM concentrations of GCV in CMV strains, the IC50 value was determined to be ≥ 8 µM for the five CMV strains, and the phenotypic presence of GCV resistance was shown. Clinical resistance associated with CMV UL97 mutation was detected in five (22.7%) of 22 patients who had HCT. GCV resistance was also demonstrated in these patients by phenotypic methods. No UL97 mutation was detected in the patients who had SOT.

Evaluation of the BD Phoenix CPO Detect Test for the detection of carbapenemase-producing Enterobacterales.

Aims: This study aimed to evaluate the performance of the BD Phoenix CPO Detect Test (BD Diagnostic Systems) for the detection and classification of carbapenemase-mediated carbapenem resistance. Methods: A total of 447 Enterobacterales strains were included in the study. All strains were tested with the BD Phoenix CPO Detect Test and the modified carbapenem inactivation method. Results: Carbapenemase production was detected in 157 of 159 carbapenemase producers, including 95.7% of class B and 99.2% of class D isolates using the BD Phoenix CPO Detect Test. BD Phoenix CPO Detect has a sensitivity of 98.7% and a specificity of 95.5% in detecting carbapenemase production. Conclusion: The classification of OXA-48 and class B carbapenemases, the most common carbapenemases circulating in Turkey, was highly accurate.

Enterobacterales are a type of bacteria that usually live harmlessly in the gut of humans. However, if the bacteria get access to the bladder or bloodstream, they can cause infection. Carbapenemase-producing Enterobacterales (CPE) are a type of bacteria that can cause carbapenem antibiotic-resistant infections, a group of powerful antibiotics. The rapid spread of CPE will pose an increasing threat to public health and medical treatment practices; therefore, rapid detection of CPE is crucial. This study assessed the performance of the BD Phoenix CPO Detect Test for the detection of carbapenemase-producing Enterobacterales. The BD Phoenix CPO Detect Test offers both the detection of carbapenemase production and antimicrobial susceptibility testing simultaneously and can be clinically useful for determining possible treatment options.

In vitro Activity of Cefiderocol and Ceftazidime-Avibactam, Against Carbapenemase-Producing Enterobacterales.

BACKGROUND: Carbapenemase production is an issue of significant clinical and public health concern, because of the shortage of effective antimicrobial agents available for treatment. Here, we present antimicrobial susceptibility data of ceftazidime-avibactam, cefiderocol, and other clinically relevant antibiotics for carbapenemase-producing Enterobacterales bloodstream isolates, in accordance with European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. METHODS: A total of 133 carbapenemase producing Enterobacterales bloodstream isolates from May 2010 to September 2018 were included in the study. Species were identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (Bruker Daltonics, Germany). The presence of the blaKPC, blaNDM, blaOXA-48, blaVIM, and blaIMP carbapenemase genes were investigated by BD Max CRE assay (Becton Dickinson, USA) and in-house PCR. Antimicrobial susceptibility testing was performed by the BD Phoenix automated system (Becton Dickinson, USA), except cefiderocol and colistin. Cefiderocol and colistin susceptibility was determined by disk diffusion and broth microdilution method, respectively. RESULTS: Except for cefiderocol and ceftazidime-avibactam, the percentage of susceptible isolates did not exceed 90% for any of the antibiotics tested. Although none of the isolates were resistant to cefiderocol, the ceftazidime-avibactam resistance rate was 9.8%. All of the ceftazidime-avibactam resistant strains were NDM (New Delhi metallo-beta-lactamases) producers. Among the other clinically relevant antibiotics tested, only amikacin, colistin, tigecycline, and fosfomycin susceptibility rates exceeded 50%. Of the 133 isolates 22.6% were resistant to colistin which is the preferred antibiotic with a second active agent for infections caused by metallo-beta-lactamase producing Enterobacterales in Turkey. CONCLUSIONS: In our study, resistance to ceftazidime-avibactam was detected only in metallo-beta-lactamase producing Enterobacterales isolates, while cefiderocol was found to be effective against all strains. It is important to monitor regional antimicrobial susceptibility data, as the emergence of antimicrobial resistant phenotypes is directly linked to the use of any given antimicrobial agent.

[Evaluation of the Methods Used for the Detection of Entamoeba histolytica in Stool Samples of Patients with Diarrhea]. / Ishalli Hastalarin Diski Örneklerinde Entamoeba histolytica'nin Saptanmasinda Kullanilan Yöntemlerin Degerlendirilmesi.

Amoebic dysentery (amebiasis) is a parasitic infection caused by Entamoeba histolytica. The diagnosis of invasive amebiasis has traditionally been based on direct and stained microscopic examination of stool samples. Stool microscopy exhibits low sensitivity and it is difficult to distinguish E.histolytica cysts and trophozoites from cells such as leukocytes, macrophages and non-pathogenic Entamoeba species in the stool by microscopy. Therefore more sensitive and specific diagnostic methods such as enzyme linked immunosorbent assay (ELISA) tests which investigate the presence of E.histolytica-specific antigen in stool, and polymerase chain reaction (PCR) are being widely used. In this study it was aimed to study stool samples of the patients who applied with the clinical signs of amebiasis by using direct and permanent stained microscopy, E.histolytica adhesin antigen ELISA test and real-time PCR-based BD Max Enteric Parasite Panel (BD Max EPP) test and to evaluate the diagnostic values of these tests. A total of 546 faecal samples with blood and/or mucus were analyzed in the study. In these samples, the presence of E.histolytica was investigated by direct and permanent stained microscopy, E.histolytica adhesin antigen ELISA and BD Max EPP PCR. Of the samples 36.3% were suspected to contain E.histolytica/dispar/moshkovskii cyst and/or trophozoite by direct microscopic examination. Trichrome staining was performed on these samples and 49 samples were found suspicious for the presence of E.histolytica/dispar/moshkovskii cysts and/or trophozoites. The presence of E.histolytica and other Entamoeba species was not confirmed in 75.2% of the samples. BD Max EPP PCR and E.histolytica adhesin antigen ELISA tests were studied in 49 faecal samples that were suspected by trichrome staining. None of these samples were positive by ELISA. Forty-four samples were negative by PCR and invalid test results were obtained in five samples. In this study, E.histolytica was not detected in the patient population. The results of this study showed that microscopic examination alone is not sufficient for the detection of E.histolytica. It is concluded that it is necessary to use a more sensitive and specific also rapid diagnostic test such as E.histolytica-specific antigen detection test or PCR in the diagnosis of amebiasis to avoid misdiagnosis and unnecessary treatment of 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.

Mortality-associated factors of candidemia: a multi-center prospective cohort in Turkey.

Candidemia may present as severe and life-threatening infections and is associated with a high mortality rate. This study aimed to evaluate the risk factors associated with 30-day mortality in patients with candidemia. A multi-center prospective observational study was conducted in seven university hospitals in six provinces in the western part of Turkey. Patient data were collected with a structured form between January 2018 and April 2019. In total, 425 episodes of candidemia were observed during the study period. Two hundred forty-one patients died within 30 days, and the 30-day crude mortality rate was 56.7%. Multivariable analysis found that SOFA score (OR: 1.28, CI: 1.154-1.420, p < 0.001), parenteral nutrition (OR: 3.9, CI: 1.752-8.810, p = 0.001), previous antibacterial treatment (OR: 9.32, CI: 1.634-53.744, p = 0.012), newly developed renal failure after candidemia (OR: 2.7, CI: 1.079-6.761, p = 0.034), and newly developed thrombocytopenia after candidemia (OR: 2.6, CI: 1. 057-6.439, p = 0.038) were significantly associated with 30-day mortality. Central venous catheter removal was the only factor protective against mortality (OR: 0.34, CI:0.147-0.768, p = 0.010) in multivariable analysis. Candidemia mortality is high in patients with high SOFA scores, those receiving TPN therapy, and those who previously received antibacterial therapy. Renal failure and thrombocytopenia developing after candidemia should be followed carefully in patients. Antifungal therapy and removing the central venous catheter are essential in the management of candidemia.

Evaluation of Aspergillus Lateral-Flow Test in Serum Samples of Pediatric Patients.

BACKGROUND: Diagnosis of invasive aspergillosis (IA) in patients with hematologic malignancies and under the risk of IA may be uncertain or may delay because of nonspecific clinical presentation of the patients and difficult application techniques of conventional methods. Early diagnosis can provide initial antifungal therapy and prevent high mortality. In this study, we investigated the performance of an Aspergillus lateral-flow device (LFD) test (OLM Diagnostics, Newcastle upon Tyne, United Kingdom) for the diagnosis of IA in pediatric febrile neutropenic patients with hematologic malignancies. METHODS: Three hundred and fourty seven serum samples of 26 febrile neutropenic episodes of 21 patients at risk for IA were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the Aspergillus LFD test at episode level and at serum level were calculated. RESULTS: According to the reference diagnostic criteria of IA, one proven and 13 probable IA episodes were defined. Twelve episodes (46.1%) did not meet the criteria for IA. The sensitivity, specificity, PPV, NPV, accuracy of the Aspergillus LFD test at episode level and at serum level were 14.3%, 100%, 100%, 50%, 53.8% and 12.1%, 100%, 100%, 50.8%, 53.9%, respectively. CONCLUSIONS: Aspergillus LFD test is an easy-to-use assay with short hands-on time; however, further study of the clinical utility in children and especially in serum samples are needed. It is a highly specific test for IA on bronchoalveolar lavage (BAL) samples but is not useful as a screening test for serum samples unless combined with galactomannan (GM) antigen test because of its potentially suboptimal sensitivity.

[Evaluation of a Commercial Multiplex Tandem Polymerase Chain Reaction Method for the Identification of the Yeasts Isolated from Blood Cultures]. / Kan Kültürlerinden Izole Edilen Mayalarin Tanimlanmasinda Ticari Bir Multipleks Tandem Polimeraz Zincir Reaksiyonu Yönteminin Degerlendirilmesi.

Candidemia is one of the most important health care-associated infections worldwide. Candida species have species-specific antifungal susceptibility profiles and it has been shown that the identification of the Candida species is necessary for the appropriate treatment of the patients with candidemia. Various methods are used to shorten the identification time for the determination of the causative species. Fungal ID multiplex tandem polymerase chain reaction (MT-PCR) (AusDiagnostics, Australia) is a test developed to identify yeasts and molds isolated from clinical specimens. In this study, we aimed to evaluate the Fungal ID MT-PCR test (AusDiagnostics, Australia) for the identification of the yeasts from positive blood cultures in Akdeniz University Hospital Central Laboratory. Between December 2016 and December 2017, blood culture samples from 92 consecutive patients with yeast cells detected in Gram stained smears were tested by Fungal ID MT-PCR and the reference method. After the subculture of the positive signaling blood culture bottles to Sabouraud dextroz agar (SDA), the identification of the yeasts were performed by morphological identification methods (Germ tube test, Corn Meal Tween® 80 agar media, etc.), BD Phoenix Yeast ID Panel (Becton Dickinson, Sparks, MD) and Bruker Biotyper matrix-assisted laser desorption ionization-time of mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Germany) systems. Identification with MALDI-TOF MS have been accepted as the reference method. Thirty-five of the isolates were identified as Candida albicans, 17 were Candida glabrata, 13 were Candida parapsilosis, 12 were Candida tropicalis, seven were Candida krusei , two were Candida guilliermondii, two were Candida dubliniensis, two were Candida inconspicua, one was Candida kefyr and one was Saprochaete capitata by the reference method. In our study, no blood culture sample yielded more than one yeast species. 94.6% of the strains were presumptively identified by the morphological identification methods. Discordant results were not detected between the BD Phoenix Yeast ID Panel and the reference method. Thirty-three of the isolates were identified as C.albicans, 15 were C.glabrata, 13 were C.parapsilosis, 11 were C.tropicalis, five were C.krusei , two were C.guilliermondii, one was C.dubliniensis, one was C.kefyr and 10 were Candida spp. by Fungal ID MT-PCR assay. Since C.inconspicua and S.capitata were not included in the test panel, C.inconspicua was identified as Candida spp. in two samples, while S.capitata could not be identified in one sample. Concordance between Fungal ID MT-PCR and the reference method were found to be 88% at the species level and 98.9% at the genus level. The sensitivity of the Fungal ID MT-PCR test in in the detection of C.krusei and C.glabrata was 71.4% and 88.2%, respectively. Fungal ID MT-PCR test has shown a high performance in the identification at the genus level, but the identification at the species level, which is important for the treatment management, was moderate. Fungal ID MT-PCR can be used as an adjunct test to the traditional identification methods for the early identification of the Candida species.

Molecular Epidemiology of Carbapenem-Resistant Enterobacterales Strains Isolated from Blood Cultures in Antalya, Turkey.

OBJECTIVE: The aim of this study was to investigate the prevalence of carbapenemase and CTX-M genes among 330 blood culture isolates of Enterobacterales with reduced susceptibility to at least 1 carbapenem, between 2010 and 2015. METHODS: BD Max CRE assay and in-house PCR were used to detect carbapenemase and CTX-M genes. RESULTS: At least 1 carbapenemase gene was detected among 113 (74.3%) of the 152 carbapenem resistant isolates. The OXA-48 (69.7%) was the most common carbapenemase followed by VIM, NDM and IMP, whereas no tested isolates were KPC-positive. Eighty-six isolates (56.6%) had CTX-M and 65 had both OXA-48 and CTX-M. Carbapenemase production in Enterobacterales was significantly increased in years (P < .05). CONCLUSION: Our study indicates that there is ongoing endemic circulation of the OXA-48 producing organism in our facility. It is noteworthy that more than half of the OXA-48 producing strains also produced CTX-M enzyme.

[Evaluation of Microscopic Examination, Culture and Polymerase Chain Reaction Tests in the Diagnosis of Trichomonas vaginalis Infection]. / Trichomonas vaginalis Enfeksiyonu Tanisinda Mikroskobik Inceleme, Kültür ve Polimeraz Zincir Reaksiyonu Testlerinin Degerlendirilmesi.

Sexually transmitted infections (STIs) are important as a public health problem all over the world. There are some difficulties in prevention and control programs of STIs due to clinical and laboratory diagnostic problems.The most common STIs are Chlamydia trachomatis infections, trichomoniasis and gonorrhea. The study aimed to investigate the direct microscopic examination, culture and polymerase chain reaction (PCR) tests in the diagnosis of Trichomonas vaginalis infection; to determine other microbiological agents that may cause vaginal discharge and to evaluate the various social variables in women with vaginal discharge admitted to the outpatient clinic of Obstetrics and Gynecology in Akdeniz University Hospital. Two hundred and fifteen patients were enrolled in the study. The socio-demographic features of the patients were recorded. Vaginal/endocervical swab specimens taken from patients were evaluated by microscopic examination. Swab specimens were inoculated into blood agar, MacConkey agar and chocolate agar for bacterial culture. Modified Trichosel broth with 5% horse blood (Becton Dickinson, USA) was used for Trichomonas spp. culture. The presence of C.trachomatis, Neisseria gonorrhoeae, and T.vaginalis in swab samples were investigated by multiplex PCR assay (BD Max CT/GC/TV, Becton Dickinson, USA). At least one pathogen was detected among 65 (30.3%) samples. T.vaginalis was detected by microscopic examination and PCR in four of 215 (1.9%) patients. Existence of yeast morphology was observed in 21 (9.8%) specimens by microscopic examination. Twenty four (11.2%) patients were diagnosed as bacterial vaginosis microscopically according to Nugent score system. Candida species grew in 32 (14.9%) and Streptococcus agalactiae grew in 2 (0.9%) of the specimens. C.trachomatis was detected in 2 (0.9%) samples and N.gonorrhoeae in 1 (0.5%) sample by PCR. In this study, 95.3% of the patients were married and 96.7% had only one sexual partner in the mean time. The rate of detection of pathogens were statistically higher in women who have had two or more pregnancies (p<0.05). In our study, T.vaginalis together with N.gonorrhoeae and C.trachomatis were investigated by PCR method in women with vaginal discharge. The use of multiplex PCR test allowed simultaneous investigation of multiple pathogens in the patient samples.

Evaluation of an Automated Yeasts Identification System for Identification of Yeast Isolates.

BACKGROUND: Invasive candidiasis is the most important health-care-associated fungal infection worldwide. In the last two decades, the cause of the increase of fungal infections is immunosuppression or serious underlying diseases. Additionally, Rhodotorula species, Blastoschizomyces capitatus, and Trichosporon species are emerging as important human pathogens in immunocompromised patients with hematological malignancy. METHODS: Between January 2012 and January 2018, a total of 603 fungal organisms were isolated from blood culture samples and included in the study. All of the isolates were identified by using standard mycological methods, MALDI TOF MS system, and the Phoenix system. Sequence analysis was performed on yeasts that could not be definitively identified by using SMM and incompatible according to the results with Phoenix and MALDI-TOF MS analysis. RESULTS: 603 fungal isolates including 594 Candida spp. and 9 other yeasts like species were analyzed. C. albicans was the most frequently isolated species. The results of identification by conventional methods and MALDI TOF MS were compared to the results of the Phoenix system. The observed concordance was 99.2%. The compatibility with other systems of the Phoenix system was 100%, 100%, 97.3%, 100%, and 96.9% for C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei, respectively. CONCLUSIONS: The BD Phoenix system was found to be a simple, reliable, and effective method to identify the main species of the genus Candida in our study.

Evaluation of Commercially Available Real-Time Polymerase Chain Reaction Assays for the Diagnosis of Invasive Aspergillosis in Patients with Haematological Malignancies.

Early diagnosis of invasive aspergillosis (IA) is a challenge. Non-specific clinical and radiologic findings, as well as difficulties in conventional diagnostic method application, may delay correct diagnosis. Nowadays, nucleic acid-based assays have reduced the need for conventional antigen detection and culture-based methods and provided new opportunities for patient care. Aspergillus PCR is now included in the latest European Cancer Research and Treatment Organization/Mycosis Study Group definition updates. We evaluated the performance of commercial real-time polymerase chain reaction (PCR) MycAssay Aspergillus PCR and Artus Aspergillus RG PCR assays and compared the results with galactomannan enzyme immunoassay. During 41 febrile neutropenic episodes, 168 serum samples were collected from 32 patients with haematological malignancies. IA diagnosis was established according to the revised guidelines of the European Organization for Research and Treatment of Cancer/Mycoses Study Group. Twenty-one probable episodes were identified. There were no proven IA cases in the study. In 20 episodes, patients did not fulfil the established criteria for the IA diagnosis. Artus Aspergillus RG PCR assay had a sensitivity of 47.6% and specificity of 100%, while those of MycAssay Aspergillus PCR were 61.9% and 100%, respectively. Two different PCR assays were used in this study. Although there are many studies that evaluated MycAssay Aspergillus PCR, data regarding Artus Aspergillus RG PCR assay are scarce. We found moderate sensitivity and high specificity in the diagnosis of IA in patients with haematological malignancy in both PCR methods. Our results demonstrated that commercial PCR assays can be applied for the early diagnosis and pre-emptive treatment of IA.

Discrimination of Aspergillus flavus from Aspergillus oryzae by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry.

BACKGROUND: Aspergillus flavus is a major cause of severe non-invasive fungal infections in the Middle Eastern countries. However, it is difficult to distinguish A flavus from A oryzae. OBJECTIVES: To assess the potential of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in discriminating between A flavus and A oryzae and compare it with ß-tubulin gene sequencing. METHODS: We used the Bruker Daltonik MALDI-TOF MS system to analyse 200 clinical and environmental A flavus isolates and one A pseudonomius and one A alliaceus (Aspergillus section Flavi) isolate a priori identified as such by sequencing of the ß-tubulin gene. RESULTS: All 200 A flavus isolates were identified at the genus level and 176 (88%) at the species levels by MALDI-TOF MS based on the spectral log-scores (≥2.0 and 1.7-1.99, respectively); among them, only 18 (10.2%) were confirmed as A flavus, whereas 35 (19.9%) were identified as A oryzae and 123 (69.9%) as A flavus/A oryzae. Aspergillus pseudonomius and A alliaceus were misidentified as A flavus and A parasiticus with log-score values of 1.39 and 1.09, respectively. CONCLUSIONS: The results indicate that the commercially available Bruker Daltonik MALDI-TOF MS score database cannot separate A flavus and A oryzae species. We also showed that establishment of an in-house library is a useful tool to discriminate closely related Aspergillus species, including A flavus and A oryzae.

[Evaluation of the BD Phoenix100 System and Colistin Broth Disk Elution Method for Antimicrobial Susceptibility Testing of Colistin Against Gram-negative Bacteria]. / Kolistinin Gram-negatif Bakterilere In Vitro Etkinliginin Belirlenmesinde BD Phoenix100 Sistemi ve Kolistin Sivi Disk Elüsyon Yöntemlerinin Degerlendirilmesi.

Infections with multidrug resistant gram-negative bacteria is a growing problem especially in health care settings. Colistin is one of the last resort antibiotics for such infections in which treatment options are limited. Increasing resistance to colistin is a global problem. Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) study groups have recommended the ISO-standard broth microdilution method (20776-1) as the reference method for the determination of colistin susceptibility. Since the broth microdilution method is not a practical method, it is rarely used in routine clinical microbiology laboratories, yet simple and accurate phenotypic detection methods for the determination of colistin resistance in routine microbiology laboratories are not precisely defined. The aim of this study was to evaluate BD Phoenix100 (Becton Dickinson, USA) system and colistin broth disk elution method for the detection of in vitro activity of colistin against gram-negative bacteria. A total of 419 gram-negative bacteria, including 199 Klebsiella pneumoniae, 163 Acinetobacter baumannii, 34 Escherichia coli, 20 Enterobacter spp., and three Citrobacter spp. isolates which were isolated from various clinical samples in our hospital between 2016-2018 were tested. The broth microdilution method was used as the reference method applying ISO-standard broth microdilution methods (20776-1) and CLSI/EUCAST recommendations. For colistin broth disk elution method, final concentrations of 0 (growth control), 1, 2 and 4 µg/ml were obtained by adding 10 µg colistin disks to four tubes containing 10 ml cation-adjusted Mueller Hinton broth per isolate. After incubation at room temperature for 30 minutes, 50 µl of standardized inoculum suspensions were added to the tubes. Colistin minimum inhibitor concentration (MIC) values were read visually after 16-20 hours of incubation at 35°C in ambient air. Manufacturer's recommendations were followed for BD Phoenix100 system. The categorical agreement between the reference broth microdilution method and the colistin broth disk elution method was 99.3%, very major error and major error rates were 0.2% and 0.5%, respectively. For BD Phoenix100 system, the categorical agreement was 95%, with a very major error rate of 5%. Our results showed that colistin broth disc elution method worked well compared to the reference broth microdilution method. The BD Phoenix100 system, with a high very major error rate, does not reliably distinguish colistin-resistant and colistin-susceptible strains.

Identification of clinical dermatophyte isolates obtained from Iran by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND AND PURPOSE: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used to discriminate among pathogenic microorganisms in clinical laboratories. The aim of this study was to assess the utility of MALDI-TOF MS in the routine identification of clinical dermatophyte isolates obtained from various geographical regions of Iran. MATERIALS AND METHODS: A total of 94 isolates, including Trichophyton interdigitale (n=44), T. rubrum (n=40), T. tonsurans (n=4), Microsporum canis (n=4), and Epidermophyton floccosum (n=1), were analyzed in this study. The identity of each isolate was determined by polymerase chani reaction amplification and sequencing of the internal transcribed spacer (ITS) region of nuclear-encoded ribosomal DNA and also MALDI-TOF MS. The obtained data by molecular approach were compared with MALDI-TOF MS. RESULTS: The MALDI-TOF MS led to the identification of 44 (47%) isolates at the species level by generating the spectral score values of ≥ 2.0. However, there was not sufficient agreement between the results of the molecular-based ITS identification methods and MALDI-TOF MS in the species identification of 16 (17%) isolates. The Bruker Daltonics database was also not able to identify protein spectra related to 12 isolates (13%), including T. interdigitale (n=5), T. rubrum (n=4), M. canis (n=2), and T. tonsurans (n=1). CONCLUSION: According to the results, the utility of MALDI-TOF MS as a routine diagnostic tool for the accurate and reliable identification of dermatophytes can be justified whenever the protein spectra of a large set of worldwide clinical isolates are included in the commercial libraries. In addition, MALDI-TOF MS can be alternatively used to construct an in-house reference database.

Evaluation of a nucleic acid amplification assay for the diagnosis of Clostridioides difficile infection.

Clostridioides difficile is the leading cause of healthcare-associated diarrhea and the laboratory diagnosis of Clostridioides difficile infection (CDI) continues to be challenging. Accurate and rapid identification of C. difficile will reduce unnecessary antibiotic use and ensure contact isolation to control the spread of CDI. In this study, diagnostic performance of BD MAX Cdiff assay (Becton Dickinson, USA) was evaluated for the detection of C. difficile in 2502 fresh stool samples from hospitalized children and adult patients and the results were compared to toxigenic culture. The frequency of CDI in adults and pediatric patients were found as 3.3% and 6.2%, respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of BD MAX Cdiff assay were found as; 100%, 99.7%, 93%, and 100% for all patients; 100%, 99.7%, 96.2%, and 100% for pediatric patients; and 100%, 99.6%, 90.2%, and 100% for adult patients, respectively. We concluded that BD MAX Cdiff assay with high sensitivity, specificity, and PPV is useful for the diagnosis of CDI. With a high NPV of 100%, BD MAX Cdiff assay is also suitable for the exclusion of CDI.

[Investigation of plasmid-mediated AmpC beta-lactamase types in Klebsiella spp. and Escherichia coli isolates resistant or intermediate to cefoxitin]. / Sefoksitine dirençli ya da az duyarli saptanan Klebsiella spp. ve Escherichia coli izolatlarinda plazmid aracili AmpC beta-laktamaz enzim tiplerinin arastirilmasi.

Plasmid mediated AmpC beta-lactamases are reported from Enterobacteriaceae with increasing frequency. There have been reports of treatment failures in patients infected with these organisms and given broad-spectrum cephalosporins. The aim of this study was to investigate the presence of plasmid mediated AmpC beta-lactamases in Escherichia coli and Klebsiella spp. A total of 41 strains of cefoxitin resistant or intermediate E. coli (n= 27) and Klebsiella spp. (n= 14) were collected from january 2005 to January 2006 at Akdeniz University Hospital Central Laboratory. Three-dimensional test was used as a phenotypic confirmatory test. Analytical isoelectric focusing electrophoresis was used to measure the pl values of the beta-lactamases. Plasmid mediated AmpC enzyme genes were amplified using multiplex polymerase chain reaction and sequenced by Beckman Coulter CEQ 8000. AmpC beta-lactamases were only detected in two isolates (7.4%) of E. coli. These isolates produced CMY-2 like enzymes and have either CTX-M or TEM enzyme. Transferable AmpC beta-lactamases are associated with multiple antibiotic resistance. Therefore detection of these enzymes in gram-negative bacteria has a clinical importance, since it can often provide valuable information to clinicians leading to more effective and appropriate use of antimicrobials.