Genotyping of 44 Isolates ofFusarium solani, the Main Agent of Fungal Keratitis in Brazil

Abstract

In a retrospective study performed over 6 years in Brazil, Fusarium solani was found to be the most common species causing mycotic keratitis. The genetic diversity of 44 isolates from 39 patients was assessed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) fingerprinting. ERIC-PCR was more discriminatory than PCR-RFLP for differentiating the strains. By combining of the results of both techniques, we identified 40 genotypes. Molecular typing revealed a high genomic heterogeneity of the strains of F. solani studied.Fusarium is a common fungus that causes ocular infections in humans (2, 12), with keratitis being the most frequent clinical manifestation. The most frequent species of Fusarium causing keratitis are Fusarium solani and Fusarium oxysporum (15,17). More rarely, Fusarium dimerum (15), Fusarium verticillioides, Fusarium sacchari (17), and very recently, Fusarium polyphialidicum (5) have also been reported. Keratitis is more common in tropical and subtropical areas. Its incidence correlates with harvest time and seasonal increases in temperature and humidity (2). A high incidence of fusariosis, especially in immunocompromised patients, has been found in different regions of the world. However, the incidence of such fungi in ocular infections are unknown. In this study, we have determined the incidence of Fusarium in Brazil. For this purpose, we have performed a retrospective study of all cases of mycotic keratitis diagnosed over a period of 6 years at a referral eye care center that treats patients from both urban and rural areas. We also carried out the molecular genotyping of F. solani, the most common species, to evaluate its genetic diversity and to determine whether some strains are prevalent in the area studied. To test the fungi, we used two common molecular techniques, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). MATERIALS AND METHODSFungal isolates and culture conditions. All cases of fungal keratitis diagnosed over a 6-year period (1996 to 2002) in the Microbiology Laboratory of the Ophthalmology Department of the Universidade Federal de São Paulo were reviewed. The Ophthalmology Department is a referral center that treats patients from all over Brazil. Diagnosis was established by direct examination of corneal scrapings positive for fungal elements and repeated cultures of the same fungus. The samples were handled as previously described (6, 12). Fungal isolates were identified according to the method of de Hoog et al. (3).A total of 44 isolates of F. solani, including 35 epidemiologically unrelated isolates, and 9 isolates obtained from 4 patients were cultured on potato dextrose agar (Pronadisa, Madrid, Spain) at 25°C and tested by ERIC-PCR and PCR-RFLP analysis of the ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, ITS2, and the D1-D2 region of the 28S rDNA.DNA extraction. Genomic DNA was extracted and purified directly from fungal colo...

Phylogenetic Diversity and Microsphere Array-Based Genotyping of Human Pathogenic Fusaria, Including Isolates from the Multistate Contact Lens-Associated U.S. Keratitis Outbreaks of 2005 and 2006

“…When this comparison was restricted to the keratitis outbreak isolates from Florida (15 corneal and 6 environmental; see Table S1 in the supplemental material), all of these isolates were identified as members of the FSSC, except for two isolates of the widespread FOSC 3-a clonal lineage (33). Similar findings that all of the outbreak isolates of 2005 and 2006 from Singapore and Hong Kong were members of the FSSC (5,20) are consistent with the results of previous studies that have documented the predominance of FSSC fusaria in causing ocular mycoses worldwide (13,26,44). The results of Chang et al (5) and the present study, however, have established that phylogenetically diverse members of the FOSC can infect the human cornea (Table 1).…”

Molecular Characterization of Fusarium Oxysporum F. Sp. Cubense of Banana

“…ERIC-PCR was more informative and less time consuming in studying closely related strains compared to PCR-RFLP of ITS + 5.8S analysis. The results were similar with the results obtained by [5,7] . The genetic variation of ITS + 5.8S within the closely related isolates was low and sometimes no variation was observed and therefore was not informative to determine the genetic variation within FOC isolates.…”

“…This suggests that ERIC-PCR is more informative in characterizing closely related strains. Godoy et al [7] also found that ERIC-PCR was more discriminatory compared to PCR-RFLP of ITS + 5.8S regions in their effort to characterize F. solani associated with mycotic keratitis. From their study, 39 ERIC groups and 13 restriction patterns were identified from 44 F. solani isolates.…”

Use of the DiversiLab System for Species and Strain Differentiation of Fusarium Species Isolates

“…Godoy et al recently showed the clinical utility of manual repetitive sequence-based PCR (rep-PCR) for genotyping Fusarium strains (7). DiversiLab, an automated rep-PCR method (5), has standardized bacterial strain typing and has been used to characterize the genotypic relatedness among Candida, Aspergillus, Dermatophytes, Zygomycetes, and Mycobacterium isolates (4,8,11,14,16 We tested 21 clinical Fusarium isolates recovered from cancer patients (M. D. Anderson mycology laboratory strain collection) and five ATCC Fusarium isolates.…”