Use of a selective medium with potassium tellurite to follow intestinal colonization of hospitalized patients by drug-resistant Enterobacteriaceae

Nosocomial infections are a relevant factor in complicating the recovery of patients interned for even minor causes. In attempt to determine their origin it is crucial to consider that their origin is of an endogenous nature. Looking for an acessible expression of intestinal colonization we analyzed fecal samples from 3 separate groups of hospital patients collected after different lenghts of time. For practical reasons one group was studied prospectively and two other groups (patients hospitalized for up to 7 days and patients hospitalized for more than 7 days) were compared to one another. We looked for the emergence of tellurite resistance among Enterobacteriaceae using a selective medium. MacConkey potassium tellurite (MCPT). The frequence of prospectively studied patients with tellurite resistant strains was significantly greater after 7 days of hospitalization. For the two other groups, patients with more than 7 days of hospitalization showed a significant increase of bacterial species and of strains with new antimicrobial resistance markers. High molecular weigth plasmids were detected in some of these strains. These data show that the MCPT medium is a useful tool for the investigation of bowel colonization in hospitalized patients by drug-resistant Enterobacteriaceae.

Use of PCR for the determination of the frequency of the deltaF508 mutation in Brasilian cistic fibrosis patients

The [Delta]F508 mutation in the cystic fibrosis (CF) gene was studied in a population of 18 Brazilian CF patients and their 17 families by use of PCR and differential hybridization with oligonucleotides. In a total of 34 chromosomes considered, 12 (35%) carried the F508 deletion, a frequency much lower than that reported in most other populations. As a consequence, CF in Brazil would be predominantly caused by mutations different from the F508 deletion

The use of ferromagnetic dacron as solid-phase in enzyme immunoassays

Ferromagnetic dacron is proposed as an alternative solid-phase for magnetic enzyme immunoassays. Human serum albumin (HSA) was covalentlyimmobilized onto ferromagnetic dacron and as enzyme immunoassay was developed using anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used as the enzymatic label and substrates, respectively. Best results were observed when particles of 63-100 µm (diameter) and 10 µg of immobilized antigen were used. Positive reactions were detected until dilutions of1:51200 of immune sera. Its reproducibility was similar to standard ELISA. Disruption of the immunocomplexes formed and recuperation of the immobilized antigen in other immunoassays also proved to be reliable.

Use of molecular probes and PCR for detection and typing of Leishmania - a mini-review

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specifics probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

Use of isozyme patterns in the identification of Biomphalaria tenagophila (D'Orbigny, 1835) and B. occidentalis (Paraense, 1981) (Gastropoda: Planorbidae)

Two sibling species of Biomphalaria, B. tenagophila and B. occidentalis were identified using isozyme patterns obtained by horizontal gel electrophoresis. Six diagnostic enzymatic loci were identified in digestive gland homogenates. The results enable us to distinguish the species, calculate the Nei's coefficient of genetic similarity, and provide a basis for making inferences about the pattern of these two planorbid species colonization and distribution.

The use and mis-use of some ecological terms and concepts in epidemiology

The article discusses the current use and mis-use of ecological terms and concepts in epidemiological literature, and in special, in works dealing with zoonotic diseases. A selection of examples was taken from papers recently published on the transmission of Chagas' disease by Triatoma sordida. Proper definitions are listed, with the intent of helping non-ecologists to use those terms and concepts correctly.

Use of the 2,3-diacyl-trehalose and the purified protein derivative in the serodiagnosis of tuberculosis in AIDS

The effect of the human immunodeficiency virus (HIV) infection on IgG production against purified protein derivative (PPD) and 2,3-diacil-trehalose (SL-IV) was investigated by an enzyme-linked immunosorbent assay (ELISA) test. Comparison between the antigens showed that immunocompetent patients produce preferentially antibodies to SL-IV than to PPD (73.3% versus 63.3%). Combination of these results showed an increase of the sensitivity to 80%, which decreased over the spectrum of immunodepression caused by HIV. In the tuberculous HIV seropositive group the sensitivities of SL-IV and PPD were 36.4% versus 40% and 0% versus 22.2% in the tuberculosis/acquired immunodeficiency syndrome (TB/AIDS) group. Combination of these results gave respectively 54.5% and 20%, showing that serological tests have limited value for diagnosis of tuberculosis in HIV infected patients. High antibody levels were observed in HIV seropositive asymptomatic group, but only two individuals were positive for both antigens. In the follow up, one of them developed tuberculous lymphadenitis, indicating that further work is needed to access the value of serological tests in predicting tuberculosis in HIV infected individuals.

The use of polyvinyl alcohol glutaraldehyde as solid-phase in ELISA for plague

Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 ¼g of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.

Use of Monoclonal Antibodies for the Identification of Leishmania spp. Isolated from Humans and Wild Rodents in the State of Campeche, Mexico

The genus Leishmania includes 30 described species which infect a wide variety of mammalian hosts. The precise identification of leishmanial parasites at the species level is very important in order to determine whether an organism, causing the disease in a given area, is of the same biotype as that found in suspected mammalian reservoirs. The objectives of the present study were (1) to identify leishmanial parasites isolated from humans and wild rodents from the State of Campeche, an endemic focus of localized cutaneous leishmaniasis (LCL) in southern Mexico, using an indirect immunofluorescent assay (IFA) with monoclonal antibodies (Mabs); and (2) to determine if the parasites of the two types of hosts were of the same biotype. All the wild rodents (six Ototylomys phyllotis, eight Oryzomys melanotis, five Peromyscus yucatanicus and two Sigmodon hispidus) and 96% (24/25) of the human isolates were identified as Leishmania (L.) mexicana confirming that this specific LCL focus is a wild zoonosis. The presence of one human isolate of L. (Viannia) braziliensis in the State of Campeche, confirmed the importance of an accurate taxonomic identification at species level.

The use of bacterial larvicides in mosquito and black fly control programmes in Brazil

Bacillus spp. based larvides are increasingly replacing, with numerous advantages, chemical insecticides in programmes for controlling black fly and mosquito populations. Brazil was among the pioneers in adopting Bacillus thuringiensis israelensis (B.t.i) to control black flies. However, the major current mosquito control programme in Brazil, the Programme for Eradication of Aedes aegypti launched in 1997, only recently decided to replace temephos by B.t.i based larvicides, in the State of Rio de Janeiro. In the last decade, works developed by research groups in Brazilian institutions have generated a significant contribution to this subject through the isolation of B. sphaericus new strains, the development of new products and the implementation of field trials of Bacillus efficacy against mosquito species under different environmental conditions.

The early use of yellow fever virus strain 17D for vaccine production in Brazil - a review

The use of yellow fever (YF) virus 17D strain for vaccine production adapted in Brazil since its introduction in 1937 was reviewed. This was possible due to the availability of official records of vaccine production. The retrieved data highlight the simultaneous use of several serially passaged 17D substrain viruses for both inocula and vaccine preparation that allowed uninterrupted production. Substitution of these substrain viruses became possible with the experience gained during quality control and human vaccination. Post-vaccinal complications in humans and the failure of some viruses in quality control tests (neurovirulence for monkeys) indicated that variables needed to be reduced during vaccine production, leading to the development of the seed lot system. The 17DD substrain, still used today, was the most frequently used substrain and the most reliable in terms of safety and efficacy. For this reason, it is possible to derive an infectious cDNA clone of this substrain combined with production in cell culture that could be used to direct the expression of heterologous antigens and lead to the development of new live vaccines.

Use of polymerase chain reaction and enzymatic cleavage in the identification of Helicobacter spp. in gastric mucosa of human beings from North Paraná, Brazil

Helicobacter pylori is the most common gastric bacteria of human beings. Animal-borne helicobacter have been associated with gastritis, ulceration, and gastric mucosa-associated lymphoid-tissue lymphoma in people. We attempted to identify the species of Helicobacter spp. that infect human beings in north Paraná, Brazil. Samples of gastric mucosa from 38 dyspeptic patients were analyzed by optic microscopy on silver stained slides, polimerase chain reaction (PCR), and enzymatic cleavage. Genus and species-specific primers to H. pylori, H. heilmannii, H. felis, and consensual primers to H. bizzozeronii or H. salomonis were used. The PCR products were submitted to enzymatic cleavage by VspI (Helicobacter spp. product) and HinfI (species products) enzymes. Thirty-two out of 38 patients evaluated had 3.2 to 5 µm long bacteria that resembled H. pylori in Warthin-Starry stained slides and were positive to the genus Helicobacter by PCR. In 30 of these patients the bacteria were identified as H. pylori. Two samples positive by silver stain were negative to all species tested by PCR. None of the 38 samples was positive to animal-origin helicobacter species. These results show that PCR and enzymatic restriction are practical methods to identify the species of helicobacters present in gastric mucosa of human beings. People in north Paraná appear to be infected mostly with H. pylori.

Use of molecular methods in identification of Candida Species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.