CUESTAS Maria Lujan
congresos y reuniones científicas
Emmonsia parva and Histoplasma capsulatum cross-species reactivity by PCR targeting the Hc100 gene
Congreso; 19° Simposio ICHS y 14° Infocus Latinoamérica.; 2016
INTRODUCTIONThe conventional diagnostic methods for histoplasmosis from clinical samples or the culture identification of Histoplasma capsulatum present difficulties since these procedures are time consuming and delay diagnosis. This situation shows for implementing molecular assays. Hc100, a distinctive target gene of H. capsulatum was sought in order to develop a diagnostic PCR assay having high specificity. This study verified the cross-species reactions between Emmonsia parva and H. capsulatum. MATERIALS AND METHODS Four isolates of H. capsulatum var. capsulatum and one strain of E. parva were obtained from the culture collection of the Mycology Center, School of Medicine, University of Buenos Aires. Isolates of H. capsulatum were adapted to yeast-like fungi with successive subcultures using Brain Heart Infusion agar at 37 ºC. At 72 -96 h post-incubation, a total of 3 or 4 mm loopful from each fungus was put on 200 µl of sterile distilled water in microcentrifuge tubes. The suspension was vortexed vigorously for 1 min and tested as template for PCR. DNA of H. capsulatum was amplified from the yeast-like form during the PCR process in the thermocycler.Molecular identification of E. parva was also based on PCR amplification and nucleotide sequencing of the ITS regions and the Hc100 gene. Genomic DNA was then extracted and purified by using the QIAmp DNA Mini Kit. DNA quality and quantity were estimated by spectrophotometry. Ten ng/µl of DNA was used as template for PCR. The experiment was performed in triplicate.After the preparation of the corresponding templates, the Hc100 gene was amplified by PCR using primers Hc I (5´-GCG TTC CGA GCC TTC CAC CTC AAC-3´) and Hc II (5´-ATG TCC CAT CGG GCG CCG TGT AGT-3´).Furthermore, the ITS region was amplified by PCR using primers ITS1 and ITS4. Primers ITS1 (5?-TCC GTA GGT GAA CCT GCG G-3?) and ITS4 (5?-TCC TCC GCT TAT TGA TAT G-3?) were universal fungal primers which contained conserved regions among fungi and it were used by molecular identification of E. parva. The PCR reaction was performed and the amplicons were purified using a QIAquick PCR Purification Kit and then bidirectionally sequenced using an ABI Prism 3100 ⁄ 3100-Avant Genetic Analyser (Applied Biosystems, USA) with the same primers used for PCR amplification. The nucleotide sequences obtained were compared with those available in the GenBank database using the BLASTn database. Primer binding sites of HcI and HcII in E. crescens KKZ59014, E. parva KLJ06212 and Ajellomyces capsulatus KC990363 were calculated using the CLC Workbench software (CLC Bio-Qiagen, Aarhus, Denmark).RESULTS This study verified the cross-species reactivity between E. parva and H. capsulatum var. capsulatum in theHc100 gene. However, a faint PCR band was distinguished in E. parva. The sequence alignment of E. parva (278 bp corresponding to the partial sequence of the Hc100 gene) showed 89% sequence similarity (query coverage 93%) to Ajellomyces capsulatus KC990363 and 88% sequence identity (query coverage 93%) to Ajellomyces capsulatus KC990367.CONCLUSIONSThis study verified the cross-species reactions between Emmonsia parva and H. capsulatum. There is not sufficient divergence targeting the Hc 100 gene in E. parva to prevent primer binding and thus subsequent amplification using the primers designed for the molecular identification of H. capsulatum. The sequence alignment of E. parva studied here (278 bp corresponding to the Hc100 gene) did not identify this species.The nucleotide sequences of the ITS region of the E. parva (621 bp) under study showed 99 % similarity with an E. parva strain (accesion number AF038327). The phylogenetic relationships from the ITS regions of the Emmonsia isolate and Ajellomyces capsulatus confirmed that the Emmonsia species (E. crescens and E. parva) are distinct from one another and from the anamorphic state of Ajellomyces capsulatus. Therefore, it is necessary to investigate a larger number of strains of related species from the genus Emmonsia causing adiaspiromycosis or systemic human mycoses in immunocompromised patients for cross-species reactivity in the PCR assay targeting the Hc100 gene.