[Evaluation of the Rapid Antigen Detection Kit with the Polymerase Chain Reaction for Detection of SARS-CoV-2 in Respiratory Samples]. / Solunum Yolu Örneklerinde SARS-CoV-2 Tespiti için Hizli Antijen Tespit Kitinin Polimeraz Zincir Reaksiyonu ile Birlikte Degerlendirilmesi.

The disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was named as coronavirus disease-2019 (COVID-19) by the World Health Organization (WHO) in February 11, 2020. The rapid diagnosis of COVID-19 patients is essential to reduce the disease spread. The reverse-transcription polymerase chain reaction (RT-PCR) is the gold standard test to diagnose SARS-CoV-2 acute infection. The rapid antigen test which can detect the presence of viral protein antigens in respiratory tract samples is being investigated as an alternative option, especially in cases where RT-PCR is not available or the test capacity is exceeded, due to its faster results, ease of application, low cost and lack of special equipment and personnel. In this study, it was aimed to evaluate the performance of a commercial rapid antigen test using nasopharyngeal samples of COVID-19 patients confirmed with RT-PCR. From the first day of the research, the first 80 consecutively SARS-CoV-2 RT-PCR positive and 40 RT-PCR negative respiratory samples sent to the Medical Microbiology Laboratory for routine SARS-CoV-2 RT-PCR testing were included in the study. RT-PCR tests of the samples were performed in routine studies with the BioSpeedy SARS-CoV-2 RT-PCR kit (Bioeksen, Turkey). Rapid antigen tests were performed with the Wesail COVID-19 antigen test kit (Guangdong, China) simultaneously with RT-PCR tests. Amongst the 80 positive RT-PCR samples, 56 were detected by the rapid antigen test. All the samples detected as positive with the rapid antigen tests were also positive with RT-PCR. There was a moderate agreement between the qualitative results of both tests (Kappa= 0.609, p<0.001). According to the PCR test, the sensitivity, specificity, positive predictive value (PPV), negative predictive value, and accuracy of the rapid antigen test were; 70%, 100%, 100%, 62.5%, and 80% (96/120), respectively. The sensitivities of the rapid antigen test were calculated as 92.6% in 54 samples with a cycle threshold (Ct) value of <17, 88.7% in 62 samples with a Ct value of <20, 77.8% in 72 samples with a Ct value of <22, and 74.7% in 75 samples with a Ct value of <25. According to our study data; the rapid antigen test was found less sensitive than the RT-PCR test. Negative results obtained with rapid antigen testing cannot exclude SARS-CoV-2 infection and must be confirmed by RT-PCR. In addition, according to the ROC analysis of rapid antigen test positivity obtained according to RT-PCR Ct values, the clinical performance of the rapid antigen test is good in samples with Ct values <20. The rapid antigen test should be evaluated as a reliable screening test in patients with high viral load. To the best of our knowledge, there is no other study in the literature performed with the Wesail COVID-19 rapid antigen test kit (Guangdong, China) used in our study. The fact that PPV was found to be 100% even at a low prevalence period of the pandemic will enable positive patients to be screened quickly and effectively with rapid antigen tests in the first step during the high prevalence period of the pandemic. In the light of these data and our results, it can be predicted that using the rapid antigen test as a screening test in the first step and confirming only negative patients with RT-PCR will contribute to the effective management of the pandemic process in terms of both time and cost. As a result of the study, the rapid antigen test with low sensitivity but high PPD can be included as a facilitating test in the first step of the diagnostic algorithm in terms of rapid identification of the patients with high viral load, initiation of treatment and providing filiation.

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 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.

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 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.

[Molecular epidemiology of beta-lactamases in ceftazidime-resistant Pseudomonas aeruginosa isolates]. / Seftazidime dirençli Pseudomonas aeruginosa izolatlarinda beta-laktamazlarin moleküler epidemiyolojisi.

Pseudomonas aeruginosa is an important opportunistic pathogen that cause mainly nosocomial infections especially in the immunocompromised patients, the elderly and patients with severe burns. The bacterial feature of developing high degree of resistance against several antibiotics leads to increased morbidity and mortality of P.aeruginosa infections. The aims of this study were to investigate the antibiotic susceptibilities of P.aeruginosa strains isolated from hospitalized patients and to determine the presence of resistance enzymes namely PER, GES, KPC, VIM, IMP and OXA. A total of 195 P.aeruginosa strains isolated from different clinical samples (29 sputum, 67 wound, 53 tracheal aspirate, 23 blood, 18 urine, 3 cerebrospinal fluid, 2 pleural fluid) of inpatients (134 male, 61 female) in Afyon Kocatepe University School of Medicine Hospital between 2010-2012, were included in the study. The isolates were identified by conventional methods and automated systems (VITEK 2, BioMerieux, France), and their antibiotic susceptibilities were detected by disk diffusion and E-test methods. Inducible beta-lactamase (IBL), extended-spectrum beta-lactamase (ESBL) and metallo-beta-lactamase (MBL) productions of the isolates were phenotypically investigated by double disk induction, double disk synergy and E-test methods, respectively. The presence of resistance genes encoding PER, GES, KPC, VIM, IMP and OXA enzymes were determined by real-time polymerase chain reaction, and sequence analysis was applied to positive samples. In our study, the antibiotic resistance rates of 195 P.aeruginosa strains were found as follows: ceftazidime 100%, tazobactam/piperacillin 90.8%, aztreonam 60.5%, cefepime 50.2%, imipenem 48.2%, meropenem 47.2%, ofloxacin 47.2%, piperacillin 44.1%, levofloxacin 31.3%, ciprofloxacin 26.2%, gentamicin 11.8%, amikacin 8.7% and tobramycin 6.2%. With the use of phenotypical methods, IBL, ESBL and MBL production rates in the isolates were detected as 89.2% (174/195), 30.7% (60/195) and 26.7% (52/195), respectively. Molecular studies showed that, five strains harboured OXA-10, four OXA-14, four VIM-2, two IMP-1, 26 GES-1 and 87 ABC transporter permease genes, while PER and KPC genes were not detected in any of the isolates. In conclusion, it was considered that the detection of beta-lactamase genes in bacteria and the identification of beta-lactamase types may provide facilities in selection of antibiotics, monitorization of therapy, prevention of resistance development of infection control programs.

Rapid detection of resistance in Staphylococcus aureus by using Quicolor ES.

Traditional microbiological methods are dependent on the growth of microorganisms, and hence require prolonged periods. The methods used to detect resistance in Staphylococcus aureus should have high sensitivity and specificity, yet provide results in a timely manner. The aim of this study was to evaluate the use of Quicolor (QC) ES(®) agar for the rapid detection of resistance in S. aureus. We evaluated 100 clinical S. aureus isolates. Resistance detection was performed using traditional microbiological methods. Methicillin resistance detection was evaluated using traditional and molecular microbiological methods. Traditional antibiotic susceptibility testing methods, such as disc diffusion, were conducted using QC ES and Mueller-Hinton (MH) media. The plates were incubated at 36 °C for 5, 6 and 24 h. Rapid results obtained using QC ES agar after 5 h of incubation were consistent with those using the overnight procedure with MH agar for 83 of the 100 S. aureus (including methicillin-susceptible S. aureus) strains. However, the correlation for oxacillin between MH (24 h) and QC ES (5 h) was not satisfactory (r = 0.770). The total agreement between QC ES and MH agar was 83% after 5 h, 89% after 6 h, and 94% after 24 h. The accurate and rapid detection of resistance in S. aureus is critical due to the associated therapeutic problems and infection control measures. We believe that the use of QC ES for S. aureus will reduce the delay in resistance detection, thus providing physicians and infection control practitioners with early information for better management.

Use of colistin with rifampicin, trimethoprim-sulfamethoxazole and teicoplanin in Acinetobacter mouse infection model.

Background: Infections with multidrug-resistant Gram-negative bacteria such as Acinetobacter baumannii are major cause of morbidity and mortality. Colistin is used commonly to treat these infections. In this study, we evaluated the efficacy of different colistin combinations in a A. baumannii infection mouse model. Materials & methods: An A. baumannii mouse infection model was developed in 150 experimental animals. Treatment groups were as follows: colistin, colistin + rifampicin, colistin + trimethoprim/sulfamethoxazole, colistin + teicoplanin and a control group. The outcome was bacterial burden in the lung and liver tissues. The treatment groups were subdivided into 24-, 48- and 72-h groups. Results: Colistin and combinations reduce the A. baumannii burden significantly in lung and liver tissues compared with the control group. Compared with colistin alone colistin + rifampicin and colistin + TMP-SMX provided significantly better reduction in the bacterial burden. Conclusion: These results may suggest that rifampicin and TMP-SMX combination with colistin may have a potential role in the treatment of A. baumannii infections.

Epidemiology of Candidemia in Children over 7 Years in a Medical Center in Turkey.

The aims of the study were to describe Candida species in children with candidemia, to determine the changing epidemiology of candidemia over time in our tertiary care hospital, and to examine the demographic and clinical characteristics of patients with candidemia caused by parapsilosis and nonparapsilosis Candida spp. From 2012 to 2018, we identified a total of 126 cases of candidemia. The most commonly isolated Candida sp. was C. parapsilosis (n = 71, 56.3%), followed by C. albicans (n = 34, 26.9%). A total of 21 candidemia episodes (16.6%) were caused by other Candida species. Patients were divided into two groups (parapsilosis and nonparapsilosis) to identify any potential differences between the groups in terms of risk factors, mortality, and antifungal resistance. The median age of the patients, the median durations of the hospital and pediatric intensive care unit stay, receipt of immunosuppressive therapy within 2 weeks of developing candidemia, the rate of using total parenteral nutrition, need for mechanical ventilation, and receipt of carbapenems were statistically significantly higher in the parapsilosis group than in the nonparapsilosis group (P = 0.020, P = 0.001, P = 0.011, P = 0.036, P = 0.002, P = 0.038, and P = 0.004, respectively). The overall 30-day mortality rates (4.2% versus 3.6%) and resistance to fluconazole (33.8% versus 32.7%) were similar between the groups (P = 0.790 and P = 0.860, respectively). The distribution of Candida strains isolated in this study was consistent with the global trend, with C. parapsilosis being the most commonly identified species. Determining local epidemiologic data at regular intervals in candidemia cases is important in terms of determining both the changing epidemiology and empirical antifungal agents. IMPORTANCE In our study, the changing epidemiology of Candida species in candidemia in children was evaluated. The dominance of Candida parapsilosis species in the changing epidemiology was remarkable. We found that fluconazole resistance was high in both parapsilosis and nonparapsilosis groups. Updating local epidemiologic data at certain intervals in candidemia cases is important in determining both the changing epidemiology and empirical antifungal agents.

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.