As infecções sistêmicas causadas por Candida spp. representam importante causa de morbidade e mortalidade em pacientes pediátricos internados em unidades de cuidados intensivos. A utilização de fármacos imunodepressores em pacientes submetidos a transplantes, antibioticoterapia prolongada, nutrição parenteral, presença de cateter venoso central, entre outros fatores, contribuem para o aumento na freqüência destas infecções oportunísticas. Embora Candida albicans permaneça como a espécie mais freqüentemente isolada de episódios de candidemia, tem ocorrido aumento do número de infecções causadas por espécies não albicans. Os métodos convencionais de cultivo apresentam sensibilidade limitada, além de demandar tempo prolongado para a identificação das espécies infectantes. A Reação em Cadeia da Polimerase (PCR) requer pequenos volumes de sangue para análise, situação ideal para crianças gravemente enfermas, propiciando diagnóstico rápido e identificação precisa das espécies de Candida. Este estudo teve como objetivos padronizar técnicas de PCR de dupla amplificação, para identificar cinco espécies de Candida em amostras de sangue de pacientes pediátricos com risco de desenvolver candidemias, e comparar os resultados com as hemoculturas. Foram analisadas amostras de sangue de 80 pacientes pediátricos internados na Unidade de Terapia Intensiva do Instituto da Criança do Hospital das Clínicas da FMUSP, alocados em dois grupos: 58 pacientes que apresentavam duas ou mais condições predisponentes que elevam o risco de infecção fúngica (Grupo 1); e 22 pacientes em condições clínicas de menor gravidade e risco mais baixo, que foram estudados para avaliação da freqüência de resultados falso-positivos por PCR (Grupo 2). A primeira amplificação identificou fragmento da região ITS do DNA ribossômico, e a segunda etapa amplificou fragmentos espécie-específicos para C.albicans, C.glabrata, C.parapsilosis, C.tropicalis e C.krusei. Para as amostras com detecção de Candida spp. somente por PCR, foram realizadas restrições enzimáticas e seqüenciamentos, para a confirmação da especificidade dos amplificados. A concordância entre os resultados das hemoculturas e das PCR foi avaliada pelo teste de McNemar, com nível de significância de 5%. As hemoculturas foram positivas em 15,5% dos pacientes do grupo 1, enquanto as amplificações por PCR detectaram DNA de Candida spp. em 25,9% dos casos, incluindo todos os pacientes com culturas positivas. Houve concordância na identificação das espécies em 88,9% dos casos. Em dois pacientes, a PCR identificou mais de uma espécie infectante, o que não ocorreu pelas hemoculturas. Nos seis casos em que somente a PCR detectou Candida spp. nas amostras de sangue, as restrições enzimáticas e os seqüenciamentos confirmaram a especificidade das amplificações. No grupo 2, as hemoculturas foram negativas em todos os 22 casos, e a PCR detectou C.glabrata em um paciente, cujos amplificados, após seqüenciamento, apresentaram 98% de homologia com o protótipo. O teste de McNemar mostrou que a discordância encontrada entre os métodos é estatisticamente significante e favorece a PCR (p = 0,031). Os resultados do presente estudo demonstraram que a técnica de PCR apresentou maior poder de detecção de Candida spp. em amostras de pacientes com risco de desenvolver candidemia, quando comparada às hemoculturas. A PCR foi ainda capaz de detectar mais de uma espécie infectante na mesma amostra de sangue / Disseminated candidiasis is an important cause of morbidity and mortality in immunocompromised and critically ill pediatric patients. Increased use of immunosupressive drugs, indwelling catheters, broad-spectrum antibiotics, parenteral nutrition and other predisposing conditions have contributed to the increment of Candida bloodstream infections. Although Candida albicans remains the leading species, a growing number of nonalbicans isolates has been reported. Laboratory diagnosis of candidemia is troublesome due to the lack of blood cultures sensitivity. Detection of Candida DNA by polymerase chain reaction (PCR) in tiny blood volumes may provide a more rapid and reliable candidemia detection in critically ill children. The present study aimed at standardizing nested-PCR amplifications for identification of five Candida species, comparing results with those obtained by blood cultures. Eighty patients admitted to the pediatric intensive care unit of the Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo were divided into two groups: 58 patients presenting with two or more predisposing conditions to develop candidemia (Group 1), and 22 patients with lower risk, enrolled to establish the frequency of PCR false-positive results (Group 2). Nested amplifications were targeted to Candida ITS sequence, the first round yielding a gender specific fragment, and the second round identified five Candida species corresponding to C. albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. krusei. When PCR positive results were not confirmed by blood cultures, enzymatic restriction and DNA sequencing were performed. Agreement between both methods was assessed by the McNemar test, adopting a level of significance of 5%. Blood cultures were positive in 15.5% of group 1 patients, whereas nested-PCR identified Candida species in 25.9%, including all culture positive patients. PCR was 88.9% concordant with blood cultures species identification, but only the molecular technique identified dual candidemia in two patients. Amplification products of six patients with negative blood cultures submitted to enzymatic restriction and sequencing analysis showed high degree of homology with Candida reference strains, confirming the specificity of PCR. Blood cultures were negative in all patients of group 2, but PCR detected C.glabrata in one case whose amplification products resulted in 98% of homology with the reference strain. The McNemar test showed disagreement between both methods, favoring PCR (p=0.031). In the present study, PCR was more sensitive to detect Candida species in comparison to blood cultures, being also able to identify dual candidemia

Louis, Tong Kwok-leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 121-130). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.III / LIST OF CONTENTS --- p.IV / LIST OF TABLES --- p.VIII / LIST OF FIGURES --- p.X / ABBREVIATIONS --- p.XII / GENERAL ABBREVIATIONS --- p.XII / VARIOUS NOMENCLATURES OR ABBREVIATIONS FOR SRSVS --- p.XIII / OBJECTIVES OF THE STUDY --- p.XIV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- HISTORICAL PERSPECTIVE --- p.2 / Chapter 1.2 --- CLINICAL FEATURES OF HUMAN SRSV INFECTION --- p.10 / Chapter 1.3 --- EPIDEMIOLOGY --- p.12 / Chapter 1.4 --- PHYSICAL CHARACTERISTICS OF SRSV --- p.14 / Chapter 1.5 --- STABILITY OF NORWALK VIRUS --- p.15 / Chapter 1.6 --- DIAGNOSTIC TOOLS FOR SRSVS --- p.15 / Chapter 1.7 --- GENOMIC ORGANIZATION OF SRSVS --- p.16 / Chapter 1.8 --- MOLECULAR TECHNOLOGIES --- p.19 / Chapter CHAPTER 2 --- MATERIALS --- p.20 / Chapter 2.1 --- FAECAL SAMPLES FROM 1986 TO 1992 --- p.21 / Chapter 2.2 --- OTHER FAECAL SAMPLES FROM 1995 TO 1998 --- p.21 / Chapter 2.3 --- AGE GROUPS OF ALL THE SAMPLES --- p.21 / Chapter CHAPTER 3 --- METHODS --- p.23 / Chapter 3.1 --- EXTRACTION OF RNA FROM PATIENT STOOL OR VOMIT SAMPLES --- p.24 / Chapter 3.2 --- REVERSE TRANSCRIPTION - POLYMERASE CHAIN REACTION (RT-PCR) FOR SRSVS --- p.25 / Chapter 3.2.1 --- Principle of RT-PCR assay for SRSV --- p.25 / Chapter 3.2.2 --- Reverse transcription (cDNA Synthesis) --- p.26 / Chapter 3.2.3 --- Polymerase chain reaction --- p.26 / Chapter 3.2.4 --- Electrophoresis (in the PCR product room) --- p.31 / Chapter 3.2.5 --- Controls for PCR assay --- p.32 / Chapter 3.2.6 --- Interpretation of SRSV polymerase gene PCR --- p.32 / Chapter 3.3 --- RT-PCR USING INOSINE CONTAINING PRIMERS FOR THE CAPSID REGIONS --- p.34 / Chapter 3.3.1 --- RT-PCR for the capsid region of SRSV genome --- p.34 / Chapter 3.3.2 --- Interpretation of SRSV capsid gene PCR --- p.36 / Chapter 3.4 --- SOLID PHASE IMMUNE ELECTRON MICROSCOPY FOR THE DETECTION OF SRSV --- p.37 / Chapter 3.5 --- OPTIMIZATION OF CONDITIONS FOR SRSV RT-PCR --- p.37 / Chapter 3.5.1 --- Titration of primers --- p.37 / Chapter 3.5.2 --- Titration of MgCl2 --- p.38 / Chapter 3.5.3 --- "Titration ofdNTPs, MgCl2 and Taq polymerase (pH 9.0)" --- p.38 / Chapter 3.6 --- SPECIFICITY OF SRSV RT-PCR --- p.38 / Chapter 3.7 --- PURIFICATION OF PCR PRODUCTS PRIOR TO CLONING …… --- p.38 / Chapter 3.8 --- CLONING OF THE PURIFIED DNA INTO pGEM-T EASY VECTOR --- p.39 / Chapter 3.8.1 --- Introduction --- p.39 / Chapter 3.8.2 --- Sequence of the pGEM-T Easy Vector --- p.42 / Chapter 3.8.3 --- Ligation --- p.44 / Chapter 3.8.4 --- Transformation of competent bacterial cells --- p.44 / Chapter 3.8.5 --- Small-scale preparations of plasmid DNA --- p.45 / Chapter 3.8.6 --- Purification of miniprep using QIAprep Miniprep --- p.45 / Chapter 3.8.7 --- Restriction analysis of small-scale preparations of plasmid DNA --- p.45 / Chapter 3.9 --- CYCLE SEQUENCING OF CLONED SRSV AMPLICONS --- p.46 / Chapter 3.9.1 --- Targets for Sequencing --- p.46 / Chapter 3.9.2 --- Procedures of cycle sequencing --- p.46 / Chapter 3.9.3 --- Gel electrophoresis --- p.48 / Chapter 3.9.4 --- Sequencing conditions --- p.49 / Chapter 3.10 --- SEQUENCE ANALYSIS --- p.49 / Chapter CHAPTER 4 --- REAGENTS AND BUFFERS --- p.51 / Chapter 4.1 --- REAGENTS AND BUFFERS FOR RNA EXTRACTION --- p.52 / Chapter 4.2 --- REAGENTS AND BUFFERS FOR REVERSE TRANSCRIPTION (cDNA SYNTHESIS) --- p.52 / Chapter 4.3 --- REAGENTS AND BUFFERS FOR PCR --- p.53 / Chapter 4.4 --- GEL ELECTROPHORESIS OF PCR PRODUCTS --- p.53 / Chapter 4.5 --- PURIFICATION OF PCR PRODUCTS --- p.54 / Chapter 4.6 --- REAGENTS FOR CLONING THE DNA INSERT INTO pGEM-T EASY VECTOR --- p.54 / Chapter 4.6.1 --- "pGEM-T Easy Vector System (Promega Corporation, USA)" --- p.54 / Chapter 4.6.2 --- Isopropylthio-β-D-galactoside (IPTG) stock solution --- p.54 / Chapter 4.6.3 --- X-Gal --- p.54 / Chapter 4.6.4 --- Luria-Bertani (LB) medium --- p.55 / Chapter 4.6.5 --- LB plates with ampicillin --- p.55 / Chapter 4.6.6 --- LB plates with ampicillin/IPTG/X-Gal --- p.55 / Chapter 4.6.7 --- SOC medium --- p.55 / Chapter 4.6.8 --- Mini-prep purification --- p.56 / Chapter 4.6.9 --- Mini-prep analysis --- p.56 / Chapter 4.6.9.1 --- Lambda DNA-Hind IIIφX-174 DNA-Hae III Digest --- p.56 / Chapter 4.6.9.2 --- Not I --- p.58 / Chapter 4.7 --- REAGENTS AND BUFFERS FOR CYCLE SEQUENCING --- p.58 / Chapter 4.7.1 --- SequiTherm EXCEĹёØ II Long-Rea´dёØ DNA Sequencing Kit-AL´FёØ --- p.58 / Chapter 4.7.2 --- Sequencing primers --- p.59 / Chapter 4.8 --- REAGENTS FOR SEQUENCING GEL CASTING --- p.59 / Chapter CHAPTER 5 --- RESULTS --- p.61 / Chapter 5.1 --- RESULTS OF RT-PCR OPTIMIZATION --- p.62 / Chapter 5.1.1 --- Magnesium chloride and pH of PCR reaction buffer --- p.62 / Chapter 5.1.2 --- Concentration of primers --- p.64 / Chapter 5.1.3 --- "Titration of dNTPs, MgCl2 and Taq polymerase (pH 9.0)" --- p.65 / Chapter 5.2 --- RESULT OF SENSITIVITY TEST --- p.66 / Chapter 5.3 --- RESULTS OF SPECIFICITY TEST --- p.67 / Chapter 5.4 --- RESULTS OF THE PCR USING INOSINE CONTAINING POL PRIMERS --- p.70 / Chapter 5.5 --- RESULTS OF PCR USING INOSINE CONTAINING CAPSID PRIMERS --- p.73 / Chapter 5.6 --- RESULTS OF SOME SAMPLES RETESTED BY SPIEM --- p.75 / Chapter 5.7 --- RESULTS OF SPORADIC OUTBREAKS --- p.77 / Chapter 5.7.1 --- A sporadic outbreak in 1996 --- p.77 / Chapter 5.7.2 --- Sporadic outbreak in a kindergarten in 1997 --- p.79 / Chapter 5.7.3 --- Sporadic outbreak at a hotel in 1998 --- p.79 / Chapter 5.7.4 --- Application of the RT-PCR to contaminated shellfish --- p.80 / Chapter 5.8 --- RESULTS OF MINI PREP ANALYSIS WITH NOT I DIGESTION --- p.85 / Chapter 5.9 --- RESULT OF ELECTROPHEROGRAM OF A SELECTED SPECIMEN FROM THE AUTOMATIC SEQUENCING --- p.86 / Chapter 5.9 --- RESULT OF ELECTROPHEROGRAM OF A SELECTED SPECIMEN FROM THE AUTOMATIC SEQUENCING --- p.86 / Chapter 5.10 --- RESULTS OF ALL TRIMMED DNA SEQUENCES --- p.87 / Chapter CHAPTER 6 --- DISCUSSION --- p.112 / REFERENCES --- p.122 / APPENDIX --- p.131 / APPENDIX I: Electron micrograph of SRSV particles --- p.132 / APPENDIX II: Confirmation for specificity test --- p.133 / APPENDIX III: Sequencing amplicons using capsid primers --- p.135 / APPENDIX IV: Sequencing amplicons (outbreak) using pol primers --- p.136 / APPENDIX V: Electropherogram (direct sequencing) --- p.138 / APPENDIX VI: Other RT-PCR results using pol primers --- p.139 / APPENDIX VII: Results of RT-PCR using capsid primers --- p.149 / APPENDIX VIII: Mini prep analysis --- p.158

by Cheng Chi Wa. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 110-120). / Abstract also in Chinese. / Title --- p.i / Abstract --- p.ii / Acknowledgement --- p.iv / Abbreviations --- p.v / Abbreviation Table for Amino Acids --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.2 / Chapter Part I --- Hypertrophic Scar / Chapter 2.1 --- Definition of hypertrophic scar --- p.2 / Chapter 2.2 --- Pathology --- p.2 / Chapter 2.3 --- Epidemiology findings --- p.3 / Chapter 2.3.1 --- Ethnicity --- p.3 / Chapter 2.3.2 --- Age --- p.3 / Chapter 2.3.3 --- Body location --- p.3 / Chapter 2.4 --- Mechanism of cutaneous wound healing --- p.4 / Chapter 2.4.1 --- Phase I - Haemostasis and inflammation --- p.4 / Chapter 2.4.1.1 --- Haemostasis --- p.6 / Chapter 2.4.1.2 --- Early phase of inflammation --- p.6 / Chapter 2.4.1.3 --- Late phase of inflammation --- p.7 / Chapter 2.4.2 --- Phase II - Re-epithelialization --- p.7 / Chapter 2.4.2.1 --- Migration of epidermal keratinocytes --- p.8 / Chapter 2.4.2.2 --- Migration of fibroblasts --- p.8 / Chapter 2.4.2.3 --- Angiogenesis --- p.9 / Chapter 2.4.3 --- Phase III - Tissue remodeling --- p.10 / Chapter 2.4.3.1 --- Cell maturation and apoptosis --- p.10 / Chapter 2.4.3.2 --- Exrtracellular matrix remodeling --- p.10 / Chapter 2.5 --- Alteration of wound healing - Possible pathogenic factors of hypertrophic scar --- p.11 / Chapter 2.5.1 --- Changes in Phase I-Inflammation --- p.13 / Chapter 2.5.2 --- Changes in Phase II - Re-epithelialization/ tissue formation --- p.14 / Chapter 2.5.3 --- Changes in Phase III - Tissue remodeling --- p.15 / Chapter 2.6 --- The Role of fibroblasts in the formation of hypertrophic scar --- p.16 / Chapter 2.6.1 --- Functions of fibroblasts in wound healing --- p.16 / Chapter 2.6.2 --- Suggested aetiological role in the formation of hypertrophic scar fibroblasts --- p.16 / Chapter 2.6.2.1 --- Fibroproliferation disorder --- p.18 / Chapter 2.6.2.2 --- Extracellular Matrix remodeling disorder --- p.18 / Chapter a) --- CoUaqen --- p.18 / Chapter b) --- Proteoglycan --- p.19 / Chapter 2.6.2.3 --- Other differentially expressed factors --- p.20 / Chapter 2.7 --- Treatment of hypertrophic scar --- p.21 / Chapter Part II --- Differential Display / Chapter 2.8 --- Current approaches for the studies of differential gene expression --- p.23 / Chapter 2.9 --- Comparison amongst different approaches --- p.23 / Chapter 2.10 --- The strategy of Differential Display RT-PCR (DDRT-PCR) --- p.24 / Chapter 2.11 --- The application of DDRT-PCR to identify differentially expressed genes --- p.26 / Chapter Chapter 3 --- Aims and Strategies --- p.27 / Chapter Chapter 4 --- Methods and Materials --- p.29 / Chapter 4.1 --- Materials --- p.29 / Chapter 4.2 --- Clinical specimen collection --- p.31 / Chapter 4.3 --- Primary explant culture --- p.31 / Chapter 4.4 --- Immunohistochemical staining --- p.32 / Chapter 4.5 --- Total RNA extraction --- p.32 / Chapter 4.6 --- DNase I digestion --- p.33 / Chapter 4.7 --- Differential display-RTPCR (DD-RTPCR) --- p.33 / Chapter 4.8 --- Polyacrylamide gel electrophoresis --- p.34 / Chapter 4.9 --- Reamplification of the differentially expressed fragments --- p.35 / Chapter 4.10 --- Molecular cloning of the DNA fragments --- p.35 / Chapter 4.11 --- Screening and miniprep of the plasmid DNA --- p.36 / Chapter 4.12 --- Cycle sequencing --- p.38 / Chapter 4.13 --- Data analysis --- p.38 / Chapter 4.14 --- RT-PCR --- p.39 / Chapter 4.15 --- Probe labeling by PCR with DIG-dUTP --- p.40 / Chapter 4.16 --- Southern blotting --- p.41 / Chapter Chapter5 --- p.42 / Chapter 5.1 --- Clinical Specimen --- p.42 / Chapter 5.2 --- Primary explant culture --- p.42 / Chapter 5.3 --- The total RNA extraction from the cultured fibroblast --- p.45 / Chapter 5.4 --- Differential display RT-PCR --- p.47 / Chapter 5.5 --- Reamplification of the DNA fragments --- p.49 / Chapter 5.6 --- Molecular cloning of the DNA fragment --- p.53 / Chapter 5.7 --- DNA sequencing of the inserts --- p.58 / Chapter 5.8 --- Analysis and identification of the DNA sequences --- p.62 / Chapter 5.9 --- Semi-quantitative RT-PCR analysis of the differentially expressed genes --- p.76 / Chapter Chapter6 --- p.87 / Chapter Part I --- Validity of the Findings / Chapter 6.1 --- The Limitation of Tissue Sampling --- p.87 / Chapter 6.2 --- Tissue Culture model --- p.88 / Chapter 6.3 --- Differential Display RT-PCR --- p.89 / Chapter 6.3.1 --- Identification of the differentially expressed genes --- p.89 / Chapter 6.3.2 --- Confirmation of the differentially expressed genes --- p.91 / Chapter 6.4 --- Technical difficulties and Limitations --- p.92 / Chapter 6.4.1 --- Sampling --- p.92 / Chapter 6.4.2 --- Primary tissue culture --- p.93 / Chapter Part II --- Significance and Future Studies / Chapter 6.5 --- Down-regulation of thrombospondin 1 (TSP 1) in the hypertrophic scar fibroblasts --- p.94 / Chapter 6.6 --- Biochemical and biological functions of TSP1 --- p.96 / Chapter 6.6.1 --- The biochemical functions of TSP1 --- p.96 / Chapter 6.6.2 --- The biochemical functions of TSP1 --- p.97 / Chapter 6.7 --- The role of TSP 1 in the pathogenesis of hypertrophic scar --- p.98 / Chapter 6.7.1 --- Down-regulation of TSP 1 may be responsible for the excessive microvessels in hypertrophic scar --- p.98 / Chapter 6.7.2 --- Down-regulation of TSP 1 may be responsible for the failure of the apoptosis of the fibroblasts in the hypertrophic scar --- p.101 / Chapter 6.8 --- Expression of TSP 1 during wound healing --- p.103 / Chapter 6.9 --- Expression of TSP 1 in hypertrophic scarring --- p.107 / Chapter 6.10 --- Cytochrome b561 and its biological function --- p.109 / Chapter 6.11 --- Future studies --- p.108 / Chapter 6.11.1 --- The expression of TSP 1 in hypertrophic scarring and normal wound healing --- p.108 / Chapter 6.11.2 --- The expression of cytochrome b561 --- p.109 / Chapter 6.11.3 --- A full scale study of differential display RT-PCR --- p.109 / References --- p.110 / Appendices --- p.121 / Chapter I --- The complete mRNA sequence of thrombospondin1 precursor --- p.121 / Chapter II --- The mRNA sequence of cytochrome b561 --- p.123

Lui Yuk Sun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 138-148). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Abberviations --- p.v / Table of Contents --- p.vii / List of Tables --- p.xiv / List of Figures --- p.xvi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Bacteriological evaluation of water --- p.1 / Chapter 1.1.1. --- Indicator organisms for water quality monitoring --- p.2 / Chapter 1.1.2. --- Properties defined for indicator organisms --- p.3 / Chapter 1.1.3. --- Example of common indicator organisms --- p.3 / Chapter 1.1.3.1. --- Total coliform group --- p.3 / Chapter 1.1.3.2. --- "Fecal coliform, Escherichia coli" --- p.4 / Chapter 1.1.3.3. --- Fecal Streptococcus --- p.5 / Chapter 1.1.3.4. --- Klebsiella --- p.5 / Chapter 1.2. --- The need for specific detection of waterborne pathogenic organisms --- p.6 / Chapter 1.3. --- Common water-borne pathogenic organisms --- p.7 / Chapter 1.3.1. --- Bacteria --- p.7 / Chapter 1.3.1.1. --- Escherichia coli 0157:H7 --- p.7 / Chapter 1.3.1.2. --- Salmonella typhimurium --- p.11 / Chapter 1.3.1.3 --- Legionella pneumophila --- p.12 / Chapter 1.3.2. --- Protozoa --- p.14 / Chapter 1.3.3. --- Viruses --- p.15 / Chapter 1.4. --- Conventional approaches for pathogens detection --- p.16 / Chapter 1.4.1. --- Examples of conventional detection methods --- p.17 / Chapter 1.4.2. --- Problems related to the conventional detection methods --- p.18 / Chapter 1.5. --- Novel approaches for pathogens detection --- p.19 / Chapter 1.5.1. --- Modifications of media --- p.19 / Chapter 1.5.2. --- Antibody-based methods --- p.20 / Chapter 1.5.3. --- Nucleic acid-based methods --- p.21 / Chapter 1.6. --- Principles of pathogens concentration by filtration and immunomagnetic separation --- p.22 / Chapter 1.7. --- Principles of pathogens detection by polymerase chain reaction --- p.24 / Chapter 1.8. --- Principles of quantitative assay of water-borne pathogens using real-time PCR --- p.26 / Chapter 1.9. --- Aims of this study --- p.28 / Chapter 2. --- Detection of water-borne bacteria by polymerase chain reaction --- p.31 / Chapter 2.1. --- Introduction --- p.31 / Chapter 2.2. --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Bacterial strains --- p.35 / Chapter 2.2.2. --- Bacterial enumeration --- p.35 / Chapter 2.2.3. --- DNA extraction and purification --- p.36 / Chapter 2.2.3.1. --- Boiling method --- p.36 / Chapter 2.2.3.2. --- Protinesae K extraction method --- p.36 / Chapter 2.2.3.3. --- Chelex extraction method --- p.37 / Chapter 2.2.4. --- Targeted sequences --- p.38 / Chapter 2.2.4.1. --- eaeA gene --- p.38 / Chapter 2.2.4.2. --- mdh gene --- p.39 / Chapter 2.2.4.3. --- flaR gene --- p.39 / Chapter 2.2.5. --- PCR amplification --- p.40 / Chapter 2.2.6. --- Gel electrophoresis --- p.41 / Chapter 2.3. --- Results --- p.42 / Chapter 2.3.1. --- Optimization of the PCR --- p.42 / Chapter 2.3.2. --- Sensitivity of PCR detection --- p.42 / Chapter 2.3.2.1. --- Boiling method --- p.42 / Chapter 2.3.2.2. --- Proteinease K method --- p.43 / Chapter 2.3.2.3. --- Chelex method --- p.43 / Chapter 2.3.3. --- Specificity of PCR detection --- p.43 / Chapter 2.3.3.1. --- primers targeted uidA gene --- p.44 / Chapter 2.3.3.2. --- primers targeted mdh gene --- p.44 / Chapter 2.3.3.3. --- primers targeted flaR gene --- p.44 / Chapter 2.4. --- Discussion --- p.57 / Chapter 3. --- Concentration and separation of water-borne bacteria by two-step-filtration and immunomagnetic separation --- p.61 / Chapter 3.1. --- Introduction --- p.61 / Chapter 3.2. --- Materials and Methods --- p.66 / Chapter 3.2.1. --- Bacterial strains --- p.66 / Chapter 3.2.2. --- Bacterial enumeration --- p.66 / Chapter 3.2.3. --- Filtration --- p.67 / Chapter 3.2.4. --- Immunomagnetic separation (IMS) --- p.68 / Chapter 3.2.4.1. --- Antibodies and Magnetic beads --- p.68 / Chapter 3.2.4.2. --- Binding of antibodies to magnetic beads --- p.68 / Chapter 3.2.4.3. --- Immunomagnetic separation of bacteria in seeded samples --- p.70 / Chapter 3.2.5. --- Determine the efficiency of filtration and immunomagnetic separation --- p.70 / Chapter 3.2.6. --- DNA extraction --- p.71 / Chapter 3.2.7. --- Multiplex PCR --- p.71 / Chapter 3.2.8. --- PCR amplification --- p.72 / Chapter 3.2.9. --- Gel electrophoresis --- p.72 / Chapter 3.3. --- Results --- p.73 / Chapter 3.3.1. --- Efficiency of filtration and immunomagnetic separation --- p.73 / Chapter 3.3.2. --- Detection limit of PCR --- p.73 / Chapter 3.3.2.1. --- Filtration and immunomagnetic separation --- p.73 / Chapter 3.3.2.2. --- Influence of background flora --- p.73 / Chapter 3.3.2.3 --- Shing Mun River and Lam Tsuen River --- p.77 / Chapter 3.3.3. --- Multiplex PCR --- p.77 / Chapter 3.4. --- Discussion --- p.91 / Chapter 4. --- Quantitative assay of water-borne pathogens using real-time PCR --- p.94 / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Materials and Methods --- p.99 / Chapter 4.2.1. --- Bacteria strains --- p.99 / Chapter 4.2.2. --- Bacterial enumeration --- p.99 / Chapter 4.2.3. --- Primers and Probes --- p.100 / Chapter 4.2.3.1. --- eaeA gene --- p.101 / Chapter 4.2.3.2. --- mdh gene --- p.102 / Chapter 4.2.3.3. --- flaR gene --- p.102 / Chapter 4.2.4. --- Targeted sequences cloning and sequencing --- p.103 / Chapter 4.2.4.1. --- Amplication of targeted sequence by PCR --- p.103 / Chapter 4.2.4.2. --- Purification of PCR product --- p.104 / Chapter 4.2.4.3. --- Ligation with cloning vector --- p.105 / Chapter 4.2.4.4. --- Transformation of E.coli DH5a cells --- p.105 / Chapter 4.2.4.5. --- Plasmid DNA isolation --- p.106 / Chapter 4.2.4.6. --- DNA quantitation and sequencing --- p.107 / Chapter 4.2.5. --- Quantitation determination using real-time PCR --- p.108 / Chapter 4.3. --- Results --- p.110 / Chapter 4.3.1. --- Determination of targeted sequences --- p.110 / Chapter 4.3.2. --- Reading of fluorescence intensity and data analysis --- p.110 / Chapter 4.3.3. --- Sensitivity of real-time PCR --- p.114 / Chapter 4.3.4. --- Specificity of real-time PCR --- p.121 / Chapter 4.3.5. --- Quantitation analysis in seeded samples --- p.121 / Chapter 4.4. --- Discussion --- p.131 / Chapter 5. --- Conclusion and future perspectives --- p.133 / Chapter 6. --- References --- p.138

Orientador: Luís Guilherme de Oliveira / Coorientador: Andressa de Souza Pollo / Banca: João Pessoa Araújo Junior / Banca: Hélio José Montassier / Resumo: O vírus da Diarreia Viral Bovina (BVDV), do gênero Pestivírus, pode ocasionar significativas perdas econômicas em bovinos. No entanto, este vírus também pode infectar os suínos, nos quais a infecção por BVDV poderá ocasionar sinais clínicos, afetar a diagnose da PSC (Peste Suína Clássica), além de torná-los fonte de disseminação viral. Tendo em vista esta problemática, este trabalho teve por objetivo verificar se leitões infectados experimentalmente por BVDV podem passar a excretar o vírus e se a disseminação viral pode ocorrer por via aerógena. Para tanto, foram realizados dois experimentos. Em cada experimento, seis leitões foram distribuídos em duplas em três câmaras de isolamento, ligadas por tubos com fluxo de ar contínuo, nas quais os animais introduzidos corresponderam ao controle, infectado e sentinela, respectivamente. Os animais da segunda câmara foram inoculados pela administração de inóculo de BVDV-1 estirpe Singer citopático por via nasal e oral. Foram avaliadas amostras de sangue, suabe nasal e retal, e no último dia foi realizada eutanásia e necropsia dos animais e coletada amostras de tecidos. No primeiro experimento, ambos os animais inoculados soroconverteram e apresentaram sinais clínicos da infecção, mas somente em um animal foi detectada excreção do vírus por via nasal e presença de RNA viral nos rins, baço e fígado, avaliadas por RT-PCR. Neste caso, a transmissão do vírus por via aerógena não foi comprovada, uma vez que o experimento foi finalizado um di... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The virus of Bovine Viral Diarrhea (BVDV), belonging to the Pestivirus genus, can cause significant economic losses in cattle. However, this virus can also infect swine, in which infection by BVDV may cause clinical signs, affect the diagnosis of CSF (Classical Swine Fever), and makes them a source of viral spread. Based on these issues, this study aimed to verify whether piglets experimentally infected by BVDV can shed the virus and whether viral airborne transmission is possible. For this, two experiments were carried out. In each experiment, six piglets were distributed in pairs in three isolation chambers, connected by continuous air flow tubes, in which the animals introduced corresponded to control, infected and sentinel, respectively. The animals present in the second chamber were inoculated with BVDV-1 strain Singer cytopathic inoculum by nasally and orally administration. Samples of blood, nasal and rectal swabs, and the last day was conducted euthanasia and necropsy of the animals and tissue samples were evaluated. In the first experiment, both inoculated animals seroconverted and showed clinical signs of infection, but only in one animal nasally virus shedding was detected, as well as, viral RNA presence in kidney, spleen and liver, both identified by RT-PCR. In this case, viral airborne transmission has not been confirmed, since the experiment was finalized one day after the viral shedding. In the second experiment, the two infected animals seroconverted with nine... (Complete abstract click electronic access below) / Mestre

A Brucella abortus causa diminuição da eficiência reprodutiva em bovinos e é também o agente de uma das zoonoses mais difundidas no mundo. A doença é alvo de programas de controle em muitos países e, no Brasil, seu combate foi melhor organizado a partir de 2001, com o lançamento de programa nacional pelo MAPA. O objetivo do presente estudo foi aperfeiçoar a detecção de B. abortus em homogeneizados de órgãos de fetos abortados por vacas infectadas. Assim, foram comparados diferentes protocolos de extração de DNA, visando à detecção de B. abortus pela PCR em amostras clínicas colhidas de fetos abortados ou bezerros oriundos de vacas experimentalmente desafiadas com B. abortus cepa 2308. Para tanto, foram construídos dois grupos padrão ouro com base na bacteriologia clássica, constituídos por: 32 pulmões (17 positivos ao isolamento), 26 baços (11 positivos ao isolamento), 23 fígados (8 positivos ao isolamento) e 22 linfonodos bronquiais (7 positivos ao isolamento). Todas essas amostras foram submetidas a três distintos protocolos de extração de DNA, seguidos do mesmo processo de amplificação com os primers B4 e B5. A análise dos resultados consolidados mostrou uma sensibilidade de 95% para o protocolo da proteinase K (PK), 86% para o do isotiocianato de guanidina (GT) e de 88% para o de Boom. Os valores encontrados de Sensibilidade para pulmão, baço, fígado e linfonodo foram, respectivamente, 100%, 100%, 100% e 71% (PK), 82%, 100%, 88% e 71% (GT) e 94%, 100%, 63% e 86% (Boom). No grupo dos animais dos quais não foi possível isolar brucellas (gold standard negativo), a PCR resultou positiva em 8 amostras para o protocolo de extração PK (4 pulmões, 1 fígado e 3 linfonodos), em 6 amostras para o protocolo de extração GT (1 pulmão, 3 fígados e 2 linfonodos) e em 37 amostras para o protocolo de extração Boom (10 pulmões, 10 baços, 10 fígados e 7 linfonodos). Esses resultados permitem afirmar que o protocolo de extração PK apresentou a melhor sensibilidade diagnóstica e que o protocolo de extração de Boom apresentou o melhor desempenho nas amostras onde o isolamento foi negativo. Dentre os órgãos estudados, o baço apresentou a maior probabilidade de detecção de B. abortus. Assim, a melhor estratégia para detecção de B. abortus em homogeneizados de órgãos de fetos abortados é a utilização do isolamento e da PCR em paralelo. / Infection by Brucella abortus diminishes the reproductive efficiency in bovines and it is also the agent of one of the most spread zoonosis in the world. In many countries control programs aim this disease, and in Brazil its combat was better organized since 2001 with the creation of the national program by the Ministry of Agriculture, Livestock and Supplies (MAPA). The objective of the present study was to improve the detection of B. abortus in aborted fetus homogenates from infected cows. For that reason, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetus or calves obtained from cows experimentally defied with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed by: 32 lungs (17 isolation positives samples), 26 spleens (11 isolation positives samples), 23 livers (8 isolation positives samples) and 22 bronchial lymph nodes (7 isolation positives samples). All samples were submitted to three distinct DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. The analysis of the consolidated results showed a 95% sensibility for the proteinase K protocol (PK), 86 % for the guanidine isotiocianate (GT) and 88% for the Boom. The sensibility values obtained for lungs, spleen, liver and lymph node were, respectively, 100%, 100%, 100% and 71% (PK), 82%, 100%, 88% and 71% (GT) and 94%, 100%, 63% and 86% (Boom). In the group of animals in which it was not possible to isolate brucellas (negative gold standard), the PCR resulted positively in 8 samples for the PK extraction protocol (4 lungs, 1 liver and 3 lymph nodes), in 6 samples for the GT extraction protocol (1 lung, 3 livers and 2 lymph nodes) and in 37 samples for the Boom extraction protocol (10 lungs, 10 spleens, 10 livers and 7 lymph nodes). These results permit affirming that the PK extraction protocol showed the best diagnostic sensibility and that the Boom extraction protocol showed the best performance in negative isolation samples. Within the studied organs, the spleen showed the highest probability of B. abortus detection. Therefore, the best strategy for B. abortus detection in organs homogenates from aborted fetus is the utilization of isolation and in parallel, the PCR.

Orientador: Henrique Nunes Oliveira / Coorientador: Márcia Cristina de Sena Oliveira / Banca: Marcos Rogério André / Banca: Janete Aparecida Desidério / Banca: Wilson Malago Junior / Banca: Fernanda de Freitas Anibal / Resumo: Entre as principais causas de perdas produtivas em bovinos criados nos trópicos está a infestação pelo carrapato Rhipicephalus (Boophilus) microplus e, consequentemente, dos hemoparasitas transmitidos por ele. A resistência dos zebuínos e de animais cruzados com raças taurinas à infestação por esse ácaro é amplamente conhecida. Entretanto, no que se refere à suscetibilidade às babesioses bovinas, existem evidências de que o grupo genético também pode interferir na resistência, seguindo o mesmo padrão observado para o carrapato vetor, com os taurinos apresentando maior sensibilidade. Assim, este estudo teve por objetivo avaliar a parasitemia porBabesia bovis e Babesia bigemina em 50 novilhas da raça Canchim ( Charolês + Zebu) naturalmente infestadas pelo R. (B.) microplus nas quatro estações do ano durante 24 meses, além de caracterizar o perfil de citocinas que podem estar associados ao fenótipo de resistência e suscetibilidade aos hemoparasitas do gênero Babesia spp. Foram realizadas contagens de fêmeas adultas de carrapatos com tamanho igual ou superior a 4,5 mm de diâmetro, presentes no lado esquerdo de cada bovino. As amostras de DNA extraídas foram submetidas à amplificação por meio da Reação em Cadeia daPolimerase Quantitativa em Tempo Real (qPCR), utilizando iniciadores que flanqueiam fragmentos dos genes mitocondriais do citocromo b (mt-cyt B), específicos para B. bovis e B. bigemina. O RNA extraído do sangue, foi usado para sintetizar o DNA complementar (cDNA) ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Among the main causes of production losses in cattle is in the tropics infestation by Rhipicephalus (Boophilus) microplus, and consequently the hemoparasites transmitted by it. The resistance of zebu and crossbred with European breeds to infestation by this mite is widely known. However, as regards susceptibility to bovine babesiosis, there is evidence that genetic group can also interfere in resistance following the same pattern observed in the tick vector, with the taurine presenting greater sensitivity. This study aimed to evaluate parasitaemia by Babesia bovis and Babesia bigemina in 50 heifers Canchim (⅝ Charolais + ⅜ Zebu) naturally infested by R. (B.) microplus in four seasons for 24 months, and characterize the profile of cytokines that may be associated with phenotype resistance and susceptibility by gender hemoparasites Babesia spp. Adult female ticks counts with size equal to or greater than 4.5 mm in diameter, present in the left side of each calf were performed. The extracted DNA samples were subjected to amplification by Reaction Polymerase Chain Quantitative Real Time (qPCR), using primers flanking fragments of mitochondrial gene cytochrome B (mt-cyt B) specific for B. bovis and B. bigemina. The RNA extracted from the blood was used to synthesize complementary DNA (cDNA) for expression analysis of genes IFN-γ, TNF-α, IL-10 and IL-12B by relative quantification (RT-qPCR). Significant differences were observed (P <0.05) between the months of reviews for the tick ... (Complete abstract click electronic access below) / Doutor

Orientador: Marcelo Vasconcelos Meireles / Banca: Sérgio Diniz Garcia / Banca: Weslen Fabrício Pires Teixeira / Resumo: Este trabalho teve como objetivos determinar a ocorrência e realizar a caracterização molecular de Cryptosporidium spp. e comparar três métodos de detecção deste protozoário em amostras fecais de canários (Serinus canaria) criados em cativeiro nas regiões Sul e Sudeste do Brasil. Um total de 498 amostras foi purificado por centrífugo-flutuação em solução de Sheather. A detecção de Cryptosporidium spp. foi realizada utilizando três métodos de diagnóstico: análise microscópica pela coloração negativa com verde malaquita, nested PCR (gene 18S rRNA), seguida de sequenciamento dos fragmentos amplificados, e PCR em duplex em tempo real (gene 18S rRNA) específica para detecção de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A positividade para Cryptosporidium spp. (total de amostras positivas em pelo menos um método de diagnóstico) obtida pela análise microscópica, nested PCR e PCR duplex em tempo real foi de 13,3% (66/498). As taxas de positividade para Cryptosporidium spp. foram 2,0% (10/498) e 4,6% (23/498) por microscopia e nested PCR, respectivamente. O sequenciamento de 20 amostras amplificadas pela nested PCR identificou C. galli (3,0%;15/498), Cryptosporidium genótipo I de aves (0,8%; 4/498) e Cryptosporidium avium (0,2%; 1/498). A PCR duplex em tempo real revelou positividade de 7,8% (39/498) para C. galli e 2,4% (12/498) para Cryptosporidium genótipo III de aves. A análise microscópica diferiu significativamente da nested PCR para detecção de Cryptosporidi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp.. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries. / Mestre

Considerable effort has recently been directed toward the miniaturization of quantitative-polymerase-chain-reaction [QPCR] instrumentation in an effort to reduce both cost and form factor for point-of-care applications. Notable gains have been made in shrinking the required volumes of PCR reagents, but resultant prototypes retain their bench-top form factor either due to heavy heating plates or cumbersome optical sensing instrumentation. In this thesis, we describe the use of complementary-metal-oxide semiconductor (CMOS) integrated circuit (IC) technology to produce a fully integrated qPCR lab-on-chip. Exploiting a 0.35-µm high-voltage CMOS process, the IC contains all of the key components for performing qPCR. Integrated resistive heaters and temperature sensors regulate the surface temperature of the chip to 0.45°C. Electrowetting-on-dielectric microfluidic pixels are actively driven from the chip surface, allowing for droplet generation and transport down to volumes of less than 1.2 nanoliters. Integrated single-photon avalanche diodes [SPAD] are used for fluorescent monitoring of the reaction, allowing for the quantification of target DNA with more than four-orders-of-magnitude of dynamic range with sensitivities down to a single copy per droplet. Using this device, reliable and sensitive real-time proof-of-concept detection of Staphylococcus aureus (S. aureus) is demonstrated.

Os membros da sub-famíla Toxoplasmatinae conhecidos são Hammondia hammondi, Toxoplasma gondii, Neospora hughesi, Neospora caninum e Hammondia heydorni. As duas primeiras espécies têm os felídeos como hospedeiro definitivo, enquanto as duas últimas têm desenvolvimento sexual em carnívoros da família dos canídeos. O ciclo biológico de N. hughesi é pouco conhecido. Foi estudado a variabilidade nucleotídica de seqüências intercaladas entre os genes codificadores das frações ribossômicas 18S, 5.8S (ITS-1). No entanto, como estas não permitem reconstruções filogenéticas com o uso de grupo externo, em virtude da inconsistência dos alinhamentos produzidos, pesquisamos uma seqüência codificadora de proteína sendo o marcador escolhido, o gene codificador da proteína de choque térmico HSP70 (heat shock protein 70KDa). Este gene é bastante utilizado para resolução de filogenias de outros organismos como, por exemplo, aqueles pertencentes ao gênero Cryptosporidium. No presente trabalho, amplificamos por PCR e seqüenciamos 951 pares de bases (pb) do gene codificador de HSP70 de oocistos T. gondii-like (oriundos de fezes gatos), de oocistos Neospora-like (de fezes de cães) e de taquizoitos de N. caninum, N. hughesi e T. gondii mantidos em laboratório. Os primers foram desenhados a partir de seqüências consenso obtidas em pesquisa de bancos de dados de seqüências EST de N. caninum e seqüências de RNAm de T. gondii. Seqüências ITS-1 destes oocistos também foram determinadas para a confirmação da espécie de parasito estudada. Os resultados mostram que os táxons H. hammondi e T. gondii são monofiléticos e geneticamente muito próximos, mas contrariando resultados anteriores, não foi demonstrada a monofilia entre os táxons H. heydorni e N. caninum. De fato, a análise de diversidade nucleotídica de gene codificador HSP70 mostra que a distância evolutiva entre H. heydorni e N. caninum é tão grande quanto a distância de cada uma destas espécies com T. gondii. Em adição, foi possível identificar dentre as amostras de oocistos, duas linhagens divergentes de H. heydorni. Paralelamente ao estudo filogenético também foi possível desenvolver um método diagnostico diferencial para oocistos tipo Hammondia. As seqüências de gene HSP70 obtidas foram alinhadas e dois pares de primers internos a estas seqüências foram desenhados. O primeiro par amplifica 771 pb de oocistos T. gondii-like e o segundo 400 pb de oocistos Neospora-like. A clivagem do fragmento de 771 pb com enzima de restrição Hin6I produz perfis eletroforéticos distintos para amostras de T. gondii e H. hammondi. A clivagem do fragmento de 400pb com a enzima de restrição MunI também produz perfis eletroforéticos distintos entre amostras de N. caninum e H. heydornii. A diferenciação dos perfis de restrição pode ser feita em eletroforese em gel de agarose a 2,5%. / Hammondia hammondi, Toxoplasma gondii, Neospora huguesi, Neospora caninum and Hammondia heydorni are the known members of the sub-family Toxoplasmatinae. H. heydorni and N. caninum use canids as definitive hosts whereas felids are the definitive hosts from T. gondii and H. hammondi. The definitive host of N. hughesi is unknown. Here, the nucleotide diversity at internal transcribed spacer (ITS-1) and heat shock protein (HSP70 kDa) loci in the subfamily toxoplasmatinae were studied. The HSP70 coding genes are widely used for phylogenetic studies in a number of other organisms specially within the genus Cryptosporidium. In the present study, it was amplified by PCR and sequenced 951 bases pairs (bp) from HSP70 coding gene from oocysts T.gondii-like (from cat), from oocysts N. caninum-like (from dog) and tachyzoites of N. hughesi grown on cell cultures. The primers were designed based on consensus sequences within EST sequences of N. caninum sequences and RNAm sequences of T. gondii. ITS-1 sequences amplified from oocysts were also obtained in order to confirm the species of the parasites. The results showed that T. gondii and H. hammondi are monophyletic and genetically very close, but the monophyletic status of H. heydorni N. caninum was not demonstrated. In fact, the nucleotide diversity at the HSP70 locus has shown that the evolutionary distance between H. heydorni and N. caninum is as high as that observed between either H. heydorni or N. caninum and T. gondii., In addition, it was possible to identify two distinct groups among the H. heydorni oocysts. Concomitantly to the phylogenetic study it was also possible to standardize a diagnostic test capable of differentiate the oocysts Hammondia-like was development a diagnostic method that differ oocysts T. gondii-like and N. caninum-like. The sequences obtained from HSP70 coding gene were aligned and two new pair of PCR primers was designed. The first pair amplifies 771bp from T. gondii-like oocysts, whereas the second pair amplifies 400 bp from N. caninum-like oocysts. The restriction enzyme Hin6I used to cleave the 771 bp amplicons generated distinct profiles with samples from H. hammondi and T. gondii. The same occurred with the restriction of the fragments of 400bp cleaved by the enzyme MunI used in N. caninum e H. heydorni samples. The profiles can be differentiated by 2.5% agarose gel electrophoresis.