Erysipelothrix rhusiopathiae, a Gram-positive bacillus, has long been an important pathogen in veterinary medicine as well as a cause of serious disease in humans. Infections caused by this organism have economic impact on animal industries, causing erysipelas in swine and morbidities in other farmed animals. Human infections are commonly erysipeloid (skin cellulitis) and occasionally septicaemia or endocarditis. Little is known of the diagnosis, epidemiology and pathogenesis of such infections in Western Australia. The aims of this thesis were to establish new diagnostic techniques for the detection and recovery of E. rhusiopathiae, to describe the epidemiology of Erysipelothrix infection in Western Australia in humans and animals, and to characterize virulence-associated characteristics, especially focusing on the neuraminidase produced by the organism. A protocol using 48 h Brain Heart Infusion enrichment followed by subculture to selective agar containing antibiotics achieved the highest recovery rate of 37% in a seafood survey. Twentyone isolates of Erysipelothrix spp., of which 19 were identified as E. rhusiopathiae, were obtained. Two published PCR assays for differentiating E. rhusiopathiae and other Erysipelothrix species were evaluated and the best PCR detection rate achieved was 67% following selective enrichment. The PCR method was 50% more sensitive than the culture method. Epidemiological surveys using the above methods showed that E. rhusiopathiae infection is present in farmed animals in Western Australia. The PCR positive frequencies (3.3-3.7%) and isolate recovery rate (2.8-3.3%) in samples from pig and sheep abattoirs and carcass washings indicate a potential threat to the economy of the farmed animal industry as well as a public health concern with the occurrence of E. rhusiopathiae in meat for consumption. Positive PCR results (1.1%) from human skin swabs of patients with cellulitis and wounds may suggest the existence of Erysipelothrix colonization in the general population. Genetic relatedness of 92 isolates of Erysipelothrix species from various sources was analyzed and a total of 64 distinct PFGE patterns identified. Isolates were further classified into 20 clonal groups based on pattern similarities, and most E. rhusiopathiae were clustered into six groups. A few patterns of other Erysipelothrix species were clustered into separate groups from E. rhusiopathiae but shared greater than 70% similarity with E. rhusiopathiae. The genetic relatedness of colonial variants was well demonstrated using this method. PFGE typing promises to be a useful tool for epidemiological and taxonomic studies of Erysipelothrix. Several virulence-associated factors were characterized in 86 isolates of Erysipelothrix spp. A rapid and sensitive peanut lectin hemagglutination assay for neuraminidase was developed and the influence of media, incubation conditions and pH on the production of the enzyme was investigated. All 61 isolates of E. rhusiopathiae produced neuraminidase in cooked meat broth with titres between 1:10 and 1:320, with no significant difference in titre among isolates from different sources. The enzyme activity was not detected in non-pathogenic Erysipelothrix spp. Capsule was produced by 78.7% of isolates of E. rhusiopathiae but not by other species, while both hyaluronidase and haemolysin were produced by non-pathogenic Erysipelothrix spp. It was concluded that neuraminidase and capsule are most likely to be virulence factors of E. rhusiopathiae. The gene encoding neuraminidase was cloned from the type strain E. rhusiopathiae ATCC 19414. The cloned fragment was a functional partial nanH gene with a mol% G+C of 39.7. The predicted amino acid sequence displayed homology with many microbial neuraminidases and contained conserved sequences found in most bacterial neuraminidases. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial genomic DNA. A neuraminidasenegative mutant vector was constructed by insertional inactivation using a tetM cassette. This has provided starting material for developing a neuraminidase-deficient E. rhusiopathiae mutant, which will permit the study of the role of neuraminidase in pathogenesis. Based on the cloned sequence, a sensitive neuraminidase-specific nested PCR technique was designed and optimized. The specificity was tested in 61 isolates of E. rhusiopathiae, 25 Erysipelothrix species, and 62 other species of neuraminidaseproducing and non-producing bacteria. All isolates of E. rhusiopathiae were PCR positive and all other bacteria were negative; thus this PCR is a highly specific method suitable for application in clinical investigations of Erysipelothrix infection. In conclusion, the present study has contributed new knowledge of the biology of Erysipelothrix spp. and current occurrence of Erysipelothrix infections in Western Australia, as well as to the understanding of pathogenesis of E. rhusiopathiae. Development of several new cultural and molecular approaches in combination with other established techniques will facilitate future studies of the epidemiology, taxonomy and pathogenesis of this bacterial species.

Thesis (M.S.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb. 19, 2009). Includes bibliographical references (p. 35-44).

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Made available in DSpace on 2014-06-11T19:30:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-11-24Bitstream added on 2014-06-13T20:00:40Z : No. of bitstreams: 1 baeza_lc_dr_arafcf.pdf: 2250737 bytes, checksum: 666c7d374ebfa4ff8507fbfbe951a4b8 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / As dermatofitoses são processos infecciosos de pele, pêlo e unhas muito comuns no mundo inteiro. Trichophyton rubrum é o dermatófito mais freqüentemente isolado em lesões superficiais de pele e unha. Estudos envolvendo este patógeno são cada vez mais importantes devido ao aparecimento de linhagens resistentes aos medicamentos antifúngicos disponíveis no mercado e ao comportamento invasivo deste agente em pacientes com o sistema imune comprometido. Estes fatos e poucos estudos levam à necessidade de se ampliar o conhecimento sobre a biologia deste agente. Visando colaborar nesse sentido, o presente trabalho teve dois objetivos centrais: 1) Realizar a tipagem molecular de isolados clínicos de T. rubrum com e sem relação epidemiológica por RAPD (Amplificação Randômica de DNA Polimórfico) com duas seqüências randômicas diferentes (denominadas de 1 e 6), bem como a análise dos elementos repetitivos (TRS-1 e TRS-2) do espaço não transcrito (NTS) do DNA ribossomal (DNAr) e 2) Identificar transcritos envolvidos na interação deste patógeno com fonte humana de queratina, através do RDA (Análise de Diferença Representacional). A aplicação do RDA permitiu pela primeira vez a identificação de alguns transcritos expressos, provavelmente relacionados à patogênese deste microrganismo. / Dermatophytosis is common infection process which occurs in skin, hair and nails and Trichophyton rubrum is the most frequently isolated dermatophyte. Studies on this pathogen are becoming increasingly important because of frequent reports on resistant strains to antifungal drugs commercially available and the invasive behavior of these agents in immunocompromised patients. These facts, associated with few studies with this agent, indicate the need to expand the information about the fungal biology. As a contribution to this goal, the present study had two central objectives: 1) To compare different methodologies for molecular typing of clinical isolates of T. rubrum epidemiologically related and unrelated for RAPD (Randomly Amplified Polymorphic DNA) with two different random primers (denominated of 1 and 6); as well as the analysis of the repetitive elements (TRS-1 and TRS-2) of the space non-transcribed (NTS) of the ribosomal DNA (DNAr) and 2) To identify transcripts involved in the interaction of this pathogen with human keratin by RDA (Representational Difference Analysis). The application of the RDA allowed for the first time the identification of expressed transcripts during the microorganism proliferation that could be related to the T. rubrum virulence.

Orientador: Maria José Soares Mendes Giannini / Banca: Clarice Queico Fujimura Leite / Banca: Celia Maria de Almeida Soares / Banca: Paulo Inácio da Costa / Banca: Mário Hiroyuki Hirata / Resumo: As dermatofitoses são processos infecciosos de pele, pêlo e unhas muito comuns no mundo inteiro. Trichophyton rubrum é o dermatófito mais freqüentemente isolado em lesões superficiais de pele e unha. Estudos envolvendo este patógeno são cada vez mais importantes devido ao aparecimento de linhagens resistentes aos medicamentos antifúngicos disponíveis no mercado e ao comportamento invasivo deste agente em pacientes com o sistema imune comprometido. Estes fatos e poucos estudos levam à necessidade de se ampliar o conhecimento sobre a biologia deste agente. Visando colaborar nesse sentido, o presente trabalho teve dois objetivos centrais: 1) Realizar a tipagem molecular de isolados clínicos de T. rubrum com e sem relação epidemiológica por RAPD (Amplificação Randômica de DNA Polimórfico) com duas seqüências randômicas diferentes (denominadas de 1 e 6), bem como a análise dos elementos repetitivos (TRS-1 e TRS-2) do espaço não transcrito (NTS) do DNA ribossomal (DNAr) e 2) Identificar transcritos envolvidos na interação deste patógeno com fonte humana de queratina, através do RDA (Análise de Diferença Representacional). A aplicação do RDA permitiu pela primeira vez a identificação de alguns transcritos expressos, provavelmente relacionados à patogênese deste microrganismo. / Abstract: Dermatophytosis is common infection process which occurs in skin, hair and nails and Trichophyton rubrum is the most frequently isolated dermatophyte. Studies on this pathogen are becoming increasingly important because of frequent reports on resistant strains to antifungal drugs commercially available and the invasive behavior of these agents in immunocompromised patients. These facts, associated with few studies with this agent, indicate the need to expand the information about the fungal biology. As a contribution to this goal, the present study had two central objectives: 1) To compare different methodologies for molecular typing of clinical isolates of T. rubrum epidemiologically related and unrelated for RAPD (Randomly Amplified Polymorphic DNA) with two different random primers (denominated of 1 and 6); as well as the analysis of the repetitive elements (TRS-1 and TRS-2) of the space non-transcribed (NTS) of the ribosomal DNA (DNAr) and 2) To identify transcripts involved in the interaction of this pathogen with human keratin by RDA (Representational Difference Analysis). The application of the RDA allowed for the first time the identification of expressed transcripts during the microorganism proliferation that could be related to the T. rubrum virulence. / Doutor

Made available in DSpace on 2015-07-22T20:49:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A metodologia do “Multilocus Sequence Typing (MLST)” tornou-se uma ferramenta importante para tipagem molecular para C. albicans porque esta metodologia pode caracterizar grande número de isolados rapidamente e está isenta da interpretação subjetiva de padrões de bandas em géis de eletroforese. Este método é muito útil no entendimento da filogenia e epidemiologia de cepas de C. albicans recuperadas de infecções fúngicas invasivas. Objetivos: 1) aplicar as metodologias de MLST e Tipagem ABC para isolados de C. albicans recuperados de infecções de corrente sanguínea em hospitais terciários no Brasil e 2) determinar se cepas indistinguíveis ou diferentes foram responsáveis pelos episódios de candidemia persistente ou candidemia recorrente em isolados sequenciais de mesmo paciente. Material e Métodos: Nós aplicamos a metodologia do MLST e Tipagem ABC em isolados de C. albicans de 61 pacientes com candidemia coletados durante um estudo multicêntrico realizado em 11 hospitais públicos terciários de 9 cidades brasileiras. Também foram avaliados os isolados sequenciais de 8 pacientes com candemia persistente ou recorrente. Candidemia persistente foi definido como um episódio de fungemia com duas ou mais culturas positivas para C. albicans, em 2 ou mais dias diferentes, a despeito da contínua terapia antifúngica adotada. Candidemia recorrente foi definida como um episódio de candidemia ocorrendo ao menos 1 mês após o episódio incidente e a negativação de duas hemoculturas sequenciais após introdução da terapia antifúngica, envolvendo a mesma espécie de Candida. Resultados: Um total de 48 únicos “diploid sequence types (DSTs)” foram observados, incluindo 10 novos genótipos e 32 novos DSTs. DST 69 foi o mais comum entre os nossos isolados. Isolados clado 1 responderam a 56% da nossa coleção. O clado 3 e clado 8 foram os clados com maior número de isolados depois de clado 1, ambos respondendo por 10% das amostras. O clado 9 e clado 17 foram responsáveis por 6,5% dos isolados cada um. Isolados clado 12 responderam por 5%. Foi isolada uma única cepa (1,5%) do clado 2, clado 4, clado 16 e um isolado categorizado como “solitário”. Para Tipagem ABC, 82% dos isolados foram classificados como tipo A, seguido por tipo B com 16,5% e tipo C com 1,5%. Quanto aos pacientes com candidemia persistente ou recorrente, para todos os pacientes exceto um, verificou-se a permanência dos mesmos DSTs encontrados entre a primeira e última amostra coletada. Um único paciente com coletas sequenciais pelo período de 10 dias apresentou 3 cepas distintas discriminadas pelo MLST. Uma destas 3 cepas foi a única representante do clado 2 em nosso estudo. Conclusão: Mais de 50% dos isolados deste estudo apresentaram novos DSTs, predominando o clado 1 em 56% das amostras. Para a Tipagem ABC, 82% dos isolados foram do tipo A. Este é o primeiro estudo de nosso conhecimento a descrever infecção de corrente sanguínea por 3 cepas distintas de C. albicans documentadas no período de 10 dias. / The DNA sequence-based genotyping technique multilocus sequence typing (MLST) has emerged as an alternative typing tool for C. albicans because can characterize large numbers of isolates rapidly, and does not require the subjective interpretation of banding patterns. This methodology is a very useful tool in understanding the phylogenetics and epidemiology of C. albicans strains from invasive candidiasis. Objective: Our goal was 1) to apply MLST and ABC typing to C. albicans strains recovered from bloodstream infection from public tertiary care hospitals in Brazil and 2) determine whether indistinguishable or different strains were responsible for persistent or recurrent fungemia by performing MLST and ABC typing on sequential C. albicans isolates from the same patient. Methods: We applied MLST and ABC typing, which is based on the presence or absence of an intron in the 25S rDNA region, to C. albicans strains from 61 patients with candidemia collected during a multicenter surveillance study in 11 public tertiary care hospitals, representative of the public health system of 9 of the largest cities in Brazil. We also analyzed C. albicans strains from 8 patients with persistent or recurrent candidemia. Persistent candidemia was defined as two or more blood cultures positive for C. albicans on 2 or more separate days. Recurrent candidemia was defined as an episode of candidemia occurring at least 1 month after the apparent complete resolution of an infectious episode caused by the same Candida species. Results: A total of 48 unique profiles or diploid sequence types (DST) were observed, with 10 new sequence types (STs) and 32 new DSTs. DST 69 was the most common DST isolated. C. albicans clade 1 accounted for 56% of the collection, clade 3 and clade 8 for 10% each, clades 9 and 17 for 6.5% each, and clade 12 for 5%. Clade 2, clade 4, clade 16 and a singleton strain had 1 isolate each (1.5%). For ABC typing, 82% of the isolates were classified as type A, followed for 16.5% type B and 1.5% type C. All the patients’ strains related to persistent or recurrent candidemia but one showed the same MLST diploid sequence type (DST), ABC type and susceptibility profile to antifungals in the first and second samples. One patient with 7 samples collected sequentially over 10 days showed 3 distinct strains, well discriminated by MLST. One of the 3 strains recovered from this patient showed a single C. albicans isolate found in our total collection classified as clade 2, although clade 2 is commonly found worldwide. Conclusion: More than 50% of isolates from this study form a unique set of DSTs and clade 1 was responsible for 56% of the isolates. For ABC typing, 82% of the isolates were type A. To the best of our knowledge, this is the first study describing a blood stream infection with 3 distinct C. albicans strains in the same patient within a short period of time. / CNPq: GM/GD 142025-2005-4 / CNPq: SWE 200669/2007-9 / TEDE / BV UNIFESP: Teses e dissertações

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No. of bitstreams: 1 franchi_eplp_dr_bot.pdf: 1593298 bytes, checksum: 660e47b876115bc8cd5e60c3800f6ecd (MD5) Previous issue date: 2016-02-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Diante da importância de S. aureus resistente à meticilina (Methicillin-resistant Staphylococcus aureus - MRSA) em feridas, este estudo objetivou estudar a prevalência, fatores de risco e epidemiologia molecular de S. aureus coletados de feridas e narinas de pacientes atendidos nas 17 Unidades Básicas de Saúde do município de Botucatu-SP, Brasil. Após a identificação dos isolados de S. aureus, foram realizados: teste de susceptibilidade à 13 drogas antimicrobianas, identificação do gene de resistência (mecA) e dos genes codificadores da Leucocidina PantonValentine- PVL (pvl), enterotoxinas A-E (sea, seb, sec, sed, e see), hemolisinas α, β e δ (hla, hlb e hld), esfoliatinas A, B e D (eta, etb e etd), biofilme (icaAD) e Toxina-1 da Sindrome do choque tóxico – TSST-1 (tst); tipagem molecular por Pulsed-Field Gel Eletroforese (PFGE), Multilocus sequence typing (MLST) e spa typing. Foram incluídos 171 pacientes, dos quais foram isolados 119 S. aureus. Amostras nasais foram coletadas apenas em 74 pacientes do total estudado. A prevalência de S. aureus e MRSA foi de 51,5% e 8,7%, respectivamente. No geral foram isolados 101 MSSA de 73 pacientes, destes 98 foram isolados de feridas e 21 de narinas; e 18 MRSA de 15 pacientes, sendo 4 isolados de narinas e 14 de feridas, com 6 MRSA com SCCmec tipo II e 12 com SCCmec tipo IV. Os isolados mostraram alto nível de resistencia a penicilina (85%), seguido pela eritromicina (27%), gentamicina (12%), clindamicina (11%), e levofloxacina (6%). Não houve resistência ao sulfametoxazol/trimetoprim, ácido fusídico, tigeciclina, quinupristina/dalfopristina e linezolida. A pesquisa por genes de virulência nos 119 isolados de S. aureus sensíveis e resistentes demonstrou que 42% possuem genes para enterotoxina A, 11% para enterotoxina B, 26% para enterotoxina C e 0,8% para enterotoxina D, 100% para o gene icaA, 95,8% para o gene icaD, 97,5% para o gene da hemolisina alfa, 65% para hemolisina beta, 95% para hemolisina delta, 4,2% para TSST-1 e 2,5% para o gene da PVL. Houve associação entre a presença de S. aureus nas narinas a nas feridas (p<0,01), o mesmo ocorreu para MRSA (p<0,01). A análise multivariada para S. aureus, demonstrou associação negativa com idade (OR: 0,94, IC95%: 0,90-0,98, p<0,01), uso de amoxicilina (OR: 0,16, IC95%: 0,04-0,60, p<0,01) e de ciprofloxacina (OR: 0,28, IC95%: 0,08-0,98, p=0,04). Por outro lado, observou-se associação positiva com uso de benzilpenicilina (OR:3,81, IC95%:1,23-11,82, p=0,02). Houve a formação de oito clusters, com predominância de MSSA que apresentaram as STs: 5, 30, 188, 1635 e spa t002. Os 18 MRSA foram caracterizados pelos STs: 5, 8 e 1176 e pelos spas t002, t008 e t062. Foram isoladas linhagens semelhantes aos clones internacionais USA300, USA500 e USA800. Nossos resultados demonstram a presença de clones importantes de MRSA resistentes e virulentos em pacientes atendidos nas diferentes UBSs estudadas. / Given the importance of methicillin resistant S. aureus (Methicillin-resistant Staphylococcus aureus - MRSA) and wounds, this study aimed to study the prevalence, risk factors and molecular epidemiology related to the presence of S. aureus sensitive and resistant to methicillin in wounds of patients who attended BHUs in a city in Sao Paulo state, Brazil. After the identification of S. aureus isolates was performed: susceptibility testing to 13 antimicrobial drugs, identification of the resistance gene (mecA) and the genes encoding Panton-Valentine Leukocidin - PVL (pvl), enterotoxins AE (sea , seb, sec, sed and see) hemolysins α, β and δ (hla, hlb and hld), esfoliatinas A, B and D (eta, etb and etd), biofilm (icaAD) and Toxin-1 syndrome of toxic shock - TSST-1 (tst); Molecular typing by Pulsed-Field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and spa typing. 171 patients were included and 119 S. aureus isolates. Nasal samples were collected only in 74 patients of the total sample. The prevalence of S. aureus and MRSA was 51.5% and 8.7%, respectively. Overall 101 MSSA were isolated from 73 patients, 98 of these were isolated from wounds and 21 nostrils; MRSA and 18 of 15 patients, 4 isolates from nostrils and 14 wounds, with six MRSA with SCCmec type II and 12 SCCmec type IV. The strains showed high-level resistance to penicillin (85%) followed by erythromycin (27%), gentamicin (12%), clindamycin (11%) and levofloxacin (6%). There was no resistance to sulfamethoxazole / trimethoprim, fusidic acid, tigecycline, quinupristin / dalfopristin and linezolid. The search for virulence genes in the 119 isolates of S. aureus sensitive and resistant, 42% demonstrated presence of genes sea, 11% to seb, 26% to sec , 0.8% to sed, 100% to icaA, 95.8% icaD, 97.5% to hla, 65% to hlb, 95% to hld, 4.2% to tst and 2.5% to pvl. There was an association between the presence of S. aureus in the nostrils to the wounds (p<0.01), the same was true for MRSA (p<0.01). Multivariate analysis for S. aureus, showed a negative association with age (OR: 0.94, 95% CI: 0.90-0.98, p<0.01), use of amoxicillin (OR: 0.16, 95% CI: 0.04-0.60, p<0.01) and ciprofloxacin (OR: 0.28, 95% CI: 0.08-0.98, p=0.04). On the other hand, there was a positive association with use of penicillin G (OR: 3.81, 95% CI: 1.23-11.82, p=0.02). There was the formation of eight clusters, with a predominance of MSSA presenting the STs: 5, 30, 188, 1635 and spa t002. The 18 MRSA were characterized by STs 5, 8 and spas 1176 and the t002, t008 and t062. Strains were isolated similar to international clones USA300, USA500 and USA800. Our results demonstrate the presence of important clones resistant and virulent MRSA patients studied in different UBS. / FAPESP: 2011/10146-7 / FAPESP: 2012/00257-9 / FAPESP: 2013/10975-9

Within the ABO system there are several blood subgroups: subgroup A, subgroup B and subgroup H, and the most frequently encountered in practice are the subgroups A1, A2, A1B and A2B. The cells of, approximately, 80% of adults in group A are A1. The remaining 20% are A2 or weaker subgroups. However, in newborns is very little literature about the frequency of the subgroups of A. At birth most of the blood group A infants seems to present itself as belonging to subgroup A2, since all the ABO antigens are not fully developed in this period. Since iron deficiency, widespread in this age group, often discussed by the scientific community is related to the disproportion between the expansion of erythroid mass and iron obtained from the diet. Around four months of age, iron stores are reduced by half, and the exogenous iron is required to maintain hemoglobin concentration during this phase of rapid growth, between four and 12 months. Our objectives were to determine the frequency of newborns belonging to subgroups A1 and A2, to identify the frequency of antigen expression A1, between six and 12 months of age, infants initially typed as belonging to subgroup A2 and check the hemoglobin levels for the detection of anemia in these children. The results showed that the frequency of newborn belonging to the A1 blood subgroup was 67% (319) and the A2 subgroup was 33% (152), from a total of 471 newborns belonging to blood group A. We found a great predominance of the A1 subgroup, contradicting the literature that reports the prevalence of subgroup A2 in newborn infants. Regarding the identification of the frequency of A1antigen expression, between six and 12 months of age (n = 40), the percentage of children who express the A1 antigen, after being with six months to one year of age was 67.5% (27), the rest remained as A2 (13). The verification of hemoglobin levels in these children (n = 71), by the method of cianometa-hemoglobin, resulting in 34% of anemic children, pointing to the presence of anemia in this age group. The rates found ranged from 6.56 g/dl to 10.8 g/dl. Thus, in relation to A1 antigen expression between six and 12 months of age further studies are needed, and for the prevalence of anemia is necessary to emphasize in public health programs, intervention measures and more effective control of this nutritional disorder. / Dentro do sistema ABO existem diversos subgrupos sanguíneos: subgrupos A, subgrupos B e subgrupo H, sendo que os mais frequentemente encontrados na prática são os subgrupos A1, A2, A1B e A2B. As células de, aproximadamente, 80% da população adulta do grupo A são A1. Os 20% restantes são A2 ou subgrupos mais fracos. Porém, em recém-nascidos é muito escassa a literatura a respeito de dados quanto à frequência dos subgrupos de A . A maioria dos lactentes do grupo sanguíneo A parece apresentar-se como pertencente ao subgrupo A2, no nascimento, já que todos os antígenos ABO não estão completamente desenvolvidos neste período. Já a carência de ferro, generalizada nesse grupo etário, muitas vezes discutida pela comunidade científica, é relacionada à desproporção entre a expansão da massa eritróide e o ferro obtido da dieta. Por volta dos quatro meses de idade, os estoques de ferro estão reduzidos pela metade, e o ferro exógeno é necessário para manter a concentração de hemoglobina durante esta fase de rápido crescimento, entre quatro e 12 meses. Os objetivos deste trabalho foram determinar a frequência de recém-nascidos pertencentes aos subgrupos A1 e A2; identificar a frequência da expressão do antígeno A1, no período entre seis e 12 meses de idade, em lactentes inicialmente tipados como pertencentes ao subgrupo A2 e verificar os níveis de hemoglobina para a detecção de anemia nestas crianças. Os resultados mostraram que a frequência de recém-nascidos pertencentes ao subgrupo sanguíneo A1 foi de 67% (319) e para o subgrupo A2 foi de 33% (152), de um total de 471 recém-nascidos pertencentes ao grupo sanguíneo A. Constatou-se uma grande predominância do subgrupo A1, contrariando a literatura que relata a prevalência de subgrupo A2 em recém-nascidos. Em relação à identificação da frequência da expressão do antígeno A1, no período entre seis e 12 meses de idade (n=40), a porcentagem de crianças que passaram a expressar o antígeno A1, depois de estarem com seis meses a um ano de idade foi de 67,5% (27), o restante permaneceu como A2 (13). A verificação dos níveis de hemoglobina nestas crianças (n=71), através do método da cianometa-hemoglobina, resultou em 34% de crianças anêmicas, apontando a presença de anemia para essa faixa etária. Os índices encontrados variaram de 6,56g/dl a 10,8g/dl. Assim, em relação à expressão do antígeno A1 entre seis e 12 meses de idade são necessários mais estudos; e, para a prevalência da anemia é necessário enfatizar, nos programas de saúde pública, medidas de intervenção mais eficazes e controle desse distúrbio nutricional.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus pneumoniae é um importante agente etiológico de infecções invasivas e não invasivas, incluindo meningite, pneumonia e otite média. A cápsula polissacarídica é o principal fator de virulência desse microrganismo, sendo também considerada um importante marcador em estudos epidemiológicos. Dentre os mais de 90 tipos capsulares conhecidos, o sorotipo 14 se destaca pela prevalência elevada em várias regiões, inclusive no Brasil. A avaliação da diversidade genética desse microrganismo também inclui a aplicação de métodos moleculares, como PFGE e MLST. Entretanto, essas metodologias são relativamente onerosas, consomem muito tempo e os resultados obtidos com a técnica de PFGE são de difícil comparação entre diferentes laboratórios. A técnica de análise do polimorfismo numérico de segmentos repetitivos em múltiplos loci [MLVA, do inglês Multiple Loci VTNR (Variable-Number Tandem Repeat) Analysis] se apresenta como uma alternativa, embora ainda necessite de padronização e avaliação mais ampla para a espécie em questão. No presente estudo, 60 amostras de Streptococcus pneumoniae pertencentes ao sorotipo 14, isoladas de diversas fontes clínicas, em diferentes locais e períodos de tempo, foram caracterizadas pelas técnicas de MLVA (baseada na análise de 18 loci distintos), MLST, PFGE e tipagem do gene pspA. O gene pspA2 predominou entre as amostras analisadas, seguido pelo gene pspA1. Os tipos de MLVA, perfis de PFGE, e STs encontrados apresentaram resultados, em geral, concordantes, indicando o elevado poder discriminatório da versão da técnica de MLVA empregada. Cinco complexos clonais (CC) de MLVA e cinco singletons puderam ser definidos. O CC de MLVA denominado de L7 foi o predominante, compreendendo 36,7% da amostragem estudada. O CC L7 mostrou-se relacionado com genes pspA da família 2, com o CC1 de MLST, com o CC Pen14-H de PFGE, e com a não susceptibilidade à penicilina, Entre os complexos clonais de MLST, o CC1 foi o prevalente e incluiu predominantemente o ST156, pertencente ao clone internacional Spain9V-3. O CC L3 e o singleton L17 de MLVA apresentaram-se associados ao CC de PFGE Eri14-A, a família 1 de PspA e ao CC2 de MLST, que por sua vez também estava relacionado com o clone internacional England14-9. O CC L15 de MLVA esteve associado ao CC de PFGE Pen14-A, ao gene pspA2, aos CC3 e CC4 de MLST e ao clone internacional do PMEN Tennessee14-18. A técnica de MLVA revelou-se significativamente mais discriminatória que as técnicas de PFGE e MLST, conforme exemplificado pela detecção de 21 perfis de MLVA, 13 perfis de PFGE e cinco STs, entre as 22 amostras pertencentes ao CC de MLVA L7. Uma versão de MLVA, compreendendo um painel com os oito loci de maior poder discriminatório, pôde ser proposta a partir da análise dos resultados obtidos. Estes aspectos, aliados ao menor tempo e custo de execução, indicam que a técnica de MLVA constitui uma alternativa importante e satisfatória para uso em estudos sobre a diversidade genética de S. pneumoniae. / Streptococcus pneumoniae is a major pathogen causing invasive and non-invasive diseases in humans, including meningitis, pneumonia and otitis media. The polysaccharide capsule of this microorganism is considered a major virulence factor and an important marker for epidemiological studies. More than 90 pneumococcal capsular serotypes are recognized, and serotype 14 is highly prevalent in many regions, including Brazil. Genotyping methods, such as PFGE and MLST, are essential to evaluate genetic diversity of this bacterium. However, these methods are expensive, time-consuming and results from different laboratories are difficult to compare. Multiple Loci VTNR (Variable-Number Tandem Repeat) Analysis (MLVA) appears as an alternative, despite the fact that standardization and wide evaluation for application to this species is still required. In the present study, a total of 60 S. pneumoniae isolates belonging to serotype 14, isolated from different sources, regions and periods of time, were analyzed by MLVA (based on the analysis of 18 distinct loci), MLST, PFGE and pspA typing methods. Gene pspA2 was the predominant, followed by pspA1. Overall, the results of PFGE, MLST and MLVA typing were congruent, and indicated the discriminatory power of the MLVA method used. Five clonal complexes (CC) and five singletons were identified by MLVA. CC L7 was the predominant MLVA CC, comprising 36.7% of all the isolates. L7 was associated with pspA2 gene and non-susceptibility to penicillin, and it was related to MLST CC1, and to PFGE Pen14-H. CC1 was the prevalent MLST CC and included mostly ST156 that belongs to international clone (IC) Spain9V-3. Another MLVA CC, named L3, and the singleton L17 were related to PFGE CC Eri14-A, MLST CC2, and IC England14-9. MLVA CC L15 was related to PFGE Pen14-A, MLST CC3 and CC4, and IC Tennessee14-18. MLVA was found to be more discriminatory than PFGE and MLST, as exemplified by detection of 21 MLVA types, 13 PFGE profiles and 5 STs among the 22 strains belonging to L7, the predominant MLVA CC. A modified version of the MLVA method, based on the analysis of 8 loci only, is proposed. This aspect, in conjunction with reducing time and costs, indicate that MLVA represents an important and satisfactory alternative method to evaluate the genetic diversity of S. pneumoniae.

Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.