Lethal effect of a bait for Rhodnius prolixus (Hemiptera: Reduviidae), the vector of Chagas' disease, containing hexachlorocyclohexane (HCH), under laboratory conditions

The lethal effect of a bait containing an aqueous hexachlorocyclohexane (HCH) suspension at the concentration of 1g/l and maintained at room temperature was studied in the laboratory over a period of 12 weeks. The suspension was placed in a latex bag hanging inside a 1000-ml beaker tightly covered with nylon netting, and left there with no changes for 85 days. Sixteen groups of R. prolixas bugs, consisting on average of 30 specimens each, were successively exposed to the bait and observed at different intervals for one week each. The mortality rate was 100% for all groups, except for the 16th, whose mortality rate was 96.7%. As the groups succeeded one another, mortality started to occur more rapidly and was more marked at the 6- and 24-h intervals. Later tests respectively started at 6:00 a.m. and 6:00 p.m. showed that diurnal and nocturnal periodicity in the offer of food had no effect on mortality. First- and 2nd- instar nymphs and adults male were more sensitive and 5th- instar nymphs were more resistant to the active principle of the bait.

Distribucion geografica de Lutzomyia verrucarum (Townsend, 1913) (Diptera, Psychodidae, Phlebotominae), vector de la batonellosis humana en el Peru

Lutzomyia verrucarum (Townsend, 1913) (Diptera: Psychodidae), vector natural de la verruga peruana o enfermedad de Carrión es una especie propia del Perú. Su distribución geográfica esta entre los paralelos 5º y 13º25' de latitud Sur, se encuentra en los valles Occidentales e Interandinos de los Andes. La distribución altitudinal de Lu. verrucarum en los diversos valles es variable; asi: Occidentales, desde 1100 hasta 2980 msnm e Interandinos, de 1200 a 3200 msnm. En ciertas áreas verrucógenas no hay correlación entre la presencia de Lu. verrucarum y la enfermedad de Carrión lo que suguiere la existencia de vectores secundarios.

Enzyme linked immunosorbent assay for rubella antibodies: a simple method of antigen production. A preliminary report

A simple method of rubella antigen production by treatment with sodium desoxycholate for use in enzyme immunoassay (IMT-ELISA) is presented. When this assay was compared with a commercial test (Enzygnost-Rubella, Behring), in the study of 108 sera and 118 filter paper blood samples, 96.9% (219/226) overall agreement and correlation coefficient of 0.90 between absorbances were observed. Seven samples showed discordant results, negative by the commercial kit and positive by our test. Four of those 7 samples were available, being 3 positive by HI.

A simple and inexprensive method to generate a microaerophilic atmosphere for the isolation of Campylobacter sp

Faeces of 138 chickens were inoculated on Blaser agar plates. One set of plates was incubated in jars with CampyPak envelopes. The others were incubated in "Zip-lock" plastic bags (7 x X in.) and a microaerophilic atmosphere was generated exhaling into the "Zip-lock" plastic bag, after holding the breath for 20 sec. Then, the bag was pressed to evacuate its atmosphere, inflated again, and pressed (4 times), and finally sealed. Campylobacter was isolated from 127 (96.2%) of samples incubated in jars with gas generator envelopes and from 129 (98%) of the specimens incubated into the bags. The proposed methodology offers good savings for cost-conscious laboratories.

Studies on prevalence of Strongyloides infection in Holambra and Maceió, Brazil, by the agar plate faecal culture method

Prevalence of Strongyloides stercoralis infection in three areas of Brazil was surveyed by a recently developed faecal culture method (an agar plate culture). The Strongyloides infection was confirmed in 11.3% of 432 subjects examined. The diagnostic efficacy of the agar plate culture was as high as 93.9% compared to only 28.5% and 26.5% by the Harada-Mori filter paper culture and faecal concentration methods, when faecal samples were examined simultaneously by these three methods. Among the 49 positive samples, about 60% were confirmed to be positive only by the agar plate culture. These results indicate that the agar plate culture is a sensitive new tool for the correct diagnosis of chronic Strongyloides infection.

A single method to stain Malassezia furfur and Corynebacterium minutissimum in scales

A single and practical method to slain Malassezia furfur and Corynebacterium minutissimum in lesions' scales is described. The scales are collected by pressing small pieces of scotch tape (about 4 cm lenght and 2 cm width) onto the lesions and following withdrawl the furfuraceous scales will remain on the glue side. These pieces are then immersed for some minutes in lactophenol-cotton blue stain. Following absorption of the stain the scales are washed in current water to remove the excess of blue stain, dried with filter paper, dehydrated via passage in two bottles containing absolute alcohol and then placed in xylene in a centrifugation tube. The xylene dissolves the scotch tape glue and the scales fall free in the tube. After centrifugation and decantation the scales concentrated on the bottom of the tube are collected with a platinum-loop, placed in Canada balsam on a microscopy slide and closed with a cover slip. The preparations are then ready to be submitted to microscopic examination. Other stains may also be used instead of lactophenol-cotton blue. This method is simple, easily performed, and offers good conditions to study these fungi as well as being useful for the diagnosis of the diseases that they cause.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

Evaluation of Etest and macrodilution broth method for antifungal susceptibility testing of Candida sp strains isolated from oral cavities of AIDS patients

A comparison of the Etest and the reference broth macrodilution susceptibility test for fluconazole, ketoconazole, itraconazole and amphotericin B was performed with 59 of Candida species isolated from the oral cavities of AIDS patients. The Etest method was performed according to the manufacturer's instructions, and the reference method was performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines. Our data showed that there was a good correlation between the MICs obtained by the Etest and broth dilution methods. When only the MIC results at ± 2 dilutions for both methods were considered, the agreement rates were 90.4% for itraconazole, ketoconazole and amphotericin B and 84.6% for fluconazole of the C. albicans tested. In contrast, to the reference method, the Etest method classified as susceptible three fluconazole-resistant isolates and one itraconazole-resistant isolate, representing four very major errors. These results indicate that Etest could be considered useful for antifungal sensitivity evaluation of yeasts in clinical laboratories.

Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

Neurocryptococcosis: diagnosis by PCR method

Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

Eco-epidemiological aspects of Trypanosoma cruzi, Trypanosoma rangeli and their vector (Rhodnius pallescens) in Panama

The eco-epidemiology of T. cruzi infection was investigated in the Eastern border of the Panama Canal in Central Panama. Between 1999 and 2000, 1110 triatomines were collected: 1050 triatomines (94.6%) from palm trees, 27 (2.4%) from periurban habitats and 33 (3.0%) inside houses. All specimens were identified as R. pallescens. There was no evidence of vector domiciliation. Salivary glands from 380 R. pallescens revealed a trypanosome natural infection rate of 7.6%, while rectal ampoule content from 373 triatomines was 45%. Isoenzyme profiles on isolated trypanosomes demonstrated that 85.4% (n = 88) were T. cruzi and 14.6% (n = 15) were T. rangeli. Blood meal analysis from 829 R. pallescens demonstrated a zoophilic vector behavior, with opossums as the preferential blood source. Seroprevalence in human samples from both study sites was less than 2%. Our results demonstrate that T. cruzi survives in the area in balanced association with R. pallescens, and with several different species of mammals in their natural niches. However, the area is an imminent risk of infection for its population, consequently it is important to implement a community educational program regarding disease knowledge and control measures.

A Triatoma maculata (Hemiptera, Reduviidae, Triatominae) population from Roraima, Amazon region, Brazil, has some bionomic characteristics of a potential Chagas disease vector

Even though Chagas disease is rare in the Brazilian Amazon, the conditions for the establishment of domiciliated cycles prevail in many areas where triatomines are of frequent occurrence. In Roraima, a previous serological and entomological survey in three agricultural settlements showed the existence of all transmission cycle elements, i.e., individuals infected by Trypanosoma cruzi, triatomine species previously found harboring T. cruzi in the broader Amazon region of neighboring countries and, domicile/ peridomicile conditions favorable to triatomine colonization. Triatoma maculata was the most frequent species, found in chicken houses in the peridomicile and sporadically within residences. Aiming to investigate the possibility of T. maculata to possess the potentiality to transmit T. cruzi in the area, bionomic characteristics were studied under laboratory conditions. These were feeding frequency, time for defecation after a blood meal, time elapsed in voluntary fasting pre- and pos-ecdysis, moulting time periods, pre-oviposition and oviposition periods and index of oviposition, incubation period, egg viability, longevity and mortality rate. Results show that the Passarão population of T. maculata should be considered a potential vector of T. cruzi since it shows a capacity to infest artificial ecotopes in the peridomicile, to carry out large number of meals during the nymphal cycle, to have a relatively short developmental cycle capable of producing 2.9 generations/year, to blood source eclecticism, to defecate immediately after the blood meal while still on the host and to the fact that has been previously found naturally infected by T.cruzi.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.