Exploring fungal diversity in Vietnam: A citizen science initiative

Abstract

Invasive fungal diseases are increasing globally, causing a large burden of disease in vulnerable populations. At the same time, antifungal resistance is rapidly emerging. Affordable nationwide and regional surveillance of fungal pathogens is needed.

We have adapted a citizen-science methodology developed by a United Kingdom research group to study six key fungi in Vietnam, where there is no existing formal surveillance. These pathogens were ranked as high or critical in the World Health Organization fungal priority pathogens list and recognized as major disease-causing agents in Vietnam. Secondary school students (n = 90) in Hanoi were our citizen scientists, collecting soil (n = 90) and air (n = 90) samples for fungal identification and characterisation of drug-susceptibility in the laboratory.

Pilot studies confirmed the effectiveness of our revised isolation procedure, which used selective culture media to improve the isolation of target fungi. Through active school and student involvement, optimized protocols, and our cost-effective sampling, the study could be scaled across Vietnam.

We demonstrate an approach to fungal surveillance which also enhances science education, and awareness of fungal diseases. It addresses critical healthcare and education challenges in Vietnam while combating the growing issues of invasive fungal diseases and antifungal resistance.

1. Introduction

Invasive fungal diseases (IFDs) are important global health issues that impact vulnerable populations globally. Cases of IFDs are rising as the at-risk population continues to expand. People most at risk are those with underlying health problems or a weakened immune system, such as chronic lung disease, prior tuberculosis (TB), human immunodeficiency virus (HIV), cancer, and diabetes mellitus. The World Health Organization (WHO) developed the first Fungal Priority Pathogens List (FPPL), aiming to drive further research and policy interventions to combat this growing issue [1]. Nineteen pathogens that can cause invasive acute and subacute systemic fungal infections for which drug resistance or other treatment and management challenges exist, were selected to rank, then categorized into three priority groups (critical, high, and medium) [1]. The critical group includes Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, and Candida albicans. The high group includes Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., eumycetoma causative agents, Mucorales, Fusarium spp., Candida tropicalis, and Candida parapsilosis. Finally, pathogens in the medium group are Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and Paracoccidioides spp [1]

In Vietnam, with a high burden of tuberculosis (172,000 annual incidence) and HIV (213,724 total cases in 2020), about 2.5 million people were estimated to be affected by various fungal infections [2,3]. Candida and Aspergillus were identified as the leading pathogens, and other concerns included Mucorales, Cryptococcus, Histoplasma, and Talaromyces due to their high mortality rates [4]. Between 2012 and 2020, the burden increased by 1.6%, mainly due to the increasing cases of leukemia, organ transplant, and chronic obstructive pulmonary disease hospitalizations [4,5]. The diagnosis and treatment of IFDs are challenged by limited access to quality diagnostics and treatment as well as emergence of antifungal resistance (AMR) in Vietnam. Environmental studies in South Vietnam in 2019 revealed very high rates of azole-resistant Aspergillus species in the environment, exceeding 50% [6,7]. A retrospective study in Cho Ray hospital showed that Candida spp. (i.e. C. tropicalis, C. albicans, C. glabrata), were resistant to amphotericin B (7.3%), triazoles (4.6%), and caspofungin (2.7%) [8]. The excessive use of anti-fungal agents in Vietnamese healthcare and agriculture likely leads to AMR in other pathogenic fungi as well. Therefore, it is important to consider One Health approaches which recognize the interconnection between people, animals, plants, and their shared environment, and conduct surveillance in the environment as well as in clinical diagnostic laboratories – unfortunately, the former is frequently overlooked; a challenge we address with the methodology presented in this paper.

Active surveillance programs are difficult to implement in lower middle-income countries due to financial and capacity restraints. Therefore, we explored a citizen science approach inspired by a national survey of the distribution of azole-resistant Aspergillus fumigatus in the UK in 2020 [9,10]. This is an appropriate methodology which can be adapted, improved, and implemented in Vietnam. In this study, we invited secondary school students as citizen scientists and broadened the scope to include 6 different genera of fungi (i.e., Aspergillus spp., Candida spp., Mucorales spp., Talaromyces marnefei, Cryptococcus neoformans, and Histoplasma capsulatum). We developed and piloted methodologies to determine the presence of fungal pathogens in the environment and their resistance rates in one province of Vietnam. Our province-wide study will explore the feasibility and cost-effectiveness of a citizen science approach as a scalable method for nationwide and potentially regional surveillance.

2. Methods

2.1. Identification of target fungal pathogens

The WHO FPPL, released in 2022, is a pivotal report designed to assist member countries in priotizing fungal pathogens based on 10 assessment criteria such as their significance to public health (e.g. fatality rate, annual incidence, among others), drug resistance, research need and epidemiology [1]. The report highlights that its findings should be localised to specific geographic areas for appropriate implementation. Therefore, we selected target pathogens based on a combination of their priority level in the WHO FPPL report and their disease burden in Vietnam (including cases incidence and mortality rate) [1,4]. Our focus was on pathogenic fungi that derive from the natural environment.

2.2. Selection of citizen scientists and approach to sampling

In collaboration with colleagues at the Ministry of Education and Training (MOET) in Vietnam, we identified sixth-grade students (n = 90) from secondary schools in Hanoi as suitable citizen scientists. The selection was made because sixth graders were already introduced to microbiology, making them well-suited for our project. Through active participation in the study, they would follow simple instructions in our video tutorial to collect soil (n = 90) and air (n = 90) samples from their surroundings (supplementary Fig. 1). Since they would not conduct any processing, their participation presents no risks to their health or well-being.

All samples would be delivered to the Hanoi Medical University laboratory where they would be further processed to isolate and identify species (according to the Standard Operating Procedures (SOPs) developed below). We targeted diverse fungal human pathogens known to be present in the environment and associated with significant burdens of disease in Vietnam. We would blend both culture and non-culture-based methods, as appropriate. Once processing is complete, we would share pictures and fun fact cards about isolated fungi to our citizen scientists to answer the question “Which fungi are present in your environment?”

2.3. Literature search

The success of this citizen science project relies on a solid and effective methodology. We conducted a comprehensive and thorough literature search in PubMed, Web of science, and Scopus to achieve 2 main objectives: (1) assessing the viability of isolating target fungi from environmental samples (air, soil) and (2) developing and refining SOPs for each specific fungus.

Our search used keywords, including “[fungus name]” and terms including “environment”, “identification”, “isolation”, “soil”, “air”, “MALDI-TOF”, “molecular identification” and “susceptibility testing”. Through these preliminary searches, we identified additional synonyms necessary for a comprehensive search, such as “anti-fungal resistance”, “fungal recovery”, “detection”, “yeast”, “mold”, “PCR assay”, and so on.

Throughout the literature review, we prioritized research articles presenting comprehensive processes that were feasible for our laboratory capacity. The final list of references served as the basis for establishing initial SOPs of sample collection, processing, and further testing for each specific fungus.

2.4. Revised SOPs for target fungus isolation

For Aspergillus, we have previously developed and fine-tuned SOPs [6,7]. Additionally, we incorporated air sampling and processing techniques inspired by the UK citizen science project [9,10]. This allowed us to benefit from established best practices and adapt them to our research effectively.

To develop SOPs for the other target fungi, we carried out pilot studies based on published methods. We assessed their effectiveness and made modifications and improvements, as necessary. These refined SOPs were adopted for the main study.

2.4.1. Fungal identification

Initial identification of fungal isolates was performed using macroscopic and microscopic observation, with reference to the textbook “Description of Medical Mycology” [11]. Preliminary morphology-based fungal identification was then validated with additional methods, such as a proteomic method (Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry [MALDI-TOF MS]) or molecular methods (Polymerase Chain Reaction [PCR] assay and DNA sequencing), enabling identification to species level [[12], [13], [14], [15], [16], [17]].

2.4.2. Anti-fungal susceptibility testing

SOPs for anti-fungal susceptibility testing against Aspergillus were well-developed in our previous studies [6,7]. These followed the European Committee on Antimicrobial Susceptibility Testing (EUCAST) microdilution method, which provided regularly updated clinical breakpoint values, to assess anti-fungal susceptibility profiles of target fungi (version E.DEF 9.4 April 2022 for molds and version E.DEF 7.4 October 2023 for yeasts) [18,19].

For susceptibility testing, we selected antifungals highlighted in Vietnamese government treatment guidelines (2021 Vietnam Ministry of Health (MOH) guidelines on the diagnosis and treatment of IFD in clinical practice) which included only licensed antifungals covered by Vietnam Health Insurance system [20].

3. Results

3.1. Identification of target fungal pathogens

Six different genera / species of pathogenic fungi (namely Aspergillus spp., Candida spp., Mucorales spp., Talaromyces marneffei, Cryptococcus spp., and Histoplasma capsulatum) were selected in this study. We chose these target fungi because they were listed as priority pathogens by WHO and have a strong evidence as major disease-causing agents in Vietnam [1,4]. As detailed in Table 1, Candida and Aspergillus were the foremost leading fungal pathogens in Vietnam, causing severe infections and high morbidity. Due to high mortality rates in vulnerable population, Mucorales, Cryptococcus, Histoplasma, and Talaromyces were also included in the study despite their low infection incidences.

Table 1.

List of target fungal pathogens in this study.

Target fungi		WHO FPPL group priority level 1	Incidence per 100 k population 4	Prevalence per 100 k population 4
Candida	C. auris	Critical	- Oral candidiasis: 83.7 - Oesophageal candidiasis: 20.1 - Candidaemia: 11.7 - Candida peritonitis: 1.4	- Recurrent vaginal candidiasis >4/times/year: 4047
	C. albicans	Critical
	C. glabrata	High
	C. tropicalis	High
	C. parapsilosis	High
	C. krusei	Medium
Aspergillus	A. fumigatus	Critical		- Chronic pulmonary aspergillosis: 120.2 - Allergic bronchopulmonary aspergillosis: 77.6 - Severe asthma with fungal sensitisation: 102.4 - Invasive aspergillosis: 24.4
Cryptococcus	C. neoformans	Critical	0.5
	C. gattii	Medium
Talaromyces marneffei		Medium	1.7
Histoplasma capsulatum		High	0.2
Mucorales spp.		High	0.2

3.2. Literature review and practical aspects

Our literature review revealed that a large number of publications have successfully isolated and identified the target fungi from environmental sources.

Aspergillus, Mucorales, Cryptococcus, and Talaromyces are commonly found in various natural environments such as air, soil, plants, and others [6,7,9,[21], [22], [23], [24], [25], [26], [27], [28], [29], [30]]. Meanwhile, Histoplasma and Candida are primarily associated with soil samples [[31], [32], [33], [34], [35], [36], [37]]. There is evidence of airborne C. auris among Candida spp., but only under conditions of aerosol turbulence [37]. Considering the distribution of these fungi in the environment, our study did not aim to recover Candida from air samples (Table 2). Histoplasma capsulatum is classified as Risk Group 3 which must be cultured at biosafety level 3; due to limited laboratory capacity, we decided to rely on molecular techniques to safely detect the presence of this fungus in the environment [38].

Table 2.

Distribution of target fungi in the environment.

No.	Target fungi	Environmental distribution
		Soil source		Air source
		Presence	Reference	Presence	Reference
1	Cryptococcus spp.	+	27, 28	+	27
2	Candida spp.	+	34–37	+⁎	37
3	Aspergillus spp.	+	6, 7, 9, 23	+	6, 7, 9, 21, 22
4	Histoplasma capsulatum	+	31–33	−
5	Mucorales spp.	+	24, 25	+	21, 26
6	Talaromyces marneffei	+	29, 30	+	29

+, positive evidence documented of isolating target fungi from sample type.

-, no evidence documented of isolating target fungi from sample type.

^⁎Only Candida auris found to be airborne under conditions of aerosol turbulence.

Air samples were collected using MicroAmp™ clear adhesive film (Applied Biosystems™, UK) following the methodology developed by Shelton et al, to isolate airborne fungi [9]. Simultaneously, soil samples were collected and processed using our previously established methods [6,7]. Two grams of soil were suspended in 18 mL of sterilized 0.85% saline supplemented with 0.05% Tween 20, vigorously vortexed, and diluted to 5 × 10⁻³ and 10⁻³ concentrations. The two diluted solutions were cultured on agar plates at 37 °C.

Many studies have recommended Sabouraud dextrose agar (SDA) supplemented with chloramphenicol (Cm) as a favorable medium for cultivating various molds and yeasts [30,39]. Therefore, we used SDA (Oxoid™, UK) supplemented with 0.1 g/L Cm (Sigma-Aldrich®, USA) (similar concentration to our previous studies [6,7]) and conducted pilot studies from March to June 2023 to test the culture medium and determine recovery rates of fungi. Preliminary results demonstrated the feasibility of the isolation SOP, however this non-selective medium allowed overgrowth of fast-growing and abundant environmental molds and yeasts (e.g., Mucorales and Aspergillus), especially in air samples, which can obscure and obstruct the isolation of Cryptococcus and Talaromyces marneffei. It was therefore necessary to modify and improve differential media in order to successfully isolate Cryptococcus (C. neoformans and C. gattii) and Talaromyces marneffei.

3.3. Revised primary isolation SOP

To increase the likelihood of successfully isolating the target fungi, we optimized the primary isolation SOP by using the following selective culture media. Fig. 1 illustrates the approach to primary isolation for each target fungi (i.e., the medium, temperature, incubation time) and outlines the adjustments we made to culture conditions based on insights from the literature review and small-scale pilots.

Fig. 1.

Optimizing SOPs for target fungus isolation.

Birdseed: Birdseed agar supplemented with 20 U/L penicillin, 40 mg/L gentamicin, 0.2 g/L chloramphenicol, 1.5 μg/L benomyl, and 0.1 g/L biphenyl;

DRBC: Dichloran-rose bengal chloramphenicol (containing 0.1 g/L chloramphenicol);

SDA + Cm: Sabouraud Dextrose Agar supplemented with 0.1 g/L chloramphenicol;

SDA + CC: Sabouraud Dextrose Agar supplemented with 0.04 g/L chloramphenicol and 0.5 g/L cycloheximide;

CHROMagar: Chromogenic agar Candida (containing 0.5 g/L chloramphenicol).

We chose Birdseed agar (Himedia®, India) supplemented with 20 U/L penicillin (Sigma-Aldrich®, USA), 40 mg/L gentamicin (Sigma-Aldrich®, USA), 0.2 g/L chloramphenicol (Sigma-Aldrich®, USA), 1.5 μg/L benomyl (Supelco, USA) and 0.1 g/L biphenyl (Sigma-Aldrich®, USA) as a suitable medium for isolating Cryptococcus [28,40]. For Talaromyces marneffei, a dimorphic fungus with slow growth rate, we selected SDA supplemented with 0.04 g/L chloramphenicol and 0.5 g/L cycloheximide at 25 °C and 37 °C [30]. Even though Candida could be successfully isolated on SDA supplemented with 0.1 g/L chloramphenicol, we preferred to use a chromogenic medium (CHROMagar™ Candida, CHROMagar, France) in order to readily allow presumptive speciation of Candida isolates based on their color characteristics [41,42]. As Candida spp. is rarely found in the air, we focused on isolating Candida spp. from soil.

The growth of certain fast-growing molds can be inhibited by using Dichloran Rose Bengal Chloramphenicol agar (DRBC agar) (Oxoid™, UK) [43]. Consequently, we added DRBC agar to isolate the target fungi from air samples, preventing their loss due to Mucorales overgrowth. Meanwhile, SDA supplemented with 0.1 g/L chloramphenicol was an option for fungal isolation from soil.

After the primary culture, all isolates were sub-cultured onto new SDA plates, identified and stored for further studies. For safety reasons, we did not develop a culture SOP for Histoplasma. Instead, genomic DNA was directly extracted from soil samples using cetyl trimethylammonium bromide extraction bufer and phenol-chloroform-isoamyl alcohol as described previously [44]. We amplified the Hc100 gene, which encodes a 100 kDa protein essential for H. capsulatum survival [45,46]. The PCR product is considered as a proof for the presence of H. capsulatum in soil.

3.4. Revised SOPs for fungal identification

• i) Morphological identification method

On birdseed agar, Cryptococcus can be identified as dark brown, yeast-like colonies [11]. Talaromyces marneffei colonies produce a red pigment diffusion on SDA, and appear in two different forms, mycelial at 25 °C and yeast-like at 37 °C [11]. As per the CHROMagar manual, certain Candida species can be differentiated based on their distinct pigmentation on Chromagar medium (https://www.chromagar.com/en/product/chromagar-candida/): green for C. albicans, mauve for C. glabrata, metallic blue for C. tropicalis, and fuzzy/pink for C. krusei. Other target fungi are preliminarily identified based on our research experience with reference to “Description of Medical Mycology” [11]. Any unidentified yeasts or molds obtained from samples would be subjected to advanced methods (MALDI-TOF, PCR or DNA sequencing).

• ii)Non-morphological identification methods

To support morphology-based fungal identification, we applied proteomic and molecular methods, as detailed in Table 3.

Table 3.

Non-morphological methods for fungal identification.

Target fungi	MALDI-TOF MS	Molecular method
Cryptococcus spp.	identifiable
Candida spp.	identifiable
Talaromyces marneffei	identifiable (for yeast form)	ITS sequencing and β-tubulin sequencing (for mycelium form)
Aspergillus spp.		ITS sequencing and β-tubulin sequencing
Mucorales spp.		ITS sequencing
Histoplasma capsulatum		Hc100 nested PCR assay

3.4.1. A proteomic method: MALDI-TOF MS

Numerous studies have demonstrated the efficacy of MALDI-TOF MS for the rapid identification of yeasts and molds [12,13,[47], [48], [49]]. However, due to the restricted mold database in our MALDI BioTyper system (MBT, Bruker Scientific), this method would only be used for yeast-like colonies like Candida spp., Cryptococcus spp., or Talaromyces marneffei. All isolates would be analysed in triplicate with the control strain Candida krusei ATCC 6258. Unidentified yeasts by MALDI-TOF MS would have their DNA extracted for sequencing.

3.4.2. Molecular method: PCR

For Histoplasma capsulatum, DNA was directly used in a nested PCR assay to detect Hc100 gene [50,51]. The PCR is performed as previously described, using two sets of primers specific for H. capsulatum: the external set HcI - HcII and the internal set HcIII – HcIV [50,51]. For other target pathogens, the internal transcribed spacer region (ITS1–5.8S-ITS2) enables the identification of many fungi to species level [[52], [53], [54]]. Among various primer sets used to target this region, we chose the primer pair ITS-1/ITS-4 [52,53,55]. For precise identification of Aspergillus spp. and Talaromyces marneffei species, β-tubulin primer pair (Bt2as/Bt2bs) would also be conducted [11,[56], [57], [58]]. Detailed primer information is provided in Table 4.

Table 4.

List of primers used in this study.

Target gene	Primers	Applied for
Hc100	HcI (5′-GCGTTCCGAGCCTTCCACCTCAAC-3′) HcII (5′-ATGTCCCATCGGGCGCCGTGTAGT-3′) HcIII (5′-GAGATCTAGTCGCGGCCAGGTTCA-3′) HcIV (5′-AGGAGAGAACTGTATCGGTGGCTTG-3′)	Histoplasma capsulatum
ITS1–5.8S-ITS2	ITS-1 (5′-TCCGTAGGTGAACCTGCGG-3′) ITS-4 (5′- TCCTCCGCTTATTGATATGC-3′)	All fungi
β-tubulin	Bt2as (5′- GGTAACCAAATCGGTGCTGCTT-3′) Bt2bs (5′-ACCCTCAGTGTAGTGACCCTTGGC-3′)	Aspergillus spp. Talaromyces marneffei

The DNA extraction and PCR SOPs, initially developed and optimized for Aspergillus in our previous study, were applied for other fungi in this research. The high yields and purity of DNA confirm the successful adaptation of this procedure. Total DNA was extracted using the MasterPure™ Yeast DNA purification kit (Lucigen® Corporation, Cambridge, UK), with the addition of a bead-beating step (1.0 mm Zirconia/Silica beads [Biospec Products, USA]) to effectively break down the cell wall [6,59]. In a 40-μL PCR reaction, the final concentration would include 1× Buffer, 2 mM MgCl₂, and 2 U Taq Polymerase (BIOTAQ™ DNA Polymerase kit, Bioline, BIO-21060), 0.4 μM of each forward and reverse primer, 0.3 mM dNTP Mix (Bioline, BIO-39028) and 50 ng DNA. PCR was performed under the following conditions: 95 °C for 5 min; followed by 30 cycles of 95 °C for 30 s, 65 °C for 20 s and 72 °C for 1 min; then final extension at 72 °C for 10 min.

PCR products, purified with QIAquick® PCR purification kit (Qiagen, 28,106), were sent for the external sequencing service (Azenta Life Sciences, USA). The nucleotide sequences obtained were compared with those already deposited in the data bank of the National Center for Biotechnology and Information (NCBI), using BLAST search tool [60]. The identification of the species was determined based on the best score.

3.5. Anti-fungal susceptibility testing

We tested susceptibility against seven anti-fungals, belonging to three classes: polyene (amphotericin B), azoles (including itraconazole, posaconazole, voriconazole, isavuconazole, fluconazole), and echinocandin (anidulafungin) (see Table 5).

Table 5.

Antifungals tested for susceptibility in target fungi.

Target fungi	Diseases	AMB	ITC	POS	VOR	ISA	FLU	AND
Cryptococcus spp.	Cryptococcosis	✓	✓	✓			✓
Candida spp.	Candidiasis	✓	✓	✓	✓		✓	✓
Aspergillus spp.	Aspergillosis	✓	✓	✓	✓	✓
Talaromyces marneffei	Talaromycosis	✓	✓		✓
Mucorales spp.	Mucormycosis	✓		✓		✓

AMB, Amphotericin B; ITC, Itraconazole; POS, Posaconazole; VOR, Voriconazole; ISA, Isavuconazole: FLU, Fluconazole; AND, Anidulafungin.

✓: Antifungal selected for the susceptibility testing of target fungi.

Candida and Aspergillus had complete treatment guidelines along with clinical breakpoints data [20,61]. For the susceptibility of Aspergillus spp., amphotericin B and 4 azoles (including itraconazole, posaconazole, voriconazole, and isavuconazole) were chosen. Candida spp. would be tested with 4 azoles (itraconazole, posaconazole, voriconazole and fluconazole) and anidulafungin. Although other echinocandins, such as micafungin and caspofungin, were also recommended for treating candidiasis, in this study we would test the susceptibility of Candida to anidulafungin only. This decision was attributed to the absence of clinical breakpoint for micafungin and caspofungin in the EUCAST database [61].

In the latest EUCAST update, Cryptococcus neoformans had only one clinical breakpoint for amphotericin B, while no clinical breakpoints have been established for Talaromyces marneffei and Mucorales [61]. Nonetheless, we still assessed their susceptibility to recommended antifungals, with the goal of determining the Minimal Inhibitory Concentration (MIC) distributions of these fungi in Vietnam. Hence, the susceptibility testing of these fungi would be conducted as followed:

• i.Cryptococcus spp.: amphotericin B, itraconazole, posaconazole and fluconazole;
• ii.Talaromyces marneffei: amphotericin B, itraconazole, voriconazole;
• iii.Mucorales spp.: amphotericin B, posaconazole and isavuconazole.

Due to biosafety concerns, we would not do the antifungal susceptibility test for Histoplasma capsulatum.

4. Discussion

To the best of our knowledge, this is the first description of a methodology for surveillance of multiple human pathogenic fungi from the environment, embracing the citizen science approach. Students' involvement in our project facilitated cost-effective collection of environmental samples at multiple sites, an approach that could easily be scaled to nationwide or regional research. Whilst conscious that involving high school students instead of employed field-workers may result in lower adherence to sampling strategies, we mitigated this risk by conducting detailed pre-sample training and providing pre-recorded online video demonstrations. Full assessment of results will reveal whether recovery rates using this approach are similar to our previous results with employed field-workers, as a marker of quality. We also balanced this risk against the benefits of engaging high school students in science projects and introducing One Health concepts to them.

We have built on the citizen science project run by Shelton and co-workers by adapting the methodology to suit the context of Vietnam and the global research trends by including six pathogenic fungal groups (namely: Aspergillus spp., Candida spp., Talaromyces marneffei, Cryptococcus spp., Mucorales spp., and Histoplasma capsulatum). These fungi are both global and local priorities [1,4]. The strategic selection of fungi, with a focus on those highly ranked and recognized as major disease-causing agents in Vietnam, is a critical factor for the project's future impact. Furthermore, integrating One Health approaches into this study promotes a sustainable research framework, fostering a comprehensive understanding of the intricate connection between environmental fungi, fungal infections, and antifungal resistance, including potential zoonotic risks in natural settings.

Including anti-fungal susceptibility testing for all species is another methodological advance. The incorporation of a simple, cost-effective sampling method, coupled with its proven effectiveness, also opens up the opportunity to conduct extensive AMR studies on a large scale, addressing an important gap, since many current AMR studies remain fragmented.

The educational aspect is also a significant highlight of our project. Specifically, in Vietnam, the Ministry of Education identifies 2 key challenges in incorporating Natural Sciences into the General Education Program: (i) facilities and equipment are lacking and (ii) teachers receive inadequate training and have limited opportunities for students to engage in practical scientific activities [62]. Therefore, our project will reward children with intellectually stimulating scientific engagement opportunities and assist them to approach science in a more interactive way. We also hope to raise public awareness of invasive fungal diseases in Vietnam.

In summary, the synergy of factors, including active school and student participation, strong collaboration from educational researchers, a well-selected list of target pathogens, and optimized SOPs, significantly enhances the project's feasibility.

CRediT authorship contribution statement

Tra-My N. Duong: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. Minh-Hang Le: Writing – review & editing, Formal analysis, Conceptualization. Thanh-Van Le: Writing – review & editing, Formal analysis, Conceptualization. Thuy T. Ha: Writing – review & editing, Investigation, Formal analysis, Conceptualization. Maryam Roudbary: Writing – review & editing, Investigation, Formal analysis. Justin Beardsley: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material: Supplementary Fig. 1 Project information and sampling instructions

Data availability

Data will be made available on request.