Genetically related micafungin-resistant Candida parapsilosis blood isolates harbouring novel mutation R658G in hotspot 1 of Fks1p: a new challenge?

BACKGROUND: Echinocandin resistance rarely occurs in clinical Candida parapsilosis isolates and the underlying mechanism is unknown. OBJECTIVES: To determine the prevalence of echinocandin resistance and the underlying mechanism for a large collection of C. parapsilosis blood isolates and to determine whether the echinocandin-resistant isolates were clonally related. METHODS: C. parapsilosis blood isolates (n = 213) were subjected to antifungal susceptibility testing (CLSI M27), for micafungin, anidulafungin, amphotericin B and, if appropriate, caspofungin. Hotspot (HS) 1 and HS2 of FKS1 were sequenced for all isolates (n = 213) and microsatellite typing was performed for echinocandin-resistant isolates. RESULTS: All isolates were susceptible to amphotericin B and two isolates were intermediate to anidulafungin (MIC = 4 mg/L), while micafungin resistance was noted in four isolates (MIC >8 mg/L); three of which were also fluconazole resistant and therefore were MDR. Interestingly, micafungin-resistant isolates, but not those intermediate to anidulafungin, carried novel mutation R658G in HS1 of Fks1p; three of which also harboured Y132F+K143R in Erg11. The first isolate (MICR1) was recovered in November 2017 from a patient admitted to paediatric gastroenterology who showed therapeutic failure under caspofungin treatment. MICR2-MICR4 were collected during 2018-19 and were recovered from three echinocandin-naive paediatric-surgery patients; the isolates shared the same genotype. CONCLUSIONS: Herein, for the first time (to the best of our knowledge), we identified micafungin-resistant C. parapsilosis blood isolates harbouring a novel mutation in HS1 of FKS1, which was likely attributable to in vitro micafungin resistance and in vivo caspofungin therapeutic failure. The acquisition of micafungin-resistant C. parapsilosis isolates in echinocandin-naive patients likely implicates clonal expansion, as supported by the close genetic relatedness of MICR2-MICR4.

[Friend or Foe? Evaluation of BCG Vaccine and Latent Tuberculosis Infection Effect in Patients Diagnosed with COVID-19 Infection]. / Dost mu Düsman mi? COVID-19 Enfeksiyonu Tanili Hastalarda BCG Asisi ve Latent Tüberküloz Enfeksiyonu Etkisinin Degerlendirilmesi.

While severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to spread rapidly worldwide, some issues such as the uncertainty of the disease progress, whether intensive care will be needed, and risk classification are still important for clinicians. It is notable that in countries where latent tuberculosis infection (LTBI) is common and participating in the national Bacillus Calmette-Guerin (BCG) vaccination program, the case-fatality rates are relatively low throughout the world. In this study, it was aimed to evaluate the effects of the BCG vaccine and LTBI status on the course of the disease in patients diagnosed with coronavirus-19 (COVID-19) infection and to compare the LTBI rate with people with and without COVID-19 infection. The patients diagnosed with COVID-19 infection who were hospitalized during a period of seven months between May 1st to December 1st, 2020 were investigated by the QuantiFERON-TB Gold Plus (QFT-Plus) test in the blood samples for the presence of LTBI. For the comparison of the patients diagnosed with COVID-19 and people without COVID-19 infections in terms of LTBI rate retrospectively; all consecutive patients who were sent blood samples to the mycobacteriology laboratory for the QFT-Plus test between January 2016 and December 2019 were included in the study. Demographic, clinical, radiological, laboratory, and follow-up data of the patients were obtained from the electronic patient file. A total of 170 patients (n= 9 8 male [57.6%], n= 72 female [42.3%], mean age= 53.5 ± 15.8 years) were enrolled. Twenty-five patients' (25/170 [14.7%]) QFT-plus tests were positive. When the cases with positive QFT-Plus test (n= 25) and the cases with negative QFT-Plus test (n = 145) were compared in terms of disease severity respectively; it was determined that mild/moderate patients were 18/25 (72%) and 108/145 (74.5%), severe patients were 7/25 (28%) and 37/145 (25.5%) (p= 0.988). When these two groups were compared in terms of the clinical course respectively; the need for intensive care was 6/25 (24%) and 34/145 (23.4%) (p= 1.00), oxygen therapy requirement was 13/25 (52%) and 49/145 (33.8%) (p= 0.128), and death was 5/25 (20%) and 18/145 (12.4%) (p= 0.341). QFT-Plus positivity was 25/170 (14.7%) in patients diagnosed with COVID-19, while in control group it was 198/496 (39.9%) (OR= 0.259, 95% CI [0.164-0.411], p<0.001). When the values were evaluated quantitatively, in the COVID-19 patient group, QFT-Plus T1/T2 (IU/ml) interferon (IFN)-É£ was 0.87 ± 1.52/0.62 ± 1.53, while in the control group it was 1.52 ± 3.69/1.50 ± 3.33 (p= 0.032, p= 0.04). There was no significant difference in the parameters investigated between 82 (48.2%) patients with BCG vaccine and those 88 (51.8%) without BCG vaccine. Although it was not statistically significant in our study, increased oxygen therapy requirement and higher mortality rates in the QFT-Plus positive group were remarkable. The detection of statistically significantly lower LTBI rates and T1-T2/IFN-É£ values in the COVID-19 group supported that SARS-CoV-2 infection may suppress lymphocyte functions in patients and IFN-É£ response. We believe that the results of our study are remarkably valuable, but more clinical studies are needed to elucidate the relationship between BCG vaccine, LTBI, and COVID-19 infection.

First Report of Candidemia Clonal Outbreak Caused by Emerging Fluconazole-Resistant Candida parapsilosis Isolates Harboring Y132F and/or Y132F+K143R in Turkey.

Clonal outbreaks of fluconazole-resistant (FLZR) Candida parapsilosis isolates have been reported in several countries. Despite its being the second leading cause of candidemia, the azole resistance mechanisms and the clonal expansion of FLZR C. parapsilosis blood isolates have not been reported in Turkey. In this study, we consecutively collected C. parapsilosis blood isolates (n = 225) from the fifth largest hospital in Turkey (2007 to 2019), assessed their azole susceptibility pattern using CLSI M27-A3/S4, and sequenced ERG11 for all and MRR1, TAC1, and UPC2 for a selected number of C. parapsilosis isolates. The typing resolution of two widely used techniques, amplified fragment length polymorphism typing (AFLP) and microsatellite typing (MST), and the biofilm production of FLZR isolates with and without Y132F were compared. Approximately 27% of isolates were FLZR (60/225), among which 90% (54/60) harbored known mutations in Erg11, including Y132F (24/60) and Y132F+K143R (19/60). Several mutations specific to FLZR isolates were found in MRR1, TAC1, and UPC2 AFLP grouped isolates into two clusters, while MST revealed several clusters. The majority of Y132F/Y132F+K143R isolates grouped in clonal clusters, which significantly expanded throughout 2007 to 2019 in neonatal wards. Candida parapsilosis isolates carrying Y132F were associated with significantly higher mortality and less biofilm production than other FLZR isolates. Collectively, we documented the first outbreak of FLZR C. parapsilosis blood isolates in Turkey. The MRR1, TAC1, and UPC2 mutations exclusively found in FLZR isolates establishes a basis for future studies, which will potentially broaden our knowledge of FLZR mechanisms in C. parapsilosis MST should be a preferred method for clonal analysis of C. parapsilosis isolates in outbreak scenarios.

Low level of antifungal resistance of Candida glabrata blood isolates in Turkey: Fluconazole minimum inhibitory concentration and FKS mutations can predict therapeutic failure.

BACKGROUND: Candida glabrata is the third leading cause of candidaemia in Turkey; however, the data regarding antifungal resistance mechanisms and genotypic diversity in association with their clinical implication are limited. OBJECTIVES: To assess genotypic diversity, antifungal susceptibility and mechanisms of drug resistance of C glabrata blood isolates and their association with patients' outcome in a retrospective multicentre study. PATIENTS/METHODS: Isolates from 107 patients were identified by ITS sequencing and analysed by multilocus microsatellite typing, antifungal susceptibility testing, and sequencing of PDR1 and FKS1/2 hotspots (HSs). RESULTS: Candida glabrata prevalence in Ege University Hospital was twofold higher in 2014-2019 than in 2005-2014. Six of the analysed isolates had fluconazole MICs ≥ 32 µg/mL; of them, five harboured unique PDR1 mutations. Although echinocandin resistance was not detected, three isolates had mutations in HS1-Fks1 (S629T, n = 1) and HS1-Fks2 (S663P, n = 2); one of the latter was also fluconazole-resistant. All patients infected with isolates carrying HS-FKS mutations and/or demonstrating fluconazole MIC ≥ 32 µg/mL (except one without clinical data) showed therapeutic failure (TF) with echinocandin and fluconazole; seven such isolates were collected in Ege (n = 4) and Gulhane (n = 3) hospitals and six detected recently. Among 34 identified genotypes, none were associated with mortality or enriched for fluconazole-resistant isolates. CONCLUSION: Antifungal susceptibility testing should be supplemented with HS-FKS sequencing to predict TF for echinocandins, whereas fluconazole MIC ≥ 32 µg/mL may predict TF. Recent emergence of C glabrata isolates associated with antifungal TF warrants future comprehensive prospective studies in Turkey.

[Evaluation of QuantiFERON®-TB Gold in Tube Test and Tuberculin Skin Test in the diagnosis of Mycobacterium tuberculosis infection]. / Mycobacterium tuberculosis enfeksiyonu tanisinda QuantiFERON®-TB Gold in Tube Testi ve Tüberkülin Deri Testinin degerlendirilmesi.

The aims of this study were to evaluate the sensitivity of QuantiFERON®-TB Gold in Tube (QFT) test and its agreement with the tuberculin skin test (TST), to investigate possible factors associated with indeterminate QFT test results and to explore the relationship between latent tuberculosis infection (LTBE) prevalence and the rate of tuberculosis (TB) cases in our region. 1455 cases with QFT test performed in Ege University Faculty of Medicine Hospital between 2013 and 2015 were included in the study and simultaneously TST results of 268 of 1455 cases were reached. TST results were evaluated according to both ≥ 10 mm and ≥ 15 mm cut-off values. The QFT results of the cases were compared according to their gender, age groups and clinical characteristics with chi-square test. Stratified analyses were also conducted according to age groups. Multivariate logistic regression was used to analyse factors associated with QFT positivity and indeterminate QFT results. Cohen's kappa was used to test the agreement between QFT and TDT, overall and stratified according to age groups. Among 1455 cases, 396 (27.2%) were QFT positive and 120 (8.2%) had an indeterminate QFT result. When the indeterminate results were excluded, QFT positivity was found as 29.7%. The highest indeterminate results were determined among 0-4 year-old and ≥ 65 year-old groups as 17.6% and 12.1%, respectively and lowest among the 55-64 age group as 4%. The comparison of the cases without any cellular immunity defect and the patients with hematologic malignancies or immune deficiency and patients under immunosuppressive treatment had two and 2.44 times more indeterminate QFT results, respectively. Among 268 cases with TST results reached, QFT positivity was 30.6%; 38.1% for TST ≥ 10 mm and 25.7% for TST ≥ 15. After the exclusion of indeterminate results, the agreement between QFT and TST ≥ 10 mm was 71.3% for positive cases and 75.5% for negative cases. The highest agreement between QFT and TST ≥ 10 mm was in the age group 35-64 and lowest in the age group ≥ 65. Among 43 culture-positive cases, 32 had QFT positive, six negative and five indeterminate results. When indeterminate results were excluded, the sensitivity of thetest was 84.2% (32/38) among culture-positive active TB cases. TST results were available for 17 of the culture-positive cases, among them QFT sensitivity was 76.5% (13/17), TST sensitivity 70.6% (12/17) and the sensitivity of both tests was 88.2% (15/17). The ratio of QFT positivity has increased as the age increased. Interestingly, QFT positivity was higher among females than males in the 15-34 age group and higher among males in the 35-64 age group. The rates of QFT positivity were lower among immunocompromised patients. When QFT and TST positivities were compared with the rate of TB cases among age groups, QFT positivity was observed as parallel to the rate of TB cases. In conclusion, although the sensitivity of QFT was higher than TST, it was found that it could not be considered as a gold standard in LTBE diagnosis. As active TB cases originate from the LTBE pool, QFT test results might be considered a better indicator of active TB development risk.

[Comparison of phenotypic methods and polymerase chain reaction for the detection of carbapenemase production in clinical Klebsiella pneumoniae isolates]. / Klinik Klebsiella pneumoniae izolatlarinda karbapenemaz üretiminin saptanmasinda polimeraz zincir reaksiyonu ve fenotipik yöntemlerin karsilastirilmasi.

Being a member of the Enterobacteriaceae family, Klebsiella pneumoniae is an opportunistic pathogen that inhabits normal human microbiota and causes predominantly hospital-acquired infections. The emergence of K.pneumoniae isolates which are resistant particularly to the carbapenem group of antibiotics has led to an increase in hospitalization period, mortality and morbidity. Although different rates of resistance are observed between countries, regions and even healthcare facilities, there has been a rapid increase in the prevalence of carbapenem-resistant strains in the last 10 years. Fast and correct identification of carbapenem-resistant strains is important for the successful treatment of infections caused by these resistant bacteria. The objective of this study was to investigate the presence and the types of carbapenemases in carbapenem-resistant K.pneumoniae strains using "MASTDISCS™ ID carbapenemase detection disc set", a commercial product that can be used for this purpose, and "Carbapenem Inactivation Method (CIM)", a relatively new method, and compare the results of these methods by polymerase chain reaction (PCR). For this purpose, we used 54 K.pneumoniae strains isolated in 2015-2016, that were resistant to any of the ertapenem, meropenem or imipenem antibiotics. The identification of the strains was performed using VITEK MS and their antibiotic susceptibility tests were carried out using the VITEK 2 Compact® automated system. For the strains that were found resistant to carbapenems in the automated system, the minimum inhibitor concentration (MIC) values were determined by the gradient testing method according to the recommendations of "The European Committee on Antimicrobial Susceptibility Testing (EUCAST)". The blaOXA-48, blaIMP, blaNDM, blaVIM, and blaSIM genes were investigated with PCR among these isolates. Phenotypic enzyme typing was performed in the carbapenem-resistant strains using the "MASTDISCS™ ID carbapenemase detection disc set" and "Carbapenem Inactivation Method (CIM)". REP-PCR was used to reveal clonal relationship of the isolates. The 54 K.pneumoniae isolates were found as resistant to carbapenem and the MIC50 and MIC90 values of imipenem, meropenem and ertapenem were 32 µg/ml. Only 33 of the strains had blaOXA-48 and two of them had only blaNDM, the remaining 19 strains had both of these two genes. The blaIMP, blaVIM and blaSIM genes were not encountered in any of the isolates. When the isolates were assessed by the REP-PCR method, six main clones were detected. The "MASTDISCS™ ID carbapenemase detection disc set" was able to detect all the carbapenemase producing strains and it remained incapable of distinguishing OXA-48 in the strains which had both OXA-48 and metallo beta lactamase (MBL) enzymes. The CIM method showed a low rate of positivity (46.15%) in the strains containing blaOXA-48, but was found much more successful in the strains containing blaNDM with a detection rate of 85.71%. In this study, it was concluded that the Mastdiscs-ID method could be successfully used to detect the presence of blaOXA-48 which has a high prevalence in our country.

Recent Increase in the Prevalence of Fluconazole-Non-susceptible Candida tropicalis Blood Isolates in Turkey: Clinical Implication of Azole-Non-susceptible and Fluconazole Tolerant Phenotypes and Genotyping.

Candida tropicalis is the fourth leading cause of candidemia in Turkey. Although C. tropicalis isolates from 1997 to 2017 were characterized as fully susceptible to antifungals, the increasing global prevalence of azole-non-susceptible (ANS) C. tropicalis and the association between high fluconazole tolerance (HFT) and fluconazole therapeutic failure (FTF) prompted us to re-evaluate azole susceptibility of C. tropicalis in Turkey. In this study, 161 C. tropicalis blood isolates from seven clinical centers were identified by ITS rDNA sequencing, genotyped by multilocus microsatellite typing, and tested for susceptibility to five azoles, two echinocandins, and amphotericin B (AMB); antifungal resistance mechanisms were assessed by sequencing of ERG11 and FKS1 genes. The results indicated that C. tropicalis isolates, which belonged to 125 genotypes grouped into 11 clusters, were fully susceptible to echinocandins and AMB; however, 18.6% of them had the ANS phenotype but only two carried the ANS-conferring mutation (Y132F). HFT was recorded in 52 isolates, 10 of which were also ANS. Large proportions of patients infected with ANS and HFT isolates (89 and 40.7%, respectively) showed FTF. Patients infected with azole-susceptible or ANS isolates did not differ in mortality, which, however, was significantly lower for those infected with HFT isolates (P = 0.007). There were significant differences in mortality (P = 0.02), ANS (P = 0.012), and HFT (P = 0.007) among genotype clusters. The alarming increase in the prevalence of C. tropicalis blood isolates with ANS and HFT in Turkey and the notable FTF rate should be a matter of public health concern.