Genotyping Analysis of Cryptococcus deuterogattii and Correlation with Virulence Factors and Antifungal Susceptibility by the Clinical and Laboratory Standards Institute and the European Committee on Antifungal Susceptibility Testing Methods.

Data about the relationship between their molecular types, virulence factors, clinical presentation, antifungal susceptibility profile, and outcome are still limited for Cryptococcus deuterogattii. This study aimed to evaluate the molecular and phenotypic characteristics of 24 C. deuterogattii isolates from the southeast region of Brazil. The molecular characterization was performed by multilocus sequence typing (MLST). The antifungal susceptibility profile was obtained according to CLSI-M27-A3 and EUCAST-EDef 7.1 methods. The virulence factors were evaluated using classic techniques. The isolates were divided into four populations. The molecular analysis suggests recombinant events in most of the groups evaluated. Resistance and susceptibility dose-dependent to fluconazole were evidenced in four isolates (16%) by EUCAST and in four isolates (16%) by CLSI methods. The agreement at ±two dilutions for both methods was 100% for itraconazole, ketoconazole, and voriconazole, 96% for amphotericin B, and 92% for fluconazole. Significant differences in virulence factor expression and antifungal susceptibility to itraconazole and amphotericin B were found. The mixed infection could be suggested by the presence of variable sequence types, differences in virulence factor production, and decreased antifungal susceptibility in two isolates from the same patient. The data presented herein corroborate previous reports about the molecular diversity of C. deuterogattii around the world.

High genetic variability of clinical and environmental Cryptococcus gattii isolates from Brazil.

Among Cryptococcus gattii genotypes, VGII has gained pivotal relevance in epidemiological, clinical and genetic contexts due to its association with several outbreaks in temperate regions and due to the high variability of this genotype. The aim of this study was to compare 25 isolates of C. gattii from the Southeast region of Brazil with previously described isolates from other regions of the country and around the world. Among the 25 isolates, 24 were VGII and one was VGI. All of them were newly identified. Three new allele types (AT) (AT47 for the URA5 locus, AT56 for the LAC1 locus, and AT96 for the IGS1 region) were also described. Compared with other Brazilian isolates, those from the Southeast region presented the greatest haplotype diversity. In general, the regions presented different sequence types (STs), and only nine STs were found in more than one location. GoeBURST analysis showed two large groups among the Brazilian isolates. The largest group consists of 59 STs predominantly from the North and Northeast regions; the other large group includes 57 STs from the Southeast and Midwest regions. In a global context the South American isolates presented the highest genetic diversity (STs = 145, haplotype diversity (Hd) = 0.999 and π = 0.00464), while the African populations showed the lowest genetic diversity (STs = 3, Hd = 0.667 and π = 0.00225). These results confirm that the Brazilian C. gattii VGII population is highly diverse and reinforce the hypothesis of dispersion of this genotype from South America.

Histoplasmosis in HIV-Infected Patients: Epidemiological, Clinical and Necropsy Data from a Brazilian Teaching Hospital.

Histoplasmosis occurs in 5-10% of HIV-infected patients in endemic areas and evolves to severe and disseminated infection with mortality rates over 50% in some regions. This report presents epidemiological, clinical and outcome data from HIV-infected patients with histoplasmosis confirmed by culture and/or at necropsy who were admitted to a Brazilian teaching hospital. Data from 65 patients were obtained from their respective medical and necropsy records. From 2005 to 2018, 36 HIV-infected patients were diagnosed with histoplasmosis confirmed by culture. At admission, most of these patients presented disseminated fungal infection, whereas 15 (41.7%) were simultaneously diagnosed with both HIV infection and histoplasmosis. Fever, weight loss, hepatosplenomegaly, respiratory and digestive symptoms were present in 86.2%, 50%, 44.4% and 41.7% of the patients, respectively. At admission, 24 patients had low CD4 T-cell count and high viral load values. Among the 30 patients who received antifungals, 16 (53.3%) were cured, 13 (43.3%) died, and one was lost to follow-up. Six patients died prior to therapy. From 1990 to 2018, 63 necropsies of patients with Histoplasma capsulatum infection were performed. Of these patients, 29 (46.0%) were HIV-infected individuals, including 21 (72.4%) who presented disseminated histoplasmosis and 21 (72.4%) who were diagnosed with histoplasmosis at necropsy. The epidemiological, clinical and outcome profiles presented herein are similar to those described elsewhere and reinforce the difficulties that are still present in limited-resource settings where advanced immunodeficiency, combined with severe fungal infection and late patient admissions, is related to poor outcomes.

Choroiditis in a HIV-infected patient with disseminated cryptococcal infection: A case report and literature review.

BACKGROUND: Ocular involvement in AIDS patients is a common event mainly caused by inflammation or infection. Despite the high prevalence rate of cryptococcosis in these individuals, ocular features have been occasionally described. CASE REPORT: A 20-year-old Brazilian female with HIV infection recently diagnosed was admitted with a respiratory profile presumptively diagnosed as Pneumocystis jirovecii pneumonia; an ophthalmologic exam suggested choroiditis by this agent as well. She was complaining of headaches and blurred vision which led to cryptococcal meningitis diagnosis by a CSF positive India ink stain and Cryptococcus neoformans positive culture. Despite therapy based on amphotericin B plus fluconazole, her clinical state progressively worsened and the patient died one week later. At necropsy, disseminated cryptococcal infection was evidenced in several organs including eyes, which presented bilateral chorioretinitis. CONCLUSIONS: Cryptococcal ocular involvement in AIDS patients has been occasionally proved among the cases already reported. Thus, the post mortem exam is still pivotal to improve the quality of the clinical diagnosis, especially in limited-resource settings.

Choroiditis in a HIV-infected patient with disseminated cryptococcal infection: a case report and literature review / Coroiditis en un paciente con VIH con criptococosis diseminada: caso clínico y revisión de la bibliografía

Background: Ocular involvement in AIDS patients is a common event mainly caused by inflammation or infection. Despite the high prevalence rate of cryptococcosis in these individuals, ocular features have been occasionally described. Case report: A 20-year-old Brazilian female with HIV infection recently diagnosed was admitted with a respiratory profile presumptively diagnosed as Pneumocystis jirovecii pneumonia; an ophthalmologic exam suggested choroiditis by this agent as well. She was complaining of headaches and blurred vision which led to cryptococcal meningitis diagnosis by a CSF positive India ink stain and Cryptococcus neoformans positive culture. Despite therapy based on amphotericin B plus fluconazole, her clinical state progressively worsened and the patient died one week later. At necropsy, disseminated cryptococcal infection was evidenced in several organs including eyes, which presented bilateral chorioretinitis. Conclusions: Cryptococcal ocular involvement in AIDS patients has been occasionally proved among the cases already reported. Thus, the post mortem exam is still pivotal to improve the quality of the clinical diagnosis, especially in limited-resource settingsl

Antecedentes: La afectación ocular en pacientes con sida es una circunstancia común provocada principalmente por procesos inflamatorios o infecciosos. A pesar de la alta prevalencia de criptococosis en estos individuos, los hallazgos oculares solo se describen ocasionalmente. Caso clínico: Una mujer brasileña de 20 años, diagnosticada poco tiempo antes de infección por el VIH, fue hospitalizada por dificultad respiratoria con el presunto diagnóstico de neumonía por Pneumocystis jirovecii; el examen oftalmológico sugirió también la existencia de coroiditis por el mismo agente etiológico. Las quejas de la paciente por cefalea y visión borrosa orientaron el diagnóstico hacia la criptococosis meníngea, confirmada por el examen directo y el crecimiento de Cryptococcus neoformans en el cultivo del líquido cefalorraquídeo. A pesar de haber comenzado un tratamiento con anfotericina B y fluconazol, el estado clínico empeoró progresivamente y la paciente falleció una semana después. La necropsia mostró criptococosis diseminada en varios órganos, incluidos los ojos, que presentaban coriorretinitis bilateral. Conclusiones: La criptococosis ocular en pacientes con VIH se ha descrito ocasionalmente en los casos publicados. Por este motivo, la necropsia todavía es fundamental para mejorar la calidad del diagnóstico clínico de esa enfermedad, especialmente en regiones con recursos limitados

Epidemiological, Clinical and Outcome Aspects of Patients with Cryptococcosis Caused by Cryptococcus gattii from a Non-endemic Area of Brazil.

Cryptococcosis by Cryptococcus gattii occurs mainly in immunocompetent hosts, however, during the last decades, a growing number of cases in immunocompromised individuals have been noticed around the world. This report presents epidemiological, clinical and outcome aspects of patients with cryptococcosis caused by this species from a non-endemic area in Brazil. Of 278 Cryptococcus spp. clinical isolates recovered during the same period, 267 (96%) were molecularly identified as Cryptococcus neoformans VNI genotype and 11 (4%) as C. gattii VGII genotype by URA-5 RFLP. Of the 11 C. gattii patients, eight were male, mean age of 47.5 years. Of these, four were HIV-infected, one was kidney transplanted, one presented low CD4+ T cells values of unknown cause, another presented chronic liver disease meanwhile the remaining four were apparently immunocompetent. Disseminated disease and cryptococcal meningitis were present in four patients each. Most patients received amphotericin B plus fluconazole. Seven out of the 11 patients cured and four died before or during the therapy. The increased number of individuals with cryptococcosis by this species during the last decades needs to be carefully evaluated specially those who are HIV-infected. Nevertheless, Cryptococcus species differentiation is currently relevant in order to better know their relation with geographical, clinical host preference and outcome particularities.

Genotypic analysis of clinical and environmental Cryptococcus neoformans isolates from Brazil reveals the presence of VNB isolates and a correlation with biological factors.

Cryptococcal infections are mainly caused by members of the Cryptococcus neoformans species complex (molecular types VNI, VNII, VNB, VNIV and the AD hybrid VNIII). PCR of the mating type loci and MLST typing using the ISHAM-MLST consensus scheme were used to evaluate the genetic relationship of 102 (63 clinical and 39 environmental) C. neoformans isolates from Uberaba, Brazil and to correlate the obtained genotypes with clinical, antifungal susceptibility and virulence factor data. All isolates were mating type alpha. MLST identified 12 known and five new sequence types (ST). Fourteen STs were identified within the VNI isolates, with ST93 (57/102, 56%) and ST77 (19/102, 19%) being the most prevalent. From the nine VNII isolates previously identify by URA5-RFLP only four (ST40) were confirmed by MLST. The remaining five grouped within the VNB clade in the phylogenetic analysis corresponding to the sequence type ST504. Other two environmental isolates also grouped within VNB clade with the new sequence type ST527. The four VNII/ST40 isolates were isolated from CSF. The two VNIV sequence types (ST11 and ST160) were isolated from blood cultures. Two of six patients evaluated with more than one isolates had mixed infections. Amongst the VNI isolates 4 populations were identified, which showed differences in their susceptibility profiles, clinical outcome and virulence factors. These results reinforce that ST93 is the most prevalent ST in HIV-infected patients in the Southeastern region of Brazil. The finding of the VNB molecular type amongst environmental Brazilian isolates highlights that this genotype is not restricted to the African continent.

Cytokine patterns in a prospective cohort of HIV-infected patients with cryptococcal meningitis following initiation of antifungal and antiretroviral therapy.

Cryptococcal meningitis (CM) is a life-threatening infection in HIV-infected patients, especially in resource-limited settings. Cytokine patterns in the cerebrospinal fluid (CSF) and sera may be related to clinical outcomes. This study aimed to evaluate cytokine patterns in the CSF and sera of HIV-infected patients with CM as well as the cytokines produced by peripheral blood mononuclear cells (PBMCs) when stimulated with LPS and cryptococcal GXM. CSF and serum levels of IL-2, IL-4, IL-8, IL-10, IL-12p40, IL-17A, INF-γ, TNF-α and CXCL-10 were measured in HIV-infected patients with CM (CM+ HIV+) at various time points. Cytokine levels were evaluated in the PBMC culture supernatants and the baseline values were compared to those of HIV-infected patients without CM (CM- HIV+) and healthy controls (CM- HIV-). CSF cytokine levels at admission (n = 33) were higher than levels among the 23 survivors at week 2, but statistically significant differences were observed for IL-8 and IFN-γ (p<0.05). CSF and serum levels of IL-4 and IL-17A at week 10 (n = 16) were lower than the baseline values, whereas IL-2 levels increased compared to week 2 (p<0.05). At week 16 (n = 15), CSF and serum levels of IL-4, IL-10 and CXCL-10 were decreased compared to the baseline values (p<0.05). PBMCs from CM- HIV- individuals produced significantly higher levels of proinflammatory cytokines in response to LPS, with the exception of TNF-α, which showed higher levels among CM+ HIV+ patients. The PBMCs of CM patients produced higher levels of IL-4 than those of CM- HIV- patients in response to GXM stimulation, and levels progressively decreased during treatment (p<0.05). Then, a progressive shift in cytokine expression favoring a Th1 pattern was observed, which is crucial in controlling cryptococcal infection. A better understanding of the protective immune response against Cryptococcus neoformans will help to develop novel strategies to improve the outcomes of patients with cryptococcosis.

Paracoccidioidomycosis in Brazilian Patients With and Without Human Immunodeficiency Virus Infection.

Paracoccidioidomycosis (PCM) is endemic to Latin America, where 10 million people may be infected with Paracoccidioides brasiliensis/Paracoccidioides lutzii and 1,600,000 individuals live with human immunodeficiency virus (HIV) infection. An epidemiological overlapping of these infections occurred early in acquired immunodeficiency syndrome era with nearly 180 published cases. This study presents epidemiological, clinical, and outcome profiles for 31 PCM patients with HIV infection diagnosed in a teaching hospital in Brazil, and includes an update of previously reported cases. Medical records were reviewed and data compared with 64 PCM patients without HIV infection. Of the 31 PCM patients with HIV infection, 23 (74.1%) were male, with a median age of 36.7 years, whereas of the 64 PCM, 45 (70.3%) were male, with a median age of 35.1 years. Both groups presented similar proportions for smoking and alcoholism. PCM patients with HIV infection presented more fever, weight loss, and the acute clinical form than the PCM patients who had more mucosal and respiratory involvement characterizing the chronic form. Most PCM patients with HIV infection exhibited overlapping symptoms from both clinical forms with median symptom duration of 4.5 months compared with 8.3 months for the PCM control. Patients received sulfonamides and/or itraconazole for a median of 15.7 and 16.7 months for PCM/HIV-infected and PCM, respectively. Relapses occurred more in PCM (12 [30%]) than PCM/HIV-infected (4 [14.8%]) patients, whose mortality rate was higher (10 [32.8%]) than PCM patients (8 [20%]). The cases of PCM/HIV infection confirm that HIV can interact with some endemic diseases without increasing their frequency, while changing their natural history, clinical presentation, and outcome. The data presented here are in agreement with those observed in other studies.

Cryptococcosis in Acquired Immunodeficiency Syndrome Patients Clinically Confirmed and/or Diagnosed at Necropsy in a Teaching Hospital in Brazil.

Cryptococcosis occurs in acquired immunodeficiency syndrome (AIDS) patients with poor compliance to antiretroviral therapy or unaware of their human immunodeficiency virus status who present severe immunosuppression at admission. Consequently, high mortality rates are observed due to disseminated fungal infection. This report presents clinical and postmortem data of AIDS patients with cryptococcosis in a teaching hospital in Brazil. Retrospectively, medical and necropsy records of AIDS patients with cryptococcosis clinically confirmed and/or postmortem verified were reviewed. Clinical data were compared with those of patients presenting a good outcome to evaluate disseminated fungal infection and the agreement between clinical and postmortem diagnosis. At admission, most of the 45 patients with cryptococcal meningitis who died, presented more altered consciousness (P = 0.0047), intracranial increased pressure (P = 0.047), and severe malnutrition (P = 0.0006) than the survivors. Of 29 (64.4%) patients with cryptococcal meningitis, 23 died before week 2 on antifungal therapy, and the other six during the next 3 months. The remaining 16 (35.6%) cases had other diagnoses and died soon after. At necropsy, 31 (68.9%) presented disseminated infection involving two or more organs, whereas 14 (31.1%) cases had meningeal or pulmonary localized infection. The agreement of 64.4% between clinical and postmortem diagnosis was similar to some studies. However, other reports have shown figures ranging from 34% to 95%. Currently, a progressive worldwide decrease of autopsies is worrying because the role of postmortem examination is pivotal to verify or identify the death causes, which contributes to improve the quality of clinical diagnosis and medical training.

Cytokine profiles at admission can be related to outcome in AIDS patients with cryptococcal meningitis.

Cryptococcal meningitis (CM) remains as common life-threatening AIDS-defining illness mainly in resource-limited settings. Previous reports suggested that baseline cytokine profiles can be associated to fungal burden and clinical outcome. This study aimed to evaluate the baseline cytokine profiles in AIDS patients with CM and its relation with the outcome at weeks 2 and 10. Thirty AIDS patients with CM diagnosed by cerebrospinal fluid (CSF) Cryptococcus neoformans positive culture, India ink stain and cryptococcal antigen test were prospectively evaluated. As controls, 56 HIV-infected patients without CM and 48 non-HIV individuals were included. Baseline CSF and sera levels of IL-2, IL-4, IL-8, IL-10, IL-12p40, IL-17A, INF-γ and TNF-α were measured by ELISA. Of 30 CM patients, 24 (80%) were male, median age of 38.1. The baseline CSF high fungal burden and positive blood culture were associated with a positive CSF culture at week 2 (p = 0.043 and 0.029). Most CSF and sera cytokines presented higher levels in CM patients than control subjects (p < 0.05). CSF levels of IL-8, IL-12p40, IL-17A, TNF-α, INF-γ and sera TNF-α were significantly higher among survivors at weeks 2 and 10 (p < 0.05). Patients with increased intracranial pression exhibited CSF IL-10 high levels and poor outcome at week 10 (p = 0.032). Otherwise, baseline CSF log10 IFN-γ and IL-17A were negatively correlated with fungal burden (r = -0.47 and -0.50; p = 0.0175 and 0.0094, respectively). The mortality rate was 33% (10/30) at week 2 and 57% (17/30) at week 10. The severity of CM and the advanced immunodeficiency at admission were related to a poor outcome in these patients. Otherwise, the predominant Th1 cytokines profile among survivors confirms its pivotal role to infection control and would be a prognostic marker in cryptococcal meningitis.

Phylogenetic analysis of phenotypically characterized Cryptococcus laurentii isolates reveals high frequency of cryptic species.

BACKGROUND: Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States. METHODS: In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region. RESULTS: BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99-100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified. CONCLUSIONS: Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.

Central nervous system paracoccidioidomycosis in an AIDS patient: case report.

Up to now, over 200 patients with paracoccidioidomycosis (PCM) associated to HIV infection have already been reported; however, the central nervous system involvement in this coinfection was rarely reported. This paper presents a 35-year-old Brazilian male AIDS patient who developed pulmonary PCM successfully treated with itraconazole. At the antiretroviral therapy starting, he had 32 CD4(+) T cells baseline count and high viral load levels. After 9 months, he presented severe fungal meningoencephalitis diagnosed by sublenticular enhanced nodular lesion at computerized tomography and magnetic resonance brain imaging and a positive Paracoccidiodes brasiliensis smear and culture from cerebrospinal fluid. At the time, a sixfold increase in CD4(+) T cell count and undetectable viral load level were evidenced. The patient received amphotericin B during 1 year presenting slow but progressive clinical improvement, and he is currently asymptomatic and without neurological disabilities. To our knowledge, this is the second case report of a patient with neuroparacoccidioidomycosis associated to HIV infection.

Susceptibility profile of clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii in Uberaba, Minas Gerais, Brazil.

Cryptococcus neoformans and C. gattii are the etiologic agents of cryptococcosis, a life-threatening disease in both immunocompromised and immunocompetent hosts. Antifungal resistance has been evaluated using different methods, breakpoints, and sizes of test populations and it is an emerging as a significant issue worldwide. A total of 176 (95 clinical and 81 environmental) C. neoformans and eight clinical C. gattii isolates were evaluated to determine the minimal inhibitory concentration (MIC) according to the Clinical and Laboratory Standards Institute method. A total of 10.5% of the C. neoformans clinical isolates were resistant to amphotericin B (AMB), and 6.2% of the environmental isolates were resistant to fluconazole (FLZ). Environmental and clinical isolates presented epidemiologic cut-off values (ECVs) of 64 and 16 to FLZ and 1 and 2 to AMB, respectively. All of the C. gattii isolates showed high susceptibility to most drugs evaluated. Clinical isolates had lower susceptibility than environmental isolates to AMB and itraconazole whereas environmental isolates had lower susceptibility than the clinical isolates to FLZ, voriconazole, and ketoconazole. However, no difference was found in the susceptibility of the two species. The MICs and ECVs to antifungals can help to select the best therapeutic option for tracking epidemiological resistance among clinical and environmental isolates of Cryptococcus spp. around the world.

RAPD analysis with the primer L15996 of Brazilian clinical and environmental Cryptococcus neoformans isolates.

Different methods have been used to perform the molecular characterization of Cryptococcus neoformans. Among them, RAPD analysis is able to separate isolates of the same species and genotypes. This study aimed to evaluate clinical and environmental C. neoformans isolates from Minas Gerais, Brazil by RAPD and correlate the genetic profiles with the ones obtained by URA5-RFLP, virulence factors and antifungal susceptibility patterns. Forty-five environmental (31 from areas surrounding hospital and 14 from captive bird droppings from pet-shops) and 29 clinical C. neoformans isolates were evaluated. Antifungal susceptibility tests (Clinical and Laboratory Standards Institute), URA5-RFLP analysis and the assessment of virulence factors were performed according to their original descriptions. RAPD profiles were obtained using the L15996 primer (5'-CTCCACCATTAGCACCCAAAGC-3'). RAPD analysis generated two to 20 bands for all studied isolates. The isolates presented similarities ranging from 10.8 to 100.0%. Considering a minimum identity score of 50%, four clusters were formed. Cluster I contained 10 pet-shops bird dropping isolates, cluster II contained 22 clinical isolates most of them recovered from cerebrospinal fluid, cluster III contained 14 isolates from hospital surroundings and cluster IV contained 12 environmental isolates most from hospital surroundings. Fourteen isolates were not grouped. The RAPD profiles were clustered according to their source and URA5-RFLP pattern. No correlation between virulence factors or antifungal susceptibility profile with the obtained RAPD profiles was observed.

Antifungal susceptibility, enzymatic activity, PCR-fingerprinting and ITS sequencing of environmental Cryptococcus laurentii isolates from Uberaba, Minas Gerais, Brazil.

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human infection have been already reported. This study aimed to evaluate the phospholipase, proteinase and hemolysins activity, the antifungal susceptibility profile, the genetic variability by M13 and (GACA)(4) fingerprinting and the internal transcribe spacer (ITS) sequencing of 38 C. laurentii isolates recovered from captive bird droppings and surrounding hospital areas. All of them exhibited phospholipase activity, while the hemolytic activity was evidenced in 34 (89.4%) isolates. None of them exhibited proteinase activity. Twenty-seven isolates (71.1%) presented susceptibility dose dependent to fluconazole. Most isolates (94.7%) were susceptible to voriconazole, while one (2.65%) was resistant to this drug. Twenty-one (55.3%) isolates showed reduced susceptibility to itraconazole while nine (23.7%) were resistant. Three (7.9%) and five (13.1%) isolates exhibited resistance to ketoconazole and amphotericin B, respectively. Most C. laurentii fingerprinting obtained with M13 and (GACA)(4) showed high heterogeneity. By using the two primers, seven (18.4%) isolates grouped as A (CL2, CL7, and CL8), B (CL35, CL38) and C (CL29, CL30) with 100% similarity. Different from most variable surrounding hospital isolates, all but one of the pet shops strains clustered with the two primers, although they had been recovered from different neighborhoods. All isolates were identified as C. laurentii phylogenetic group I by ITS sequencing. Thus, the presence of virulence factors, a decreased antifungal susceptibility and a heterogeneous molecular pattern of the C. laurentii isolates here described suggests this species can be a potential pathogen in the context of the immunocompromised population.

Clinical, epidemiological and outcome features of patients with cryptococcosis in Uberaba, Minas Gerais, Brazil.

Nearly one million of cryptococcosis cases occur yearly around the world, involving mainly HIV-infected patients who are not receiving antiretroviral therapy (ART) or present poor adherence. This study aims to evaluate epidemiological, clinical and outcome aspects of patients with cryptococcosis from 1998-2010. Patients were prospectively recruited, and their medical and laboratory records were reviewed. A total of 131 cases were included, and of these, 119 (90.83%) had AIDS, 4 received a renal transplant, 2 presented systemic lupus erythematosus and 6 (4.6%) were apparently immunocompetent. Ninety-one (69.46%) were men, and the median age was 38.7 years. Cryptococcal meningitis (CM) was diagnosed in 103 (78.62%), whereas 28 (21.38%) had cryptococcal infection in other sites. Of patients with CM, 94 (91.26%) had AIDS being cryptococcosis the first defining illness in 61 (64.9%), while 37 (60.65%) of them presented simultaneously both diagnosis. Headache, altered mental status, papilledema and seizures at admission were significatively associated with a poor outcome. Of 163 different isolates, 155 (95.09%) were Cryptococcus neoformans and eight (4.88%) Cryptococcus gattii. Antifungal therapy was warranted in 8 (87.4%) patients with CM, but 46 (51.1%) died during the first days or weeks. Of 28 patients without CM, 21 (75%) received treatment, but 6 (28.6%) died. The poor outcome among this case series was similar to that reported from other developing countries, but it is paradoxal in Brazil where the ART is at free disposal in the public health services. Despite, at least 60-70% of patients present advanced immunosuppression when they receive the AIDS diagnosis.

Primary cutaneous cryptococcosis caused by Cryptococcus gattii in an immunocompetent host.

This paper presents the case of a 75-year-old Brazilian man who developed inflammatory skin lesions with nodules and ulcerations on the right forearm after an injury caused by handling barbed-wire and Eucalyptus spp. logs. Histopathological assessment of the lesions showed granulomatous processes with yeasts similar to Cryptococcus spp. Tissue fragments yielded yeasts when cultured that were identified as Cryptococcus gattii VGII through biochemical reactions and URA5-RFLP genotype. No evidence of systemic involvement or any underlying immunosuppressive diseases were identified, which supported the diagnosis of primary cutaneous cryptococcosis. After 5 months on therapy with high fluconazole doses, the skin lesions had fully healed.

Genotyping of Cryptococcus neoformans isolated from captive birds in Uberaba, Minas Gerais, Brazil.

To evaluate Cryptococcus spp. molecular types isolated from captive birds' droppings, an epidemiological survey was carried out in Uberaba, Minas Gerais, Brazil, from December 2006 to September 2008. A total of 253 samples of bird excreta (120 fresh and 133 dry) were collected from pet shop cages and houses in different neighbourhoods. Cryptococcus neoformans was isolated in 19 (14.28%) dry samples and one fresh sample (0.84%). Cryptococcus laurentii was recovered from seven (5.26%) dry samples, but not in the fresh samples. The canavanine-glycine-bromothymol blue test was positive in all but one of the C. laurentii isolates. Cryptococcus neoformans molecular typing was performed using URA5-RFLP and the mating type locus using mating type specific PCR. Nineteen (95.0%) presented genotype VNI and one VNII (5.0%). In addition, all isolates presented mating type α. Thus, the genotype of the environmental C. neoformans isolates observed in this study is in accordance with others already reported around the world and adds information about its distribution in Brazil. Cryptococcus laurentii strains were typed using URA5-RFLP and M13 fingerprinting, which showed similar profiles among them. Thus, despite the low number of C. laurentii isolates analysed, their molecular profile is different from another already reported.

Immune reconstitution inflammatory syndrome (IRIS) associated with Cryptococcus neoformans infection in AIDS patients.

UNLABELLED: Cryptococcosis is frequently associated to the immune reconstitution inflammatory syndrome (IRIS) in AIDS patients on highly active antiretroviral therapy (HAART). This study aimed to evaluate clinical and evolutive features of IRIS associated cryptococcosis patients in Uberaba, Brazil. PATIENTS: Eighty-one AIDS individuals admitted at the teaching hospital with cryptococcal meningitis were evaluated and from these, 40 were prospectively followed. Of 40 patients with cryptococcosis, nine (22.5%) presented clinical and laboratory features of IRIS. Six (66.6%) were male, with a mean age of 37.2. Five (55.5%) presented cryptococcosis as first AIDS defining condition. In seven (77.9%) IRIS was characterised as a relapse of meningeal symptoms after 10 weeks, mean time of 72 days, of starting HAART whereas, two asymptomatic patients developed the syndrome as an unmasked cryptococcosis after 10 and 12 weeks on HAART. Lymphadenitis as isolated finding associated with IRIS was evidenced in three cases. All patients presented low CD4(+) and high RNA viral load baseline values. Cultures of cerebrospinal fluid and lymph-node fragments tissues of these cases were negative. Six of nine individuals developed high intracranial pressure requiring a daily relief lumbar puncture. No deaths occurred during the evolution of these patients. The incidence and clinical evolutive profile observed in this case series are in accordance with other reports elsewhere.