Epidemiological, clinical, and genomic landscape of coccidioidomycosis in northeastern Brazil.

Coccidioidomycosis, listed as a priority mycosis by the WHO, is endemic in the United States but often overlooked in Central and South America. Employing a multi-institutional approach, we investigate how disease characteristics, pathogen genetic variation, and environmental factors impact coccidioidomycosis epidemiology and outcomes in South America. We identified 292 cases (1978-2021) and 42 outbreaks in Piauí and Maranhão states, Brazil, the largest series outside the US/Mexico epidemic zone. The male-to-female ratio was 57.4:1 and the most common activity was armadillo hunting (91.1%) 4 to 30 days before symptom onset. Most patients (92.8%) exhibited typical acute pulmonary disease, with cough (93%), fever (90%), and chest pain (77%) as predominant symptoms. The case fatality rate was 8%. Our negative binomial regression model indicates that reduced precipitation levels in the current (p = 0.015) and preceding year (p = 0.001) predict heightened incidence. Unlike other hotspots, acidic soil characterizes this region. Brazilian strains differ genomically from other C. posadasii lineages. Northeastern Brazil presents a distinctive coccidioidomycosis profile, with armadillo hunters facing elevated risks. Low annual rainfall emerges as a key factor in increasing cases. A unique C. posadasii lineage in Brazil suggests potential differences in environmental, virulence, and/or pathogenesis traits compared to other Coccidioides genotypes.

Antigens of Coccidioides posadasii as an important tool for the immunodiagnosis of coccidioidomycosis.

Serologic diagnosis has been presented as a safe alternative for coccidioidomycosis. However, commercial kits based on coccidioidal antibodies available in the USA are considered too expensive for laboratories outside that country. In this study, we describe the preparation of antigens for detection of human coccidioidal antibodies by the immunodiffusion test (ID) and enzyme immunoassay (EIA). Antigens were tested against serum samples from patients with coccidioidomycosis, histoplasmosis and paracoccidioidomycosis, as well as healthy individuals. The highest reactivity in the ID tests was seen in the F0-90 antigen. In the EIAs, the best results were obtained with the F60-90 antigen. None of the serum samples from healthy individuals were recognized by any of the antigen extracts tested by ID or EIA. In conclusion, the F0-90 and F60-90 antigens have the potential to be commercially employed in presumptive diagnosis of coccidioidomycosis by ID or EIA, respectively. The tests could improve serological diagnosis of coccidioidomycosis in South America.

Molecular identification of Coccidioides spp. in soil samples from Brazil.

BACKGROUND: Since 1991 several outbreaks of acute coccidioidomycosis (CM) were diagnosed in the semi-arid Northeast of Brazil, mainly related to disturbance of armadillo burrows caused by hunters while digging them for the capture of these animals. This activity causes dust contaminated with arthroconidia of Coccidioides posadasii, which, once inhaled, cause the mycosis. We report on the identification of C. posadasii in soil samples related to outbreaks of CM. RESULTS: Twenty four soil samples had their DNA extracted and subsequently submitted to a semi-nested PCR technique using specific primers. While only 6 (25%) soil samples were positive for C. posadasii by mice inoculation, all (100%) were positive by the molecular tool. CONCLUSION: This methodology represents a simple, sensitive and specific molecular technique to determine the environmental distribution of Coccidioides spp. in endemic areas, but cannot distinguish the species. Moreover, it may be useful to identify culture isolates. Key-words: 1. Coccidioidomycosis. 2. Coccidioides spp. 3. C. posadasii. 4. Semi-arid. 5. Semi-nested PCR.

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil).

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7%) were human immunodeficiency virus (HIV)-positive and 26 (41.3%) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7%) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5%), and 24/63 (38.1%) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8%). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6%) of the HIV-positive subjects and in 13/26 (50%) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.

[Histoplasmosis in the northeast of Brazil. Report of three cases]. / Histoplasmose no Nordeste do Brasil. Relato de três casos.

Histoplasmosis is an endemic mycosis in specific areas of the U.S. and Latin America, caused by dimorphic geophilic fungus H. capsulatum. The description of cases in Brazil's northeast, considering that this region isn't an endemic area, indicates the existence of small outbreaks in several regions. Precise reasons for this pattern of distribution are still unknown. Activities related to the removal of soil in these places are associated with exposure to H. capsulatum. The objective of this work is to describe three isolated cases of histoplasmosis which occurred in Brazil's northeast region in individuals exposed to different activities (demolition of houses, wells diggers and exposure to bats).

Novel recombinant multiepitope proteins for the detection of anti-Cryptococcus antibodies.

AIM: To produce and test recombinant multiepitope proteins as an alternative assay for the serological diagnosis of cryptococcosis. MATERIALS & METHODS: Previously, synthetic peptides were used to detect anti-Cryptococcus antibodies, and in silico analyses showed that the union of peptides would improve the results. Here, the coding sequences of these peptides were assembled into synthetic genes. Four genes have been cloned and expressed in Escherichia coli, producing recombinant multiepitope proteins: proteins A, B, C and D. RESULTS: All constructs yielded good results; however, protein D showed the best results, with a sensitivity of 88.57% and specificity of 100%. CONCLUSION: The multiepitope proteins were shown to be potential antigens for the diagnosis of cryptococcosis in an attempt to detect anti-Cryptococcus antibodies.

Genotypic and Phenotypic Diversity of Cryptococcus gattii VGII Clinical Isolates and Its Impact on Virulence.

The Cryptococcus gattii species complex harbors the main etiological agents of cryptococcosis in immunocompetent patients. C. gattii molecular type VGII predominates in the north and northeastern regions of Brazil, leading to high morbidity and mortality rates. C. gattii VGII isolates have a strong clinical relevance and phenotypic variations. These phenotypic variations among C. gattii species complex isolates suggest that some strains are more virulent than others, but little information is available related to the pathogenic properties of those strains. In this study, we analyzed some virulence determinants of C. gattii VGII strains (CG01, CG02, and CG03) isolated from patients in the state of Piauí, Brazil. The C. gattii R265 VGIIa strain, which was isolated from the Vancouver outbreak, differed from C. gattii CG01, CG02 and CG03 isolates (also classified as VGII) when analyzed the capsular dimensions, melanin production, urease activity, as well as the glucuronoxylomannan (GXM) secretion. Those differences directly reflected in their virulence potential. In addition, CG02 displayed higher virulence compared to R265 (VGIIa) strain in a cryptococcal murine model of infection. Lastly, we examined the genotypic diversity of these strains through Multilocus Sequence Type (MLST) and one new subtype was described for the CG02 isolate. This study confirms the presence and the phenotypic and genotypic diversity of highly virulent strains in the Northeast region of Brazil.

Population Genetic Analysis Reveals a High Genetic Diversity in the Brazilian Cryptococcus gattii VGII Population and Shifts the Global Origin from the Amazon Rainforest to the Semi-arid Desert in the Northeast of Brazil.

Cryptococcus neoformans and Cryptococcus gattii are responsible globally for almost one million cryptococcosis cases yearly, mostly in immunocompromised patients, such as those living with HIV. Infections due to C. gattii have mainly been described in tropical and subtropical regions, but its adaptation to temperate regions was crucial in the species evolution and highlighted the importance of this pathogenic yeast in the context of disease. Cryptococcus gattii molecular type VGII has come to the forefront in connection with an on-going emergence in the Pacific North West of North America. Taking into account that previous work pointed towards South America as an origin of this species, the present work aimed to assess the genetic diversity within the Brazilian C. gattii VGII population in order to gain new insights into its origin and global dispersal from the South American continent using the ISHAM consensus MLST typing scheme. Our results corroborate the finding that the Brazilian C. gattii VGII population is highly diverse. The diversity is likely due to recombination generated from sexual reproduction, as evidenced by the presence of both mating types in clinical and environmental samples. The data presented herein strongly supports the emergence of highly virulent strains from ancestors in the Northern regions of Brazil, Amazonia and the Northeast. Numerous genotypes represent a link between Brazil and other parts of the world reinforcing South America as the most likely origin of the C. gattii VGII subtypes and their subsequent global spread, including their dispersal into North America, where they caused a major emergence.

Immunoreactivity of synthetic peptides derived from proteins of Cryptococcus gattii.

AIM: To determine the immunoreactivity of synthetic Cryptococcus-derived peptides. MATERIALS & METHODS: A total of 63 B-cell epitopes from previously identified Cryptococcus gattii immunoreactive proteins were synthesized and evaluated as antigens in ELISAs. The peptides were first evaluated for their ability to react against sera from immunocompetent subjects carrying cryptococcal meningitis. Peptides that yielded high sensitivity and specificity in the first test were then retested with sera from individuals with other fungal pathologies for cross-reactivity determination. RESULTS: Six of 63 synthetic peptides were recognized by antibodies in immunoassays, with a specificity of 100%, sensitivity of 78% and low cross-reactivity. CONCLUSION: We successfully determined the immunoreactivity of selected synthetic peptides of C. gattii derived proteins.

Immunoproteomics and immunoinformatics analysis of Cryptococcus gattii: novel candidate antigens for diagnosis.

AIM: To identify immunoreactive proteins of Cryptococcus gattii genotype VGII and their B-cell epitopes. MATERIALS & METHODS: We combined 2D gel electrophoresis, immunoblotting and mass spectrometry to identify immunoreactive proteins from four strains of C. gattii genotype VGII (CG01, CG02, CG03 and R265). Next, we screened the identified proteins to map B-cell epitopes. RESULTS: Sixty-eight immunoreactive proteins were identified. The strains and the number of proteins we found were: CG01 (12), CG02 (12), CG03 (18) and R265 (26). In addition, we mapped 374 peptides potentially targeted by B cells. CONCLUSION: Both immunoreactive proteins and B-cell epitopes of C. gattii genotype VGII that were potentially targeted by a host humoral response were identified. Considering the evolutionary relevance of the identified proteins, we may speculate that they could be used as the initial targets for recombinant protein and peptide synthesis aimed at the development of immunodiagnostic tools for cryptococcosis.

Restriction enzyme analysis of the hsp65 gene in clinical isolates from patients suspected of having pulmonary tuberculosis in Teresina, Brazil.

OBJECTIVE: To identify mycobacterial species in the sputum of patients suspected of having pulmonary tuberculosis and to determine the impact that the acquisition of this knowledge has on the therapeutic approach. METHODS: We evaluated 106 patients suspected of having pulmonary tuberculosis and referred to the pulmonology department of a public hospital in the city of Teresina, Brazil. Morning sputum specimens were evaluated for the presence of mycobacteria by sputum smear microscopy and culture. We used PCR and restriction enzyme analysis of the hsp65 gene (PRA-hsp65) to identify the strains of mycobacteria isolated in culture. RESULTS: A total of 206 sputum samples were analyzed. Patient ages ranged from 15 to 87 years, and 67% were male. There was cough in 100% of the cases. The predominant radiographic pattern was moderate disease, observed in 70%. Smear positivity was 76%, and isolation in culture occurred in 91% of the cultures. Traditional tests identified nontuberculous mycobacteria (NTM) in 9% of the isolates. The PRA-hsp65 method confirmed these data, showing seven band patterns that were able to identify the isolated species of NTM: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5; and M. gordonae 7. All of the patients with NTM were over 60 years of age, and bronchiectasis was seen in 88% of the X-rays. There were two cases of reinfection, initially attributed to M. abscessus and M. kansasii. CONCLUSIONS: In immunocompetent patients, NTM can infect the lungs. It is important to identify the specific NTM in order to establish the correct diagnosis and choose the most appropriate therapeutic regimen. The PRA-hsp65 method is useful in identifying NTM species and can be implemented in molecular biology laboratories that do not specialize in the identification of mycobacteria.

Análise de restrição enzimática do gene hsp65 de isolados clínicos de pacientes com suspeita de tuberculose pulmonar em Teresina, Piauí / Restriction enzyme analysis of the hsp65 gene in clinical isolates from patients suspected of having pulmonary tuberculosis in Teresina, Brazil

OBJETIVO: Identificar as espécies de micobactérias encontradas no escarro de pacientes com suspeita de tuberculose pulmonar e analisar o impacto dessas identificações na abordagem terapêutica. MÉTODOS: Foram avaliados 106 pacientes com suspeita de tuberculose pulmonar encaminhados para o serviço de pneumologia de um hospital público em Teresina, Piauí. Espécimes de escarro matinal foram avaliados quanto à presença de micobactérias por baciloscopia e cultura. Foram utilizadas PCR e análise de restrição enzimática do gene hsp65 (PRA-hsp65) para a identificação das cepas de micobactérias isoladas em cultura. RESULTADOS: Foram analisadas 206 amostras de escarro. A idade dos pacientes variou de 15 a 87 anos, sendo 67 por cento do gênero masculino. Tosse ocorreu em 100 por cento dos casos. O padrão radiográfico predominante foi de lesão moderada, observada em 70 por cento. A positividade no esfregaço foi de 76 por cento, e isolamento em cultura ocorreu em 91 por cento das culturas executadas. Testes tradicionais identificaram micobactérias não tuberculosas (MNT) em 9 por cento dos isolados. O método PRA-hsp65 confirmou esses dados, mostrando sete padrões de bandas capazes de identificar as espécies de MNT isoladas: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5 e M. gordonae 7. Todos os pacientes com MNT tinham mais de 60 anos, e observaram-se bronquiectasias em 88 por cento das radiografias. Houve dois casos de reinfecção, identificados inicialmente como infecção por M. abscessus e M. kansasii. CONCLUSÕES: As MNT causam infecção pulmonar em pacientes imunocompetentes, e a identificação das MNT é importante para estabelecer o diagnóstico correto e a decisão terapêutica mais adequada. O método PRA-hsp65 é útil para identificar espécies de MNT e pode ser implantado em laboratórios de biologia molecular não especializados em micobactérias.

OBJECTIVE: To identify mycobacterial species in the sputum of patients suspected of having pulmonary tuberculosis and to determine the impact that the acquisition of this knowledge has on the therapeutic approach. METHODS: We evaluated 106 patients suspected of having pulmonary tuberculosis and referred to the pulmonology department of a public hospital in the city of Teresina, Brazil. Morning sputum specimens were evaluated for the presence of mycobacteria by sputum smear microscopy and culture. We used PCR and restriction enzyme analysis of the hsp65 gene (PRA-hsp65) to identify the strains of mycobacteria isolated in culture. RESULTS: A total of 206 sputum samples were analyzed. Patient ages ranged from 15 to 87 years, and 67 percent were male. There was cough in 100 percent of the cases. The predominant radiographic pattern was moderate disease, observed in 70 percent. Smear positivity was 76 percent, and isolation in culture occurred in 91 percent of the cultures. Traditional tests identified nontuberculous mycobacteria (NTM) in 9 percent of the isolates. The PRA-hsp65 method confirmed these data, showing seven band patterns that were able to identify the isolated species of NTM: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5; and M. gordonae 7. All of the patients with NTM were over 60 years of age, and bronchiectasis was seen in 88 percent of the X-rays. There were two cases of reinfection, initially attributed to M. abscessus and M. kansasii. CONCLUSIONS: In immunocompetent patients, NTM can infect the lungs. It is important to identify the specific NTM in order to establish the correct diagnosis and choose the most appropriate therapeutic regimen. The PRA-hsp65 method is useful in identifying NTM species and can be implemented in molecular biology laboratories that do not specialize in the identification of mycobacteria.

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil)

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7 percent) were human immunodeficiency virus (HIV)-positive and 26 (41.3 percent) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7 percent) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5 percent), and 24/63 (38.1 percent) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8 percent). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6 percent) of the HIV-positive subjects and in 13/26 (50 percent) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.

Coccidioidomycosis: an unusual cause of acute respiratory distress syndrome

A male farmer, 20 years old, from the countryside of the State of Piauí, developed acute respiratory infection. Despite adequate antimicrobial therapy, his conditions worsened, requiring mechanical ventilation. His X-rays showed diffuse pulmonary infiltrates. His PaO2/FiO2 ratio was 58. Direct microscopy and culture of tracheal aspirates showed the presence of Coccidioides immitis. Autochthonous cases of coccidioidomycosis have only recently been described in Brazil, most of them from the State of Piauí. C. immitis has been isolated from humans, dogs and armadillos (Dasypus novemcinctus), and also from soil samples of armadillo's burrows. Failure to respond to antimicrobial therapy and a patient's origin from recognized endemic areas should alert to the possibility of acute pulmonary coccidioidomycosis.