Anderson Messias Rodrigues

Phylogenetic Analysis Reveals a High Prevalence of Sporothrix brasiliensis in Feline Sporotrichosis Outbreaks

Sporothrix schenckii, previously assumed to be the sole agent of human and animal sporotrichosis, is in fact a species complex. Recently recognized taxa include S. brasiliensis, S. globosa, S. mexicana, and S. luriei, in addition to S. schenckii sensu stricto. Over the last decades, large epidemics of sporotrichosis occurred in Brazil due to zoonotic transmission, and cats were pointed out as key susceptible hosts. In order to understand the eco-epidemiology of feline sporotrichosis and its role in human sporotrichosis a survey was conducted among symptomatic cats. Prevalence and phylogenetic relationships among feline Sporothrix species were investigated by reconstructing their phylogenetic origin using the calmodulin (CAL) and the translation elongation factor-1 alpha (EF1α) loci in strains originated from Rio de Janeiro (RJ, n = 15), Rio Grande do Sul (RS, n = 10), Paraná (PR, n = 4), São Paulo (SP, n = 3) and Minas Gerais (MG, n = 1). Our results showed that S. brasiliensis is highly prevalent among cats (96.9%) with sporotrichosis, while S. schenckii was identified only once. The genotype of Sporothrix from cats was found identical to S. brasiliensis from human sources confirming that the disease is transmitted by cats. Sporothrix brasiliensis presented low genetic diversity compared to its sister taxon S. schenckii. No evidence of recombination in S. brasiliensis was found by split decomposition or PHI-test analysis, suggesting that S. brasiliensis is a clonal species. Strains recovered in states SP, MG and PR share the genotype of the RJ outbreak, different from the RS clone. The occurrence of separate genotypes among strains indicated that the Brazilian S. brasiliensis epidemic has at least two distinct sources. We suggest that cats represent a major host and the main source of cat and human S. brasiliensis infections in Brazil.

Emergence of pathogenicity in the Sporothrix schenckii complex

Sporothrix schenckii sensu lato is a complex of thermally dimorphic species whose natural habitats are soil and plant materials. However, the traumatic implantation of the species into human skin is traditionally thought to be the route leading to the fungal disease sporotrichosis. The complex contains Sporotrhix mexicana, S. globosa, S. brasiliensis, S. luriei, in addition to S. schenckii sensu stricto. In this study we evaluated the differences among these species relative to their frequency in the environment and in human hosts, as well as discuss their remarkable diverse pathogenicity. Today, S. brasiliensis is epidemic in and geographically restricted to Brazil. In contrast, S. mexicana and S. globosa have rarely been reported over the decades. We discovered that the species have been present in collections from clinical cases since 1955 and were able to re-identify six isolates originally classified as S. schenckii as Sporothrix mexicana (three isolates) and Sporothrix globosa (three isolates). Despite their long presence as potential human pathogens they have not shown any increase in frequency as etiologic agents of human infections.

Characterization of virulence profile, protein secretion and immunogenicity of different Sporothrix schenckii sensu stricto isolates compared with S. globosa and S. brasiliensis species

A comparative study about protein secretion, immunogenicity and virulence was performed in order to characterize and to compare eight Sporothrix schenckii sensu stricto isolates. For virulence characterization, a murine model, based on survival assay and CFU counting was used. S. brasiliensis and S. globosa, a highly virulent and a non-virulent isolates, respectively were used as external controls. Exoantigen profiles showed different secreted molecules; the 46- and 60-kDa molecules were commonly secreted by all three species. The S. schenckii s. str. isolates could be classified as non-virulent or presenting low, medium or high virulence, based on survival times after infection and recovery of viable fungi. The humoral response profiles of mice infected with S. schenckii s. str., S. globosa and S. brasiliensis were heterogeneous; five virulent isolates (S. schenckii s. str., n = 4 and S. brasiliensis, n = 1) had in common the recognition of the 60-kDa molecule by their respective antisera, suggesting that this antigen may be involved in virulence. Furthermore, the 110-kDa molecule was secreted and recognized by antisera from four virulent isolates (S. schenckii s. str., n = 3 and S. brasiliensis, n = 1), so there is a possibility that this molecule is also related to virulence. Our findings reveal different degrees of virulence in S. schenckii s. str. isolates and suggest the correlation of protein secretion and immunogenicity with virulence of S. schenckii complex. These findings provide new insights into the pathogenesis of S. schenckii s. str. and improve the knowledge about immunogenicity and protein profiles in S. schenckii complex.

Global ITS diversity in the Sporothrix schenckii complex

Molecular phylogeny has revealed that sporotrichosis is caused by several Sporothrix species which differ in clinical behavior. The complex is embedded within Ophiostoma, a genus mainly comprising fungi that live in association with bark beetles, but differs by a high virulence towards humans and other mammals. The different ecology is corroborated by phylogenetic separation. The aim of the present study was to determine the validity of the rDNA internal transcribed spacer (ITS) region as a marker for diagnostics of species in the clinical group, using beta-tubulin sequences to calibrate species delimitations. The topology of the two gene trees was concordant, and all clinically relevant Sporothrix species could easily be recognized by means of the ITS region. An increased geographic sampling did not affected delimitation success in the clinical clade of the S. schenckii complex.

Phylogeography and evolutionary patterns in Sporothrix spanning more than 14 000 human and animal case reports.

Pathology to vertebrate hosts has emerged repeatedly in the order Ophiostomatales. Occasional infections have been observed in Sporothrix mexicana at a low level of virulence, while the main pathogenic species cluster in a derived clade around S. schenckii s.str. In this paper, phylogeny and epidemiology of the members of this clade were investigated for 99 clinical and 36 environmental strains using four genetic loci, viz. rDNA ITS and partial CAL, TEF1, and TEF3; data are compared with amplified fragment length polymorphism (AFLP) genotyping. The four main species of the pathogenic clade were recognised. The species proved to show high degrees of endemicity, which enabled interpretation of literature data where live material or genetic information is lacking. The clade of four species comprised nine subclusters, which often had limited geographic distribution and were separate from each other in all partitions, suggesting low degrees of interbreeding between populations. In contrast, S. globosa exhibited consistent global distribution of identical AFLP types, suggesting another type of dispersal. Sporothrix brasiliensis is known to be involved in an expanding zoonosis and transmitted by cats, whereas S. globosa infections originated from putrid plant material, causing a sapronosis. Sporothrix schenckii s.str., the most variable species within the clade, also had a plant origin, with ecological similarities to that of S. globosa. A hypothesis was put forward that highly specific conditions in the plant material are required to promote the growth of Sporothrix. Fermented, self-heated plant debris may stimulate the thermodependent yeast-like invasive form of the fungus, which facilitates repeated infection of mammals.

Disseminated Sporothrix brasiliensis infection with endocardial and ocular involvement in an HIV-infected patient.

Disseminated sporotrichosis occurs in individuals with impaired cellular immunity, such as in cases of neoplasia, transplantation, diabetes, and especially, acquired immunodeficiency syndrome. This report presents a 32-year-old Brazilian human immunodeficiency virus (HIV)-infected patient who developed a protracted condition of disseminated sporotrichosis with endocarditis, bilateral endophthalmitis, and lymphatic involvement. He needed cardiac surgery to replace the mitral valve. Sporothrix brasiliensis isolates were recovered from cultures of subcutaneous nodules and mitral valve fragments. Species identification was based on classical and molecular methods. The patient received amphotericin B for 52 days and subsequently, oral itraconazole. He remains asymptomatic, and he is on maintenance therapy with itraconazole. Despite his positive clinical outcome, he developed bilateral blindness. To our knowledge, this case is the first report of endocarditis and endophthalmitis caused by S. brasiliensis.

Emerging sporotrichosis is driven by clonal and recombinant Sporothrix species

Sporotrichosis, caused by agents of the fungal genus Sporothrix, occurs worldwide, but the infectious species are not evenly distributed. Sporothrix propagules usually gain entry into the warm-blooded host through minor trauma to the skin from contaminated plant debris or through scratches or bites from felines carrying the disease, generally in the form of outbreaks. Over the last decade, sporotrichosis has changed from a relatively obscure endemic infection to an epidemic zoonotic health problem. We evaluated the impact of the feline host on the epidemiology, spatial distribution, prevalence and genetic diversity of human sporotrichosis. Nuclear and mitochondrial markers revealed large structural genetic differences between S. brasiliensis and S. schenckii populations, suggesting that the interplay of host, pathogen and environment has a structuring effect on the diversity, frequency and distribution of Sporothrix species. Phylogenetic data support a recent habitat shift within S. brasiliensis from plant to cat that seems to have occurred in southeastern Brazil and is responsible for its emergence. A clonal structure was found in the early expansionary phase of the cat–human epidemic. However, the prevalent recombination structure in the plant-associated pathogen S. schenckii generates a diversity of genotypes that did not show any significant increase in frequency as etiological agents of human infection over time. These results suggest that closely related pathogens can follow different strategies in epidemics. Thus, species-specific types of transmission may require distinct public health strategies for disease control.

Feline sporotrichosis due to Sporothrix brasiliensis: an emerging animal infection in São Paulo, Brazil

BackgroundSporotrichosis is a mycotic infectious disease that is generally acquired by traumatic inoculation of contaminated materials especially from plant debris or through bites and scratches from diseased animals, such as domestic cats. It affects the skin, lymphatic system, and other organs in the warm-blooded host. Etiological agents are embedded in the plant-associated order Ophiostomatales. With essential differences between possible outbreak sources and ecological niche, host-environment interactions are classic determinants of risk factors for disease acquisition. Sporotrichosis outbreaks with zoonotic transmission, such as those that are ongoing in southern and southeastern Brazil, have highlighted the threat of cross-species pathogen transmission. Sporothrix brasiliensis has emerged as a human threat owing to the intimate contact pattern between diseased cats and humans in endemic areas.ResultsWe describe the recent emergence of feline sporotrichosis in the metropolitan region of São Paulo, Brazil, with an overwhelming occurrence of S. brasiliensis as the etiological agent. A phylogenetic and a haplotype approach were used to investigate the origin of this epidemic and the impact of feline transmission on genetic diversity. During the last 3-year period, 163 cases of feline sporotrichosis were reported in São Paulo with proven S. brasiliensis culture. The haplotype diversity of feline S. brasiliensis isolates revealed the expansion of a clonal population with low genetic diversity. Haplotype analysis confirmed that isolates from São Paulo shared the haplotype originated in the long-lasting outbreak of cat-transmitted sporotrichosis in Rio de Janeiro, which differed from the haplotype circulating in the Rio Grande do Sul epidemic.ConclusionsThe fast spread of sporotrichosis in a short period of time highlights the potential for outbreaks and suggests that the mycosis may affect an urban population with a high concentration of susceptible felines. The feline sporotrichosis epidemic shows no signs of slowing, and this epidemiological pattern may require specific public health strategies to control future outbreaks.

Susceptibility of Sporothrix brasiliensis isolates to amphotericin B, azoles, and terbinafine

The in vitro activity of the antifungal agents amphotericin B (AMB), itraconazole (ITC), posaconazole (PSC), voriconazole (VRC), and terbinafine (TRB) against 32 Brazilian isolates of Sporothrix brasiliensis, including 16 isolates from a recent (2011-2012) epidemic in Rio de Janeiro state, was examined. We describe and genotype new isolates and clustered them with 16 older (from 2004 or earlier) S. brasiliensis isolates by phylogenetic analysis. We tested both the yeast and the mycelium form of all isolates using broth microdilution methods based on the reference protocols M38-A2 and M27-A3 (recommended by the Clinical and Laboratory Standards Institute). Considering minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs), TRB was found to be the most active drug in vitro for both fungal forms, followed by PSC. Several isolates showed high MICs for AMB and/or ITC, which are currently used as first-line therapy for sporotrichosis. VRC displayed very low activity against S. brasiliensis isolates. The primary morphological modification observed on treated yeasts by transmission electron microscopy analysis was changes in cell wall. Our results indicate that TRB is the antifungal with the best in vitro activity against S. brasiliensis and support the use of TRB as a promising option for the treatment of cutaneous and/or lymphocutaneous sporotrichosis.

Genetic diversity of medically important and emerging Candida species causing invasive infection

BackgroundGenetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis.MethodsWe selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C® system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis.ResultsAmong 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C® profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp.ConclusionsMolecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.