ABSTRACT The yeast phase of 22 Histoplasma capsulatum clinical isolates from Mexico, Argentina, Colombia, and Guatemala and three reference strains, one from Panama and two from the United States of America (USA), were screened for thermosensitivity characteristics using different analyses. Growth curves at 0, 3, 6, 12, 24, and 30 h of incubation at 37 and 40 °C, the growth inhibition percentage at 40 °C, and the doubling time at 37 and 40 °C were determined for all yeasts studied. Most of the isolates examined exhibited thermotolerant phenotypes at 40 °C, whereas a thermosensitive phenotype at 40 °C was only detected in the Downs reference strain from the USA. Growth inhibition values lower than 33.8% supported the predominance of the thermotolerant phenotype at 40 °C. The doubling time means found for the different isolates were 5.14 h ± 1.47 h at 37 °C and 5.55 h ± 1.87 h at 40 °C. This is the first report to underscore the predominance of thermotolerant and delayed doubling time phenotypes in H. capsulatum clinical isolates from different regions of Latin America.

ABSTRACT At present, there is no standardized marker that is routinely used in clinical laboratories to diagnose coccidioidomycosis. Thus, the goals of this study were to obtain a sequence characterized amplified region (SCAR) marker for the identification of Coccidioides spp., evaluate its specificity and sensitivity in fungal DNA-spiked blood and sputum samples, and compare it with previously described molecular markers. Specific amplified fragment length polymorphism (AFLP) amplicons for Coccidioides immitis and Coccidioides posadasii were cloned into the vector pGEM® -T Easy vector and sequenced to develop a SCAR marker. Oligonucleotides were designed to identify Coccidioides spp. by polymerase chain reaction (PCR), and the specificity and sensitivity of these oligonucleotides were tested with the DNA from related pathogens. The specificity and sensitivity of the SCAR marker was evaluated with blood and sputum samples spiked with Coccidioides DNA and compared with other previously described markers (621, GAC2, and Ag2/PRA). In addition, the conditions for its use were established using biological samples. A specific marker named SCAR300 was obtained to identify Coccidioides spp. that exhibited good sensitivity and specificity. The results showed that all of the markers tested in this study can identify Coccidioides spp. However, the SCAR300 and 621 markers were the most sensitive, whereas the SCAR300 marker was the most specific. Thus, the SCAR300 marker is a useful tool to identify Coccidioides spp.

Abstract Metarhizium anisopliae is an anamorphic fungus widely studied due to its use as a biological control agent. In Mexico, it is mainly used as an integrated management strategy to control populations of spittlebugs (Hemiptera: Cercopidae) that affect sugarcane fields and cattle pastures. In this study, 13 strains isolated from Aeneolamia spp. (Hemiptera: Cercopidae) from the main areas of sugarcane production of Mexico and 11 reference strains of Metarhizium from different insect hosts and geographic origins were evaluated based on their conidial dimensions, thermotolerance at 15, 25, 30, and 35 ºC, random amplification of polymorphic DNA (RAPD) patterns using a combined analysis with 14 different oligonucleotides and diversity estimators, and a phylogenetic analysis with the ribosomal RNA internal transcribed spacer (ITS) region. All strains used in this study showed typical morphological characteristics corresponding to M. anisopliae and were able to grow at 25 and 30 ºC with restricted growth at low (15 ºC) and high temperatures (35 ºC). Polymorphism analysis clustered all strains from Aeneolamia sp. in one well-defined group with low variability among them. Phylogenetic relationships based on nuclear ribosomal ITS region sequences recovered a subclade within M. anisopliae formed by 10 of the Mexican strains.

Resumen Metarhizium anisopliae es un hongo anamórfico ampliamente estudiado debido a su uso como agente de control biológico. En México se utiliza principalmente como una estrategia de manejo integrado para controlar poblaciones de mosca pinta (Hemiptera: Cercopidae) que afectan los campos de caña de azúcar y pastizales de ganado. Estudios previos muestran evidencia de variabilidad fisiológica y filogenética entre las cepas de Metarhizium anisopliae. En este estudio, 13 cepas aisladas de Aeneolamia spp. (Hemiptera: Cercopidae) que provienen de las principales áreas de producción de caña de azúcar de México y 11 cepas de referencia de Metarhizium de diferentes insectos hospederos y orígenes geográficos, fueron evaluadas en función de sus dimensiones conidiales, su termotolerancia a 15, 25, 30 y 35 ºC, amplificación aleatoria de patrones de ADN polimórfico (RAPD) usando un análisis combinado con 14 oligonucleótidos diferentes, análisis de estimadores de diversidad y un análisis filogenético con la región de los espaciadores internos transcritos (ITS) del gen del ARN ribosómico. Todas las cepas utilizadas en este estudio mostraron características morfológicas típicas correspondientes a M. anisopliae, crecieron a 25 y 30 ºC con restricción a bajas (15 ºC) y altas (35 ºC) temperaturas. El análisis del polimorfismo agrupó todas las cepas de Aeneolamia sp. en un grupo bien definido con baja variabilidad entre ellos. El análisis filogenético evidenció un subclado dentro de M. anisopliae formado por 10 de las cepas mexicanas.

ABSTRACT The aim of this study was genotypically characterize Leptospira sp. clinical isolates from Mexico which were previously identified as Leptospira interrogans serovar Pomona (POM) by phenotypic methods. The Random Amplified Polymorphic DNA (RAPD) method was used for DNA amplification with five oligonucleotides. A dendrogram was constructed using the Unweighted Pair Group Method Analysis (UPGMA). During the genotypic characterization, the studied isolates constituted a group which was associated with the reference strain L. interrogans serovar Pomona. The Minimum Spanning Networks (MST) analysis revealed the same cluster between Mexican isolates and the reference strain POM. Clinical isolates identified as L. interrogans serovar POM have a clonal reproduction type, suggesting that this clone is distributed in different regions of Mexico.

ABSTRACT Candida glabrata complex includes three species identified through molecular biology methods: C. glabrata sensu stricto , C. nivariensis and C. bracarensis . In Mexico, the phenotypic methods are still used in the diagnosis; therefore, the presence of C. nivariensis and C. bracarensis among clinical isolates is still unknown. The aim of this study was to evaluate the utility of a multiplex PCR for the identification of the C. glabrata species complex. DNA samples from 92 clinical isolates that were previously identified through phenotypic characteristics as C. glabrata were amplified by four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f) that generate amplicons of 397, 293 and 223-bp corresponding to C. glabrata sensu stricto , C. nivariensis , and C. bracarensis , respectively. The amplicon sequences were used to perform a phylogenetic analysis through the Maximum Likelihood method (MEGA6), including strains and reference sequences of species belonging to C. glabrata complex. In addition, recombination and linkage disequilibrium were estimated (DnaSP version 5.0) for C. glabrata sensu stricto isolate s . Eighty-eight isolates generated a 397-bp fragment and only in one isolate a 223-bp amplicon was observed. In the phylogenetic tree, the sequences of 397-bp were grouped with C. glabrata reference sequences , and the sequence of 223-bp was grouped with C. bracarensis reference sequences, corroborating the PCR identification. The number of recombination events for the isolates of C. glabrata sensu stricto was zero, suggesting a clonal population structure. Three isolates that did not amplify any of the expected fragments were identified as Saccharomyces cerevisiae through the sequencing of the D1/D2 domain region within the 28S rDNA gene. The multiplex PCR is a fast, cost-effective and reliable tool that can be used in clinical laboratories to identify C. glabrata complex species.

La infección causada por el complejo Candida parapsilosis puede presentarse esporádicamente o en forma de brotes, por lo que el estudio de la variabilidad genética de los aislados clínicos puede revelar la presencia de genotipos endémicos y la ocurrencia de transmisión horizontal. Este estudio analizó, mediante Amplificación al Azar del ADN Polimórfico (RAPD) con cuatro oligonucleótidos (M13, AP3, T3B y R108), la variabilidad genética de 11 aislados clínicos del complejo C. parapsilosis, obtenidos en los servicios de Medicina Interna y Cirugía General de un hospital de la Ciudad de México, e identificar si los pacientes fueron infectados por el mismo genotipo. La cepa ATCC® 22019™ fue incluida en el análisis. Los aislados se identificaron por VITEK 2 Compact® y PCR. Con base en los perfiles polimórficos,se construyó un dendrograma por UPGMA y se calcularon el coeficiente de correlación cofenética(CCCr), el índice de asociación (I A) y los indicadores de diversidad genética. Todos los aislados fueron identificados como C. parapsilosis sensu stricto. El dendrograma mostró dos grupos (I y II), en el I se encontraron tres genotipos integrados por la cepa 22019 y cinco aislados asociados en su mayoría a candidiasis invasiva; el II mostró seis genotipos integrados por seis aislados asociados en su mayoría a candidiasis mucocutánea. El I A y los indicadores de diversidad genética obtenidos revelaron un sistema de reproducción recombinante. El RAPD con los oligonucleótidos M13, AP3, T3B y R108 es útil en la investigación de posibles brotes causados por C. parapsilosis y en la determinación de su variabilidad genética.

The infection caused by the Candida parapsilosis complex may occur sporadically or in the form of outbreaks, so the study of the genetic variability in clinical isolates may reveal the presence of endemic genotypes and the occurrence of horizontal transmission. This study analyzed the genetic variability of 11 clinical isolates of the C. parapsilosis complex obtained from Internal Medicine and General Surgery services of a hospital in Mexico City, using Random Amplification of Polymorphic DNA (RAPD) with four oligonucleotides (M13, AP3, T3B and R108), also evaluated whether the patients were infected by the same genotype. The strain ATCC® 22019 ™ was included in the analysis. Isolates were identified by VITEK 2 Compact® and PCR. With the polymorphic profiles, a dendrogram was constructed by UPGMA and the cophenetic correlation coefficient (CCCr), the association index (I A), and the indicators of genetic diversity were calculated. All isolates were identified as C. parapsilosis sensu stricto. The dendrogram showed two groups (I and II). Three genotypes integrated by the strain 22019 and five isolates, mostly associated with invasive candidiasis, were found in the group I. The group II showed six genotypes composed of six isolates, mostly associated with mucocutaneous candidiasis. The I A and the indicators of genetic diversity obtained, revealed a recombinant reproduction system. The RAPD with the oligonucleotides M13, AP3, T3B and R108 is useful in the investigation of possible outbreaks caused by C. parapsilosis and in the determination of their genetic variability.

The infection process whereby Isaria fumosorosea colonizes whitefly (Trialeurodes vaporariorum) nymphs was investigated using light and scanning electron microscopy. The fungal growth development index was used to determine pathogenicity of the isolates studied. The ultrastructural findings allowed us to examine the course of colonization of T. vaporariorum by this fungus through the formation of cuticular penetration structures. I. fumosorosea produced structures that resemble appressoria in shape, and isolates cause serious cuticular damage suggesting of enzymatic action. The results of this study suggest isolate EH-506/3 as suitable for whitefly biocontrol.

El proceso de infección por el cual Isaria fumosorosea coloniza a las ninfas de la mosquita blanca (Trialeurodes vaporariorum) fue investigado usando microscopía de luz y electrónica de barrido. El índice de crecimiento y desarrollo fúngico fue usado para determinar la patogenicidad de los aislados estudiados. Los hallazgos ultraestructurales nos permitieron examinar el curso de la colonización de este hongo en T. vaporariorum mediante la formación de estructuras de penetración en la cutícula. Los aislados de I. fumosorosea produjeron estructuras que recuerdan apresorios en su forma y causan serios daños cuticulares en el hospedante que sugieren la acción enzimática. Los resultados de este estudio sugieren que el aislado EH-506/3 es adecuado para el control biológico de la mosquita blanca.

Diabetes mellitus (DM) is a chronic-degenerative disease with a high incidence and prevalence around the world. Chronic hyperglycemia, a feature of diabetes, induces a higher production of superoxide free radicals, which play a major role in the mechanisms of tissue damage responsible for the clinical complications of DM: advanced glycosylation and activation of sorbitol, hexosamines, and protein kinase C pathways. Although different drugs that inhibit the damaging pathways have been developed, their actual benefit in diabetic patients has not been proved yet. Therefore, adequate glycemia control is still the fundamental factor to prevent or delay tissue damage.

La diabetes mellitus (DM) es una enfermedad crónico-degenerativa de elevada incidencia y prevalencia a nivel mundial. La hiperglucemia crónica, característica de la diabetes, genera mayor producción de radicales de superóxido, que juegan un papel principal en los mecanismos de daño tisular responsables de las complicaciones clínicas de la DM: la glucosilación de proteínas y acumulación de productos de la glucosilación avanzada, y la activación de las vías del sorbitol, de las hexosaminas y de la proteína cinasa C. Aunque se han desarrollado diferentes fármacos para inhibir las vías de daño, aún no se ha demostrado su verdadero beneficio en diabéticos. Por lo tanto, el buen control de la glucemia sigue siendo el factor fundamental para evitar o retardar el daño tisular.

Fifty-five clinical and environmental Aspergillus fumigatus isolates from Mexico, Argentina, France and Peru were analyzed to determine their genetic variability, reproductive system and level of differentiation using amplified fragment length polymorphism markers. The level of genetic variability was assessed by measuring the percentage of polymorphic loci, number of effective alleles, expected heterozygocity and by performing an association index test (I A). The degree of genetic differentiation and variation was determined using analysis of molecular variance at three levels. Using the paired genetic distances, a dendrogram was built to detect the genetic relationship among alleles. Finally, a network of haplotypes was constructed to determine the geographic relationship among them. The results indicate that the clinical isolates have greater genetic variability than the environmental isolates. The I A of the clinical and environmental isolates suggests a recombining population structure. The genetic differentiation among isolates and the dendrogram suggest that the groups of isolates are different. The network of haplotypes demonstrates that the majority of the isolates are grouped according to geographic origin.