| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1228-1 | ||||
Resumo:Histoplasmosis, caused by the thermodimorphic fungus Histoplasma capsulatum and related species, is a systemic mycosis commonly found in nitrogenous bird droppings and bat guano. The disease is transmitted through inhalation of fungal propagules in the air, leading to pulmonary clinical manifestations that may disseminate and result in fatalities. Although histoplasmosis is widespread globally, accurately estimating the number of cases can be challenging due to misdiagnosis, often confused with tuberculosis due to clinical similarities. Histoplasmosis is highly prevalent in the United States, particularly in the Mississippi and Ohio river regions, where it is the most common fungal disease. In Brazil, the disease accounts for 2.19 out of every 1,000 hospitalizations. Despite the scarcity of molecular epidemiological data for histoplasmosis, recent reports indicate its growing recognition in regions beyond the traditional endemic areas in the Americas. This study aimed to investigate the genetic diversity of Histoplasma from different regions in Brazil using AFLP (Amplified fragment length polymorphism) markers. Genome sequences (n=9) of Histoplasma spp. were obtained from Genbank and in silico-digested with EcoRI and MseI using ISIF software, considering fragments of 50-500 bp. A total of 256 combinations of selective primers were applied to generate 2,304 fingerprint profiles with varying fragment numbers and sizes (3 to 406 fragments per combination). In silico analysis recognized six primer pair combinations for in vitro testing (C1–C6). The protocol was implemented for a subset of Histoplasma isolates (n=16). In vitro AFLP analysis produced 398 fragments in the six tested combinations, which were further analyzed using BioNumerics v.7.6. Dendrograms constructed based on the Jaccard coefficient and UPGMA showed well-supported branches with excellent global cophenetic values (75–84%). The isolates were grouped into two main clusters according to their band profiles. Genetic diversity indices were calculated for each combination, revealing great polymorphism information content (PIC) values ranging from 0.3670 to 0.3750, marker index (MI) values from 0.0124 to 0.0166, effective multiplex ratio (E) values from 26.315 to 35.3125, discriminating power (D) values from 0.7149 to 0.8312, expected heterozygosity (H) values from 0.4842 to 0.5000, and mean heterozygosity (Havp) values from 0.0004 to 0.0005. We assessed the quality of standard fluorescent AFLP genotyping by analyzing our dataset’s error rate and reproducibility. The recommended and generally acceptable error rate for AFLP data falls within the 2–5% range. Remarkably, our analysis demonstrated error rate values ranging from 0.2375% to 1.0619%. Thus, four combinations (C1, C4, C5, and C6) were identified as the most suitable for exploring the genetic variability in Histoplasma due to their superior diversity indices and lower error rates. The mating-type identity of 16 isolates revealed a nearly equal distribution (1:1 ratio) of MAT1-1 and MAT1-2 alleles, suggesting random mating. Overall, this study recommends using the four combinations to investigate genetic variability in Histoplasma, offering a promising technique for epidemiological investigations of histoplasmosis. Moreover, our findings open avenues for comparative genomic studies, which will contribute to achieving a consensus taxonomy, testing reproductive barriers, and exploring the significance of this diversity. Palavras-chave: AFLP, Genetic diversity, Histoplasma, Histoplasmosis Agência de fomento:CAPES (88887.159096/2017-00), CNPq (433276/2018-5), FAPESP (2017/27265-5) | |||||