Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

EVALUATION OF A RAPID SCREENING ASSAY FOR BACTERIAL IDENTIFICATION (DOT-ELISA) IN FECAL SAMPLES FROM CHILDREN

With the objective of standardizing a Dot Enzyme-Linked Immunosorbent Assay (Dot-ELISA) to detect antigens of fecal bacterial enteropathogens, 250 children, aged under 36 months and of both sexes, were studied; of which 162 had acute gastroenteritis. The efficacy of a rapid screening assay for bacterial enteropathogens (enteropathogenic Escherichia coli "EPEC", enteroinvasive Escherichia coli "EIEC", Salmonella spp. and Shigella spp.) was evaluated. The fecal samples were also submitted to a traditional method of stool culture for comparison. The concordance index between the two techniques, calculated using the Kappa (k) index for the above mentioned bacterial strains was 0.8859, 0.9055, 0.7932 and 0.7829 respectively. These values express an almost perfect degree of concordance for the first two and substantial concordance for the latter two, thus enabling this technique to be applied in the early diagnosis of diarrhea in infants. With a view to increasing the sensitivity and specificity of this immunological test, a study was made of the antigenic preparations obtained from two types of treatment: 1) deproteinization by heating; 2) precipitation and concentration of the lipopolysaccharide antigen (LPS) using an ethanol-acetone solution, which was then heated in the presence of sodium EDTA

QUESTIONNAIRES IN THE SCREENING FOR Schistosoma mansoni INFECTION: A STUDY OF SOCIO DEMOGRAPHIC AND WATER CONTACT VARIABLES IN FOUR COMMUNITIES IN BRAZIL

The use of questionnaires has been recommended for identifying, at a lower cost, individuals at risk for schistosomiasis. In this study, validity of information obtained by questionnaire in the screening for Schistosoma mansoni infection was assessed in four communities in the State of Minas Gerais, Brazil. Explanatory variables were water contact activities, sociodemographic characteristics and previous treatment for schistosomiasis. From 677, 1474, 766 and 3290 individuals eligible for stool examination in the communities, 89 to 97% participated in the study. The estimated probability of individuals to be infected, if they have all characteristics identified as independently associated with S.mansoni infection, varied from 15% in Canabrava, to 42% in Belo Horizonte, 48% in Comercinho and 80% in São José do Acácio. Our results do not support the hypothesis that a same questionnaire on risk factors could be used in screening for S.mansoni infection in different communities.

FATAL GROUP A STREPTOCOCCAL TOXIC SHOCK-LIKE SYNDROME IN A CHILD WITH VARICELLA: REPORT OF THE FIRST WELL DOCUMENTED CASE WITH DETECTION OF THE GENETIC SEQUENCES THAT CODE FOR EXOTOXINS SPE A AND B, IN SÃO PAULO, BRAZIL

A previously healthy seven-year-old boy was admitted to the intensive care unit because of toxaemia associated with varicella. He rapidly developed shock and multisystem organ failure associated with the appearance of a deep-seated soft tissue infection and, despite aggressive treatment, died on hospital day 4. An M-non-typable, spe A and spe B positive Group A Streptococcus was cultured from a deep soft tissue aspirate. The criteria for defining Streptococcal toxic shock-like syndrome were fulfilled. The authors discuss the clinical and pathophysiological aspects of this disease as well as some unusual clinical findings related to this case.

Genetic variation between susceptible and non-susceptible snails to Schistosoma infection using random amplified polymorphic DNA analysis (RAPDs)

Susceptibility of snails to infection by certain trematodes and their suitability as hosts for continued development has been a bewildering problem in host-parasite relationships. The present work emphasizes our interest in snail genetics to determine what genes or gene products are specifically responsible for susceptibility of snails to infection. High molecular weight DNA was extracted from both susceptible and non-susceptible snails within the same species Biomphalaria tenagophila. RAPD was undertaken to distinguish between the two types of snails. Random primers (10 mers) were used to amplify the extracted DNA by the polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis (PAGE) and silver staining. The results suggest that RAPD represents an efficient means of genome comparison, since many molecular markers were detected as genetic variations between susceptible and non-susceptible snails.

Genetic relationships among serogroup B: serotype 4 Neisseria meningitidis strains

We compared the results obtained by serotyping of PorB epitopes using an expanded panel of monoclonal antibodies (mAb) including mAb 7 and mAb 10, with results obtained by RFLP of rRNA genes (ribotyping). The purpose of this study was to assess the correlation between phenotypic- and genotypic- methods for typing N. meningitidis. The ribotypes obtained using ClaI or EcoRV endonucleases grouped the strains in seven and two different patterns, respectively. This additional characterization of PorB epitopes improved the correlation between these two methods of typing N. meningitidis.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Analysis of genetic relatedness between populations of Aedes aegypti from different geographic regions of São Paulo state, Brazil

RAPD markers have been used for the analysis of genetic differentiation of Aedes aegypti, because they allow the study of genetic relationships among populations. The aim of this study was to identify populations in different geographic regions of the São Paulo State in order to understand the infestation pattern of A. aegypti. The dendrogram constructed with the combined data set of the RAPD patterns showed that the mosquitoes were segregated into two major clusters. Mosquitoes from the Western region of the São Paulo State constituted one cluster and the other was composed of mosquitoes from a laboratory strain and from a coastal city, where the largest Latin American port is located. These data are in agreement with the report on the infestation in the São Paulo State. The genetic proximity was greater between mosquitoes whose geographic origin was closer. However, mosquitoes from the coastal city were genetically closer to laboratory-reared mosquitoes than to field-collected mosquitoes from the São Paulo State. The origin of the infestation in this place remains unclear, but certainly it is related to mosquitoes of origins different from those that infested the West and North region of the State in the 80's.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.

Screening for pulmonary tuberculosis in Teresópolis, RJ, Brazil: the search for respiratory symptomatic patients in emergency service of "Hospital das Clínicas de Teresópolis Costantino Ottaviano, Fundação Educacional Serra dos Órgãos"

The aim of the present study was to investigate the detection percentage of tuberculosis among patients that are respiratory symptomatic (TB suspects). In this work, we present the preliminary results of research carried out at "Hospital das Clínicas de Teresópolis Costantino Ottaviano da Fundação Educacional Serra dos Órgãos (FESO)" from November 2003 to April 2004. Among the 40 respiratory symptomatic individuals identified and referred to the Tuberculosis Control Program in Teresópolis, two (5.0%) were characterized as smear-positive. These results confirm reports in the literature and underscore the need for and importance of this strategy.

Analysis of the genetic polymorphism of Paracoccidioides brasiliensis and Paracoccidioides cerebriformis "Moore" by random amplified polymorphic DNA (RAPD) and 28S ribosomal DNA sequencing: Paracoccidioides cerebriformis revisited

Our purpose was to compare the genetic polymorphism of six samples of P. brasiliensis (113, 339, BAT, T1F1, T3B6, T5LN1), with four samples of P. cerebriformis (735, 741, 750, 361) from the Mycological Laboratory of the Instituto de Medicina Tropical de São Paulo, using Random Amplified Polymorphic DNA Analysis (RAPD). RAPD profiles clearly segregated P. brasiliensis and P. cerebriformis isolates. However, the variation on band patterns among P. cerebriformis isolates was high. Sequencing of the 28S rDNA gene showed nucleotide conservancy among P. cerebriformis isolates, providing basis for taxonomical grouping, and disclosing high divergence to P. brasiliensis supporting that they are in fact two distinct species. Moreover, DNA sequence suggests that P. cerebriformis belongs in fact to the Aspergillus genus.

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Ten isolates of Paracoccidioides brasiliensis were examined for differences in virulence in outbred mice intravenously inoculated with the fungus, associated with mycelial morphology, and genetic patterns measured by random amplified polymorphic DNA (RAPD). Virulence was evaluated by viable yeast cell recovery from lungs and demonstration of histopathologic lesions in different organs. The results showed that the isolates presented four virulence degrees: high virulence, intermediate, low and non-virulence. RAPD clustered the isolates studied in two main groups with 56% of genetic similarity. Strains with low virulence, Pb265 or the non-virulent, Pb192, showed glabrous/cerebriform morphology and high genetic similarity (98.7%) when compared to the other isolates studied. The same was observed with Bt79 and Bt83 that shared 96% genetic similarity, cottony colonies and high virulence. The RAPD technique could only discriminate P. brasiliensis isolates according to glabrous/cerebriform or cottony colonies, and also high from low virulence strains. Isolates with intermediate virulence such as Pb18, Pb18B6, Bt32 and Bt56 showed variability in their similarity coefficient suggesting that RAPD was able to detect genetic variability in this fungal specie. Virulence profile of P. brasiliensis demonstrated that both mycelial morphologic extreme phenotypes may be associated with fungal virulence and their in vitro subculture time. Thus, RAPD technique analysis employed in association with virulence, morphologic and immunologic aspects might prove adequate to detect differences between P. brasiliensis isolates.

Optimization of random amplified polymorphic DNA techniques for use in genetic studies of Cuban triatominae

Random amplified polymorphic DNA (RAPD) technique is a simple and reliable method to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of assay. In this study, we analyzed the effect of changing the concentration of primer, magnesium chloride, template DNA and Taq DNA polymerase with the objective of determining their optimum concentration for the standardization of RAPD technique for genetic studies of Cuban Triatominae. Reproducible amplification patterns were obtained using 5 pmoL of primer, 2.5 mM of MgCl2, 25 ng of template DNA and 2 U of Taq DNA polymerase in 25 µL of the reaction. A panel of five random primers was used to evaluate the genetic variability of T. flavida. Three of these (OPA-1, OPA-2 and OPA-4) generated reproducible and distinguishable fingerprinting patterns of Triatominae. Numerical analysis of 52 RAPD amplified bands generated for all five primers was carried out with unweighted pair group method analysis (UPGMA). Jaccard's Similarity Coefficient data were used to construct a dendrogram. Two groups could be distinguished by RAPD data and these groups coincided with geographic origin, i.e. the populations captured in areas from east and west of Guanahacabibes, Pinar del Río. T. flavida present low interpopulation variability that could result in greater susceptibility to pesticides in control programs. The RAPD protocol and the selected primers are useful for molecular characterization of Cuban Triatominae.

Genetic diversity and antimicrobial resistance of enterococcal isolates from Southern region of Brazil

In the present study, a total of 455 enterococcal isolates, recovered from patients living in the city of Porto Alegre, State of Rio Grande do Sul, Brazil, during the period from July 1996 to June 1997, were identified to the species level by conventional biochemical and microbiological tests, and assayed for their susceptibilities to antimicrobial agents. The genetic diversity of antimicrobial resistant strains was evaluated by pulsed-field gel electrophoresis (PFGE) analysis of SmaI restricted chromosomal DNA. The most frequent species was Enterococcus faecalis (92.8%). Other species identified were: E. faecium (2.9%), E. gallinarum (1.5%), E. avium (1.1%), E. hirae (0.7%), E. casseliflavus (0.4%), E. durans (0.4%) and E. raffinosus (0.2%). The overall prevalence of isolates with high-level resistance (HLR) to aminoglycosides was 37.8%. HLR to gentamicin was found in 24.8%. No strains with acquired resistance to vancomycin were found. PFGE analysis showed the predominance of clonal group A, comprising strains isolated from different clinical specimens obtained from patients in three hospitals. These results suggest intra and inter-hospital dissemination of one predominant clonal group of E. faecalis isolates with HLR to gentamicin in the hospitals included in this study.