Antifungal susceptibilities of clinical and environmental isolates of Cryptococcus neoformans in goiânia city, Goiás, Brasil

We evaluated the antifungal activities of amphotericin B, fluconazole, itraconazole and voriconazole in 70 Cryptococcus neoformans strains obtained from cerebrospinal fluid from AIDS patients and 40 C. neoformans strains isolated from the environment. Four clinical isolates were identified as C. neoformans var. gattii. The susceptibility test was done using a broth microdilution method according to NCCLS M27-A2. Range minimal inhibitory concentrations (MICs) for C. neoformans clinical isolates were 0.06-1.0 µg/mL for amphotericin B, 0.125-8 µg/mL for fluconazole, 0.03-0.5 µg/mL for itraconazole and 0.03-0.25 µg/mL for voriconazole. C. neoformans environmental isolates showed range MICs 0.015-0.125 µg/mL, 0.25-2.0 µg/mL, 0.007-0.125 µg/mL and 0.03-0.25 µg/mL for amphotericin B, fluconazole, itraconazole and voriconazole respectively. The MICs results obtained from clinical and environmental isolates showed similar pattern of susceptibility and no resistance has been found in our isolates.

Adulticidal activity against Stegomyia aegypti (Diptera: Culicidae) of three Piper spp.

Three Piper species, Piper longum, P. ribesoides and P. sarmentosum, were selected for investigation of adulticidal potential against Stegomyia aegypti, a main vector of dengue and dengue haemorrhagic fever. Successive extraction by maceration with 95% ethanol showed percentage yields of ethanolic extracts, which derived from P. longum, P. ribesoides and P. sarmentosum, of 8.89, 3.21 and 5.30% (w/w), respectively. All Piper extracts illustrated an impressive adulticidal activity when tested against female mosquitoes by topical application. The susceptibility of St. aegypti females to ethanol-extracted Piper was dose dependent and varied among the plant species. The highest adulticidal effect was established from P. sarmentosum, followed by P. ribesoides and P. longum, with LD50 values of 0.14, 0.15 and 0.26 µg/female, respectively. The potential of these Piper species, as possible mosquitocides, established convincing activity for further researches to develop natural substances for combat against adult mosquitoes.

Cryptococcus neoformans strains isolated from church towers in Rio de Janeiro City, RJ, Brazil

Cryptococcosis has been a significant cause of morbidity and mortality in patients with Aids. Many reservoirs of the agent Cryptococcus neoformans have been reported, but the ecology of this yeast must be elucidated in order to establish surveillance programs and to prevent infections. The objective of this study was to evaluate the presence of C. neoformans in Rio de Janeiro City, RJ, Brazil. Ten churches were selected for sampling and detection of the yeast collecting pigeon dropping, air samples from church towers and neighboring areas during one year. The data demonstrated that C. neoformans has been present in every church selected and was present in 37.8% of 219 pigeon dropping samples. As well as, the yeast was isolated from soil, insects, eggs, pigeon nests and feathers. Fifteen air samples (4.9%) were positive. The growth on C.G.B. medium showed that all strains belonged to C. neoformans var. neoformans, with 98.8% of the strains belonging to serotype A.

New data on Echinococcus spp. in Southern Brazil

40 Echinococcus isolates from sheep and cattle in Southern Brazil were genetically analysed in order to obtain further data on the presence of different taxa of the Echinococcus granulosus complex. Differentiation was done using a PCR technique and sequencing of mitochondrial cytochrome c oxidase subunit 1 (CO1). Most samples (38) could be allocated to the sheep strain (G1) of E. granulosus, while two samples belonged to E. ortleppi, previously known as cattle strain (G5) of E. granulosus. Due to the shorter prepatent period in dogs of the latter taxon, this records have important implications for the design of control measures in this endemic region.

Aeromonas spp. isolated from oysters (Crassostrea rhizophorea) from a natural oyster bed, Ceará, Brazil

Between April and October 2002, thirty fortnightly collections of oysters (Crassostrea rhizophorea) from a natural oyster bed at the Cocó River estuary in the Sabiaguaba region (Fortaleza, Ceará, Brazil) were carried out, aiming to isolate Aeromonas spp. strains. Oyster samples were submitted to the direct plating (DP) and the presence/absence (P/A) methods. Aeromonas were identified in 15 (50%) samples analyzed by the DP method and in 13 (43%) analyzed by the P/A method. A. caviae, A. eucrenophila, A. media, A. sobria, A. trota, A. veronii bv. sobria, A. veronii bv. veronii and Aeromonas sp. were isolated. The predominant species was A. veronii (both biovars), which was identified in 13 (43%) samples, followed by A. media in 11 (37%) and A. caviae in seven (23%). From the 59 strains identified, 28 (48%) presented resistance to at least one of the eight antibiotics tested.

Bites by coral snakes (Micrurus spp.) in Campinas, State of São Paulo, Southeastern Brazil

Coral snakes (Micrurus spp.) are the main representatives of the Elapidae in South America. However, bites by these snakes are uncommon. We retrospectively reviewed the data from 11 individuals bitten by coral snakes over a 20-year period; four were confirmed (snake brought for identification) and seven were highly suspected (neuromuscular manifestations) cases of elapid envenoming. The cases were classified as dry-bite (n = 1, caused by M. lemniscatus; did not receive antivenom), mild (n = 2, local manifestations with no acute myasthenic syndrome; M. frontalis and Micrurus spp.), moderate (n = 5, mild myasthenia) or severe (n = 3, important myasthenia; one of them caused by M. frontalis). The main clinical features upon admission were paresthesia (local, n = 9; generalized, n = 2), local pain (n = 8), palpebral ptosis (n = 8), weakness (n = 4) and inability to stand up (n = 3). No patient developed respiratory failure. Antivenom was used in ten cases, with mild early reactions occurring in three. An anticholinesterase drug was administered in the three severe cases, with a good response in two. No deaths were observed. Despite the high toxicity of coral snake venoms, the prognosis following envenoming is good. In serious bites by M. frontalis or M. lemniscatus, the venom of which acts postsynaptically, anticholinesterases may be useful as an ancillary measure if antivenom is unavailable, if there is a delay in obtaining a sufficient amount, or in those patients given the highest recommended doses of antivenom without improvement of the paralysis or with delayed recovery.

An evaluation of manual and mechanical methods to identify Candida spp. from human and animal sources

To compare two yeast identification methods, i. e, the manual and the VITEK mechanical methods, 62 clinical samples from hemocultures and animal sources were analyzed. After identification as Candida yeasts by the VITEK method, the strains were recharacterized using manual assimilation methods and sugar fermentation tests. Our findings reveal 58% concurrent identification between the two methods for animal strains, and 51% for human hemoculture strains.

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (> 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.

The "in vitro" antifungal activity evaluation of propolis G12 ethanol extract on Cryptococcus neoformans

Cryptococcosis is a worldwide disease caused by the etiological agent Cryptococcus neoformans. It affects mainly immunocompromised humans. It is relatively rare in animals only affecting those that have received prolonged antibiotic therapy. The propolis is a resin that can present several biological properties, including antibacterial, antifungal and antiviral activities. The standard strain C. neoformans ATTC 90112 was used to the antifungal evaluation. The tests were realized with propolis ethanol extract (PEE) G12 in concentrations from 0.1 to 1.6 mg mL-1. The evaluation of MIC and MFC were done according to DUARTE (2002)5. The inhibitory effect of PEE G12 on the fungal growing was seen at the concentration of 0.2 mg mL-1 and 1.6 mg mL-1 was considered a fungicidal one.

Serotype and mating type characterization of Cryptococcus neoformans by Multiplex PCR

Cryptococcus neoformans is an encapsulated yeast, etiological agent of cryptococcosis. The species is commonly associated with pigeon droppings and plant materials. The aim of the present work was to verify the presence of the yeast in pigeon droppings, and to identify the isolates obtained in serotypes and mating types (MAT). Ten samples of pigeon droppings were collected in the rural area of the city of Alfenas, Brazil. Samples were inoculated in agar Niger medium for fungal isolation and 22 isolates with characteristics of C. neoformans were obtained. The serotypes and MAT were determined by multiplex PCR using specific primers. Serotypes were also determined by using the Kit Crypto Check. Among the 22 samples evaluated, eight were identified as C. neoformans by classic identification tests. These samples were characterized as serotype A by the Kit Crypto check and as serotype A MAT alpha by the multiplex PCR. The present study reinforces the evidence that pigeon droppings are a reservoir for C. neoformans and confirms the prevalence of C. neoformans var. grubii (Aalpha) among environmental isolates. It also demonstrates that multiplex PCR is an acceptable alternative for serotype analysis because it reduces the costs for each reaction and analyses serotype and MAT simultaneously.

Cryptococcus gattii fungemia: report of a case with lung and brain lesions mimicking radiological features of malignancy

A 64-year-old apparently immunocompetent white man developed lung and brain lesions of disseminated cryptococcosis. The radiologic features mimicked those of lung cancer metastatic to the central nervous system. C. gattii was recovered from cultures of bronchoalveolar lavage fluid, brain biopsy, and blood. The same fungus was recovered from pulmonary and brain specimens at autopsy. Serum and cerebrospinal fluid cryptococcal antigen tests were diagnostic in our case and should be included in the diagnostic evaluation of unexplained pulmonary and cerebral lesions. A literature search showed few reports of fungemia by this species of Cryptococcus, contrasting to C. neoformans.

Occurrence of antibodies against Leptospira spp. in horses of the urban area of Londrina, Paraná, Brazil

A total of 320 horses were studied in this paper, both male and female, between two and 17 years of age, which were used for traction of wagons in the urban area of the municipality of Londrina (PR). These animals were kept, after their daily work, in abandoned areas or plots, in the outskirts of the urban area of the city. When these animals were attended by the veterinarians, between 1996 and 2005, none of them presented symptoms suggesting leptospirosis. The most frequent reasons for the visit were loss of weight, unwillingness for work, parasitism, laminess, and wounds. Microscopic Seroagglutination Test (SAM), with 22 Leptospira serovars, was performed in sera sample from all these animals. The aim of this study was to investigate the occurrence of antibodies against Leptospira spp. in horses from the urban area of Londrina (PR). From the samples tested, 214 (66.88%) were considered positive, with titers between 100 and 3200, being that 49 (22.90%) presented antibodies against a single serovar of Leptospira, and 165 (77.10%) samples presented antibodies against two or more serovars simultaneously, where in 88 (53.33%) it was possible to characterize the most likely probable serovar. Antibodies against the serovar Icterohaemorrhagiae were detected in 32 (23.36%) animals.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 °C for up to six years and the low number of oocysts may be overcomed by repetitions of extraction.