Selection of Specific Peptides for Coccidioides spp. Obtained from Antigenic Fractions through SDS-PAGE and Western Blot Methods by the Recognition of Sera from Patients with Coccidioidomycosis.

Antigenic fractions of 100, 50, 37, and 28 kDa obtained through the SDS-PAGE method that were more frequently recognized by anti-Coccidioides antibodies in the sera of coccidioidomycosis patients were selected using western blotting. Subsequently, these bands were sequenced, and the obtained proteins were analysed by BLAST to choose peptides specific for Coccidioides spp. from among the shared aligned sequences of related fungi. A peptide specific for C. immitis was selected from the "GPI anchored serine-threonine rich protein OS C. immitis", while from the "uncharacterized protein of C. immitis", we selected a peptide for C. immitis and C. posadasii. These proteins arose from the 100 kDa antigenic fraction. From the protein "fatty acid amide hydrolase 1 of C. posadasii" that was identified from the 50 kDa antigenic fraction, a peptide was selected that recognized C. immitis and C. posadasii. In addition, the analysis of all the peptides (353) of each of the assembled proteins showed that only 35 had 100% identity with proteins of C. immitis and C. posadasii, one had 100% identity with only C. immitis, and one had 100% identity with only C. posadasii. These peptides can be used as diagnostic reagents, vaccines, and antifungals.

The habitat of Coccidioides spp. and the role of animals as reservoirs and disseminators in nature.

BACKGROUND: Coccidioidomycosis, a potentially fatal fungal infection, is considered an emergent mycotic disease because of the increased incidence of fungal infections registered over recent years. Infection occurs through the inhalation of arthroconidia from two main species of Coccidioides: Coccidioides immitis and C. posadasii, which are both endemic to arid and semi-arid regions of North America. Coccidioides species not only infect humans but can also infect other mammals (land, aquatic, wild or domestic), reptiles and birds. OBJECTIVE: To obtain information regarding the habitat of Coccidioides spp. and the animals infected by this fungus and to identify the role that infected animals play as reservoirs and disseminators of this fungus in nature. MATERIALS: A literature review was conducted to identify the habitat of Coccidioides spp. and the infected non-human animal species targeted by this fungus. RESULTS AND CONCLUSIONS: This review allows us to suggest that Coccidioides spp. may be classified as halotolerant organisms; nevertheless, to perpetuate their life cycle, these organisms depend on different animal species (reservoirs) that serve as a link with the environment, by acting as disseminators of the fungi in nature.

Development of SCAR Markers for Genetic Authentication of Metarhizium acridum.

In this study, molecular typing using Randomly Amplified Polymorphic DNA (RAPD-PCR) was conducted on 16 original isolates of Metarhizium acridum obtained from locusts (Schistocerca piceifrons ssp. piceifrons.) in Mexico (MX). The analysis included reference strains of the genus Metarhizium sourced from various geographical regions. The isolates were identified by phenotypic (macro and micromorphology) and genotypic methods (RAPD-PCR and Amplified Fragment Length Polymorphisms (AFLP), through a multidimensional analysis of principal coordinates (PCoA) and a minimum spanning network (MST). Subsequently, Sequences-Characterized Amplified Region (SCAR) markers were developed for the molecular detection of M. acridum, these markers were chosen from polymorphic patterns obtained with 14 primers via RAPD-PCR. Phenotypic and genotypic characterization identified the MX isolates as M. acridum. Of all the polymorphic patterns obtained, only OPA04 and OPA05 were chosen, which presented species-specific bands for M. acridum, and further utilized to create SCAR markers through cloning and sequencing of the specific bands. The specificity of these two markers was confirmed via Southern hybridization. The SCAR markers (Ma-160OPA-05 and Ma-151OPA-04) exhibit remarkable sensitivity, detecting down to less than 0.1 ng, as well as high specificity, as evidenced by their inability to cross-amplify or generate amplification with DNAs from other strains of Metarhizium (as Metarhizium anisopliae) or different genera of entomopathogenic fungi (Cordyceps fumosorosea and Akanthomyces lecanii). These SCAR markers yield readily detectable results, showcasing high reproducibility. They serve as a valuable tool, especially in field applications.

AFLP analysis reveals high genetic diversity but low population structure in Coccidioides posadasii isolates from Mexico and Argentina.

BACKGROUND: Coccidioides immitis and C. posadasii cause coccidioidomycosis, a disease that is endemic to North and South America, but for Central America, the incidence of coccidioidomycosis has not been clearly established. Several studies suggest genetic variability in these fungi; however, little definitive information has been discovered about the variability of Coccidioides fungi in Mexico (MX) and Argentina (AR). Thus, the goals for this work were to study 32 Coccidioides spp. isolates from MX and AR, identify the species of these Coccidioides spp. isolates, analyse their phenotypic variability, examine their genetic variability and investigate the Coccidioides reproductive system and its level of genetic differentiation. METHODS: Coccidioides spp. isolates from MX and AR were taxonomically identified by phylogenetic inference analysis using partial sequences of the Ag2/PRA gene and their phenotypic characteristics analysed. The genetic variability, reproductive system and level of differentiation were estimated using AFLP markers. The level of genetic variability was assessed measuring the percentage of polymorphic loci, number of effective allele, expected heterocygosity and Index of Association (IA). The degree of genetic differentiation was determined by AMOVA. Genetic similarities among isolates were estimated using Jaccard index. The UPGMA was used to contsruct the corresponding dendrogram. Finally, a network of haplotypes was built to evaluate the genealogical relationships among AFLP haplotypes. RESULTS: All isolates of Coccidioides spp. from MX and AR were identified as C. posadasii. No phenotypic variability was observed among the C. posadasii isolates from MX and AR. Analyses of genetic diversity and population structure were conducted using AFLP markers. Different estimators of genetic variability indicated that the C. posadasii isolates from MX and AR had high genetic variability. Furthermore, AMOVA, dendrogram and haplotype network showed a small genetic differentiation among the C. posadasii populations analysed from MX and AR. Additionally, the IA calculated for the isolates suggested that the species has a recombinant reproductive system. CONCLUSIONS: No phenotypic variability was observed among the C. posadasii isolates from MX and AR. The high genetic variability observed in the isolates from MX and AR and the small genetic differentiation observed among the C. posadasii isolates analysed, suggest that this species could be distributed as a single genetic population in Latin America.

Phenotypic and Genotypic Identification of Dermatophytes from Mexico and Central American Countries.

Dermatophytes are fungi included in the genera Trichophyton, Microsporum, Epidermophyton, Nannizzia, Paraphyton, Lophophyton, and Arthroderma. Molecular techniques have contributed to faster and more precise identification, allowing significant advances in phylogenetic studies. This work aimed to identify clinical isolates of dermatophytes through phenotypic (macro- and micromorphology and conidia size) and genotypic methods (sequences of ITS regions, genes of ß tubulin (BT2), and elongation factor α (Tef-1α)) and determine the phylogenetic relationships between isolates. Ninety-four dermatophyte isolates from Costa Rica, Guatemala, Honduras, Mexico, and the Dominican Republic were studied. The isolates presented macro- and micromorphology and conidia size described for the genera Trichophyton, Microsporum, and Epidermophyton. Genotypic analysis classified the isolates into the genera Trichophyton (63.8%), Nannizzia (25.5%), Arthroderma (9.6%), and Epidermophyton (1.1%). The most frequent species were T. rubrum (26 isolates, 27.6%), T. interdigitale (26 isolates, 27.6%), and N. incurvata (11 isolates, 11.7%), N. gypsea and A. otae (nine isolates, 9.6%), among others. The genotypic methods clarified the taxonomic status of closely related species. For instance, the ITS and BT2 markers of T. rubrum/T. violaceum did not differ but the Tef-1α gene did. On the other hand, the three markers differed in T. equinum/T. tonsurans. Therefore, the ITS, BT2, and Tef-1α genes are useful for typing in phylogenetic analyses of dermatophytes, with Tef-1α being the most informative locus. It should be noted that isolate MM-474 was identified as T. tonsurans when using ITS and Tef-1α, but when using BT2, it was identified as T. rubrum. On the other hand, no significant difference was found when comparing the methods for constructing phylogenies, as the topologies were similar.

Clinical Cases of Coccidioidomycosis in the Americas in the Period 1950-2021: Epidemiology Data, Diagnosis, and Treatment.

Coccidioidomycosis, caused by Coccidioides immitis and C. posadasii, causes significant morbidity and mortality, both in immunocompetent and immunocompromised people, mainly in endemic areas. The present work analyzed its epidemiology, diagnostic methods, and treatment by reviewing clinical cases published from 1950 to 2021. Fifty-nine articles were included, corresponding to 275 clinical cases. The results showed a higher incidence of coccidioidomycosis in the male gender than the female gender. The most affected age group was 31-40 years, and the most reported clinical presentation was disseminated with greater involvement in cutaneous and subcutaneous tissue, followed by the CNS, bone system, and peritoneum. The species most frequently reported was C. immitis. The most used treatment was azoles, followed by their combination with amphotericin B, monotherapy with amphotericin B, and alternative medicine. This work shows that epidemiological data outside the USA are still scarce. Serological tests are the preferred diagnostic method in daily medical practice, and cultures remain the gold standard. The treatment for coccidioidomycosis is ketoconazole and amphotericin B, individually or in combination.

Vaccines Against COVID-19: A Review.

As a result of the COVID-19 pandemic, various joint efforts have been made to support the creation of vaccines. Different projects have been under development, of which some are in the clinical evaluation stage and others in are in phase III with positive results. The aim of this paper was to describe the current situation of the development and production of vaccines available to the population to facilitate future research and continue developing and proposing ideas for the benefit of the population. So, we carried out a systematic review using databases such as PubMed, ScienceDirect, SciELO, and MEDLINE, including keywords such as "vaccines," "COVID-19," and "SARS-CoV-2". We reviewed the development and production of the anti-COVID vaccine and its different platforms, the background leading to the massive development of these substances, and the most basic immune aspects for a better understanding of their physiological activity and the immune response in those who receive the vaccine. We also analyzed immunization effects in populations with any medical or physiological conditions (such as immunosuppression, people with comorbidities, and pregnancy), as well as the response to immunization with heterologous vaccines and the hybrid immunity (the combination of natural immunity to SARS-CoV-2 with immunity generated by the vaccine). Likewise, we address the current situation in Mexico and its role in managing the vaccination process against SARS-CoV-2 at the national and international levels. There are still many clinical and molecular aspects to be described, such as the duration of active immunity and the development of immunological memory, to mention some of the most important ones. However, due to the short time since the global vaccination roll-out and that it has been progressive (not counting children and people with medical conditions), it is premature to say whether a second vaccination schedule will be necessary for the near future. Thus, it is essential to continue with health measures.

Selection of Polymorphic Patterns Obtained by RAPD-PCR through Qualitative and Quantitative Analyses to Differentiate Aspergillus fumigatus.

The objective of this work was to use the random amplification of the polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique to select polymorphic patterns through qualitative and quantitative analyses to differentiate the species A. flavus, A. fumigatus, A. niger and A. tubingensis. Twenty-seven Aspergillus isolates from different species were typified using phenotypic (macro- and micromorphology) and genotypic (partial BenA gene sequencing) methods. Thirty-four primers were used to obtain polymorphic patterns, and with these a qualitative analysis was performed to select the primers that presented species-specific patterns to distinguish each species. For the quantitative selection, a database was built from the polymorphic patterns and used for the construction of logistic regression models; later, the model that presented the highest value of sensitivity against specificity was evaluated through ROC curves. The qualitative selection showed that the primers OPA-19, P54, 1253 and OPA-02 could differentiate the species. A quantitative analysis was carried out through logistic regression, whereby a species-specific correlation of sensitivity and specificity greater than 90% was obtained for the primers: OPC-06 with a 96.32% match to A. flavus; OPF-01 with a 100% match to A. fumigatus; OPG-13 with a 98.01% match to A. tubingensis; and OPF-07 with a 99.71% match to A. niger. The primer OPF-01 discriminated the four species as well as closely related species. The quantitative methods using the selected primers allowed discrimination between species and showed their usefulness for genotyping some of the species of medical relevance belonging to the genus Aspergillus.

Detection and Molecular Identification of Eight Candida Species in Clinical Samples by Simplex PCR.

Systemic candidiasis is a frequent opportunistic mycosis that can be life-threatening. Its main etiological agent is Candida albicans; however, the isolation of non-albicans Candida species has been increasing. Some of these species exhibit greater resistance to antifungals, so the rapid and specific identification of yeasts is crucial for a timely diagnosis and optimal treatment of patients. Multiple molecular assays have been developed, based mainly on polymerase chain reaction (PCR), showing high specificity and sensitivity to detect and identify Candida spp. Nevertheless, its application in diagnosis has been limited due to specialized infrastructure or methodological complexity. The objective of this study was to develop a PCR assay that detects and identifies some of the most common pathogenic Candida species and evaluate their diagnostic utility in blood samples and bronchial lavage. A pair of oligonucleotides was designed, CandF and CandR, based on sequence analysis of the 18S-ITS1-5.8S-ITS2-28S region of the rDNA of Candida spp., deposited in GenBank. The designed oligonucleotides identified C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei/Pichia kudriazevii, C. guilliermondii/Meyerozyma guilliermondii, C. lusitaniae/Clavispora lusitaniae, and C. dubliniensis using simplex PCR based on the amplicon size, showing a detection limit of 10 pg/µL of DNA or 103 yeasts/mL. Based on cultures as the gold standard, it was determined that the sensitivity (73.9%), specificity (96.3%), and the positive (94.4%) and negative (81.2%) predictive values of the PCR assay with the designed oligonucleotides justify their reliable use in diagnosis.

Aspergillus-SARS-CoV-2 Coinfection: What Is Known?

COVID-19-associated pulmonary aspergillosis (CAPA) has had a high incidence. In addition, it has been associated with prolonged hospital stays, as well as several predisposing risk factors, such as fungal factors (nosocomial organism, the size of the conidia, and the ability of the Aspergillus spp. of colonizing the respiratory tract), environmental factors (remodeling in hospitals, use of air conditioning and negative pressure in intensive care units), comorbidities, and immunosuppressive therapies. In addition to these factors, SARS-CoV-2 per se is associated with significant dysfunction of the patient's immune system, involving both innate and acquired immunity, with reduced CD4+ and CD8+ T cell counts and cytokine storm. Therefore, this review aims to identify the factors influencing the fungus so that coinfection with SARS-CoV-2 can occur. In addition, we analyze the predisposing factors in the fungus, host, and the immune response alteration due to the pathogenicity of SARS-CoV-2 that causes the development of CAPA.

Phenotypic characteristics of isolates of Aspergillus section Fumigati from different geographic origins and their relationships with genotypic characteristics.

BACKGROUND: Epidemiological studies worldwide have shown that A. fumigatus exhibits important phenotypic and genotypic diversity, and these findings have been of great importance in improving the diagnosis and treatment of diseases caused by this fungus. However, few studies have been carried out related to the epidemiology of this fungus in Latin America. This study's aim is to report on the epidemiology of the fungus by analyzing the phenotypic variability of Aspergillus section Fumigati isolates from different Latin American countries and the relationship between this variability, the geographical origin and genotypic characteristics. METHODS: We analyzed the phenotypic characteristics (macro- and micromorphology, conidial size, vesicles size, antifungal susceptibility and thermotolerance at 28, 37 and 48°C) of A. section Fumigati isolates from Mexico (MX), Argentina (AR), Peru (PE) and France (FR). The results were analyzed using analysis of variance (ANOVA) and Tukey's multiple comparison test to detect significant differences. Two dendrograms among isolates were obtained with UPGMA using the Euclidean distance index. One was drawn for phenotypic data, and the other for phenotypic and genotypic data. A PCoA was done for shown isolates in a space of reduced dimensionality. In order to determine the degree of association between the phenotypic and genotypic characteristics AFLP, we calculated the correlation between parwise Euclidean distance matrices of both data sets with the nonparametric Mantel test. RESULTS: No variability was found in the macromorphology of the studied isolates; however, the micromorphology and growth rate showed that the PE isolates grew at a faster rate and exhibited the widest vesicles in comparison to the isolates from MX, AR and FR. The dendrogram constructed with phenotypic data showed three distinct groups. The group I and II were formed with isolates from PE and FR, respectively, while group III was formed with isolates from MX and AR. The dendrogram with phenotypic and genotypic data showed the same cluster, except for an isolate from FR that formed a separate cluster. This cluster was confirmed using PCoA. The correlation between the phenotypic and genotypic data of the isolates revealed a statistically significant association between these characteristics. CONCLUSIONS: The PE isolates showed specific phenotypic characteristics that clearly differentiate them from the rest of the isolates, which matches the genotypic data. The correlation between the phenotypic and genotypic characteristics showed a statistically significant association. In conclusion, phenotypic and genotypic methods together increase the power of correlation between isolates.

Sporotrichosis in Mexico.

Sporotrichosis is an endemic mycosis caused by the species of the Sporothrix genus, and it is considered one of the most frequent subcutaneous mycoses in Mexico. This mycosis has become a relevant fungal infection in the last two decades. Today, much is known of its epidemiology and distribution, and its taxonomy has undergone revisions. New clinical species have been identified and classified through molecular tools, and they now include Sporothrix schenckii sensu stricto, Sporothrix brasiliensis, Sporothrix globosa, and Sporothrix luriei. In this article, we present a systematic review of sporotrichosis in Mexico that analyzes its epidemiology, geographic distribution, and diagnosis. The results show that the most common clinical presentation of sporotrichosis in Mexico is the lymphocutaneous form, with a higher incidence in the 0-15 age range, mainly in males, and for which trauma with plants is the most frequent source of infection. In Mexico, the laboratory diagnosis of sporotrichosis is mainly carried out using conventional methods, but in recent years, several researchers have used molecular methods to identify the Sporothrix species. The treatment of choice depends mainly on the clinical form of the disease, the host's immunological status, and the species of Sporothrix involved. Despite the significance of this mycosis in Mexico, public information about sporotrichosis is scarce, and it is not considered reportable according to Mexico's epidemiological national system, the "Sistema Nacional de Vigilancia Epidemiológica." Due to the lack of data in Mexico regarding the epidemiology of this disease, we present a systematic review of sporotrichosis in Mexico, between 1914 and 2019, that analyzes its epidemiology, geographic distribution, and diagnosis.

Phylogenetic Identification, Diversity, and Richness of Aspergillus from Homes in Havana, Cuba.

Aspergillus is one of the most common fungal genera found indoors; it is important because it can cause a wide range of diseases in humans. Aspergillus species identification is based on a combination of morphological, physiological, and molecular methods. However, molecular methodologies have rarely been used for the identification of environmental isolates of Aspergillus in Cuba. Therefore, the objective of this work was to identify the species of the genus Aspergillus obtained from houses in Havana, Cuba, through the construction of phylogeny from a partial sequence of the benA gene region, and to analyze the diversity and richness of Aspergillus in the studied municipalities. Isolates of Aspergillus spp. included in this study presented the typical macro- and micromorphology described for the genus. According to this polyphasic characterization, A. niger, A. flavus, A. welwitschiae, A. heteromorphus, A. sydowii, A. tamarii, A. fumigatus, A. clavatus, and A. tubingensis were the most abundant species. Most of the identified species constitute new records for outdoor and indoor environments in Cuba and contribute to the knowledge of fungal biodiversity in the country. These results constitute an alert for the health authorities of the country, since prolonged exposure of the inhabitants to Aspergillus spores can cause severe persistent asthma, among other diseases.

Molecular identification of yeasts from the order Trichosporonales causing superficial infections.

BACKGROUND: The molecular reclassification of the order Trichosporonales placed the medically relevant Trichosporon species into three genera of the family Trichosporonaceae: Cutaneotrichosporon, Trichosporon, and Apiotrichum. From the clinical and epidemiological standpoint, it is important to identify any species of the family Trichosporonaceae because they present different antifungal susceptibility profiles. In Mexico, little is known about trichosporonosis etiology because the fungi are identified through phenotypic methods. AIMS: To identify at a molecular level 12 yeast isolates morfologically compatible with Trichosporon, obtained from patients with superficial infections. METHODS: The yeast isolates were obtained from patients with white piedra, onychomycosis, and hand and foot dermatomycosis, and were identified morphologically and genotypically (sequencing of the IGS1 region and phylogenetic analysis using the Maximum Likelihood Method). The phylogenetic analysis included 40 yeast sequences from the order Trichosporonales and one from Cryptococcus neoformans as outgroup. RESULTS: Based on the molecular analysis, we identified three (25%) Trichosporon inkin isolates, two (16.7%) Trichosporon asteroides, two (16.7%) Cutaneotrichosporon mucoides, and one each (8.3%) of Trichosporon aquatile, Trichosporon asahii, Apiotrichum montevideense, Cutaneotrichosporon cutaneum, and Cutaneotrichosporon jirovecii. CONCLUSIONS: The molecular characterization of the isolates showed a broad diversity of species within the order Trichosporonales, particularly among onychomycosis. It is essential to identify these yeasts at the species level to delve into their epidemiology.

Diagnostic accuracy of antigen detection in urine and molecular assays testing in different clinical samples for the diagnosis of progressive disseminated histoplasmosis in patients living with HIV/AIDS: A prospective multicenter study in Mexico.

BACKGROUND: The progressive disseminated histoplasmosis (PDH) has been associated with severe disease and high risk of death among people living with HIV (PLWHIV). Therefore, the purpose of this multicenter, prospective, double-blinded study done in ten Mexican hospitals was to determine the diagnostic accuracy of detecting Histoplasma capsulatum antigen in urine using the IMMY ALPHA Histoplasma EIA kit (IAHE), clarus Histoplasma GM Enzyme Immunoassay (cHGEI IMMY) and MiraVista Histoplasma Urine Antigen LFA (MVHUALFA); as well as the Hcp100 and 1281-1283220SCAR nested PCRs in blood, bone-marrow, tissue biopsies and urine. METHODOLOGY/PRINCIPAL FINDINGS: We included 415 PLWHIV older than 18 years of age with suspicion of PDH. Using as diagnostic standard recovery of H. capsulatum in blood, bone marrow or tissue cultures, or histopathological exam compatible, detected 108 patients (26%, [95%CI, 21.78-30.22]) with proven-PDH. We analyzed 391 urine samples by the IAHE, cHGEI IMMY and MVHUALFA; the sensitivity/specificity values obtained were 67.3% (95% CI, 57.4-76.2) / 96.2% (95% CI, 93.2-98.0) for IAHE, 91.3% (95% CI, 84.2-96.0) / 90.9% (95% CI, 87.0-94.0) for cHGEI IMMY and 90.4% (95% CI, 83.0-95.3) / 92.3% (95% CI, 88.6-95.1) for MVHUALFA. The Hcp100 nested PCR was performed on 393, 343, 75 and 297, blood, bone marrow, tissue and urine samples respectively; the sensitivity/specificity values obtained were 62.9% (95%CI, 53.3-72.5)/ 89.5% (95%CI, 86.0-93.0), 65.9% (95%CI, 56.0-75.8)/ 89.0% (95%CI, 85.2-92.9), 62.1% (95%CI, 44.4-79.7)/ 82.6% (95%CI, 71.7-93.6) and 34.9% (95%CI, 24.8-46.2)/ 67.3% (95%CI, 60.6-73.5) respectively; and 1281-1283220SCAR nested PCR was performed on 392, 344, 75 and 291, respectively; the sensitivity/specificity values obtained were 65.3% (95% CI, 55.9-74.7)/ 58.8% (95%CI, 53.2-64.5), 70.8% (95%CI, 61.3-80.2)/ 52.9% (95%CI, 46.8-59.1), 71.4% (95%CI, 54.7-88.2)/ 40.4% (95%CI, 26.4-54.5) and 18.1% (95%CI, 10.5-28.1)/ 90.4% (95%CI, 85.5-94.0), respectively. CONCLUSIONS/SIGNIFICANCE: The cHGEI IMMY and MVHUALFA tests showed excellent performance for the diagnosis of PDH in PLWHIV. The integration of these tests in clinical laboratories will certainly impact on early diagnosis and treatment.

Identification of CSP Types and Genotypic Variability of Clinical and Environmental Isolates of Aspergillus fumigatus from Different Geographic Origins.

The CSP (cell surface protein) microsatellite marker is useful for typing Aspergillus fumigatus isolates and determining relationships at the subpopulation level because it has shown high discriminatory power. In the present study, 90 A. fumigatus isolates from Mexico (MX), Argentina (AR), France (FR), and Peru (PE) were identified through a phylogenetic analysis using the benA gene fragment and were typed with the CSP microsatellite, and the types were identified using the nomenclature recommended in the literature. Genetic variability was analyzed through haplotype diversity, nucleotide diversity, polymorphic sites, and nucleotide differences between pairs of sequences. The population structure was evaluated using the Tajima's D statistic. No new CSP types were recorded in the MX, FR, and PE isolates, while in the AR isolates, two new CSP types were identified (t25 and t26). The most common CSP types in the studied populations were t01, t02, t03, and t04A; these results are consistent with findings in other countries. In addition, the genetic diversity parameters we obtained revealed that the greatest genetic diversity was found in the MX population, followed by AR and FR. No population structure was identified among the isolates studied.

Population structure and diversity of the pathogenic fungus Aspergillus fumigatus isolated from different sources and geographic origins.

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.

Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples.

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.

Genotyping of Leptospira interrogans isolates from Mexican patients.

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.

Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates.

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.