Nilma Cintra Leal

Fundação Oswaldo Cruz, Rio de Janeiro, Rio de Janeiro, Brazil

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Publications (51)72.59 Total impact

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    ABSTRACT: Introduction Methicillin-resistant Staphylococcus aureus (MRSA) strains have been responsible for many nosocomial outbreaks. Within hospitals, colonized employees often act as reservoirs for the spread of this organism. This study collected clinical samples of 91 patients admitted to the intensive care unit (ICU), hemodialysis/nephrology service and surgical clinic, and biological samples from the nasal cavities of 120 professionals working in those environments, of a University Hospital in Recife, in the State of Pernambuco, Brazil. The main objective of this study was to determine the occurrence and dissemination of methicillin- and vancomycin-resistant Staphylococcus spp. Methods The isolates obtained were tested for susceptibility to oxacillin and vancomycin and detection of the mecA gene. In addition, the isolates were evaluated for the presence of clones by ribotyping-polymerase chain reaction (PCR). Results MRSA occurrence, as detected by the presence of the mecA gene, was more prevalent among nursing technicians; 48.1% (13/27) and 40.7% (11/27) of the isolates were from health professionals of the surgical clinic. In patients, the most frequent occurrence of mecA-positive isolates was among the samples from catheter tips (33.3%; 3/9), obtained mostly from the hemodialysis/nephrology service. Eight vancomycin-resistant strains were found among the MRSA isolates through vancomycin screening. Based on the amplification patterns, 17 ribotypes were identified, with some distributed between patients and professionals. Conclusions Despite the great diversity of clones, which makes it difficult to trace the source of the infection, knowledge of the molecular and phenotypic profiles of Staphylococcus samples can contribute towards guiding therapeutic approaches in the treatment and control of nosocomial infections.
    Revista da Sociedade Brasileira de Medicina Tropical 07/2014; 47(4):437-446. · 0.94 Impact Factor
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    ABSTRACT: Abstract The marine clam Anomalocardia brasiliana is a candidate as a sentinel animal to monitor the contamination levels of coliforms in shellfish-harvesting areas of Brazil's northeastern region. The aim of the present study was to search enterotoxin-encoding genes plus the mecA gene among coagulase-negative staphylococci (CNS) isolates from shellfish meats of A. brasiliana. The specimen clam (n=48; 40 clams per sample) was collected during low tide in the bay area of Mangue Seco from April through June 2009, and random samples of chilled and frozen shelled clam meat (n=33; 250 g per sample) were obtained from retail shops from January through March 2012. Seventy-nine CNS isolates were identified, including Staphylococcus xylosus, S. cohnii spp. urealyticus, S. sciuri, and S. lentus. A high percentage of isolates resistant to erythromycin (58.5%), penicillin (51.2%), and tetracycline (43.9%), and the fluoroquinolones levofloxacin (39%) and ciprofloxacin (34.1%) were recorded from those environmental samples. Isolates from retail shops were particularly resistant to oxacillin (55.3%) and penicillin (36.8%). All CNS resistant to oxacillin and/or cefoxitin were positive for the presence of the mecA gene, but phenotypically susceptible to vancomycin. Also, the enterotoxin-encoding genes seg and seh were detected through multiplex-polymerase chain reaction in 77.7% and 88.8% of the isolates from environmental samples, versus 90.5% and 100% of the isolates from retail shops, respectively. The data reveal the risk to public health due to consuming raw or undercooked shellfish containing enterotoxigenic plus methicillin-resistant CNS.
    Foodborne Pathogens and Disease 10/2013; · 2.28 Impact Factor
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    ABSTRACT: Introduction The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio. Methods A duplex-polymerase chain reaction (PCR) was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans. Results The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans. Conclusions This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.
    Revista da Sociedade Brasileira de Medicina Tropical 05/2013; · 0.93 Impact Factor
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    ABSTRACT: After the worldwide cholera epidemic in 1993, permanent environmental monitoring of hydrographic basins was established in Pernambuco, Brazil, where cholera is endemic. After a quiescent period, 4 rfbN (serogroup O1) positive water samples that were culture negative were detected by multiplex single-tube nested PCR (MSTNPCR); 2 of these were also ctxA (cholera toxin) positive. From May to June 2012, 30 V. cholerae O1 isolates were obtained by culturing samples. These isolates were analyzed for the presence of virulence genes by PCR, intergenic spacer region 16S-23S PCR (ISR-PCR), and pulsed field gel electrophoresis (PFGE). The isolates were positive for the rfbN gene and negative for the assessed pathogenic genes and were classified into 2 groups by ISR and the same profile by PFGE. Close genetic similarity was observed between them (2012) and environmental strains from 2004 to 2005, indicating the permanence of endemic V. cholerae O1 in the region.
    The Scientific World Journal 01/2013; 2013:746254. · 1.22 Impact Factor
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    ABSTRACT: This work aimed to assess pathogenic potential and clonal relatedness of Aeromonas sp. and Vibrio cholerae isolates recovered during a diarrhea outbreak in Brazil. Clinical and environmental isolates were investigated for the presence of known pathogenic genes and clonal relatedness was assessed by intergenic spacer region (ISR) 16S-23S amplification. Four Aeromonas genes (lip, exu, gcat, flaA/B) were found at high overall frequency in both clinical and environmental isolates although the lip gene was specifically absent from selected species. A fifth gene, aerA, was rarely found in A. caviae, the most abundant species. The ISR profile revealed high heterogeneity among the Aeromonas isolates and no correlation with species identification. In contrast, in all the V. cholerae isolates the four genes investigated (ctxA, tcpA, zot and ace) were amplified and revealed homogeneous ISR and RAPD profiles. Although Aeromonas isolates were the major enteric pathogen recovered, their ISR profiles are not compatible with a unique cause for the diarrhea events, while the clonal relationship clearly implicates V. cholerae in those cases from which it was isolated. These results reinforce the need for a better definition of the role of aeromonads in diarrhea and whether they benefit from co-infection with V. cholerae.
    Revista do Instituto de Medicina Tropical de São Paulo 12/2012; 54(6):299-304. · 0.96 Impact Factor
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    Advances in Experimental Medicine and Biology 01/2012; 954:69-77. · 2.01 Impact Factor
  • Advances in Experimental Medicine and Biology 01/2012; 954:143-7. · 2.01 Impact Factor
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    ABSTRACT: Plague outbreaks are occasionally reported in Brazil. Unfortunately, due to great genetic similarity, molecular subtyping of Yersinia pestis strains is difficult. Analysis of multiple-locus variable number of tandem repeats (VNTR), also known as MLVA, has been found to be a valuable tool to discriminate among strains. To check for genetic differences, strains obtained from two different ecological complexes in Brazil collected during two different epidemiological events, an epizootic in Sítio Alagoinha in 1967 and an outbreak in Planalto da Borborema in 1986, were subtyped through MLVA using 12 VNTR loci. Three clusters (A, B and C) were observed. Of the 20 strains from the epizootic, 18 fit into cluster A. Cluster A was divided into two subgroups: A(1) (15 strains) and A(2) (3 strains). Of the 17 strains from the outbreak, 15 fit into cluster B. Cluster B was divided into three subgroups: B(1) (4 strains), B(2) (4 strains) and B(3) (7 strains). Cluster C is a singleton with one epizootic strain. The external standards, Y. pestis CO92 and Y. pseudotuberculosis IP32953, formed two clusters of singletons. The stability of 12 VNTR loci of three unrelated cultures included in this study was assessed. The 12 VNTR loci were stable through multiple serial subcultures in the laboratory. MLVA revealed that Y. pestis populations in Brazil are not monomorphic, and that there is intraspecific genetic diversity among Brazilian plague strains. We conclude that there is some correlation among genetic groups of this species, related to the temporal and geographic origin of isolates.
    Genetics and molecular research: GMR 01/2012; 11(3):3414-24. · 0.85 Impact Factor
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    ABSTRACT: Traditional methods of typing Vibrio cholerae define virulent strains according to their recognition by sera directed against the known epidemic serogroups O1 and O139, overlooking potentially virulent non-O1/non-O139 strains. Here, we have undertaken the characterization of eight clinical isolates of non-O1/non-O139 V. cholerae, collected during cholera outbreaks in Brazil. Seven of these were typed as O26 and one, 17155, was defined as non-typable. A PCR-based approach has previously detected in these strains several virulence genes derived from the CTXvarphi prophage and generally associated with pathogenic strains. Here, the presence of the O1-specific wbeN gene was investigated through PCR and found to be restricted to strain 17155, as well as one of the O26 strains, 4756, although neither strain was recognized by O1-specific antisera. The same two isolates were the only strains able to express the cholera toxin in culture, assayed by western blotting. They also possessed four repeats of the heptanucleotide TTTTGAT upstream of the ctxAB genes encoding the cholera toxin. The remaining strains possessed only two intact repeats, whereas pathogenic O1 possessed four to six repeats. To define their evolutionary relationships, selected 16S-23S intergenic rRNA spacer regions were sequenced from the various strains and the resulting sequences used to build phylogenetic trees. Strains 4756 and 17155 always clustered with control O1 strains, whereas the remaining O26 strains clustered separately. These results confirm that, despite their serological phenotype, these two strains are genotypically related to O1 strains and potentially able to produce epidemic cholera.
    Clinical Microbiology and Infection 06/2009; 16(1):62-7. · 4.58 Impact Factor
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    ABSTRACT: 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. Eighteen samples of the O3:K6 and O3:KUT serotypes of V. parahaemolyticus were analysed by multiplex polymerase chain reaction (m-PCR) for detection of the tl, tdh and trh genes, by random-amplified polymorphic DNA (RAPD) using two primers, and by amplification of the rDNA 16S-23S region. The gene tl was amplified in all the samples, tdh in 16 while trh in none; amplification of rDNA 16S-23S generated only one profile; each RAPD primer produced two amplification patterns allowing grouping two tdh(-) Kanagawa-negative isolates. V. parahaemolyticus with characteristics of the pandemic clone appears to be widely disseminated in the studied region. Because of the genetic uniformity of the isolates, elucidation of outbreaks or tracking the source of contamination by the present molecular techniques seems useless. Detection of V. parahaemolyticus with virulence potential of pandemic clone from two outbreaks and from several isolated gastroenteritis cases points out the need for inclusion of this micro-organism in the Brazilian routine monitoring of the diarrhoeas for elucidation of their aetiology.
    Journal of Applied Microbiology 04/2008; 105(3):691-7. · 2.39 Impact Factor
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    ABSTRACT: Through a continuous bacteriological monitoring programme carried out by the Health Secretariat of the State of Pernambuco, Brazil, two isolates of Vibrio cholerae O1 El Tor Ogawa were discovered in an endemic area in 2001, during a cholera inactive period, along with six V. cholerae non-O1/non-O139 strains and two Aeromonas veronii biovar sobria strains showing an unusual characteristic of agglutination with O1 antiserum. Between that time and 2005, eight other O1 isolates were found. The virulence genes present in the V. cholerae differed among strains, with only three O1 strains harboring the ctxA gene. The O1 and some non-O1/non-O139 strains displayed identical patterns of amplification of the 16S-23S intergenic spacer region. RAPD of the 10 V. cholerae O1 strains, with the two primers used, revealed heterogeneity. The presence of V. cholerae carrying virulence genes in the aquatic basins examined confirms that they constitute a vibrio reservoir during a cholera inactive period, thus strengthening the argument for a continuous monitoring programme and preventative measures for cholera, mainly in the areas where the supply of drinking water is deficient.
    Transactions of the Royal Society of Tropical Medicine and Hygiene 04/2008; 102(3):272-6. · 1.93 Impact Factor
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    ABSTRACT: 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.
    Journal of Microbiological Methods 03/2008; 72(2):191-6. · 2.10 Impact Factor
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    ABSTRACT: The performance of a single-tube nested-PCR (STNPCR) technique was evaluated for plague diagnosis in comparison to conventional (one step) and two step nested PCR (NPCR). Assays were carried out with primers targeting the gene caf1 that encodes the Yersinia pestis F1 antigen. For STNPCR inner primers were immobilized onto the inside of the microtube caps and after the first amplification they were eluted by inversion of the tube. This procedure avoids opening the tube, reducing the risks of false-positive results by cross-contamination. The immobilized primers are stable for several months at -20 degrees C, thus, the tubes can be prepared beforehand and stored until use. STNPCR was more sensitive than conventional PCR, and less sensitive than NPCR. This drawback is compensated by a lower risk of cross-contamination. The experiments with infected animals showed that NPCR and STNPCR were able to produce positive results in all samples tested, despite contamination with other organisms. In contrast, conventional PCR yielded positive results in a smaller number of samples. Three out of 62 culture-negative rodents from plague areas, were positive by STNPCR. In conclusion, the PCR approaches evaluated, particularly NPCR and STNPCR have potential to be used as alternative tools in epidemiological surveys of plague. Furthermore, as the results can be obtained quickly (less than 24 hour), these techniques could be useful in emergency situations in which the rapidity in diagnosis is essential for adoption of immediate measures of control.
    Advances in Experimental Medicine and Biology 02/2007; 603:351-9. · 2.01 Impact Factor
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    ABSTRACT: One hundred seventy nine Vibrio cholerae non-O1/non-O139 strains from clinical and different environmental sources isolated in Brazil from 1991 to 2000 were serogrouped and screened for the presence of four different virulence factors. The Random Amplification of Polymorphic DNA (RAPD) technique was used to evaluate the genetic relatedness among strains. Fifty-four different serogroups were identified and V. cholerae O26 was the most common (7.8%). PCR analysis for three genes (ctxA, zot, ace) located of the CTX genetic element and one gene (tcpA) located on the VPI pathogenicity island showed that 27 strains harbored one or more of these genes. Eight (4.5%) strains possessed the complete set of CTX element genes and all but one of these belonged to the O26 serogroup suggesting that V. cholerae O26 has the potential to be an epidemic strain. The RAPD profiles revealed a wide variability among strains and no genetic correlation was observed.
    Revista do Instituto de Medicina Tropical de São Paulo 01/2006; 48(2):65-70. · 0.91 Impact Factor
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    ABSTRACT: The pigmentation (pgm) locus is a large unstable area of the Yersinia pestis chromosome composed of a segment of iron acquisition (HPI) linked to a pigmentation segment. In this work we examined the mobility of HPI and the pigmen-tation segment in three Y. pestis isolates using successive subcultures on Congo red agar (CRA) plates. Strain P. CE 882 was shown to be highly stable while strains P. Exu 340 and P. Peru 375 dissociated into several phenotypes, PCR analysis showing evidence of changes in the pgm locus of the derived cultures. Strains P. Exu 340 and P. Peru 375 produced previously unreported cultures positive for the pesticin/yersiniabactin outer membrane receptor (psn+) but negative for the iron-regulated protein (irp2-), suggesting the occurrence of rearrangements in this chromosomal region and either a sequential loss or the loss of separated segments. These results provide evidence that besides deletion en bloc, specific rearrangements are also involved in the deletion events for that locus.
    Genetics and Molecular Biology 01/2006; 29(1). · 0.88 Impact Factor
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    ABSTRACT: Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.
    Memórias do Instituto Oswaldo Cruz 12/2004; 99(7):727-32. · 1.57 Impact Factor
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    ABSTRACT: Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.
    Revista da Sociedade Brasileira de Medicina Tropical 03/2004; 37(2):143-7. · 0.94 Impact Factor
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    ABSTRACT: 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. Seventy-nine strains were examined by PCR for the toxin genes (ctx A, zot and ace), virulence-associated genes (tcp A and tox T) and RAPD sequences. Except for one strain (no. 1123) from the Amazonas State, all the strains analysed carried the genes ctx A, zot, ace, tcp A and tox T. RAPD fingerprinting revealed variability but no correlation with serotype, biotype or geographical origin of the isolates was found. A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V. cholerae O1 strains. The simplicity and discriminative capacity of this technique make it useful for detecting genetic diversity among micro-organisms from a defined group or for outbreak investigation.
    Journal of Applied Microbiology 02/2004; 96(3):447-54. · 2.39 Impact Factor
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    ABSTRACT: The study was developed to analyze the plasmidial DNA, research virulence genes and identify genetic diversity of 31 strains of Vibrio cholerae non-O1 isolated from zooplankton of the Bacanga and Anil rivers in São Luis-MA. The study of plasmidial DNA showed 2 or 3 plasmids from 10 strains between 5.5 and 40 kilobasis. There was only single ribotype pattern. PCR methods did not show the genes ctxA, ace and zot, while RADP-PCR identified genetic diversity in the strains, showing the potential for variability in this species.
    Revista da Sociedade Brasileira de Medicina Tropical 01/2004; 37(4):324-8. · 0.94 Impact Factor
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    Revista Da Sociedade Brasileira De Medicina Tropical - REV SOC BRAS MED TROP. 01/2004; 37(4).

Publication Stats

227 Citations
72.59 Total Impact Points

Institutions

  • 1989–2014
    • Fundação Oswaldo Cruz
      • • Aggeu Magalhães Research Center (CPqAM)
      • • Departamento de Bacteriologia (INCQS)
      • • Department of Microbiology (INCQS)
      Rio de Janeiro, Rio de Janeiro, Brazil
  • 2008
    • National Council for Scientific and Technological Development, Brazil
      Palmyra, Minas Gerais, Brazil
  • 2006
    • Federal University of Pernambuco
      Arrecife, Pernambuco, Brazil
  • 2004
    • Universidade Federal do Maranhão
      • Departamento de Patologia
      Maranhão, Maranhão, Brazil
  • 2002
    • Universidade Federal Rural de Pernambuco
      Arrecife, Pernambuco, Brazil