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.

[Comparison of Cytomegalovirus Polymerase Chain Reaction Test Results Obtained with Fully Automated Systems: Roche Cobas 6800 and Qiagen NeuMoDx Experience]. / Tam Otomatize Sistemlerde Çalisilan Sitomegalovirüs Polimeraz Zincir Reaksiyonu Test Sonuçlarinin Karsilastirilmasi: Roche Cobas 6800 ile Qiagen NeuMoDx Deneyimi.

Viral load monitoring is important in identifying patients at risk of developing cytomegalovirus (CMV) related complications after transplantation and for this purpose, quantitative real-time polymerase chain reaction (Rt-qPCR) tests are most commonly used. The main problem in CMV DNA Rt-qPCR tests that make quantitative measurements is that there are significant differences in measurements performed with different kits in different laboratories. Comparability of viral load measurements between laboratories has increased with the introduction of quantitative PCR tests calibrated with the CMV International Quantitation Standard (IQS) developed by the World Health Organization (WHO). However, quantitative agreement between measurements made with different kits has still not been fully achieved. In this study, it was aimed to investigate the quantitative compatibility between measurements made with Cobas 6800 (Roche Diagnostics, Mannheim, Germany) and NeuMoDx (Qiagen, Ann Arbor, USA) CMV DNA Rt-qPCR tests, which are fully automated new generation systems calibrated with the WHO CMV IQS. The results of 214 plasma samples, which were studied simultaneously with Cobas 6800 CMV Rt-qPCR and NeuMoDx CMV Rt-qPCR tests were analyzed. In the tests, the extraction, amplification and detection stages were carried out fully automatically. CMV DNA was detected in 144 (67.28%) samples in both tests and was not detected in 53 (24.76%) samples. Incompatible results were obtained in a total of 17 (7.94%) samples. Good agreement was found between the qualitative results of both tests (kappa= 0.80, p< 0.001). When the quantitative results (n= 129) obtained in the dynamic measurement range of both tests were examined, the median viral load values measured by Cobas 6800 CMV Rt-qPCR and NeuMoDx CMV Rt-qPCR tests were 513 IU/mL (range= 35-37000) and 741 IU/mL (range= 68-48978), respectively. According to the correlation analysis, a very strong correlation was found between the results of both tests (r= 0.94, p< 0.001). According to Bland-Altman analysis; the average difference between the results of the NeuMoDx CMV Rt-qPCR test and the Cobas 6800 CMV Rt-qPCR test was found to be -0.14 log10 [standard deviation (SD)= 0.23] IU/mL and it was determined that the Cobas 6800 CMV Rt-qPCR test had lower measurements than the NeuMoDx CMV Rt-qPCR test. In 120 of 129 samples (93%) whose results were within the dynamic measurement range of both tests, the measurement difference was within ± 0.5 log10 IU/mL and in 9 (7%), it was detected as more than ± 0.5 log10 (median 0.54 log10 IU/ml; range= 0.51-0.81). No measurement difference of more than ± 1.0 log10 was detected in any sample. In this study, quantitative agreement was found in the measurements made in plasma samples with the fully automated Cobas 6800 CMV Rt-qPCR and NeuMoDx CMV Rt-qPCR tests calibrated with the CMV IQS. To the best of our knowledge, a study comparing viral load measurements made with Cobas 6800 and NeuMoDx fully automated systems in the detection of CMV DNA has not yet been conducted, and this is the first study on this subject.

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.

[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.

[Distribution of Hepatitis C Virus (HCV) Genotypes Among Intravenous Drug and Non-Drug User Patients]. / Damar Içi Madde Bagimliligi Olan ve Madde Bagimlisi Olmayan Hastalar Arasinda Hepatit C Virus (HCV) Genotiplerinin Dagilimi.

Genotype distribution of hepatitis C virus (HCV) can vary over the years between different patient groups and regions. The prevalence of intravenous drug users (IVDU) is known to increase in our country, yet there are a limited number of studies investigating the distribution of HCV genotypes in this group. These data are essential for monitorization of the changes in HCV epidemiology. The present study aimed to evaluate the five-year results of HCV genotyping among patients infected with HCV related to IVDU and unrelated to drug use. Plasma samples of 720 patients (HCV antibody, HCV RNA positive), which were sent to our laboratory for HCV genotyping between January 2014-March 2019 were analyzed. HCV RNA extraction from plasma samples was performed in the automated-extraction system of EZ1 advanced (Qiagen, Germany) using the EZ1 virus mini kit v2.0 (Qiagen, Germany). Amplicons were obtained by amplifying the 5'NCR and core gene region in the Rotorgene 6000 real-time PCR (Qiagen, Germany) device with the HCV RNA real-time quantitative 2.0 (NLM, Italy) kit. For the genotyping, a commercial line probe assay (LIPA) based on in vitro reverse hybridization GEN-C2.0 kit (NLM, Italy) which can distinguish 1, 2, 3, 4, 6 genotypes and 1a, 1b, 2a/c, 2b, 3a, 3b, 3c, 3k, 4a, 4b, 4c/d, 4e, 4f, 4h, 5a, 6a/b, 6g, 6f/q, 6m, 7a subtypes of HCV, based on variations in the 5'-NCR and core regions was used. HCV genotype distribution of 266 IVDU (93.2%: male; median age: 25 ± 6.82) and 454 non-drug users (51.3%: male; median age: 56.5 ± 16.06) were examined. In order of frequency in the group with IVDU; genotype 1a, 3a, 1b, 4c/d, 2b, 4, 3 were observed and genotype 1, 2a/c and mixed genotype (1+3a) were detected in one patient. In the group without IVDU, in order of frequency; genotype 1b, 1a, 3a, 1, 2a/c, 4 were observed and genotype 2b, 4c/d, 5a, 6a/b, 6 and mixed genotype (3+4) were detected in one patient. Genotypes 1a and 3a were significantly higher in the IVDU group (p< 0.00001, p< 0.00001), while 1b was significantly higher in patients without IVDU (p< 0.00001). Genotypes 1a and 3a were more common in young men (p< 0.00001, p= 0.000163), while 1b was higher in middleaged women (p< 0.00001). The incidence of genotypes 1b (p= 0.021) and 3a (p= 0.012) was higher in foreign nationals than the Turkish patients. When the HCV genotype distribution was examined by years, it was observed that the percentages of genotype 1b and 1a were decreasing, while the percentage of genotype 3a was increasing. As a result, in this study, HCV genotype distribution among IVDU was observed to be different from the general population without IVDU. It was found that genotypes 1a and 3a were more common in the IVDU group. As in the other regions of our country, genotype 1b was found most frequently in the general population. Genotype 3a increases significantly compared to years. In our study, the determination of genotypes existing in different parts of the world may be due to the foreign nationals living in our city and our region is a tourism center. It is also necessary to investigate whether there is an increase in IVDU over the years.

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.

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 Enzyme Immunoassay (EIA), Immunoblot and HIV RNA Polymerase Chain Reaction Test Results in the Diagnosis of Human Immunodeficiency Virus (HIV) Infection]. / Insan Immün Yetmezlik Virüsü (HIV) Enfeksiyonu Tanisinda Enzim 'Immunoassay' (EIA), Immünoblot ve HIV RNA Polimeraz Zincir Reaksiyonu Test Sonuçlarinin Degerlendirilmesi.

Acquired Immunodeficiency syndrome (AIDS) is an important global public health issue. Increasing HIV/AIDS cases reported each year has become a serious health problem for our country. The fourth generation enzyme immunoassay (EIA) test is the first step in the laboratory diagnosis of human immunodeficiency virus (HIV) infection. When the EIA test is repeatedly reactive, antibody-based tests such as immuno blot (IB), line immunoassay (LIA), HIV 1-2 antibody differentiation immunoassay, and HIV RNA tests for the early period of infection are used as confirmatory tests. The aim of this study was to evaluate the results of three different methods for the diagnosis of HIV infection. HIV 1-2 IB and quantitative HIV-1 RNA PCR tests were performed in 199 patient samples. These samples were detected as the reactive or gray zone with HIV 1-2 Ab+Ag EIA test between 2010 and 2015 at Akdeniz University Hospital, Microbiology Laboratory. HIV 1-2 Ab+Ag determination in serum samples was performed with the EIA method (Elecsys HIV combi PT test, Roche Diagnostics, Germany). A commercial kit (INNO-LIA HIV I-II Score, Innogenetics, Belgium) was used for HIV 1-2 IB method. The presence of HIV-1 RNA was investigated by automated nucleic acid extraction and real-time PCR method (Ampliprep/COBAS Tagman HIV-1 Test, Roche Diagnostics, Germany) in plasma samples. For statistical analysis, SPSS, Mann Whitney U test was used, ROC analysis was performed and p<0.05 value was considered statistically significant. HIV 1-2 Ab+Ag EIA COI (cut-off index) median value was higher with positive HIV 1-2 IB and HIV-1 RNA results than negative HIV 1-2 IB and HIV-1 RNA results. These values were 394 (range: 11.5-2272) and 1.79 (range: 1.01-83.3) respectively and this difference was statistically significant (p< 0.001). HIV-1 RNA test results were positive in one patient with gray zone and two patients with negative HIV 1-2 IB result (viral loads were > 10.000.000, > 10.000.000 and 5.040.000 copies/ml, respectively). For the kit that we used for HIV 1-2 Ab+Ag EIA COI ratio of >16.45 had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 97.6%, 98.1%, 97.6% and 98.1%, respectively for the detection of HIV infection (r= 0.994, p< 0.001). HIV 1-2 Ab+Ag EIA S/CO ratio of < 9.26 had a sensitivity, specificity, PPV and NPV of 100%, 92.5%, 91.1% and 100% (p< 0.001). HIV infection is diagnosed if HIV 1-2 Ab+Ag EIA test result is repeatedly reactive and HIV 1-2 IB test and HIV-1 RNA tests are positive. In our study, HIV 1-2 Ab+Ag EIA COI median value was 394 (range: 11.5-2272) in this group of patients (p< 0.001). HIV-1 RNA PCR test was positive in three patients with > 10.000.000, 5.040.000 and > 10.000.000 copies/ml whose EIA tests were repeatedly reactive. HIV IB test was detected as the gray zone in one of them and as negative in the remaining two (HIV EIA S/CO values were 265, 9.5 and 131.8, respectively). These patients were diagnosed as acute HIV infection with clinical and laboratory findings. In conclusion, HIV RNA should also be performed and included in the diagnostic algorithm for acute HIV infection.

[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 Samples with DFS Staining Pattern Detected by Indirect Immunofluorescence Assay.

BACKGROUND: In this study, we aimed to determine the presence of anti DFS70 antibody by a specific IB method in samples showing the DFS pattern and to determine the distribution of DFS pattern in different patient groups. METHODS: 2,401 serum samples, which were received for ANA screening, were tested by IIF method at Akdeniz University Hospital Diagnostic Laboratory. Out of 139 samples with DFS pattern, 75 samples were tested for the presence of anti DFS70 antibody by IB and were included in the study. Patients' clinical diagnoses were obtained retrospectively from medical records. RESULTS: 63 (84%) of 75 samples, which showed DFS patern by IIF, were found to have anti DFS70 antibody by IB. Five of these patients were diagnosed with SARD while the rest (58) had diseases other than SARD. CONCLUSIONS: DFS pattern detected by IIF and isolated anti DFS70 antibody positivity detected by IB show high concordance. However IIF results should be confirmed because of the patterns that can be misidentified as DFS pattern. The presence of anti-DFS70 antibodies, which help to exclude SARD, prevent further unnecessary referral demands.

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.

[Determination of cytomegalovirus glycoprotein B genotypes in different geographical regions and different patient groups in Turkey]. / Türkiye'de farkli cografi bölgelerde ve farkli hasta gruplarindaki sitomegalovirus izolatlarinin glikoprotein B genotiplerinin belirlenmesi.

Cytomegalovirus (CMV), a common virus found all around the world, usually causes asymptomatic infections in immunocompetent hosts, however it may lead to serious complications in immunodeficient patients and in the fetus. CMV is divided into four genotypes according to the polymorphisms in UL55 gene that encodes for envelope glycoprotein B. Nucleotide polymorphisms of CMV gB gene can affect the cell tropism of the virus and host immune response and believed to have important changes in the pathogenesis of CMV. The aim of this study was to determine the gB genotypes of CMV isolates from different patient groups selected from different regions of Turkey. A total of 136 clinical specimens from patients (66 female, 70 male; age range: 0-65 years, mean age: 24.03 ± 17.17) who were diagnosed to have CMV infection by polymerase chain reaction (PCR) and/or antigenemia tests, between 2001-2014, in the medical school hospitals of Akdeniz, Ege, Istanbul Cerrahpasa and Erciyes Universities (located at Mediterranean, Aegean, northwest and central Anatolia regions, respectively), were included in the study. The patient group consisted of 80 renal transplant (RT) recipients, 35 stem cell transplant (SCT) recipients, 13 newborns, seven heart transplant (HT) recipients and one pregnant woman. CMV gB genotypes were determined by PCR-RFLP (restriction fragment length polymorphism) method, and DNA sequencing and phylogenetic analysis were performed for the randomly selected 15 isolates with different genotypes. Among 136 (135 plasma, 1 amnion fluid) samples, the most frequent genotype was gB1 (n= 44, 32.4%), followed by gB2 (n= 39, 28.6%), gB3 (n= 36, 26.5%) and gB4 (n= 8, 5.9%); however nine (6.6%) samples could not be genotyped. When analysis were interpreted according to the patient groups, it was determined that the genotypes in RT recipients were gB1 32.3%, gB2 28.7%, gB3 26.5% and gB4 5.9%; in SCT recipients gB1 34.3%, gB2 28.6%, gB3 22.9% and gB4 5.7%; in HT recipients gB3 57.1%, gB1 14.3% and gB2 14.3%; in newborns gB1 38.4%, gB3 30.8%, gB2 15.4% and gB4 7.7%, and gB2 genotype in the pregnant woman. As our study was a descriptive study to determine the genotypes of CMV gB, the relationship between the genotypes and the variants such as viral load, symptomatic disease and prognosis were not analyzed. As a result, the isolation of different gB genotypes in various case groups from four distinctive provinces, underlines the diversity of CMV gB genotypes in Turkey.