Do initial hematologic indices predict the severity of COVID-19 patients?

Background/aim: In this study, we aimed to evaluate the initial hematological findings analyzed on admission in confirmed COVID-19 patients who were transferred to the intensive care unit (ICU), to predict possible hematological indices. Materials and methods: Initial neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), monocyte to lymphocyte ratio (MLR), red cell distribution width to platelet ratio (RPR), mean platelet volume to platelet ratio, and lymphocyte multiplied by platelet count (LYM × PLT), of 695 patients with laboratory-confirmed COVID-19 were investigated and comparisons were made between the mild/moderate and severe groups. Results: The proportion of COVID-19 cases admitted to the ICU was 3.9%. The median age of patients admitted to the ICU was significantly higher than those who were not; [68.5 (interquartile range (IQR); 21.5] years vs. 41.0 (IQR; 15.7) years; P < 0.001. Severe cases had higher NLR (6.6 vs. 2.4; P < 0.001), and MLR (0.40 vs. 0.28; P = 0.004) and lower PLR (180.0 vs. 129.0; P < 0.001) compared to that of mild or moderate patients. Among all of the parameters, the ROC curve of NLR gave us the best ability to distinguish serious patients at an early stage (AUC = 0. 819, 95% confidence interval 0.729­0.910; P < 0.001). Conclusion: These data showed that age, initial NLR, PLR, and LYM × PLT were associated with the severity of COVID-19 disease and patients' need for the ICU. Therefore, initial hemogram parameters may be essential to predict the prognosis of COVID-19 patients.

[Comparison of Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution methods for determining the susceptibilities of Candida isolates]. / Candida izolatlarinin antifungal duyarliliginin belirlenmesinde Klinik Laboratuvar Standartlari Enstitüsü (CLSI) ve Avrupa Antimikrobiyal Duyarlilik Testleri Komitesi (EUCAST) sivi mikrodilüsyon yöntemlerinin karsilastirilmasi.

Candida species are among the top 10 pathogens causing bloodstream infections associated with high morbidity, mortality. In spite of the development of new antifungal drugs, epidemiological studies have shown that resistance to antifungal drugs among Candida isolates is becoming a serious problem. The aim of this study was to compare the antifungal broth microdilution methods of the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) for amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole and anidulafungin susceptibility of Candida blood isolates. The study consisted of 74 Candida albicans, 67 Candida parapsilosis, 30 Candida glabrata, and 18 Candida tropicalis isolates. The minimum inhibitory concentrations were determined after 24 and 48 hour of incubation with CLSI method and only 24 hour of incubation with EUCAST method except anidulofungin. The MIC values obtained by both methods were considered to be compatible within ± 2 dilution limits. The categorical agreement between methods for each antifungal agent was assessed using clinical break points and epidemiological cut-off values. The agreement (± 2 dilution) between the methods was found to be species, drug, and incubation time dependent. After 24 hour incubation, good agreement category (> 90%) was detected between amphotericin B, itraconazole, posaconazole and anidulofungin, but was lower category (< 85%) was determined with fluconazole and voriconazole especially for relatively slow growing C.glabrata and C.parapsilosis isolates. Excellent categorical agreement (100%) was observed for amfoterisin B/C.parapsilosis, C.glabrata, C.tropicalis and anidulofungin/C.albicans, C.glabrata, C.tropicalis but least category was determined for posaconazole and C.albicans (71.6% at 24 hour; 73% at 48 hour). In vitro resistance of therapeutically used fluconazole and anidulafungin determined by both methods was rare among C.albicans (1.3%, 2.7% respectively), C.glabrata (0%, 3.3% respectively) and C.tropicalis (0%, 5.6% respectively) isolates but, an increase of non-susceptible isolates were observed among C.parapsilosis (11.9% at 24 hour of incubation; 17.9% at 48 hour of incubation) for fluconazole. There was also a cross resistance between fluconazole and voriconazole for three C.parapsilosis isolates and one multidrug resistant (fluconazole, itraconazole, posaconazole and anidulofungin) C.albicans isolate (fluconazole, itraconazole, posaconazole and anidulofungin). As a result in this study, it was determined thatboth methods were similar and can be used according to preference of laboratories. The CLSI antifungal susceptibility test results can be assessed at the end of 24 hour incubation, but sometimes it is important that the evaluation should be performed as a result of 48 hour incubation in slow growing species such as C.glabrata.

[Development of a real-time polymerase chain reaction method for the identification of Candida species]. / Candida türlerini tanimlayan bir gerçek zamanli polimeraz zincir reaksiyonu yönteminin gelistirilmesi.

Candida species are one of the major causes of nosocomial infections and are the fourth most common agent involved in bloodstream infections. The impact of non-albicans Candida species is increasing, however C.albicans is still the most common species. Since the antifungal susceptibility pattern among Candida spp. may be different, rapid diagnosis and identification of non-albicans Candida spp. are important for the determination of antifungal agents that will be used for treatment. The aim of the study was to describe a real-time polymerase chain reaction (Rt-PCR) assay that rapidly detects, identifies and quantitates Candida species from blood culture samples. A total of 50 consecutive positive blood culture bottles (BACTEC, Beckton Dickinson, USA) identified at our laboratory between June-November 2013, were included in the study. Reference strains of Candida spp. (C.albicans ATCC 10231, C.glabrata ATCC 90030, C.tropicalis ATCC 1021, C.krusei ATCC 6258, C.parapsilosis ATCC 22019 and C. dubliniensis CD36) grown on Sabouraud dextrose agar were used for quality control. BACTEC bottles that were positive for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were also studied to search the cross-reactivity. A commercial kit (Zymo Research, USA) was used for DNA extraction. Real-time PCR was performed on LightCycler 480 (Roche, Germany) with primers and probes specific for 18S rRNA of Candida species. Twenty microlitres of the reaction mix contained 2 µl of extracted DNA, 2 µl of LightCycler Fast Start DNA Master Probe (Roche Diagnostics, Germany), 2 µl of MgCl(2) (5 mmol), 2 µl of 10x PCR buffer (Roche Diagnostics, Germany), 0.5 µl of each primer (0.01 nmol/µl) and 1 µl of each probe (0.1 µmol/µl) (TibMolBiol, Germany). Amplification was performed using the following conditions; 95°C for 10 mins and 50 cycles of denaturation at 95°C for 10 secs, annealing at 62°C for 10 secs and polymerisation at 72°C for 20 secs. A melting curve was created by cooling the producs at 50°C for 30 secs and then heating to 80°C at a rate of 0.1°C/sec measuring of the fluorescence simultaneously. For the quantitation of fungal DNA according to the standard curve, serial dilutions of C.albicans ATCC 10231 DNA from 3 x 10(5) to 3 x 10(2) ng/µl were used. All of the strains were also identified by conventional methods and sequence analysis in order to compare the results obtained by Rt-PCR. In our study, all patient and standard samples could be amplified, identified and quantitated by this developed Rt-PCR method. A total of 50 strains, of them 26 were C.parapsilosis, 15 were C.glabrata, 6 were C.albicans, and 3 were C.tropicalis have been detected and identified among patient samples. The results were completely concordant with the sequencing and conventional methods, so the sensitivity and specificity of this method were estimated as 100 percent. In conclusion, it was novel Rt-PCR developed and evaluated in this study is considered as a rapid, accurate, reproducible, sensitive and specific method for the detection, identification and quantitation of commonly observed Candida spp. strains.

[Comparison of Phoenix™ Yeast ID Panel and API® ID 32C commercial systems for the identification of Candida species isolated from clinical samples]. / Klinik örneklerden izole edilen Candida türlerinin tanimlanmasinda Phoenix™ Yeast ID Panel ile API(®) ID 32C ticari sistemlerinin karsilastirilmasi

Opportunistic fungal pathogens are one of the important causes of nosocomial infections, and several different types of yeasts, especially Candida species are increasingly recovered from immunocompromised patients. Since many of the yeasts are resistant to the commonly used antifungal agents, the introduction of appropriate therapy depends on rapid and accurate identification. The aims of this study were to compare the commercial identification systems namely API® ID 32C (bioMerieux, France) and Phoenix™ Yeast ID Panel (Becton Dickinson Diagnostics, USA) for the identification of Candida species and to evaluate the effect of morphological findings in the identification process. A total of 211 yeast strains isolated from different clinical samples (111 urine, 34 blood/vascular catheter, 27 upper/lower respiratory tract, 16 abscess/pus, 13 throat/vagina swabs and 10 sterile body fluids) of 137 patients hospitalized in Uludag University Health and Research Center between October 2013 to January 2014, were included in the study. Samples were cultured on blood agar, chromogenic agar (CHROMagar Candida, BD, USA) and Saboraud's dextrose agar (SDA), and isolated yeast colonies were evaluated with germ tube test and morphological examination by microscopy on cornmeal/Tween-80 agar. The isolates were identified as well by two commercial systems according to the manufacturers' recommendations. Discrepant results between the systems were tried to be resolved by using morphological characteristics of the yeasts. Of the isolates 159 were identified identical by both of the systems, and the concordance between those systems were estimated as 75.4%. According to the concordant identification, the most frequently isolated species was C.albicans (44.1%) followed by C.tropicalis (9.9%), C.glabrata (9.5%), C.parapsilosis (8.5%) and C.kefyr (8.1%). The concordance rate was 81.7% in identification of frequently isolated species (C.albicans, C.tropicalis, C.parapsilosis, C.glabrata, C.kefyr), however it was 38.7% for the rarely isolated ones (C.krusei, C.lusitaniae, C.inconspicua/C.norvagensis, C.catenulata), representing statistical significance (p= 0.034; x2 test). Although not significant (p= 0.31; x2 test), the rate of concordance was increased (88.1%), when adding the morphological findings to the identification process. Of 211 isolates 37 (17.5%), 50 (23.7%) and 124 (58.8%) were identified according to their growth characteristics on chromogenic agar, blood agar and SDA, respectively, indicating no statistically significant difference between the media (p> 0.05). Although genotypic identification is essential, phenotypic methods are more commonly used in routine laboratories for the identification of yeast species. However, since genotypic identification could not be performed in this study, none of the systems were accepted as the standard method and therefore the sensitivity and specificity of the systems were not calculated. On the other hand, our data indicated that the two identification systems were comparable and careful observation of yeast morphology could add confidence to the identification. In conclusion, since the Phoenix™ Yeast ID system was found more practical with easier interpretation, and the results were obtained earlier than those of the API® ID 32C system (16 hours versus 48 hours), it was thought that Phoenix™ Yeast ID system may be used reliably in the routine laboratories. However, as none of the methods evaluated was completely reliable as a stand-alone, careful evaluation is necessary for species identification.

[Determination of Salmonella serotypes by conventional and molecular methods]. / Salmonella Serotiplerinin Konvansiyonel ve Moleküler Yöntemler ile Belirlenmesi.

Determination of Salmonella enterica serotypes is crucial for epidemiological studies. Salmonella serotypes are defined on the basis of somatic (O) and flagellar (H) antigens, both of which are present in the cell wall of Salmonella. The aim of this study was to compare the results of molecular serotyping obtained by multiplex polymerase chain reaction (mPCR) with conventional serotyping results. Conventional serotyping has been performed in Ministry of Health Refik Saydam Hygiene Center as part of the National Laboratory of Enteric Pathogenes Surveillance Network (UEPLA). A total of 100 Salmonella strains, thay comprise 14 different serotypes by the reference laboratory have been investigated by using specific primers for Salmonella serogroups (A, B, C1, D and E) and Vi antigen gene clusters via mPCR method. Serotypes have been determined by applying four sequential mPCR targeting the fliC and fliB genes encoding the H1 antigens (H1: a, -b, -d, -g,m, -i, -r, -z10) and H2 antigen complexes (H2: 1,2, -1,5, -1,6, -1,7 and H: enx, enz15). The results of mPCR showed 100% consistency with the serogroups determined by the conventional method. Both sensitivity and specificity of mPCR according to each serogroups were found to be 100%. Results of serotyping that have been determined with the molecular antigenic formula showed accurate results for 2 (2%), probable results for 91 (91%) and incomplete formula for 7 (7%) isolates. Molecular serotyping results of the most common isolated Salmonella serotypes of which S.Enteritidis, S.Typhimurium and S.Paratyphi from clinical microbiology laboratories have been determined as probable results. Antigenic formula of these serotypes that detected using mPCR were considered to be consistent with the results of conventional serotyping when interpreted with epidemiologic data. The sensitivity of mPCR to identify S.Typhi which have been determined as accurate result with molecular serotyping was 100% for serogrouping and serotyping. Multiplex PCR is cheaper and faster for the serotyping of strains isolated in clinical laboratories, compared to the conventional methods. However since it is not possible to detect all serotypes by using molecular typing, this technique can not be currently considered as an alternative for conventional serotyping. Nevertheless molecular typing could be beneficial in providing the preliminary results earlier.

Species Distribution and Antifungal Susceptibilities of Candida Species Isolated From Blood Culture.

Introduction Candida species (spp.) are among the leading agents of bloodstream infections. Candidemias are a major cause of morbidity and mortality. Having an understanding of Candida epidemiology and antifungal susceptibility patterns in each center is crucial in guiding the management of candidemia. In this study, the species distribution and antifungal susceptibility of Candida spp. isolated from blood culture at the University of Health Sciences, Bursa Yuksek Ihtisas Training & Research Hospital were examined and the first data on the epidemiology of candidemia in our center were presented. Methods A total of 236 Candida strains isolated from blood cultures in our hospital over a four-year period were analyzed and their antifungal susceptibilities were studied retrospectively. Strains were identified at the species complex (SC) level by the germ tube test, morphology in cornmeal-tween 80 medium, and the automated VITEK 2 Compact (bioMérieux, Marcy-l'Étoile, France) system. Antifungal susceptibility tests were performed on VITEK 2 Compact (bioMérieux, Marcy-l'Étoile, France) system. The susceptibilities of the strains to fluconazole, voriconazole, micafungin, and amphotericin B were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines and epidemiologic cut-off values. Results Of the Candida (C.) strains, 131 were C. albicans (55.5%), 40 were C. parapsilosis SC (16.9%), 21 were C. tropicalis (8.9%), 19 were C. glabrata SC (8.1%), eight were C. lusitaniae (3.4%), seven were C. kefyr (3.0%), six were C. krusei (2.6%), two were C. guilliermondii (0.8%) and two were C. dubliniensis (0.8%). Amphotericin B resistance was not detected in Candida strains. Micafungin susceptibility was 98.3%, and four C. parapsilosis SC strains (10%) were intermediate (I) to micafungin. Fluconazole susceptibility was 87.2%. Apart from C. krusei strains which intrinsically resistant to fluconazole, three C. parapsilosis (7.5%), one C. glabrata SC (5.3%) strain were resistant (R) to fluconazole, and one C. lusitaniae (12.5%) strain was wild-type (WT). Voriconazole susceptibility of Candida strains was 98.6%. Two C. parapsilosis SC strains were I to voriconazole, while one strain was R. Conclusion In this study, the first epidemiological data of candidemia agents in our hospital were presented. It was determined that rare and naturally resistant species did not cause any problem in our center yet. C. parapsilosis SC strains showed decreased susceptibility to fluconazole, whereas Candida strains were highly susceptible to the four antifungals tested. Close monitoring of these data will help guide the treatment of candidemia.

Comparison of Clinical Laboratory Standards Institute (CLSI) Microdilution Method and VITEK 2 Automated Antifungal Susceptibility System for the Determination of Antifungal Susceptibility of Candida Species.

Introduction Changes in the epidemiology of Candida infections, increasing resistance, and advances in treatment have increased the need to perform antifungal susceptibility testing in clinical laboratories. Standardized reference, the microbroth dilution method, and various commercial antifungal susceptibility test systems are used to determine antifungal susceptibility. This study aims to determine and compare the antifungal susceptibility of various Candida species isolated from blood cultures in our laboratory with the CLSI M27 microdilution reference method and VITEK 2 automated system (bioMérieux, Marcy-l'Étoile, France). Methods The antifungal susceptibility of a total of 140 Candida strains to fluconazole, voriconazole, and amphotericin B, and a total of 92 strains to anidulafungin was tested with the CLSI M27 method and the VITEK 2 automated system. For fluconazole, voriconazole, and amphotericin B, essential and categorical agreement percentages were calculated between the two methods. Because there is no anidulafungin in the VITEK 2 system, anidulafungin results obtained with CLSI were compared with micafungin only in terms of categorical agreement. In the category comparison, CLSI clinical breakpoints were used; the epidemiological cut-off values were used when they were not available. Very major error, major error, and minor error rates were calculated. Results In general, the minimum inhibitory concentration (MIC) values obtained with VITEK 2 for azole group drugs were found to be one-fold higher than the CLSI MICs read at the 24th hour. While the essential agreement between the two methods was >90% for amphotericin B and voriconazole, it remained at 85% for fluconazole. Overall, the best categorical agreement was obtained with amphotericin B (99.3%), and the least categorical agreement was obtained with voriconazole (85.7%). A very major error was seen with amphotericin B (0.7%) and fluconazole (0.7%) in one C. parapsilosis strain each. No resistance was detected with VITEK 2 in one C. glabrata strain found to be resistant to fluconazole by the reference method. Major and minor error rates were higher for azole drugs than amphotericin B and anidulafungin/micafungin. Conclusion The VITEK 2 system is a fast and highly applicable system, and with these features, it is advantageous for routine laboratories. In this study, although the error rate was not very high, one fluconazole-resistant C. parapsilosis and C. glabrata strain could not be detected with VITEK 2. The increase in data on the antifungal performance of the VITEK 2 system, which is available in many routine laboratories due to its ability to be used for bacteria identification and sensitivity, will contribute to the usability of the system for this purpose. In this study, data that will support the literature information in terms of the antifungal performance of the VITEK 2 system are presented.

Biofilm Formation and Resistance to Fungicides in Clinically Relevant Members of the Fungal Genus Fusarium.

Clinically relevant members of the fungal genus, Fusarium, exhibit an extraordinary genetic diversity and cause a wide spectrum of infections in both healthy individuals and immunocompromised patients. Generally, Fusarium species are intrinsically resistant to all systemic antifungals. We investigated whether the presence or absence of the ability to produce biofilms across and within Fusarium species complexes is linked to higher resistance against antifungals. A collection of 41 Fusarium strains, obtained from 38 patients with superficial and systemic infections, and three infected crops, were tested, including 25 species within the Fusarium fujikuroi species complex, 14 from the Fusarium solani species complex (FSSC), one Fusarium dimerum species complex, and one Fusarium oxysporum species complex isolate. Of all isolates tested, only seven strains from two species of FSSC, five F. petroliphilum and two F. keratoplasticum strains, recovered from blood, nail scrapings, and nasal biopsy samples, could produce biofilms under the tested conditions. In the liquid culture tested, sessile biofilm-forming Fusarium strains exhibited elevated minimum inhibitory concentrations (MICs) for amphotericin B, voriconazole, and posaconazole, compared to their planktonic counterparts, indicating that the ability to form biofilm may significantly increase resistance. Collectively, this suggests that once a surface adherent biofilm has been established, therapies designed to kill planktonic cells of Fusarium are ineffective.