Instability and Subpopulation Dynamics of Azole Trailing Phenotypes in Clinical Candida tropicalis Isolates

Instability and Subpopulation Dynamics of Azole Trailing Phenotypes in Clinical Candida tropicalis Isolates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Instability and Subpopulation Dynamics of Azole Trailing Phenotypes in Clinical Candida tropicalis Isolates Snigdha Reddy, Saikat Paul, Shivaprakash M Rudramurthy, Harsimran Kaur, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8484141/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Apr, 2026 Read the published version in Mycopathologia → Version 1 posted 9 You are reading this latest preprint version Abstract Azole antifungal "trailing growth" in Candida spp., a tolerance phenotype distinct from resistance, complicates interpretation of in vitro susceptibility and may contribute to persistent candidemia, especially in resource-limited settings. This study investigated the stability, characteristics, and mechanisms of azole trailing, primarily in the emerging pathogen Candida tropicalis . From 847 clinical C. tropicalis isolates (2022–2024) screened by CLSI broth microdilution (BMD), 6.85% (n = 58) exhibited trailing, most frequently with fluconazole. Disk diffusion with elevated drug concentrations revealed microcolonies within inhibition zones, confirming tolerance to supra-MIC levels. Longitudinal monitoring revealed phenotypic instability: trailing was lost in 37.5% of C. tropicalis , 50% of C. albicans , and 66.7% of C. glabrata isolates after 8–12 months of storage at -80°C. This loss was also induced by repetitive freeze-thaw cycles in some isolates. Trailing was pH-dependent, consistently observed only at pH 6–8. Serial induction under fluconazole pressure successfully revived the phenotype in some isolates. Population Analysis Profiling (PAP) of nine trailing C. tropicalis isolates confirmed the presence of drug-tolerant subpopulations, akin to a heteroresistance reference strain. These findings demonstrate that azole trailing in C. tropicalis is a reversible, pH-dependent tolerance phenotype mediated by a minor subpopulation, which is unstable under routine laboratory storage. The discrepancy between in vitro susceptibility results and this tolerant phenotype underscores the need for adjunctive assays, like PAP, to better characterize antifungal response and improve clinical correlation. C. tropicalis Azole Resistance Tolerance Trailing Antifungal Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Invasive fungal infections represent a significant global public health threat, with Candida species ranking among the foremost fungal pathogens associated with high morbidity and mortality worldwide ( 1 ). While C. albicans has historically been the most prevalent agent of fungal sepsis, the epidemiological landscape has shifted considerably ( 2 , 3 ). Non- albicans Candida (NAC) species, including C. glabrata , C. tropicalis , C. parapsilosis , and C. krusei , now contribute substantially to the global burden of candidemia ( 4 ). Among these, Candida tropicalis has emerged as a leading cause of invasive candidiasis, with notably high prevalence in the Asia-Pacific and Latin American regions ( 5 ). This species is of particular concern in high-risk populations, such as patients with hematologic malignancies, and is associated with alarmingly high mortality rates ( 5 , 6 , 7 ). The management of invasive candidiasis relies on a limited resource of antifungal drug classes. Azoles, particularly fluconazole, remain a cornerstone of therapy due to their efficacy and favourable bioavailability ( 8 , 9 ). However, the rising incidence of azole resistance threatens their clinical utility. A key challenge is the frequent discordance between in vitro susceptibility, as determined by standardized antifungal susceptibility testing (AFST), and clinical outcomes, that are predicted by the “90/60” rule, where ~ 90% of susceptible isolates and ~ 60% of resistant isolates respond to therapy ( 9 ). This discrepancy raises critical concerns about the adequacy of current testing methodologies and interpretive criteria ( 10 ). It also implies the influence of host factors as well as the characteristics of the pathogen not reflected by the MIC on clinical outcomes ( 9 ). Standardized AFST guidelines from the CLSI and EUCAST recommend determining the minimum inhibitory concentration (MIC) at 24 hours based on a 50% growth inhibition threshold. A significant complication in applying this standard to azoles is the phenomenon of "trailing growth", a reduced but persistent growth observed at drug concentrations above the MIC ( 11 ). This is especially common in C. tropicalis and C. albicans , with one large survey reporting a mean residual growth of approximately 10% in these species, a rate substantially higher than in others ( 12 ). Trailing growth obscures MIC endpoint determination, often leading to the misclassification of isolates as resistant or susceptible depending on the reading time and method ( 11 , 12 ). Critically, trailing growth is distinct from stable, genetic resistance. It is considered a form of antifungal tolerance, where a subpopulation of cells survives drug exposure without an increase in the population's MIC ( 9 ). Genetic studies further support that azole resistance and trailing are distinct phenotypes in C. tropicalis , with the latter being a reversible, non-heritable tolerance mechanism ( 13 ). This phenomenon shares features with, but is mechanistically different from, heteroresistance, where a small subpopulation (< 1%) exhibits growth at very high drug concentrations (≥ 8×MIC). Both trailing and heteroresistance evade detection in routine AFST due to the use of low inocula that measure only the dominant population response ( 9 , 14 – 16 ). The population analysis profile (PAP) assay, which plates a dense culture onto a gradient of drug concentrations, is specifically designed to detect and quantify these minority subpopulations. This method provides a continuous measure of tolerance, offering a more nuanced view than the binary result of a standard MIC ( 17 ). PAP-based studies have been pivotal in revealing the clinical significance of heteroresistance; for example, in C. glabrata , heteroresistant strains were linked to persistent infection in murine models despite in vitro "susceptibility," explaining some clinical treatment failures ( 18 ). The application of PAP to C. albicans is more recent but equally insightful ( 19 ). The molecular basis of trailing is an area of active investigation. Trailing isolates share profiles with resistant strains, including the upregulation of secreted aspartyl proteases (Saps), which may aid in both virulence and antifungal evasion ( 20 ). Evidence suggesting that histone deacetylase inhibitors can reduce trailing further points to a role for epigenetic regulation in this phenotype ( 21 ). Nevertheless, the precise genetic and epigenetic determinants remain to be fully elucidated. Despite growing research, the trailing phenomenon remains underexplored in its biological basis and clinical impact. Whether it constitutes a true resistance mechanism, a transient adaptive response, or an in vitro artifact is still debated ( 9 ). Clarifying its epidemiological trends and biological drivers is essential for refining AFST protocols and therapeutic strategies. This is especially urgent given fluconazole's critical role in clinical practice, particularly as step-down therapy. The recurring reports of discordance between in vitro susceptibility and clinical response in trailing isolates underscore the need for more sophisticated interpretations of AFST. In this context, we conducted a comprehensive study to characterize the trailing phenomenon in C. tropicalis and other relevant species. Our investigation aims to determine the phenotypic stability of trailing, explore its potential mechanisms, and assess the constancy of this trait, with the ultimate goal of improving diagnostic accuracy and patient outcomes in invasive candidiasis. Methods Clinical Isolates and Identification Non-duplicate clinical isolates of Candida tropicalis from invasive candidiasis cases, collected from 2022 to 2024, were included in this study. The isolates were obtained from the Mycology Laboratory, Department of Medical Microbiology, PGIMER, Chandigarh, India. Species-level identification was performed using MALDI-TOF MS (bioMérieux, France) according to the manufacturer’s instructions, and all isolates were preserved in 20% glycerol at − 80°C. Primary Antifungal Susceptibility Screening (AFST) Antifungal susceptibility testing (AFST) against five azoles (fluconazole, voriconazole, itraconazole, posaconazole, and isavuconazole) was performed in biological triplicates using the CLSI M27-A4 broth microdilution method. MICs were read after 24 and 48 hours of incubation at 35°C. Trailing growth was defined as persistent, partial growth at supra-MIC concentrations (≥ 2 dilutions above the MIC) without a distinct endpoint, per CLSI guidelines and literature. Quality control employed Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258. Isolates exhibiting trailing were preserved for further analysis, and their prevalence was calculated relative to the total isolates received. Secondary Screening: Disk Diffusion Method In contrast to liquid culture assays where cell mixing obscures tolerant subpopulations, making them difficult to detect, these fractions are readily apparent on agar plates where individual clonal progeny can be distinguished ( 9 ). Disk diffusion testing for fluconazole and voriconazole was therefore conducted according to CLSI M44-A2 guidelines using sterile blank filter paper disks (HiMedia, India) impregnated with 25 µg fluconazole and 1 µg voriconazole, which were stored at 4°C in airtight containers. Plates were examined for the zone of inhibition and for microcolonies within the zone (designated "i") as indicative of tolerance, in addition to semi-confluent colonies outside the inhibition zone (designated "o"). To further evaluate dose-dependent tolerance, additional disks with higher concentrations were used: fluconazole (50 µg and 100 µg) and voriconazole (2 µg and 4 µg). All microcolonies ("i") and semi-confluent growths ("o") were then subcultured and re-tested with a 25 µg fluconazole disk to evaluate their phenotypic consistency with the parental isolate. Each assay, performed in duplicate, included C. parapsilosis ATCC 22019 as a quality control strain. Assessment of Trailing Phenotype Stability To assess the stability of the trailing phenotype, we revived archived azole-trailing isolates from glycerol stocks and re-tested them using broth microdilution (BMD). This included a panel of 32 C. tropicalis isolates, as well as trailing isolates from other Candida species ( C. albicans, C. parapsilosis, C. glabrata, C. viswanathii , and C. kefyr ). Longitudinal Trailing Retention The chronological stability of the trailing phenotype was assessed in 14 freshly isolated C. tropicalis strains over a 12-month period. Twelve replicate cryovials were prepared per isolate and stored at − 80°C. Monthly, one vial per isolate was revived for AFST by BMD to document the persistence of the trailing phenotype, MIC values, and phenotypic reproducibility, thereby defining the duration of trait retention. Assessing Trailing Growth Dynamics by Repetitive Freeze-Thaw To systematically investigate the underlying cause of the loss of the trailing phenomenon, three highly trailing fungal isolates (Tr3, Tr22, and Tr23) were selected alongside control isolates comprising an azole-resistant (CtR) and an azole-susceptible (CtS) strain of C. tropicalis. Each isolate was preserved in 20% glycerol stocks prepared with both distilled water and Yeast Peptone Dextrose (YPD) broth, with fifteen vials prepared per isolate; each vial was inoculated with 10^8 cells from either log-phase (8-hour YPD growth) or stationary-phase (18-hour YPD growth) cultures, labeled sequentially from Vial1 to Vial15, and stored at − 80°C. A rigorous revival schedule was implemented to evaluate viability and phenotypic stability over repeated freeze-thaw cycles: on Day 1, five vials per isolate were thawed, with Vial1 used for viability testing and broth microdilution (BMD) and Vials2–5 refrozen; one week later (Day 8), Vials2–5 were re-thawed, Vial2 was tested, and Vials3–5 were returned to storage; this sequential weekly thawing and testing continued until all vials were processed, ensuring consistent handling and minimal additional freeze-thaw cycles. Viability testing confirmed post-thaw cell survival, while BMD assays, performed according to standard protocols, determined MIC values to assess antifungal susceptibility and phenotypic reproducibility throughout the study. Effect of pH on Trailing Phenotype To assess the environmental modulation of the trailing phenotype, AFST was performed across a pH gradient ranging from 3.0 to 11.0 ( 20 , 22 ). RPMI-1640 media was buffered with sterile HCl or NaOH solutions prior to autoclaving, and the pH was confirmed using a calibrated digital pH meter. Both trailing C. tropicalis and non- tropicalis isolates were tested via BMD across this spectrum. The trailing intensity and MIC values were recorded at each pH point to analyze pH-dependent phenotypic variation. Revival of trailing phenotype in isolates that lost the trait An in vitro induction experiment was performed in an attempt to revive the trailing phenotype in five clinical Candida tropicalis isolates (Tr32, Tr34, Tr36, Tr37, Tr38) that had lost it. Each isolate was grown overnight in azole-free YPD broth at 35°C with shaking (180 rpm) and adjusted to ~ 10⁷ CFU/mL. From this, an aliquot containing 10⁶ cells was transferred into fresh YPD broth containing a sub-inhibitory concentration of fluconazole (0.125 µg/mL) and incubated for 48 hours under the same conditions. This culture was then serially passaged; every 48 hours, 10⁶ cells were subcultured into fresh medium containing either the same or an incrementally increased (log₂, i.e., doubled every two passages) concentration of fluconazole, up to a maximum of 8 µg/mL over 12 passages. At each passage, viability and susceptibility were assessed by BMD-based AFST. The induction process was terminated for a given isolate upon confirmed revival of the trailing phenotype via AFST. Population Analysis Profiling (PAP) Assay The PAP assay was employed to investigate the presence of fluconazole-resistant subpopulations capable of growth at supra-minimum inhibitory concentration (MIC) levels. Yeast extract agar with 2% glucose (YAG) plates were prepared with two-fold serial dilutions of fluconazole, encompassing a concentration range from 0.12 to 128 µg/ml; a drug-free YAG plate was included as a growth control for each isolate. Standardized inocula were prepared by adjusting cell suspensions to an optical density at 600 nm (OD₆₀₀) of 0.1, corresponding to approximately 1×10⁶ CFU/ml, followed by the preparation of serial log₁₀ dilutions down to 10³ CFU/ml. Each agar plate was divided into quadrants and inoculated with six replicate 10 µl drops of each cell dilution. Following incubation at 30°C for 24–48 hours, colony-forming units (CFU) were enumerated for each quadrant. The average CFU/ml was calculated and plotted against the corresponding fluconazole concentration to generate PAP curves. The area under each PAP curve (PAP-AUC) was computed using GraphPad Prism version 9.5 (GraphPad Software, USA), and statistical comparisons between groups were performed using two-way analysis of variance (ANOVA). A known heteroresistant strain, Candida glabrata 1646 (GenBank Accession No: KX270996), which was kindly provided by Tel Aviv University, Israel, served as the reference control in all assays ( 18 ). This reference strain has previously defined MIC values as follows: fluconazole, 1 µg/ml; voriconazole, 0.25 µg/ml; itraconazole, 0.5 µg/ml; posaconazole, 0.12 µg/ml; and isavuconazole, 0.03 µg/ml. The PAP-AUC of this reference strain provided a standard for characterizing and quantifying the degree of fluconazole heteroresistance in the test isolates. Results Primary Screening of Antifungal Susceptibility In this study, between 2022 and 2024, 847 clinical Candida tropicalis isolates from invasive candidiasis cases were screened for susceptibility to five azole antifungals using broth microdilution (BMD). The majority of isolates (745, 87.96%) were susceptible to all azoles tested. Non-susceptible isolates included 90 (10.62%) that were resistant to one or more azoles and 12 (1.42%) that exhibited susceptibility dose-dependent (SDD) to fluconazole. Among the susceptible isolates, 58 (6.85% of the total) exhibited the trailing growth phenomenon to one or more azoles. The MIC distribution for the isolates presented in Fig. 1 . The incidence of trailing growth was highest for fluconazole (Flu) (58/58; 100%), followed by voriconazole (Vori) (33/58; 56.89%), itraconazole (Itra) (24/58; 41.37%), posaconazole (Posa) (23/58; 39.65%), and isavuconazole (Isavu) (21/58; 36.21%). The MIC ranges for the trailing isolates were as follows: Flu, 0.12–2 µg/mL; Vori, 0.03–1 µg/mL; Itra and Isavu, 0.03–0.12 µg/mL; Posa, 0.03–0.25 µg/mL (Table 1 , Table S1 ). Table 1 The range of MICs to azoles as observed in the C. tropicalis trailing isolates under study. Azoles Fluconazole(µg/ml) Voriconazole (µg/ml) Itraconazole (µg/ml) Posaconazole (µg/ml) Isavuconazole (µg/ml) Susceptible range 0.12–2 0.03–1 0.03–1 0.03–0.5 0.03–0.5 Susceptible, trailing 0.12–2 0.03–1 0.03–0.12 0.03–0.25 0.03–0.12 Number of isolates, % 58, 100% 33, 56.89% 24, 41.37% 23, 39.65% 21, 36.21% We also determined the trailing in presence of one (Only flu), two (Flu + vori ), more than two and all five azoles tested (Fig. 2 , Table S1 ). Notably, 22.4% (13/58) of the isolates exhibited trailing against all five azoles, suggestive of a broad-spectrum azole tolerance. Secondary Screening by Disk Diffusion (DD) To further characterize the trailing phenotype, we performed disk diffusion assays on a selected isolates (Table S2) using fluconazole (25, 50, and 100 µg) and voriconazole (1, 2, and 4 µg) disks. For all tested isolates, the zone diameters increased with higher drug concentrations. However, the consistent presence of microcolonies within the inhibition zones across all disk potencies confirmed the phenotypic tolerance associated with trailing (Fig. 3 ; Table S2). Additionally, we also checked the microcolonies (" i ") and semi-confluent growths (" o ") among the same isolates used for disk diffusion assay. The selected “ i ” and “ o ” colonies showed similar phenotypic consistency with the parental isolate (Table 2 ). Table 2 Zone diameters (in mm) of the ‘ i ’ and ‘ o ’ colonies in comparison with the parental isolate Isolate ID Fluconazole 25 µg Parental ‘ i ’ ‘ o ’ Tr1 26 27 27 Tr2 20 21 20 Tr3 24 25 25 Tr4 26 25 25 Tr5 29 27 27 Tr6 15 16 17 Tr7 14 16 16 Tr8 15 15 14 Tr9 15 15 15 Tr10 24 22 22 Loss of Trailing Phenomenon To check if the isolates can retain trailing phenotype, 18 isolates revived and re-tested for antifungal susceptibility (AFST) by broth microdilution (BMD), the majority exhibited clear, interpretable endpoints for azole susceptibility. The MICs for these isolates did not exceed the clinical breakpoint of 8 µg/ml, indicating a susceptible phenotype without a trailing effect (data not shown). Only four isolates (22.22%) maintained the trailing phenotype, which was specific to fluconazole, while the remainder reverted to a non-trailing, susceptible phenotype (Table 3 ). Table 3 The list of isolates that retained trailing from the first set of isolates tested and their respective MICs. Isolate Fluconazole MIC (µg/ml) Voriconazole MIC (µg/ml) Itraconazole MIC (µg/ml) Posaconazole MIC (µg/ml) Isavuconazole MIC (µg/ml) Tr3 0.12 0.03 0.03 0.03 0.03 Tr22 0.25 0.03 0.06 0.03 0.03 Tr23 0.5 1 0.12 0.06 0.03 Tr24 0.12 0.03 0.06 0.03 0.03 Driven by this surprising observation, we tested an additional 14 C. tropicalis isolates. To determine if this loss of trailing phenotype was species-specific, we also extended our investigation to other Candida species. Repeat antifungal susceptibility testing (AST) confirmed the loss of trailing, revealing clear MIC endpoints. When data from all trailing isolates were combined, the loss of trailing was observed in 62.5% of C. tropicalis , 50% of C. albicans , and 33.3% of C. glabrata isolates (Table 5 ). These results demonstrate that the loss of trailing is not unique to C. tropicalis but occurs across multiple Candida species. Table 4 Species-specific distribution of the loss of trailing. Species No of isolates tested for presence of trailing No of isolates with loss of trailing effect on repeat testing C. tropicalis 32 (18 + 14) 62.5% (n = 20) C. albicans 20 50% (n = 10) C. glabrata 6 33.33% (n = 2) C. viswanathii 2 100% (n = 2) C. parapsilosis 4 0 C. kefyr 1 0 Time-Dependent Retention of Trailing To determine the long-term stability of the trailing phenotype, a batch of 14 trailing C. tropicalis isolates was aliquoted into monthly glycerol stocks. Each month, one vial per isolate was revived and tested. The trailing phenotype remained stable for the first seven months. However, three isolates lost trailing at month 8, and an additional three at month 11, resulting in a cumulative loss of 42.85% over one year (Fig. 4 & Table S3). The concomitant stability of MICs suggests this loss is a phenotypic, rather than genotypic, change, potentially influenced by freeze-thaw cycles or epigenetic shifts. This experiment was conducted exclusively on C. tropicalis . Influence Storing Conditions on Cell Viability and Susceptibility We next investigated the influence of repeated freeze-thaw cycles on cell viability and phenotypic stability. Weekly revivals of frozen glycerol stocks from trailing isolates (Tr3, Tr22, Tr23) and controls [ C. tropicalis resistant (CtR) & susceptible (CtS) isolates] were performed. While all isolates maintained sufficient viability and stable MICs throughout the experiment, two of the three trailing isolates (Tr22 and Tr23) transitioned to a non-trailing phenotype by the 15th revival. Given that the trailing phenotype is likely mediated by a small subpopulation, this loss may be due to the cumulative, selective depletion of these cells during the freeze-thaw process (Table 5 ). Table 5 Cell viability and MIC stability of isolates post freeze-thaw cycles Isolate [MIC(µg/ml)] 20% glycerol in water 20% glycerol in YPD MIC (µg/ml)/ phenotype Log phase cells Stationary phase cells Log phase cells Stationary phase cells Tr3 [0.12] 80% 80.31% 81.53% 81.53% 0.12/ susceptible, trailing Tr22 [0.25] 91.55% 89.45% 91.89% 91.89% 0.12/ susceptible, non-trailing Tr23 [0.5] 77.56% 77.56% 78.52% 78.84% 0.5/ susceptible, non-trailing CtR [64] 90.3% 90.1% 90.4% 90.4% 64 / resistant CtS [0.25] 89.5% 90.2% 90.5% 90.2% 0.25/ susceptible pH-Dependent Modulation of Trailing The influence of environmental pH on the trailing phenotype was evaluated by determining fluconazole susceptibility in RPMI media adjusted to pH levels from 3 to 11. Trailing growth was not observed at the acidic (pH ≤ 3) or alkaline (pH ≥ 8) extremes, which allowed for definitive endpoint determination. The phenotype was consistently present within the pH 6 to 8 range, with its incidence greatest at physiological pH (Table 6 ). Table 6 Fluconazole MICs at different pH among the isolates retained trailing pH Tr3 Tr22 Tr23 Tr24 Tr25 Tr26 Tr27 Tr28 Tr29 Tr30 Tr31 Tr32 Tr33 Tr34 Tr35 Tr36 Tr37 Tr38 pH 3 & 4 5 0.5 0.12 0.12 0.12 0.25 0.25 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.12 0.5 0.5 5.5 0.5 0.12 0.12 0.12 0.25 0.25 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.12 0.5 0.5 6 0.5 0.12 0.12 0.12 0.25 0.25 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.12 0.5 0.5 6.5 0.5 0.12 0.12 0.12 0.25 0.25 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.12 0.5 0.5 7 0.5 0.12 0.12 0.12 0.25 0.25 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.12 0.5 0.5 7.5 0.5 0.12 0.12 0.12 0.25 0.25 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.12 0.5 0.5 8 0.5 0.12 0.12 0.12 0.25 0.25 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.12 0.5 0.5 8.5 0.5 0.12 0.12 0.12 0.25 0.25 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.12 0.5 0.5 9 0.5 0.12 0.12 0.12 0.25 0.25 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.12 0.5 0.5 pH 10 & 11 Purple cells indicate the presence of trailing growth, while uncolored (white) cells indicate its absence at the corresponding pH. We also investigated whether the pH-dependent trailing phenotype, previously observed in C. tropicalis , occurred in other non- tropicalis species. Similar to C. tropicalis , isolates of C. albicans , C. glabrata , C. parapsilosis , and C. viswanathii exhibited trailing growth within a neutral-to-basic pH range (pH 6–8.5), but not under acidic or alkaline conditions. Specifically, trailing was most prominent between pH 6 and 8 for C. albicans and between pH 6 and 8.5 for the other species (Table S4A & S4B). Revival of trailing phenotype in isolates that lost the trait We next sought to determine whether the trailing phenotype could be restored in strains that had lost it. The induction protocol successfully restored the trailing phenotype in two of the five C. tropicalis isolates (Tr 37 and Tr 38) that had lost it. This revival occurred following 12 passages under high fluconazole stress (4 µg/mL). For the remaining three isolates, escalating the drug concentration to 8 µg/mL did not induce trailing (Table 7 ). No significant changes in MIC values were observed post-induction, indicating revival of trailing without full resistance development. Table 7 Details of isolates for revival of trailing Isolate Initial MIC to Flu Lost trailing phenotype after No of passages of Flu induction Current Flu MIC Tr 32 0.12 t 8 months 14 0.25 Tr 34 0.25 t 8 months 14 0.25 Tr 36 0.12 t 11 months 14 0.25 Tr 37 0.25 t 11 months 12 0.25 t Tr 38 0.5 t 11 months 12 0.5 t The “ t ” after MIC value indicates trailing to fluconazole. Population Analysis Profiling (PAP) Assay To assess subpopulation-level azole tolerance, we performed population analysis profiling (PAP) on all trailing isolates using fluconazole-gradient Yeast Extract Agar with Glucose (YAG) plates. Log₁₀-diluted inocula (10³ to 10⁶ CFU/mL) were spotted, and the resulting colony-forming units (CFU/mL) were plotted against drug concentrations. As anticipated, growth was suppressed at the MIC, yet low-level growth persisted at supra-MIC concentrations in all isolates (Fig. 5 and Fig. S1 ), confirming the presence of heteroresistance. We quantified this tolerance by calculating the area under the curve (AUC) for each isolate and normalizing it against the control C. glabrata 1646 strain to generate an Area under the Curve Ratio (AUCR). The AUCR analysis provided a sensitive measure of heteroresistance, revealing that while MIC values did not predict the size of the tolerant subpopulation, all isolates exhibited AUCR values indicative of fluconazole tolerance. Future studies will expand PAP to a larger cohort to establish a definitive heteroresistance threshold for C. tropicalis . It is known that 1% − 95% of subpopulations can grow at supra-MIC. The MICs however do not correlate with the fraction of subpopulations growing at higher concentrations of the drug. All the isolates tested so far have shown “tolerance”. However, the PAP assay being a known standard for in vitro detection of tolerance/heteroresistance, gives an AUCR unique to every isolate and does not correlate with the MIC of the respective isolate, i.e., isolates with similar MICs do not however have the similar AUCRs. Discussion The phenomenon of trailing growth, clinically conceptualized as antifungal tolerance, presents a persistent and confounding challenge in medical mycology. Originally characterized as a technical limitation of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method M27-A, trailing describes the ambiguous growth pattern where an isolate appears susceptible at the standard 24-hour reading but exhibits significant, often resistant-range growth at 48 hours. This "low-high" MIC phenotype complicates definitive susceptibility reporting ( 22 ). Crucially, emerging clinical and experimental evidence suggests this in vitro artifact may obscure a more complex biological reality with direct therapeutic implications. Studies have demonstrated that isolates displaying pronounced trailing can remain clinically susceptible, responding effectively to fluconazole therapy in both animal infection models and human cases, thereby implying that the earlier 24-hour MIC may offer a more accurate reflection of in vivo drug efficacy ( 23 ). This discordance is clinically significant, as highlighted by cases where infections with trailing isolates persist despite an apparently susceptible MIC, underscoring the critical need for more discriminative diagnostic assays to reliably separate true susceptibility from phenotypic tolerance ( 12 ). The clinical interpretation of trailing growth remains a subject of active debate, rooted in seemingly contradictory data. Some clinical reports posit that infections caused by trailing isolates typically respond to standard fluconazole regimens, reinforcing the validity of the 24-hour reading for predicting patient outcomes ( 26 ). Experimental models have sought to clarify this correlation. One pivotal study using a murine model of invasive candidiasis demonstrated that fluconazole treatment significantly improved survival rates in animals infected with both susceptible and trailing isolates, but not in those infected with genetically resistant strains, a finding corroborated by reduced fungal kidney burden ( 27 ). Mechanistically, the trailing phenotype shares features with resistance; for example, C. albicans isolates with strong trailing to fluconazole have been found to harbor mutations in ERG11 and show upregulation of efflux pumps, hallmarks of azole resistance ( 29 ). However, in vivo evidence frequently contradicts this, showing that such trailing isolates retain responsiveness to azole therapy ( 30 – 33 ). This paradox is further complicated by drug-specific effects, as illustrated by a Galleria mellonella larvae model, where voriconazole treatment failed to improve survival against trailing C. albicans , suggesting that for this triazole, trailing might indeed mask clinically relevant resistance ( 28 ). Population-based studies add another layer of nuance. Data from the extensive CANDIPOP study indicate that in vitro trailing does not necessarily translate to clinical failure, with isolates from successfully treated patients showing no association between trailing and increased mortality ( 24 ). Other patient data support this, indicating that trailing offers no significant survival advantage to yeast during azole therapy ( 30 – 33 ). A prevailing hypothesis is that the partial growth inhibition characteristic of trailing may be sufficient for a competent host immune system to eradicate the infection, effectively decoupling the phenotype from therapeutic failure in immunocompetent hosts. Yet, this relationship falters in vulnerable populations. Rosenberg et al., who framed the phenomenon as "tolerance," found that isolates from immunocompromised patients with persistent candidemia exhibited higher tolerance levels than those from patients who responded to fluconazole, implicating high tolerance in infection persistence despite treatment ( 9 ). The trailing/tolerance phenotype is remarkably plastic, influenced by a network of extrinsic factors beyond genetics. A meticulous investigation demonstrated that commonly co-administered non-antifungal medications can significantly modulate C. albicans susceptibility to fluconazole, with some drugs exerting antagonistic effects that exacerbate both resistance and tolerance, the latter defined here as the growth of microcolonies within an inhibition zone ( 34 ). This underscores that susceptibility interpretations for trailing isolates require extreme caution, as both fungal adaptation and ultimate treatment response are difficult to predict ( 28 , 34 ). Fundamentally, tolerance operates as a concentration-independent subpopulation effect. As demonstrated via disc diffusion, increasing drug concentrations enlarge the zone of inhibition, but the fraction of the population that grows as microcolonies remains constant, a finding consistent across studies ( 9 ). This tolerance is inversely correlated with intracellular drug accumulation, distinguishing it from classical resistance ( 9 ). Adding to this complexity, we report for the first time that the trailing phenotype itself is unstable and can be lost. Environmental factors significantly modulate its expression. Long-term storage led to a loss of trailing in 62.5% of C. tropicalis , 50% of C. albicans , and 33.3% of C. glabrata isolates, with a 43% overall loss noted after 12 months. Furthermore, extreme pH conditions (beyond pH 5–8) were observed to disrupt trailing expression. This aligns with prior work by Ribeiro et al., who found C. tropicalis loses its trailing ability under acidic conditions ( 20 ), and Rosenberg et al., who noted tolerance levels decrease at lower pH ( 9 ). Temperature shifts also exert influence, with higher temperatures (37–41°C) potentially reducing the trailing subpopulation, while lower temperatures may induce stress responses that enhance it, without altering the core MIC or inhibition zone diameter ( 9 ). This collective evidence positions trailing not as a fixed genetic trait, but as a form of phenotypic heterogeneity subject to environmental regulation ( 9 ). A central mechanistic hypothesis for tolerance involves the slow growth dynamics of a subpopulation under antifungal stress. This reduced metabolic rate may enable these cells to endure drug exposure more effectively than their faster-growing counterparts, representing a bet-hedging survival strategy. The inverse correlation between tolerance and intracellular drug accumulation suggests an active cellular process, potentially involving efflux pumps or altered membrane permeability, that mitigates cytotoxic drug effects ( 9 ). This survival strategy has direct clinical ramifications: the persistence of these tolerant subpopulations is believed to contribute to infection recurrence and therapeutic failure, as evidenced in multiple clinical cases ( 18 ). To address the diagnostic shortfall, Population Analysis Profiling (PAP) has emerged as a pivotal tool. This assay quantifies heteroresistance by plotting the proportion of a population that grows across a gradient of drug concentrations. The resulting Area Under the Curve (AUC) serves as a quantitative measure of tolerance. Crucially, two isolates with identical fluconazole MICs can have vastly different PAP-AUCs; the isolate with the higher AUC (indicating a larger tolerant subpopulation) is prognostically more likely to be difficult to eradicate in vivo ( 9 , 18 , 19 ). Ben-Ami et al. revealed that 57.6% of clinical C. glabrata isolates displayed fluconazole heteroresistance via PAP, with higher AUC values correlating with higher MICs and a greater risk of treatment failure ( 18 ). Similarly, applying PAP to C. tropicalis has successfully identified tolerant subpopulations that survive fluconazole exposure without acquiring stable genetic resistance ( 25 ). The derivative metric, the Area Under the Concentration-Response curve (AUCR), further refines this analysis by integrating drug exposure and biological response, revealing distinct, clinically relevant tolerance patterns ( 19 ). Conclusion In conclusion, the phenotypic landscape of trailing and tolerance in Candida spp., particularly C. tropicalis , is multidimensional. It arises from a confluence of stable subpopulation dynamics, adaptive slow growth, active drug efflux mechanisms, and profound responsiveness to environmental cues like pH, temperature, and concomitant drugs. These factors collectively fuel the persistence of infections and pose formidable challenges for therapy. The clinical implications are profound, necessitating a paradigm shift beyond standard MIC-based approaches. Incorporating functional assays like PAP into translational research frameworks is essential to distinguish tolerance from resistance, guide tailored therapeutic strategies, and ultimately improve outcomes for patients with recalcitrant or relapsing candidiasis. Future research must prioritize elucidating the molecular regulators of this phenotypic heterogeneity to identify novel targets for adjuvant therapies designed to disarm these fungal survival strategies. Declarations Conflict of Interest The authors declare no competing interests to disclose. Funding This study was supported by the Indian Council of Medical Research (ICMR Grant ID No. Myco/Adhoc/4/2022-ECD-II) Author Contribution Snigdha Reddy contributed to the conception and design of the study, data acquisition and analysis, manuscript drafting, and approved the final version.Saikat Paul contributed to the conception and design of the study, data analysis, manuscript drafting and critical revision, and approved the final version.Shivaprakash M. Rudramurthy contributed to critical revision of the manuscript and approved the final version.Harsimran Kaur contributed to critical revision of the manuscript and approved the final version.Anup K. Ghosh contributed to the conception and design of the study, manuscript drafting and critical revision, approved the final version, and takes responsibility for the integrity of the work. Acknowledgement The authors duly acknowledge the Indian Council of Medical Research (ICMR), Government of India for financial supports. 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18:16:48","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152122,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8484141/v1/b58c44d4169ae9136a442eb0.html"},{"id":101203593,"identity":"f08da6da-d687-48a2-be1c-388079dd89b2","added_by":"auto","created_at":"2026-01-27 09:40:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111094,"visible":true,"origin":"","legend":"\u003cp\u003eGraph depicting MIC distribution of the isolates exhibiting trailing phenomenon against each azole antifungal. Multiple data points are not visible in the graph due to same MIC values.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8484141/v1/e2d61aa768c3d8dd16ea4857.png"},{"id":101007457,"identity":"e0ef411f-d4aa-4930-94a0-ea5c873ea5d0","added_by":"auto","created_at":"2026-01-23 18:16:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55885,"visible":true,"origin":"","legend":"\u003cp\u003ePresence of trailing growth among the isolates (n=58) in the presence of Flu and Flu + other azoles.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8484141/v1/7e0b474b82db9e363fd71194.png"},{"id":101204068,"identity":"f21348c9-2be9-43e1-bd0c-9c7bb9ab17ac","added_by":"auto","created_at":"2026-01-27 09:41:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":674266,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative disk diffusion assay depicting the zones of inhibition with micro colonies inside the zone.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8484141/v1/babe59bd0914fd4752c8baf9.png"},{"id":101204017,"identity":"1fd7bee0-aa64-458e-b2ac-ae1380fb60df","added_by":"auto","created_at":"2026-01-27 09:41:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePersistence of the trailing phenotype over a 12-month period\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8484141/v1/7901863f69059d035a193045.png"},{"id":101203807,"identity":"2d027589-a1fa-4dbd-ab90-d2aa818ba832","added_by":"auto","created_at":"2026-01-27 09:40:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":212694,"visible":true,"origin":"","legend":"\u003cp\u003ePAP assay of trailing isolates with controls – fluconazole resistant (CtR), fluconazole susceptible (CtS) and known reference strain (CG).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8484141/v1/8bbe772d35d2e4341c627a74.png"},{"id":108437545,"identity":"65cbcf61-0169-4879-b4cd-6e2b2e761a34","added_by":"auto","created_at":"2026-05-04 15:58:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1538713,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8484141/v1/e3043709-f443-478c-a4d0-3993eef05359.pdf"},{"id":101007463,"identity":"8d3cf016-39c3-4217-a828-74f489afa062","added_by":"auto","created_at":"2026-01-23 18:16:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":139642,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation12292025.docx","url":"https://assets-eu.researchsquare.com/files/rs-8484141/v1/ee1cfe3bbe8fc7392a9fc55f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Instability and Subpopulation Dynamics of Azole Trailing Phenotypes in Clinical Candida tropicalis Isolates","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInvasive fungal infections represent a significant global public health threat, with \u003cem\u003eCandida\u003c/em\u003e species ranking among the foremost fungal pathogens associated with high morbidity and mortality worldwide (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). While \u003cem\u003eC. albicans\u003c/em\u003e has historically been the most prevalent agent of fungal sepsis, the epidemiological landscape has shifted considerably (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Non-\u003cem\u003ealbicans Candida\u003c/em\u003e (NAC) species, including \u003cem\u003eC. glabrata\u003c/em\u003e, \u003cem\u003eC. tropicalis\u003c/em\u003e, \u003cem\u003eC. parapsilosis\u003c/em\u003e, and \u003cem\u003eC. krusei\u003c/em\u003e, now contribute substantially to the global burden of candidemia (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Among these, \u003cem\u003eCandida tropicalis\u003c/em\u003e has emerged as a leading cause of invasive candidiasis, with notably high prevalence in the Asia-Pacific and Latin American regions (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). This species is of particular concern in high-risk populations, such as patients with hematologic malignancies, and is associated with alarmingly high mortality rates (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe management of invasive candidiasis relies on a limited resource of antifungal drug classes. Azoles, particularly fluconazole, remain a cornerstone of therapy due to their efficacy and favourable bioavailability (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, the rising incidence of azole resistance threatens their clinical utility. A key challenge is the frequent discordance between \u003cem\u003ein vitro\u003c/em\u003e susceptibility, as determined by standardized antifungal susceptibility testing (AFST), and clinical outcomes, that are predicted by the \u0026ldquo;90/60\u0026rdquo; rule, where ~\u0026thinsp;90% of susceptible isolates and ~\u0026thinsp;60% of resistant isolates respond to therapy (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This discrepancy raises critical concerns about the adequacy of current testing methodologies and interpretive criteria (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). It also implies the influence of host factors as well as the characteristics of the pathogen not reflected by the MIC on clinical outcomes (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStandardized AFST guidelines from the CLSI and EUCAST recommend determining the minimum inhibitory concentration (MIC) at 24 hours based on a 50% growth inhibition threshold. A significant complication in applying this standard to azoles is the phenomenon of \"trailing growth\", a reduced but persistent growth observed at drug concentrations above the MIC (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This is especially common in \u003cem\u003eC. tropicalis\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, with one large survey reporting a mean residual growth of approximately 10% in these species, a rate substantially higher than in others (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Trailing growth obscures MIC endpoint determination, often leading to the misclassification of isolates as resistant or susceptible depending on the reading time and method (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCritically, trailing growth is distinct from stable, genetic resistance. It is considered a form of antifungal tolerance, where a subpopulation of cells survives drug exposure without an increase in the population's MIC (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Genetic studies further support that azole resistance and trailing are distinct phenotypes in \u003cem\u003eC. tropicalis\u003c/em\u003e, with the latter being a reversible, non-heritable tolerance mechanism (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This phenomenon shares features with, but is mechanistically different from, heteroresistance, where a small subpopulation (\u0026lt;\u0026thinsp;1%) exhibits growth at very high drug concentrations (\u0026ge;\u0026thinsp;8\u0026times;MIC). Both trailing and heteroresistance evade detection in routine AFST due to the use of low inocula that measure only the dominant population response (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The population analysis profile (PAP) assay, which plates a dense culture onto a gradient of drug concentrations, is specifically designed to detect and quantify these minority subpopulations. This method provides a continuous measure of tolerance, offering a more nuanced view than the binary result of a standard MIC (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). PAP-based studies have been pivotal in revealing the clinical significance of heteroresistance; for example, in \u003cem\u003eC. glabrata\u003c/em\u003e, heteroresistant strains were linked to persistent infection in murine models despite \u003cem\u003ein vitro\u003c/em\u003e \"susceptibility,\" explaining some clinical treatment failures (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The application of PAP to \u003cem\u003eC. albicans\u003c/em\u003e is more recent but equally insightful (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe molecular basis of trailing is an area of active investigation. Trailing isolates share profiles with resistant strains, including the upregulation of secreted aspartyl proteases (Saps), which may aid in both virulence and antifungal evasion (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Evidence suggesting that histone deacetylase inhibitors can reduce trailing further points to a role for epigenetic regulation in this phenotype (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Nevertheless, the precise genetic and epigenetic determinants remain to be fully elucidated.\u003c/p\u003e \u003cp\u003eDespite growing research, the trailing phenomenon remains underexplored in its biological basis and clinical impact. Whether it constitutes a true resistance mechanism, a transient adaptive response, or an \u003cem\u003ein vitro\u003c/em\u003e artifact is still debated (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Clarifying its epidemiological trends and biological drivers is essential for refining AFST protocols and therapeutic strategies. This is especially urgent given fluconazole's critical role in clinical practice, particularly as step-down therapy. The recurring reports of discordance between \u003cem\u003ein vitro\u003c/em\u003e susceptibility and clinical response in trailing isolates underscore the need for more sophisticated interpretations of AFST.\u003c/p\u003e \u003cp\u003eIn this context, we conducted a comprehensive study to characterize the trailing phenomenon in \u003cem\u003eC. tropicalis\u003c/em\u003e and other relevant species. Our investigation aims to determine the phenotypic stability of trailing, explore its potential mechanisms, and assess the constancy of this trait, with the ultimate goal of improving diagnostic accuracy and patient outcomes in invasive candidiasis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eClinical Isolates and Identification\u003c/p\u003e \u003cp\u003eNon-duplicate clinical isolates of \u003cem\u003eCandida tropicalis\u003c/em\u003e from invasive candidiasis cases, collected from 2022 to 2024, were included in this study. The isolates were obtained from the Mycology Laboratory, Department of Medical Microbiology, PGIMER, Chandigarh, India. Species-level identification was performed using MALDI-TOF MS (bioM\u0026eacute;rieux, France) according to the manufacturer\u0026rsquo;s instructions, and all isolates were preserved in 20% glycerol at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003cp\u003ePrimary Antifungal Susceptibility Screening (AFST)\u003c/p\u003e \u003cp\u003eAntifungal susceptibility testing (AFST) against five azoles (fluconazole, voriconazole, itraconazole, posaconazole, and isavuconazole) was performed in biological triplicates using the CLSI M27-A4 broth microdilution method. MICs were read after 24 and 48 hours of incubation at 35\u0026deg;C. Trailing growth was defined as persistent, partial growth at supra-MIC concentrations (\u0026ge;\u0026thinsp;2 dilutions above the MIC) without a distinct endpoint, per CLSI guidelines and literature. Quality control employed \u003cem\u003eCandida parapsilosis\u003c/em\u003e ATCC 22019 and \u003cem\u003eCandida krusei\u003c/em\u003e ATCC 6258. Isolates exhibiting trailing were preserved for further analysis, and their prevalence was calculated relative to the total isolates received.\u003c/p\u003e \u003cp\u003eSecondary Screening: Disk Diffusion Method\u003c/p\u003e \u003cp\u003eIn contrast to liquid culture assays where cell mixing obscures tolerant subpopulations, making them difficult to detect, these fractions are readily apparent on agar plates where individual clonal progeny can be distinguished (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Disk diffusion testing for fluconazole and voriconazole was therefore conducted according to CLSI M44-A2 guidelines using sterile blank filter paper disks (HiMedia, India) impregnated with 25 \u0026micro;g fluconazole and 1 \u0026micro;g voriconazole, which were stored at 4\u0026deg;C in airtight containers. Plates were examined for the zone of inhibition and for microcolonies within the zone (designated \"i\") as indicative of tolerance, in addition to semi-confluent colonies outside the inhibition zone (designated \"o\"). To further evaluate dose-dependent tolerance, additional disks with higher concentrations were used: fluconazole (50 \u0026micro;g and 100 \u0026micro;g) and voriconazole (2 \u0026micro;g and 4 \u0026micro;g). All microcolonies (\"i\") and semi-confluent growths (\"o\") were then subcultured and re-tested with a 25 \u0026micro;g fluconazole disk to evaluate their phenotypic consistency with the parental isolate. Each assay, performed in duplicate, included C. \u003cem\u003eparapsilosis\u003c/em\u003e ATCC 22019 as a quality control strain.\u003c/p\u003e \u003cp\u003eAssessment of Trailing Phenotype Stability\u003c/p\u003e \u003cp\u003eTo assess the stability of the trailing phenotype, we revived archived azole-trailing isolates from glycerol stocks and re-tested them using broth microdilution (BMD). This included a panel of 32 \u003cem\u003eC. tropicalis\u003c/em\u003e isolates, as well as trailing isolates from other \u003cem\u003eCandida\u003c/em\u003e species (\u003cem\u003eC. albicans, C. parapsilosis, C. glabrata, C. viswanathii\u003c/em\u003e, and \u003cem\u003eC. kefyr\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eLongitudinal Trailing Retention\u003c/p\u003e \u003cp\u003eThe chronological stability of the trailing phenotype was assessed in 14 freshly isolated \u003cem\u003eC. tropicalis\u003c/em\u003e strains over a 12-month period. Twelve replicate cryovials were prepared per isolate and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Monthly, one vial per isolate was revived for AFST by BMD to document the persistence of the trailing phenotype, MIC values, and phenotypic reproducibility, thereby defining the duration of trait retention.\u003c/p\u003e \u003cp\u003eAssessing Trailing Growth Dynamics by Repetitive Freeze-Thaw\u003c/p\u003e \u003cp\u003eTo systematically investigate the underlying cause of the loss of the trailing phenomenon, three highly trailing fungal isolates (Tr3, Tr22, and Tr23) were selected alongside control isolates comprising an azole-resistant (CtR) and an azole-susceptible (CtS) strain of C. tropicalis. Each isolate was preserved in 20% glycerol stocks prepared with both distilled water and Yeast Peptone Dextrose (YPD) broth, with fifteen vials prepared per isolate; each vial was inoculated with 10^8 cells from either log-phase (8-hour YPD growth) or stationary-phase (18-hour YPD growth) cultures, labeled sequentially from Vial1 to Vial15, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. A rigorous revival schedule was implemented to evaluate viability and phenotypic stability over repeated freeze-thaw cycles: on Day 1, five vials per isolate were thawed, with Vial1 used for viability testing and broth microdilution (BMD) and Vials2\u0026ndash;5 refrozen; one week later (Day 8), Vials2\u0026ndash;5 were re-thawed, Vial2 was tested, and Vials3\u0026ndash;5 were returned to storage; this sequential weekly thawing and testing continued until all vials were processed, ensuring consistent handling and minimal additional freeze-thaw cycles. Viability testing confirmed post-thaw cell survival, while BMD assays, performed according to standard protocols, determined MIC values to assess antifungal susceptibility and phenotypic reproducibility throughout the study.\u003c/p\u003e \u003cp\u003eEffect of pH on Trailing Phenotype\u003c/p\u003e \u003cp\u003eTo assess the environmental modulation of the trailing phenotype, AFST was performed across a pH gradient ranging from 3.0 to 11.0 (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). RPMI-1640 media was buffered with sterile HCl or NaOH solutions prior to autoclaving, and the pH was confirmed using a calibrated digital pH meter. Both trailing \u003cem\u003eC. tropicalis\u003c/em\u003e and non-\u003cem\u003etropicalis\u003c/em\u003e isolates were tested via BMD across this spectrum. The trailing intensity and MIC values were recorded at each pH point to analyze pH-dependent phenotypic variation.\u003c/p\u003e \u003cp\u003eRevival of trailing phenotype in isolates that lost the trait\u003c/p\u003e \u003cp\u003eAn \u003cem\u003ein vitro\u003c/em\u003e induction experiment was performed in an attempt to revive the trailing phenotype in five clinical \u003cem\u003eCandida tropicalis\u003c/em\u003e isolates (Tr32, Tr34, Tr36, Tr37, Tr38) that had lost it. Each isolate was grown overnight in azole-free YPD broth at 35\u0026deg;C with shaking (180 rpm) and adjusted to ~\u0026thinsp;10⁷ CFU/mL. From this, an aliquot containing 10⁶ cells was transferred into fresh YPD broth containing a sub-inhibitory concentration of fluconazole (0.125 \u0026micro;g/mL) and incubated for 48 hours under the same conditions. This culture was then serially passaged; every 48 hours, 10⁶ cells were subcultured into fresh medium containing either the same or an incrementally increased (log₂, i.e., doubled every two passages) concentration of fluconazole, up to a maximum of 8 \u0026micro;g/mL over 12 passages. At each passage, viability and susceptibility were assessed by BMD-based AFST. The induction process was terminated for a given isolate upon confirmed revival of the trailing phenotype via AFST.\u003c/p\u003e \u003cp\u003ePopulation Analysis Profiling (PAP) Assay\u003c/p\u003e \u003cp\u003eThe PAP assay was employed to investigate the presence of fluconazole-resistant subpopulations capable of growth at supra-minimum inhibitory concentration (MIC) levels. Yeast extract agar with 2% glucose (YAG) plates were prepared with two-fold serial dilutions of fluconazole, encompassing a concentration range from 0.12 to 128 \u0026micro;g/ml; a drug-free YAG plate was included as a growth control for each isolate. Standardized inocula were prepared by adjusting cell suspensions to an optical density at 600 nm (OD₆₀₀) of 0.1, corresponding to approximately 1\u0026times;10⁶ CFU/ml, followed by the preparation of serial log₁₀ dilutions down to 10\u0026sup3; CFU/ml. Each agar plate was divided into quadrants and inoculated with six replicate 10 \u0026micro;l drops of each cell dilution. Following incubation at 30\u0026deg;C for 24\u0026ndash;48 hours, colony-forming units (CFU) were enumerated for each quadrant. The average CFU/ml was calculated and plotted against the corresponding fluconazole concentration to generate PAP curves. The area under each PAP curve (PAP-AUC) was computed using GraphPad Prism version 9.5 (GraphPad Software, USA), and statistical comparisons between groups were performed using two-way analysis of variance (ANOVA). A known heteroresistant strain, \u003cem\u003eCandida glabrata\u003c/em\u003e 1646 (GenBank Accession No: KX270996), which was kindly provided by Tel Aviv University, Israel, served as the reference control in all assays (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). This reference strain has previously defined MIC values as follows: fluconazole, 1 \u0026micro;g/ml; voriconazole, 0.25 \u0026micro;g/ml; itraconazole, 0.5 \u0026micro;g/ml; posaconazole, 0.12 \u0026micro;g/ml; and isavuconazole, 0.03 \u0026micro;g/ml. The PAP-AUC of this reference strain provided a standard for characterizing and quantifying the degree of fluconazole heteroresistance in the test isolates.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003ePrimary Screening of Antifungal Susceptibility\u003c/p\u003e \u003cp\u003eIn this study, between 2022 and 2024, 847 clinical \u003cem\u003eCandida tropicalis\u003c/em\u003e isolates from invasive candidiasis cases were screened for susceptibility to five azole antifungals using broth microdilution (BMD). The majority of isolates (745, 87.96%) were susceptible to all azoles tested. Non-susceptible isolates included 90 (10.62%) that were resistant to one or more azoles and 12 (1.42%) that exhibited susceptibility dose-dependent (SDD) to fluconazole. Among the susceptible isolates, 58 (6.85% of the total) exhibited the trailing growth phenomenon to one or more azoles. The MIC distribution for the isolates presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe incidence of trailing growth was highest for fluconazole (Flu) (58/58; 100%), followed by voriconazole (Vori) (33/58; 56.89%), itraconazole (Itra) (24/58; 41.37%), posaconazole (Posa) (23/58; 39.65%), and isavuconazole (Isavu) (21/58; 36.21%). The MIC ranges for the trailing isolates were as follows: Flu, 0.12\u0026ndash;2 \u0026micro;g/mL; Vori, 0.03\u0026ndash;1 \u0026micro;g/mL; Itra and Isavu, 0.03\u0026ndash;0.12 \u0026micro;g/mL; Posa, 0.03\u0026ndash;0.25 \u0026micro;g/mL (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe range of MICs to azoles as observed in the \u003cem\u003eC. tropicalis\u003c/em\u003e trailing isolates under study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzoles\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFluconazole(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVoriconazole\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eItraconazole\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePosaconazole\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIsavuconazole\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSusceptible range\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026ndash;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026ndash;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.03\u0026ndash;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03\u0026ndash;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSusceptible, trailing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026ndash;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026ndash;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.03\u0026ndash;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03\u0026ndash;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of isolates, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58, 100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33, 56.89%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24, 41.37%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23, 39.65%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e21, 36.21%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe also determined the trailing in presence of one (Only flu), two (Flu\u0026thinsp;+\u0026thinsp;vori ), more than two and all five azoles tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, 22.4% (13/58) of the isolates exhibited trailing against all five azoles, suggestive of a broad-spectrum azole tolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecondary Screening by Disk Diffusion (DD)\u003c/p\u003e \u003cp\u003eTo further characterize the trailing phenotype, we performed disk diffusion assays on a selected isolates (Table S2) using fluconazole (25, 50, and 100 \u0026micro;g) and voriconazole (1, 2, and 4 \u0026micro;g) disks. For all tested isolates, the zone diameters increased with higher drug concentrations. However, the consistent presence of microcolonies within the inhibition zones across all disk potencies confirmed the phenotypic tolerance associated with trailing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, we also checked the microcolonies (\"\u003cem\u003ei\u003c/em\u003e\") and semi-confluent growths (\"\u003cem\u003eo\u003c/em\u003e\") among the same isolates used for disk diffusion assay. The selected \u0026ldquo;\u003cem\u003ei\u003c/em\u003e\u0026rdquo; and \u0026ldquo;\u003cem\u003eo\u003c/em\u003e\u0026rdquo; colonies showed similar phenotypic consistency with the parental isolate (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eZone diameters (in mm) of the \u0026lsquo;\u003cem\u003ei\u003c/em\u003e\u0026rsquo; and \u0026lsquo;\u003cem\u003eo\u003c/em\u003e\u0026rsquo; colonies in comparison with the parental isolate\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIsolate ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFluconazole 25 \u0026micro;g\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParental\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lsquo;\u003cem\u003ei\u003c/em\u003e\u0026rsquo;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lsquo;\u003cem\u003eo\u003c/em\u003e\u0026rsquo;\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLoss of Trailing Phenomenon\u003c/p\u003e \u003cp\u003eTo check if the isolates can retain trailing phenotype, 18 isolates revived and re-tested for antifungal susceptibility (AFST) by broth microdilution (BMD), the majority exhibited clear, interpretable endpoints for azole susceptibility. The MICs for these isolates did not exceed the clinical breakpoint of 8 \u0026micro;g/ml, indicating a susceptible phenotype without a trailing effect (data not shown). Only four isolates (22.22%) maintained the trailing phenotype, which was specific to fluconazole, while the remainder reverted to a non-trailing, susceptible phenotype (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe list of isolates that retained trailing from the first set of isolates tested and their respective MICs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFluconazole MIC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVoriconazole MIC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eItraconazole MIC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePosaconazole MIC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIsavuconazole MIC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDriven by this surprising observation, we tested an additional 14 \u003cem\u003eC. tropicalis\u003c/em\u003e isolates. To determine if this loss of trailing phenotype was species-specific, we also extended our investigation to other \u003cem\u003eCandida\u003c/em\u003e species. Repeat antifungal susceptibility testing (AST) confirmed the loss of trailing, revealing clear MIC endpoints. When data from all trailing isolates were combined, the loss of trailing was observed in 62.5% of \u003cem\u003eC. tropicalis\u003c/em\u003e, 50% of \u003cem\u003eC. albicans\u003c/em\u003e, and 33.3% of \u003cem\u003eC. glabrata\u003c/em\u003e isolates (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results demonstrate that the loss of trailing is not unique to \u003cem\u003eC. tropicalis\u003c/em\u003e but occurs across multiple \u003cem\u003eCandida\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecies-specific distribution of the loss of trailing.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo of isolates tested for presence of trailing\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo of isolates with loss of trailing effect on repeat testing\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. tropicalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (18\u0026thinsp;+\u0026thinsp;14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.5% (n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50% (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. glabrata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.33% (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. viswanathii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100% (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. parapsilosis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. kefyr\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTime-Dependent Retention of Trailing\u003c/p\u003e \u003cp\u003eTo determine the long-term stability of the trailing phenotype, a batch of 14 trailing \u003cem\u003eC. tropicalis\u003c/em\u003e isolates was aliquoted into monthly glycerol stocks. Each month, one vial per isolate was revived and tested. The trailing phenotype remained stable for the first seven months. However, three isolates lost trailing at month 8, and an additional three at month 11, resulting in a cumulative loss of 42.85% over one year (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; Table S3). The concomitant stability of MICs suggests this loss is a phenotypic, rather than genotypic, change, potentially influenced by freeze-thaw cycles or epigenetic shifts. This experiment was conducted exclusively on \u003cem\u003eC. tropicalis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInfluence Storing Conditions on Cell Viability and Susceptibility\u003c/p\u003e \u003cp\u003eWe next investigated the influence of repeated freeze-thaw cycles on cell viability and phenotypic stability. Weekly revivals of frozen glycerol stocks from trailing isolates (Tr3, Tr22, Tr23) and controls [\u003cem\u003eC. tropicalis\u003c/em\u003e resistant (CtR) \u0026amp; susceptible (CtS) isolates] were performed. While all isolates maintained sufficient viability and stable MICs throughout the experiment, two of the three trailing isolates (Tr22 and Tr23) transitioned to a non-trailing phenotype by the 15th revival. Given that the trailing phenotype is likely mediated by a small subpopulation, this loss may be due to the cumulative, selective depletion of these cells during the freeze-thaw process (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell viability and MIC stability of isolates post freeze-thaw cycles\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate [MIC(\u0026micro;g/ml)]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e20% glycerol in water\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e20% glycerol in YPD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMIC (\u0026micro;g/ml)/ phenotype\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLog phase cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStationary phase cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLog phase cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStationary phase cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr3 [0.12]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80.31%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81.53%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.53%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.12/ susceptible, trailing\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr22 [0.25]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91.55%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.45%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.89%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e91.89%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.12/ susceptible, non-trailing\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr23 [0.5]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.56%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77.56%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e78.84%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5/ susceptible, non-trailing\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCtR [64]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64 / resistant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCtS [0.25]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.25/ susceptible\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003epH-Dependent Modulation of Trailing\u003c/p\u003e \u003cp\u003eThe influence of environmental pH on the trailing phenotype was evaluated by determining fluconazole susceptibility in RPMI media adjusted to pH levels from 3 to 11. Trailing growth was not observed at the acidic (pH\u0026thinsp;\u0026le;\u0026thinsp;3) or alkaline (pH\u0026thinsp;\u0026ge;\u0026thinsp;8) extremes, which allowed for definitive endpoint determination. The phenotype was consistently present within the pH 6 to 8 range, with its incidence greatest at physiological pH (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFluconazole MICs at different pH among the isolates retained trailing\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"19\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c18\" colnum=\"18\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c19\" colnum=\"19\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTr3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTr22\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTr23\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTr24\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTr25\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTr26\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTr27\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTr28\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eTr29\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eTr30\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTr31\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eTr32\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eTr33\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003eTr34\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e \u003cp\u003eTr35\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c17\"\u003e \u003cp\u003eTr36\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c18\"\u003e \u003cp\u003eTr37\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c19\"\u003e \u003cp\u003eTr38\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"19\" nameend=\"c19\" namest=\"c1\"\u003e \u003cp\u003epH 3 \u0026amp; 4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e 0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"19\"\u003epH 10 \u0026amp; 11\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePurple cells indicate the presence of trailing growth, while uncolored (white) cells indicate its absence at the corresponding pH.\u003c/p\u003e \u003cp\u003eWe also investigated whether the pH-dependent trailing phenotype, previously observed in \u003cem\u003eC. tropicalis\u003c/em\u003e, occurred in other non-\u003cem\u003etropicalis\u003c/em\u003e species. Similar to \u003cem\u003eC. tropicalis\u003c/em\u003e, isolates of \u003cem\u003eC. albicans\u003c/em\u003e, \u003cem\u003eC. glabrata\u003c/em\u003e, \u003cem\u003eC. parapsilosis\u003c/em\u003e, and \u003cem\u003eC. viswanathii\u003c/em\u003e exhibited trailing growth within a neutral-to-basic pH range (pH 6\u0026ndash;8.5), but not under acidic or alkaline conditions. Specifically, trailing was most prominent between pH 6 and 8 for \u003cem\u003eC. albicans\u003c/em\u003e and between pH 6 and 8.5 for the other species (Table S4A \u0026amp; S4B).\u003c/p\u003e \u003cp\u003eRevival of trailing phenotype in isolates that lost the trait\u003c/p\u003e \u003cp\u003eWe next sought to determine whether the trailing phenotype could be restored in strains that had lost it. The induction protocol successfully restored the trailing phenotype in two of the five \u003cem\u003eC. tropicalis\u003c/em\u003e isolates (Tr 37 and Tr 38) that had lost it. This revival occurred following 12 passages under high fluconazole stress (4 \u0026micro;g/mL). For the remaining three isolates, escalating the drug concentration to 8 \u0026micro;g/mL did not induce trailing (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). No significant changes in MIC values were observed post-induction, indicating revival of trailing without full resistance development.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetails of isolates for revival of trailing\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial MIC to Flu\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLost trailing phenotype after\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo of passages of Flu induction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCurrent Flu MIC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr 32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12 t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr 34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25 t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr 36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12 t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr 37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25 t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25 t\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTr 38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5 t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5 t\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe \u0026ldquo;\u003cb\u003et\u003c/b\u003e\u0026rdquo; after MIC value indicates trailing to fluconazole.\u003c/p\u003e \u003cp\u003ePopulation Analysis Profiling (PAP) Assay\u003c/p\u003e \u003cp\u003eTo assess subpopulation-level azole tolerance, we performed population analysis profiling (PAP) on all trailing isolates using fluconazole-gradient Yeast Extract Agar with Glucose (YAG) plates. Log₁₀-diluted inocula (10\u0026sup3; to 10⁶ CFU/mL) were spotted, and the resulting colony-forming units (CFU/mL) were plotted against drug concentrations. As anticipated, growth was suppressed at the MIC, yet low-level growth persisted at supra-MIC concentrations in all isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), confirming the presence of heteroresistance.\u003c/p\u003e \u003cp\u003eWe quantified this tolerance by calculating the area under the curve (AUC) for each isolate and normalizing it against the control \u003cem\u003eC. glabrata\u003c/em\u003e 1646 strain to generate an Area under the Curve Ratio (AUCR). The AUCR analysis provided a sensitive measure of heteroresistance, revealing that while MIC values did not predict the size of the tolerant subpopulation, all isolates exhibited AUCR values indicative of fluconazole tolerance. Future studies will expand PAP to a larger cohort to establish a definitive heteroresistance threshold for \u003cem\u003eC. tropicalis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is known that 1% \u0026minus;\u0026thinsp;95% of subpopulations can grow at supra-MIC. The MICs however do not correlate with the fraction of subpopulations growing at higher concentrations of the drug. All the isolates tested so far have shown \u0026ldquo;tolerance\u0026rdquo;. However, the PAP assay being a known standard for in vitro detection of tolerance/heteroresistance, gives an AUCR unique to every isolate and does not correlate with the MIC of the respective isolate, i.e., isolates with similar MICs do not however have the similar AUCRs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe phenomenon of trailing growth, clinically conceptualized as antifungal tolerance, presents a persistent and confounding challenge in medical mycology. Originally characterized as a technical limitation of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method M27-A, trailing describes the ambiguous growth pattern where an isolate appears susceptible at the standard 24-hour reading but exhibits significant, often resistant-range growth at 48 hours. This \"low-high\" MIC phenotype complicates definitive susceptibility reporting (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Crucially, emerging clinical and experimental evidence suggests this in vitro artifact may obscure a more complex biological reality with direct therapeutic implications. Studies have demonstrated that isolates displaying pronounced trailing can remain clinically susceptible, responding effectively to fluconazole therapy in both animal infection models and human cases, thereby implying that the earlier 24-hour MIC may offer a more accurate reflection of \u003cem\u003ein vivo\u003c/em\u003e drug efficacy (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This discordance is clinically significant, as highlighted by cases where infections with trailing isolates persist despite an apparently susceptible MIC, underscoring the critical need for more discriminative diagnostic assays to reliably separate true susceptibility from phenotypic tolerance (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe clinical interpretation of trailing growth remains a subject of active debate, rooted in seemingly contradictory data. Some clinical reports posit that infections caused by trailing isolates typically respond to standard fluconazole regimens, reinforcing the validity of the 24-hour reading for predicting patient outcomes (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Experimental models have sought to clarify this correlation. One pivotal study using a murine model of invasive candidiasis demonstrated that fluconazole treatment significantly improved survival rates in animals infected with both susceptible and trailing isolates, but not in those infected with genetically resistant strains, a finding corroborated by reduced fungal kidney burden (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Mechanistically, the trailing phenotype shares features with resistance; for example, \u003cem\u003eC. albicans\u003c/em\u003e isolates with strong trailing to fluconazole have been found to harbor mutations in \u003cem\u003eERG11\u003c/em\u003e and show upregulation of efflux pumps, hallmarks of azole resistance (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). However, \u003cem\u003ein vivo\u003c/em\u003e evidence frequently contradicts this, showing that such trailing isolates retain responsiveness to azole therapy (\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). This paradox is further complicated by drug-specific effects, as illustrated by a \u003cem\u003eGalleria mellonella\u003c/em\u003e larvae model, where voriconazole treatment failed to improve survival against trailing \u003cem\u003eC. albicans\u003c/em\u003e, suggesting that for this triazole, trailing might indeed mask clinically relevant resistance (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePopulation-based studies add another layer of nuance. Data from the extensive CANDIPOP study indicate that \u003cem\u003ein vitro\u003c/em\u003e trailing does not necessarily translate to clinical failure, with isolates from successfully treated patients showing no association between trailing and increased mortality (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Other patient data support this, indicating that trailing offers no significant survival advantage to yeast during azole therapy (\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). A prevailing hypothesis is that the partial growth inhibition characteristic of trailing may be sufficient for a competent host immune system to eradicate the infection, effectively decoupling the phenotype from therapeutic failure in immunocompetent hosts. Yet, this relationship falters in vulnerable populations. Rosenberg et al., who framed the phenomenon as \"tolerance,\" found that isolates from immunocompromised patients with persistent candidemia exhibited higher tolerance levels than those from patients who responded to fluconazole, implicating high tolerance in infection persistence despite treatment (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe trailing/tolerance phenotype is remarkably plastic, influenced by a network of extrinsic factors beyond genetics. A meticulous investigation demonstrated that commonly co-administered non-antifungal medications can significantly modulate \u003cem\u003eC. albicans\u003c/em\u003e susceptibility to fluconazole, with some drugs exerting antagonistic effects that exacerbate both resistance and tolerance, the latter defined here as the growth of microcolonies within an inhibition zone (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). This underscores that susceptibility interpretations for trailing isolates require extreme caution, as both fungal adaptation and ultimate treatment response are difficult to predict (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Fundamentally, tolerance operates as a concentration-independent subpopulation effect. As demonstrated via disc diffusion, increasing drug concentrations enlarge the zone of inhibition, but the fraction of the population that grows as microcolonies remains constant, a finding consistent across studies (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This tolerance is inversely correlated with intracellular drug accumulation, distinguishing it from classical resistance (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdding to this complexity, we report for the first time that the trailing phenotype itself is unstable and can be lost. Environmental factors significantly modulate its expression. Long-term storage led to a loss of trailing in 62.5% of \u003cem\u003eC. tropicalis\u003c/em\u003e, 50% of \u003cem\u003eC. albicans\u003c/em\u003e, and 33.3% of \u003cem\u003eC. glabrata\u003c/em\u003e isolates, with a 43% overall loss noted after 12 months. Furthermore, extreme pH conditions (beyond pH 5\u0026ndash;8) were observed to disrupt trailing expression. This aligns with prior work by Ribeiro et al., who found \u003cem\u003eC. tropicalis\u003c/em\u003e loses its trailing ability under acidic conditions (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), and Rosenberg et al., who noted tolerance levels decrease at lower pH (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Temperature shifts also exert influence, with higher temperatures (37\u0026ndash;41\u0026deg;C) potentially reducing the trailing subpopulation, while lower temperatures may induce stress responses that enhance it, without altering the core MIC or inhibition zone diameter (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This collective evidence positions trailing not as a fixed genetic trait, but as a form of phenotypic heterogeneity subject to environmental regulation (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA central mechanistic hypothesis for tolerance involves the slow growth dynamics of a subpopulation under antifungal stress. This reduced metabolic rate may enable these cells to endure drug exposure more effectively than their faster-growing counterparts, representing a bet-hedging survival strategy. The inverse correlation between tolerance and intracellular drug accumulation suggests an active cellular process, potentially involving efflux pumps or altered membrane permeability, that mitigates cytotoxic drug effects (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This survival strategy has direct clinical ramifications: the persistence of these tolerant subpopulations is believed to contribute to infection recurrence and therapeutic failure, as evidenced in multiple clinical cases (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo address the diagnostic shortfall, Population Analysis Profiling (PAP) has emerged as a pivotal tool. This assay quantifies heteroresistance by plotting the proportion of a population that grows across a gradient of drug concentrations. The resulting Area Under the Curve (AUC) serves as a quantitative measure of tolerance. Crucially, two isolates with identical fluconazole MICs can have vastly different PAP-AUCs; the isolate with the higher AUC (indicating a larger tolerant subpopulation) is prognostically more likely to be difficult to eradicate \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Ben-Ami et al. revealed that 57.6% of clinical \u003cem\u003eC. glabrata\u003c/em\u003e isolates displayed fluconazole heteroresistance via PAP, with higher AUC values correlating with higher MICs and a greater risk of treatment failure (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Similarly, applying PAP to \u003cem\u003eC. tropicalis\u003c/em\u003e has successfully identified tolerant subpopulations that survive fluconazole exposure without acquiring stable genetic resistance (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The derivative metric, the Area Under the Concentration-Response curve (AUCR), further refines this analysis by integrating drug exposure and biological response, revealing distinct, clinically relevant tolerance patterns (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the phenotypic landscape of trailing and tolerance in \u003cem\u003eCandida\u003c/em\u003e spp., particularly \u003cem\u003eC. tropicalis\u003c/em\u003e, is multidimensional. It arises from a confluence of stable subpopulation dynamics, adaptive slow growth, active drug efflux mechanisms, and profound responsiveness to environmental cues like pH, temperature, and concomitant drugs. These factors collectively fuel the persistence of infections and pose formidable challenges for therapy. The clinical implications are profound, necessitating a paradigm shift beyond standard MIC-based approaches. Incorporating functional assays like PAP into translational research frameworks is essential to distinguish tolerance from resistance, guide tailored therapeutic strategies, and ultimately improve outcomes for patients with recalcitrant or relapsing candidiasis. Future research must prioritize elucidating the molecular regulators of this phenotypic heterogeneity to identify novel targets for adjuvant therapies designed to disarm these fungal survival strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by the Indian Council of Medical Research (ICMR Grant ID No. Myco/Adhoc/4/2022-ECD-II)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSnigdha Reddy contributed to the conception and design of the study, data acquisition and analysis, manuscript drafting, and approved the final version.Saikat Paul contributed to the conception and design of the study, data analysis, manuscript drafting and critical revision, and approved the final version.Shivaprakash M. Rudramurthy contributed to critical revision of the manuscript and approved the final version.Harsimran Kaur contributed to critical revision of the manuscript and approved the final version.Anup K. Ghosh contributed to the conception and design of the study, manuscript drafting and critical revision, approved the final version, and takes responsibility for the integrity of the work.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors duly acknowledge the Indian Council of Medical Research (ICMR), Government of India for financial supports.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets analyzed and used during the study are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFiracative C. Invasive fungal disease in humans: are we aware of the real impact? Mem Inst Oswaldo Cruz. 2020 Oct 9;115:e200430. \u003c/li\u003e\n\u003cli\u003eDeorukhkar SC, Saini S, Mathew S. Non-albicans Candida Infection: An Emerging Threat. Interdiscip Perspect Infect Dis. 2014;2014:615958. \u003c/li\u003e\n\u003cli\u003eAbdel-Hamid RM, El-Mahallawy HA, Abdelfattah NE, Wassef MA. The impact of increasing non-albicans Candida trends on diagnostics in immunocompromised patients. Braz J Microbiol. 2023 Dec;54(4):2879-2892. \u003c/li\u003e\n\u003cli\u003eGuinea J. Global trends in the distribution of Candida species causing candidemia. Clin Microbiol Infect. 2014 Jun;20 Suppl 6:5-10. \u003c/li\u003e\n\u003cli\u003eChen P, Chuang Y, Wu U, et al. Clonality of Fluconazole-Nonsusceptible Candida tropicalis in Bloodstream Infections, Taiwan, 2011\u0026ndash;2017. \u003cem\u003eEmerging Infectious Diseases\u003c/em\u003e. 2019;25(9):1668-1675. \u003c/li\u003e\n\u003cli\u003eChen PY, Chuang YC, Wang JT, Sheng WH, Yu CJ, Chu CC, Hsueh PR, Chang SC, Chen YC. Comparison of epidemiology and treatment outcome of patients with candidemia at a teaching hospital in Northern Taiwan, in 2002 and 2010. J Microbiol Immunol Infect. 2014 Apr;47(2):95-103. \u003c/li\u003e\n\u003cli\u003eTan BH, Chakrabarti A, Li RY, Patel AK, Watcharananan SP, Liu Z, Chindamporn A, Tan AL, Sun PL, Wu UI, Chen YC; Asia Fungal Working Group (AFWG). Incidence and species distribution of candidaemia in Asia: a laboratory-based surveillance study. Clin Microbiol Infect. 2015 Oct;21(10):946-53. \u003c/li\u003e\n\u003cli\u003eRobbins N.Wright GD.Cowen LE. Antifungal Drugs: The Current Armamentarium and Development of New Agents. Microbiol Spectr. 2016 Oct;4(5).\u003c/li\u003e\n\u003cli\u003eRosenberg A, Ene IV, Bibi M, Zakin S, Segal ES, Ziv N, Dahan AM, Colombo AL, Bennett RJ, Berman J. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat Commun. 2018 Jun 25;9(1):2470.\u003c/li\u003e\n\u003cli\u003eFranconi I, Lupetti A. In Vitro Susceptibility Tests in the Context of Antifungal Resistance: Beyond Minimum Inhibitory Concentration in \u003cem\u003eCandida\u003c/em\u003e spp. J Fungi (Basel). 2023 Dec 12;9(12):1188. \u003c/li\u003e\n\u003cli\u003eWiederhold NP. Antifungal Susceptibility Testing: A Primer for Clinicians. Open Forum Infect Dis. 2021 Sep 9;8(11):ofab444. \u003c/li\u003e\n\u003cli\u003eMarcos-Zambrano LJ, Escribano P, Sanchez-Carrillo C, Bouza E, Guinea J. Scope and frequency of fluconazole trailing assessed using EUCAST in invasive Candida spp. isolates. Med Mycol. 2016;54(7):733\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eYang Y-L, Lin C-C, Chang T-P, Lauderdale T-L, Chen H-T, Lee C-F, et al. (2012) Comparison of Human and Soil \u003cem\u003eCandida tropicalis\u003c/em\u003e Isolates with Reduced Susceptibility to Fluconazole. PLoS ONE 7(4): e34609. \u003c/li\u003e\n\u003cli\u003eEl-Halfawy OM, Valvano MA. Antimicrobial heteroresistance: an emerging field in need of clarity. Clin Microbiol Rev. 2015;28(1):191\u0026ndash;207.\u003c/li\u003e\n\u003cli\u003eBand VI, Weiss DS. Heteroresistance: A cause of unexplained antibiotic treatment failure? PLoS Pathog. 2019;15(6):e1007726.\u003c/li\u003e\n\u003cli\u003eHeyman G, Jonsson S, Fatsis-Kavalopoulos N, Hjort K, Nicoloff H, Furebring M, et al. Prevalence, misclassification, and clinical consequences of the heteroresistant phenotype in Escherichia coli bloodstream infections in patients in Uppsala, Sweden: a retrospective cohort study. Lancet Microbe. 2025;6(4):101010.\u003c/li\u003e\n\u003cli\u003eStone NR, Rhodes J, Fisher MC, Mfinanga S, Kivuyo S, Rugemalila J, et al. Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis. J Clin Invest. 2019;129(3):999\u0026ndash;1014.\u003c/li\u003e\n\u003cli\u003eBen-Ami R, Zimmerman O, Finn T, Amit S, Novikov A, Wertheimer N, Lurie-Weinberger M, Berman J. Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence. mBio. 2016 Aug 2;7(4):e00655-16. \u003c/li\u003e\n\u003cli\u003eGautier C, Maciel EI, Ene IV. Approaches for identifying and measuring heteroresistance in azole-susceptible Candida isolates. Microbiol Spectr. 2024;12(4):e0404123.\u003c/li\u003e\n\u003cli\u003eDornelas-Ribeiro M, Pinheiro EO, Guerra C, Braga-Silva LA, Carvalho SM, Santos AL, et al. Cellular characterisation of Candida tropicalis presenting fluconazole-related trailing growth. Mem Inst Oswaldo Cruz. 2012;107(1):31\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eSmith WL, Edlind TD. Histone deacetylase inhibitors enhance Candida albicans sensitivity to azoles and related antifungals: correlation with reduction in CDR and ERG upregulation. Antimicrob Agents Chemother. 2002;46(11):3532\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eMarr KARustad TR, Rex JH, White TC. The Trailing End Point Phenotype in Antifungal Susceptibility Testing Is pH Dependent. Antimicrob Agents Chemother. 1999 Jun;43(6):1383-6. \u003c/li\u003e\n\u003cli\u003eOstrosky-Zeichner L, Rex JH, Pfaller MA, Diekema DJ, Alexander BD, Andes D, Brown SD, Chaturvedi V, Ghannoum MA, Knapp CC, Sheehan DJ, Walsh TJ. Rationale for reading fluconazole MICs at 24 hours rather than 48 hours when testing Candida spp. by the CLSI M27-A2 standard method. Antimicrob Agents Chemother. 2008 Nov;52(11):4175-7. \u003c/li\u003e\n\u003cli\u003eRueda C, Puig-Asensio M, Guinea J, Almirante B, Cuenca-Estrella M, Zaragoza O; CANDIPOP Project from GEIH-GEMICOMED (SEIMC) and REIPI. Evaluation of the possible influence of trailing and paradoxical effects on the clinical outcome of patients with candidemia. Clin Microbiol Infect. 2017 Jan;23(1):49.e1-49.e8. \u003c/li\u003e\n\u003cli\u003eChen P-Y, Chuang Y-C, Wu U-I, Sun H-Y, Wang J-T, Sheng W-H, Chen Y-C, Chang S-C. Mechanisms of Azole Resistance and Trailing in \u003cem\u003eCandida tropicalis\u003c/em\u003e Bloodstream Isolates. \u003cem\u003eJournal of Fungi\u003c/em\u003e. 2021; 7(8):612. \u003c/li\u003e\n\u003cli\u003eRevankar SG, Kirkpatrick WR, McAtee RK, Fothergill AW, Redding SW, Rinaldi MG, Patterson TF. Interpretation of trailing endpoints in antifungal susceptibility testing by the National Committee for Clinical Laboratory Standards method. J Clin Microbiol. 1998 Jan;36(1):153-6. \u003c/li\u003e\n\u003cli\u003eArthington-Skaggs BA, Warnock DW, Morrison CJ. Quantitation of Candida albicans ergosterol content improves the correlation between in vitro antifungal susceptibility test results and in vivo outcome after fluconazole treatment in a murine model of invasive candidiasis. Antimicrob Agents Chemother. 2000 Aug;44(8):2081-5. \u003c/li\u003e\n\u003cli\u003eBinder U, Aigner M, Risslegger B, H\u0026ouml;rtnagl C, Lass-Fl\u0026ouml;rl C, Lackner M. Minimal Inhibitory Concentration (MIC)-Phenomena in \u003cem\u003eCandida albicans\u003c/em\u003e and Their Impact on the Diagnosis of Antifungal Resistance. J Fungi (Basel). 2019 Sep 4;5(3):83.\u003c/li\u003e\n\u003cli\u003eLee MK, Williams LE, Warnock DW, Arthington-Skaggs BA. Drug resistance genes and trailing growth in Candida albicans isolates. J Antimicrob Chemother. 2004 Feb;53(2):217-24. \u003c/li\u003e\n\u003cli\u003eArthington-Skaggs BA, Lee-Yang W, Ciblak MA, Frade JP, Brandt ME, Hajjeh RA, Harrison LH, Sofair AN, Warnock DW; Candidemia Active Surveillance Group. Comparison of visual and spectrophotometric methods of broth microdilution MIC end point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of fluconazole and itraconazole against trailing and nontrailing Candida isolates. Antimicrob Agents Chemother. 2002 Aug;46(8):2477-81.\u003c/li\u003e\n\u003cli\u003eLee MK, Kim HR, Kang JO, Kim MN, Kim EC, Kim JS, Kim JJ, Park YJ, Song W, Shin JH, Lee KM, Lee NY, Lee M, Lee WG, Lee CK, Lee HJ, Chang CL, Choi TY. Susceptibility and trailing growth of Candida albicans to fluconazole: results of a Korean multicentre study. Mycoses. 2007 Mar;50(2):148-9. \u003c/li\u003e\n\u003cli\u003eArthington-Skaggs BA, Warnock DW, Morrison CJ. Quantitation of Candida albicans ergosterol content improves the correlation between in vitro antifungal susceptibility test results and in vivo outcome after fluconazole treatment in a murine model of invasive candidiasis. Antimicrob Agents Chemother. 2000 Aug;44(8):2081-5.\u003c/li\u003e\n\u003cli\u003eOdabasi Z, Paetznick VL, Rodriguez JR, Chen E, Rex JH, Leitz GJ, Ostrosky-Zeichner L. Lack of correlation of 24- vs. 48-h itraconazole minimum inhibitory concentrations with microbiological and survival outcomes in a guinea pig model of disseminated candidiasis. Mycoses. 2010 Sep;53(5):438-42. \u003c/li\u003e\n\u003cli\u003eObermeier M, Esparza-Mora MA, Heese O, Cohen N, Varma SJ, Tober-Lau P, Hartl J, Kurth F, Berman J, Ralser M. Non-antifungal medications administered during fungal infections drive drug tolerance and resistance in \u003cem\u003eCandida albicans\u003c/em\u003e. J Med Microbiol. 2025 Jul;74(7):002046. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"C. tropicalis, Azole, Resistance, Tolerance, Trailing, Antifungal","lastPublishedDoi":"10.21203/rs.3.rs-8484141/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8484141/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAzole antifungal \"trailing growth\" in \u003cem\u003eCandida\u003c/em\u003e spp., a tolerance phenotype distinct from resistance, complicates interpretation of \u003cem\u003ein vitro\u003c/em\u003e susceptibility and may contribute to persistent candidemia, especially in resource-limited settings. This study investigated the stability, characteristics, and mechanisms of azole trailing, primarily in the emerging pathogen \u003cem\u003eCandida tropicalis\u003c/em\u003e. From 847 clinical \u003cem\u003eC. tropicalis\u003c/em\u003e isolates (2022\u0026ndash;2024) screened by CLSI broth microdilution (BMD), 6.85% (n\u0026thinsp;=\u0026thinsp;58) exhibited trailing, most frequently with fluconazole. Disk diffusion with elevated drug concentrations revealed microcolonies within inhibition zones, confirming tolerance to supra-MIC levels. Longitudinal monitoring revealed phenotypic instability: trailing was lost in 37.5% of \u003cem\u003eC. tropicalis\u003c/em\u003e, 50% of \u003cem\u003eC. albicans\u003c/em\u003e, and 66.7% of \u003cem\u003eC. glabrata\u003c/em\u003e isolates after 8\u0026ndash;12 months of storage at -80\u0026deg;C. This loss was also induced by repetitive freeze-thaw cycles in some isolates. Trailing was pH-dependent, consistently observed only at pH 6\u0026ndash;8. Serial induction under fluconazole pressure successfully revived the phenotype in some isolates. Population Analysis Profiling (PAP) of nine trailing \u003cem\u003eC. tropicalis\u003c/em\u003e isolates confirmed the presence of drug-tolerant subpopulations, akin to a heteroresistance reference strain. These findings demonstrate that azole trailing in \u003cem\u003eC. tropicalis\u003c/em\u003e is a reversible, pH-dependent tolerance phenotype mediated by a minor subpopulation, which is unstable under routine laboratory storage. The discrepancy between \u003cem\u003ein vitro\u003c/em\u003e susceptibility results and this tolerant phenotype underscores the need for adjunctive assays, like PAP, to better characterize antifungal response and improve clinical correlation.\u003c/p\u003e","manuscriptTitle":"Instability and Subpopulation Dynamics of Azole Trailing Phenotypes in Clinical Candida tropicalis Isolates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 18:16:43","doi":"10.21203/rs.3.rs-8484141/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-12T13:13:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-05T08:20:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-30T13:59:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289396492844188959238361578532381437343","date":"2026-01-26T06:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162704583384632174388383298683133305494","date":"2026-01-22T06:52:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T15:46:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-06T07:11:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-06T07:08:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycopathologia","date":"2025-12-30T20:26:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"810fd44f-779f-4ac0-8bc6-4178225b1ad5","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T15:58:47+00:00","versionOfRecord":{"articleIdentity":"rs-8484141","link":"https://doi.org/10.1007/s11046-026-01074-5","journal":{"identity":"mycopathologia","isVorOnly":false,"title":"Mycopathologia"},"publishedOn":"2026-04-27 15:56:50","publishedOnDateReadable":"April 27th, 2026"},"versionCreatedAt":"2026-01-23 18:16:43","video":"","vorDoi":"10.1007/s11046-026-01074-5","vorDoiUrl":"https://doi.org/10.1007/s11046-026-01074-5","workflowStages":[]},"version":"v1","identity":"rs-8484141","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8484141","identity":"rs-8484141","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}