Padronização e validação de marcadores moleculares para o diagnóstico da Leishmaniose Tegumentar

O diagnóstico da leishmaniose tegumentar (LT) baseia-se em critérios clínicos e epidemiológicos podendo ser confirmado por exames laboratoriais de rotina como a pesquisa direta do parasito por microscopia e a intradermorreação de Montenegro. Atualmente, os métodos moleculares, principalmente a reação da cadeia da polimerase (PCR), têm sido considerados para aplicação em amostras clínicas, devido a sua alta sensibilidade e especificidade. Este trabalho teve como objetivo a padronização e validação das técnicas de PCR-RFLP com diferentes iniciadores (kDNA, its1, hsp70 e prp1), visando o diagnóstico e a identificação das espécies de Leishmania presentes em amostras de DNA provenientes de lesões de pele ou mucosa de 140 pacientes com suspeita de leishmaniose tegumentar. Para tal, realizamos ensaios de: 1) sensibilidade das PCRs com os diferentes iniciadores, 2) especificidade dos ensaios utilizando DNAs de diferentes espécies de referência de Leishmania, de tripanossomatídeos inferiores e de fungos, 3) validação das técnicas de PCR-RFLP com os iniciadores estudados em amostras de DNA de lesões de pele ou mucosas de pacientes com LT. Os resultados dos ensaios de limiar de detecção das PCR (sensibilidade) mostraram que os quatro iniciadores do estudo foram capazes de detectar o DNA do parasito, porém em quantidades distintas: até 500 fg com os iniciadores para kDNA e its1, até 400 fg com hsp70 e até 5 ng com prp1. Quanto à especificidade dos iniciadores, não houve amplificação dos DNAs fúngicos. Por outro lado, nos ensaios com os iniciadores para kDNA e hsp70, verificamos amplificação do fragmento esperado em amostras de DNA de tripanossomatídeos. Nos ensaios com its1 e prp1, o padrão de amplificação com os DNAs de tripanossomatídeos foi diferente do apresentado pelas espécies de Leishmania. Verificou-se nos ensaios de validação que o PCR-kDNA detectou o parasito em todas as 140 amostras de DNA de pacientes e, assim, foi utilizado como critério de inclusão das amostras. A PCR-its1 apresentou menor sensibilidade, mesmo após a reamplificação com o mesmo iniciador (85,7% ou 120/140 amostras). Para os ensaios da PCR-hsp70, as amostras de DNA foram amplificadas com Repli G, para obter uma sensibilidade de 68,4% (89/140 amostras). A PCR-prp1 não detectou o parasito em amostras de DNA dos pacientes. Quanto aos ensaios para a identificação da espécie presente na lesão, a PCR-kDNA-RFLP-HaeIII e a PCR-its1- RFLP-HaeIII permitem a distinção de L. (L.) amazonensis das outras espécies pertencentes ao subgênero Viannia. A PCR-hsp70-RFLP-HaeIII-BstUI, apesar de potencialmente ser capaz de identificar as seis espécies de Leishmania analisadas, quando utilizada na avaliação das amostras humanas permitiu apenas a identificação de L. (V.) braziliensis. No entanto, é uma técnica com várias etapas e de difícil execução, o que pode inviabilizar o seu uso rotineiro em centros de referência em diagnósticos. Assim, recomendamos o uso dessa metodologia apenas em locais onde várias espécies de Leishmania sejam endêmicas. Finalmente, os resultados indicam que o kDNA-PCR devido à alta sensibilidade apresentada e facilidade de execução pode ser empregada como exame de rotina nos centros de referência, permitindo a confirmação ou exclusão da LT.

Genetic characterization of clinical isolates of Clostridium difficile using an optimized RAPD protocol and PCR ribotyping reveals strain diversity between two tertiary referral Trusts in the West Midlands, UK

Epidemiological investigations of Clostridium difficile often focus on differences between separate geographical areas. In this investigation, two populations of C. difficile recovered from separate tertiary referral Trusts within the West Midlands, UK, were characterized using both PCR ribotyping and an optimized RAPD (random amplification of polymorphic DNA) protocol. The PCR ribotyping and RAPD methodologies identified differences between the two C. difficile populations, in both the prevalence and the diversity of types identified. The use of PCR ribotyping in conjunction with RAPD further categorized different types within defined PCR ribotypes, identifying different types within the same PCR ribotype and therefore providing a greater discriminatory power than either of the methods when used alone. The differences observed in this study between the two Trusts in the distribution of both RAPD 'type' and PCR ribotype demonstrate the diversity that is present amongst isolates of C. difficile within a relatively small geographical area and warrants a need for further investigation into the local epidemiology of C. difficile.

Analysis of clinical isolates of Propionibacterium acnes by optimised RAPD

Random amplification of polymorphic DNA (RAPD) was evaluated as a genotypic method for typing clinical strains of Propionibacterium acnes. RAPD can suffer from problems of reproducibility if parameters are not standardised. In this study the reaction conditions were optimised by adjusting template DNA concentration and buffer constituents. All isolates were typeable using the optimised RAPD protocol which was found to be highly discriminatory (Simpson's diversity index, 0.98) and reproducible. Typing of P. acnes by optimised RAPD is an invaluable tool for the epidemiological investigation of P. acnes for which no other widely accepted method currently exists. © 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Phenotypic and genotypic characterisation of Clostrum Difficile

Clostridium difficile is at present one of the most common nosocomial infections in the developed world. Hypervirulent strains (PCR ribotype 027) of C. difficile which produce enhanced levels of toxins have also been associated with other characteristics such as a greater rate of sporulation and resistance to fluoroquinolones. Infection due to C. difficile PCR ribotype 027 has also been associated with greater rates of morbidity and mortality. The aim of this thesis was to investigate both the phenotypic and genotypic characteristics of two populations of toxigenic clinical isolates of C. difficile which were recovered from two separate hospital trusts within the UK. Phenotypic characterisation of the isolates was undertaken using analytical profile indexes (APIs), minimum inhibitory concentrations(MICs) and S-layer protein typing. In addition to this, isolates were also investigated for the production of a range of extracellular enzymes as potential virulence factors. Genotypic characterisation was performed using a random amplification of polymorphic DNA(RAPD) PCR protocol which was fully optimised in this study, and the gold standard method, PCR ribotyping. The discriminatory power of both methods was compared and the similarity between the different isolates also analysed. Associations between the phenotypic and genotypic characteristics and the recovery location of the isolate were then investigated. Extracellular enzyme production and API testing revealed little variation between the isolates; with S-layer typing demonstrating low discrimination. Minimum inhibitory concentrations did not identify any resistance towards either vancomycin or metronidazole; there were however significant differences in the distribution of antibiogram profiles of isolates recovered from the two different trusts. The RAPD PCR protocol was successfully optimised and alongside PCR ribotyping, effectively typed all of the clinical isolates and also identified differences in the number of types defined between the two locations. Both PCR ribotyping and RAPD demonstrated similar discriminatory power; however, the two genotyping methods did not generate amplicons that mapped directly onto each other and therefore clearly characterised isolates based on different genomic markers. The RAPD protocol also identified different subtypes within PCR ribotypes, therefore demonstrating that all isolates defined as a particular PCR ribotype were not the same strain. No associations could be demonstrated between the phenotypic and genotypic characteristics observed; however, the location from which an isolate was recovered did appear to influence antibiotic resistance and genotypic characteristics. The phenotypic and genotypic characteristics observed amongst the C. difficile isolates in this study, may provide a basis for the identification of further targets which may be potentially incorporated into future methods for the characterisation of C. difficile isolates.

Characterisation of propionibacterium acnes

Propionibacterium acnes forms part of the normal flora of the skin, oral cavity, large intestine and the external ear. Historically, P. acnes is considered to be of low virulence; however, in recent years it has been found as the aetiological agent in various pathologies including acne vulgaris, endophthalmitis, endocarditis, osteomyelitis, sarcoidosis, prosthetic hip infections and sciatica. It currently remains unclear why this normally harmless commensal can cause infection and contribute to a number of clinically significant conditions. This thesis has sought to investigate the phenotypic, genetic and antigenic properties of P.acnes strains isolated from sciatica patients undergoing microdiscectomy, normal skin, blood cultures, prosthetic hips and acne lesions. Isolates' phenotype was examined by determining their biotype by analytical profile index, antimicrobial susceptibility, virulence factor expression and serotype. A molecular typing method for P.acnes was developed using random amplification of polymorphic DNA (RAPD). Patient serum was used to screen P.acnes strains for antigens expressed in vivo and the chemical composition determined. The serodiagnostic potential and inflammatory properties of identified antigens were assessed. The optimised and reproducible RAPD protocol classified strains into three major clusters and was found to distinguish between the serotypes I and II for a large number of clinical isolates. Molecular typing by RAPD also enabled the identification of a genotype that did not react with the type I or II monoclonal antibodies and these strains may therefore constitute a previously undiscovered subspecies of P.acnes with a genetic background different from the type I and II serotypes. A major cell associated antigen produced by all strains was identified and characterised. A serological assay based on the antigen was used to measure IgG and IgM levels in serum from patients with acne, sciatica and controls. No difference in levels of antibodies was detected. Inflammatory properties of the antigen were measured by exposing murine macrophage-like cells and measuring the release of nitric oxide and tumour necrosis factor-alpha (TNF-α). Only TNF-α was elicited in response to the antigen. The phenotypic, genotypic and antigenic properties of this organism may provide a basis for future studies on P.acnes virulence and provide an insight into its mechanisms of pathogenesis.

A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterisation of type 1 cell surface-associated antigens

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64?% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

RAPD for the typing of coagulase-negative staphylococci implicated in catheter-related bloodstream infection

Objectives: A rapid random amplification of polymorphic DNA (RAPD) technique was developed to distinguish between strains of coagulase-negative staphylococci (CoNS) involved in central venous catheter (CVC)-related bloodstream infection. Its performance was compared with that of pulsed-field gel electrophoresis (PFGE). Methods: Patients at the University Hospital Birmingham NHS Foundation Trust, U.K. who underwent stem cell transplantation and were diagnosed with CVC-related bloodstream infection due to CoNS whilst on the bone marrow transplant unit were studied. Isolates of CoNS were genotyped by PFGE and RAPD, the latter employing a single primer and a simple DNA extraction method. Results: Both RAPD and PFGE were highly discriminatory (Simpson's diversity index, 0.96 and 0.99, respectively). Within the 49 isolates obtained from blood cultures of 33 patients, 20 distinct strains were identified by PFGE and 25 by RAPD. Of the 25 strains identified by RAPD, nine clusters of CoNS contained isolates from multiple patients, suggesting limited nosocomial spread. However, there was no significant association between time of inpatient stay and infection due to any particular strain. Conclusion: The RAPD technique presented allows CoNS strains to be genotyped with high discrimination within 4 h, facilitating real-time epidemiological investigations. In this study, no single strain of CoNS was associated with a significant number of CVC-related bloodstream infections. © 2005 Published by Elsevier Ltd on behalf of the British Infection Society.

Diversity and biotechnological potential of culturable bacteria from Brazilian mangrove sediment.

Mangrove ecosystems are environments subject to substantial degradation by anthropogenic activities. Its location, in coastal area, interfacing the continents and the oceans makes it substantially important in the prospection for biotechnological applications. In this study, we assessed the diversity of culturable bacteria present over the seasons at two depths (0-10 and 30-40 cm) in a mangrove sediment and in a transect area from the land to the sea. In total, 238 bacteria were isolated, characterized by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and further identified, by Fatty Acid Methyl Esther (FAME-MIDI), into the orders of Vibrionales, Actinomycetales and Bacillales. Also the ability of the isolates in producing economically important enzymes (amylases, proteases, esterases and lipases) was evaluated and the order Vibrionales was the main enzymatic source.

Microsatellite analysis of pacu broodstocks used in the stocking program of Paranapanema River, Brazil

O monitoramento da diversidade genética é fundamental em um programa de repovoamento. Avaliouse a diversidade genética de pacu Piaractus mesopotamicus (Holmberg, 1887) em duas estações de piscicultura em Andirá -Paraná, Brasil, utilizadas no programa de repovoamento do Rio Paranapanema. Foram amplificados seis loci microssatélite para avaliar 60 amostras de nadadeira. O estoque de reprodutores B apresentou maior número de alelos e heterozigose (alelos: 22 e H O: 0,628) que o estoque de reprodutores A (alelos: 21 e H O: 0,600). Alelos com baixos níveis de frequência foram observados nos dois estoques. Os coeficientes positivos de endogamia no locus Pme2 (estoque A: F IS = 0,30 e estoque B: F IS = 0,20), Pme5 (estoque B: F IS = 0,15), Pme14 (estoque A: F IS = 0,07) e Pme28 (estoque A: F IS = 0,24 e estoque B: F IS = 0,20), indicaram deficiência de heterozigotos. Foi detectada a presença de um alelo nulo no lócus Pme2. As estimativas negativas nos loci Pme4 (estoque A: F IS = -0,43 e estoque B: F IS= -0,37), Pme5 (estoque A: F IS = - 0,11), Pme14 (estoque B: F IS = - 0,15) e Pme32 (estoque A: F IS = - 0,93 e estoque B: F IS = - 0,60) foram indicativas de excesso de heterozigotos. Foi evidenciado desequilíbrio de ligação e riqueza alélica baixa só no estoque A. A diversidade genética de Nei foi alta nos dois estoques. A distância (0,085) e identidade (0,918) genética mostraram similaridade entre os estoques, o qual reflete uma possível origem comum. 6,05% da variância genética total foi devida a diferenças entre os estoques. Foi observado um recente efeito gargalo nos dois estoques. Os resultados indicaram uma alta diversidade genética nos estoques de reprodutores e baixa diferenciação genética entre eles, o que foi causado pelo manejo reprodutivo das pisciculturas, redução do tamanho populacional e intercâmbio genético entre as pisciculturas.

Analysis of genetic diversity in Cassia brewsteri with randomly amplified DNA fingerprints (RAFS)

Genetic diversity in Cassia brewsteri (F. Muell.) F. Muell. ex Benth. was assessed with Randomly Amplified DNA Fingerprints (RAFs). Thirty accessions of C. brewsteri collected from throughout its natural distribution were analysed with three random decamer primers, along with three accessions of C. tomentella (Benth.) Domin and a single accession of each of C. queenslandica C. T. White and C. marksiana (F. M. Bailey) Domin. The three primers yielded a reproducible amplification profile of 265 scorable polymorphic fragments for the 35 accessions. These molecular markers were used to calculate Nei and Li similarity coefficients between each pair of individuals. A matrix of dissimilarity of each pair of individuals was examined by multidimensional scaling (MDS). The analysis supports the division of C. brewsteri into two subspecies and the suggestion that intergradation of C. brewsteri and C. tomentella can occur where the distributions of these species meet.

Intrinsically bent DNA sites in the Drosophila melanogaster third chromosome amplified domain

Bent DNA sites promote the curvature of DNA in both eukaryotic and prokaryotic chromosomes. Here, we investigate the localization and structure of intrinsically bent DNA sites in the extensively characterized Drosophila melanogaster third chromosome DAFC-66D segment (Drosophila amplicon in the follicle cells). This region contains the amplification control element ACE3, which is a replication enhancer that acts in cis to activate the major replication origin ori-beta. Through both electrophoretic and in silico analysis, we have identified three major bent DNA sites in DAFC-66D. The bent DNA site (b1) is localized in the ACE3 element, whereas the other two bent DNA sites (b2 and b3) are localized in the ori-beta region. Four additional bent DNA sites were identified in the intron of the S18 gene and near the TATA box of the S15, S19, and S16 genes. The identification of DNA bent sites in genomic regions previously characterized as functionally relevant for DNA amplification further supports a function for DNA bent sites in DNA replication in eukaryotes.

EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs' and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.

Comparative Analysis by Polymerase Chain Reaction Amplified Minicircles of Kinetoplast DNA of a Stable Strain of Trypanosoma cruzi from São Felipe, Bahia, its Clones and Subclones: Possibility of Predominance of a Principal Clone in this Area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%.This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

The polymorphic nature of HIV type 1 env V4 affects the patterns of potential N-glycosylation sites in proviral DNA at the intrahost level.

We have previously shown that env V4 from HIV-1 plasma RNA is highly heterogeneous within a single patient, due to indel-associated polymorphism. In this study, we have analyzed the variability of V4 in proviral DNA from unfractionated PBMC and sorted T and non-T cell populations within individual patients. Our data show that the degree of sequence variability and length polymorphism in V4 from HIV provirus is even higher than we previously reported in plasma. The data also show that the sequence of V4 depends largely on the experimental approach chosen. We could observe no clear trend for compartmentalization of V4 variants in specific cell types. Of interest is the fact that some variants that had been found to be predominant in plasma were not detected in any of the cell subsets analyzed. Consistently with our observations in plasma, V3 was found to be relatively conserved at both interpatient and intrapatient level. Our data show that V4 polymorphism involving insertions and deletions in addition to point mutations results in changes in the patterns of sequons in HIV-1 proviral DNA as well as in plasma RNA. These rearrangements may result in the coexistence, within the same individual, of a swarm of different V4 regions, each characterized by a different carbohydrate surface shield. Further studies are needed to investigate the mechanism responsible for the variability observed in V4 and its role in HIV pathogenesis.