Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents.

AIMS: A new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. METHODS: Spiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: No qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%-100% (69/72-72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. CONCLUSIONS: The developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.

Epidemiology of Nosocomial Candidemia, Mortality, and Antifungal Resistance: 7-Year Experience in Turkey.

Candidemia is an important clinical condition that prolongs hospital stays and increases morbidity, mortality, and hospital costs. The aim of this retrospective study was to evaluate the epidemiological and microbiological characteristics of patients with candidemia between January 2013 and December 2019. Two hundred forty-one episodes of candidemia were observed in 230 patients, 45% of whom were female. The median age was 63 years, and 53.9% of the episodes were in the intensive care unit (ICU). Commonly observed predisposing factors for candidemia included antibiotic use (71.3%), urinary catheterization (56.3%), central venous catheter placement (50.3%), total parenteral nutrition (47.9%), solid-organ malignancy (46%), surgery (48.6%), chemotherapy (37%), and steroid treatment (25.5%). The crude mortality rate was 52.7%. A significant difference was found between survivors and non-survivors (P = 0.007) according to the Charlson Comorbidity Index. However, no statistically significant association was found between mortality and age, sex, surgical procedure, catheter-related candidemia, or Candida spp. infection. The most frequently isolated Candida sp. was C. albicans (51%). Overall resistance rates to fluconazole, voriconazole, caspofungin, micafungin, and flucytosine were 3.7%, 0%, 2.5%, 1.8%, and 1.8%, respectively. Consequently, there is a need for tests that provide higher success rates, rapid diagnosis of candidemia, and local epidemiological data on antifungal resistance.

Candida auris Fungemia and a local spread taken under control with infection control measures: First report from Turkey.

Candidaauris, draws attention as a new emerging antifungal resistant pathogen, leading to healthcare-associated infections and outbreaks. This is the first report of C. auris fungemia in a 81-year-old patient, confirmed by sequential analysis, from Turkey. Although the source of the isolate could not be identified, its spread in the hospital has been taken under control by effective infection control measures.

Development of a fast and low-cost qPCR assay for diagnosis of acute gas pharyngitis.

BACKGROUND: Group A streptococci (GAS) are the most common bacterial cause of acute pharyngitis and account for 15-30 % of cases of acute pharyngitis in children and 5-10 % of cases in adults. In this study, a real-time quantitative PCR (qPCR) based GAS detection assay in pharyngeal swab specimens was developed. METHODS: The qPCR assay was compared with the gold standard bacterial culture and a rapid antigen detection test (RADT) to evaluate its clinical performance in 687 patients. The analytical sensitivity of the assay was 240 cfu/swab. Forty-five different potential cross-reacting organisms did not react with the test. Four different laboratories for the reproducibility studies were in 100 % (60/60) agreement for the contrived GAS positive and negative swab samples. RESULTS: The relative sensitivities of the RADT and the qPCR test were 55.9 and 100 %; and the relative specificities were 100 and 96.3 %, respectively. Duration of the total assay for 24 samples including pre-analytical processing and analysis changed between 42 and 55 min depending on the type of qPCR instrument used. A simple DNA extraction method and a low qPCR volume made the developed assay an economical alternative for the GAS detection. CONCLUSION: We showed that the developed qPCR test is rapid, cheap, sensitive and specific and therefore can be used to replace both antigen detection and culture for diagnosis of acute GAS pharyngitis.