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

ABSTRACT 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.

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Rapid Detection ofFKS-Associated Echinocandin Resistance in Candida glabrata

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01574-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6573-6577 ◽

Cited By ~ 36

Author(s):

Yanan Zhao ◽

Yoji Nagasaki ◽

Milena Kordalewska ◽

Ellen G. Press ◽

Ryan K. Shields ◽

...

Keyword(s):

Candida Glabrata ◽

Molecular Beacon ◽

Rapid Identification ◽

Molecular Diagnostic ◽

Wild Type ◽

Asymmetric Pcr ◽

Content Type ◽

Allele Specific ◽

Echinocandin Resistance

ABSTRACTA novel and highly accurate diagnostic assay platform was established for rapid identification ofFKSmutations associated with echinocandin resistance inCandida glabrata. The assay platform uses allele-specific molecular beacon and DNA melt analysis following asymmetric PCR. A dual assay forFKS1andFKS2was developed to identify within 3 h the most common and clinically relevant resistance-associated mutations, including 8FKS1HS1 (wild type [WT], S629P, F625S, D632Y, D632E [T1896G], D632E [T1896A], I634V, and F625F) and 7FKS2HS1 (WT, F659del, F659S, F659V, F659L, S663P, and S663F) genotypes. A blinded panel of 188C. glabrataclinical isolates was tested by both assays. The molecular diagnostic results from the dual assay were 100% concordant with data obtained from DNA sequencing. This platform has the potential to overcome the deficiencies of existingin vitrosusceptibility-based assays to identify echinocandin-resistantC. glabrataand holds promise as a surrogate diagnostic method to better direct echinocandin therapy.

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Detection of Candida auris Antifungal Drug Resistance Markers Directly from Clinical Skin Swabs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01754-19 ◽

2019 ◽

Vol 63 (12) ◽

Cited By ~ 4

Author(s):

Milena Kordalewska ◽

Annie Lee ◽

Yanan Zhao ◽

David S. Perlin

Keyword(s):

Drug Resistance ◽

Antifungal Susceptibility ◽

Rapid Assessment ◽

Rapid Identification ◽

Antifungal Drug ◽

Infection Prevention And Control ◽

Molecular Diagnostic ◽

Candida Auris ◽

Content Type ◽

Antifungal Drug Resistance

ABSTRACT Accurate and rapid assessment of Candida auris antifungal drug resistance is crucial for effective infection prevention and control actions, as well as for 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 the gold standard. All swabs were correctly categorized as harboring wild-type or mutant C. auris.

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Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01083-17 ◽

2017 ◽

Vol 61 (12) ◽

Cited By ~ 9

Author(s):

L. Bernal-Martínez ◽

H. Gil ◽

O. Rivero-Menéndez ◽

S. Gago ◽

M. Cuenca-Estrella ◽

...

Keyword(s):

High Resolution ◽

Aspergillus Fumigatus ◽

Tandem Repeats ◽

High Resolution Melting ◽

Screening Method ◽

Melting Curve ◽

Health Concern ◽

Azole Resistance ◽

Content Type ◽

Selection Of

ABSTRACT The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A. Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus. PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR34, TR46, and TR53. Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

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Reversal of Azole Resistance inCandida albicansby Sulfa Antibacterial Drugs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00701-17 ◽

2017 ◽

Vol 62 (3) ◽

Cited By ~ 15

Author(s):

Hassan E. Eldesouky ◽

Abdelrahman Mayhoub ◽

Tony R. Hazbun ◽

Mohamed N. Seleem

Keyword(s):

Treatment Options ◽

Antifungal Drugs ◽

Infection Model ◽

Azole Resistance ◽

Global Public Health ◽

Sulfa Drugs ◽

Antibacterial Drugs ◽

Content Type

ABSTRACTInvasive candidiasis presents an emerging global public health challenge due to the emergence of resistance to the frontline treatment options, such as fluconazole. Hence, the identification of other compounds capable of pairing with fluconazole and averting azole resistance would potentially prolong the clinical utility of this important group. In an effort to repurpose drugs in the field of antifungal drug discovery, we explored sulfa antibacterial drugs for the purpose of reversing azole resistance inCandida. In this study, we assembled and investigated a library of 21 sulfa antibacterial drugs for their ability to restore fluconazole sensitivity inCandida albicans. Surprisingly, the majority of assayed sulfa drugs (15 of 21) were found to exhibit synergistic relationships with fluconazole by checkerboard assay with fractional inhibitory concentration index (ΣFIC) values ranging from <0.0312 to 0.25. Remarkably, five sulfa drugs were able to reverse azole resistance in a clinically achievable range. The structure-activity relationships (SARs) of the amino benzene sulfonamide scaffold as antifungal agents were studied. We also identified the possible mechanism of the synergistic interaction of sulfa antibacterial drugs with azole antifungal drugs. Furthermore, the ability of sulfa antibacterial drugs to inhibitCandidabiofilm by 40%in vitrowas confirmed. In addition, the effects of sulfa-fluconazole combinations onCandidagrowth kinetics and efflux machinery were explored. Finally, using aCaenorhabditis elegansinfection model, we demonstrated that the sulfa-fluconazole combination does possess potent antifungal activityin vivo, reducingCandidain infected worms by ∼50% compared to the control.

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The Fungal Cyp51-Specific Inhibitor VT-1598 DemonstratesIn VitroandIn VivoActivity againstCandida auris

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02233-18 ◽

2018 ◽

Vol 63 (3) ◽

Cited By ~ 10

Author(s):

Nathan P. Wiederhold ◽

Shawn R. Lockhart ◽

Laura K. Najvar ◽

Elizabeth L. Berkow ◽

Rosie Jaramillo ◽

...

Keyword(s):

Specific Inhibitor ◽

Candida Auris ◽

Once Daily ◽

Content Type ◽

Fungal Burden ◽

Invasive Infections ◽

Trough Concentrations ◽

Dose Dependent

ABSTRACTCandida aurisis an emerging pathogen associated with significant mortality and often multidrug resistance. VT-1598, a tetrazole-based fungal CYP51-specific inhibitor, was evaluatedin vitroandin vivoagainstC. auris. Susceptibility testing was performed against 100 clinical isolates ofC. aurisby broth microdilution. Neutropenic mice were infected intravenously withC. auris, and treatment began 24 h postinoculation with a vehicle control, oral VT-1598 (5, 15, and 50 mg/kg of body weight once daily), oral fluconazole (20 mg/kg once daily), or intraperitoneal caspofungin (10 mg/kg once daily), which continued for 7 days. Fungal burden was assessed in the kidneys and brains on day 8 in the fungal burden arm and on the days the mice succumbed to infection or on day 21 in the survival arm. VT-1598 plasma trough concentrations were also assessed on day 8. VT-1598 demonstratedin vitroactivity againstC. auris, with a mode MIC of 0.25 μg/ml and MICs ranging from 0.03 to 8 μg/ml. Treatment with VT-1598 resulted in significant and dose-dependent improvements in survival (median survival, 15 and >21 days for VT-1598 at 15 and 50 mg/kg, respectively) and reductions in kidney and brain fungal burden (reductions of 1.88 to 3.61 log10CFU/g) compared to the control (5 days). The reductions in fungal burden correlated with plasma trough concentrations. Treatment with caspofungin, but not fluconazole, also resulted in significant improvements in survival and reductions in fungal burden compared to those with the control. These results suggest that VT-1598 may be a future option for the treatment of invasive infections caused byC. auris.

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In Vitro Antifungal Combination of Flucytosine with Amphotericin B, Voriconazole, or Micafungin against Candida auris Shows No Antagonism

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01393-19 ◽

2019 ◽

Vol 63 (12) ◽

Cited By ~ 8

Author(s):

A. L. Bidaud ◽

F. Botterel ◽

A. Chowdhary ◽

E. Dannaoui

Keyword(s):

Amphotericin B ◽

Inhibitory Concentration ◽

Geometric Mean ◽

Multidrug Resistant ◽

Candida Auris ◽

Content Type ◽

Hospital Acquired Infections ◽

Resistant Mutants ◽

Hospital Acquired

ABSTRACT Candida auris is an emerging, multidrug-resistant pathogen responsible for invasive hospital-acquired infections. Flucytosine is an effective anti-Candida species drug, but which cannot be used as a monotherapy because of the risk of development of resistant mutants during treatment. It is, therefore, noteworthy to test possible combinations with flucytosine that may have a synergistic interaction. In this study, we determined the in vitro interaction between flucytosine and amphotericin B, micafungin, or voriconazole. These combinations have been tested against 15 C. auris isolates. The MIC ranges (geometric mean [Gmean]) of flucytosine, amphotericin B, micafungin, and voriconazole were 0.125 to 1 μg/ml (0.42 μg/ml), 0.25 to 1 μg/ml (0.66 μg/ml), 0.125 to 0.5 μg/ml (0.3 μg/ml), and 0.03 to 4 μg/ml (1.05 μg/ml), respectively. When tested in combination, indifferent interactions were mostly observed with fractional inhibitory concentration index values from 0.5 to 1, 0.31 to 1.01, and 0.5 to 1.06 for the combinations of flucytosine with amphotericin B, micafungin, and voriconazole, respectively. A synergy was observed for the strain CBS 10913 from Japan. No antagonism was observed for any combination. The combination of flucytosine with amphotericin B or micafungin may be relevant for the treatment of C. auris infections.

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The Novel Arylamidine T-2307 Demonstrates In Vitro and In Vivo Activity against Candida auris

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02198-19 ◽

2019 ◽

Vol 64 (3) ◽

Author(s):

Nathan P. Wiederhold ◽

Laura K. Najvar ◽

Rosie Jaramillo ◽

Marcos Olivo ◽

Hoja Patterson ◽

...

Keyword(s):

The Novel ◽

Candida Auris ◽

Once Daily ◽

Content Type ◽

Fungal Burden

ABSTRACT The in vitro and in vivo activity of the arylamidine T-2307 against Candida auris was evaluated. T-2307 demonstrated in vitro activity (MIC ranges ≤ 0.008 to 0.015 μg/ml at 50% inhibition; 0.125 to >4 μg/ml at 100% inhibition). Treatment with T-2307 (3 mg/kg subcutaneous [SC] once daily) also significantly improved survival (70% at 21 days postinfection) and reduced kidney fungal burden (5.06 log10 CFU/g) compared to control (0% survival and 7.09 log10 CFU/g) (P < 0.01).

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In Vitro Activity of a Novel Antifungal Compound, MYC-053, against Clinically Significant Antifungal-Resistant Strains of Candida glabrata, Candida auris, Cryptococcus neoformans, and Pneumocystis spp.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01975-18 ◽

2019 ◽

Vol 63 (4) ◽

Cited By ~ 7

Author(s):

G. Tetz ◽

M. Collins ◽

D. Vikina ◽

V. Tetz

Keyword(s):

Cryptococcus Neoformans ◽

Candida Glabrata ◽

Fungal Infections ◽

Antifungal Compound ◽

Candida Auris ◽

Content Type ◽

Treatment And Prevention ◽

Resistant Strains ◽

Clinically Significant

ABSTRACT An urgent need exists for new antifungal compounds to treat fungal infections in immunocompromised patients. The aim of the current study was to investigate the potency of a novel antifungal compound, MYC-053, against the emerging yeast and yeast-like pathogens Candida glabrata, Candida auris, Cryptococcus neoformans, and Pneumocystis species. MYC-053 was equally effective against the susceptible control strains, clinical isolates, and resistant strains, with MICs of 0.125 to 4.0 μg/ml. Notably, unlike other antifungals such as azoles, polyenes, and echinocandins, MYC-053 was effective against Pneumocystis isolates, therefore being the only synthetic antifungal that may potentially be used against Pneumocystis spp., Candida spp., and Cryptococcus spp. MYC-053 was highly effective against preformed 48-h-old C. glabrata and C. neoformans biofilms, with minimal biofilm eradication concentrations equal to 1 to 4 times the MIC. Together, these data indicated that MYC-053 may be developed into a promising antifungal agent for the treatment and prevention of invasive fungal infections caused by yeasts and yeast-like fungi.

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Updating Molecular Diagnostics for Detecting Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Isolates in Blood Culture Bottles

Journal of Clinical Microbiology ◽

10.1128/jcm.01195-19 ◽

2019 ◽

Vol 57 (11) ◽

Cited By ~ 5

Author(s):

Fred C. Tenover ◽

Isabella A. Tickler ◽

Victoria M. Le ◽

Scott Dewell ◽

Rodrigo E. Mendes ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Blood Culture ◽

False Negative ◽

The United States ◽

Molecular Diagnostic ◽

Methicillin Resistant ◽

Content Type ◽

Target Nucleic Acid ◽

Antimicrobial Resistance Genes

ABSTRACT Molecular diagnostic tests can be used to provide rapid identification of staphylococcal species in blood culture bottles to help improve antimicrobial stewardship. However, alterations in the target nucleic acid sequences of the microorganisms or their antimicrobial resistance genes can lead to false-negative results. We determined the whole-genome sequences of 4 blood culture isolates of Staphylococcus aureus and 2 control organisms to understand the genetic basis of genotype-phenotype discrepancies when using the Xpert MRSA/SA BC test (in vitro diagnostic medical device [IVD]). Three methicillin-resistant S. aureus (MRSA) isolates each had a different insertion of a genetic element in the staphylococcal cassette chromosome (SCCmec)-orfX junction region that led to a misclassification as methicillin-susceptible S. aureus (MSSA). One strain contained a deletion in spa, which produced a false S. aureus-negative result. A control strain of S. aureus that harbored an SCCmec element but no mecA (an empty cassette) was correctly called MSSA by the Xpert test. The second control contained an SCCM1 insertion. The updated Xpert MRSA/SA BC test successfully detected both spa and SCCmec variants of MRSA and correctly identified empty-cassette strains of S. aureus as MSSA. Among a sample of 252 MSSA isolates from the United States and Europe, 3.9% contained empty SCCmec cassettes, 1.6% carried SCCM1, <1% had spa deletions, and <1% contained SCCmec variants other than those with SCCM1. These data suggest that genetic variations that may interfere with Xpert MRSA/SA BC test results remain rare. Results for all the isolates were correct when tested with the updated assay.

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