First Report of Fluconazole‐Resistant Candida parapsilosis Harbouring the G458S ERG11p Substitution in a Brazilian Hospital

ABSTRACT

Background

Fluconazole‐resistant Candida parapsilosis is a matter of concern.

Objectives

To investigate fluconazole resistance among Candida parapsilosis complex isolates from patients admitted to three Brazilian hospitals, and to characterise resistance mechanisms and clonal relatedness.

Patients/Methods

A total of 76 C. parapsilosis complex isolates were collected from 60 patients hospitalised at three medical centres (H1–H3) located in Vitória, Espírito Santo state (2017–2023). Of these, 38 isolates were obtained from bloodstream cultures, four from catheter tips and 34 from an ICU surveillance study conducted at H1. In vitro susceptibility testing was performed according to the E.Def 7.4 EUCAST method. C. parapsilosis sensu stricto isolates were genotyped using microsatellite markers, and the ERG11 gene was sequenced in resistant isolates.

Results

Candida parapsilosis sensu stricto was the most common species (65/76 isolates; 85.5%), followed by C. orthopsilosis (n = 8, 10.5%), and C. metapsilosis (n = 3, 4%). Among the C. parapsilosis sensu stricto isolates, 5/65 (7.7%) were fluconazole‐resistant, harboured the G458S ERG11p substitution, and were detected in 2019 and 2020. Only one fluconazole‐resistant isolate was also voriconazole‐resistant; all isolates were susceptible to the remaining agents. Fluconazole‐resistant isolates sourced from H2 were grouped into two unrelated genotypes, and recovered from patients with haematological malignancies, tumours and central nervous system disorders. Only two patients had a history of azole exposure.

Conclusions

This is the first report of fluconazole‐resistant C. parapsilosis sensu stricto isolates harbouring the G458S ERG11p substitution in Brazil. Here reported isolates showed resistance to fluconazole alone, which is an unusual pattern among C. parapsilosis sensu stricto isolates harbouring the G458S substitution.

1. Introduction

The Candida parapsilosis species complex is part of the gastrointestinal tract and skin microbiota and may act as a transient coloniser. It has a high capacity to form biofilms on abiotic surfaces and to cause hospital outbreaks through horizontal transmission via healthcare workers' hands [1, 2, 3, 4].

Over the past two decades, the C. parapsilosis complex has emerged as an increasingly common cause of invasive disease, accounting for 19%–35% [5, 6] of candidemia cases among neonates, intensive care unit (ICU) patients, transplant recipients and patients with COVID‐19 [1]. In Brazil, the C. parapsilosis complex has been identified as a leading cause of candidemia, responsible for 20%–30% [7] of bloodstream infections and ranking second only to C. albicans in most surveillance studies [8].

Moreover, therapeutic options against the C. parapsilosis complex remain restricted to a few antifungal classes. Although echinocandins are recommended as first‐line therapy for invasive candidiasis, their minimum inhibitory concentration (MICs) values against the C. parapsilosis complex are typically higher than those against other Candida species [9, 10]. In recent years, this situation has become more concerning due to the rising resistance to azoles in C. parapsilosis sensu stricto, a class of antifungals widely used worldwide for the treatment and prophylaxis of candidaemia [11]. Azole resistance is particularly alarming in low‐ and middle‐income countries, where fluconazole remains the most widely used antifungal for the management of Candida spp. infections, and alternative therapeutic options are either prohibitively expensive or not readily accessible [12, 13].

Fluconazole resistance in C. parapsilosis sensu stricto is mainly driven by mutations in the ERG11 gene, particularly the Y132F substitution, among others, which impair drug‐target binding [14, 15]. Genotyping of C. parapsilosis sensu stricto is essential for monitoring clonal spread, especially in isolates harbouring the Y132F substitution, which are more prone to clonal transmission than other fluconazole‐resistant strains [16]. Isolates with the Y132F substitution have been increasingly reported worldwide, including in Brazil, where a retrospective study (2011–2012) identified nine C. parapsilosis sensu stricto bloodstream isolates with elevated fluconazole MICs and harbouring the Y132F ERG11p substitution [14]. Subsequent studies have found that fluconazole‐resistant isolates in Brazil mostly harbour the Y132F substitution [15, 17]. Clonal spread of isolates has been confirmed as 60% of bloodstream episodes in two Brazilian referral centres during the COVID‐19 pandemic were caused by a genotype carrying this substitution [3]. Collectively, these findings underscore that clonal dissemination of isolates harbouring the Y132F substitution is a key driver of fluconazole resistance within Brazilian healthcare settings.

Given the rising number of Brazilian hospitals reporting fluconazole‐resistant strains, we consider it particularly important to investigate, for the first time, the epidemiology of resistance to the C. parapsilosis complex in the metropolitan region of Vitória, capital of Espírito Santo, located in the Southeastern Region, the most populous Brazilian geographic region. Here, we report for the first time fluconazole‐resistant C. parapsilosis sensu stricto isolates harbouring the G458S ERG11p substitution in Brazil.

2. Materials and Methods

2.1. Study Design, Setting and Isolates Studied

The study included 76 C. parapsilosis complex isolates collected between 2017 and 2023 at three hospitals (H1–H3) in the city of Vitória, Espírito Santo state, Brazil. Out of the 76 isolates, 60 were from patients (one isolate per patient), 15 from hospital workers and one from a surface sample. Of these, 38 isolates were obtained from bloodstream infections and four from catheter tips of patients without candidaemia. Additionally, 34 isolates were sourced from an ICU surveillance study conducted at hospital H1 in 2021 and 2022, aimed at evaluating oral colonisation by Candida spp. and invasive candidiasis [18]. The surveillance study screened 209 patients, of whom 18 yielded C. parapsilosis complex isolates (17 from the oral mucosa and one from a corneal fragment). Among the 19 healthcare professionals screened, colonisation was detected in 15 (eight from ear lobes, seven from hands); one isolate was also recovered from the arm of a patient's bed.

Presumptive identification of isolates was performed using classical phenotypic methods, including microscopic morphology on cornmeal–Tween 80 agar (Oxoid, Basingstoke, Hampshire, England) and carbohydrate fermentation and assimilation tests, as previously described [19]. Final species‐level identification was confirmed by Matrix‐Assisted Laser Desorption Ionisation–Time of Flight Mass Spectrometry (MALDI‐TOF MS) using Biotyper 3.3 software and a Microflex LT spectrometer (Bruker Daltonics, Massachusetts, USA), according to the manufacturer's instructions. Isolates were stored and frozen at −70°C in Yeast‐Extract‐Peptone‐Dextrose broth (Sigma‐Aldrich, St. Louis, Missouri, USA) supplemented with 20% glycerol at the Collection of Fungi of the Medical Mycology Research Centre (CIMM)/Federal University of Espirito Santo (UFES).

2.2. In Vitro Antifungal Susceptibility Testing

Stored isolates were revived on Sabouraud Dextrose Agar (SDA) (Oxoid, Basingstoke, Hampshire, England) and on CHROMagar Candida (Becton, Dickinson, Heidelberg, Baden‐Württemberg, Germany) and incubated for 48 h at 35°C ± 2°C to ensure purity [19]. Antifungal susceptibility testing was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution reference method (E.Def 7.4) using tissue‐culture–treated microplates (CELLSTAR) (Greiner Bio‐One, Frickenhausen, Germany) [20]. The antifungal agents tested included amphotericin B, fluconazole, voriconazole, posaconazole, micafungin and anidulafungin (Sigma‐Aldrich, Madrid, Spain), as well as isavuconazole (Basilea Pharmaceutica, Basel, Switzerland) and ibrexafungerp (Scynexis Inc., Durham, North Carolina, USA). C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as quality control strains. Epidemiological cutoff values (ECOFFs) or wild‐type upper limits (for isavuconazole and ibrexafungerp) were used to classify isolates as non‐wild type [11, 21, 22]. If EUCAST breakpoints were available, isolates were then classified as resistant or susceptible. All isolates were tested once, but resistant or non‐wild‐type isolates were retested for confirmation.

2.3. ERG11 Gene Sequencing and Microsatellite Genotyping

The ERG11 gene was sequenced in fluconazole‐resistant C. parapsilosis sensu stricto isolates, as previously described [11]. All C. parapsilosis sensu stricto isolates, regardless of azole susceptibility profile, were subjected to microsatellite genotyping using four species‐specific markers (CP1, CP4a, CP6, and B) according to established protocols [23]. Amplicons were resolved by capillary electrophoresis using the 3130xl Genetic Analyser (Thermo Fisher Scientific/Applied Biosystems, Carlsbad, USA), with the GeneScan ROX size standard. Electropherograms were analysed using GeneMapper v4.0 software (Thermo Fisher Scientific/Applied Biosystems, Foster City, USA). Genetic relationships among isolates were inferred by constructing a minimum spanning tree with BioNumerics version 7.6 (Applied Maths, Sint‐Martens‐Latem, Belgium). The allelic composition of each locus was converted into binary data by recording the presence or absence of each allele. Singletons were defined as genotypes observed only once. Isolates were considered identical when sharing the same alleles at all loci and were grouped into clusters when the same genotype was identified in two or more patients; genotypes differing at only one microsatellite marker were classified as clonally related [24]. Distinct genotypes were coded as CP‐X ( C. parapsilosis ), where X corresponds to the internal code assigned in our collection. Genotypes involving fluconazole‐resistant isolates were compared with 141 genotypes comprising 727 fluconazole‐resistant isolates with different molecular mechanisms previously characterised; only two genotypes included isolates harbouring the Y132F substitution.

2.4. Ethical Committee Approval

This study was approved by the Ethics and Research Committee of the Federal University of Espírito Santo (UFES) and the Capixaba Institute for Health Education, Research and Innovation (ICEPi)/SESA (protocol no. 4374111).

3. Results

The distribution of isolates across participating hospitals was as follows: H1 (n = 55, 72.4%), H2 (n = 19, 25%) and H3 (n = 2, 2.6%). C. parapsilosis sensu stricto was the most frequently identified species (n = 65/76; 85.5%), followed by C. orthopsilosis (n = 8/76, 10.5%) and C. metapsilosis (n = 3/76.4%), which exclusively sourced H1 (Figure 1).

FIGURE 1.

Distribution of species within the Candida parapsilosis complex, identified by MALDI‐TOF MS, among the 76 isolates recovered from the Medical Mycology Research Centre collection included in this study.

All C. orthopsilosis and C. metapsilosis isolates were susceptible or wild type to all antifungal agents tested. All C. parapsilosis sensu stricto isolates were susceptible to amphotericin B, posaconazole, micafungin, anidulafungin and ibrexafungerp wild type (Table 1). However, six C. parapsilosis sensu stricto isolates were fluconazole‐non‐wild‐type. Of these, five isolates (n = 5/65; 7.7%) were fluconazole‐resistant and voriconazole non‐wild type (one of which was voriconazole‐resistant), and three were non‐wild‐type for isavuconazole (Table 1); the remaining fluconazole‐susceptible isolate (MIC = 4 mg/L) was also susceptible to the other azoles and exhibited a wild‐type ERG11 gene sequence.

TABLE 1.

In vitro susceptibility profile of the 65 C. parapsilosis sensu stricto isolates to eight antifungal agents, as determined by the EUCAST broth microdilution method.

	MIC distributions (no. of isolates at each MIC, in mg/L)																No. isolates (%)
	0.001	0.002	0.004	0.008	0.016	0.03	0.06	0.125	0.25	0.5	1	2	4	8	16	32	Non‐wild type	Resistance
Amphotericin B	—	—	0	0	0	0	0	0	0	40	25	0	—	—	—	—	0%	0%
Fluconazole	—	—	—	—	—	—	1	1	3	22	24	8	1	3	2	0	9.2%	7.7%
Voriconazole	—	2	2	19	27	6	4	1	3	1	0	0	—	—	—	—	7.7%	1.5%
Posaconazole	—	1	2	32	28	0	0	0	0	0	0	0	—	—	—	—	0%	0%
Isavuconazole	0	1	18	29	10	4	2	1	0	0	—	—	—	—	—	—	4.6%	ND
Micafungin	—	—	—	—	1	0	0	0	0	0	47	17	0	0	—	—	0%	0%
Anidulafungin	—	—	—	—	1	0	0	0	0	1	14	38	11	0	—	—	0%	0%
Ibrexafungerp	—	—	—	—	0	0	0	0	1	30	34	0	0	0	—	—	0%	ND

Note: Numbers in bold indicate resistant isolates, while underlined values denote non‐wild‐type phenotypes according to ECOFFs or previously reported wild‐type upper limits. Cells marked with “—” indicate antifungal concentrations not tested; ND indicates data not determined.

The 65 C. parapsilosis sensu stricto isolates were grouped into 51 genotypes, of which 44 were singletons and seven were clusters. None of the clusters involved isolates from different hospitals. Singleton genotypes and five clusters consisted exclusively of fluconazole‐susceptible isolates. These clusters were found in hospitals H1 or H2, involved between two and five isolates each, and were associated with blood cultures from ICU patients (three clusters); an oral mucosa isolate from an ICU patient and an environmental isolate from the bed he occupied (CP‐0670); or blood cultures and a catheter tip isolate from unrelated patients admitted to three wards (CP‐0915) (Table 2).

TABLE 2.

Overview of fluconazole‐susceptible clusters C. parapsilosis sensu stricto, indicating clinical source, and ward of patient admission at the moment of isolation.

Genotypes	Hospital	Isolate code	Source	Unit	Isolation data
CP‐0919	H2	BR12‐245	Bloodstream	Oncology ward	17 April 2019
		BR12‐246		ICU	17 April 2019
		BR12‐247		ICU	17 April 2019
CP‐0930	H1	BR12‐268	Bloodstream	ICU	03 August 2021
		BR13‐317			01 November 2021
CP‐0670 a	H1	BR12‐290	Bed arm2	ICU	01 November 2021
		BR12‐294	Oral mucosa1		01 November 2021
CP‐0915	H1	BR12‐261	Bloodstream	General surgery	22 September 2020
		BR12‐278	Bloodstream	ICU	06 July 2023
		BR12‐238	Bloodstream	Cardiology	04 June 2018
		BR12‐239	Bloodstream	General surgery	22 September 2020
		BR12‐241	Catheter tip	ICU	24 September 2018
CP‐0922	H2	BR13‐251	Bloodstream	ICU	18 December 2019
		BR13‐258	Bloodstream	ICU	14 February 2020

Abbreviations: H1, Hospital 1; H2, Hospital 2; ICU, intensive care unit.

^a Cluster CP‐0670 involved isolates from ¹an oral mucosa sample of one patient (BR12‐294), admitted to the ICU and ²an arm of the bed (BR12‐290) where he stayed.

Resistant isolates harbouring the G458S ERG11p substitution were grouped into two unrelated clusters, both involving blood cultures from patients admitted to hospital H2. Cluster CP‐0918 comprised four fluconazole‐resistant isolates from the oncology ward. The first candidaemia episode caused by this genotype was identified in February 2019; another episode occurred in July 2019, and two additional episodes were diagnosed in February 2020 (Table 3). Cluster CP‐0917 included three isolates from blood cultures of ICU patients: one fluconazole‐resistant isolate detected in February 2019 and two fluconazole‐susceptible isolates detected in February 2019 and February 2020, respectively (Table 3 and Figure 2).

TABLE 3.

Overview of fluconazole‐resistant C. parapsilosis sensu stricto isolates harbouring the G458S substitution from hospital H2, the genotypes found, and clinical data from patients involved.

Genotype	Isolate code	Isolation date	AMB	FLU	VOR	POS	ISA	MFG	AFG	IBX	Underlying conditions	Inpatient unit	Previous exposure to antifungals
CP‐0918	BR13‐243	15 February 2019	1	8	0.25	0.008	0.06	1	2	1	Acute lymphoblastic leukaemia	Oncology ward	No
	BR12‐249	03 July 2019	0.5	16	0.25	0.008	0.125	2	2	1	Acute lymphoblastic leukaemia		Micafungin, voriconazole, and amphotericin B
	BR13‐254	14 February 2020	1	8	0.25	0.016	0.03	1	4	1	Ependymoma		Fluconazole and micafungin
	BR13‐256	14 February 2020	1	16	0.5	0.008	0.06	1	2	1	Acute myeloid leukaemia		Micafungin
CP‐0917 a	BR13‐252	18 December 2019	1	8	0.125	0.008	0.016	1	4	1	Congenital hydrocephalus and Ventriculitis	ICU	Micafungin and amphotericin B

Abbreviations: AFG, anidulafungin; AMB, amphotericin B; FLU, fluconazole; IBX, ibrexafungerp; ICU, intensive care unit; ISA, isavuconazole; MFG, micafungin; POS, posaconazole; VOR, voriconazole.

^a Cluster CP‐0917 involved one fluconazole‐resistant C. parapsilosis sensu stricto isolate (BR13‐252), and two additional fluconazole‐susceptible C. parapsilosis sensu stricto isolates recovered on 15 February 2019 and 14 February 2020.

FIGURE 2.

Microsatellite dendrogram showing the clustering and fluconazole‐susceptibility profiles (resistant or susceptible) of C. parapsilosis sensu stricto isolates obtained from three hospitals in Espírito Santo. Isolates within the same cluster are shown in distinct colours. Green squares represent fluconazole‐susceptible isolates, while red squares indicate fluconazole‐resistant isolates. ¹Isolates from samples collected from healthcare professionals.

Patients with candidaemia caused by resistant isolates had the following underlying conditions: acute lymphoblastic leukaemia (40%), acute myeloid leukaemia (20%), ependymoma (20%), hydrocephalus and ventriculitis (20%). Only two of these patients had received azole treatment before the blood culture isolation (Table 3).

4. Discussion

This study reports, for the first time, fluconazole‐resistant C. parapsilosis sensu stricto isolates harbouring the G458S ERG11p substitution causing candidaemia in patients admitted to a Brazilian hospital. These resistant isolates were grouped into two unrelated genotypes and were detected over a relatively short period.

Candida parapsilosis complex exhibits intrinsically reduced susceptibility to echinocandins, and the emergence of azole resistance further restricts the therapeutic options available for treating affected patients. Azole resistance in C. parapsilosis sensu stricto is particularly concerning, given its role as a leading cause of candidemia globally, especially among vulnerable populations such as neonates and immunocompromised individuals. This issue is especially critical in low‐ and middle‐income countries, where fluconazole remains the most accessible antifungal agent for both treatment and prophylaxis. In contrast, C. orthopsilosis and C. metapsilosis are less prone to developing antifungal resistance, as demonstrated in our study and consistent with previous reports [25].

Azole‐resistant C. parapsilosis sensu stricto isolates have been reported in several countries, including Brazil. However, the impact appears to vary across hospitals, suggesting localised outbreaks rather than widespread dissemination. A recent study conducted in Madrid exemplifies this, showing that fluconazole‐resistant isolates affected certain hospitals while others remained unaffected [11].

Azole resistance in C. parapsilosis sensu stricto is predominantly associated with the Y132F substitution, first reported in 2004 and now documented in an increasing number of countries [26]. Isolates harbouring Y132F have demonstrated strong potential for clonal spread and for causing hospital outbreaks. Routine screening for Y132F is not commonly conducted in Brazilian clinical laboratories, limiting the ability to assess the true extent of the problem. Thomaz et al. [27] described a large outbreak in a reference oncology hospital, where approximately 68% of isolates were fluconazole‐resistant, many harbouring the Y132F substitution. These isolates were recovered from blood cultures, hospital surfaces, and healthcare workers' hands, and microsatellite typing confirmed their clonal nature. Such findings highlight the critical role of horizontal transmission in the spread of resistant clones, contributing to persistent and difficult‐to‐control hospital outbreaks.

During the COVID‐19 pandemic, an inter‐hospital outbreak was reported in which a cardiology‐specialised hospital received patients from, or connected to, an oncology hospital experiencing a persistent outbreak of fluconazole‐resistant C. parapsilosis sensu stricto. Many isolates from the cardiology hospital also harboured the Y132F substitution, indicating cross‐institutional dissemination [3]. To our knowledge, this is the first report here of the G458S substitution in azole‐resistant C. parapsilosis sensu stricto isolates in Brazil, expanding the understanding of antifungal resistance in the country.

Given the potential for resistant isolates to spread, genotyping is essential for tracking clonal dissemination and informing infection control strategies. Outbreaks involving fluconazole‐resistant C. parapsilosis sensu stricto clones in intensive care and oncology units have been reported in Europe and Asia, often involving isolates with the Y132F substitution [24, 28]. A recent set of microsatellite markers has been helpful for genotyping fluconazole‐resistant C. parapsilosis sensu stricto isolates [23]. Using these markers, we identified two genotypes involving five fluconazole‐resistant isolates harbouring the G458S substitution. These isolates were recovered from patients with haematological malignancies over 2 years, suggesting clonal dissemination.

Outbreaks involving G458S‐harbouring isolates are rarely reported, likely because of their lower transmissibility. The small outbreak described here reflects lapses in infection control at hospital H2. All patients in the oncology ward were hospitalised in the same open‐plan ward, which consisted of a large, shared area separated only by partitions and a single entrance door. The patients' beds were located in close physical proximity to one another. In addition, healthcare professionals were shared among all patients, and each child was allowed to be accompanied by an adult caregiver. As a result, there was substantial circulation of caregivers, visitors and healthcare staff within the ward, creating a high level of interpersonal contact and movement. This is supported by the fact that four out of the seven clusters identified—including those with fluconazole‐susceptible isolates—were found in hospital H2, and 12 of the 19 (63%) isolates from this hospital were involved in clusters.

Despite the more extensive sampling at hospital H1, horizontal transmission appeared to be lower than at H2, with only 9 of the isolates 55 (16%) involved in clusters. The lower horizontal transmission may be attributable to educational activities implemented at hospital H1 shortly after the surveillance study began. Nevertheless, both fluconazole‐susceptible and ‐resistant clusters were identified, indicating some degree of nosocomial transmission, particularly in the ICU. For example, genotype CP‐0919 involved patients from different wards within a short timeframe, while genotype CP‐0915 appears to be endemic to the hospital. Given the small size of hospitals H1 and H2, healthcare professionals may have been circulating between the ICU and other wards.

The G458S isolates were grouped into two unrelated genotypes, neither of which was related to previously studied G458S genotypes. These genotypes may have emerged independently and affected patients who had not received prior azole treatment, suggesting limited hospital transmission. This limited transmission in the hospital environment aligns with previous observations of G458S‐harbouring isolates, which appear less prone to widespread dissemination. The detection of genetically unrelated isolates with the same mutation may reflect local selective pressures—potentially due to antifungal use, environmental residues, disinfectants, or veterinary applications. The presence of G458S‐harbouring strains in H2 may reflect the presence of that strain in the region. However, since no epidemiological surveillance studies or environmental studies were simultaneously conducted in the area, such hypotheses are to be explored.

While Y132F‐harbouring isolates typically exhibit resistance to fluconazole and voriconazole, G458S‐harbouring isolates are generally associated with pan‐azole resistance [11, 22]. However, in our study, the five G458S isolates were fluconazole‐resistant, with only one showing voriconazole resistance; all remained susceptible to posaconazole and wild‐type for isavuconazole. This unexpected resistance phenotype suggests a more complex genetic background, warranting further investigation through whole‐genome sequencing.

This study has several limitations. No systematic surveillance was conducted across the three participating hospitals during the study period, meaning the isolates studied may not fully represent the broader epidemiological situation. This limitation is particularly relevant for hospital H2, where G458S isolates were detected in candidaemia patients, and other reservoirs of resistant isolates may exist. Additionally, the small number of resistant isolates and their confinement to a single hospital limit the generalisability of our findings. Despite these limitations, our work provides valuable insights into the molecular epidemiology of antifungal resistance in C. parapsilosis in Brazil. It underscores the urgent need for multicentre studies to assess the actual burden and dissemination of resistant strains.

In conclusion, this study reports two relevant observations. This is the first case series of fluconazole‐resistant C. parapsilosis sensu stricto isolates harbouring the G458S Erg11p substitution in patients treated at a Brazilian hospital. Secondly, these isolates exhibited an unexpected resistance profile since they were not pan‐azole resistant. Their detection in candidaemia patients, clonal relatedness and persistence within a hospital setting underscore failures in infection control that may facilitate the spread of resistant strains.

Conflicts of Interest

J.G. has received funds for participating in educational activities organised on behalf of Gilead, Pfizer and Mundipharma; he has also received research funds from FIS, Gilead, F2G, Shionogi, Scynexis, Mundipharma and Cidara outside the submitted work. P.E. has received funds for participating in educational activities organised on behalf of Gilead, Pfizer and has also received research funds from FIS. The remaining authors do not disclose any conflicts of interest within the scope of this manuscript.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.