SUSCEPTIBILITY TEST FOR FUNGI: CLINICAL AND LABORATORIAL CORRELATIONS IN MEDICAL MYCOLOGY

SUMMARYDuring recent decades, antifungal susceptibility testing has become standardized and nowadays has the same role of the antibacterial susceptibility testing in microbiology laboratories. American and European standards have been developed, as well as equivalent commercial systems which are more appropriate for clinical laboratories. The detection of resistant strains by means of these systems has allowed the study and understanding of the molecular basis and the mechanisms of resistance of fungal species to antifungal agents. In addition, many studies on the correlation of in vitro results with the outcome of patients have been performed, reaching the conclusion that infections caused by resistant strains have worse outcome than those caused by susceptible fungal isolates. These studies have allowed the development of interpretative breakpoints for Candida spp. and Aspergillus spp., the most frequent agents of fungal infections in the world. In summary, antifungal susceptibility tests have become essential tools to guide the treatment of fungal diseases, to know the local and global disease epidemiology, and to identify resistance to antifungals.

SEROVARS AND ANTIMICROBIAL RESISTANCE OF Salmonella spp. ISOLATED FROM TURKEY AND BROILER CARCASSES IN SOUTHERN BRAZIL BETWEEN 2004 AND 2006

Salmonella spp. causes diseases in fowls, when species-specific serovars (Salmonella Pullorum and S.Gallinarum) are present in flocks, and public health problems, when non-typhoid serovars are isolated, as well as possible bacterial resistance induced by the preventive and therapeutic use of antimicrobials in animal production. This study describes the serovars and bacterial resistance of 280Salmonella spp. strains isolated from turkey and broiler carcasses in Southern Brazil between 2004 and 2006. SalmonellaEnteritidis was the most prevalent serovar (55.7%), followed by Heidelberg (5.0%), Agona (4.3%), Bredeney (3.9%), Hadar (3.2%), and Typhimurium (2.9%). Tennessee and S. Enterica subspecies enterica(O: 4.5) were isolated only in turkeys, and Hadar (18.6%) was the most prevalent serovar in this species. Antimicrobial susceptibility tests were performed in 178 isolates (43 from turkeys and 135 from broilers). All isolates were sensitive to amoxicillin + clavulanic acid, polymyxin B, ciprofloxacin, and norfloxacin, and were resistant to bacitracin and penicillin. Broiler carcass isolates showed resistance to nalidixic acid (48.9%), nitrofurantoin (34.3%), neomycin (9.6%), tetracycline (5.2%), and kanamycin (8.9%); and turkey carcass isolates were resistant to nalidixic acid (62.8%), tetracycline (34.9%), and neomycin (30.2%), with a significant difference in turkeys when compared to broiler carcass isolates. These results indicate the need for judicious use of antimicrobials in livestock production, given that the serovars identified are potential causes of food poisoning.

ISOLATION AND GENOTYPING OF Toxoplasma gondii IN SERONEGATIVE URBAN RATS AND PRESENCE OF ANTIBODIES IN COMMUNICATING DOGS IN BRAZIL

The role of rodents in the epidemiology of toxoplasmosis was investigated inLondrina, Paraná State, Brazil. One hundred and eighty-one Rattus rattus and one Mus musculus were caught in 37 places. Blood and tissues were collected and submitted to the indirect fluorescence antibody test (IFAT) and the bioassay. Serum samples from 61 contacting dogs were also collected. Sixteen rats (8.8%) were positive for Toxoplasma gondii, but just two of them were positive by serology and bioassay test. Antibodies were found in nine (4.9%) rats. Tissues of nine rats bioassayed were positive and four isolates were obtained. Restriction fragment length polymorphism (RFLP) analysis was performed using 12 markers (SAG1, SAG2, SAG2-alt, C22-8, C29-2, L358, PK1, BTUB, GRA6, SAG3, Apico, CS3). Genotyping revealed that the four strains isolated from this study have been isolated before in cats and chickens from Brazil. None of the isolates was identified like clonal archetypal T-types I, II, and III. The rats presented lower serologic Toxoplasma gondii prevalence (8.8%) compared to contacting dogs (70.5%).

Classification of varieties of pasteurella pestis strains isolated in the interior of the state of Pernambuco-Brazil

Studies were made on the biochemical behavior of 100 strains of P.pestis isolated in Northeastern Brazil with regard to production of nitrous acid, reduction of nitrates to nitrltes, and aciáification of glycerol. Results showed that 98 strains can be classified as "orientalis variety", while the remaining two could not be included in any of the existing "varieties".

Trypanosoma (herpetosoma) Rangeli tejera, 1920: influence of host weight, size of inoculum, and route of infection upon experimental parasitemia

To study the influence of host age, inoculum size, and route of infection on Trypanosoma (Herpetosoma) rangeli, 12 lots of 6.0 g albino mice (NMRI strain) were infected up. with from 25x10¹ to 25x10(6) trypomastigotes/gram body weight harvested from LIT medium. The lower inocula produced low but persistent parasitemias, while the higher inocula produced high levels of parasitemia that fell quickly, suggesting the mobilization of resistance mechanisms. In other experiments, i.p. inoculation produced higher parasitemias than s.c. inoculation, and 6.0 g mice had higher parasitemias than 16.0 or 26.0 g mice. Thus, a standard methodology would seem to be necessary in the study of the various strains and/or species that may make up the T. rangeli complex.

Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160), whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

Effects of severe protein restriction in levels of parasitemia and in mortality of mice accutely infected with Trypanosoma cruzi

Adult mice were submitted to different degrees of protein restriction for five weeks (4.75, 9.5,14.25 and 19% of protein in isocaloric diets with normal content of mineral and vitamins), being subsequently infected with two strains of Trypanosoma cruzi: 10(5) trypomastigotes of Y strain or 14(5) trypomastigotes of CL strain. The same diet was maintained for all animals and the infection wasfollowed up by evaluation of blood parasites, mortality and intensity of lesions in the heart and skeleton muscle. Only severe protein restriction (4.75%) induced decrease in resistance to the infection with both the Y and CL strains of T. cruzi, which resulted in higher parasitemia and mortality. The inflammatory lesions in heart and skeleton muscle were less extensive in groups with severe protein restriction despite the increased number of parasite in muscle cells. Depression of immune mechanisms could be responsiblefor the reduced resistance and reduced inflammatory reaction after T. cruzi infection in severely protein restricted animals.

The hamster (mesocricetus auratus) as experimental model in chagas' disease: parasitological and histopathological studies in acute and chronic phases of Trypanosoma cruzi infection

This research characterizes the acute and chronic phases of Chagas ' disease in hamster through parasitological and histopathological studies. The acute phase was achieved with 44 young hamsters injected intraperitoneally with 100.000 blood trypomastigotes of Benedito and Y strains of T. cruzi. The chronic phase was induced in 46 hamsters injected intraperitoneally with 35.000 trypomastigotes ofVicentina, Benedito and Y strains. Animals were sacrificed at regular intervals of 24 hours of acute phase and from the 3rd to the 10th month of infection ofchronic phase. In the acute phase, parasites were easily recoveredfrom all animals and there was an inflammatory reaction characterized by mononuclear and polymorphous leukocyte infiltration of variable degree in the majority of tissues and organs, specially in the connective loose and fatty tissues, smooth muscle myocardium and skeletal muscle. In the chronic phase the lesions occurred in the same tissues and organs, but the inflammatory response was less severe and characterized by mononuclear infiltration mainly with focal or zonalfibrosis in the myocardiun. In 50% of infected animals parasites were found inmyocardiun and recoveredfrom pericardic, peritoneal and ascitic fluids in some animals. Signs of heart failure, sudden death and enlargement of bowel were observed regularly. We concluded that the hamster is an useful model for Chagas' disease studies.

Biochemical behavior of Trypanosoma cruzi strains isolated from mice submitted to specific chemotherapy

To investigate the influence of chemotherapy on the biochemical beha vior of Trypanosoma cruzi strains, three groups of mice were infected with one of three strains of T. cruzi of different biological and isoenzymic patterns (Peruvian, 21 SF and Colombian strains). Each group was subdivided into subgroups: 1 - treated with nifurtimox; 2 - treated with benznidazole and 3 - untreated infected controls. At the end of treatment, that lasted for 90 days, xenodiagnosis, sub inoculation of blood into new born mice and haemoculture were performed as tests of cure. From the positive tests, 22 samples of T. cruzi were isolated from all subgroups. Electrophoretic analysis of the isoenzymes PGM, GP1, ALAT and AS AT failed to show any difference between parasite strains isolated from treated and untreated mice, which indicates that no detectable clonal selection or parasite genetic markers alterations concerning the isoenzymes analysed have been determined by treatment with drugs of recognized antiparasitic effect, suggesting stability of the phenotypic characteristics of the three biological types of T. cruzi strains.

Cardiac plexus of dogs experimentally infected with Trypanosoma cruzi: inflammatory lesions and quantitative studies

Qualitative and quantitative aspects of the superficial and profound cardiac plexus of dogs experimentally infected with Be-62 and Be-78 strains of Trypanosoma cruzi were studied. Animals were autopsied in the acute phase of infection. The inflammatory process, lesions and number of parasites were more intense and frequent in animals infected with the Be-78 strain than in those infected with Be-62. Despite this, no statistically significant differences could be found between the number of neuron bodies in the ganglia of infected and control dogs.

Immunodiagnosis of chronic Chagas' disease using the Tc 46 and Tc 58 antigens

The polypeptides of 46 and 58kDa were recognized in different T. cruzi strains (Y, WSL and Colombiana) by serum of all chagasic patients studied. These polypeptides were isolated from T. cruzi Y strain and used in ELISA. The sensitivity and specificity were 97.6% [CI 95%: 86-100%] and 100% [CI 95%: 89.3-100%], respectively when Tc 46 was used. When Tc 58 was used the sensitivity and specificity were 100% [CI 95%: 89.6-100%] and 90.2% [CI 95%: 75.9-96.8%], respectively.

In vitro evaluation of verapamil and other modulating agents in Brazilian chloroquine-resistant Plasmodium falciparum isolates

Verapamil, was assayed to record its modulating effect upon Brazilian Plasmodium falciparum isolates resistant to chloroquine. Other cardiovascular drugs known to be modulating agents in resistant malaria and/or multidrug-resistant neoplasias, including nifedipine, nitrendipine, diltiazem and propranolol, were also evaluated. Concentrations similar to those for cardiovascular therapy were used in the in vitro microtechnique for antimalarial drug susceptibility. Intrinsic antiplasmodial activity was observed from the lowest concentrations without a significant modulating action. Other reported modulating agents, such as the antipsychotic drug trifluoperazine and the antidepressants desipramine and imipramine, demonstrated similar responses under the same experimental conditions. Results suggest a much higher susceptibility of Brazilian strains, as well as an indifferent behaviour in relation to modulating agents.

Epidemiological and molecular characteristics of HIV-1 infection among female commercial sex workers, men who have sex with men and people living with AIDS in Paraguay

An HIV seroprevalence and molecular study was conducted among 935 subjects: 723 female commercial sex workers, 92 men who have sex with men and 120 HIV-positive volunteers. The reported injection drug use rates were 0.7% in female commercial sex workers and 3% in men who have sex with men. Sexually transmitted infections were reported in 265 (37%) of the female commercial sex workers and 38 (41%) of the men who have sex with men. A total of 20 (2.8%) female commercial sex workers and 12 (13%) men who have sex with men became HIV infected during the study period. A history of sexually transmitted infection increased the risk of subsequent HIV infection twofold (adjusted odds ratio of 2.5) among the female commercial sex workers, while cocaine use had an adjusted odds ratios of 6.61 among men who have sex with men. From 130 samples, and based on heteroduplex mobility assaying for the env gene, with sequencing of part of pol and/or full genomes, subtype B was the predominant subtype identified (66%); followed by subtype F (22%) and subtype C (4%). Recombinant CRF12-BF strains were identified in 6% and CRF17_BF was identified in 2%.

Chemical composition, toxicity and larvicidal and antifungal activities of Persea americana (avocado) seed extracts

The present study had the aim of testing the hexane and methanol extracts of avocado seeds, in order to determine their toxicity towards Artemia salina, evaluate their larvicidal activity towards Aedes aegypti and investigate their in vitro antifungal potential against strains of Candida spp, Cryptococcus neoformans and Malassezia pachydermatis through the microdilution technique. In toxicity tests on Artemia salina, the hexane and methanol extracts from avocado seeds showed LC50 values of 2.37 and 24.13mg mL-1 respectively. Against Aedes aegypti larvae, the LC50 results obtained were 16.7mg mL-1 for hexane extract and 8.87mg mL-1 for methanol extract from avocado seeds. The extracts tested were also active against all the yeast strains tested in vitro, with differing results such that the minimum inhibitory concentration of the hexane extract ranged from 0.625 to 1.25mg L-¹, from 0.312 to 0.625mg mL-1 and from 0.031 to 0.625mg mL-1, for the strains of Candida spp, Cryptococcus neoformans and Malassezia pachydermatis, respectively. The minimal inhibitory concentration for the methanol extract ranged from 0.125 to 0.625mg mL-1, from 0.08 to 0.156mg mL-1 and from 0.312 to 0.625mg mL-1, for the strains of Candida spp., Cryptococcus neoformans and Malassezia pachydermatis, respectively.

PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.