Nilma Cintra Leal

Acute attacks in the extremities of persons living in an area endemic for bancroftian filariasis: differentiation of two syndromes

The natural history of lymphatic disease in human filariasis remains unclear, but recurrent episodes of acute lymphangitis are believed to constitute a major risk factor for the development of chronic lymphoedema and elephantiasis. Prospective analysis of 600 patients referred to the filariasis clinic of the Centro de Pesquisas Aggeu Magalhães/FIOCRUZ in Recife, Brazil, indicated that 2 distinct acute syndromes accompanied by lymphangitis occur in residents of this filariasis-endemic area. One syndrome, which we call acute filarial lymphangitis (AFL), is caused by the death of adult worms. It is relatively uncommon in untreated persons, usually is asymptomatic or has a mild clinical course, and rarely causes residual lymphoedema. The second syndrome, of acute dermatolymphangioadenitis (ADLA), is not caused by filarial worms per se, but probably results from secondary bacterial infections. ADLA is a common cause of chronic lymphoedema and elephantiasis in Recife as well as in other areas of Brazil where lymphatic filariasis is not present. The syndromes of AFL and ADLA can be readily distinguished from each other by simple clinical criteria.

Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Evaluation of a RAPD-based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil.

Aims:  To evaluate the utility of random amplification of polymorphic DNA (RAPD) technique for routine practice in public health laboratories for epidemiological studies of Vibrio cholerae O1 isolates.

Prevalence of cagA and vacA genes in isolates from patients with Helicobacter pylori-associated gastroduodenal diseases in Recife, Pernambuco, Brazil

Geographical differences in the prevalence of Helicobacter pylori genes and their association with disease severity have been identified. This study analyzes the prevalences of the cagA gene and alleles of the vacA gene in H. pylori-associated gastroduodenal diseases in isolates from Recife, PE, Brazil. Gastric biopsy of 61 H. pylori-positive patients were submitted to DNA extraction and gene amplification by polymerase chain reaction. Among the 61 patients, 21 suffered from duodenal ulcer (DU) and 40 from gastritis (GT). The prevalence of H. pylori strains harbouring the cagA gene was higher in the DU group (90.5%) than in the GT group (60%) (p=0.02). The vacA gene was amplified in 56 out of 61 biopsies, of which 43 (76.8%) contained bacteria carrying the s1 allele and 13 (23.2%) the s2. However, the prevalence of the vacA s1 genotyping was the same in either DU or GT group. The majority of the s1-typed strains, 39 (90.7%) out of 43, were subtype s1b. In resume there was a strong association between the H. pylori cagA+ gene and DU. However, there were no differences between the DU and GT groups in relation to the vacA s1 and s2 alleles distribution, albeit the subtype s1b was predominant.

Typing of human and bovine Staphylococcus aureus by RAPD-PCR and ribotyping-PCR

Aims:  To investigate genetic diversity among Staphylococcus aureus and to delineate the geographical distribution of the strains found.

Estudos bacteriológicos e sorológicos de um surto de peste no Estado da Paraíba, Brasil

During a plaque outbreak in the Borborema Plateau focus (Paraiba), bacteriological and serological studies were carried out in material from 452 patients (48 positives), 1,938 rodents and other small mammals (75 positives), 4,756 dogs (141 positives) and 2,047 cats (57 positives) obtained from 41 counties (out of which, 21 produced positive samples). Twenty Yersinia pestis strains isolated from material from 3 patients and 17 rodents, displayed biochemical reactions, virulence factors, antibiotic susceptibility and animal experimental pathogenicity similar to those observed in strains previously isolated. According to our findings this recent plague outbreak did not exhibit different factors from those observed during prior outbreaks in other plague foci in the northeast of Brazil.During a plague outbreak in the Borborema Plateu focus (Paraiba), bacteriological and serological studies were carryed out in material from 452 patients (48 positives), 1,938 rodents and other small mammals (75 positives), 4,756 dogs (141 positives) and 2,047 cats (57 positives) obtained from 41 counties (out of which, 21 produced positive samples). Twenty Yersinia pestis strains isolated from material from 3 patients ans 17 rodents, displayed biochemical reactions, virulence factors, antibiotic susceptibility and animal experimental pathogenicity similar to those observed in strains previously isolated. According to our findings this recent plague outbreak did not exhibite different factors from those observed during prior outbreaks in other plague foci in the northeast of Brazil.

Vibrio parahaemolyticus serovar O3:K6 gastroenteritis in northeast Brazil.

Aims:  To examine the virulence factors and the genetic relationship isolates of the serogroup O3 of Vibrio parahaemolyticus in outbreaks of diarrhoea in the northeast region of Brazil.

Development of a multiplex single-tube nested PCR (MSTNPCR) assay for Vibrio cholerae O1 detection.

A multiplex nested PCR method for detection of Vibrio cholerae O1 using a single tube was developed (MSTNPCR). Firstly, single-tube nested PCR (STNPCR) with primers directed to ctxA gene was standardized, and its detection limit was compared to simple PCR and two-step nested PCR. Secondly, primers directed to rfbN gene were added to the reaction. The detection limit of the multiplex reaction was determined using V. cholerae O1 DNA and V. cholerae O1 grown in alkaline peptone water (APW). STNPCR was shown to be approximately 100-fold more sensitive than simple PCR and 10 times less sensitive than two-step nested PCR. This drawback is compensated by a lower risk of cross-contamination. The addition of a second target did not impair the detection limit of STNPCR (as little as 1 pg of V. cholerae O1 DNA detected). MSTNPCR could specifically detect up to three V. cholerae O1 cells or colony forming units (cfu) directly from the APW growth. A diagnostic kit consisting of a set of microtubes having the inner primers fixed onto the inside of the tube cap and a set of tubes containing the reaction mixture was evaluated for stability, and it proved to be stable for five months at -20 degrees C. Therefore, MSTNPCR would be useful in the detection of V. cholerae O1 directly from environmental waters in cholera endemic areas and in complementing the identification of toxigenic strains isolated by culture.

RAPD-PCR typing of Yersinia enterocolitica (Enterobacteriaceae) O:3 serotype strains isolated from pigs and humans

Sixteen strains of Yersinia enterocolitica serotype O:3, isolated from apparently healthy pigs collected in Rio de Janeiro, and four human strains of serotypes O:4, O:5, O:6 and O:13 were analyzed by RAPD-PCR. The strains were grouped into five genotypic profiles according to the amplification patterns obtained with three random primers. Fifteen of the 16 pig strains had identical amplification patterns, which was named genotypic profile 1. The one different profile was named genotypic profile 2. Genotypic profile 1 was also exhibited by the O:6 human serotype strain. The O:4 and O:13 human serotype strains showed similar amplification profiles with two primers. However, the third primer induced a distinct profile in each strain. Therefore, these two strains were placed into genotypic profile 3 and 4, respectively. Each primer produced a completely different amplification profile in the O:5 human serotype strain; therefore, it was named genotypic profile 5. The presence or absence of plasmids in the strains studied did not affect the amplification results. These results show that genetic variations can exist within a serotype, and strains of different serotypes can exhibit the same amplification profile when compared using other primers.

A simple PCR-based procedure for plaque diagnosis

Supernatant of boiled spleen saline-suspensions of Yersinia pestis experimentally infected animals were used as template for PCR amplification without DNA extraction. PCR sensitivity was enhanced by a second round of amplification (Nested). No amplification was observed from non-infected animals.