Species Identification and Mycotoxigenic Potential of Aspergillus Section Flavi Isolated from Maize Marketed in the Metropolitan Region of Asunción, Paraguay.

Zea mays var. amylacea and Zea mays var. indurata are maize ecotypes from Paraguay. Aspergillus section Flavi is the main spoilage fungus of maize under storage conditions. Due to its large intraspecific genetic variability, the accurate identification of this fungal taxonomic group is difficult. In the present study, potential mycotoxigenic strains of Aspergillus section Flavi isolated from Z. mays var. indurata and Z. mays var. amylacea that are marketed in the metropolitan region of Asunción were identified by a polyphasic approach. Based on morphological characters, 211 isolates were confirmed to belong to Aspergillus section Flavi. A subset of 92 strains was identified as Aspergillus flavus by mass spectrometry MALDI-TOF and the strains were classified by MALDI-TOF MS into chemotypes based on their aflatoxins and cyclopiazonic acid production. According to the partial sequencing of ITS and CaM genes, a representative subset of 38 A. flavus strains was confirmed. Overall, 75 A. flavus strains (86%) were characterized as producers of aflatoxins. The co-occurrence of at least two mycotoxins (AF/ZEA, FUM/ZEA, and AF/ZEA/FUM) was detected for five of the Z. mays samples (63%). Considering the high mycological bioburden and mycotoxin contamination, maize marketed in the metropolitan region of Asunción constitutes a potential risk to food safety and public health and requires control measures.

Presence of Aflatoxin M1 in Commercial Milk in Paraguay.

ABSTRACT: The presence of aflatoxin M1 (AFM1) in milk is a public health concern because milk is a significant part of human diets worldwide. In economies where AFM1 monitoring is low or nonexistent, the possibility of AFM1 contamination might be increased. Our study was conducted to detect and quantify AFM1 in fluid milk and milk drinks of various brands, fat concentrations, packages, and heat treatments sold in the Metropolitan Area of Asunción, Paraguay. Eighty samples were collected from supermarkets in the Metropolitan Area of Asunción following a nonprobability sampling method. An enzyme-linked immunosorbent assay for AFM1 (25 to 500 ppt) was used to quantify the toxin, and results were analyzed with nonparametric methods. All samples were positive values for AFM1 (above the detection limit of 25 ng/kg); 85% of the samples had 30 to 50 ng/kg, and 15% had >500 ng/kg. No significant difference in AFM1 concentration was found based on fat concentration, heat treatment, or type of packaging of these milk products; however, significant differences were found between brands.

First Report of a Leaf Blight Caused by Pyricularia pennisetigena on Cenchrus echinatus in Paraguay.

The genus Pyricularia contains several fungal species known to cause diseases on plants in the Poaceae family (Klaubauf et al. 2014; Wang et al. 2019). While sampling for P. oryzae during March-2015 and April-2018, common weed Cenchrus echinatus L. was observed with leaf lesions in and around experimental wheat fields in the departments of Canindeyú and Itapúa. C. echinatus samples from both locations displayed similar leaf lesions, varying from small light brown pinpoint to elliptical brown lesions with greyish center. Symptomatic leaves were surface disinfested and cultured on potato dextrose agar (PDA) amended with 1% gentamicin at 25°C. Two monosporic isolates were obtained, one from Itapúa (ITCeh117) and the other from Canindeyú (YCeh55). The isolates were subsequently grown on oatmeal agar (OA) and PDA under a 12-h photoperiod at 25°C and evaluated after ten days for colony diameter, sporulation, macroscopic and microscopic features. Colonies on OA reached up to 4.8 cm diameter and were light grey, whereas colonies on PDA reached up to 5.3 cm diameter and were brown with grey centers, with cottony mycelium and broad white rims. Mycelium consisted of smooth, hyaline, branched, septate hyphae 4-4.5 µm diameter. Conidiophores were erect, straight or curved, unbranched, medium brown and smooth. Conidia were solitary, pyriform, pale brown, smooth, granular, 2-septate, 32-33 × 9-10 µm; truncated with protruding hilum and varied in length from 1.0 to 1.5 µm and diameters from 2.0 to 2.2 µm. Both isolates were similar and identified as Pyricularia pennisetigena, according to morphological and morphometric characteristics (Klaubauf et al. 2014). Subsequently, genomic DNA was extracted from each isolate using the primers described in Klaubauf et al. (2014) to amplify and sequence the internal transcribed spacers (ITS), partial large subunit (LSU), partial RNA polymerase II large subunit gene (RPB1), partial actin gene (ACT), and partial calmodulin gene (CAL). Sequences from each isolate (YCeh55/ITCeh117) were deposited in GenBank with the following submission ID for ITS: MN947521/MN947526, RPB1: MN984710/MN984715, LSU: MN944829/MN944834, ACT: MN917177/MN917182, and CAL: MN984688/MN984693. Phylogenetic analysis was conducted using the software Beast v1.10.4. The results obtained from the concatenated matrix of the five loci placed these isolates in the P. pennisetigena clade. To confirm pathogenicity, each isolate was adjusted to 5×104 conidia/ml of sterile water and C. echinatus plants were sprayed with the conidial suspension for isolate YCeh55, ITCeh117 or sterile water using an oilless airbrush sprayer until runoff. The three treatments were kept in the greenhouse at 25-28°C and about 75% relative humidity under natural daylight. Each treatment included three to five inoculated plants and 10 leaves were evaluated per treatment. Symptoms were observed 8-15 days after inoculation and were similar to those originally observed in the field for both isolates, whereas the control plants remained asymptomatic. P. pennisetigena was re-isolated from the inoculated leaves fulfilling Koch's postulates. To our knowledge, this is the first report of leaf blight on C. echinatus caused by P. pennisetigena in Paraguay. The occurrence of P. pennisetigena in the region and its ability to infect economically important crops such as wheat and barley (Klaubauf et al. 2014; Reges et al., 2016, 2018) pose a potential threat to agriculture in Paraguay.

Presencia de aflatoxina M1 en fórmulas lácteas infantiles comercializadas en el área metropolitana a Asunción, Paraguay

RESUMEN Introducción: La aflatoxina M1 (AFM1) es un metabolito tóxico derivado de la aflatoxina B. Su ingestión en lactantes se ha relacionado con retraso en el crecimiento, aumento de susceptibilidad a enfermedades infecciosas, reducción de la eficiencia en la inmunización y cirrosis. En Paraguay se recomienda lactancia exclusiva hasta los 6 meses, sin embargo se comercializan diferentes marcas de fórmulas infantiles dirigidas a lactantes menores de 6 meses. Objetivo: Detectar y cuantificar la presencia de AFM1 en fórmulas para lactantes comercializadas en el Área Metropolitana. Materiales y Métodos: Se adquirieron fórmulas fluidas (n=18) y en polvo (n=91) para lactantes de 0 a 12 meses de farmacias y supermercados del Área Metropolitana de Asunción y fueron analizados mediante el ensayo de inmunoafinidad ligado a enzimas (ELISA). Resultados: 9,75% (0 a 6 meses) y 2% (6 a 12 meses) de las fórmulas lácteas en polvo y 100% de las fórmulas fluidas resultaron positivas para AFM1. La mediana de contenido de AFM1 en formulas en polvo fue de 1820 ng/kg y 510 ng/kg en las marcas A y B. En las fórmulas líquidas fue de 31,8 ng/kg y 33,6 ng/kg para las dos marcas analizadas respectivamente, p=0,0001. Conclusiones: Se detectó AFM1 en todas las fórmulas líquidas analizadas, y en el 2 y 9,7% de las fórmulas en polvo de las marcas Ay B respectivamente Los niveles de AFM1 fueron mayores en las fórmulas en polvo.

ABSTRACT Introduction: Aflatoxin M1 (AFM1) is a toxic metabolite derived from aflatoxin B. Its ingestion in infants has been related to growth retardation, increased susceptibility to infectious diseases, reduced immunization efficiency, and cirrhosis. In Paraguay, exclusive breastfeeding is recommended up to 6 months of age, however, different brands of infant formulas targeted at this age range are marketed. Objective: To detect and quantify the presence of AFM1 in infant formulas marketed in the Metropolitan Area. Materials and Methods: Fluid (n = 18) and powder (n = 91) formulas for infants aged 0 to 12 months were purchased from pharmacies and supermarkets in the Metropolitan Area of Asunción and were analyzed using the enzyme-linked immunoaffinity assay (ELISA). Results: 9.75% (0 to 6 months) and 2% (6 to 12 months) of the powdered milk formulas and 100% of the fluid formulas were positive for AFM1. The median content of AFM1 in powder formulas was 1820 ng/kg and 510 ng/kg in brands A and B, while in liquid formulas it was 31.8 ng/kg and 33.6 ng/kg for the two brands analyzed respectively (p = 0.0001). Conclusions: AFM1 was detected in all the liquid formulas analyzed, and in 2 and 9.7% of the powder formulas of brands A and B, respectively. The levels of AFM1 were higher in the powder formulas.

Occurrence of Deoxynivalenol and Ochratoxin A in Beers and Wines Commercialized in Paraguay.

Alcoholic beverages can be contaminated with mycotoxins. Ochratoxin A (OTA) is the most frequently detected mycotoxinin wine and is produced by several species of Aspergillus. This mycotoxin is nephrotoxic and carcinogenic. In beer, the most commonly identified mycotoxin is deoxynivalenol (DON). Ingestion of food contaminated with DON has been associated with adverse gastrointestinal effects. Despite the harmful effects of mycotoxins on health, there are no regulations regarding their limits in alcoholic beverages in Paraguay. Here we determine the presence of OTA and DON in wine and beer, respectively. Four commercial brands of wine and twenty-nine brands of craft and industrial beerwere tested by the Agra quant ELISA method. One brand of wine was positive for OTA and seven brands of beer (one of them craft) were positive for DON. The values found for both toxins are below the recommended maximum intake proposed by international standards. Giving the high consumption of these products in the country, regulations and monitoring systems mustbe established to check the maximum levels of mycotoxins allowed in alcoholic beverages.

Presencia de micotoxinas en preparados y colados comerciales para lactantes en el área metropolitana de Asunción, Paraguay

Introducción: Las micotoxicosis son enfermedades producidas por micotoxinas, metabolitos secundarios tóxicos producidos por hongos filamentosos. Los lactantes son especialmente susceptibles a este tipo de toxinas debido a la inmadurez anatómica y funcional de sus sistemas digestivo e inmune, lo que se refleja en la relación entre la cantidad de alimento ingerida y su peso. Objetivo: Determinar la presencia de micotoxinas en alimentos para lactantes comercializados en farmacias y supermercados del Área Metropolitana. Materiales y métodos: Se colectaron al azar 66 unidades de productos de seis marcas diferentes de preparados y colados comerciales importados, dulces y salados, de farmacias y supermercados del Área Metropolitana. Posteriormente, fueron analizados mediante el ensayo de inmunoafinidad ligado a enzimas (ELISA). Se realizó el análisis de varianza y la posterior comparación de medias de las concentraciones de micotoxinas mediante la prueba de Tukey (IC= 95%), con el estadístico InfoStat®. Resultados: Las micotoxinas prevalentes fueron aflatoxinas (AF) y Toxina T2, que se presentaron en 39% de las unidades muestrales analizadas, tanto en preparados como en colados. En tercer orden de importancia se encuentra ocratoxina A (OTA), detectada en 18% de las mismas. En cuarto lugar, deoxinivalenol (DON) se detectó en 4% los productos. Se presentaron diferencias significativas entre los preparados y los colados, siendo los colados los que en media presentaron niveles más altos de concentraciones de todas las micotoxinas estudiadas. Conclusiones: Se constataron niveles variables de AF, OTA, T2 y DON en los alimentos para lactantes comercializados en el Área Metropolitana. Los colados presentaron concentraciones más elevadas de micotoxinas en media en todos los productos analizados. Tanto AF como OTA superaron los límites máximos permitidos por las normas internacionales.

Introduction: Mycotoxicoses are diseases caused by mycotoxins, secondary toxic metabolites produced by filamentous fungi. Infants are especially susceptible to this type of toxins due to the anatomical and functional immaturity of their digestive and immune systems, which is related to the amount of food eaten and their weight. Objective: To determine the presence of mycotoxins in foods for infants sold in pharmacies and supermarkets in the Metropolitan Area. Materials and methods: 66 units of products from six different brands of imported commercial sweet and salty preparations and strained foods from pharmacies and supermarkets in the Metropolitan Area were randomly collected. Subsequently, they were analyzed by the enzyme-linked immunoaffinity test (ELISA). The variance analysis and the subsequent comparison of means of mycotoxin concentrations were performed using the Tukey test (95% CI), with the InfoStat® statistic. Results: The prevalent mycotoxins were aflatoxins (AF) and T-2 Toxin, which were present in 39% of the sample units analyzed, both in preparations and in strains. In third order of importance we detected Ochratoxin A (OTA) in 18% of the units. Fourth, deoxinivalenol (DON) products were detected in 4%. There were significant differences between the preparations and the strains, with the strains having the highest concentration levels of all the mycotoxins studied. Conclusions: Variable levels of AF, OTA, T-2 and DON were found in infant foods marketed in the Metropolitan Area. The strains showed a higher average concentrations of mycotoxins in all the products analyzed. Both AF and OTA exceeded the maximum limits allowed by international standards.

Penfigoide ampollar en lactante / Bullous pemphigoid in an infant

El penfigoide ampollar es una enfermedad autoinmune rara en la niñez, que afecta usualmente a los adultos. Clínicamente puede ser indistinguible de otras dermatosis ampollares. Presentamos un caso de penfigoide ampollar en un niño de 6 meses de vida con ampollas generalizadas y revisamos los principales diagnósticos diferenciales en este grupo etario.

Bullous pemphigoid is an immunological disorder that predominantly affects the elderly. It may be clinically indistinguishable from other immunobullous dermatoses. We present a case of bullous pemphigoid in a 6-month-old boy who presented generalized bullae and review the main differential diagnoses.