Drug resistance and tolerance in fungi.

Drug uptake and efflux appear to contribute at least partially to tolerance, based on studies with fluorescent azole probes and strains with similar MIC levels but different tolerance levels.

“In addition to affecting resistance, drug uptake and efflux appears to contribute, at least partially, to tolerance9, based on studies with fluorescent azole probes and strains with similar MIC levels but different tolerance levels (measured as FoG and SMG, see Box ##)89,90”

• cites [9] Rosenberg, A. et al. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 9, 2470 (2018). This article provides a quantitative characterization of fluconazole tolerance in C. albicans, distinguishing it from resistance and highlighting its phenotypic heterogeneity and association with subpopulation growth, and sensitivity to adjuvant drugs. sourceDisputed

  In the Discussion, the paper says higher tolerance is associated with more cells having lower intracellular drug levels, but explicitly states that tolerance is not a function of either uptake or efflux rates. The section “Decreased intracellular drug levels underlie increased tolerance” likewise reports that measured efflux rates did not correlate with FoG. Thus, the fluorescent-probe findings do not support the attributed conclusion that uptake and efflux contribute to tolerance. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [89] Benhamou, R. I. et al. Real-time imaging of the azole class of antifungal drugs in live Candida cells. ACS Chem. Biol. 12, 1769–1777 (2017). sourcePartly supported

  The Introduction describes fluorescent azole probes for studying drug uptake, and Results and Discussion reports that increased efflux is associated with fluconazole resistance in one Candida strain. The paper does not report that uptake or efflux contributes to tolerance, or compare strains with similar MICs but different FoG or SMG tolerance levels. No page numbers are available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [90] Benhamou, R. I., Bibi, M., Berman, J. & Fridman, M. Localizing antifungal drugs to the correct organelle can markedly enhance their efficacy. Angew. Chem. 57, 6230–6235 (2018). sourceNot checked — no full text of the source

Infections from Candida isolates that test susceptible frequently do not respond to available treatments.

“For example, the rate of fluconazole and echinocandin resistance in Candida. albicans, the most prevalent Candida species, is generally less than 1%7, yet existing therapies often fail to treat infections caused by susceptible Candida isolates.”

• cites [7] Pfaller, M. A., Diekema, D. J., Turnidge, J. D., Castanheira, M. & Jones, R. N. Twenty years of the SENTRY antifungal surveillance program: results for Candida species from 1997–2016. Open Forum Infect. Dis. 6, S79–S94 (2019). sourceCould not verify

  The Results report very low echinocandin resistance in C. albicans, and the Conclusions mention breakthrough infections. Neither passage says that infections caused by isolates testing susceptible frequently fail to respond to treatment. Searches for treatment failure despite in vitro susceptibility found no such statement in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

In C. albicans, resistance to fluconazole and echinocandins usually occurs in under 1% of isolates.

“For example, the rate of fluconazole and echinocandin resistance in Candida. albicans, the most prevalent Candida species, is generally less than 1%7, yet existing therapies often fail to treat infections caused by susceptible Candida isolates.”

• cites [7] Pfaller, M. A., Diekema, D. J., Turnidge, J. D., Castanheira, M. & Jones, R. N. Twenty years of the SENTRY antifungal surveillance program: results for Candida species from 1997–2016. Open Forum Infect. Dis. 6, S79–S94 (2019). sourceSupported

A recent quantitative study of seven diverse C. albicans isolates described four features of cells tolerant to fluconazole.

“A recent quantitative study9 identified four characteristics of fluconazole-tolerant cells across a set of seven diverse C. albicans isolates.”

• cites [9] Rosenberg, A. et al. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 9, 2470 (2018). This article provides a quantitative characterization of fluconazole tolerance in C. albicans, distinguishing it from resistance and highlighting its phenotypic heterogeneity and association with subpopulation growth, and sensitivity to adjuvant drugs. sourcePartly supported

  The Discussion (page not provided) describes quantitative characterization of fluconazole tolerance in C. albicans isolates and lists four cellular features, followed by a fifth point: tolerance is mechanistically distinct from resistance. The claim's restriction to seven diverse isolates is not supported: the paper also describes seven non-persistent clinical isolates and isolates from 12 patients with persistent candidemia (Candida albicans isolates, page not provided). (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Genome changes such as whole-chromosome aneuploidy and shorter amplifications can raise resistance.

“Copy number variations, including whole-chromosome aneuploidy and shorter-range amplifications, are genome changes that can increase resistance32,43–49.”

• cites [32] Prasad, R., Rawal, M. K. & Shah, A. H. Candida efflux ATPases and antiporters in clinical drug resistance. Adv. Exp. Med. Biol. 892, 351–376 (2016). sourceNot checked — no full text of the source
• cites [43] Kordalewska, M. et al. Understanding echinocandin resistance in the emerging pathogen Candida auris. Antimicrob Agents Chemother. 62, e00238–18 (2018). sourceNot checked — no full text of the source
• cites [44] Selmecki, A., Forche, A. & Berman, J. Aneuploidy and isochromosome formation in drug-resistant Candida albicans. Science 313, 367–370 (2006). sourceNot checked — no full text of the source
• cites [45] Anderson, M. Z., Saha, A., Haseeb, A. & Bennett, R. J. A chromosome 4 trisomy contributes to increased fluconazole resistance in a clinical isolate of Candida albicans. Microbiology 163, 856–865 (2017). sourceSupported
• cites [46] Yang, F. et al. Tolerance to caspofungin in Candida albicans is associated with at least three distinctive mechanisms that govern expression of FKS genes and cell wall remodeling. Antimicrob. Agents Chemother. 61, e00071–17 (2017). sourceNot checked — no full text of the source
• cites [47] Yang, F., Kravets, A., Bethlendy, G., Welle, S. & Rustchenko, E. Chromosome 5 monosomy of Candida albicans controls susceptibility to various toxic agents, including major antifungals. Antimicrob. Agents Chemother. 57, 5026–5036 (2013). sourceNot checked — no full text of the source
• cites [48] Yang, F. et al. Aneuploidy enables cross-adaptation to unrelated drugs. Mol. Biol. Evol. 36, 1768–1782 (2019). sourceSupported
• cites [49] Todd, R. T., Wikoff, T. D., Forche, A. & Selmecki, A. Genome plasticity in Candida albicans is driven by long repeat sequences. eLife. 8, e45954 (2019). sourcePartly supported

  The Introduction (page not provided) says that amplification of a chromosome arm confers fluconazole resistance, and the Results report increased resistance with another segmental amplification. The paper also associates aneuploidy broadly with resistant isolates, but the passages do not establish that whole-chromosome aneuploidy itself increases resistance. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

In Candida auris, ERG11 polymorphisms and extra ERG11 gene copies are thought to underlie its mostly intrinsic azole resistance.

“Finally, in Candida auris, ERG11 polymorphisms as well as an increased number of ERG11 gene copies are thought to be responsible for its largely intrinsic azole resistance37–40”

• cites [37] Xiang, M. J. et al. Erg11 mutations associated with azole resistance in clinical isolates of Candida albicans. FEMS Yeast Res. 13, 386–393 (2013). sourceNot checked — no full text of the source
• cites [38] Flowers, S. A., Colon, B., Whaley, S. G., Schuler, M. A. & Rogers, P. D. Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans. Antimicrob. Agents Chemother. 59, 450–460 (2015). sourceNot checked — no full text of the source
• cites [39] Healey, K. R. et al. Limited ERG11 mutations identified in isolates of Candida auris directly contribute to reduced azole susceptibility. Antimicrob Agents Chemother. 62, e01427–18 (2018). sourcePartly supported

  The paper’s Results (TEXT; page unavailable) show that two C. auris ERG11 substitutions, Y132F and K143R, raised azole MICs, while several other substitutions did not. It does not establish that C. auris has extra ERG11 gene copies or that these mechanisms explain largely intrinsic azole resistance; its gene-dosage discussion concerns the experimental constructs and a comparison with C. albicans. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [40] Chowdhary, A. et al. 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. J. Antimicrob. Chemother. 73, 891–899 (2018). sourcePartly supported

  The Discussion (page unavailable) links ERG11 substitutions Y132F and K143R to fluconazole resistance in the C. auris isolates studied. It does not establish that C. auris has extra ERG11 gene copies or that its azole resistance is largely intrinsic. The reported increase after fluconazole exposure concerns ERG11 expression, not gene copy number. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Epidemiological cut-off values rely on MICs to detect acquired resistance, typically caused by mutations.

“Epidemiological cut-off values use MICs to identify isolates with acquired resistance, usually via mutations24.”

• cites [24] Meletiadis, J., Curfs-Breuker, I., Meis, J. F. & Mouton, J. W. In vitro antifungal susceptibility testing of Candida isolates with the EUCAST methodology, a new method for ECOFF determination. Antimicrob. Agents Chemother. 61, e02372–16 (2017). sourcePartly supported

  The paper’s introductory text (indexed as ABSTRACT; page unavailable) says ECOFFs use MIC distributions to detect non-wild-type isolates that may have reduced susceptibility. It does not establish that those isolates have acquired resistance or that resistance is usually caused by mutations; it notes that testing isolates with known resistance mechanisms would be useful. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

For a given isolate, tolerant cells form a subpopulation that generally makes up 5%-90% of cells, varying by isolate.

“Second, within a given isolate, tolerant cells (those that do grow slowly in the presence of the drug, while the remaining cells do not grow) represent a subpopulation (generally 5%-90% of the cells, depending on the isolate) (Fig. 1B).”

• cites [9] Rosenberg, A. et al. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 9, 2470 (2018). This article provides a quantitative characterization of fluconazole tolerance in C. albicans, distinguishing it from resistance and highlighting its phenotypic heterogeneity and association with subpopulation growth, and sensitivity to adjuvant drugs. sourcePartly supported

  The Subpopulation growth dynamics section reports that the fraction of cells growing in drug varies by isolate and represents a subpopulation. It gives an observed range of 2%–98%, however, rather than establishing the claimed ‘generally 5%–90%’ range (page not available in our copy). (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Candida species are the leading cause of human fungal infections.

“In this Review, we explore the molecular mechanisms that contribute to antifungal drug resistance, with a focus on Candida spp., the most common cause of fungal infections in humans7.”

• cites [7] Pfaller, M. A., Diekema, D. J., Turnidge, J. D., Castanheira, M. & Jones, R. N. Twenty years of the SENTRY antifungal surveillance program: results for Candida species from 1997–2016. Open Forum Infect. Dis. 6, S79–S94 (2019). sourcePartly supported

  The paper’s discussion of invasive fungal infections (section labeled Abstract > Conclusions; page unavailable) calls candidemia and other forms of invasive candidiasis “the most important of the invasive mycoses.” That supports Candida’s prominence in invasive disease, but not the broader claim that it is the most common cause of all fungal infections in humans. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

On drug-containing plates, colonies become visible sooner for highly tolerant isolates than for less tolerant ones.

“Fourth, the time required for cells to appear as visible colonies on a petri plate containing drug is shorter for isolates with high tolerance levels than for isolates with lower tolerance levels (Fig. 1C).”

• cites [9] Rosenberg, A. et al. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 9, 2470 (2018). This article provides a quantitative characterization of fluconazole tolerance in C. albicans, distinguishing it from resistance and highlighting its phenotypic heterogeneity and association with subpopulation growth, and sensitivity to adjuvant drugs. sourcePartly supported

  In “Subpopulation growth dynamics” (page unavailable), the paper reports that highly tolerant isolates had a shorter drug-induced delay in colony appearance than less tolerant isolates. Because colony appearance times also differed without drug, this does not establish that highly tolerant isolates’ colonies became visible sooner in absolute time on drug-containing plates. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Drug concentration has little effect on tolerance.

“Third, tolerance is largely independent of drug concentration (Box 1).”

• cites [9] Rosenberg, A. et al. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 9, 2470 (2018). This article provides a quantitative characterization of fluconazole tolerance in C. albicans, distinguishing it from resistance and highlighting its phenotypic heterogeneity and association with subpopulation growth, and sensitivity to adjuvant drugs. sourceSupported

For bactericidal drugs, tolerant bacteria show a longer minimum duration to killing than susceptible bacteria.

“Tolerant bacterial and fungal isolates have MICs in the susceptible range, but tolerant bacteria are characterized by longer minimum duration to killing (MDK) than susceptible isolates10, based on responses to bactericidal drugs10–12.”

• cites [10] Brauner, A., Fridman, O., Gefen, O. & Balaban, N. Q. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat. Rev. Microbiol. 14, 320–330 (2016). sourceNot checked — no full text of the source
• cites [11] Balaban, N. Q. et al. Definitions and guidelines for research on antibiotic persistence. Nat. Rev. Microbiol. 17, 441–448 (2019). sourceSupported
  Experimental — accuracy not yet measured
• cites [12] Brauner, A., Shoresh, N., Fridman, O. & Balaban, N. Q. An experimental framework for quantifying bacterial tolerance. Biophys. J. 112, 2664–2671 (2017). sourceSupported
  Experimental — accuracy not yet measured

Candida biofilms cause a temporary physiological change in drug responses.

“Candida spp. biofilms induce a transient, physiological alteration in drug responses22 (Box 3); that is, fungal biofilms exhibit reduced antifungal drug susceptibility via mechanisms that are dependent on cells growing within a biofilm, in which cells adhere to a substrate and form a thick mat of cells and secreted extracellular matrix that holds them together and reduces drug efficacy.”

• cites [22] Taff, H. T., Mitchell, K. F., Edward, J. A. & Andes, D. R. Mechanisms of Candida biofilm drug resistance. Future Microbiol. 8, 1325–1337 (2013). sourceSupported

Fungal biofilms are less susceptible to antifungals through mechanisms requiring biofilm growth, with cells adhering to a surface and forming a dense mat plus secreted matrix that lowers drug efficacy.

“Candida spp. biofilms induce a transient, physiological alteration in drug responses22 (Box 3); that is, fungal biofilms exhibit reduced antifungal drug susceptibility via mechanisms that are dependent on cells growing within a biofilm, in which cells adhere to a substrate and form a thick mat of cells and secreted extracellular matrix that holds them together and reduces drug efficacy.”

• cites [22] Taff, H. T., Mitchell, K. F., Edward, J. A. & Andes, D. R. Mechanisms of Candida biofilm drug resistance. Future Microbiol. 8, 1325–1337 (2013). sourceSupported

In non-standard assays, greater drug-phase growth than a reference strain may reflect tolerance or trailing, not resistance, since the 24-hour MIC is unchanged and increased growth appears at 48 hours.

“Further effects could be due to tolerance or trailing, rather than resistance9,25–28 because the MIC was not altered at 24 h. Instead, increased growth in the presence of the drug (relative to a reference strain) was usually detected at 48 h.”

• cites [9] Rosenberg, A. et al. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 9, 2470 (2018). This article provides a quantitative characterization of fluconazole tolerance in C. albicans, distinguishing it from resistance and highlighting its phenotypic heterogeneity and association with subpopulation growth, and sensitivity to adjuvant drugs. sourceSupported
• cites [25] Lockhart, S. R., Ghannoum, M. A. & Alexander, B. D. Establishment and use of epidemiological cutoff values for molds and yeasts by use of the Clinical and Laboratory Standards Institute M57 standard. J. Clin. Microbiol. 55, 1262–1268 (2017). This review provides an important and accessible over-review of the concept of epidemiological cut-off values as well as the methodology that underlies their establishment. sourceNot checked — no full text of the source
• cites [26] Onyewu, C., Wormley, F. L. Jr., Perfect, J. R. & Heitman, J. The calcineurin target, Crz1, functions in azole tolerance but is not required for virulence of Candida albicans. Infect. Immun. 72, 7330–7333 (2004). sourcePartly supported

  The fluconazole Etest passage (labeled Abstract in our copy; page unavailable) describes growth within an inhibition halo as a trailing effect, and the assay passage says plates were read after 48 hours. The paper does not report an unchanged 24-hour MIC or increased drug-phase growth relative to the reference strain; it observed trailing in the reference strain too. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [27] Sanglard, D., Ischer, F., Marchetti, O., Entenza, J. & Bille, J. Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence. Mol. Microbiol. 48, 959–976 (2003). sourceNot checked — no full text of the source
• cites [28] Luna-Tapia, A., Tournu, H., Peters, T. L. & Palmer, G. E. Endosomal trafficking defects can induce calcium-dependent azole tolerance in Candida albicans. Antimicrob. Agents Chemother. 60, 7170–7177 (2016). sourceNot checked — no full text of the source

These LOH events included homozygosis of a chromosome 5 left-arm region containing an ERG11 point mutation.

“These LOH events included homozygosis of a region of the left arm of chromosome 5 that includes a point mutation in ERG1155.”

• cites [55] Ford, C. B. et al. The evolution of drug resistance in clinical isolates of Candida albicans. eLife 4, e00662 (2015). sourceSupported

Exposure to azoles promotes mitotic defects that yield tetraploid and aneuploid cells.

“Importantly, exposure to azoles promotes mitotic defects that yield tetraploid and aneuploid cells58.”

• cites [58] Forche, A. et al. The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains. PLoS Biol. 6, e110 (2008). sourcePartly supported

  The Results section reports aneuploid progeny arising from chromosome loss in tetraploid C. albicans strains grown on pre-spo or sorbose medium. It does not show that azole exposure promotes mitotic defects or produces those cells; searches for azole and fluconazole found references but no passage establishing that causal claim. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Growth conditions influence the degree of tolerance exhibited by a strain.

“In addition to genetic determinants of tolerance level, growth conditions influence the degree of tolerance exhibited by a strain.”

• cites [76] Walker, L. A., Gow, N. A. & Munro, C. A. Elevated chitin content reduces the susceptibility of Candida species to caspofungin. Antimicrob. Agents Chemother. 57, 146–154 (2013). This study finds that the ability of Candida species to grow in the presence of sub-MIC levels of caspofungin is associated with increased chitin levels in the cell wall. Pretreatment of cells with CaCl 2 and calcofluor white induces a similar increase in chitin and ability to grow in sub-MIC caspofungin levels. Whether this effect is an alteration in tolerance or resistance levels remains to be determined; higher cell sourceNot checked — no full text of the source
• cites [77] Lee, K. K. et al. Elevated cell wall chitin in Candida albicans confers echinocandin resistance in vivo. Antimicrob. Agents Chemother. 56, 208–217 (2012). sourceSupported
• cites [68] Cowen, L. E., Sanglard, D., Howard, S. J., Rogers, P. D. & Perlin, D. S. Mechanisms of antifungal drug resistance. CSH Perspect. Med. 5, a019752 (2015). sourcePartly supported

  The Biofilms section reports that biofilm growth makes normally susceptible planktonic cells highly resistant, supporting the broader point that growth state affects drug response. The Adaptive Cellular Factors section discusses drug tolerance, but our copy does not directly establish that growth conditions change the degree of tolerance exhibited by a given strain. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [78] Singh-Babak, S. D. et al. Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog. 8, e1002718 (2012). sourcePartly supported

  The Discussion reports that Hsp90 is required for basal echinocandin tolerance in C. glabrata and describes methionine-mediated repression of HSP90 in a conditional strain. That supports a condition-dependent change in this engineered setting, but the paper does not establish that ordinary changes in growth conditions alter the tolerance of a strain generally. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

Extended echinocandin treatment is linked to the appearance of resistant strains.

“Of note, long periods of echinocandin therapy are associated with the emergence of echinocandin-resistant strains65.”

• cites [65] Perlin, D. S. Echinocandin resistance in Candida. Clin. Infect. Dis. 61, S612–S617 (2015). sourceSupported

Certain drugs increase genome instability, promoting aneuploidy and whole-chromosome LOH.

“Finally, some drugs enhance genome instability and thus promote aneuploidy and whole-chromosome LOH57,58.”

• cites [57] Cowen, L. E. et al. Population genomics of drug resistance in Candida albicans. Proc. Natl Acad. Sci. USA 99, 9284–9289 (2002). sourceCould not verify

  The Discussion (page unavailable) suggests that mutations, broadly including chromosomal rearrangements, may underlie changes in gene expression during adaptation to fluconazole. It does not say that the drug increases genome instability or promotes aneuploidy or whole-chromosome LOH; searches for those terms found no supporting passage. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [58] Forche, A. et al. The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains. PLoS Biol. 6, e110 (2008). sourcePartly supported

  The Results section (Analysis of the Pattern of Concerted Chromosome Loss during the Parasexual Cycle; page unavailable) reports more trisomies and a different distribution of whole-chromosome LOH in progeny grown on sorbose medium than on pre-spo medium. It attributes the difference tentatively to selective pressure from the growth medium, not to drugs; the claimed drug-induced genome instability is therefore unsupported by this paper. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

In heterozygous diploids like C. albicans, loss of heterozygosity is another common resistance mechanism.

“For heterozygous diploids such as C. albicans, loss of heterozygosity (LOH) is another frequent mechanism of resistance (reviewed in Refs. 52 and 53).”

• cites [52] Mount, H. O. et al. Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs. PLoS Genet. 14, e1007319 (2018). This article screens a library of C. albicans deletion strains and identifies two genes that affect azole responses, have roles in stress response pathways and functions that are are suppressed by the acquisition of aneuploid chromosomes. Whether the mutations are affecting resistance or tolerance is not clear, but activities of the identified genes (vacuolar retrograde trafficking and a cell wall and polarity GTPase activating pro sourceCould not verify

  The Introduction describes aneuploidy as frequent in fluconazole-resistant isolates, while the Results section on chromosome 3 and 7 reports structural changes in four evolved resistant isolates. Neither passage says that loss of heterozygosity is a frequent resistance mechanism in C. albicans. A search for “loss of heterozygosity” found the term in a reference title, not in a finding that establishes the claim; page numbers were unavailable in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [53] Todd, R. T., Forche, A. & Selmecki, A. Ploidy variation in fungi: polyploidy, aneuploidy, and genome evolution. Microbiol. Spectr. 5, https://doi.org/10.1128/microbiolspec.FUNK-0051-2016 (2017). sourcePartly supported

  The Introduction describes C. albicans as generally diploid, and section 4 reports loss of heterozygosity (LOH) in C. albicans cells (page numbers unavailable). The paper does not say LOH is a frequent resistance mechanism; its discussion of fluconazole resistance in section 6 instead concerns aneuploidy and gene amplification. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Conditions that increase cell wall chitin promote cell survival in the presence of echinocandin.

“For example, conditions that increase cell wall chitin promote cell survival in the presence of echinocandin76,77, whereas calcineurin inhibitors reduce echinocandin tolerance (measured using MIC assays analyzed after 72 h)68,78.”

• cites [76] Walker, L. A., Gow, N. A. & Munro, C. A. Elevated chitin content reduces the susceptibility of Candida species to caspofungin. Antimicrob. Agents Chemother. 57, 146–154 (2013). This study finds that the ability of Candida species to grow in the presence of sub-MIC levels of caspofungin is associated with increased chitin levels in the cell wall. Pretreatment of cells with CaCl 2 and calcofluor white induces a similar increase in chitin and ability to grow in sub-MIC caspofungin levels. Whether this effect is an alteration in tolerance or resistance levels remains to be determined; higher cell sourceNot checked — no full text of the source
• cites [77] Lee, K. K. et al. Elevated cell wall chitin in Candida albicans confers echinocandin resistance in vivo. Antimicrob. Agents Chemother. 56, 208–217 (2012). sourceSupported

Many infections caused by echinocandin-susceptible C. glabrata or C. albicans isolates do not clear easily.

“In addition, a substantial number of infections with echinocandin-susceptible C. glabrata or C. albicans isolates are not readily cleared68, and the degree to which echinocandin tolerance may play a role in this remains to be determined.”

• cites [68] Cowen, L. E., Sanglard, D., Howard, S. J., Rogers, P. D. & Perlin, D. S. Mechanisms of antifungal drug resistance. CSH Perspect. Med. 5, a019752 (2015). sourceCould not verify

  The Adaptive Cellular Factors section discusses echinocandin tolerance but says these stress responses are not typically associated with clinical failures. The Drug Resistance Mechanisms section describes growth of susceptible fungi at high drug concentrations, not whether infections with susceptible C. glabrata or C. albicans isolates clear. A search for clinical outcomes and clearance found no passage establishing the claimed substantial number of difficult-to-clear infections. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

More recent work found no correlation between MSH2 mutations and antifungal resistance in vitro or in vivo.

“however, more recent work did not find a correlation between MSH2 mutations and antifungal resistance in vitro or in vivo69,70.”

• cites [69] Bordallo-Cardona, M. A. et al. MSH2 gene point mutations are not antifungal resistance markers in Candida glabrata. Antimicrob. Agents Chemother. 63, e01876–18 (2019). sourceSupported
• cites [70] Singh, A. et al. Absence of azole or echinocandin resistance in Candida glabrata isolates in India despite background prevalence of strains with defects in the DNA mismatch repair pathway. Antimicrob. Agents Chemother. 62, e00195–18 (2018). sourceNot checked — no full text of the source

Segmental trisomy of chromosome 3 conferred increased fitness in the presence of fluconazole, attributable at least partially to extra copies of NPR2.

“For example, segmental trisomy of chromosome 3 conferred an increased fitness in the presence of fluconazole that was attributable, at least partially, to extra copies of NPR2, which encodes a urea transporter51.”

• cites [51] Selmecki, A. M., Dulmage, K., Cowen, L. E., Anderson, J. B. & Berman, J. Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance. PLoS Genet. 5, e1000705 (2009). sourcePartly supported

  The Results section (Figure 6; page unavailable) reports that clones carrying a supernumerary chromosome with a Chr3R segment were slightly more fit in fluconazole than clones without it. The Discussion (‘Other aneuploidies in FluR strains’; page unavailable) also identifies segmental Chr3 trisomy, but discusses CDR1 and MRR1, not NPR2. Our copy does not support attributing the fitness benefit to extra NPR2 copies or identifying NPR2 as a urea transporter. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Sphingolipid biosynthesis is an important mechanism by which cells survive azole-induced ergosterol depletion.

“this approach identified sphingolipid biosynthesis as an important mechanism by which cells survive azole-induced ergosterol depletion73.”

• cites [73] Hickman, M. A. et al. The ‘obligate diploid’ Candida albicans forms mating-competent haploids. Nature 494, 55–59 (2013). sourceCould not verify

  The ‘Detection of haploid C. albicans cells’ section mentions a haploid isolate found among colonies growing near fluconazole, but does not discuss how cells survive azole treatment. Searches for sphingolipid biosynthesis and ergosterol depletion found no passage addressing the claimed mechanism; the ‘Haploid strains as genetic tools’ section concerns gene-knockout phenotyping instead. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Aneuploidy can alter fluconazole tolerance in Candida albicans.

“Aneuploidy can alter fluconazole tolerance in C. albicans drug38,51.”

• cites [38] Flowers, S. A., Colon, B., Whaley, S. G., Schuler, M. A. & Rogers, P. D. Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans. Antimicrob. Agents Chemother. 59, 450–460 (2015). sourceNot checked — no full text of the source
• cites [51] Selmecki, A. M., Dulmage, K., Cowen, L. E., Anderson, J. B. & Berman, J. Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance. PLoS Genet. 5, e1000705 (2009). sourceSupported

In clinical C. albicans isolates from one HIV patient on fluconazole, LOH events kept in all later isolates appeared alongside acquired bona fide resistance.

“For example, in a series of clinical C. albicans isolates from the same patient with HIV treated with fluconazole54,55, LOH events that were retained in all subsequent isolates arose concomitant with the acquisition of bona fide resistance.”

• cites [54] Bennett, R. J., Forche, A. & Berman, J. Rapid mechanisms for generating genome diversity: whole ploidy shifts, aneuploidy, and loss of heterozygosity. Cold Spring Harb. Perspect. Med. 4, a019604 (2014). sourceNot checked — no full text of the source
• cites [55] Ford, C. B. et al. The evolution of drug resistance in clinical isolates of Candida albicans. eLife 4, e00662 (2015). sourceSupported

Higher copy numbers of chromosome 3's right arm plus unknown genes on trisomic chromosome 7 correlated with increased efflux.

“In agreement with this, increased copies of the right arm of chromosome 3, together with unidentified genes on trisomic chromosome 7 were associated with increased efflux51.”

• cites [51] Selmecki, A. M., Dulmage, K., Cowen, L. E., Anderson, J. B. & Berman, J. Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance. PLoS Genet. 5, e1000705 (2009). sourcePartly supported

  The Discussion section, “Other aneuploidies in FluR strains” (page unavailable), reports extra copies of chromosome 3’s right-arm segment containing CDR1 and MRR1, genes linked to drug efflux. The Results section, “Multiple aneuploidies are evident in drug-evolved populations” (page unavailable), reports chromosome 7 trisomy but suggests it may enhance fitness, not efflux. The claim’s attribution of increased efflux to unidentified genes on trisomic chromosome 7 is therefore unsupported. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Aneuploidy and/or loss of heteroresistance are rapidly acquired genome changes that can result in increased tolerance.

“Aneuploidy and/or loss of heteroresistance are rapidly acquired genome changes that can result in increased tolerance. and are likely to affect one or several genes that alter tolerance level.”

• cites [38] Flowers, S. A., Colon, B., Whaley, S. G., Schuler, M. A. & Rogers, P. D. Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans. Antimicrob. Agents Chemother. 59, 450–460 (2015). sourceNot checked — no full text of the source
• cites [51] Selmecki, A. M., Dulmage, K., Cowen, L. E., Anderson, J. B. & Berman, J. Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance. PLoS Genet. 5, e1000705 (2009). sourcePartly supported

  The Introduction and Results report that aneuploidies appeared during early fluconazole exposure and were associated with improved growth and drug resistance. The paper does not establish that loss of heteroresistance produces increased tolerance; its Discussion instead says MTL heterozygosity was maintained throughout the three evolution experiments. Its demonstrated outcome is fluconazole resistance and fitness, rather than tolerance generally. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Fluconazole exposure drives whole-chromosome LOH through atypical mitotic divisions with chromosome gains and losses.

“For example, exposure to fluconazole promotes whole-chromosome LOH via unusual mitotic divisions (chromosome duplications and losses)58–60.”

• cites [58] Forche, A. et al. The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains. PLoS Biol. 6, e110 (2008). sourcePartly supported

  The Results section on concerted chromosome loss reports whole-chromosome LOH and chromosome re-duplication in parasexual progeny grown on sorbose medium. It does not show that fluconazole exposure promotes those events or establish the claimed mechanism under fluconazole exposure; a fluconazole-related study appears only in the References. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [59] Harrison, B. D. et al. A tetraploid intermediate precedes aneuploid formation in yeasts exposed to fluconazole. PLoS Biol. 12, e1001815 (2014). sourcePartly supported

  The Discussion (page not provided) reports that fluconazole exposure increases loss of heterozygosity (LOH), which can arise through chromosome missegregation or recombination. The Results show unusual mitotic divisions and unequal chromosome segregation, but the paper does not establish that those divisions cause whole-chromosome LOH specifically. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [60] Forche, A. et al. Stress alters rates and types of loss of heterozygosity in Candida albicans. mBio 2, e00129–11 (2011). sourcePartly supported

  The Results report that fluconazole increased the proportion of whole-chromosome LOH about fivefold. The Introduction explains how chromosome nondisjunction and subsequent reduplication can produce whole-chromosome LOH, but the Discussion says the mechanism under fluconazole is unclear. The claim presents that proposed mechanism as established. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Echinocandin resistance results almost entirely from FKS gene mutations, which alter the drug target 1,3-β-glucan synthase.

“Echinocandin resistance is almost exclusively due to mutations in FKS genes (Table 1), which encode the target enzyme of the drug, 1,3-β-glucan synthase65.”

• cites [65] Perlin, D. S. Echinocandin resistance in Candida. Clin. Infect. Dis. 61, S612–S617 (2015). sourceSupported

Calcineurin inhibitors reduce echinocandin tolerance.

“whereas calcineurin inhibitors reduce echinocandin tolerance (measured using MIC assays analyzed after 72 h)68,78.”

• cites [68] Cowen, L. E., Sanglard, D., Howard, S. J., Rogers, P. D. & Perlin, D. S. Mechanisms of antifungal drug resistance. CSH Perspect. Med. 5, a019752 (2015). sourcePartly supported

  The Adaptive Cellular Factors section says calcineurin signaling helps fungi survive echinocandin-induced stress, and Drug Resistance Mechanisms reports that calcineurin inhibitors eliminate paradoxical growth at high echinocandin concentrations. This supports reduced tolerance, but our copy does not establish the specified measurement by MIC assays analyzed after 72 hours. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [78] Singh-Babak, S. D. et al. Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog. 8, e1002718 (2012). sourcePartly supported

  The Results section reports that the calcineurin inhibitors cyclosporin A and FK506 reduced caspofungin resistance in a C. glabrata clinical isolate. The Methods describe MIC assays using these inhibitors, but our copy does not establish a 72-hour readout for those assays; it specifies a 24-hour endpoint for clinical caspofungin MICs. No page numbers are available in our copy. (LLM, conf=0.86)

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Mutations at either of two FKS hot-spot regions can raise an echinocandin's in vitro IC50 by as much as 1,000-fold.

“Point mutations in one of two specific hot-spot regions of FKS genes increase the half maximal inhibition concentration (IC50) of an echinocandin in vitro by up to 3 orders of magnitude67.”

• cites [67] Garcia-Effron, G., Lee, S., Park, S., Cleary, J. D. & Perlin, D. S. Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-β-D-glucan synthase: implication for the existing susceptibility breakpoint. Antimicrob. Agents Chemother. 53, 3690–3699 (2009). sourceSupported
  Experimental — accuracy not yet measured

Multi-drug resistant Candida glabrata isolates are increasing at an alarming rate.

“The emergence of multi-drug resistant (MDR) C. glabrata isolates is increasing at an alarming rate.”

• cites [71] Shor, E., Schuyler, J. & Perlin, D. S. A novel, drug resistance-independent, fluorescence-based approach to measure mutation rates in microbial pathogens. mBio 10, e00120–19 (2019). sourcePartly supported

  The Introduction says that C. glabrata infections have become more prevalent and that studies have documented the emergence of multidrug-resistant infections. It does not say that the number of multidrug-resistant isolates is increasing, or establish an alarming rate of increase; those claims go beyond the passage. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Inhibitors of Hsp90, calcineurin, TOR and mutants affecting Rim101 effectively eliminate tolerance and greatly increase the fungicidal activity of fluconazole across diverse isolates with different tolerance levels.

“Inhibitors of heat shock protein 90 (Hsp90)82, calcineurin21,25,26, target of rapamycin (TOR)83 and mutants that affect Rim10184 (a transcription factor involved in the pH response pathway) effectively eliminate tolerance and greatly increase the fungicidal activity [G] of fluconazole across a diverse set of isolates with different tolerance levels (Fig. 1E; Box 5).”

• cites [82] Cowen, L. E. & Lindquist, S. Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309, 2185–2189 (2005). sourceNot checked — no full text of the source
• cites [21] Marchetti, O., Moreillon, P., Glauser, M. P., Bille, J. & Sanglard, D. Potent synergism of the combination of fluconazole and cyclosporine in Candida albicans. Antimicrob. Agents Chemother. 44, 2373–2381 (2000). sourcePartly supported

  The Discussion (page unavailable in our copy) reports that cyclosporine or FK506 combined with fluconazole eliminated trailing growth, and identifies calcineurin as a possible cyclosporine target. It does not establish that calcineurin inhibition caused the effect. The fluconazole–cyclosporine combination was fungicidal in two of three fluconazole-susceptible isolates but only fungistatic in the third; the paper does not establish the claimed effect across isolates with different tolerance levels. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [25] Lockhart, S. R., Ghannoum, M. A. & Alexander, B. D. Establishment and use of epidemiological cutoff values for molds and yeasts by use of the Clinical and Laboratory Standards Institute M57 standard. J. Clin. Microbiol. 55, 1262–1268 (2017). This review provides an important and accessible over-review of the concept of epidemiological cut-off values as well as the methodology that underlies their establishment. sourceNot checked — no full text of the source
• cites [26] Onyewu, C., Wormley, F. L. Jr., Perfect, J. R. & Heitman, J. The calcineurin target, Crz1, functions in azole tolerance but is not required for virulence of Candida albicans. Infect. Immun. 72, 7330–7333 (2004). sourcePartly supported

  The passage describing fluconazole Etest assays (indexed as Abstract; page unavailable) reports that FK506, a calcineurin inhibitor, abolished the trailing effect in the strains tested. The paper also reports enhanced inhibition with FK506 and fluconazole, but it does not establish increased fungicidal activity across diverse isolates with different tolerance levels. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [83] Khandelwal, N. K. et al. Azole resistance in a Candida albicans mutant lacking the ABC transporter CDR6/ROA1 depends on TOR signaling. J. Biol. Chem. 293, 412–432 (2018). sourcePartly supported

  The Introduction (p. 2) reports that Tor1 inhibition leads to azole hypersensitivity, and the discussion (p. 15) links hyperactive Tor1 to azole resistance in the NKKY101 mutant. The paper does not show that TOR inhibitors eliminate fluconazole tolerance or greatly increase its fungicidal activity across diverse isolates; its experiments focus on the mutant and related strains. The other targets in the citing sentence have separate citations and are not assessed here. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Mutations in Tac1 increase efflux activity.

“For example, mutations in Tac1, a positive transcriptional regulator of efflux pump genes, increases efflux activity49.”

• cites [49] Todd, R. T., Wikoff, T. D., Forche, A. & Selmecki, A. Genome plasticity in Candida albicans is driven by long repeat sequences. eLife. 8, e45954 (2019). sourcePartly supported

  The Introduction identifies Tac1 as a transcriptional activator of drug efflux pumps. It attributes fluconazole resistance to amplification of TAC1 and ERG11, but does not say that mutations in Tac1 increase efflux activity. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Organelles such as the vacuole make important contributions to tolerance and/or trailing growth.

“Organelles such as the vacuole make important contributions to tolerance and/or trailing growth.”

• cites [27] Sanglard, D., Ischer, F., Marchetti, O., Entenza, J. & Bille, J. Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence. Mol. Microbiol. 48, 959–976 (2003). sourceNot checked — no full text of the source
• cites [51] Selmecki, A. M., Dulmage, K., Cowen, L. E., Anderson, J. B. & Berman, J. Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance. PLoS Genet. 5, e1000705 (2009). sourceCould not verify

  The Results section on i(5L) and fitness discusses the advantage conferred by aneuploidy during fluconazole exposure, not a contribution from organelles. Searches for “vacuole” and “trailing” found no passage in our copy addressing the claim; page numbers were unavailable. (LLM, conf=0.75)

  Experimental — accuracy not yet measured
• cites [94] Roemer, T. & Krysan, D. J. Antifungal drug development: challenges, unmet clinical needs, and new approaches. Cold Spring Harb. Perspect. Med. 4, a019703 (2014). sourceNot checked — no full text of the source
• cites [95] Cornet, M. et al. Deletions of endocytic components VPS28 and VPS32 affect growth at alkaline pH and virulence through both RIM101-dependent and RIM101-independent pathways in Candida albicans. Infect. Immun. 73, 7977–7987 (2005). sourceNot checked — no full text of the source

Inter-isolate heterogeneity of tolerance is likely due to genetic differences that contribute to several other clinically relevant phenotypes, including biofilm formation, filamentous growth responses and temperature sensitivity.

“The inter-isolate heterogeneity is likely due to genetic differences that contribute to several other clinically relevant phenotypes, including biofilm formation, filamentous growth responses and sensitivity to temperature52,60,79,80.”

• cites [52] Mount, H. O. et al. Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs. PLoS Genet. 14, e1007319 (2018). This article screens a library of C. albicans deletion strains and identifies two genes that affect azole responses, have roles in stress response pathways and functions that are are suppressed by the acquisition of aneuploid chromosomes. Whether the mutations are affecting resistance or tolerance is not clear, but activities of the identified genes (vacuolar retrograde trafficking and a cell wall and polarity GTPase activating pro sourceCould not verify

  The Results section on basal fluconazole tolerance identifies genes affecting tolerance, and the clinical-isolate Results section reports differing effects of gene deletions on azole resistance. Neither connects inter-isolate tolerance heterogeneity to genetic differences affecting biofilm formation, filamentous growth, or temperature sensitivity. Searches for those phenotypes found no passage making that connection; page numbers were unavailable in our copy. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [60] Forche, A. et al. Stress alters rates and types of loss of heterozygosity in Candida albicans. mBio 2, e00129–11 (2011). sourcePartly supported

  The Introduction (page unavailable) says genetic variation occurs among C. albicans clinical isolates and that different alleles can confer different benefits under stress. That supports a general genetic basis for differences in stress response, but the paper does not connect inter-isolate tolerance to biofilm formation, filamentous growth, or temperature sensitivity. Its temperature experiments measure loss of heterozygosity under heat stress, not differences in temperature sensitivity among isolates. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [79] Selmecki, A., Forche, A. & Berman, J. Genomic plasticity of the human fungal pathogen Candida albicans. Eukaryot. Cell 9, 991–1008 (2010). sourceNot checked — no full text of the source
• cites [80] Sitterle, E. et al. Within-host genomic diversity of Candida albicans in healthy carriers. Sci. Rep. 9, 2563 (2019). sourcePartly supported

  The Discussion (page unavailable) reports genetic heterogeneity among isolates from healthy carriers and says genomic variation can facilitate adaptation to environmental changes and affect virulence factors. It does not establish that genetic differences explain inter-isolate tolerance heterogeneity or specifically link those differences to biofilm formation, filamentous growth responses, and temperature sensitivity. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Isolates that are more tolerant have lower average intracellular drug than less tolerant isolates.

“For example, isolates that are more tolerant have lower average intracellular drug than less tolerant isolates9 (Fig 1D).”

• cites [9] Rosenberg, A. et al. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 9, 2470 (2018). This article provides a quantitative characterization of fluconazole tolerance in C. albicans, distinguishing it from resistance and highlighting its phenotypic heterogeneity and association with subpopulation growth, and sensitivity to adjuvant drugs. sourceSupported

Many core stress response pathways in C. albicans, including Hsp90, Ca2+-calmodulin-calcineurin, lysine deacetylases, protein kinase C and HOG pathways, also affect echinocandin responses.

“Importantly, many of these same core stress response pathways in C. albicans (Hsp90, Ca2+-calmodulin-calcineurin, lysine deacetylases, protein kinase C and HOG (high osmolarity glycerol responses) pathways) t also affect echinocandin responses68,86,87.”

• cites [68] Cowen, L. E., Sanglard, D., Howard, S. J., Rogers, P. D. & Perlin, D. S. Mechanisms of antifungal drug resistance. CSH Perspect. Med. 5, a019752 (2015). sourcePartly supported

  The Hsp90, Chitin Synthesis, and Cell Integrity Circuit sections link Hsp90, calcineurin, PKC, and HOG signaling to echinocandin responses in C. albicans. The Hsp90 and Related Factors section discusses lysine deacetylases in relation to C. albicans azole resistance, but does not establish their effect on its echinocandin responses. Page numbers are not available in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [86] Shapiro, R. S., Zaas, A. K., Betancourt-Quiroz, M., Perfect, J. R. & Cowen, L. E. The Hsp90 co-chaperone Sgt1 governs Candida albicans morphogenesis and drug resistance. PLoS One 7, e44734 (2012). sourcePartly supported

  The Introduction (page unavailable) says that Hsp90 affects C. albicans echinocandin resistance and links Hsp90-mediated drug resistance to calcineurin and the PKC pathway. It does not establish the claimed roles of lysine deacetylases or the HOG pathway in echinocandin responses; searches for those terms found no supporting passage in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [87] Cowen, L. E. et al. Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc. Natl Acad. Sci. USA 106, 2818–2823 (2009). sourcePartly supported

  The Discussion links Hsp90 and calcineurin to echinocandin responses in Aspergillus, but links those pathways to azole responses in C. albicans. It does not establish that the listed pathways affect echinocandin responses in C. albicans, and it does not address lysine deacetylases, protein kinase C, or HOG in that context. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The involvement of lysine deacetylases and calcineurin implies a connection between tolerance and the metabolic anti-stress machinery.

“Indeed, the involvement of lysine deacetylases and calcineurin implies a connection between tolerance and the metabolic anti-stress machinery (for example Ref. 88).”

• cites [88] Olin-Sandoval, V. et al. Lysine harvesting is an antioxidant strategy and triggers underground polyamine metabolism. Nature 572, 249–253 (2019). sourceSupported

pH, temperature and nutrient availability affect the level of fluconazole tolerance.

“Similarly, pH, temperature and nutrient availability affect the level of fluconazole tolerance9.”

• cites [9] Rosenberg, A. et al. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 9, 2470 (2018). This article provides a quantitative characterization of fluconazole tolerance in C. albicans, distinguishing it from resistance and highlighting its phenotypic heterogeneity and association with subpopulation growth, and sensitivity to adjuvant drugs. sourceSupported

An isolate that is resistant because of increased efflux has a proportion of cells with very low levels of intracellular drug, about tenfold less than the moderate drug levels seen in tolerant isolates.

“By contrast, an isolate that is resistant because of increased efflux has a proportion of cells with very low levels of intracellular drug (10X less than those with moderate drug levels compared to tolerant isolates)9 (Fig. 1D).”

• cites [9] Rosenberg, A. et al. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 9, 2470 (2018). This article provides a quantitative characterization of fluconazole tolerance in C. albicans, distinguishing it from resistance and highlighting its phenotypic heterogeneity and association with subpopulation growth, and sensitivity to adjuvant drugs. sourcePartly supported

  The Fig. 3 caption compares intracellular drug levels in resistant strain T101 with those in isolates of differing tolerance and defines low and mid drug-level categories whose lower bounds differ by about tenfold. The Discussion notes the role of efflux in drug resistance, but our copy does not establish that T101 is resistant because of increased efflux or explicitly quantify a tenfold difference between its cells and tolerant isolates. No page number is available in our copy. (LLM, conf=0.79)

  Experimental — accuracy not yet measured

Studies using inhibitors or mutant strains showed that these pathways reduce fungal growth in combination with caspofungin or anidulafungin.

“Specifically, studies of these pathways using either inhibitors or mutant strains showed they reduce fungal growth in combination with caspofungin or anidulafungin.”

• cites [68] Cowen, L. E., Sanglard, D., Howard, S. J., Rogers, P. D. & Perlin, D. S. Mechanisms of antifungal drug resistance. CSH Perspect. Med. 5, a019752 (2015). sourcePartly supported

  The Cell Integrity Circuit section reports that deleting pathway genes increases susceptibility to echinocandins and that kinase inhibitors act synergistically with echinocandins against fungi. The Hsp90 section likewise reports reduced echinocandin tolerance after genetic or pharmacological compromise. These passages support the general finding, but do not establish the claimed growth reduction specifically in combination with caspofungin or anidulafungin; they describe inhibiting the pathways, not the pathways themselves reducing growth. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [86] Shapiro, R. S., Zaas, A. K., Betancourt-Quiroz, M., Perfect, J. R. & Cowen, L. E. The Hsp90 co-chaperone Sgt1 governs Candida albicans morphogenesis and drug resistance. PLoS One 7, e44734 (2012). sourcePartly supported

  The Results section on Sgt1 depletion reports that genetically reducing Sgt1 increased Candida albicans susceptibility to the echinocandin micafungin and created a fungicidal combination. It does not report the claimed combinations with caspofungin or anidulafungin, or show an inhibitor-based combination in those experiments. No page number is available in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [87] Cowen, L. E. et al. Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc. Natl Acad. Sci. USA 106, 2818–2823 (2009). sourcePartly supported

  The Results section reports that inhibiting Hsp90 enhanced caspofungin’s efficacy against A. fumigatus. Our copy does not report a corresponding result with anidulafungin or show that mutant strains reduced fungal growth in combination with either drug; it describes genetic depletion of Hsp90 in combination with fluconazole instead. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Colonies from higher tolerance isolates appear faster than those from low tolerance isolates.

“Furthermore, colonies from higher tolerance isolates appear faster than those from low tolerance isolates (Fig. 1C).”

The authors propose that tolerance is a pathogenic-fungal property separable from resistance by one in vitro assay measuring susceptibility at 24 hours and growth at 48 hours.

“We posit that antifungal tolerance is a property of pathogenic fungi that can be distinguished from resistance using the same in vitro assay that can quantify both susceptibility [G] (measured as MIC at 24 h) and growth properties at 48 h (the fraction of growth [G] or supra-MIC growth [G]) (Box 1).”

Non-mycologic factors-host factors, immune status, and pharmacologic issues-have been proposed to explain treatment failure against susceptible Candida isolates.

“This has been attributed to non-mycologic causes such as host factors, immune status, and/or pharmacologic issues8.”

• cites [8] Lepak, A. J. & Andes, D. R. Antifungal pharmacokinetics and pharmacodynamics. Cold Spring Harb. Perspect. Med. 5, a019653 (2014). sourceNot checked — no full text of the source

Antifungal tolerance may explain why overall treatment outcomes do not match the low rates of clinical resistance.

“Specifically, the discordance between overall treatment outcome and low levels of clinical resistance may be attributable to antifungal drug tolerance.”

When resistance removes a single antifungal agent, patient care is substantially affected.

“Thus, the loss of one antifungal drug through resistance has a major impact on patient care.”

Fungal infections that resist drugs have become a notable clinical issue.

“Drug-resistant fungal infections are emerging as an important clinical problem1,2.”

• cites [1] Arendrup, M. C. & Patterson, T. F. Multidrug-resistant Candida: epidemiology, molecular mechanisms, and treatment. J. Infect. Dis. 216, S445–S451 (2017). sourceNot checked — no full text of the source
• cites [2] Perlin, D. S., Rautemaa-Richardson, R. & Alastruey-Izquierdo, A. The global problem of antifungal resistance: prevalence, mechanisms, and management. Lancet Infect. Dis. 17, e383–e392 (2017). sourceNot checked — no full text of the source

Three main antifungal classes-polyenes, azoles, and echinocandins-are the only agents currently used against invasive fungal infections.

“Currently, only three primary classes of agents are used to treat invasive fungal infections5: polyenes (for example, amphotericin B); azoles (for example, fluconazole) and echinocandins (for example, caspofungin) (Table 1).”

• cites [5] Krysan, D. J. The unmet clinical need of novel antifungal drugs. Virulence 8, 135–137 (2017). sourceNot checked — no full text of the source

Drug-fungus interactions other than the MIC may influence how a patient responds to therapy.

“However, specific interactions between the drug and the fungus, aside from the MIC, may affect therapeutic responses as well.”

The rise in at-risk patients for invasive fungal infections is mainly due to complex surgical procedures, immune suppression, or reduced immune function.

“This is mainly due to an increase in patients at-risk for invasive fungal infections from complex surgical procedures, immune-suppression or reduced immune function3.”

• cites [3] Alangaden, G. J. Nosocomial fungal infections: epidemiology, infection control, and prevention. Infect. Dis. Clin. North Am. 25, 201–225 (2011). sourceNot checked — no full text of the source

For MRSA, six separate antibiotic classes exist as treatment options.

“By comparison, six distinct classes of antibiotics are currently available to treat methicillin-resistant Staphylococcus aureus (MRSA), an important and widespread drug-resistant bacterium6.”

• cites [6] Bassetti, M., Carnelutti, A., Castaldo, N. & Peghin, M. Important new therapies for methicillin-resistant Staphylococcus aureus. Expert Opin. Pharmacother. 20, 2317–2334 (2019). sourceNot checked — no full text of the source

Tolerance reflects intrinsic phenotypic heterogeneity within a fungal isolate.

“Thus, drug tolerance is a manifestation of phenotypic heterogeneity intrinsic to a given fungal isolate.”

Multiple genetic factors that vary among isolates are linked to tolerance.

“Studies are beginning to provide evidence that tolerance is associated with multiple genetic components that differ between isolates.”

The core molecular pathways of antifungal resistance are shared across species.

“Although resistance is emerging in non-Candida species such as Aspergillus spp.,29 the general molecular mechanisms of resistance are conserved; therefore, we will focus primarily on Candida spp., the most prevalent species and the most common cause of both life-threatening invasive disease and clinically important mucosal diseases, such as vulvovaginal candidiasis, thrush and esophagitis.”

Antifungal activity is often measured with non-standard assays that yield no MIC value.

“In addition, non-standard assays are frequently used to measure antifungal activity but do not generate an MIC value.”

An isolate's genetic composition may influence how much its isogenic cells vary in their responses.

“The genetic make up of an individual isolate may also affect the degree of variability between the responses of different isogenic cells in the population.”

Because antifungal tolerance is most apparent with fungistatic drugs, cell-death measurements are less useful.

“By contrast, antifungal tolerance is most evident with fungistatic drugs [G], and thus measuring cell death is less relevant.”

Resistance is appearing in non-Candida fungi, including Aspergillus species.

“Although resistance is emerging in non-Candida species such as Aspergillus spp.,29 the general molecular mechanisms of resistance are conserved; therefore, we will focus primarily on Candida spp., the most prevalent species and the most common cause of both life-threatening invasive disease and clinically important mucosal diseases, such as vulvovaginal candidiasis, thrush and esophagitis.”

• cites [29] Luna-Tapia, A., Butts, A. & Palmer, G. E. Loss of C-5 sterol desaturase activity in Candida albicans: azole resistance or merely trailing growth? Antimicrob. Agents Chemother. 63, e00129–11 (2019). sourceNot checked — no full text of the source

In C. albicans, the specific aneuploidy isochromosome (5L) creates two extra copies of chromosome 5's left arm, giving extra TAC1 and ERG11 copies that confer azole resistance.

“For example, isochromosome (5L), a specific aneuploidy in C. albicans, results in the formation of two extra copies of the left arm of chromosome 543, and, consequently, extra copies of TAC1 and ERG11, genes that confer resistance to azoles in these strains50.”

• cites [43] Kordalewska, M. et al. Understanding echinocandin resistance in the emerging pathogen Candida auris. Antimicrob Agents Chemother. 62, e00238–18 (2018). sourceNot checked — no full text of the source
• cites [50] Coste, A. et al. A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans. Genetics 172, 2139–2156 (2006). sourceNot checked — no full text of the source

In C. albicans, gain-of-function Upc2 mutations, which affect a key regulator of ergosterol biosynthesis genes, raise ERG11 expression and lower azole susceptibility.

“In C. albicans, gain-of-function mutations in Upc2, a key transcriptional regulator of ergosterol biosynthetic genes34, lead to increased ERG11 expression and reduced azole susceptibility.”

• cites [34] Holmes, A. R. et al. Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2. Mol. Microbiol. 62, 170–186 (2006). sourceNot checked — no full text of the source

Both bacterial and fungal isolates that are tolerant have MIC values within the susceptible range.

“Tolerant bacterial and fungal isolates have MICs in the susceptible range, but tolerant bacteria are characterized by longer minimum duration to killing (MDK) than susceptible isolates10, based on responses to bactericidal drugs10–12.”

• cites [10] Brauner, A., Fridman, O., Gefen, O. & Balaban, N. Q. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat. Rev. Microbiol. 14, 320–330 (2016). sourceNot checked — no full text of the source

Candida glabrata and Candida tropicalis show the highest resistance rates for both azoles and echinocandins.

“The highest rates of both azole and echinocandin resistance are observed in Candida glabrata and Candida tropicalis30 (Table 1).”

• cites [30] van der Linden, J. W. et al. Aspergillosis due to voriconazole highly resistant Aspergillus fumigatus and recovery of genetically related resistant isolates from domiciles. Clin. Infect. Dis. 57, 513–520 (2013). sourceNot checked — no full text of the source

Serial dilution agar plating assays often label isolates or mutants as resistant when they outgrow the reference strain.

“For example, serial dilution agar plating assays frequently compare the susceptibility of one isolate or a mutant to its appropriate reference strain; isolates and mutants that grow better than the reference strain in these assays are often referred to as resistant.”

Additional copies of chromosome 3's right arm may raise drug efflux because CDR1 and MRR1 reside there.

“Similarly, extra copies of the right arm of chromosome 3 may increase drug efflux because CDR1 and MRR1, which encode a major ABC transporter and a transcriptional activator of MDR1 (the MFS transporter), respectively, are found on this chromosome (Candida Genome Database [http://www.candidagenome.org/]).”

• cites Candida Genome DatabaseNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Resistance can also result from drug-target changes, including higher target expression or mutations in the target protein sequence.

“Alterations in antifungal drug targets, due to increased target expression or mutations within the target protein sequence, also mediate antifungal drug resistance (Table 1).”

Many such transcription factors also control other genes involved in resistance, virulence, or general yeast fitness.

“Of note, many of these transcription factors also regulate other genes that also contribute to resistance, virulence or general yeast cell fitness32.”

• cites [32] Prasad, R., Rawal, M. K. & Shah, A. H. Candida efflux ATPases and antiporters in clinical drug resistance. Adv. Exp. Med. Biol. 892, 351–376 (2016). sourceNot checked — no full text of the source

Azole resistance most commonly arises from increased efflux pump activity, mainly through gain-of-function mutations in transcription factors controlling ABC transporters (TAC1 in C. albicans, PDR1 in C. glabrata) and MFS pumps (MRR1 in C. albicans).

“The most prevalent mechanism of azole resistance involves increased drug efflux pump activity, primarily due to gain-of-function mutations in transcription factors that regulate ATP-binding cassette (ABC) transporters (TAC1 in C. albicans and PDR1 in C. glabrata) and major facilitator super family (MFS) pumps (MRR1 in C. albicans)31,32.”

• cites [31] McCarty, T. P. & Pappas, P. G. Invasive candidiasis. Infect. Dis. Clin. North Am. 30, 103–124 (2016). sourceNot checked — no full text of the source
• cites [32] Prasad, R., Rawal, M. K. & Shah, A. H. Candida efflux ATPases and antiporters in clinical drug resistance. Adv. Exp. Med. Biol. 892, 351–376 (2016). sourceNot checked — no full text of the source

When susceptible fungal subpopulations grow above the MIC, the cause may be tolerance, heteroresistance, or persistence, depending on drug, organism, and subpopulation size.

“Growth of subpopulations of susceptible fungal isolates in the presence of drugs at concentrations above the MIC can be due to tolerance, heteroresistance [G] or persistence (see Box 2), depending on the drug, organism and the size of the subpopulation.”

These genetic factors influence how isogenic cells in one isolate respond physiologically or metabolically to environmental or nutritional cues.

“These genetic components, that differ between isolates, affect the ability of isogenic cells within a single isolate to respond physiologically or metabolically (to environmental or nutritional signals).”

A single aneuploid chromosome can confer more than one unrelated phenotype.

“Notably, a single aneuploid chromosome can confer more than one unrelated phenotype.”

Trisomy of chromosome 2 also promotes cell survival in the presence of hydroxyurea.

“Interestingly, trisomy of chromosome 2 also promotes cell survival in the presence of hydroxyurea, a cancer chemotherapy drug.”

• cites [47] Yang, F., Kravets, A., Bethlendy, G., Welle, S. & Rustchenko, E. Chromosome 5 monosomy of Candida albicans controls susceptibility to various toxic agents, including major antifungals. Antimicrob. Agents Chemother. 57, 5026–5036 (2013). sourceNot checked — no full text of the source

Trisomy of chromosome 2 promotes the growth of C. albicans cells in the presence of caspofungin without altering the MIC.

“Trisomy of chromosome 2 promotes the growth of C. albicans cells in the presence of the antifungal drug caspofungin47 without altering the MIC.”

• cites [47] Yang, F., Kravets, A., Bethlendy, G., Welle, S. & Rustchenko, E. Chromosome 5 monosomy of Candida albicans controls susceptibility to various toxic agents, including major antifungals. Antimicrob. Agents Chemother. 57, 5026–5036 (2013). sourceNot checked — no full text of the source

Tolerance depends on a shared set of critical pathways across diverse isolates.

“This implies that tolerance depends on a shared set of critical pathways across diverse isolates.”

Tolerance is due to the slow growth of some cells in the population.

“This highlights the idea that tolerance is due to the slow growth of some cells in the population and raises questions about how such cells differ from those that do not grow (or grow much more slowly).”

Differences in tolerance levels measured as FoG or SMG between isolates have a genetic component.

“In this sense, the differences in tolerance levels measured as FoG or SMG (Box 1) between isolates have a genetic component.”

Tolerance seems to depend on intracellular processes that facilitate adaptation in response to drug stresses.

“Thus, tolerance seems to depend on intracellular processes that facilitate adaptation in response to drug stresses.”

In resistant isolates, mutations affecting efflux would enable most or all cells to reduce intracellular drug concentration to very low levels.

“In resistant isolates, mutations (for example, gain-of-function alleles in drug efflux pumps or their positive regulators) affecting efflux would enable most (or all) cells to reduce intracellular drug concentration to very low levels.”

Many types of mutations could lead to changes in tolerance because many central cellular pathways affect tolerance.

“The large number of central cellular pathways that affect tolerance also implies that many types of mutations could lead to changes in tolerance.”

Mutations in some pathways seem to affect resistance as well as tolerance.

“Mutations in some pathways seem to affect resistance as well as tolerance.”

Combination therapies with adjuvant drugs that interfere with the compensatory stress responses required for tolerance have the potential to increase the lethality of an inhibitory antifungal drug.

“Combination therapies with adjuvant drugs that interfere with the compensatory stress responses required for tolerance have the potential to increase the lethality of an inhibitory antifungal drug.”

Variations in the function of the critical pathways listed above are candidate drivers of differences in tolerance levels between isolates.

“Thus, variations on the function of the critical pathways listed above are candidate drivers of differences in tolerance levels between isolates.”

Tolerance in all strains is dependent on all of the major hubs of cell homeostasis, irrespective of subpopulation size and ability to grow in the presence of the drug.

“This implies that tolerance in all strains is dependent on all of these major hubs of cell homeostasis, irrespective of both the subpopulation size and its ability to in the presence of the drug.”

There is a mechanistic distinction between resistance and tolerance.

“This suggests a mechanistic distinction between resistance and tolerance.”

At least for azoles, cell-to-cell differences in drug responses within a single isolate seem to be connected to differences in intracellular drug concentration between cells.

“The mechanisms that drive cell-to-cell differences in drug responses within a single isolate remain to be determined; but, at least for azoles, they seem to be connected to differences in intracellular drug concentration between different cells.”

Pharmacological or genetic disruption of Hsp90, calcineurin, TOR, lysine deacetylases or Rim101 enhances the effect of fluconazole.

“Consistent with this, either pharmacological or genetic disruption of Hsp90, calcineurin, TOR, lysine deacetylases or Rim101 function enhance the effect of fluconazole (reviewed in Ref. 85).”

• cites [85] Robbins, N., Caplan, T. & Cowen, L. E. Molecular evolution of antifungal drug resistance. Annu. Rev. Microbiol. 71, 753–775 (2017). sourceNot checked — no full text of the source

Drug pressure leads to positive selection of mutants that exhibit improved fitness.

“In addition, drug pressure will lead to the positive selection of mutants that exhibit improved fitness.”

In tolerant isolates, physiological differences would enable some cells to reduce intracellular drug concentrations modestly relative to others, facilitating slow growth in cells with higher efflux gene expression.

“In tolerant isolates, physiological differences (for example, normal distribution in the amount of efflux gene expression) would enable some cells to reduce intracellular drug concentrations modestly relative to others, thereby facilitating slow growth in those cells with higher efflux gene expression.”