RESUMO
Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection acquired after inhalation of Paracoccidioides propagules from the environment. The main agents include members of the P. brasiliensis complex (phylogenetically-defined species S1, PS2, PS3, and PS4) and P. lutzii. DNA-sequencing of protein-coding loci (e.g., GP43, ARF, and TUB1) is the reference method for recognizing Paracoccidioides species due to a lack of robust phenotypic markers. Thus, developing new molecular markers that are informative and cost-effective is key to providing quality information to explore genetic diversity within Paracoccidioides. We report using new amplified fragment length polymorphism (AFLP) markers and mating-type analysis for genotyping Paracoccidioides species. The bioinformatic analysis generated 144 in silico AFLP profiles, highlighting two discriminatory primer pairs combinations (#1 EcoRI-AC/MseI-CT and #2 EcoRI-AT/MseI-CT). The combinations #1 and #2 were used in vitro to genotype 165 Paracoccidioides isolates recovered from across a vast area of South America. Considering the overall scored AFLP markers in vitro (67-87 fragments), the values of polymorphism information content (PIC = 0.3345-0.3456), marker index (MI = 0.0018), effective multiplex ratio (E = 44.6788-60.3818), resolving power (Rp = 22.3152-34.3152), discriminating power (D = 0.5183-0.5553), expected heterozygosity (H = 0.4247-0.4443), and mean heterozygosity (H avp = 0.00002-0.00004), demonstrated the utility of AFLP markers to speciate Paracoccidioides and to dissect both deep and fine-scale genetic structures. Analysis of molecular variance (AMOVA) revealed that the total genetic variance (65-66 %) was due to variability among P. brasiliensis complex and P. lutzii (PhiPT = 0.651-0.658, P < 0.0001), supporting a highly structured population. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for P. brasiliensis s. str. (χ2 = 1.025; P = 0.3113), P. venezuelensis (χ2 = 0.692; P = 0.4054), and P. lutzii (χ2 = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found for P. americana (χ2 = 8.909; P = 0.0028) and P. restrepiensis (χ2 = 4.571; P = 0.0325) with a preponderance of MAT1-1. Geographical distributions confirmed that P. americana, P. restrepiensis, and P. lutzii are more widespread than previously thought. P. brasiliensis s. str. is by far the most widely occurring lineage in Latin America countries, occurring in all regions of Brazil. Our new DNA fingerprint assay proved to be rapid, reproducible, and highly discriminatory, to give insights into the taxonomy, ecology, and epidemiology of Paracoccidioides species, guiding disease-control strategies to mitigate PCM.
RESUMO
Sporothrix (Ophiostomatales) comprises species that are pathogenic to humans and other mammals as well as environmental fungi. Developments in molecular phylogeny have changed our perceptions about the epidemiology, host-association, and virulence of Sporothrix. The classical agent of sporotrichosis, Sporothrix schenckii, now comprises several species nested in a clinical clade with S. brasiliensis, S. globosa, and S. luriei. To gain a more precise view of outbreaks dynamics, structure, and origin of genetic variation within and among populations of Sporothrix, we applied three sets of discriminatory AFLP markers (#3 EcoRI-GA/MseI-TT, #5 EcoRI-GA/MseI-AG, and #6 EcoRI-TA/MseI-AA) and mating-type analysis to a large collection of human, animal and environmental isolates spanning the major endemic areas. A total of 451 polymorphic loci were amplified in vitro from 188 samples, and revealed high polymorphism information content (PIC = 0.1765-0.2253), marker index (MI = 0.0001-0.0002), effective multiplex ratio (E = 15.1720-23.5591), resolving power (Rp = 26.1075-40.2795), discriminating power (D = 0.9766-0.9879), expected heterozygosity (H = 0.1957-0.2588), and mean heterozygosity (Havp = 0.000007-0.000009), demonstrating the effectiveness of AFLP markers to speciate Sporothrix. Analysis using the program structure indicated three genetic clusters matching S. brasiliensis (population 1), S. schenckii (population 2), and S. globosa (population 3), with the presence of patterns of admixture amongst all populations. AMOVA revealed highly structured clusters (PhiPT = 0.458-0.484, P < 0.0001), with roughly equivalent genetic variability within (46-48 %) and between (52-54 %) populations. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for S. schenckii (χ2 = 2.522; P = 0.1122), supporting random mating. In contrast, skewed distributions were found for S. globosa (χ2 = 9.529; P = 0.0020) with a predominance of MAT1-1 isolates, and regional differences were highlighted for S. brasiliensis with the overwhelming occurrence of MAT1-2 in Rio de Janeiro (χ2 = 14.222; P = 0.0002) and Pernambuco (χ2 = 7.364; P = 0.0067), in comparison to a higher prevalence of MAT1-1 in the Rio Grande do Sul (χ2 = 7.364; P = 0.0067). Epidemiological trends reveal the geographic expansion of cat-transmitted sporotrichosis due to S. brasiliensis via founder effect. These data support Rio de Janeiro as the centre of origin that has led to the spread of this disease to other regions in Brazil. Our ability to reconstruct the source, spread, and evolution of the ongoing outbreaks from molecular data provides high-quality information for decision-making aimed at mitigating the progression of the disease. Other uses include surveillance, rapid diagnosis, case connectivity, and guiding access to appropriate antifungal treatment.
RESUMO
Histoplasmosis is a serious infectious disease in humans caused by Histoplasma spp. (Onygenales), whose natural reservoirs are thought to be soil enriched with bird and bat guano. The true global burden of histoplasmosis is underestimated and frequently the pulmonary manifestations are misdiagnosed as tuberculosis. Molecular data on epidemiology of Histoplasma are still scarce, even though there is increasing recognition of histoplasmosis in recent years in areas distant from the traditional endemic regions in the Americas. We used multi-locus sequence data from protein coding loci (ADP-ribosylation factor, H antigen precursor, and delta-9 fatty acid desaturase), DNA barcoding (ITS1/2+5.8s), AFLP markers and mating type analysis to determine the genetic diversity, population structure and recognise the existence of different phylogenetic species among 436 isolates of Histoplasma obtained globally. Our study describes new phylogenetic species and the molecular characteristics of Histoplasma lineages causing outbreaks with a high number of severe outcomes in Northeast Brazil between 2011 and 2015. Genetic diversity levels provide evidence for recombination, common ancestry and clustering of Brazilian isolates at different geographic scales with the emergence of LAm C, a new genotype assigned to a separate population cluster in Northeast Brazil that exhibited low diversity indicative of isolation. The global survey revealed that the high genetic variability among Brazilian isolates along with the presence of divergent cryptic species and/or genotypes may support the hypothesis of Brazil being the center of dispersion of Histoplasma in South America, possibly with the contribution of migratory hosts such as birds and bats. Outside Brazil, the predominant species depends on the region. We confirm that histoplasmosis has significantly broadened its area of occurrence, an important feature of emerging pathogens. From a practical point of view, our data point to the emergence of histoplasmosis caused by a plethora of genotypes, and will enable epidemiological analysis focused on understanding the processes that lead to histoplasmosis. Further, the description of this diversity opens avenues for comparative genomic studies, which will allow progress toward a consensus taxonomy, improve understanding of the presence of hybrids in natural populations of medically relevant fungi, test reproductive barriers and to explore the significance of this variation.
RESUMO
Autosomal recessive (AR) CARD9 (caspase recruitment domain-containing protein 9) deficiency underlies invasive infections by fungi of the ascomycete phylum in previously healthy individuals at almost any age. Although CARD9 is expressed mostly by myeloid cells, the cellular basis of fungal infections in patients with inherited CARD9 deficiency is unclear. Therapy for fungal infections is challenging, with at least 20% premature mortality. We report two unrelated patients from Brazil and Morocco with AR CARD9 deficiency, both successfully treated with hematopoietic stem cell transplantation (HSCT). From childhood onward, the patients had invasive dermatophytic disease, which persisted or recurred despite multiple courses of antifungal treatment. Sanger sequencing identified homozygous missense CARD9 variants at the same residue, c.302G>T (p.R101L) in the Brazilian patient and c.301C>T (p.R101C) in the Moroccan patient. At the ages of 25 and 44 years, respectively, they received a HSCT. The first patient received a HLA-matched HSCT from his CARD9-mutated heterozygous sister. There was 100% donor chimerism at D + 100. The other patient received a T cell-depleted haploidentical HSCT from his CARD9-mutated heterozygous brother. A second HSCT from the same donor was performed due to severe amegakaryocytic thrombocytopenia despite achieving full donor chimerism (100%). At last follow-up, more than 3 years after HSCT, both patients have achieved complete clinical remission and stopped antifungal therapy. HSCT might be a life-saving therapeutic option in patients with AR CARD9 deficiency. This observation strongly suggests that the pathogenesis of fungal infections in these patients is largely due to the disruption of leukocyte-mediated CARD9 immunity.
Assuntos
Candidíase Mucocutânea Crônica/terapia , Transplante de Células-Tronco Hematopoéticas , Adulto , Antifúngicos/uso terapêutico , Candidíase Mucocutânea Crônica/diagnóstico por imagem , Candidíase Mucocutânea Crônica/imunologia , Pré-Escolar , Humanos , Masculino , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Resultado do TratamentoRESUMO
Cryptococcosis, caused by Cryptococcus gattii sensu lato, is an emerging disease that was initially found in (sub)tropical regions but recently expanded to temperate regions. Cryptococcus gattii s.l. infections are mostly encountered in healthy individuals, frequently affecting both lungs and the central nervous system (CNS). Usually, C. gattii s.l. is less susceptible to antifungal compounds than its counterpart, C. neoformans s.l. We studied 18 clinical C. gattii s.l. isolates with amplified fragment length polymorphism (AFLP) fingerprinting, mating-typing, multi-locus sequence typing (MLST) and antifungal susceptibility testing. All isolates were C. deuterogattii (genotype AFLP6/VGII), 14 were mating-type α and four were type a. Amphotericin B, itraconazole, voriconazole, posaconazole and isavuconazole showed high activity, with minimum inhibitory concentration (MIC) ranges of 0.063-0.25, 0.031-0.25, 0.031-0.25, 0.031-0.25 and <0.016-0.25 µg mL-1, respectively. Fluconazole and flucytosine had high geometric mean MICs of 2.07 and 3.7 µg mL-1, respectively. Most cases occurred in immunocompetent patients (n = 10; 55.6 %) and CNS involvement was the most common clinical presentation (n = 14; 77.8 %). Three patients (16.7 %) showed sequelae, hyperreflexia, dysarthria, diadochokinesia, anosmia and upper limb weakness. In conclusion, all infections were caused by C. deuterogattii (AFLP6/VGII) and the majority of patients were immunocompetent, with the CNS as the most affected site. All antifungal drugs had high in vitro activity against C. deuterogattii isolates, except fluconazole and flucytosine.
Assuntos
Antifúngicos/farmacologia , Criptococose/microbiologia , Cryptococcus gattii/classificação , Cryptococcus gattii/efeitos dos fármacos , Farmacorresistência Fúngica , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Brasil/epidemiologia , Criptococose/epidemiologia , Cryptococcus gattii/genética , Cryptococcus gattii/isolamento & purificação , Impressões Digitais de DNA , Feminino , Genes Fúngicos Tipo Acasalamento , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Tipagem de Sequências MultilocusRESUMO
Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 µg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 µg/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 µg/ml (VGII); itraconazole, 0.25 µg/ml (VNI), 0.5 µg/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 µg/ml (VGIV); posaconazole, 0.25 µg/ml (C. neoformans nontyped and VNI) and 0.5 µg/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 µg/ml (VNIV), 0.25 µg/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 µg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.
Assuntos
Antifúngicos/uso terapêutico , Criptococose/tratamento farmacológico , Criptococose/epidemiologia , Cryptococcus gattii/efeitos dos fármacos , Fluconazol/uso terapêutico , Itraconazol/uso terapêutico , Pirimidinas/uso terapêutico , Triazóis/uso terapêutico , Antifúngicos/farmacologia , Austrália/epidemiologia , Criptococose/microbiologia , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/isolamento & purificação , Farmacorresistência Fúngica/efeitos dos fármacos , Europa (Continente)/epidemiologia , Fluconazol/farmacologia , Humanos , Índia/epidemiologia , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana , América do Norte/epidemiologia , Pirimidinas/farmacologia , África do Sul/epidemiologia , América do Sul/epidemiologia , Triazóis/farmacologia , VoriconazolAssuntos
Acinonyx/microbiologia , Criptococose/veterinária , Cryptococcus gattii/isolamento & purificação , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Animais , Animais de Zoológico , Criptococose/microbiologia , Criptococose/patologia , Cryptococcus gattii/classificação , Cryptococcus gattii/genética , Cuba , DNA Fúngico/genética , Tipagem Molecular , Técnicas de Tipagem Micológica , RecidivaRESUMO
The genotypic diversity of Brazilian Cryptococcus neoformans strains was analyzed. The majority of the samples were alphaA (65%), followed by alphaB (17.5%), alphaD (9%), alphaAaD hybrids (5%), and alphaC (3.5%). A considerable genotypic diversity occurred within C. neoformans var. grubii, and a new amplified fragment length polymorphism genotype, 1B, was recognized.