In vivo emergence of high-level resistance during treatment reveals the first identified mechanism of amphotericin B resistance in Candida auris.

OBJECTIVE: Candida auris has emerged as a health-care-associated and multidrug-resistant fungal pathogen of great clinical concern. As many as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, but no mechanisms contributing to this resistance have been identified. Here we describe a clinical case in which high-level amphotericin B resistance was acquired in vivo during therapy and undertake molecular and genetic studies to identify and characterize the genetic determinant of resistance. METHODS: Whole-genome sequencing was performed on four C. auris isolates obtained from a single patient case. Cas9-mediated genetic manipulations were then used to generate mutant strains harbouring mutations of interest, and these strains were subsequently subjected to amphotericin B susceptibility testing and comprehensive sterol profiling. RESULTS: A novel mutation in the C. auris sterol-methyltransferase gene ERG6 was found to be associated with amphotericin B resistance, and this mutation alone conferred a >32-fold increase in amphotericin B resistance. Comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harbouring the clinically derived ERG6 mutation. CONCLUSIONS: Together these findings definitively demonstrate mutations in C. auris ERG6 as the first identified mechanism of clinical amphotericin B resistance in C. auris and represent a significant step forward in the understanding of antifungal resistance in this emerging public health threat.

Molecular characterisation of Candida auris isolates from immunocompromised patients in a tertiary-care hospital in Kuwait reveals a novel mutation in FKS1 conferring reduced susceptibility to echinocandins.

BACKGROUND: Candida auris is an emerging, potentially multidrug-resistant pathogen that exhibits clade-specific resistance to fluconazole and also develops resistance to echinocandins and amphotericin B easily. This study analysed 49 C auris isolates for alterations in hotspot-1 and hotspot-2 of FKS1 for the detection of mutations conferring reduced susceptibility to echinocandins. METHODS: C auris isolates (n = 49) obtained from 18 immunocompromised patients during June 2016-December 2018 were analysed. Antifungal susceptibility testing was performed by Etest and broth microdilution-based MICRONAUT-AM assay. Mutations in hotspot-1 and hotspot-2 regions of FKS1 were detected by PCR sequencing and fingerprinting of the isolates was done by short tandem repeat typing. RESULTS: The patients had multiple comorbidities/risk factors for Candida spp. infection including cancer/leukaemia/lymphoma/myeloma (n = 16), arterial/central line (n = 17), urinary catheter (n = 17), mechanical ventilation (n = 14) and major surgery (n = 9) and received antifungal drugs as prophylaxis and/or empiric treatment. Seven patients developed C auris candidemia/breakthrough candidemia, nine patients had candiduria with/without candidemia and four patients developed surgical site/respiratory infection. Resistance to fluconazole and amphotericin B was detected in 44 and four isolates, respectively. Twelve C auris isolates from eight patients showed reduced susceptibility to echinocandins. Seven isolates contained hostspot-1 mutations and three isolates from a candidemia patient contained R1354H mutation in hotspot-2 of FKS1. Ten patients died, five were cured, two were lost to follow-up and treatment details for one patient were not available. CONCLUSIONS: Our findings describe development of a novel mutation in FKS1 conferring reduced susceptibility to echinocandins in one patient during treatment and unfavourable clinical outcome for many C auris-infected patients.

Health Care-Associated Infections in a Neurocritical Care Unit of a Developing Country.

BACKGROUND: Health care-associated infections (HAIs) in intensive care units (ICUs) specialized for neurocritical care (neurocritical care units [NCCUs]) are serious yet preventable complications that contribute significantly to morbidity and mortality worldwide. However, reliable data are scarcely available from the developing world. We aimed to analyze the incidence, epidemiology, microbial etiology, and outcomes of HAIs in an NCCU of a tertiary care teaching hospital in a high-income, developing country. METHODS: In this 3-year retrospective cohort study, all patients admitted to the NCCU at the Ibn Sina Hospital in Kuwait for ≥ 2 calendar days were included. Patient demographics, hospitalization, and details of ICU-acquired infections were evaluated. Patient-related outcomes included hospital and ICU length of stay (LOS) and in-hospital mortality. RESULTS: Among 913 patients with a total of 4921 ICU days, 79 patients had 109 episodes of HAIs. The overall incidence rate and incidence density of HAIs were 11.9/100 patients and 22.1/1000 ICU days, respectively. Multiple episodes of infection were documented in 29% of patients. The most prevalent infections were urinary tract infections (UTIs; 40/109 [37%]), bloodstream infections (30/109 [28%]), and pneumonia (16/109 [15%]). Seventy-six percent of infections were device-associated infections. A total of 158 pathogens were isolated, of which 109 were Gram-negative bacteria. Of the 40 Gram-positive bacteria, 22 were staphylococci. Seven infections were due to Clostridium difficile. There were 15 Staphylococcus aureus isolates, 47% of which were methicillin resistant. Two episodes of UTIs were due to Candida species. There were 84 Enterobacteriaceae isolates, 24% of which were extended-spectrum ß-lactamase producers. All Pseudomonas aeruginosa isolates were susceptible to aminoglycosides and carbapenems. Klebsiella species were the most common pathogen (45/158 [28%]), causing pneumonia (11/33 isolates [33%]), bloodstream infections (12/37 isolates [32%]), and UTIs (16/52 isolates [31%]). One episode of bloodstream infection was due to multidrug resistant Acinetobacter baumanii which was susceptible only to colistin. Only pneumonia was independently associated with mortality, while all HAIs that occurred were significantly associated with a prolonged ICU LOS. CONCLUSIONS: This is the first HAI surveillance study in an NCCU in Kuwait, and our results demonstrate the burden of HAIs on the neurologically injured patient, regardless of the site of infection. The high prevalence and resistant profile of HAIs in an NCCU in a developing country relative to a developed country has important implications for patient safety and emphasizes the need to strengthen collaboration between NCCU teams and infection control teams to prevent serious complications in this setting.

Invasive Candida auris infections in Kuwait hospitals: epidemiology, antifungal treatment and outcome.

PURPOSE: Candida auris is a recently recognized yeast pathogen, which has attracted worldwide attention due to its multidrug-resistant nature and associated high mortality rates. Its persistence in hospital environment and propensity of nosocomial transmission underscores the need of continuous monitoring to prevent outbreaks. Since the first case of C. auris candidemia in May, 2014, we have identified 17 additional invasive cases, which are described here. METHODS: Identity of 17 isolates originating from proven or possible cases of invasive C. auris infection and identified as Candida haemulonii by Vitek 2 yeast identification system was confirmed by PCR-sequencing of rDNA. Information about risk factors, treatment and outcomes were retrospectively retrieved from case files. Antifungal susceptibility testing was performed by Etest. RESULTS: Thirteen cases of candidemia and 4 cases of other invasive infections were detected in 6 hospitals across Kuwait. Major risk factors included adult patients with cancer, diabetes, gastrointestinal/liver diseases and extended (> 25 days) hospital stay. All isolates were resistant to fluconazole. Additionally, 5 and 4 isolates were also resistant to voriconazole and amphotericin B, respectively. Despite antifungal treatment, 9 of 15 patients died. Most patients (n = 12) were hospitalized in 2 hospitals that are in close proximity, whereas 5 other patients were from 3 hospitals that are situated > 10 km apart. CONCLUSIONS: Occurrence of successive cases of invasive C. auris infections with resulting mortality in nine patients suggests persistence of this multidrug-resistant yeast in major hospitals in Kuwait. Early detection by continuous surveillance and enforcement of infection control measures are recommended.

Development of Echinocandin Resistance in Candida tropicalis following Short-Term Exposure to Caspofungin for Empiric Therapy.

Isolation of two echinocandin-resistant Candida tropicalis strains from endotracheal secretions of a patient following short-term exposure to caspofungin is described. Both strains exhibited resistance to echinocandins by Etest and reference broth microdilution, showing a homozygous S645P mutation within the hot spot 1 (HS-1) region of FKS1 and belonging to a unique multilocus sequence type. Other C. tropicalis isolates collected from patients in the same intensive care unit within a 60-day period were susceptible to echinocandins and contained wild-type FKS1 sequences.

A 4-year prospective study of septicemia in pediatric surgical patients at a tertiary care teaching hospital in Kuwait.

BACKGROUND: Critically ill children are at high risk for developing nosocomial infections that contributes to death in 4% of all pediatric intensive care unit admissions. This prospective study was undertaken to determine the prevalence of septicemia in the pediatric surgery department of a large tertiary care teaching hospital in Kuwait and to evaluate the risk factors, the microbial etiology, and the antimicrobial susceptibility pattern of the isolated microorganisms. METHODS: All patients admitted to the pediatric surgery department from January 2001 until December 2004 with the diagnosis of septicemia were included in the study, and the microbiologically proven cases were then analyzed. The patients' demographics and risk factors for sepsis were recorded. All positive blood cultures were subjected to identification and antimicrobial susceptibility testing by VITEK 2(bioMerieux, Marcy l'Etoile, France). RESULTS: Of 3408 patients suspected to have septicemia, 78 (2.3%) patients developed microbiologically documented septicemias, 26% of those were low-birth weight patients, and 82% were patients with congenital anomalies; 87% of those needed surgical intervention. More than 50% were admitted to the intensive care unit, and 80.5% needed ventilatory support. Fifty-seven percent had early onset septicemia. Gram-positive and gram-negative bacteria accounted for 54% and 39% of the septicemia cases, respectively, whereas Candida spp was responsible for 7%. More than 50% of the staphylococci were resistant to cloxacillin, and all gram-positives were uniformly susceptible to glycopeptides and linezolid. Gram-negative bacteria showed variable resistance to cephalosporins (65%), piperacillin/tazobactam (29%), and carbapenems (11%). The attributable mortality rate for these septic episodes was 19% mainly because of gram-negative bacteria and Candida. CONCLUSION: The main etiologic agents of neonatal septicemia were coagulase-negative Staphylococcus, Pseudomonas aeruginosa, and members of the family Enterobacteriaceae. Empirical therapy with piperacillin/tazobactam or carbapenems for gram-negative septicemia and glycopeptides for gram-positive septicemia was effective.