Aflatoxin profiles of Aspergillus flavus isolates in Sudanese fungal rhinosinusitis

Abstract Aspergillus flavus is a commonly encountered pathogen responsible for fungal rhinosinusitis (FRS) in arid regions. The species is known to produce aflatoxins, posing a significant risk to human health. This study aimed to investigate the aflatoxin profiles of A. flavus isolates causing FRS in Sudan. A total of 93 clinical and 34 environmental A. flavus isolates were studied. Aflatoxin profiles were evaluated by phenotypic (thin-layer and high-performance chromatography) and genotypic methods at various temperatures and substrates. Gene expression of aflD and aflR was also analyzed. A total of 42/93 (45%) isolates were positive for aflatoxin B1 and AFB2 by HPLC. When the incubation temperature changed from 28°C to 36°C, the number of positive isolates decreased to 41% (38/93). Genetic analysis revealed that 85% (79/93) of clinical isolates possessed all seven aflatoxin biosynthesis-associated genes, while 27% (14/51) of non-producing isolates lacked specific genes (aflD/aflR/aflS). Mutations were observed in aflS and aflR genes across both aflatoxin-producers and non-producers. Gene expression of aflD and aflR showed the highest expression between the 4th and 6th days of incubation on the Sabouraud medium and on the 9th day of incubation on the RPMI (Roswell Park Memorial Institute) medium. Aspergillus flavus clinical isolates demonstrated aflatoxigenic capabilities, influenced by incubation temperature and substrate. Dynamic aflD and aflR gene expression patterns over time enriched our understanding of aflatoxin production regulation. The overall findings underscored the health risks of Sudanese patients infected by this species, emphasizing the importance of monitoring aflatoxin exposure.


Introduction
Aspergillus flavus, a common airborne species, is frequently encountered in agricultural settings, where it is known for contaminating crops, particularly grains and peanuts.However, its significance extends beyond agriculture, as it plays a pivotal role in causing invasive aspergillosis (IA), notably fungal rhinosinusitis (FRS). 1 , 2 This fungus is responsible for 86% of FRS cases in Sudan, 3 highlighting its importance in arid regions compared to A. fumigatus , the global primary cause of IA and a top concern on the WHO's fungal priority pathogens list.FRS places a significant burden in arid regions, 4 with around 1.5 million cases annually reported in India and an estimated incidence of 200 cases per 100 000 in Sudan. 5 , 6An elevated incidence rate has also been observed beyond arid regions, evidenced by 392 cases per 100 000 individuals documented in Turkey . 7Notably , epidemiologic data on the disease in other countries of the world is scarce.FRS encompasses two distinct forms: invasive and non-invasive.The latter is milder and often chronic, primarily confined to the sinuses, while the invasive form is severe, but can also manifest as a chronic condition that can last for years. 8spergillus flavus is notorious for producing toxic metabolites and primarily mycotoxins that pose a severe threat to human health.Aflatoxin B1 (AFB1), a prominent mycotoxin produced by A. flavus , has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). 9Prolonged exposure to AFB1 can compromise cellmediated immunity, increasing susceptibility to various diseases. 10A study conducted in Japan demonstrated that aflatoxin could suppress leukocyte function and immune mechanisms, further emphasizing its detrimental effects. 11Although the detection of aflatoxin in humans in vivo has not been extensively explored, two studies in Japan have reported the presence of aflatoxin in autopsied materials from patients infected with A. flavus , 11 , 12 providing compelling evidence that the species can indeed produce aflatoxin in vivo .Given the capacity of A. flavus to produce aflatoxins, which can be chronically exposed to humans, there might be a potential influence on pathogenicity, and to possible predisposition of chronically infected individuals to nasopharyngeal carcinoma.Consequently, the investigation of the regularity of aflatoxin production and a deeper exploration of the mechanism governing this production in clinical settings remains imperative.
Aflatoxins are products of a complex biosynthesis pathway involving a minimum of 27 enzymatic reactions, known as the polyketide pathway. 13The genes responsible for encoding these enzymes are clustered, encompassing a total of 30 genes, and their expression is coordinated by two clusterspecific regulators: aflR and aflS . 14 , 15While aflR governs both aflatoxin and sterigmatocystin production, aflS (also known as aflJ ) is exclusively associated with aflatoxin regulation.A key structural gene in the biosynthetic pathway, aflD ( nor-1 ), encodes an enzyme responsible for catalyzing the conversion of the initial aflatoxin biosynthesis intermediate, norsolorinic acid, to averantin. 16Additional other key genes include aflM ( ver-1 ), aflO ( omtB ), and aflP ( omtA ), among others. 179][20][21] AflR, in particular, a positive regulator of the aflatoxin gene cluster, has been demonstrated to induce a significant upregulation, resulting in a 50fold increase in aflatoxin production in A. flavus . 22Furthermore, studies have shown that overexpression of aflR could influence the transcriptional and aflatoxigenic patterns in A. flavus . 23Notably, expression of those genes is largely affected by ecological factors and nutritional conditions, with studies indicating that climate changes, particularly exposure to elevated CO 2 concentrations, can significantly upregulate aflD and aflR gene expression resulting in higher production of toxin. 24 , 25While these studies addressed aflatoxin production in agricultural contexts, comprehensive investigations focusing on strains from clinical sources remain limited.Analysis of the aflatoxin characterization of clinical A. flavus may contribute to the understanding of pathogenicity and disease progression.
Several methods are currently employed for aflatoxin detection, including thin-layer chromatography (TLC), enzymelinked immunosorbent assay (ELISA), high-performance liquid chromatography with fluorescence detector (HPLC-FLD) or with tandem mass spectrometry (HPLC-MS/MS).TLC and ELISA are widely utilized to detect aflatoxins in various foods, offering sensitivity around 1-20 μg/kg and higher sensitivity but lower stability , respectively . 26 , 279][30] A comprehensive analysis of aflatoxins can be achieved by combining quantitative and qualitative methods.
Our investigation aimed to explore aflatoxin production in clinical and environmental isolates.An assessment was conducted to investigate the impact of different environmental variables, including temperature and growth media on aflatoxin production, by employing both qualitative and quantitative methods.Furthermore, aflatoxigenic genes were detected, thereby providing insights into the genetic mechanisms behind aflatoxin biosynthesis and gene expression profiles of aflR and aflD , with particular emphasis on settings that simulate the host environment.

Strains source, isolation, and identification of environmental strains
Ninety-three clinical Aspergillus flavus isolates (88 clinical isolates have been published previously 3 and five are shown in Supplementary Table 1 ) were acquired from patients diagnosed with FRS in Sudan maintained in 20% glycerol solution and stored at −80 • C. To enable a comparison between clinical and environmental sources, environmental isolates were originally designed to be collected from local surroundings.Unfortunately, access to the isolates from Sudan was not possible due to safety concerns.Therefore, A. flavus from other geographic regions were included as follows: 16 were extracted from crops, such as peanuts and corn, in China, 13 were obtained from the CBS reference collection (origin: England, Japan, South Africa, Uganda, and Brazil), and five were achieved from the Mycology Reference Laboratory at Radboudumc (The Netherlands).As negative controls for aflatoxin detection, three isolates ( A. terreus , Penicillium citrinum , and Trichoderma asperellum ) were included.Identification of the isolates involved a combination of morphological 31 and molecular analysis (GenBank accession number shown in Supplementary Table 1 ), 32 comprising sequencing of the ITS (Internal transcribed spacer) rDNA region, a segment of the BenA gene encoding β-tubulin, 33 and partial CaM gene encoding calmodulin. 34

Morphology and growth
Spore suspensions were prepared at a concentration of 1 × 10 6 spores/ml using a hemocytometer and observed with a light microscope (Zeiss Axio Scope, Carl Zeiss, Göttingen, Germany).A volume of 20 μl of the spore suspension was inoculated centrally on 9-cm Petri dishes with various media, including Sabouraud glucose agar (SDA, Thermo Fisher Scientific, MA, USA), Water agar (WA), RPMI agar, WA + 4% glucose, and Blood agar (BA: containing 5% sheep blood).Three replicates per sample were incubated under two distinct temperatures (28 ± 1 • C and 36 ± 1 • C) for 6 days.Micro-morphology was examined using a light microscope (Zeiss Axio Scope, Carl Zeiss, Göttingen, Germany).The images were annotated in Adobe Photoshop 2022.Diameters of the colonies were recorded after the termination of the incubation to compare the growth on different media.These cultures were applied for aflatoxin detection following the observation of morphology and growth.

Aflatoxin extraction
For aflatoxin extraction, agar plugs (5 plugs per plate, approx.0.5 g) were excised using a five-point sampling method. 21hose plugs were then mixed with 600 μl methanol (high grade) and subjected to 30 min of shaking using a TissueLyser (Xinyi-48, Ningbo, China).After centrifugation at 6000 rpm for 10 min, the supernatant was carefully transferred to a new tube. 35The extracts were directly filtered through a Nylon 13 mm, 0.22 μm filter (BKMAM-Lab Ltd, Changde, China) into amber HPLC vials for further analysis.

Quantitative analysis by TLC
Silica gel-coated plates (100 mm × 200 mm, GF254) were prepared with a marked origin line approximately 3 cm from the bottom.Standard samples of AFB1/AFB2/AFG1 and AFG2 (1 μg/ml) were spotted on the origin line and placed in development tanks containing a development solution [chloroform: acetone 92:8 (v/v)] for a duration of 30 min.Plates were then examined under UV light (WFH-203B, Shanghai, China) to detect aflatoxins based on characteristic fluorescence. 35 , 36alitative analysis by HPLC Standard stock solutions (10 μg/ml) were prepared.A mixture of standard working solution (AFB1 and AFG1: 100 ng/ml, AFB2 and AFG2: 30 ng/ml), as well as a standard series working solution were prepared in accordance with the standards outlined in the aflatoxin detection protocol (GB 5009.22-2016,https:// food.chemlinked.com/database/ view/ 555 ).Sample extracts were subsequently subjected to HPLC using an Ultimate 3000 system controller, 470 fluorescence detector (Thermo Scientific, MA, USA) (lexc, 360 nm; lem, 450 nm) and a C18 column (Agilent, 250 mm × 4.6 mm, 5 μm) all controlled by Ultimate 3000 software.A 50 μl of each sample was injected and column temperature was maintained at 35 • C. The analysis was performed at a flow rate of 1 ml/min of the mobile phase (40% methanol: 60% water) and the run time was set at 30 min.Post-column photochemistry derivatization (Ruipin Biology Ltd.Shanghai, China) was applied to the samples.Standard series working solutions were injected into HPLC to establish a standard curve ( Supplementary Table 2 ).The detection limit (LOD) and quantification limit (LOQ) were established using the standard deviation (SD) of the response (Sb) and the calibration curve's slope (m), as outlined by LOD = 3 Sb/m, LOQ = 10 Sb/m ( Supplementary Table 2 ).

Detection of aflatoxin biosynthesis-associated genes
Seven aflatoxin biosynthesis-associated genes ( aflD, aflM, aflO, aflP , aflQ aflR, and aflS ) were detected.DNA extraction, PCR (Polymerase Chain Reaction) amplification and gel electrophoresis of all isolates were performed following standard procedures. 37The seven pairs of primers used were in accordance with the specification outlined by Gallo et al. 38 PCR products of 10 randomly selected aflatoxin-producing isolates and 10 non-aflatoxin-producing isolates were chosen for Sanger Sequencer-based sequencing.Subsequently, the acquired sequences were assembled using SnapGene and aligned with the reference strains through Benchling ( https: // www.benchling.com/ ) (GenBank accession numbers shown in Supplementary Table 3 ).The graph of gene presence vs. absence was presented by chiplot ( https:// www.chiplot.online/).

Strain incubation
Ten high-level aflatoxin-producing A. flavus isolates including five from the environment and five from clinical origin were selected for gene expression study .Additionally , two aflatoxin-non-producing isolates that possessed all genes associated with aflatoxin biosynthesis were also included for comparison.The spore suspension was prepared as described previously.Each spore suspension (1 ml) was cultured in 12-well microplates using Sabouraud dextrose broth (SDB, Thermo Fisher Scientific, MA, USA) and RPMI 1640.All microplates were cultured under normal CO 2 (0.04%) and 5% CO 2 incubation conditions at 36 ± 1 • C for durations of 2, 4, 6, 9, and 12 days, respectively.Experiments were conducted in triplicate.RPMI 1640 was selected due to its common use in clinical research, and the 5% CO 2 concentration aimed to mimic human tissue.

RNA extraction and cDNA
Biomass harvested from the cultures was stored at −80 • C until RNA extraction.RNA was extracted using Trizol reagents (Thermo Fisher Scientific, MA, USA) and purified following established protocols. 39RNA concentration and quality were determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).Reversetranscription PCR (RT-PCR) was carried out using a two-step reverse-transcription real-time quantitative PCR (qPCR) kit.Initially, cDNA was synthesized from 1000 ng of total RNA, following the PrimeScript RT Reagent kit protocol (New England BioLabs Inc.).The resulting cDNA served as template amplification using LightCycler ® 480 SYBR Green I Master kit (Thermo Fisher Scientific, Wilmington, DE, USA).

qPCR analysis
The qPCR reactions were carried out using the Roche LightCycler ® 480 II instrument (Roche Exiqon, Mannheim, Germany).The assays were prepared in triplicate in optical 96-well reaction plates and sealed with optical adhesive covers.The SYBR Green methodology was performed, with each reaction containing 10 μl of SYBR mix 2 ×, 0.5 μl of each 10 μM primer, and 5 μl of each cDNA template, resulting in a final volume of 20 μl.A negative control without cDNA was included in every run.The thermal cycling protocol consisted of an initial pre-incubation at 95 • C for 5 min, followed by 45 cycles of 95 • C for 10 s and 55 • C for 20 s.Melting curve analysis was performed by heating to 95 • C for 5 s, 65 • C for 1 min and 97 • C with continuous fluorescence measurement to quantify the PCR product.The threshold cycle (Ct) value was determined using LightCycler ® 480 Software.Primer pairs F/R-aflD, aflR and β-tubulin were used to amplify the structural gene ( aflD ), the regulatory gene ( aflR ), and the housekeeping gene ( BenA ), respectively. 25elative quantification of the expression of aflD and aflR genes was carried out using BenA gene.The expression ratio was calculated following the previous method. 40Log2 values of the relative expression of the aflD and aflR genes were graphically represented.

Data analysis
The data analyses were conducted using analysis of variance ( Anova ) with the IBM SPSS 27 statistical analysis software.The results were reported as the mean ± SD.Statistically significant differences were observed by employing Duncan's novel multiple-range test at the significance levels of P = .05.

Morphology and growth
Isolates cultured on SDA showed yellow-green spores and typical conidia structure, with conidial emerging from the phialides located on the conidiophore vesicles (Fig. 1 ).On WA and RPMI agar, the isolates exhibited limited growth and de- generated sporulation, with observation of sparse conidia and an absence of characteristic Aspergillus head.Blood agar resulted in slow growth and a lack of sporulation, despite a notable increase in hyphal biomass thickness.

Aflatoxin detection by TLC
Of the 93 clinical isolates tested, 40% (37/93) demonstrated positive aflatoxin production when incubated on an SDA medium at 28 • C. Incubation at 36 • C resulted in a reduction in the proportion of aflatoxin-producing isolates, with only 25% (23/93) exhibiting positivity (Fig. 2 , Supplementary Table 4 ).For the environmental isolates, 47% (16/34) displayed aflatoxin production at 28 • C. The TLC proved to be somewhat constrained in its capacity to differentiate between various aflatoxin types, as certain isolates exhibited positive bands for all types, albeit with varying sizes.The aflatoxin-producing capacity of Aspergillus flavus isolates by TLC following 6 days of incubation revealed a lack of production among isolates when cultured on W A, W A + G, BA, and RPMI media.

Aflatoxin detection by HPLC
Following the preliminary detection via TLC, all samples were subjected to HPLC analysis for a more comprehensive and quantitative evaluation of aflatoxin presence.The HPLC results remained consistent with those obtained through TLC and showed higher sensitivity.It was noteworthy that 45% (42/93) of clinical isolates and 50% (17/34) of environmental isolates were positive with HPLC, representing a significant increase of 5% ( P < .001)and 3%, respectively, compared to TLC findings (Fig. 3 B).Further analysis of the positive results unveiled that these isolates exclusively produced B-type aflatoxins, with no evidence of G-type.Among these isolates, the majority were capable of producing AFB1 and AFB2, although 12% (5/42) of clinical isolates exclusively synthesized AFB2, while 10% (4/42) exclusively produced AFB1 ( Table 2 ), and 79% (33/42) of clinical isolates could generate both AFB1 and AFB2, with AFB1 levels significantly surpassing AFB2 at 28 • C.
The range of the total aflatoxin production for clinical isolates spanned from 0.01 ± 0 to 34.17 ±0 mg/kg at 28 • C and 0.01 ± 0 to 20.75 ± 0.13 mg/kg at 36 • C. In contrast, environmental isolates displayed a range of 0.002 ± 0 to 19.59 ± 0.22 mg/kg at 28 • C. As depicted in Figure 3 A, with a temperature increase to 36 • C, only 41% (38/93) of the clinical isolates were capable of producing aflatoxin.Within these strains, 55% (23/42) exhibited markedly lower levels of aflatoxin production ( P < .001),while 29% (12/42) demonstrated enhanced production.The findings provided evidence of temperature-dependent variation in aflatoxin production among A. flavus isolates.Other media failed to exhibit the target peaks associated with aflatoxins on the chromatography.
To discern the genetic distinctions between aflatoxinproducing and non-producing isolates, targeted sequencing, and alignment of three pivotal aflatoxin biosynthesisassociated genes ( aflD , aflR , and aflS ) were conducted.The result is summarized in Table 2 .Notably, both aflatoxinproducing and non-producing isolates exhibited mutations within the aflS gene in the coding regions.Specifically, two aflatoxin-non-producing isolates displayed a nonsynonymous mutation (E193S) in this gene.No mutations were observed in the aflD gene of aflatoxin-producing isolates, while a single non-synonymous mutation (A86P) was identified in one aflatoxin-non-producing isolate.Interestingly, mutations within the aflR gene were prevalent among the isolates, except for a solitary aflatoxin-non-producing isolate.These mutations in the aflR gene were also identified in two aflatoxin-producing isolates.

Relative gene expression analysis of aflatoxin biosynthesis-associated genes
To explore the gene expression trends of aflD and aflR , four aflatoxin-producing isolates were incubated for 12 days.The results of relative expression analysis of aflD and aflR are shown in Figure 5 A. When 4 aflatoxin-producing isolates were cultured on RPMI medium at 36 • C under both 0.04% and 5% CO 2 , a consistent trend emerged for the expression of aflD and aflR genes over time.Initially, there was an upregulation, reaching its peak after 9 days of incubation, followed by a sharp decrease.In contrast, for four aflatoxinproducing isolates incubated on SDB medium at 36 • C, a different pattern was noted, the expression of both genes reaching its maximum between the 4th and 6th days of incubation and then began to decline.By the 9th day, both genes exhibited downregulation.
To compare aflD and aflR gene expression of the different types of the medium during 6th and 9th days' incubation, 12 isolates including each of 10 aflatoxin-producing, and 2 non-producing isolates were assessed (Fig. 5 B).The results during the 6-day incubation period revealed that all 12 tested isolates exhibited the downregulated expression of aflD and aflR, ranging from 0.01 to 3.8 folds when incubated in RPMI, 5% CO 2 SDB, and 5% CO 2 RPMI compared to SDB (36 • C, 0.04% CO 2 ).When 12 isolates were incubated for 9 days in RPMI and 5% CO 2 RPMI, the expression in 12 isolates showed an upregulation with the range from 0.01 to 2.6 folds compared to SDB (36 • C, 0.04% CO 2 ).

Discussion
In our study, characterization of the aflatoxin from 93 isolates obtained from FRS in Sudan and 34 environmental strains was performed.Our results revealed that 45% (42/93) of clinical Aspergillus flavus isolates were aflatoxigenic, producing AFB1 and AFB2, similar to the environmental isolates.Furthermore, a significant proportion, i.e., 85% (79/93) of clinical isolates and 91.2% (31/34) of environmental isolates harboured all seven aflatoxin biosynthesis-associated genes.
A comprehensive evaluation of aflatoxin profiles was performed using both phenotypic (TLC and HPLC) and genotypic methods.Using TLC, with its simplicity and costeffectiveness, served as a preliminary screening tool, we identified aflatoxin presence in 40% of clinical strains.This initial screening prompted further quantitative analysis via HPLC, which unveiled significant variability in aflatoxin concentrations-ranging from 0.01 to 34.17 mg/kg in clinical isolates and from 0.002 to 19.59 mg/kg in environmental samples, suggesting that both clinical and environmental strains possess a significant pathogenic potential. 41 , 42Being an opportunistic pathogen, A. flavus infections are primarily acquired through the inhalation of airborne conidia, posing a risk to humans in environments with high aflatoxin levels.The primary intake pathways may include inhalation, ingestion (such as consumption of aflatoxin-contaminated food products) and cutaneous contact (direct contact with contaminated materials). 43flatoxin production was significantly influenced by our choice of culture medium.Previous research by Liu et al. 44 demonstrated that A. flavus tended to produce higher levels of aflatoxin when cultivated on solid compared to liquid media.Accordingly, SDA was selected as the standard medium for aflatoxin detection in our study, as it provided ample nutrients for fungal growth. 45The absence of aflatoxin production in nutrient-poor media (WA) explained that aflatoxin is considered a secondary metabolite, and likely demanded substantial energy investment for biosynthesis. 46This process relied on a ready supply of diverse nutrients and precursors, encompassing carbon sources (e.g., sugars), nitrogen sources (e.g., amino acids), as well as vitamins and minerals. 47In resource-limited environments, the fungus may prioritize essential metabolic functions necessary for survival and growth, potentially at the expense of secondary metabolite production, such as aflatoxins. 48This absence of aflatoxin production is consistent with observations of sporulation degeneration and the presence of thin hyphae in nutrient-poor media.
Our findings also highlighted the relationship between mycotoxin production and sporulation, a phenomenon emphasized by Mostafa et al. 49 who demonstrated that most of the toxins were produced after the fungus had completed its ini-  tial growth phase and began its development stage, characterized by sporulation, and in case of A. flavus , sclerotia formation.Existing literature has mentioned that conidia contained a range of secondary metabolites, including melanin and mycotoxins, which played pivotal roles in pathogenesis and development. 50 , 51A series of studies reported aflatoxins using different substrates including natural and synthetic media like potato dextrose agar (PD A), SD A, malt extract agar (MEA), and yeast extract sucrose agar (YES). 45 , 47In contrast, RPMI medium is a common substrate for cell incubation, the first time to be used for analysis of aflatoxin profiles.We encountered a puzzling scenario where aflatoxins were conspicuously absent in the RPMI medium despite the presence of typical conidial heads and dense sporulation during incubation.In contrast to the SDA medium, the RPMI 1640 medium distinguishes itself as the sole option that includes glutathione as a reducing agent and notably higher levels of essential vitamins, including choline, vitamin B12, biotin, and p-aminobenzoic acid (PABA).
Given the limitation of the phenotypic method in detecting aflatoxins and characterizing aflatoxin profiles, the genotypic method emerged as a valuable tool to validate our findings.As emphasized by Dehghan et al., the presence of the aflR gene served as a distinctive marker for aflatoxin production in clinical isolates. 52This insight provided a critical reference for our current study .Notably , our genotypic result revealed that 85% (79/93) of clinical and 91% of environmental isolates possessed all seven tested genes, indicating the potential risks for aflatoxin production by these isolates.The higher proportion in the environmental isolates possessed all seven tested genes suggested that the host environment, potentially subjected to oxidative stress may stimulated the absence of the major regulators in response to long-term adaption to adverse conditions. 53Another study reported that 73% of the isolates contained both aflR and aflD genes, while 67% possessed the aflM gene.This genetic profile was found in both aflatoxigenic and non-aflatoxigenic isolates, 54 aligning with our findings.However, the result of mutations in aflatoxin biosynthesisassociated genes ( aflD/aflR/aflR ) did not provide clear markers to distinguish aflatoxin-producing and non-producing isolates, as both non-synonymous and synonymous mutations were detected in our study.Existing literature does not provide conclusive insights into this matter.It's essential to clarify that the genotypic method is dedicated to the identification of aflatoxin-related genes, without directly quantifying aflatoxin production.For a precise assessment of specific aflatoxin production, the phenotypic method is employed.Consequently, the integration of both methods is imperative to achieve a comprehensive and research-focused evaluation of the aflatoxin profiles in A. flavus isolates.
The gene expression analysis of aflD and aflR revealed that the highest expression levels occurred between the 4th and 6th day of incubation on the SDB medium and on the 9th day of incubation on the RPMI medium.The variations in substrate led to differences in gene expression levels, possibly explaining why aflatoxins could not be detected by TLC and HPLC in isolates cultured in RPMI for 6 days.It is plausible that the accumulation of aflatoxins was insufficient for detection.This requires further analysis for confirmation.Additionally, a strong correlation between the expression of aflatoxin genes, as analyzed by RT-PCR, and aflatoxin production in 10 aflatoxin-producing isolates was identified.However, two aflatoxin-non-producing strains displayed transcription of all relevant genes but did not produce aflatoxin in the medium, a phenomenon also noted by Degola et al. 55 In our study, to mimic the host environment and for the first time, 5% CO 2 was applied to investigate the relative expression of aflD and aflR .Baazeem et al. explored the effect of climate changes on gene expression and showed that AFB1 production and relative expression of aflR was significantly stimulated ( P < .05)when exposed to 0.1% CO 2 at 0.98-0.95aw and 35 • C in vitro . 25We observed that the relative  gene expression of aflR and aflD was down-regulated in 5% CO 2 in both RPMI and SDB media during 6-day incubation.However, as time progressed, this expression level showed no significant difference beyond 0.04% CO 2 , suggesting that the isolates had adapted to 5% CO 2 over time.Peromingo et al. 39 found that gene expression of aflR and aflD reached its peak when isolates were grown on dry-cured ham during a 6-day incubation period, which was in line with our study when isolates were cultured in an SDB medium.A study conducted by Norlia et al. 56 has highlighted that environmental factors can potentially facilitate fungal infection.It has been suggested that exposure to host-induced stress may trigger adaptation, leading to gene expression for mycotoxin production and sexual recombination in A. flavus .Moreover, Barakat et al. 41 detected a significant correlation between the degree of severity of keratitis and the quantity of produced AFB1.Hence, in cases of chronic A. flavus infection, it is crucial to consider the buildup of aflatoxin.Evaluating the presence or absence of aflatoxins holds significant importance in clinical settings.
Although our study indicated that aflatoxin production depended on the substrate, it is important to note that aflatoxin might be present in the host in cases of aspergillosis.The expression levels of aflD and aflR varied across different media and time periods, suggesting that accumulation required time.For instance, Klich et al. 57 reported that A. flavus isolates produced aflatoxin in YES but none of them produced toxin on brain heart infusion agar (BHA), a medium that simulates human tissue.However, it's worth noting that the TLC method used may have missed very low levels of toxin.While the authors believed that aflatoxin production is unlikely to play a role in human mycosis, it cannot be ruled out entirely.In 1998, Mori et al. 58 documented a human case of systemic infection caused by an A. flavus isolate that produced aflatoxins both in vitro and in vivo ; extracts from lung lesions and culture filtrates contained aflatoxins B1, B2, and aflatoxin M1.More recently, Mori et al. reported the presence of aflatoxin in the lung tissue of a leukemia patient infected by A. flavus . 11Although the patient positively responded to leukemia chemotherapy, the primary attribution for their unfortunate demise was A. flavus infection and probably the immunosuppressive effects of aflatoxin.Additionally, aflatoxin was detected in the urine of a patient with A. flavus pulmonary aspergilloma in Ecuador, although it could not be conclusively established whether the aspergilloma was the definitive source. 59he significance of A. flavus has been consistently underestimated, despite its prevalence in many regions worldwide and the potential for toxin production by its isolates.It is imperative to draw greater attention to this issue.This necessitates heightened awareness, rigorous assessments of its true impact, and in-depth exploration of the toxin's role in pathogenicity, as well as its possible associations with conditions like nasopharyngeal carcinoma.In light of this, further research and vigilance are warranted to unravel the full extent of its implications and to address the potential health concerns associated with it.

Conclusions
In conclusion, our study offered comprehensive insights into the characteristics of aflatoxin profiles of Aspergillus flavus isolates originating from FRS cases in Sudan.The combined use of phenotypic and genotypic methods was essential for the comprehensive characterization of aflatoxin production profiles.The large number of clinical isolates demonstrated aflatoxigenic capabilities, underscore the potential health risks associated with A. flavus infections and the importance of monitoring aflatoxin exposure.A positive correlation between the expression of aflR and aflD , and aflatoxin production was established, shedding light on the regulation of this mycotoxin.Importantly, dynamic patterns of aflD and aflR gene expression over time and across different substrates were observed, further enriching our understanding of aflatoxin production regulation.Further research is warranted to elucidate the complex regulation of aflatoxin production and its implications for public health.

Figure 1 .
Figure 1.Morphology and growth of isolate M.064-44 cultured in different media at 28 • C and 36 • C for 6 days of incubation, respectively.(A-I) Macroand micromorphology of isolates incubated at 36 • C, first row: Colony, second row: Micromorphology, third row: Micromorphology of isolates incubated on WA, RPMI, and SDA from left to right; (J) The growth diameter of isolates.WA Water agar; WA + G Water agar with 4% glucose; BA Blood agar; SDA Sabouraud dextrose agar.

Figure 3 .
Figure 3.The proportion and amount of aflatoxins produced by Aspergillus flavus clinical isolates incubated at 28 and 36 • C by HPLC.(A) The proportion of aflatoxins produced by clinical isolates and (B) the amount of aflatoxins.

Figure 5 .
Figure 5.The relative expression of aflD and aflR of isolate in different media during different incubation time points incubated at 36 • C. (A) The relative expression of aflD and aflR of aflatoxin-producing isolates [the control conditions (calibrator) used for comparisons between treatments 2 days' incubation]; (B) The relative expression of aflD and aflR of 10 aflatoxin-producing and two non-producing isolates (E37 and E38) in different media during 6-and 9-da y s incubation [the control conditions (calibrator) used for comparisons between treatments was the 36 • C, SDB, 0.04% CO 2 ].