The impact of COVID on bacterial sepsis.

PURPOSE: To identify the predictors of morbidity and mortality in matched COVID-19 positive and negative patients who were septic with Gram positive or Gram negative infections. METHODS: We conducted a retrospective review, from March to October 2020, of matched septic patients at five Hackensack Meridian Health hospitals who had bacteremia with Staphylococcus aureus, Klebsiella pneumoniae or Escherichia coli with and without COVID-19. We extracted patient demographics, comorbidities and clinical outcomes data using ICD-10 codes. Bacterial isolates were compared by whole genome sequencing analysis. Multivariate logistic regression was used to analyze independent predictors of morbidity and mortality. RESULTS: A total of 208 patients were grouped by positive bloodstream infection (BSI) with COVID-19 (n = 104) and without COVID-19 (n = 104). Most patients were over age 50 (90% vs. 89%) and Caucasian (78% vs. 86%). Inpatient mortality was higher in patients with COVID-19 for both GP (35% vs. 8%, p < 0.05) and GN (28% vs. 10%, p < 0.05) BSIs. Patients with Gram positive (GP) BSIs had a significant increase in mortality risk (OR 4.5, CI 1.4-14.5, p < 0.05) in contrast to those with Gram negative (GN) infections (OR 0.4, CI 0.4-4.0, p = 0.4). CONCLUSION: Concurrent COVID-19 infection is associated with a significant increase in morbidity and mortality in patients with GP and GN BSIs. Patients with S. aureus BSIs with COVID-19 are more likely to develop shock and respiratory failure and have higher rates and odds of mortality than those without COVID-19. These findings provide an essential insight into the care of these patients, especially those co-infected with Staphylococcus aureus.

Novel Non-Hot Spot Modification in Fks1 of Candida auris Confers Echinocandin Resistance.

We determined the echinocandin susceptibility and FKS1 genotypes of 13 clinical isolates of Candida auris that were recovered from 4 patients at a tertiary care center in Salvador, Brazil. Three isolates were categorized as echinocandin-resistant, and they harbored a novel FKS1 mutation that led to an amino acid change W691L located downstream from hot spot 1. When introduced to echinocandin-susceptible C. auris strains by CRISPR/Cas9, Fks1 W691L induced elevated MIC values to all echinocandins (anidulafungin, 16 to 32×; caspofungin, >64×; micafungin, >64×).

Detection and Identification of Candida auris from Clinical Skin Swabs.

Candida auris is a recently emerged pathogenic fungus posing an urgent global health threat. Given its propensity to cause nosocomial outbreaks, rapid detection and accurate identification of C. auris have become essential for an effective implementation of infection prevention and control measures in healthcare facilities. Unfortunately, the requirement of culturing a yeast colony from a patient sample, central for most of the available diagnostic tools, results in substantial delays (several days or longer) in diagnosis. However, nucleic acid-based techniques can deliver accurate diagnostic results within several hours since they are based on analysis of DNA extracted from patient specimen (no need of culture).Here we describe a real-time PCR assay protocol which can be applied for C. auris detection and identification directly from patient swabs.

Deciphering Candida auris Paradoxical Growth Effect (Eagle Effect) in Response to Echinocandins.

The paradoxical growth effect (PGE; also known as Eagle effect) is an in vitro phenomenon observed during antifungal susceptibility testing (AFST). In PGE, some fungal isolates grow in medium containing high concentrations of an echinocandin, above the minimal inhibitory concentration (MIC), despite being fully susceptible at lower concentrations. The presence of PGE complicates the assignment of isolates to susceptible or resistant category, especially in the case of newly emerged pathogens like Candida auris, for which susceptibility breakpoints are not established.Here we describe a protocol aiding in the determination of whether a given C. auris isolate is echinocandin-resistant or echinocandin-susceptible but exhibiting paradoxical growth.

Impact of Erg11 Amino Acid Substitutions Identified in Candida auris Clade III Isolates on Triazole Drug Susceptibility.

ERG11 sequencing of 28 Candida auris clade III isolates revealed the presence of concomitant V125A and F126L substitutions. Heterologous expression of Erg11-V125A/F126L in Saccharomyces cerevisiae led to reduced fluconazole and voriconazole susceptibilities. Generation of single substitution gene variants through site-directed mutagenesis uncovered that F126L primarily contributes to the elevated triazole MICs. A similar yet diminished pattern of reduced susceptibility was observed with the long-tailed triazoles posaconazole and itraconazole for the V125A/F126L, F126L, Y132F, and K143R alleles.

Antifungal Drug Susceptibility and Genetic Characterization of Fungi Recovered from COVID-19 Patients.

Fungal infections are common complications of respiratory viral infections and are associated with the increased need for intensive care and elevated mortality. Data regarding microbiological and molecular characteristics of such infections in COVID-19 patients are scarce. Here, we performed a comprehensive analysis, including species identification, antifungal susceptibility testing, molecular resistance determinants analysis, typing, and retrospective clinical data review, of fungal isolates recovered from 19 COVID-19 patients, who were hospitalized at the Hackensack University Medical Center (HUMC) in Hackensack, New Jersey, USA, in the initial phase of the pandemic from April-May 2020. In total, 17 Candida albicans, two C. parapsilosis, and two Aspergillus fumigatus were analyzed. All Candida spp. isolates were susceptible to micafungin and azole drugs (fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole). A. fumigatus isolates were susceptible to micafungin and all triazole drugs except fluconazole (intrinsic resistance). Multilocus sequence typing (MLST) of C. albicans isolates revealed 15 different sequence types (STs), which clustered below the clade-defining limit of p-distance < 0.04. Pulsed-field gel electrophoresis (PFGE) karyotyping revealed no chromosomal rearrangements in these isolates. A. fumigatus isolates were of different, non-related genotypes. We speculate that virus- and drug-induced immunosuppression (94.7% of the patients received corticosteroids), together with prolonged hospital stay (median duration of 29 days) and mechanical ventilation (median duration of 24 days) likely increased the susceptibility to secondary respiratory and bloodstream infections in the studied patient population. The presence of fungi in blood or respiratory tract fluid was a prognosticator for poor clinical outcome, which presented as an 89.5% 30-day mortality in our patient cohort.

Emergence of Multiple SARS-CoV-2 Antibody Escape Variants in an Immunocompromised Host Undergoing Convalescent Plasma Treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), harboring spike protein N-terminal domain (NTD) or receptor-binding domain (RBD) mutations, exhibit reduced in vitro susceptibility to convalescent-phase serum, commercial antibody cocktails, and vaccine neutralization and have been associated with reinfections. The accumulation of these mutations could be the consequence of intrahost viral evolution due to prolonged infection in immunocompromised hosts. In this study, we document the microevolution of SARS-CoV-2 recovered from sequential tracheal aspirates from an immunosuppressed patient on steroids and convalescent plasma therapy and identify the emergence of multiple NTD and RBD mutations. SARS-CoV-2 genomes from the first swab (day 0) and from three tracheal aspirates (days 7, 21, and 27) were compared at the sequence level. We identified a mixed viral population with five different S protein mutations (141 to 144 deletion, 243 to 244 deletion, E484K, Q493K, and Q493R) at the NTD or RBD region from the second tracheal aspirate sample (day 21) and a predominance of the S protein 141 to 144 LGVY deletion and E484K mutant on day 27. The neutralizing antibodies against various S protein lentiviral pseudovirus mutants, as well as the anti-SARS-CoV-2 total Ig and IgG, showed "U" shape dynamics, in support of the endogenous development of neutralizing antibodies. The patient's compromised immune status, the antirejection regiment, convalescent plasma treatment, and the development of neutralizing antibodies may have resulted in unique selective pressures on the intrahost genomic evolution, and this observation supports the hypotheses that VOCs can independently arise and that immunocompromised patients on convalescent plasma therapy are potential breeding grounds for immune escape mutants. IMPORTANCE Over a year of the COVID-19 pandemic, distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages have arisen in multiple geographic areas around the world. SARS-CoV-2 variants of concern (VOCs), i.e., B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), and B.1.617.2 (delta), harboring mutations and/or deletions in spike protein N-terminal domain (NTD) or receptor-binding domain (RBD) regions showed evidence of increased transmissibility and disease severity and possible reduced vaccine efficacy. In this study, we report the emergence of five different NTD and RBD mutations in an uncommon SARS-CoV-2 B.1.369 lineage from an immunosuppressed patient undergoing steroid and convalescent plasma therapy. The observation highlighted that VOCs can independently arise in immunocompromised populations undergoing anti-SARS-CoV-2 therapy, and enhanced measures will be required to reduce the transmission.

Novel FKS1 and FKS2 modifications in a high-level echinocandin resistant clinical isolate of Candida glabrata.

Echinocandin resistance in Candida glabrata poses a serious clinical challenge. The underlying resistance mechanism of a pan-echinocandin-resistant C. glabrata isolate (strain L74) was investigated in this study. FKS mutants carrying specific mutations found in L74 were reconstructed by the Alt-R CRISPR-Cas9 system (Fks1 WT/Fks2-E655K, strain CRISPR 31) and site-directed mutagenesis (strain fks1Δ/Fks2-E655K). Sequence analysis of strain L74 revealed a premature stop codon W508stop in FKS1 and an E655K mutation preceding the hotspot 1 region in FKS2. Introduction of the Fks2-E655K mutation in ATCC 2001 (strain CRISPR 31) conferred a modest reduction in susceptibility. However, the same FKS2 mutation in the fks1Δ background (strain fks1Δ/Fks2-E655K) resulted in high levels of resistance to echinocandins. Glucan synthase isolated from L74 was dramatically less sensitive to micafungin (MCF) relative to ATCC 2001. Both FKS1/FKS2 transcript ratios and Fks1/Fks2 protein ratios were significantly lower in L74 and fks1Δ/Fks2-E655K compared to ATCC 2001 and CRISPR 31 (P <0.05). Mice challenged with CRISPR 31 and fks1Δ/Fks2-E655K mutants failed to respond to MCF. In conclusion, the high-level of echinocandin resistance in the clinical isolate of C. glabrata L74 was concluded to result from the combination of null function of Fks1 and the point mutation E655K in Fks2.

Detection of Candida auris antifungal drug resistance markers directly from clinical skin swabs.

Accurate and rapid assessment of Candida auris antifungal drug resistance is crucial for effective infection prevention and control actions, and patient management. Here, performance of a molecular diagnostic platform, enabling rapid identification of FKS1 and ERG11 mutations conferring echinocandin and azole resistance, respectively, was evaluated on a panel of clinical skin swabs. Gene sequencing and antifungal susceptibility testing were used as "gold standard". All swabs were correctly categorized as harboring wild-type or mutant C. auris.

Identification of Drug Resistant Candida auris.

Candida auris is a multidrug resistant yeast, recognized as a cause of invasive infections and health care associated outbreaks around the world. C. auris is of great public health concern, due to its propensity for drug resistance, mode and pace of its transmission, and the possibility that biologic and epidemiologic factors could exacerbate worldwide emergence of C. auris infections. Currently, outbreak response is complicated by limited treatment options and inadequate disinfection strategies, as well as by issues (misidentification, long turnaround time) associated with application of commonly used diagnostic tools. Misdiagnosis of C. auris is common since many diagnostic platforms available in clinical and public health laboratories depend on reference databases that have not fully incorporated C. auris. Moreover, the correlation between minimal inhibitory concentration values (MICs) and clinical outcomes is poorly understood resulting in the absence of C. auris-specific breakpoints. New, accurate and fast diagnostic methods have emerged to facilitate effective patient management and improve infection control measures, ultimately reducing the potential for C. auris transmission. This review provides an overview of available C. auris detection/identification and antifungal susceptibility determination methods and discusses their advantages and limitations. A special emphasis has been placed on culture-independent methods that have recently been developed and offer faster turnaround times.

Molecular Diagnostics in the Times of Surveillance for Candida auris.

Recently, global health professionals have been significantly challenged by the emergence of Candida auris and its propensity to colonize human skin, persist in the healthcare environment, and cause healthcare-associated outbreaks. Additionally, C. auris isolates are often characterized by elevated minimal inhibitory concentration (MIC) values for antifungal drugs. Thus, rapid detection and accurate identification of C. auris together with an assessment of potential antifungal drug resistance has become essential for effective patient management, and infection prevention and control in healthcare facilities. Surprisingly, almost all of the commonly available diagnostic tools rely on recovery (growth) of yeast colonies from collected samples, which delays the diagnostic result by several days or longer. To circumvent these issues, molecular-based DNA amplification assays have been developed to identify C. auris DNA directly from patient samples. Moreover, allele discriminating detection probes can be used to rapidly assess validated mechanisms of echinocandin and azole resistance.

PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera.

Fungi of the Scopulariopsis genus, commonly found in the environment, are opportunistic pathogens that can cause various types of human infections. So far, no efficient molecular method has been developed for species differentiation among Scopulariopsis and related genera. In order to advance this field, we have evaluated performance of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, based on cytochrome c oxidase subunit 1 and ß-tubulin genes. The assays resulted in 2-10 restriction patterns, depending on the gene amplified and restriction enzyme applied. Pooled analysis of the patterns allowed to propose an algorithm, that can be successfully used for an accurate species-specific identification of 21 species of the Scopulariopsis-like fungi.

Rapid Detection of ERG11-Associated Azole Resistance and FKS-Associated Echinocandin Resistance in Candida auris.

Candida auris is an emerging multidrug-resistant yeast that can cause serious invasive infections. The accurate and rapid assessment of antifungal resistance is important for effective patient management. A novel and highly accurate diagnostic platform was established for the rapid identification of ERG11 mutations conferring azole resistance and FKS1 mutations associated with echinocandin resistance in C. auris Using allele-specific molecular beacons and DNA melting curve analysis following asymmetric PCR, a duplex ERG11 assay and a simplex FKS1 HS1 assay were developed to identify the most prominent resistance-associated mutations (Y132F and K143R in ERG11; S639F in FKS1 HS1) within 2 h. Assays were validated by testing a panel of 94 C. auris clinical isolates in a blind manner. The molecular diagnostic results from the assays were 100% concordant with DNA sequencing results. This platform has the potential to overcome the deficiencies of existing in vitro susceptibility-based assays to identify azole- and/or echinocandin-resistant C. auris, and thus, it holds promise as a surrogate diagnostic method to direct antifungal therapy more effectively.

Spontaneous Mutational Frequency and FKS Mutation Rates Vary by Echinocandin Agent against Candida glabrata.

Echinocandins are front-line agents for treatment of invasive candidiasis. There are no reported agent-specific differences in Candida mutational frequency of resistance or propensity to develop FKS mutations. The objective of this study was to measure spontaneous and FKS mutation rates among Candida glabrata strains. Twenty bloodstream isolates from patients with or without prior echinocandin exposure were included. Minimum inhibitory concentrations (MICs), minimum fungicidal concentrations (MFCs), and mutation prevention concentrations were higher for caspofungin than for anidulafungin (P < 0.0001) and micafungin (P < 0.0001). Mutational frequencies of resistance at 3× the baseline MIC were highest for caspofungin and lowest for micafungin. A total of 247 isolates were recovered at or above the MFC for caspofungin (n = 159), anidulafungin (n = 74), or micafungin (n = 14). Agent-specific MIC increases were noted for anidulafungin and caspofungin, but not micafungin. Thirty-three percent of isolates harbored hot spot mutations in FKS1 (n = 6) or FKS2 (n = 76). Mutations at the Ser629 (Fks1) or Ser663 (Fks2) loci were more common after selection with anidulafungin or micafungin than with caspofungin (P = 0.003). Four isolates demonstrated >4-fold increases in MICs without FKS hot spot mutations; three of these harbored Fks2 mutations upstream of hot spot 1. The final isolate was FKS1 and FKS2 wild-type, but the 50% inhibitory concentrations of caspofungin and micafungin were increased 2.7- and 8-fold, respectively. In conclusion, micafungin may be superior in vitro to the other agents in limiting the emergence of resistance among C. glabrata Caspofungin exposure may be most likely to promote resistance development. These data provide a foundation for future investigations of newly developed echinocandin agents.

Direct Detection of Emergent Fungal Pathogen Candida auris in Clinical Skin Swabs by SYBR Green-Based Quantitative PCR Assay.

The recent emergence of the multidrug-resistant and pathogenic yeast Candida auris continues to cause public health concern worldwide. C. auris is alarming because it causes health care-associated outbreaks and can establish invasive infections with high mortality rates. Transmission between patients is facilitated by the ability of C. auris to persistently colonize multiple body sites, including the skin, and survive for weeks on surfaces in health care settings. Rapid identification of colonized patients is needed to implement timely infection control measures. Currently, CDC laboratories use an enrichment culture-based approach that can take up to 2 weeks to identify C. auris from composite swabs from the bilateral axillae and groin. A rapid SYBR green quantitative PCR (qPCR) assay that can identify C. auris in a single day was recently described. In this study, we developed the SYBR green qPCR assay further by incorporating a DNA extraction procedure for skin swabs and by including an internal amplification control based on the distinguishable melt curve of a lambda DNA amplicon. The assay was conducted using 103 clinical axilla/groin skin swab samples. Using the enrichment culture-based approach as a gold standard, we determined that the SYBR green C. auris qPCR has a sensitivity of 0.93 and specificity of 0.96. Overall, we found that the SYBR green C. auris qPCR assay can be successfully applied for rapid and accurate detection of C. auris in patient skin swabs, thereby increasing diagnostic options for this emerging pathogen.

Limited ERG11 Mutations Identified in Isolates of Candida auris Directly Contribute to Reduced Azole Susceptibility.

Multiple Erg11 amino acid substitutions were identified in clinical isolates of Candida auris originating from India and Colombia. Elevated azole MICs were detected in Saccharomyces cerevisiae upon heterologous expression of C. aurisERG11 alleles that encoded for Y132F or K143R substitutions; however, expression of alleles encoding I466M, Y501H, or other clade-defined amino acid differences yielded susceptible MICs. Similar to other Candida species, specific C. aurisERG11 mutations resulted directly in reduced azole susceptibility.

Understanding Echinocandin Resistance in the Emerging Pathogen Candida auris.

Candida auris has simultaneously emerged on five continents as a fungal pathogen causing nosocomial outbreaks. The challenges in the treatment of C. auris infections are the variable antifungal susceptibility profiles among clinical isolates and the development of resistance to single or multiple classes of available antifungal drugs. Here, the in vitro susceptibility to echinocandin antifungal drugs was determined and FKS1 sequencing was performed on 106 C. auris clinical isolates. Four isolates were identified to be resistant to all tested echinocandins (MIC ≥ 4 mg/liter) and harbored an S639F mutation in FKS1 hot spot region 1. All remaining isolates were FKS1 wild type (WT) and echinocandin susceptible, with micafungin being the most potent echinocandin (MIC50 = 0.125 mg/liter). Antifungal susceptibility testing with caspofungin was challenging due to the fact that all FKS1 WT isolates exhibited an Eagle effect (also known as the paradoxical growth effect), which occurred at various intensities. To assess whether the Eagle effect resulted in pharmacodynamic resistance, 8 representative isolates were evaluated for their in vivo drug response in a murine model of invasive candidiasis. All isolates were susceptible to caspofungin at a human therapeutic dose, except for those harboring the S639F mutation. The data suggest that only isolates carrying mutations in FKS1 are echinocandin resistant and that routine in vitro testing of C. auris isolates for susceptibility to caspofungin by the broth microdilution method should be viewed cautiously or avoided.

A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates (2009-17) in India: role of the ERG11 and FKS1 genes in azole and echinocandin resistance.

Background: Candida auris has emerged globally as an MDR nosocomial pathogen in ICU patients. Objectives: We studied the antifungal susceptibility of C. auris isolates (n = 350) from 10 hospitals in India collected over a period of 8 years. To investigate azole resistance, ERG11 gene sequencing and expression profiling was conducted. In addition, echinocandin resistance linked to mutations in the C. auris FKS1 gene was analysed. Methods: CLSI antifungal susceptibility testing of six azoles, amphotericin B, three echinocandins, terbinafine, 5-flucytosine and nystatin was conducted. Screening for amino acid substitutions in ERG11 and FKS1 was performed. Results: Overall, 90% of C. auris were fluconazole resistant (MICs 32 to ≥64 mg/L) and 2% and 8% were resistant to echinocandins (≥8 mg/L) and amphotericin B (≥2 mg/L), respectively. ERG11 sequences of C. auris exhibited amino acid substitutions Y132 and K143 in 77% (n = 34/44) of strains that were fluconazole resistant whereas WT genotypes, i.e. without substitutions at these positions, were observed in isolates with low fluconazole MICs (1-2 mg/L) suggesting that these substitutions confer a phenotype of resistance to fluconazole similar to that described for Candida albicans. No significant expression of ERG11 was observed, although expression was inducible in vitro with fluconazole exposure. Echinocandin resistance was linked to a novel mutation S639F in FKS1 hot spot region I. Conclusions: Overall, 25% and 13% of isolates were MDR and multi-azole resistant, respectively. The most common resistance combination was azoles and 5-flucytosine in 14% followed by azoles and amphotericin B in 7% and azoles and echinocandins in 2% of isolates.

FKS2 and FKS3 Genes of Opportunistic Human Pathogen Candida albicans Influence Echinocandin Susceptibility.

Candida albicans, a prevailing opportunistic fungal pathogen of humans, has a diploid genome containing three homologous FKS genes that are evolutionarily conserved. One of these, the essential gene FKS1, encodes the catalytic subunit of glucan synthase, which is the target of echinocandin drugs and also serves as a site of drug resistance. The other two glucan synthase-encoding genes, FKS2 and FKS3, are also expressed, but their roles in resistance are considered unimportant. However, we report here that expression of FKS1 is upregulated in strains lacking either FKS2 or FKS3 Furthermore, in contrast to what is observed in heterozygous FKS1 deletion strains, cells lacking FKS2 or FKS3 contain increased amounts of cell wall glucan, are more resistant to echinocandin drugs, and consistently are tolerant to cell wall-damaging agents. Our data indicate that C. albicansFKS2 and FKS3 can act as negative regulators of FKS1, thereby influencing echinocandin susceptibility.