Absence of norovirus contamination in shellfish harvested and commercialized in the Northeast coast of Brazil.

Norovirus (NoV) is the main cause of gastroenteritis outbreaks worldwide. Although NoV spreads mainly from person to person, it is estimated that a large proportion of NoV outbreaks are caused by foodborne transmission. Bivalve mollusks are one of the most important foods involved in NoV transmission to humans. Little is known about NoV prevalence in shellfish harvested and commercialized in Brazil. The aim of this study was to map, for the first time, the distribution of NoV contamination in oysters and mussels harvested and commercialized in the coast of Pernambuco state, northeast Brazil. A total of 380 mollusks (260 oysters and 120 mussels) were collected between February and August 2017 either directly from harvesting areas or obtained from beach vendors at 17 sites in Pernambuco. Samples were processed and tested for NoV contamination using a SYBR Green real-time PCR assay. All samples were negative for NoV GI or GII contamination, suggesting a low risk of NoV contamination from this food source during the study period. Additional surveys in different areas of the Brazilian coast are warranted to monitor the risk of NoV infection upon seafood consumption.

Absence of norovirus contamination in shellfish harvested and commercialized in the Northeast coast of Brazil

Norovirus (NoV) is the main cause of gastroenteritis outbreaks worldwide. Although NoV spreads mainly from person to person, it is estimated that a large proportion of NoV outbreaks are caused by foodborne transmission. Bivalve mollusks are one of the most important foods involved in NoV transmission to humans. Little is known about NoV prevalence in shellfish harvested and commercialized in Brazil. The aim of this study was to map, for the first time, the distribution of NoV contamination in oysters and mussels harvested and commercialized in the coast of Pernambuco state, northeast Brazil. A total of 380 mollusks (260 oysters and 120 mussels) were collected between February and August 2017 either directly from harvesting areas or obtained from beach vendors at 17 sites in Pernambuco. Samples were processed and tested for NoV contamination using a SYBR Green real-time PCR assay. All samples were negative for NoV GI or GII contamination, suggesting a low risk of NoV contamination from this food source during the study period. Additional surveys in different areas of the Brazilian coast are warranted to monitor the risk of NoV infection upon seafood consumption.

Chronic renal failure in equine due to ascending pyelonephritis predisposed by cauda equina syndrome: case report / Insuficiência renal crônica em equino devido à pielonefrite ascendente predisposta por síndrome da cauda equina: relato de caso

Descreve-se o caso de uma égua, da raça Campeiro, utilizada como doadora de embriões, que apresentava quadros de cistite recorrente e incontinência urinária. Os sinais clínicos evoluíram para emagrecimento progressivo, anorexia, apatia e isolamento do plantel. Ao exame físico, foi identificada hipotonia da cauda, hipoalgesia da região perineal, flacidez retal e vesical, compatíveis com sinais relacionados à síndrome da cauda equina. Exames complementares laboratoriais, exame ultrassonográfico e necropsia confirmaram o diagnóstico de insuficiência renal crônica (IRC), a qual foi atribuida à pielonefrite ascendente. O exame de urocultura demonstrou crescimento de bactérias do gêneroStreptococcus sp. Este é um caso raro em equinos em que a disfunção de neurônio motor inferior propiciou o desenvolvimento de processo infeccioso no trato urinário, progredindo para um quadro crônico renal incompatível com a vida.(AU)

Descreve-se o caso de uma égua, da raça Campeiro, utilizada como doadora de embriões, que apresentava quadros de cistite recorrente e incontinência urinária. Os sinais clínicos evoluíram para emagrecimento progressivo, anorexia, apatia e isolamento do plantel. Ao exame físico, foi identificada hipotonia da cauda, hipoalgesia da região perineal, flacidez retal e vesical, compatíveis com sinais relacionados à síndrome da cauda equina. Exames complementares laboratoriais, exame ultrassonográfico e necropsia confirmaram o diagnóstico de insuficiência renal crônica (IRC), a qual foi atribuida à pielonefrite ascendente. O exame de urocultura demonstrou crescimento de bactérias do gênero Streptococcus sp. Este é um caso raro em equinos em que a disfunção de neurônio motor inferior propiciou o desenvolvimento de processo infeccioso no trato urinário, progredindo para um quadro crônico renal incompatível com a vida.(AU)

Prevalence and antifungal susceptibility of Candida parapsilosis complex isolates collected from oral cavities of HIV-infected individuals.

At present, few data are available on the prevalence and antifungal susceptibility of Candida parapsilosis complex isolates from HIV-infected individuals. The C. parapsilosis complex comprises three species, C. parapsilosis sensu stricto, C. metapsilosis and C. orthopsilosis. Fifteen of 318 Candida isolates were identified as members of the C. parapsilosis complex by PCR and restriction fragment length polymorphism (RFLP). The prevalence of C. parapsilosis complex isolates was 4.7 %, 2.2 % being identified as C. parapsilosis sensu stricto and 2.5 % as C. metapsilosis, while no C. orthopsilosis was isolated. This is believed to be the first study that has identified isolates of C. metapsilosis obtained from the oral cavity of HIV-infected individuals. Antifungal susceptibility tests indicated that all the isolates were susceptible to amphotericin B (AMB), fluconazole (FLC), ketoconazole (KTC), itraconazole (ITC), voriconazole (VRC) and caspofungin (CASPO). Although isolates of C. parapsilosis sensu stricto and C. metapsilosis were susceptible to FLC, isolates of C. metapsilosis showed a tendency for higher MICs (≥1.0 µg ml(-1)). Based upon the frequency of candidiasis and the fact that certain isolates of the C. parapsilosis complex respond differently to FLC therapy, our data may be of therapeutic relevance with respect to susceptibility and potential resistance to specific antifungal agents. Our data suggest that C. metapsilosis can be a human commensal; its importance as a pathogen has yet to be confirmed.

Accuracy of routine diagnostic tests used in paracoccidioidomycosis patients at a university hospital.

The identification of appropriate laboratory measures to confirm clinical hypotheses is important in routine paracoccidioidomycosis medical care. The clinical records and laboratory reports of 401 paracoccidioidomycosis patients attended at the Tropical Diseases Area, Faculdade de Medicina de Botucatu, from 1974 to 2008 were reviewed. Direct mycological (DM), cell block (CB), histopathological (HP), and double immunodiffusion (DID) tests were evaluated before treatment. Typical Paracoccidioides brasiliensis yeast forms were observed in clinical specimens of 86% of the patients, but 14% were detected only by serological test. DM of 51 different tissue specimens produced 74.5% sensitivity, and 62.5% sensitivity was observed in 112 sputum samples. CB in 483 sputum samples generated 55.3% sensitivity. HP performed in 239 samples from different tissues revealed 96.7% sensitivity. Serology carried out in 351 patients and 200 healthy controls provided 90.0% sensitivity, 100.0% specificity, 100.0% positive predictive value, 85.1% negative predictive value and 93.6% accuracy. Comparisons of laboratory measurements performed in the same patient showed that sensitivity decreases from HP to DID to CB and DM, with the last two assays providing similar sensitivities. This study demonstrated that P. brasiliensis identification by HP, CB, and/or DM associated with DID is sufficient to establish the laboratorial diagnosis of paracoccidioidomycosis in practically all cases.

Oral Candida spp. colonization in human immunodeficiency virus-infected individuals: [review]

Several yeast species of Candida genus can colonize the skin as well as the mucous membrane of the vagina and the digestive tract for short or long periods. Depending on the host's immunological state and the yeast's virulence, colonization can become an infection, invading the colonized tissues and also disseminating. AIDS is characterized by the host's intensive and progressive immunodepression which manifests as diverse symptoms, mainly lesions in the mouth. Oral candidiasis is the most prevalent opportunistic infection in individuals infected with human immunodeficiency virus (HIV) and is an important indicator of the disease progress and the immunosuppression increase. The factors involved in the equilibrium between Candida spp. and HIV-infected subjects are sometimes contradictory and were evaluated in the present study specially for colonization.

Dysphonia and laryngeal sequelae in paracoccidioidomycosis patients: a morphological and phoniatric study.

The larynx is the third most commonly involved organ in paracoccidioidomycosis (PCM). While a few studies have evaluated laryngeal sequelae, there have not been any investigations of voice abnormalities in PCM patients. To evaluate persistent dysphonia and laryngeal lesions, we studied 15 normal subjects and 30 post-treatment PCM patients, i.e., 15 with only pulmonary and 15 with both laryngeal and pulmonary involvement. Perceptual and acoustic voice analysis were performed with all patients, while endoscopic studies were also conducted with the 15 laryngeal patients. Voice analysis showed instability by perceptual analysis (P<0.01) in both groups, but more severe dysphonia was noted in the laryngeal group (P<0.01). The dysponia, seen in 66.7% of these patients (dysphonia index < 7.0), was characterized by roughness and breathness. The Dr. Speech (Tiger Electronics) analysis program did not accept five voices from the laryngeal group due to the severe dysphonia. Jitter was elevated in five laryngeal lesion patients. Endoscopy showed that 80% of patients with laryngeal lesion had two or more laryngeal structures involved. Vocal fold alterations were seen in all laryngeal lesion patients, which included involvement of the arythenoids, epiglottis, and vestibular folds. This first functional study of laryngeal sequelae in PCM revealed frequent and severe dysphonia that may have important social consequences for patients.

Infiltrative myelopathy by paracoccidioidomycosis. A review and report of nine cases with emphasis on bone marrow morphology.

AIMS: To report nine additional well-defined cases with infiltrative myelopathy by paracoccidioidomycosis (PCM), to describe the specific lesions and infection-related stromal abnormalities, to review the literature on this type of involvement and to introduce a new cause of granulomatous lesions of bone marrow. METHODS AND RESULTS: Different bone marrow specimens were studied (aspirated smears, aspirated clots, biopsy imprints and biopsies) from nine patients with acute or subacute forms of PCM known to have PCM infiltrative myelopathy. CONCLUSIONS: The biopsy specimens were the best for demonstrating bone marrow involvement by PCM. The lesions varied from compact and focal granulomas with few fungal cells to numerous disseminated fungal cells within a loose granulomatous inflammatory reaction, with a continuum between these extremes suggesting a spectrum of immune response to the fungi. Other findings such as bone marrow fibrosis, parenchymal coagulative necrosis and bone necrosis were also observed in the affected areas.

Mycoses associated with AIDS in the Third World.

Despite advances in diagnosis and treatment, the epidemiological status of the human immunodeficiency virus (HIV) infection is far from under control in most of the developing world. Sub-Saharan Africa, Southeast Asia and India show increased rates of new infections. In Latin America and the Caribbean there were 1.6 million estimated cases of HIV-infected patients at the end of 1997. Fungal diseases have been one of the most relevant diagnoses in relation to the acquired immunodeficiency syndrome (AIDS). Infections due to Candida species and Cryptococcus neoformans var. neoformans are common worldwide. Histoplasma capsulatum, Coccidioides immitis and Penicillium marneffei are important causes of disease in endemic areas. Infection due to Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis are uncommon even where they are endemic. Phaeohyphomycetes, hyalohyphomycetes and zygomycetes are still rare as a cause of disease among AIDS patients. However, agents pertaining to these groups, such as Aspergillus spp., have an increasing incidence. Superficial mycoses due to dermatophytes have special features from epidemiological, clinical and therapeutic points of view.

Correlation between antigenemia of Paracoccidioides brasiliensis and inhibiting effects of plasma in patients with paracoccidioidomycosis.

Metabolites produced by pathogenic fungi may be involved in the pathogenesis of fungal infections consequently altering the defence mechanisms of the host. In this study the levels of Paracoccidioides brasiliensis antigens detected in the plasma of patients with paracoccidioidomycosis correlated with the suppression index detected by the low mitogenic response of peripheral blood mononuclear cells (PBMC) to phytohaemaglutinin (PHA). This inhibitory effect on lymphoproliferation was observed in the plasma of 58% of the patients, suggesting the presence of inhibitory factors. Plasma samples from paracoccidioidomycosis patients having or not having inhibitory factors showed no significant effect on chromosomes of lymphocytes from healthy individuals. However, these plasmas had a suppressive activity on the blastogenic response of these lymphocytes stimulated with PHA, that was independent of a cytotoxic effect. P. brasiliensis antigens added to the proliferative response of PBMC from healthy individuals stimulated or not stimulated with PHA showed a dose-dependent suppressor effect, reproducing the inhibitory effect of patients' plasma. We suggest that the antigens of P. brasiliensis present in the plasma of patients, even at low concentrations, can play an important role in the reduction of the cellular immune response and in the genesis of the immunoregulatory disturbances observed in paracoccidioidomycosis.

Paracoccidioides brasiliensis isolated from armadillos is virulent to Syrian hamsters.

Isolates of Paracoccidioides brasiliensis may vary in virulence according to time of in vitro subcultivation. The present study compared the morphology and pathogenicity to hamsters of two P. brasiliensis isolates: one obtained from human lesions and maintained in the laboratory for several years (Pb-18) and the other isolate recovered from hamsters inoculated with organ homogenates from armadillos (Pb-T). The microscopic morphology of Pb-18 and Pb-T showed yeast cells with similar diameter. However, Pb-T produced a significantly higher number of buds per mother cell than Pb-18. Besides, the mycelial form of Pb-T developed abundant sporulation during 8 weeks of culture which was absent in the Pb-18 isolate. Virulence studies demonstrated that mortality rates, antibody levels, fungal load and extent of lesions in the organs were significantly higher in animals infected with Pb-T. The results demonstrated that Pb-T recently isolated from an animal was more virulent than Pb-18. These differences between the two P. brasiliensis isolates may be indicators of virulence attenuation in this fungal species.

Detection of circulating Paracoccidioides brasiliensis antigen in urine of paracoccidioidomycosis patients before and during treatment.

For the diagnosis and follow-up of paracoccidioidomycosis patients undergoing therapy, we evaluated two methods (immunoblotting and competition enzyme immunoassay) for the detection of circulating antigen in urine samples. A complex pattern of reactivity was observed in the immunoblot test. Bands of 70 and 43 kDa were detected more often in urine samples from patients before treatment. The immunoblot method detected gp43 and gp70 separately or concurrently in 11 (91.7%) of 12 patients, whereas the competition enzyme immunoassay detected antigenuria in 9 (75%) of 12 patients. Both tests appeared to be highly specific (100%), considering that neither fraction detectable by immunoblotting was present in urine samples from the control group. gp43 remained present in the urine samples collected during the treatment period, with a significant decrease in reactivity in samples collected during clinical recovery and increased reactivity in samples collected during relapses. Reactivity of some bands was also detected in urine specimens from patients with "apparent cure." The detection of Paracoccidioides brasiliensis antigens in urine appears to be a promising method for diagnosing infection, for evaluating the efficacy of treatment, and for detecting relapse.

Serological diagnosis of Chagas' disease: a potential confirmatory assay using preserved protein antigens of Trypanosoma cruzi.

The diagnosis of Chagas' disease relies mostly on data provided by immunologic tests, but inconclusive results often require elucidation, especially in blood banks. When six different types of Trypanosoma cruzi epimastigote antigens were studied by an immunoblotting assay (IBA), a preserved protein antigen (Ag PP) was found to present the most interesting immunochemical features because of its high reactivity with anti-T. cruzi antibodies. Thus, the IBA with Ag PP (PP IBA) was assessed with panels of coded and noncoded serum samples prepared in different laboratories, including the Brazilian Reference Laboratory for Chagas' Disease. It was found that serum samples from patients proved (clinically, eletrocardiographically, serologically, and epidemiologically) to have Chagas' disease consistently recognized 12 bands (140, 100, 85, 78, 59, 57, 46, 35, 27, 23, 20, and 18 kDa) of Ag PP. In contrast, sera from nonchagasic patients, including patients with mucocutaneous leishmaniasis, were negative or reacted weakly, and one serum sample did not have more than five different bands. These bands were 78, 57, 46, 35, 27, 23, 20, or 18 kDa. A criterion was adopted to interpret the results obtained in the PP IBA. The criterion considered positive a serum sample recognizing all 12 bands and considered negative a serum sample that did not recognize any of the bands except the eight nonspecific bands mentioned above. The PP IBA indicated maximum sensitivity and specificity as well as high positive and negative predictive values. The data demonstrate that the PP IBA discriminates chagasic from nonchagasic infections and seems to be applicable as a confirmatory assay for elucidating inconclusive results obtained by standard serology.

Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu, SP, Brazil.

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 degrees C (yeast form) and as cottonous colonies at 25 degrees C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp 43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population.

Isolation of fungi from nature in the region of Botucatu, State of Sao Paulo, Brazil, an endemic area of Paracoccidioidomycosis

In an attempt to isolate Paracoccidioides brasiliensis from nature 887 samples of soil from Botucatu, SP, Brazil, were collected cultured in brain heart infusion agar supplement with dextrose, in potato dextrose agar and in yeast extract starch dextrose agar, all with antibiotics, at 25º and 37ºC. Five thermo-dependent dimorphic fungi morphologically resembling P. brasiliensis were isolated; two from armadillo holes; further studies of the biology, antigenicity and genetic features of the five dimorphic fungi are necessary to clarify their taxonomy and their possible relation to P.brasiliensis. In addition, 98 dematiaceous fungi and 581 different soecies of Aspergillus spp. were also isolated. Our findings emphasize that armadillos and their environment are associated with thermo-dimorphic fungi and confirm the ubiquity of pathogenic dematiaceous fungi and Aspergillus spp.

The use of glucan as immunostimulant in the treatment of paracoccidioidomycosis.

A group of 10 patients, nine of them seriously infected with Paracoccidioides brasiliensis (G1), received glucan (beta-1,3 polyglucose) as an immunostimulant intravenously once a week for one month, followed by monthly doses (10 mg) over an 11-month period, together with a specific anti-fungal agent as an immunostimulant. A second group of eight moderately infected patients (G2) was treated with only the anti-fungal agent. Among the patients in G1, there was only one case of relapse compared with five in G2. Values for the erythrocyte sedimentation rate (ESR) showed a significant difference (P < 0.001) post-treatment in G1 patients, when compared with the pretreatment levels. There was also a significant reduction (P < 0.001) in the level of serum antibodies to P. brasiliensis in the G1 patients in post-treatment examinations. The phytohemagglutinin (PHA) skin test showed a positive reaction among the patients in G1 (P < 0.01) post-treatment and there was a tendency towards an increase in the number of CD4+ T lymphocytes in both groups after treatment. The serum level of tumor necrosis factor (TNF) proved to be significantly higher (P < 0.02) in the G1 patients during treatment. In the G1 patients, the correlation between ESR and TNF tended to be negative whereas that between ESR and serum antibodies was positive. The present results indicate that the patients who received glucan, in spite of being more seriously ill, had a stronger and more favorable response to therapy.

Isolation of fungi from nature in the region of Botucatu, state of São Paulo, Brazil, an endemic area of paracoccidioidomycosis.

In an attempt to isolate Paracoccidioides brasiliensis from nature 887 samples of soil from Botucatu, SP, Brazil, were collected cultured in brain heart infusion agar supplemented with dextrose, in potato dextrose agar and in yeast extract starch dextrose agar, all with antibiotics, at 25 degrees and 37 degrees C. Five thermo-dependent dimorphic fungi morphologically resembling P. brasiliensis were isolated; two from armadillo holes; further studies of the biology, antigenicity and genetic features of the five dimorphic fungi are necessary to clarify their taxonomy and their possible relation to P. brasiliensis. In addition, 98 dematiaceous fungi and 581 different species of Aspergillus spp. were also isolated. Our findings emphasize that armadillos and their environment are associated with thermo-dimorphic fungi and confirm the ubiquity of pathogenic dematiaceous fungi and Aspergillus spp.

Evaluation of Paracoccidioides brasiliensis exoantigen in the detection of delayed dermal hypersensitivity in experimental and human paracoccidioidomycosis.

The exoantigen of Paracoccidioides brasiliensis standardized by Camargo et al. (AgR) was used to evaluate the in vivo and in vitro cell immune response of experimental animals and of patients with paracoccidioidomycosis (PBM). Fava Netto antigen (AgF) was tested in parallel as a control antigen. The study was conducted with mice and guinea pigs infected with P. brasiliensis or immunized with its fungal antigens, on patients with PBM and on their respective control groups. The cell immune response was analysed by skin tests, and by the macrophage and leucocyte migration inhibition tests (MMIT and LMIT) in the animals and in the patients, respectively. The skin test with AgR as paracoccidioidin was positive in infected or immunized mice and guinea pigs and negative in control animals. The skin tests with AgR (24 h) showed 96.7% positivity in patients with PBM and were negative in control individuals. Histopathological study of the in vivo tests in the different experimental models was consistent with a delayed hypersensitivity response (DHR). Immunohistochemical study of the skin tests of PBM patients demonstrated a predominance of T lymphocytes, confirming the nature of a DHR to the fungal antigens. The in vitro cell immune response showed variable results for the various experimental models, i.e. significant rates of MMIT in immunized mice, a tendency to positivity in infected guinea pigs, and the absence of migration inhibition in PBM patients. Taken together, the data indicate that the AgR is efficient as paracoccidioidin in the evaluation of DHR in PBM, with an optimum time of reading the test of 24 h.