Section Flavi Research Articles

Comparative analysis of the genomes and aflatoxin production patterns of three species within the Aspergillus section Flavi reveals an undescribed chemotype and habitat-specific genetic traits

Aflatoxins are the most dangerous mycotoxins for food safety. They are mainly produced by Aspergillus flavus, A. parasiticus, and A. minisclerotigenes. The latter, an understudied species, was the main culprit for outbreaks of fatal aflatoxicosis in Kenya in the past. To determine specific genetic characteristics of these Aspergillus species, their genomes are comparatively analyzed. Differences reflecting the typical habitat are reported, such as an increased number of carbohydrate-active enzymes, including enzymes for lignin degradation, in the genomes of A. minisclerotigenes and A. parasiticus. Further, variations within the aflatoxin gene clusters are described, which are related to different chemotypes of aflatoxin biosynthesis. These include a substitution within the aflL gene of the A. parasiticus isolate, which leads to the translation of a stop codon, thereby switching off the production of the group 1 aflatoxins B1 and G1. In addition, we demonstrate that the inability of the A. minisclerotigenes isolates to produce group G aflatoxins is associated with a 2.2 kb deletion within the aflF and aflU genes. These findings reveal a relatively high genetic homology among the three Aspergillus species investigated. However, they also demonstrate consequential genetic differences that have an important impact on risk-assessment and food safety.

Two new qPCR assays for detecting and quantifying the Aspergillus flavus and Aspergillus parasiticus clades in maize kernels

AbstractThe fungi of Aspergillus section Flavi can produce carcinogenic mycotoxins—aflatoxins (AFs)—of two types: types B and G (AFBs and AFGs). AFs are highly hazardous for human and animal health. Their levels in food and feed are therefore highly regulated, with a low acceptable limit for AF content. In France, climate change has led to the detection of AFs in maize harvests since 2015. Mycoflora analyses have identified two species, A. flavus (producing AFBs) and A. parasiticus (producing both AFBs and AFGs), as responsible for this AF contamination. However, mycoflora analysis is a time‐consuming method that cannot readily be applied to large numbers of samples. We propose here an alternative clade‐specific functional TaqMan quantitative PCR method based on the calmodulin gene for distinguishing between the A. flavus clade (AfC) and the A. parasiticus clade (ApC). We applied this method to 553 maize samples collected in three different harvest years (2018–2020). Both clades were detected in about 40% of the samples tested. As expected, we observed significant positive correlations between AFBs and AfC DNA (R2 = 0.708), and between AFGs and ApC DNA (R2 = 0.885). This method will be useful for the rapid, simple and cheap characterization of maize grain contamination with Aspergillus section Flavi. This method will make it possible to study the relationship between agroclimatic conditions, AF content and species prevalence, to facilitate the anticipation of AF risks due to global warming in France.

Evaluation of Primers OPF-01, P54, and 1253 to Identify A. fumigatus, A. flavus, and A. niger from Polymorphic Patterns Obtained by RAPD-PCR.

We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns' polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively.

Apple mycotoxins: From orchard to processed apple puree

Apples and apple-derived products can be contaminated with patulin and, to a lesser extent, aflatoxin B1 and fumonisins. Fruits were collected from Golden Delicious and Imperatore trees in three orchards in Veneto, Northern Italy, and analysed for the presence of fungi and mycotoxins. Sampling and analyses were also carried out from storage bins to final puree tanks along the apple-puree production chain. Alternaria spp. and Penicillium spp. were commonly isolated from apples collected in the field, but their presence varied with cultivar and orchard location. In contrast, Aspergillus section Flavi spp. and Fusarium spp. were only detected via direct isolation from chopped fruits. Patulin and Alternaria toxins were frequently identified in fresh fruits, while aflatoxins were also present in some fruits albeit at very low levels. Fungi were present in fruits entering the apple-puree production line, but not in apple puree after treatment at 95–105 °C. In contrast, the abovementioned mycotoxins were detected at all stages along the production line. Surprisingly, fumonisins were only detected after the apple puree was subjected to thermal treatment. Implications of these findings were discussed and few unresolved scientific questions were posed to stimulate further lines of study.

A polyphasic characterisation of Aspergillus section Flavi isolated from Malaysian and Nigerian food grains and poultry feeds by phenotypic, chemotypic, and molecular methods

The taxonomy and toxigenicity of Aspergillus section Flavi (ASF) in various regions are still under-reported, as many potentially toxic species are continuously discovered. This study aims to determine the aflatoxigenicity and identify 74 isolates of ASF from Malaysian and Nigerian food grains and feeds using phenotypic, chemotypic, and molecular methods. Isolates were screened for sclerotium morphotype and aspergillic acid production phenotypically, while an ultra-fast reverse-phase high-performance liquid chromatography (HPLC) was optimised to quantify the cyclopiazonic acid and aflatoxins produced by the isolated ASF, based on which, they were characterised into phenotypes L-strains, S-strains, and NS-strains, and chemotypes I to VI. Chemotypic data and nucleotide sequences of β-tubulin and internal transcribed spacer (ITS) genes were utilised to identify the isolates’ species and determine their phylogenetic relationships. Aflatoxigenesis on solid media and multiplex polymerase chain reactions of four essential genes (aflR, aflM, aflD, and aflP) of aflatoxin biosynthesis were used to confirm the aflatoxigenicity of each isolate. All the six chemotypes of ASF were present among the isolated strains, with the aflatoxigenic chemotypes (55.41%) and CPA-positive isolates (68.92%) being significantly higher (p < 0.05) than the non-aflatoxigenic and CPA negative chemotypes. Aflatoxins and CPA were produced in the 0.25–19.58 ng/g range and 29.89–46.55 ng/g, respectively. Chemotypic data accurately differentiate toxigenic from atoxigenic species but only identify 90% of species names. Molecular data accurately resolved species but not toxigenicity. The results highlight the need for robust regulatory measures in the concerned regions to mitigate the risk of aflatoxicosis.

First report of Aspergillus species in green pistachio of Bronte

Aspergillus contamination of pistachios causes significant product losses and potential presence of mycotoxins, particularly aflatoxin B1 (AFB1), and ochratoxin A (OTA). These toxins, which threaten human health, are strictly monitored by most nations. Italian pistachios produced in Bronte, Sicily, have high nutritional value and unique organoleptic properties, but the extent to which they contain these contaminants is unknown. Aspergillus spp. isolated from Bronte pistachios (cultivar Napoletana) were assessed for their ability to synthesize OTA or AFB1. Aspergillus occurrence in pistachio samples was measured at 1137 cfu g-1 for in shell pistachios and 770 cfu g-1 for kernels. The predominant isolated Aspergillus species was A. niger representing 74% of section Nigri (black isolates) and 47% of all Aspergillus isolates. Within section Flavi, A. flavus comprised 83% of green isolates. Only one black isolate (identified as A. carbonarius) had high OTA production, but all the A. flavus isolates had potential to produce AFG1 and AFB1, with AFB1 produced amount ranging from 0.1 to 8498 ng mL-1 of culture filtrate.

Maize Aspergillus section Flavi isolate diversity may be distinct from that of soil and subsequently the source of aflatoxin contamination

Aspergillus section Flavi (Flavi) is a diverse group of fungal species whose common members include A. flavus and A. parasiticus. These are well-known for the production of aflatoxin (AF) B and G and other toxic metabolites, like cyclopiazonic acid (CPA). They are saprophytic soil dwellers and also become crop opportunistic epiphytes. The consequence is contamination of the crop with mycotoxins, such as carcinogenic AF. We investigated the Flavi community structure of maize and that of their surrounding soil, including their mycotoxigenicity. Furthermore, we investigated the link of the maize Flavi diversity with preharvest maize AF levels. The study was carried out in four selected districts of Zambia, in a low rainfall zone. The Flavi characterisation was triphasic, involving morphological (colony colour and sclerotia formation), metabolic (AF and CPA production) and genetic (calmodulin gene polymorphism) analyses. Flavi abundance was determined by dilution plate technique on modified rose Bengal agar. Results showed that Flavi communities on maize and in soil differed. Maize had a higher Flavi species diversity than soil. A. parasiticus dominated the soil community by frequency of field appearance (85%), while maize was dominated by A. minisclerotigenes (45%). CPA-producers with or without AF production dominated the maize (65%) while producers of only AF (B/G) dominated the soil (88%). The ratio between maize A. parasiticus and A. minisclerotigenes abundance seemed to have had a bearing on the levels of AF in maize, with a ratio close to 1:1 having higher levels than a pure community of either A. parasiticus or A. minisclerotigenes.

Review on novel approaches for controlling aflatoxin B1: Harnessing nature's defenses against food toxins

Aflatoxin B1 is the most harmful food toxin for humans and animals. According to the International Agency for Research on Cancer, it is ranked as one of the most carcinogenic substances in the world, directly connected to hepatocarcinoma in both humans and animals. Aflatoxin B1 is also involved in the occurrence of pathologies, and aflatoxicoses. Aflatoxin B1 can be produced by fungi from the genus Aspergillus section Flavi in a variety of foods during storage. Once aflatoxins are present, food processing cannot eradicate them. The damage and lack of complete elimination of aflatoxins from foodstuffs make them the most monitored and controlled substances in the world. Given the high-risk of Aflatoxin B1 on both animal and human health, it is important to study their worldwide distribution and existing control methods through a systematic review. Articles published from 1945 to 2022 found on several databases as Scopus, PubMed, Dimensions, Google Scholar and an extraction sieve was used to select the relevant articles. Of the two hundred and twenty-eight (228) French and English scientific articles on aflatoxins identified, forty of the most relevant original articles were selected for inclusion in this review following a rigorous selection process. Several genes are involved in the synthesis of aflatoxin B1. Moreover, certain environmental conditions, in particular oxidative stress are propitious for fungus by over-expressing aflatoxins. However, the fungi defences can be controlled by several methods. Articles showed efficiency of various of them. Good cultural practices and awareness raising are part of the preventive control. Synthetic chemicals such as insecticides and fungicides are chronically used in chemical control of fungal growth and prevent aflatoxins from being produced. Biological control is based on allelic recombination between toxic and atoxic strains. The use of microbial competition is focused on the natural predators of aflatoxic molds, most often lactic acid bacteria, and the natural control relies on the use of natural plant substances. Natural substances like aqueous or organic plant extracts that contain proteins, polyphenols, tannins, antioxidants, flavonoids, terpenes, and chelating ion as well as caffeic acid, gallic acid and ascorbic acid can be used to control fungal contamination currently. These molecules interfere with free radicals to slow down or even inhibit the production of reactive oxygen species significantly reducing the production of aflatoxins making them inactive. Implementation of the means of controlling fungal growth and producing aflatoxins will help make agriculture globally competitive and ensure food security. Key words: Natural substances, Aflatoxin B1, food poisoning, alternative control, biocontrol

Bioburden, phenotypic and spectroscopic characterisation of toxigenic and atoxigenic Aspergillus section Flavi from poultry feeds in Kelantan, Malaysia and Katsina, Nigeria

Bioburden, phenotypic and spectroscopic characterisation of toxigenic and atoxigenic Aspergillus section Flavi from poultry feeds in Kelantan, Malaysia and Katsina, Nigeria

Aflatoxins in the nut chains: strategies to reduce their impact on consumer’s health and economic losses

Abstract The presence of Aspergillus section Flavi and aflatoxins (AFs) in nut chains are of great concern due to the toxic effects of AFs on human and animal health as well as the economic losses during commercialisation. Extreme events related to climate change like increased temperature, severe rainfalls and droughts are modulating factors of fungal growth and mycotoxin production. The Expert Working Group (EWG) on nuts that worked under the frame of the Horizon 2020 Mycokey Project prepared this review based on discussions about the situation of contamination with AFs in nuts and the available strategies to reduce the impact of these toxins in the nut chains. The EWG selected as examples peanuts, pistachios and Brazil nuts, and discussed in the review the importance of nut value chains in the production, commercialisation and regulations that establish maximum levels for AFs. Also data on the Rapid Alert System for Food and Feed (RASFF) was considered and showed several notifications for mycotoxins, mainly AFs for nuts and nut products, such as pistachio nuts and dried mix nuts. Under a scenario of climate change, we elaborated strategies to prevent AFs contamination with emphasis on biocontrol and decision support systems. Also, the relevance of strategies to prevent and reduce both fungal infection and AFs accumulation in peanuts, Brazil nuts and pistachio are included as guidelines.

Aspergillus population diversity and its role in aflatoxin contamination of cashew nuts from coastal Kenya.

Cashew nuts are among the main cash crops in coastal Kenya, due in large part to their high nutritional value. Unfortunately, they also make them highly susceptible to mold contamination, resulting in biodeterioration of the nutritional value and potential contamination with toxic secondary metabolites, such as aflatoxins, that cause them to be rejected for sale at the market. We determined the population diversity of the Aspergillus species and their role in aflatoxin contamination in cashew nuts in selected coastal regions of Kenya. Fifty raw cashew nut samples were collected from post-harvest storage facilities across three counties in Kenya's coastal region and examined for moisture content and the presence of Aspergillus fungi. About 63 presumptive isolates were recovered from the cashew nuts. ITS and 28S rDNA regions were sequenced. The aflD, aflM and aflR genes were amplified to identify the potentially aflatoxigenic from the Aspergillus isolates. The Aflatoxins' presence on the isolates was screened using UV and the ammonia vapour test on coconut milk agar and validated using ELISA assay. A comparison of cashew moisture content between the three counties sampled revealed a significant difference. Sixty-three isolates were recovered and identified to section based on morphological characters and their respective ITS regions were used to obtain species identifications. Three sections from the genus were represented, Flavi and Nigri, and Terrei with isolates from the section Nigri having slightly greater abundance (n = 35). The aflD, aflM and aflR genes were amplified for all isolates to assess the presence of the aflatoxin biosynthesis pathway, indicating the potential for aflatoxin production. Less than half of the Aspergillus isolates (39.68%) contained the aflatoxin pathway genes, while 22.22% isolates were aflatoxigenic, which included only the section Flavi isolates. Section Flavi isolates identification was confirmed by calmodulin gene. The presence of species from Aspergillus section Flavi and section Nigri indicate the potential for aflatoxin or ochratoxin in the cashew nuts. The study established a foundation for future investigations of the fungi and mycotoxins contaminating cashew nuts in Kenya, which necessitates developing strategies to prevent infection by mycotoxigenic fungi, especially during the storage and processing phases.

Assessment of waste workers occupational risk to microbial agents and cytotoxic effects of mixed contaminants present in the air of waste truck cabin and ventilation filters

ABSTRACT Workers in the waste-processing industry are potentially exposed to high concentrations of biological contaminants, leading to respiratory and digestive problems and skin irritations. However, few data on the exposure of waste collection truck (WCT) drivers are available. The goal was to document the microbial risk of the waste collection truck (WCT) workers while in the vehicle cab. Long-period sampling using the truck air filters (CAF) and short time ambient air sampling in the cab were used. The potential release of microbial particles from CAFs was also investigated since it could contribute to the microbial load of the cabin air. A combination of analytical methods also helped assess the complex mixture of the biological agents. Aspergillus sections Fumigati and Flavi, E. coli, Enterobacter spp. and Legionella spp. were detected in the CAF of trucks collecting three types of waste. The highest levels of bacteria and fungi were found in the CAF from organic WCT. The highest endotoxin concentrations in CAF were 300 EU/cm2. Most of the CAF showed cytotoxic effects on both lung cells and hepatocytes. Only one mycotoxin was detected in a CAF. The maximal concentrations in the ambient WCT air varied according to the type of waste collected. The highest proportion (84%) of the air samples without cytotoxic effects on the lungs cells was for the recyclable material WCTs. The results revealed the potential microbial risk to workers from a complex mixture of bio-contaminants in the cabs of vehicles collecting all types of waste. The sustained cytotoxic effect indicates the potential adverse health-related impact of mixed contaminants (biological and non-biological) for the workers. Overall, this study highlights the benefits of using complementary sampling strategy and combined analytical methods for a the assessment of the microbial risk in work environments and the need to implement protective measures for the workers. Implications: Exposure to microbial agents is a well-known occupational hazard in the waste management sector. No previous study had evaluated the cytotoxicity of ambient air and ventilation filters to document worker exposure to a combination of contaminants during waste collection. This research confirms the usefulness of ventilation filters for long-term characterization of exposure to infectious agents, azole-resistant fungi, coliform bacteria and mycotoxin. Overall, this study highlights the importance of using several sampling and analysis methods for a comprehensive assessment of microbial risk in work environments, as well as the need to implement appropriate protective measures for collection workers.

Inhibition of Aflatoxin B1 by Aqueous extract from Green Cardamom

Aflatoxin B1 is a secondary metabolite produced by Aspergillus section Flavi during their development, is one of the most dangerous mycotoxins identified to date, that may contaminate many commodities, especially in tropical and sub-tropical regions. Aflatoxin B1 is believed to be the most essential member of this family of mycotoxins, due to its carcinogenic properties in humans and animals. However, there are many strategies to prevent mycotoxin contamination in which some of them are based on the use of natural compounds that may able to minimize the toxin synthesis pathways. Aims: The current work aimed to identify specific compounds from Green Cardamom that expected to interfere with aflatoxin production pathway. Methodology: The adapted method was based on the incubation of fungal culture with different concentrations of the aqueous extraction of Green Cardamom. Results: The aqueous extract of Green Cardamom was able to inhibit Fungal growth and Aflatoxin B1 production by the toxigenic strain of Aspergillus Flavus in a dose-dependent manner. Discussion: the reduction of Aflatoxin B1 production was significantly higher (94.34%) in comparison with other plant extracts such as Caffeine (50%), and reduced fungal growth with an average of (4%) compared to control. Conclusion: Aqueous Extracts of Green Cardamom show an obvious reduction in inhibition synthesis of Aflatoxin B1 by toxigenic Aspergillus strain, the adapted method was effective, but even further investigation is still recommended.

Characterization of Aspergillus section Flavi associated with stored grains

Increased frequencies of Aspergillus section Flavi and aflatoxins in cereal grains have been seen in recent years due to changes in climate circumstances, such as high temperatures and drought. To assess the microbiological risks of contamination, it is critical to have a reliable and accurate means of identifying the fungi. The main goal of this study was to characterize Aspergillus species from section Flavi obtained from twenty-three samples of barley and maize grains, gathered from different markets in Qena, Egypt, using morphological and molecular techniques. Twenty-three isolates were chosen, one isolate from each sample; they were identified as A. aflatoxiformans (4 isolates), A. flavus (18), and A. parasiticus (1). The existence of four aflatoxin biosynthesis genes was also investigated in relation to the strains’ ability to produce total aflatoxins and aflatoxin B1, focusing on the regulatory gene aflR and the structural genes aflD and aflM. All strains producing aflatoxins were linked to the presence of aflR1 and/or aflR2, except two isolates that exhibited aflatoxins but from which aflR1 or aflR2 were not detected, which may be due to one or more missing or unstudied additional genes involved in aflatoxin production. AflD and aflM genes were amplified by 10 and 9 isolates, respectively. Five samples of barley and maize were contaminated by aflatoxins. Fifteen isolates were positive for producing total aflatoxins in the range of 0.1–240 ppm. Antagonistic activity of Trichoderma viride against A. flavus (F5) was assessed at 31.3%. Trichoderma reduced total aflatoxins in all treated seeds, particularly those subjected to Trichoderma formulation.

Aflatoxin Decontamination in Maize Steep Liquor Obtained from Bioethanol Production Using Laccases from Species within the Basidiomycota Phylum.

Maize (Zea mays L.) is an important crop in Argentina. Aspergillus section Flavi can infect this crop at the pre-harvest stage, and the harvested grains can be contaminated with aflatoxins (AFs). During the production of bioethanol from maize, AF levels can increase up to three times in the final co-products, known as, dry and wet distiller's grain with solubles (DDGS and WDGS), intended for animal feed. Fungal enzymes like laccases can be a useful tool for reducing AF contamination in the co-products obtained from this process. The aim of the present study was to evaluate the ability of laccase enzymes included in enzymatic extracts (EE) produced by different species in the Basidiomycota phylum to reduce AF (AFB1 and AFB2) accumulation under the conditions of in vitro assays. Four laccase activities (5, 10, 15, and 20 U/mL) exerted by nine isolates were evaluated in the absence and presence of vanillic acid (VA), serving as a laccase redox mediator for the degradation of total AFs. The enzymatic stability in maize steep liquor (MSL) was confirmed after a 60 h incubation period. The most effective EE in terms of reducing AF content in the buffer was selected for an additional assay carried out under the same conditions using maize steep liquor obtained after the saccharification stage during the bioethanol production process. The highest degradation percentages were observed at 20 U/mL of laccase enzymatic activity and 1 mM of VA, corresponding to 26% for AFB1 and 26.6% for AFB2. The present study provides valuable data for the development of an efficient tool based on fungal laccases for preventing AF accumulation in the co-products of bioethanol produced from maize used for animal feed.

Simultaneous detection of mycotoxigenic Aspergillus species of sections Circumdati and Flavi using multiplex digital PCR.

Populations of ochratoxin-producing Aspergillus section Circumdati species and aflatoxin-producing Aspergillus section Flavi species frequently coexist in soil and are the main sources of mycotoxin contamination of tree nuts. Identification of mycotoxigenic Aspergillus species in these sections is difficult using traditional isolation and culture methods. We developed a multiplex digital PCR (dPCR) assay to detect and quantify Aspergillus ochraceus, Aspergillus westerdijkiae, and Aspergillus steynii (section Circumdati), as well as Aspergillus flavus and Aspergillus parasiticus (section Flavi), in environmental samples based on species-specific calmodulin gene sequences. Relative quantification of each species by dPCR of mixed-species templates correlated with corresponding DNA input ratios. Target species could be detected in soil inoculated with conidia from each species. Non-target species of sections Circumdati, Flavi, and Nigri were generally not detectable using this dPCR method. Detected non-target species (Aspergillus fresenii, Aspergillus melleus, Aspergillus sclerotiorum, and Aspergillus subramanianii) were discernible from A. ochraceus in dual-template dPCR reactions based on differential fluorescence intensity.

Fungal and Mycotoxin Contamination of Green Leaf Spices Commercialized in Romania: A Food Choice Perspective

A healthy, sustainable diet contributes massively to the general well-being of a population. Nowadays, people have started to significantly improve their diet by reducing processed products, as well as including a higher number of fruits, vegetables, cereals, and spices to flavor their food. However, making the right food choice, without any harmful pathogens that pose a risk to human health, can remain quite challenging. Despite prioritizing food safety in its processing, production, and distribution, food contamination remains a prevalent and undesirable occurrence. This study aims to detect the fungal load of commonly used green leaf spices commercialized in Romania and the impact of those microorganisms and their secondary metabolites on consumers. Six (28.57%) out of the twenty-one tested samples showed different degrees of fungal contamination, mostly with Aspergillus section Flavi, followed by Aspergillus section Nigri and other fungi in lower amounts. Three main fungal secondary metabolites with potential impact on consumers, namely mycotoxins, were determined with high-performance liquid chromatography (HPLC): Aflatoxin B1, Ochratoxin A, and Zearalenone. Moreover, their legal limits (5 μg/kg, 15 μg/kg, and 50 μg/kg, respectively) were exceeded by 95.24%, 100%, and 85.71%, respectively. Environmental factors that affect the processing and packaging of these spices did not show any relation to fungal contamination, conversely to price, which significantly correlates with the mycological quality of the products.

Antifungal susceptibility of Aspergillusgenus determined by the Etest® method: eleven years of experience at the Instituto Médico La Floresta. Caracas, Venezuela.

Abstract. This research aimed to determine the susceptibility of Aspergil-lus spp. to four antifungal agents using the Etest® method in several clinical samples (respiratory samples, soft tissue, otic tissue, and ocular tissue, among others) from a private health center in Venezuela. Thirty-three strains were evaluated: 11 Aspergillus section Flavi, eight Aspergillus section Fumigati, six Aspergillus section Nigri, four Aspergillus section Terrei, and four Aspergillus spp. A 0.5 McFarland standard suspension of a 5-day culture of each Aspergillusstrain was prepared on Potato Dextrose agar and then inoculated on Sabouraud agar plates with 2% glucose. Voriconazole (VCZ), amphotericin B (AMB), caspo-fungin (CAS), and posaconazole (PCZ) were tested. Minimal inhibitory concen-trations (MIC) in μg/mL were determined after 24 and 48 hours of incubation at 35 °C and th range (R), geometric mean (GM), MIC50, and MIC90 were calcu-lated. The results for the 33 Aspergillus spp. tested after 24 h were the follow-ing: VCZ (R = 0.031- 16; GM = 0.145; MIC50 = 0.125 and MIC90 = 0.5), AMB (R = 0.031-16; GM = 0.644; MIC50 = 0.5 and MIC90 = 8), CAS (R = 0.031-16; GM = 0.1076; MIC50 = 0.063 and MIC90 = 1), PCZ (R =0.031 - 0.5; GM = 0.0755; MIC50 = 0.063 and MIC90 = 0.25). This investigation allowed assessing the an-tifungal susceptibility profiles of Aspergillus spp. isolated from clinical samples by the Etest® method, which is practical, reproducible and easy to perform in microbiology laboratories.

Surveillance of Amphotericin B and Azole Resistance in Aspergillus Isolated from Patients in a Tertiary Teaching Hospital.

The genus Aspergillus harbors human infection-causing pathogens and is involved in the complex one-health challenge of antifungal resistance. Here, a 6-year retrospective study was conducted with Aspergillus spp. isolated from patients with invasive, chronic, and clinically suspected aspergillosis in a tertiary teaching hospital. A total of 64 Aspergillus spp. clinical isolates were investigated regarding molecular identification, biofilm, virulence in Galleria mellonella, antifungal susceptibility, and resistance to amphotericin B and azoles. Aspergillus section Fumigati (A. fumigatus sensu stricto, 62.5%) and section Flavi (A. flavus, 20.3%; A. parasiticus, 14%; and A. tamarii, 3.1%) have been identified. Aspergillus section Flavi clinical isolates were more virulent than section Fumigati clinical isolates. Furthermore, scant evidence supports a link between biofilm formation and virulence. The susceptibility of the Aspergillus spp. clinical isolates to itraconazole, posaconazole, voriconazole, and amphotericin B was evaluated. Most Aspergillus spp. clinical isolates (67.2%) had an AMB MIC value equal to or above 2 µg/mL, warning of a higher probability of therapeutic failure in the region under study. In general, the triazoles presented MIC values above the epidemiological cutoff value. The high triazole MIC values of A. fumigatus s.s. clinical isolates were investigated by sequencing the promoter region and cyp51A locus. The Cyp51A amino acid substitutions F46Y, M172V, N248T, N248K, D255E, and E427K were globally detected in 47.5% of A. fumigatus s.s. clinical isolates, and most of them are associated with high triazole MICs. Even so, the findings support voriconazole or itraconazole as the first therapeutic choice for treating Aspergillus infections. This study emphasizes the significance of continued surveillance of Aspergillus spp. infections to help overcome the gap in knowledge of the global fungal burden of infections and antifungal resistance, supporting public health initiatives.

Aflatoxin levels in poultry feed: a comparison of mash and pellet forms

The current research was conducted to determine the aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates and the frequency of aflatoxin levels (B1, B2, G1, G2), in main feed ingredients (corn and soybean meal) and poultry finished feed (in mash and pellet forms). Eighty-five samples of corn, soybean meal, and poultry finished feed was randomly collected from feed mills in Iran. Regarding macro and microscopic morphological criteria, Aspergillus isolates were identified, and aflatoxins were determined by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). All of poultry feed samples were contaminated with different levels of aflatoxins, ranging from ND (they were not detected in those samples) to 5.58 µg/kg. At all stages of processing, the poultry feed had lower levels of aflatoxins in comparison with the accepted/residue levels of poultry feed mills. Higher amounts of aflatoxins (B1, B2, G1, G2, and total) were detected in pelleted feed, compared to other poultry samples (P < 0.05). The total toxin level in mash feed samples reached a maximum of 3.31 ppb. The results indicate that finished feed samples in pellet form may pose a greater risk than their individual ingredients in poultry feed, particularly when suboptimal conditions exist for eliminating fungal populations. So, the prevention and reduction of (Aspergillus section Flavi) are highly important in maintaining quality control of poultry feed, as the production of aflatoxins can occur during the process of converting raw ingredients into finished feed.