Drug Resistance Patterns among Hospitalized Tuberculous Patients in Rio de Janeiro, Brazil, 1993-1994

The purpose of this study was to analyze the prevalence and risk factors for drug resistance among hospitalized patients in two tertiary care centers, an acquired immunodeficiency syndrome (AIDS) reference center and a sanatorium, in Rio de Janeiro, Brazil. From 1993-1994, 389 patients were diagnosed as having tuberculosis (TB). Isolates from 265 patients were tested for in vitro susceptibility to rifampin and isoniazid. Resistance to one or more drugs was detected in 44 patients (16.6%) and was significantly more common among recurrent cases in both hospitals (p=0.03 in the AIDS center and p=0.001 in the sanatorium). Twenty seven patients (10.2%) had isolates resistant to both isoniazid and rifampin. Multi-drug resistance was associated with human immunodeficiency virus (HIV) infection among patients who had never been treated for TB. In conclusion, drug-resistant TB is high in hospitalized patients in Rio de Janeiro, especially among HIV infected patients. Therefore, measures to control TB and prevent nosocomial transmission need urgently to be set up in the Brazilian hospitals.

Hepatitis C virus prevalence among an immigrant community to the Southern Amazon, Brazil

A community-based random survey was conducted in a southern Brazilian Amazonian county aiming to investigate hepatitis C virus (HCV) infection prevalence and the association of demographic variables and lifestyle behaviours. Seven hundred eighty individuals were serologically screened with a third generation enzyme-linked immunosorbent assay to detect anti-HCV antibodies between 1994/1995. Positive samples were retested for confirmation with a line immunoassay (LIA, Inno-LIA HCV Ab III). Most of these subjects were low income and came from southern Brazilian states (65.8). Two point four percent (IC 95% 1.2%- 4.6%) of the subjects had LIA-confirmed anti-HCV antibodies reactivity. The age-specific prevalence of HCV antibodies slightly increased with age, with the highest prevalence after the age of 40 years. The results of multivariate analysis indicate a strong association between HCV antibodies and previous surgery and history of intravenous drug use. There were no apparent association with gender, hepatitis B virus markers, blood transfusion, and sexual activity. Mean time living in Amazon did not differ between confirmed and negative anti-HCV individuals. The present data point out an intermediate endemicity of HCV infection among this immigrant community to the Amazon region and that few HCV infected participants presented known risk factors.

Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method

From March 1996 to August 1997, a study was carried out in a malaria endemic area of the Brazilian Amazon region. In vivo sensitivity evaluation to antimalarial drugs was performed in 129 patients. Blood samples (0.5 ml) were drawn from each patient and cryopreserved to proceed to in vitro studies. In vitro sensitivity evaluation performed using a radioisotope method was carried out with the cryopreserved samples from September to December 1997. Thirty-one samples were tested for chloroquine, mefloquine, halofantrine, quinine, arteether and atovaquone. Resistance was evidenced in 96.6% (29/30) of the samples tested for chloroquine, 3.3% (1/30) for quinine, none (0/30) for mefloquine and none for halofantrine (0/30). Overall low sensitivity was evidenced in 10% of the samples tested for quinine, 22.5% tested for halofantrine and in 20% tested for mefloquine. Means of IC 50 values were 132.2 (SD: 46.5) ng/ml for chloroquine, 130.6 (SD: 49.6) ng/ml for quinine, 3.4 (SD: 1.3) ng/ml for mefloquine, 0.7 (SD: 0.3) ng/ml for halofantrine, 1 (SD: 0.6) ng/ml for arteether and 0.4 (SD: 0.2) ng/ml for atovaquone. Means of chloroquine IC 50 of the tested samples were comparable to that of the chloroquine-resistant strain W2 (137.57 ng/ml) and nearly nine times higher than that of the chloroquine-sensitive strain D6 (15.09 ng/ml). Means of quinine IC 50 of the tested samples were 1.7 times higher than that of the low sensitivity strain W2 (74.84 ng/ml) and nearly five times higher than that of the quinine-sensitive strain D6 (27.53 ng/ml). These results disclose in vitro high resistance levels to chloroquine, low sensitivity to quinine and evidence of decreasing sensitivity to mefloquine and halofantrine in the area under evaluation.

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuber-culosis infection.

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott®), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95%) sera from patients in the study group and negative in 38 of the 39 (97%) sera from those in the control group, showing an accuracy of 96%. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay.

Population genetic analysis of Colombian Trypanosoma cruzi isolates revealed by enzyme electrophoretic profiles

Although Colombia presents an enormous biological diversity, few studies have been conducted on the population genetics of Trypanosoma cruzi. This study was carried out with 23 Colombian stocks of this protozoa analyzed for 13 isoenzymatic loci. The Hardy-Weinberg equilibrium, the genetic diversity and heterogeneity, the genetic relationships and the possible spatial structure of these 23 Colombian stocks of T. cruzi were estimated. The majority of results obtained are in agreement with a clonal population structure. Nevertheless, two aspects expected in a clonal structure were not discovered in the Colombian T. cruzi stocks. There was an absence of given zymodemes over-represented from a geographical point of view and the presumed temporal stabilizing selective phenomena was not observed either in the Colombian stocks sampled several times through the years of the study. Some hypotheses are discussed in order to explain the results found.

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.

Enzyme-linked immunosorbent assay for detection and quantification of Cryptococcus neoformans antigen

An enzyme-linked immunosorbent assay was standardized for the detection of cryptococcal antigen in serum and cerebrospinal fluid. The system was evaluated in clinical samples from patients infected by human immunodeficiency virus with and without previous cryptococcosis diagnosis. The evaluated system is highly sensitive and specific, and when it was compared with latex agglutination there were not significant differences. A standard curve with purified Cryptococcus neoformans antigen was settled down for the antigen quantification in positive samples.

Anesthesia of Biomphalaria spp. (Mollusca, Gastropoda): sodium pentobarbital is the drug of choice

The anesthetic effect of some water-soluble anesthesic or narcotic drugs currently used in mice was tested in molluscs of the Biomphalaria genus. Sodium thiopental was very toxic to the snails resulting in high rates of mortality in all the treatment schedules tested. Cetamine base, at concentration of 0.25 mg/ml of water, resulted in partial snail anesthesia (40% of snails were anesthetized) only after 20 h of exposition. The association of Cetamine base with Tiazine chloridrate did not improve the anesthesic effect, and higher concentrations of these drugs were toxic to the snails. Sodium pentobarbital at 0.4 mg/ml in water for 8 h was the best treatment schedule to anesthetize Biomphalaria snails. In this schedule, the snails were anesthetized without any toxic effect. The procedure provides a powerful tool for in vivo studies that demande a complete state of snail anesthesia.

Genome comparison of progressively drug resistant Plasmodium falciparum lines derived from drug sensitive clone

Chloroquine has been the mainstay of malaria chemotherapy for the past five decades, but resistance is now widespread. Pyrimethamine or proguanil form an important component of some alternate drug combinations being used for treatment of uncomplicated Plasmodium falciparum infections in areas of chloroquine resistance. Both pyrimethamine and proguanil are dihydrofolate reductase (DHFR) inhibitors, the proguanil acting primarily through its major metabolite cycloguanil. Resistance to these drugs arises due to specific point mutations in the dhfr gene. Cross resistance between cycloguanil and pyrimethamine is not absolute. It is, therefore, important to investigate mutation rates in P. falciparum for pyrimethamine and proguanil so that DHFR inhibitor with less mutation rate is favored in drug combinations. Hence, we have compared mutation rates in P. falciparum genome for pyrimethamine and cycloguanil. Using erythrocytic stages of P. falciparum cultures, progressively drug resistant lines were selected in vitro and comparing their RFLP profile with a repeat sequence. Our finding suggests that pyrimethamine has higher mutation rate compared to cycloguanil. It enhances the degree of genomic polymorphism leading to diversity of natural parasite population which in turn is predisposes the parasites for faster selection of resistance to some other antimalarial drugs.

Detection of the acute phase of abdominal angiostrongyliasis with a parasite-specific IgG enzyme linked immunosorbent assay

Angiostrongylus costaricensis may cause intestinal lesions of varied severity when it accidentally infects man in Central and South America. First-stage larvae have never been detected in stools. Therefore, a parasite-specific IgG ELISA was evaluated for the determination of the acute phase of infection. The specificity and the sensitivity of the immunoassay was shown to be 76.2% and 91.1%, respectively. Eight serum samples taken from patients with histopathological diagnosis, at different time points (3 to 15 months) after surgical treatment, showed a sharp and early decline in antibody reactivity. The titration of anti-A. costaricensis antibodies has proved to be a useful method for the diagnosis of acute abdominal angiostrongyliasis.