Molecular identification of clinical isolates of Fusarium in Colombia

Abstract

Objective Identifying Fusarium isolates from mycosis symptomatic patients through molecular techniques as PCR and sequencing. Methods In this study, samples were taken from 101 mycosis symptomatic patients in-between [2004][2005][2006]. To determine isolates belonging to the Fusarium genus, the DNAr 28S region was amplified through PCR and specific PCR primers further confirmed their identity to the species level. Additionally, in order to confirm the identity of the species of the isolates, 75 isolates of these were analyzed by partial sequencing of the 28S rDNA and the TEF1-α gene. Results The 28S rDNA portion detected all 101 isolates as belonging to Fusarium and the PCR specific primers detected 52 and 29 isolates as F. oxysporum and F. solani, respectively; 34 and 41 of these, afterwards studied by partial sequencing of the 28S rDNA and TEF1-α genes respectively, were effectively identified by the technique. Conclusion From all the molecular markers used to identify Fusarium isolates, the sequence of the TEF1-α gene provided the best resolution in the identification of species level; however it is possible to discriminate between F. oxysporum and F. solani isolates by PCR, in most of the cases, what is important considering the simplicity of the technique and a faster diagnosis.Key Words: PCR; rDNA; sequence analysis; elongation factor (source: MeSH, NLM). RESUMENObjetivo Identificar aislamientos de Fusarium en pacientes con micosis por medio de las técnicas moleculares de PCR y secuenciación. Métodos Se tomaron 101 muestras de pacientes con micosis sintomática, entre los años 2004 y 2006. Para la detección de aislamientos como pertenecientes al género Fusarium, se amplificó parcialmente por PCR la región 28S del DNAr; y posteriormente -para la detección de la especie de Fusarium-se utilizaron cebadores específicos para F. oxysporum y F. solani. La verificación de la identidad de la especie de los aislamientos se hizo por secuenciación parcial de los genes 28S DNAr y TEF1-α. Resultados El total de 101 aislamientos fueron detectados como pertenecientes al género Fusarium utilizando un cebador universal de la region 28S DNAr; 52 y 29 aislamientos se detectaron como F. oxysporum o F. salani, respectivamente con los cebadores específicos, y la secuenciación parcial de los genes 28S rDNA o TEF1-α confirmó la identidad de las especies. Conclusión La secuenciación parcial del gen TEF1-α es aún el mejor marcador molecular para identificar aislamientos de Fusarium a nivel de especie. Sin embargo, en la mayoría de los casos es posible discriminar entre aislamientos de F. oxysporum y F. solani por PCR con cebadores específicos, lo que proporciona una ventaja importante considerando la simplicidad de la técnica y el rápido diagnóstico.Palabras Clave: ADN ribosómico; PCR; secuenciación; elongación (fuente: DeCS, BIREME).

First Comprehensive Report of Clinical Fusarium Strains Isolated in the State of Sao Paulo (Brazil) and Identified by MALDI-TOF MS and Molecular Biology

“…There is an increasing incidence of infections related to immunosuppressed patients (e.g., elderly, AIDS, solid organ, and bone marrow transplantation; oncological, hematological, and autoimmune diseases) [10,11]. The number of subcutaneous fusarioses has increased at alarming levels, making fusariosis, after aspergillosis, the second most common filamentous fungal infection in patients with hematologic neoplasms [12][13][14].…”

Assessment of genotyping markers in the molecular characterization of a population of clinical isolates of Fusarium in Colombia

“…However, the large amount of information about fusariosis shows the importance of keeping the FSSC classification for medical professionals (16). In Colombia, the identification of Fusarium at the species level for epidemiological purposes is not common (18,19), although, in the last years, there have been more studies to characterize clinical isolates at this level (20)(21)(22)(23). The lack of this type of characterization has been a serious problem for physicians as proper identification is necessary for an adequate treatment (24).…”

“…Currently, the highest resolution marker is the translation elongation factor 1-α (TEF1-α) gene (31) now complemented with multilocus sequence typing (MLST) through the addition of two partial sequences from the two largest DNA-directed RNA polymerase subunits (RPB1 and RPB2) (29,32). Despite these resources, there is still a need for other molecular methods to complement current approaches and provide more information about the genetic diversity of the isolates and their correlation with phenotypical traits (23). Therefore, it is necessary to evaluate molecular markers for the identification of Fusarium species supplying enough information for phylogenetic inference, population genetics, and epidemiology (8).…”

Identification structure of the mating‐type locus and distribution of Kabatiella zeae in China

“…After incubation for 16 hr, individually germinated spores were picked from the plate using a sterile needle under a dissection microscope, and the spores were sub-cultured at 25°C for 7 days on PDA. Single spore cultures of 51 isolates were identified as K. zeae with morphological method and ITS subunit sequences alignment; in addition, JLMHK-9 strain was identified as 28S rDNA subunit sequences (Arny et al, 1971;Gaviria-Rivera et al, 2018;Meng et al, 2016) ( Figure S1; Table S1). The phylogenetic tree of the 28S rDNA and ITS region was constructed by maximum-likelihood with 1000 replicates, using PhyML 3.0 online software ( Figure S1; Table S1).…”