Radiometric detection of metabolic activity of Paracoccidioides brasiliensis and its susceptibility to amphotericin B and diethylstilbestrol

Paracoccidioidomycosis (South American blastomycosis) is a systemic disease, strikingly more frequent in males, caused by the dimorphic fungus Paracoccidioides brasiliensis. A radiometric assay system has been applied to study the metabolic activity and the effect of drugs on this fungus "in vitro". The Y form of the yeast, grown in liquid Sabouraud medium was inoculated into sterile reaction vials containing the 6B aerobic medium along with 2.0 μCi of 14C-substrates. Control vials, prepared in the same way, contained autoclaved fungi. To study the effects of amphotericin B (AB) (0.1 and 10 μg/ml) and diethylstilbestrol (DSB) (1.0, 5.0 and 10 μg/ml) extra controls with live fungi and no drug were used. All vials were incubated at 35°C and metabolism measured daily with a Bactec instrument. 14CO2 production by P. brasiliensis was slow and could be followed for as long as 50 days. AB at 10mg/ml and DSB at 5 μg/ml inhibited the metabolism and had a cidal effect on this fungus. The results with DSB might explain the low incidence of the disease in females. This technique shows promise for studying metabolic pathways, investi gating more convenient 14C-substrates to expedite radiometric detection and for monitoring the effects of other drugs and factors on the metabolism of P. brasiliensis "in vitro".

Blood groups and malaria

The possible relationship between erythrocyte antigens and the presence of malaria infection by P. vivax and P. falciparurn was sought in four different ethnic groups of two departments of Colombia. Malaria infection by P. falciparum was found in 91.4% of malaria infected blacks. No significant differences were found between the presence of malaria infection and ABO antigens. In the other blood groups, it was observed that groups MNSs conferred black people a greater Rr for malaria by both species of Plasmodium and that Duffy-negative blacks and indians appeared to be resistant to P. vivax infection. A predominance of P. vivax infection was observed in Katio indians while P.falciparum was predominant in Kuna indians; the reason for this finding still needs to be explored.

Paracoccidioides brasilienses isolates obtained from patients with acute and chronic disease exhibit morphological differences after animal passage

The basis for virulence in Paracoccidioides brasiliensis is not completely understood. There is a consensus that the sequencial in vitro subcultivation of P. brasiliensis leads to loss of its pathogenicity, which can be reverted by reisolation from animal passage. Attention to morphological and biochemical properties that are regained or demonstrated after animal passage may provide new insights into factors related to the pathogenicity and virulence of P. brasiliensis. We evaluated morphological characters: the percentage of budding cells, number of buds by cell and the diameter of 100 mother cells of yeast-like cells of 30 P. brasiliensis isolates, before and after animal passage. The isolates were obtained from patients with different clinical forms of paracoccidioidomycosis (PCM): acute form (group A, n=15) and chronic form (group C, n=15). The measurement of the yeast cell sizes was carried out with the aid of an Olympus CBB microscope coupled with a micrometer disc. We measured the major transverse and longitudinal axes of 100 viable cells of each preparation. The percentage of budding cells as also the number of buds by cell was not influenced by animal passage, regardless of the source of the strain (acute or chronic groups). The size values of P. brasiliensis isolates from groups A and C, measured before the animal passage exhibited the same behavior. After animal passage, there was a statistically significant difference between the cell sizes of P. brasiliensis isolates recovered from testicles inoculated with strains from groups A and C. The maximum diameter of mother cells from group A isolates exhibited a size of 42.1mm in contrast with 32.9mm exhibited by mother cells from group C (p<0.05). The diameter of 1500 mother cells from group A isolates exhibited a medium size of 16.0mm (SD ± 4.0), a value significantly higher than the 14.1mm (SD = ± 3.3) exhibited by 1500 mother cells from group C isolates (p<0.05). Our results reinforce the polymorphism exhibited by P. brasiliensis in biological material and the need for further investigations to elucidate the role of morphological parameters of the fungus in the natural history of the disease.

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.

High prevalence of malaria infection in Amazonas state, Venezuela

This study was carried out to determine the incidence of malaria in an endemic region of Amazonas State, Venezuela. For this, 200 random samples were collected from symptomatic and asymptomatic individuals from San Fernando de Atabapo and Santa Barbara. Epidemiological factors were related to malaria infection, which was diagnosed by microscopy observation and amplification of the 18S rDNA sequence by PCR. Malaria prevalence in these populations was 28.5%, whilst P. vivax and P. falciparum prevalences were 12 and 17%, respectively. No infection by P. malariae was found. A mixed infection was found on an asymptomatic individual. Prevalence patterns differed between age groups depending on the Plasmodium species. We found that 34.8% of the P. vivax and 15.2% of the P. falciparum infections were asymptomatic. The use of nets was helpful to prevent P. vivax infection, but did not protect against P. falciparum infection. The results suggest the presence of more than one mosquito vector in the area, displaying a differential pattern of infection for each Plasmodium species. There appear to be risk factors associated with malaria infections in some individuals. The population based approach and PCR diagnosis improved the accuracy of the statistical analysis in the study.

Trypanosoma cruzi in marsupial didelphids (Philander frenata and Didelhis marsupialis): differences in the humoral immune response in natural and experimental infections

Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence.

Plasmodium falciparum infection in pregnant women attending antenatal care in Luanda, Angola

INTRODUCTION: Malaria during pregnancy remains a serious public health problem. The aim of this study was to establish the prevalence and possible risk factors for malaria in pregnant women attending antenatal care at Augusto Ngangula Specialized General Hospital in Luanda, Angola. METHODS: Pregnant women (679 total) who attended antenatal care from April to September 2008 were included in the study after signing informed consent. For each participant, the social-demographic profile and malaria and obstetric histories were investigated via a questionnaire. Diagnosis was made by optic microscopy, and hemoglobin concentration measured. The associations between age, parity, gestational age, residence, schooling, malaria during gravity, anemia and treatment with incidence of Plasmodium falciparum infection were analyzed through logistic regression. RESULTS: During the period of study, 74 (10.9%) out of 679 women were infected by P. falciparum. The average concentration of hemoglobin was 11.1 ± 0.07g/dL, and there were significant associations between the history of malaria during pregnancy, P. falciparum infection (p<0.01) and anemia at the time of observation (p<0.001). CONCLUSIONS: Previous history of malaria during pregnancy represents a risk factor for current infection and anemia was an important complication associated with malaria, even in women who were treated with sulfadoxine-pyrimethamine during pregnancy.

Phenotypic and molecular characterization of antimicrobial resistance and virulence factors in Pseudomonas aeruginosa clinical isolates from Recife, State of Pernambuco, Brazil

INTRODUCTION: The emergence of carbapenem resistance mechanisms in Pseudomonas aeruginosa has been outstanding due to the wide spectrum of antimicrobial degradation of these bacteria, reducing of therapeutic options. METHODS: Sixty-one clinical strains of P. aeruginosa isolated from five public hospitals in Recife, Pernambuco, Brazil, were examined between 2006 and 2010, aiming of evaluating the profiles of virulence, resistance to antimicrobials, presence of metallo-β-lactamase (MBL) genes, and clonal relationship among isolates. RESULTS: A high percentage of virulence factors (34.4% mucoid colonies; 70.5% pyocyanin; 93.4% gelatinase positives; and 72.1% hemolysin positive) and a high percentage of antimicrobial resistance rates (4.9% pan-resistant and 54.1% multi-drug resistant isolates) were observed. Among the 29 isolates resistant to imipenem and/or ceftazidime, 44.8% (13/29) were MBL producers by phenotypic evaluation, and of these, 46.2% (6/13) were positive for the blaSPM-1 gene. The blaIMP and blaVIM genes were not detected. The molecular typing revealed 21 molecular profiles of which seven were detected in distinct hospitals and periods. Among the six positive blaSPM-1 isolates, three presented the same clonal profile and were from the same hospital, whereas the other three presented different clonal profiles. CONCLUSIONS: These results revealed that P. aeruginosa is able to accumulate different resistance and virulence factors, making the treatment of infections difficult. The identification of blaSPM-1 genes and the dissemination of clones in different hospitals, indicate the need for stricter application of infection control measures in hospitals in Recife, Brazil, aiming at reducing costs and damages caused by P. aeruginosa infections.

Trends in male contraception

Methods that are available for male contraception, namely coitus interruptus, condoms, and vasectomy, have been used since the 19th century. With the exceptions of a few improvements of these methods, no major progress has been made with respect to introducing new male contraceptives since then. It is extremely urgent to develop new, safe, effective, and reversible male contraceptive methods. Among all male contraceptive methods that are being investigated, the hormonal approach is the closest to clinical application. Hormonal contraception provides pregnancy protection by means of spermatogenic suppression. Androgen-progestin regimens currently represent the best available hormonal combination for induction of a profound suppression of spermatogenesis. Further development of new steroids is mandatory for increasing the choices of available contraceptive formulations and to optimize long-term safety of these regimens.

Geological characteristics of the nesting areas of the giant Amazon river turtle (Podocnemis expansa) in the Crixás-Açu river in Goiás State, Brazil

A large population of the giant Amazon river turtle (Podocnemis expansa) nests along the beaches of the Crixás-Açu River in the central western region of Brazil. In spite of the existence of several point bars in the area, only a selected group of beaches is used for nesting by P. expansa. Geological aspects, such as river width and depth, beach height above 200 cm with sandy sediments, were indispensable for the choice of these nesting sites. The relatively reduced dimensions of the point bars and the great number of turtles, which nest in the same local, contributed to the existence of a high nest concentration. The rapid rise of the Crixás-Açu River caused the flooding of the beaches and the drowning of hatchlings and embryos. It is estimated that nearly all the nests were lost. The height of the nesting place and the time of flooding related to the incubation period are decisive in embryo survivorship. The Retiro, Júnior, Assombrado and Limoeiro beaches, which are situated at heights of 183 to 310 cm, were inundated on 8 November 2000. The Barreira Branca beach, with a height of up to 380 cm was completely inundated on 13 December 2000. All of these beaches were flooded before the hatchlings emerged.

Cinética da reação de redução de nitrato por actinomicetos isolados do solo II - Influência dos elementos minerais

Some strains of Actinomycetes showed variation in its activity to reduce nitrate to nitrite when the nutrient solution was modified in relation to the major elements. One of these strains incapable of !this reaction showed a strong activity in nutrient solution without K and little activity without P. Otherwise strains capable of reducing nitrate to nitrite had this capacity decreased in absence of each one of the following elements: K, P, Mg, e S. K showed to be the most important of them, followed by P, Mg e S. Without any of these elements the pH of the nutrient solution has to be increased from 5,5 to 6,5 for the strains capable to reduce nitrate to nitrite preserve its capacity. The time for the reaction is increased from 5 to 8 days and the amount of nitrite obtained is small.

The influence of shell species and size on the shell selection pattern of Paguristes tortugae (Decapoda, Diogenidae) from Anchieta Island (Ubatuba, Brazil)

Shell selection by the hermit crab Paguristes tortugae Schmitt, 1933 from Anchieta Island (Brazil) was analyzed using the six most frequently occupied shell species in the field and taking into account the sexual condition of the individuals, the shell size and the shell species. The experiments were conducted under laboratory conditions and the shell species preference was estimated on the basis of the frequency that each species was chosen by the individuals. The preferred shell species and size were determined by regression analysis. The highest correlation coefficients were obtained for the relations between the hermit dimensions and shell dry weight. The ovigerous females preferred shells with larger internal volume: Leucozonia nassa (Gmelin, 1791) and Cerithium atratum (Born, 1778). In the experiment of shell size, males preferred heavier shells whereas females selected the shape characteristics of the shell, such as the aperture and the internal volume, which are probably related to the growth and offspring guarantee, respectively. In general, and independent of sex condition, P. tortugae showed significant selection among all shells utilized. The results suggest that shell selection by P. tortugae involves sexual and reproductive condition preferences.

Culex saltanensis Dyar, 1928: natural vector of Plasmodium juxtanucleare in Rio de Janeiro, Brazil

Searching for the natural vector of Plasmodium juxtanucleare in an enzootic locality: Granjas Calábria (33% of the chickens infected), Jacarepaguá, in Rio de Janeiro, Brazil, 13 comparative captures of mosquitoes were carried out, simultaneously on man (out-doors) and on chiken (in a poultry-yard), between 6 and 9 p.m., from September to March 1989. Culex saltanensis was the most frequent species in captures on chicken, accounting for 41.7% of the mosquitoes collected on this bait, showing to be highly ornithophilic (90% captured on chicken versus 10% on man). Seven specimens of Cx. saltanensis were found naturally infected in granjas Calábria: five with mature pedunculate oocysts and two with sporozoites (on in the haemocoele and one in the salivary glands). These sporozoites porudced an infection by P. juxtanucleare in a chick, which had parasitemia on day 41 after inoculation. One Cx. coronator was found with mature pedunculate oocysts. Culex saltanensis was regarded as primary vector of P. juxtanucleare in Rio de Janeiro for being highly ornithophilic and in enough density to maintain the transmission, having been found with infective sporozoites in its salivary glands, and being susceptible to the parasite and able to transmit experimentally it by the bite.

Attempts to Improve Xenodiagnosis: Comparative Test of Sensibility Using Rhodnius neglectus, Panstrongylus megistus, Triatoma vitticeps and Triatoma infestans in Endemic Areas of Brazil

From June 1984 to July 1992, 392 xenodiagnostic tests were applied on 264 patients with chronic Chagas disease from Brazilian endemic areas of Virgem da Lapa and Coronel Murta, situated in the Jequitinhonha Valley, in the State of Minas Gerais. The susceptibilities of Rhodnius neglectus, Panstrongylus megistus, Triatoma vitticeps and Triatoma infestans were compared. Most of the time 20 nymphs (fourth instar) of each species were applied to 161 women and 103 men aged between 5 and 83 years of age. The tests were prepared to compare the susceptibilities of two species at a time, using the same patients for each test. Results showed a xenopositiveness of 26.28% (103 tests) being 27.98% in women (68 positive in 243 applied tests) and 23.49% in men (35 positive in 149 applied tests). The relative frequency of xenopositiveness displayed a great superiority of P. megistus and T. vitticeps. In tests from type I, for example, P. megistus was the unique responsible for 10.73% of positive xenodiagnosis vs. only 0.98% in T. infestans. Other parameters analized in this work confirm this superiority, and corroborate that T. infestans can be replaced by P. megistus and /or T. vitticeps in order to upgrade the efficacy of xenodiagnosis

A dhfr-ts- Leishmania major Knockout Mutant Cross-protects against Leishmania amazonensis

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65% smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75% in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57% by SC and 49% by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization.