Diagnóstico e epidemiologia molecular de cepas de Mycobacterium tuberculosis no Estado de Santa Catarina

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em FarmáciaResumo: A Tuberculose (TB), doença infectocontagiosa causada pelo M. tuberculosis, é um grave problema de saúde pública, sendo responsável por cerca de 1,5 milhões de mortes/ano no mundo. Embora o Estado de Santa Catarina apresente uma das mais baixas taxas de incidência (27,6/100.000 habitantes) do país, alguns municípios, como Itajaí (77,3 casos novos/100.000 habitantes) apresentam taxas iguais e/ou superiores às do Brasil (43/100.000 habitantes) e de outros países, nos quais a situação é muito grave. Em relação ao controle da TB, o Estado de Santa Catarina conta com o Hospital Nereu Ramos (hospital de referência) e com o LACEN/SC (laboratório de referência). A oportunidade de associar informações diagnósticas rápidas e dados de epidemiologia molecular representa um avanço importante no controle da doença. Diante disso, este trabalho teve como objetivos avaliar, por meio da PCR (IS6110 e 16S rRNA), a inclusão de pérolas de vidro como modificação no protocolo de rotina utilizado no Laboratório de Biologia Molecular e Micobactérias/UFSC para tratamento das amostras de escarro; e estudar a epidemiologia molecular de cepas circulantes de M. tuberculosis no Estado de Santa Catarina pela metodologia Spoligotyping. De março/2010 a março/2011 foram obtidos 406 isolados clínicos, sendo que 120 amostras de escarro foram utilizadas para a avaliação do protocolo modificado para tratamento das amostras. A utilização das pérolas de vidro demonstrou um incremento na sensibilidade da PCR para detecção de M. tuberculosis, especialmente nas amostras paucibacilares. Entre os casos incluídos no estudo, 85% dos indivíduos residiam nas regiões do Vale do Itajaí, da Grande Florianópolis e do Nordeste Catarinense, sendo predominante os indivíduos brancos, adultos jovens, do sexo masculino, com baixa escolaridade, desempregados ou com baixa qualificação profissional. Cerca de 50% da população apresentou algum tipo de agravo associado, sendo 23,1% coinfectados com HIV, 37,6% etilistas e 35,2% dependentes químicos. Nas análises pelo Spoligotyping, observou-se grande variedade de genótipos circulantes, dos quais 13,3% ainda não haviam sido descritos no SpolDB4. A linhagem LAM foi a mais frequente (40,1%), seguida da T (23,4%), da Harleem (12,8%), da U (3,7%), da X (2,7%) e da S (1,7%), sendo as subfamílias LAM9 (20,0%), H3 (9,6%) e T2/T3 (8,4%) as mais frequentemente identificadas. Os perfis mais frequentes são similares com cepas circulantes em Portugal, Itália e outros países europeus, o que reflete a influência da colonização do Estado na população de cepas de M. tuberculosis. Algumas subfamílias e SITs (Spoligotyping International Types), encontrados em baixa frequência, ocorreram exclusivamente em certas regiões do Estado. A utilização do protocolo modificado para tratamento das amostras de escarro possibilitou uma melhora no diagnóstico molecular da TB realizado no Laboratório de Biologia Molecular e Micobactérias/UFSC em parceira com o Hospital Nereu Ramos e o LACEN/SC. Além disso, o estudo epidemiológico molecular forneceu, pela primeira vez, dados sobre a estrutura populacional de M. tuberculosis circulantes no Estado de Santa Catarina

Genomic epidemiology of a national outbreak of post-surgical Mycobacterium abscessus wound infections in Brazil.

An epidemic of post-surgical wound infections, caused by a non-tuberculous mycobacterium, has been on-going in Brazil. It has been unclear whether one or multiple lineages are responsible and whether their wide geographical distribution across Brazil is due to spread from a single point source or is the result of human-mediated transmission. 188 isolates, collected from nine Brazilian states, were whole genome sequenced and analysed using phylogenetic and comparative genomic approaches. The isolates from Brazil formed a single clade, which was estimated to have emerged in 2003. We observed temporal and geographic structure within the lineage that enabled us to infer the movement of sub-lineages across Brazil. The genome size of the Brazilian lineage was reduced relative to most strains in the three subspecies of Mycobacterium abscessus and contained a novel plasmid, pMAB02, in addition to the previously described pMAB01 plasmid. One lineage, which emerged just prior to the initial outbreak, is responsible for the epidemic of post-surgical wound infections in Brazil. Phylogenetic analysis indicates that multiple transmission events led to its spread. The presence of a novel plasmid and the reduced genome size suggest that the lineage has undergone adaptation to the surgical niche

Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil.

Submitted by Nuzia Santos ([email protected]) on 2016-02-22T18:00:26Z No. of bitstreams: 1 Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil..pdf: 292609 bytes, checksum: 4bea0aaa468b44fe8ec1bba4d8017a9e (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-02-22T18:06:23Z (GMT) No. of bitstreams: 1 Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil..pdf: 292609 bytes, checksum: 4bea0aaa468b44fe8ec1bba4d8017a9e (MD5)Made available in DSpace on 2016-02-22T18:06:23Z (GMT). No. of bitstreams: 1 Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil..pdf: 292609 bytes, checksum: 4bea0aaa468b44fe8ec1bba4d8017a9e (MD5) Previous issue date: 2015Universidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilLaboratório Central de Saúde Pública de Santa Catarina. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilDrug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosis clinical isolates were analysed by spoligotyping and a partial region of the rpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoB in RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations within rpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil

Acute phase proteins for the diagnosis of bacterial infection and prediction of mortality in acute complications of cirrhosis

Introduction. Bacterial infection is a frequent complication in patients with decompensated liver cirrhosis and is related to high mortality rates during follow-up of these individuals. We sought to evaluate the diagnostic value of C-reactive protein (CRP) and procalcitonin (PCT) in diagnosing infection and to investigate the relationship between these biomarkers and mortality after hospital admission.Material and methods. Prospective study that included cirrhotic patients admitted to the hospital due to complications of the disease. The diagnostic accuracy of CRP and PCT for the diagnosis of infection was evaluated by estimating the sensitivity and specificity and by measuring the area under the receiver operating characteristics curve (AUROC).Results. A total of 64 patients and 81 hospitalizations were analyzed during the study. The mean age was 54.31 ± 11.87 years with male predominance (68.8%). Significantly higher median CRP and PCT levels were observed among infected patients (P 29.5 exhibited sensitivity of 82% and specificity of 81% for the diagnosis of bacterial infection. Similarly, PCT levels > 1.10 showed sensitivity of 67% and specificity of 90%. Significantly higher levels of CRP (P = 0.026) and PCT (P = 0.001) were observed among those who died within three months after admission.Conclusion. CRP and PCT were reliable markers of bacterial infection in subjects admitted due to complications of liver cirrhosis and higher levels of these tests are related to short-term mortality in those patients

Mycobacterium franklinii sp. nov., a species closely related to members of the Mycobacterium chelonae-Mycobacterium abscessus group

Two isolates from water, D16Q19 and D16R27, were shown to be highly similar in their 16S rRNA, 16S-23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences to 'Mycobacterium franklinii' DSM 45524, described in 2011 but with the name not validly published. They are all nonpigmented rapid growers and are related phenotypically and genetically to the Mycobacterium chelonae Mycobacterium abscessus group. Extensive characterization by phenotypic analysis, biochemical tests, drug susceptibility testing, PCR restriction enzyme analysis of the hsp65 gene and ITS, DNA sequencing of housekeeping genes and DNA-DNA hybridization demonstrated that 'M. franklinii DSM 45524, D16Q19 and D16R27 belong to a single species that is separated from other members of the M. chelonae M. abscessus group. On the basis of these results we propose the formal recognition of Mycobacterium franklinii sp. nov. Strain DSM 45524(T) (=ATCC BAA-2149(T)) is the type strain

IGF-I and IGFBP-3 serum levels in patients hospitalized for complications of liver cirrhosis

Background. IGF-I and IGFBP-3 are part of IGF system and, due to their predominantly hepatic synthesis, they seem to correlate with hepatic dysfunction intensity. Aims. To investigate the significance of IGF-I and IGFBP-3 in patients with decompensated liver disease.Material and methods. Cross-sectional study that included cirrhotic patients admitted to hospital due to complications of the disease, in whom IGF-I and IGFBP-3 serum levels were measured by chemiluminescence. Results. Seventy-four subjects with a mean age of 53.1 ± 11.6 years were included in the study, 73% were males. IGF-I levels were positively correlated with IGFBP-3 and albumin, and negatively correlated with Child-Pugh, MELD, creatinine, INR and aPTT ratio. IGFBP-3 levels were positively correlated with IGF-I and albumin, and negatively correlated with Child-Pugh, MELD, creatinine, INR, total bilirubin and aPTT ratio. Significantly lower scores of IGF-I and IGFBP-3 were observed in patients with higher MELD values and higher Child-Pugh classes (P < 0.05).Conclusions. In cirrhotic patients admitted to hospital due to complications of the disease, IGF-I and IGFBP-3 serum levels were associated with variables related to liver dysfunction and to more advanced liver disease. The levels of these markers seem to undergo little influence from other clinical and laboratory variables, therefore mainly reflecting hepatic functional status

External quality assurance with dried tube specimens (DTS) for point-of-care syphilis and HIV tests: experience in an indigenous populations screening programme in the Brazilian Amazon.

OBJECTIVES: The availability of point-of-care (POC) tests for infectious diseases has revolutionised the provision of healthcare for remote rural populations without access to laboratories. However, quality assurance for POC tests has been largely overlooked. We have evaluated the use and stability of dry tube specimens (DTS) for External Quality Assurance (EQA) for HIV and syphilis screening in remote indigenous populations in the Amazon region of Brazil. METHODS: All healthcare workers (HCWs) participating in the community-screening were trained. We used HIV and syphilis DTS panels developed by the reference laboratory, containing samples with negative and positive results at different antibody concentrations, for both infections. DTS panels were distributed to HCWs in the communities for reconstitution and testing using POC HIV and syphilis tests. The results of testing were sent to the reference laboratory for marking and remedial action taken where necessary. RESULTS: In total 268 HCWs tested 1607 samples for syphilis and 1608 samples for HIV. Results from HCWs showed a concordance rate of 90% for syphilis and 93% for HIV (κ coefficients of 0.74 and 0.78, respectively) with reference laboratories. Most false negatives were in samples of very low antibody concentration. DTS syphilis specimens produced the expected results after storage at 2-8°C or at 18-24°C for up to 3 weeks. CONCLUSIONS: The results show that POC tests for syphilis and HIV give valid results in environments where traditional tests do not work, but errors in the interpretation of POC test results were identified by the EQA programme using DTS. EQA using DTS can help to improve the quality of screening programmes using POC tests in remote regions

Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae--Mycobacterium abscessus group

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results,demonstrated that they share characteristics with M. chelonae–M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T)