Can the pan-immune-inflammation value predict gram negative bloodstream infection-related 30-day mortality in solid organ transplant patients?

BACKGROUND: The recently used pan-immune-inflammation value (PIV) has not been adequately studied as a predictive marker for mortality in immunosuppressed patients. The aim of this study was to evaluate the usefulness of baseline PIV level as a predictor of 30-day mortality in solid organ transplant (SOT) recipients with gram negative bloodstream infections (GN-BSI). METHODS: This retrospective, cross-sectional study was conducted between January 1, 2019, and December 31, 2022, in 1104 SOT recipients. During the study period, 118 GN-BSI were recorded in 113 patients. Clinical, epidemiological, and laboratory data were collected, and mortality rates (30-day and all-cause) were recorded. RESULTS: The 113 recipients had a median age of 50 years [interquartile range (IQR) 37.5-61.5 years] with a male predominance (n = 72, 63.7%). The three most common microorganisms were as follows: 46 isolates (38.9%) of Escherichia coli, 41 (34.7%) of Klebsiella pneumoniae, and 12 (10.2%) of Acinetobacter baumannii. In 44.9% and 35.6% of the isolates, production of extended-spectrum beta-lactamases and carbapenem resistance were detected, respectively. The incidence of carbapenem-resistant GN-BSI was higher in liver recipients than in renal recipients (n = 27, 69.2% vs n = 13, 17.6%, p < 0.001). All-cause and 30-day mortality rates after GN-BSI were 26.5% (n = 30), and 16.8% (n = 19), respectively. In the group with GN-BSI-related 30-day mortality, the median PIV level was significantly lower (327.3, IQR 64.8-795.4 vs. 1049.6, IQR 338.6-2177.1; p = 0.002). The binary logistic regression analysis identified low PIV level [hazard ratio (HR) = 0.93, 95% confidence interval (CI) 0.86-0.99; p = 0.04], and increased age (HR = 1.05, 95% CI 1.01-1.09; p = 0.002) as factors associated with 30-day mortality. The receiver operating characteristic analysis revealed that PIV could determine the GN-BSI-related 30-day mortality with area under curve (AUC): 0.723, 95% CI 0.597-0.848, p = 0.0005. CONCLUSIONS: PIV is a simple and inexpensive biomarker that can be used to estimate mortality in immunosuppressed patients, but the results need to be interpreted carefully.

Molecular Detection of Dermatophytes and Nondermatophytes in Onychomycosis in Antalya, Turkey.

BACKGROUND: Onychomycosis is a chronic nail infection, and dermatophytes, yeasts, and nondermatophytic molds may be the causative agents. This study aimed to determine the etiological agents of onychomycosis by using conventional and molecular methods. METHODS: Between June 2020 and July 2021, 37 patients with a presumptive diagnosis of onychomycosis and mycological evidence (culture and/or EUROArray Dermatomycosis assay) were included in the study. Organisms detected in cultured nail specimens were identified by combined phenotypic characteristics and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). An EUROarray Dermatomycosis assay was used for molecular detection of fungal pathogens. RESULTS: The EUROArray Dermatomycosis assay was positive for a single fungal target in 23 samples, and 14 samples were positive by culture. The most common pathogen was Trichophyton rubrum in both methods. Coinfection was detected in 14 samples by using molecular methods, and Trichophyton rubrum and Fusarium solani (9 samples) were the most common pathogens detected together. Trichophyton spp., nondermatophyte molds, and Candida spp. were detected in 33 (89.2%), 16 (43.2%), and 6 (16.2%) samples, respectively, when the two methods were evaluated together. CONCLUSIONS: Our results revealed that fungal culture allows the diagnosis of onychomycosis, but it is not as sensitive as the EUROArray Dermatomycosis test, especially in patients receiving antifungal therapy.

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.

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.

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.

[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.

Incidence of BKV in the urine and blood samples of pediatric patients undergoing HSCT.

The aims were to investigate the incidence of BKV infection and the presence of HC in pediatric patients undergoing HSCT. Twenty-four children patients (M/F: 17/7) undergoing HSCT in a single center over a period of 1 year were included in the study. The presence of BKV DNA was determined by quantitative real-time PCR in plasma and urine samples at the following times: before transplantation, twice a week until engraftment time, and weekly for + 100 days. The mean age of the patients was 7.79 ± 5.03 years, the mean follow-up time was 95.6 ± 25.9 days, and the average number of samples per patient was 15.8 ± 3.2. BKV DNA was detected in at least one urine sample in 91.6% (n: 22) and at least one plasma sample in 75% (n:18) of the patients. The median time to the first BKV DNA positivity in urine and plasma samples was 11 (range: 1-80) and 32 days (range: 2-79), respectively. The median value of BKV DNA copies in urine and plasma were 1.7 × 106 (range: 2.8 × 101 -1.2 × 1014 ) and 1.9 × 103 copies/mL (range: 3-2.1 × 106 ), respectively. Thirteen patients (54.2%) had hematuria with BKV viruria; 8 (33.3%) patients had viremia. The median value of the BKV DNA copies in urine and plasma was 4.4 × 107 (range: 65-1 × 1011 ) and 2.9 × 103 (range: 7-7.8 × 104 ) copies/mL in these patients. Two (15.4%) of the 13 patients with BKV viruria and hematuria were diagnosed with BKV-related HC. BKV DNA viral load monitoring of urine and plasma in pediatric HSCT patients with a high risk for viral infections is valuable for understanding the development of BKV-related HC.

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.

The Evaluation of the Performance of C. Diff Quik Chek Complete and Toxin A + B (Clostridium difficile) DUO Diagnostic Tests Compared with Toxigenic Culture in the Diagnosis of Clostridium difficile Infection.

BACKGROUND: Clostridium difficile is an important cause of nosocomial diarrhea and the best standard laboratory method for the diagnosis of C. difficile infection is controversial. In this study, we aimed to investigate the performance of Toxin A + B (Clostridium difficile) DUO kit which detects C. difficile toxin A and B by the immunochromatographic method and C. Diff Quik Chek Complete (QCC) rapid membrane immunoassay kit which determines the presence of glutamate dehydrogenase (GDH) and C. difficile toxin A and B in stool samples, compared with toxigenic culture in the diagnosis of C. difficile infection. METHODS: One hundred ninety-three stool samples from patients suspected of having C. difficile infection were included in the study. The performances of two commercial tests were compared with toxigenic culture which was accepted as the reference method. RESULTS: The sensitivity and specificity of the GDH component of QCC were 94.4% and 97.7%, the sensitivity and specificity of the toxin component were 92.3% and 100%, respectively. The sensitivity and specificity of Toxin A + B (Clostridium difficile) DUO test were found as 53.8% and 87.8%, respectively. CONCLUSIONS: C. Diff Quik Chek Complete test, which is a rapid test with high sensitivity and specificity, can be used alone for the diagnosis of C. difficile infection while Toxin A + B (Clostridium difficile) DUO test cannot be used for the same purpose due to the low sensitivity and specificity of the test.

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.

[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.

[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.

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 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.

[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.

[Evaluation of blood culture practices: Use of system (Epicenter) data]. / Kan kültürü uygulamalarinin degerlendirilmesi: Isletim sistemi (Epicenter) verilerinin kullanimi.

Sepsis is a serious clinical problem and estimated to be responsible for 18 million annual deaths worldwide. Therefore, the use and the rapid processing of blood cultures are important for the transition from empiric therapy to directed therapy. The aim of this study was to assess the best blood culture practices in Turkey. We have examined the collection practices and techniques at four different hospitals, and a total of 165.443 blood culture bottles were evaluated (2013-2015). At the preanalytical phase most of the data which were important and which could support hospital quality systems/practices were not entered into the HIS and EpiCenter system. At the analytical phase loading of the bottles and removal of positive bottles primarily occurred between 6:00 and 9:00 AM but the positivity rate of the bottles showed a homogeneous distribution throughout the day. In other words, there were significant delays at processing positive blood culture bottles related to laboratory workers. The effect of education regarding best practices, transition from single bottle to two bottle cultures was successful in all hospitals. Single bottle usage decreased below 10% in all hospitals. Significantly more positive cultures were detected at multiple cultures when compared with the single bottle collection practice. In retrospective patient records, it was seen that all the laboratories reported the results of Gram staining to the clinics. However, these data were not recorded to the EpiCenter. The contamination rates of Ankara Numune Hospital and Akdeniz University Faculty of Medicine Hospital are 6.2% and 5.4% respectively, contamination rates were not reported in other hospitals. The most common isolates detected in blood cultures were Escherichia coli, Klebsiella pneumoniae, Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii. The mean time for the detection of these organisms were less than 20 hours in the aerobic bottle and anaerobic bottles. A total of 79.6% of facultative anaerobic isolates were detected in both bottles; 9.8% were detected only in the aerobic bottles; 10.6% of the isolates were detected only in the anaerobic bottles. As a result, the educational efforts in Turkey have met with success for transition from collecting single bottle blood culture sets to two bottle blood cultures. However, further efforts are needed to increase the number of blood culture sets collected during a 24 hours' period. In addition, errors at the preanalytical, analytical and postanalytical periods (taking samples, loading bottles into the system and processing positive blood cultures) should be eliminated.

Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI- TOF MS) for Early Identification of Septic Patients.

BACKGROUND: Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful technique for the rapid identification of bacteria from growing colonies in routine cultures. In this study, we evaluated the feasibility of a 5-hour incubation on solid medium after sub-cultivation of positive blood culture broth without any preparation steps in order to speed up the identification of bacteria. METHODS: In addition to standard laboratory protocols, a Columbia agar plate with 5% sheep blood was inoculated with 1 drop from the blood culture broth. After a 5-hour incubation period, a colony from the culture plate was submitted to MALDI-TOF MS. RESULTS: A total of 1351 positive blood cultures (1299 monomicrobial and 51 polymicrobial) were analyzed. When compared to routine identification procedure results for positive blood cultures, 79.3% of isolates were correctly identified to the species level. When manufacturer-recommended score values were taken into account, MALDITOF MS correctly identified 98.4% of the isolates to the species level with a score of > 2.0, 89.1% with a score between 1.7 and 2.0, and 75.4% with a score of < 1.7. CONCLUSIONS: ln our evaluation, a large majority of the S. aureus (91.5%) and Enterobacteriaceae (87.6%) were correctly identified at the species level. A 5-hour incubation period was found to be associated with moderate identification results for CoNS, Enterococcus spp., and nonfermentative gram negative bacilli, with failure being mostly observed with Streptococcus spp., Candida spp., and other gram positive bacteria. We believe that the performance of MALDI-TOF MS identification after short-term culture is directly related to the sufficient growth of microorganisms at 5 hours.

Evaluation of MALDI-TOF-MS for the Identification of Yeast Isolates Causing Bloodstream Infection.

BACKGROUND: Infections due to Candida species are major causes of morbidity and mortality in humans, causing a diverse spectrum of clinical disease ranging from superficial and mucosal infections to invasive disease. Several authors have demonstrated that mortality is closely linked to both timing of therapy and/or source control. The rapid identification of pathogenic species is helpful to start timely and effective antifungal therapy. The aim of this study was to assess the performance of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system for the correct and rapid identification of yeast isolates causing bloodstream infection. METHODS: Between January 2014 and January 2015, a total of 117 yeast like organisms isolated from blood culture samples of 117 episodes from 102 patients who had blood stream infections were included in the study. The isolates were identified by MALDI-TOF MS. The results were compared with those obtained by the standard mycological methods and/or sequence analysis. RESULTS: One hundred and seventeen yeast isolates including 115 Candida spp and two non-Candida yeasts were analysed. The Biotyper correctly identified 115 (98.3%) isolates to the genus level and 102 (87.2%) isolates to the species level using the manufacturer's recommended cutoff scores. CONCLUSIONS: The Bruker Biotyper is a rapid, easy, inexpensive, and highly reliable system for the identification of yeast isolates. Early identification with MALDI-TOF MS would save time for determination of antifungal susceptibility and proper treatment strategy. The expansion of the database of the library by addition of less common species will improve the performance of the system.

[Results of a multicenter study investigating plasmid mediated colistin resistance genes (mcr-1 and mcr-2) in clinical Enterobacteriaceae isolates from Turkey]. / Ülkemizde klinik Enterobacteriaceae izolatlarinda plazmit aracili kolistin direnç genlerini (mcr-1 ve mcr-2) arastiran çok merkezli çalismaya ait sonuçlar.

Colistin is a polymyxin antibiotic which is considered as one of the last line agents against infections due to multidrug resistant or carbapenem resistant gram-negative pathogens. Colistin resistance is associated with chromosomal alterations which can usually cause mutations in genes coding specific two component regulator systems. The first plasmid-mediated colistin resistance gene, mcr-1 was described in Escherichia coli and Klebsiella pneumoniae isolates in December 2015 and followed by another plasmid-mediated colistin resistance gene mcr-2 in 2016. The rapid and interspecies dissemination of plasmid-mediated resistance mechanisms through horizontal gene transfer, have made these genes considerably threatening. After the first reports, although mcr-1/mcr-2 producing Enterobacteriaceae isolates have been reported from many countries, there have been no reports from Turkey. Thus, the aim of this study was to investigate the presence of mcr-1/mcr-2 in clinical Enterobacteriaceae isolates from different parts of our country. A total of 329 Enterobacteriaceae isolates from 22 laboratories were collected which were isolated between March, 2015 and February, 2016. mcr-1/mcr-2 were investigated by polymerase chain reaction during February-March, 2016. Two hundred and seventeen of Klebsiella pneumoniae (66%), 75 of Salmonella spp. (22.8%), 31 of Esherichia coli (9.4%), 3 of Enterobacter cloacae (0.9%), 2 of Klebsiella oxytoca (0.6%) and 1 of Enterobacter aerogenes (0.3%) isolates were included to the study. Agarose gel electrophoresis results of PCR studies have shown expected band sizes for positive control isolates as 309 bp for mcr-1 and 567 bp for mcr-2. However, the presence of mcr-1/mcr-2 genes was not detected among the tested study isolates of Enterobacteriaceae. Although mcr-1/mcr-2 were not detected in our study isolates, it is highly important to understand the mechanism of resistance dissemination and determine the resistant isolates by considering that colistin is a last-line antibiotic against infections of multidrug or carbapenem resistant gram-negative bacteria. Thus, it is suggested that these mechanisms should be followed-up in both clinical and non-clinical (e.g. isolates from food animals, raw meats and environment) isolates of special populations.