Avaliação dos efeitos da conversão do plasma sobre reatividade e estabilidade de painéis sorológicos para diagnóstico da sífilis

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Biologia.Os casos de sífilis vêm aumentando no mundo, no Brasil e em Santa Catarina. A OMS estima que 937 mil pessoas são infectadas a cada ano no país. Em Santa Catarina foram registrados 5.427 novos casos de sífilis em 2015, um aumento de quase 50% comparado com o total notificado em 2014. O diagnóstico da sífilis é baseado nas manifestações clínicas, pesquisa direta do patógeno e testes sorológicos. Para que os testes realizados no diagnóstico da sífilis possuam confiabilidade no seu resultado, é necessário que os laboratórios e os serviços que realizam o diagnóstico participem de programas de controle de qualidade. A unidade de saúde ou laboratório deve garantir que no processo analítico os resultados atendam aos critérios de qualidade exigidos por lei. O presente trabalho pretende avaliar os efeitos da conversão do plasma em soro e a adição de azida de sódio como conservante. A desfibrinação transforma o plasma, de bolsas de doadores, em soro, por meio da recuperação da cascata de coagulação com trombina e cálcio. Além disso, o trabalho pretende verificar possíveis alterações na reatividade das amostras submetidas a diferentes temperaturas de armazenamento e ciclos de congelamento e descongelamento durante 90 dias. Foram utilizadas duas bolsas reagentes para sífilis e uma bolsa não reagente. Após o tratamento das bolsas, as amostras foram separadas em 4 grupos (G): G1: Plasma sem azida de sódio, G2: Soro sem de azida de sódio, G3: Plasma com azida de sódio, G4: Soro com azida de sódio. Foram distribuídas alíquotas desses quatro grupos em cinco condições de armazenamento: 2 a 8ºC, 30ºC, -20ºC (duas alíquotas) e -80ºC. Uma das alíquotas armazenadas em -20ºC foi descongelada em banho-maria (37ºC) por 30 minutos e a outra foi descongelada em temperatura ambiente de laboratório (20 a 26ºC). A partir do armazenamento das alíquotas em cada condição, foram realizados 12 ciclos de testagem, um a cada sete dias, totalizando 12 semanas. As amostras foram avaliadas com um teste rápido treponêmico (Bioeasy) e um teste não-treponêmico (RPR Wama), a fim de comparar os tratamentos realizados nas bolsas de plasma e os efeitos das diferentes condições às quais as amostras foram expostas, por meio da comparação dos resultados da reatividade dessas alíquotas nos respectivos testes. Esse estudo demonstrou que apesar do tempo de armazenamento e constante manipulação das alíquotas, os resultados obtidos na bolsa 1 (reagente) não demonstram diferença entre a reatividade inicial e final nos testes realizados. Já os resultados relacionados à bolsa 2 (reagente) tiveram diferenças nos testes não-treponêmicos e principalmente nas alíquotas armazenadas na temperatura de 30°C, que também sofreram alterações no teste treponêmico, porém, as alíquotas dessa bolsa armazenadas em -20°C não sofreram alterações em sua reatividade.Syphilis cases have been increasing in the world, in Brazil and in Santa Catarina. WHO estimates that 937 thousand people are infected each year in the country. In Santa Catarina, 5.427 new cases of syphilis were registered in 2015, an increase of almost 50% compared to the number of cases reported in 2014. The diagnosis of syphilis is based on clinical manifestations, direct pathogen research and serological tests. In order for the tests performed in the diagnosis of syphilis to have reliability in their results, the laboratories and services that perform the diagnosis must participate in quality control programs. The health unit or laboratory must ensure that in the analytical process the results meet the quality criteria required by law. The present work intends to evaluate the effects of the conversion of plasma to serum and the addition of sodium azide as a preservative. Defibrination transforms plasma, from donor bags, into serum by means of recovery the plasma coagulation cascade with thrombin and calcium. In addition, the work intends to verify possible changes in the reactivity of samples submitted to different storage temperatures and freezing and thawing cycles for 90 days. Two different syphilis reagent bags and one non-reagent bag were used. After treatment of this bags, the samples were separated into 4 groups (G): G1: Plasma without sodium azide, G2: Serum without sodium azide, G3: Plasma with sodium azide, G4: Serum with sodium azide. Aliquots of these four groups were distributed under five storage conditions: 2-8°C, 30°C, -20°C (two aliquots) and -80°C. One of the aliquots stored at -20°C was thawed in a warm bath (37 °C) for 30 minutes. The remainder was thawed at laboratory room temperature (20 to 26 °C). From the storage of the aliquots in each condition, 12 cycles of testing were performed, one every seven days, totaling 12 weeks. The samples were evaluated with a rapid treponemal test (Bioeasy) and a non-treponemal test (RPR Wama), in order to compare the treatments performed in the plasma bag and the effects of the different conditions to which samples were exposed, by means of the comparison of the reactivity results of these rates in the respective tests. This study demonstrated that despite storage time and constant manipulation of aliquots, the results obtained in bag 1 (reagent) did not show any difference between initial and final reactivity in the tests performed. However, the results related to bag 2 (reagent) had differences in the non-treponemal tests, mainly in the aliquots stored at 30°C, which also underwent alterations in the treponemal test, but the aliquots of this bag stored at -20°C did not change this reactivity

Genomic epidemiology of Mycobacterium tuberculosis in Santa Catarina, Southern Brazil

Mycobacterium tuberculosis (M.tb), the pathogen responsible for tuberculosis (TB) poses as the major cause of death among infectious diseases. The knowledge about the molecular diversity of M.tb enables the implementation of more effective surveillance and control measures and, nowadays, Whole Genome Sequencing (WGS) holds the potential to produce high-resolution epidemiological data in a high-throughput manner. Florianópolis, the state capital of Santa Catarina (SC) in south Brazil, shows a high TB incidence (46.0/100,000). Here we carried out a WGS-based evaluation of the M.tb strain diversity, drug-resistance and ongoing transmission in the capital metropolitan region. Resistance to isoniazid, rifampicin, streptomycin was identified respectively in 4.0% (n = 6), 2.0% (n = 3) and 1.3% (n = 2) of the 151 studied strains by WGS. Besides, resistance to pyrazinamide and ethambutol was detected in 0.7% (n = 1) and reistance to ethionamide and fluoroquinolone (FQ) in 1.3% (n = 2), while a single (0.7%) multidrug-resistant (MDR) strain was identified. SNP-based typing classified all isolates into M.tb Lineage 4, with high proportion of sublineages LAM (60.3%), T (16.4%) and Haarlem (7.9%). The average core-genome distance between isolates was 420.3 SNPs, with 43.7% of all isolates grouped across 22 genomic clusters thereby showing the presence of important ongoing TB transmission events. Most clusters were geographically distributed across the study setting which highlights the need for an urgent interruption of these large transmission chains. The data conveyed by this study shows the presence of important and uncontrolled TB transmission in the metropolitan area and provides precise data to support TB control measures in this region.publishersversionpublishe

In vitro selection of Neisseria gonorrhoeae unveils novel mutations associated with extended-spectrum cephalosporin resistance

The emergence of Neisseria gonorrhoeae strains resistant to extended-spectrum cephalosporins (ESCs) is a worldwide concern because this class of antibiotics represents the last empirical treatment option for gonorrhea. The abusive use of antimicrobials may be an essential factor for the emergence of ESC resistance in N. gonorrhoeae. Cephalosporin resistance mechanisms have not been fully clarified. In this study, we mapped mutations in the genome of N. gonorrhoeae isolates after resistance induction with cefixime and explored related metabolic pathways. Six clinical isolates with different antimicrobial susceptibility profiles and genotypes and two gonococcal reference strains (WHO F and WHO Y) were induced with increasing concentrations of cefixime. Antimicrobial susceptibility testing was performed against six antimicrobial agents before and after induction. Clinical isolates were whole-genome sequenced before and after induction, whereas reference strains were sequenced after induction only. Cefixime resistance induction was completed after 138 subcultures. Several metabolic pathways were affected by resistance induction. Five isolates showed SNPs in PBP2. The isolates M111 and M128 (ST1407 with mosaic penA-34.001) acquired one and four novel missense mutations in PBP2, respectively. These isolates exhibited the highest minimum inhibitory concentration (MIC) for cefixime among all clinical isolates. Mutations in genes contributing to ESC resistance and in other genes were also observed. Interestingly, M107 and M110 (ST338) showed no mutations in key determinants of ESC resistance despite having a 127-fold increase in the MIC of cefixime. These findings point to the existence of different mechanisms of acquisition of ESC resistance induced by cefixime exposure. Furthermore, the results reinforce the importance of the gonococcal antimicrobial resistance surveillance program in Brazil, given the changes in treatment protocols made in 2017 and the nationwide prevalence of sequence types that can develop resistance to ESC

Análise do transcriptoma de isolados de Neisseria gonorrhoeae: investigação sobre o perfil de sensibilidade à ceftriaxona

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2020.Neisseria gonorrhoeae (NG) é o agente etiológico da gonorreia, infecção sexualmente transmissível que afeta cerca de 78 milhões de pessoas por ano. Desde a implementação do tratamento, o gonococo desenvolveu rapidamente diversos mecanismos de resistência às múltiplas classes de antimicrobianos utilizadas para tratar a infecção. Atualmente, a ceftriaxona (CRO) é o fármaco utilizado no tratamento de primeira linha recomendado pelo Organização Mundial da Saúde, apesar de já existir casos de resistência descritos. Considerando a possibilidade da infecção gonocóccica tornar-se intratável, existe a necessidade de compreender mecanismos moleculares e fenotípicos relacionados a resistência antimicrobiana, principalmente à CRO. Neste estudo, sequenciamos alguns genes (mtrR, penA, ponA e porB) de 16 isolados com diferentes MIC para CRO e realizamos RNA-Seq para avaliar os mecanismos de resistência. Para realizar a análise do transcriptoma, um isolado clínico de NG (MIC CRO 0.125mg/L) foi cultivado em placas de meio GC (R) e em placas com meio GC acrescido da concentração subinibitória de CRO (0.06mg/L) (RC) e um isolado com MIC CRO 0.0005mg/L foi cultivado em placas de meio GC (S). Foi realizado o sequenciamento de RNA Illumina (Novaseq 100bp paired-end) em triplicatas biológicas. A análise dos dados do RNA- Seq foi realizada no sistema Rockhopper. A análise das mutações nos genes sequenciados foi feita na plataforma NGSTAR. Os padrões encontrados para os isolados com MIC CRO (0,03 ? 0,125mg/L) foram os seguintes: mtrR: -35A del, ponA: L421P, penA: mosaico, porB: G120K / A121D. Os isolados com MIC < 0,004mg/L não apresentaram mutações em sua maioria. Os padrões de mutação encontrados nos isolados com redução de sensibilidade são idênticos ao de isolados que possuem resistência à CRO (MIC = 0,250mg/L), demonstrando a falta de diferenciação genotípica em cepas com sensibilidade distinta à CRO. Ao analisar os dados de RNA-Seq, foi possível identificar 244 genes diferencialmente expressos entre o isolado S e o isolado R. Entre os 20 genes com maior diferença na expressão, 7 estão relacionados a proteínas associadas ao fago, 5 são RNAs antissenso e 4 estão relacionados ao sistema de modificação- restrição do tipo III. Na comparação entre o isolado RC e o isolado R, foi possível observar 116 genes diferencialmente expressos, e entre os 20 genes com maior diferença na expressão encontramos 10 RNAs antissenso e 3 genes de proteínas hipotéticas. Foi possível observar que o sistema de restrição-modificação do tipo III pode ter relevância no fenótipo de resistência à CRO, bem como a transcrição de genes associados ao fago, presentes no transcriptoma dos 3 experimentos, o que carece de validação experimental. Além disso, a presença de CRO parece induzir uma resposta ao dano do DNA em NG, mediada por DinB e RecA. Ainda, verificamos que os componentes genômicos de resistência não são exclusivamente responsáveis por determinar a resistência à CRO.Observamos numerosas proteínas hipotéticas e a presença de RNAs antissenso nos experimentos sugerindo regulações transcricionais. Devido à ausência de informação revisada do genoma de NG, não foi possível realizar o enriquecimento de dados do conjunto de genes diferencialmente expressos, destacando um ponto nevrálgico das análises in silico.Abstract: Neisseria gonorrhoeae (NG) is the etiological agent of gonorrhea, a sexually transmitted infection that affects about 78 million people per year. Since the implementation of the treatment, gonococcus rapidly developed resistance mechanisms to the multiple classes of antimicrobials used to treat the infection. Nowadays, ceftriaxone (CRO) is the drug used as a first-line treatment recommended by the World Health Organization (WHO), although resistance to this drug was already described. Owing to the possibility of the gonococcal infection becoming untreatable, there is a need to understand the molecular and phenotypic mechanisms related to antimicrobial resistance mainly to ceftriaxone. In this study, we sequenced some genes (mtrR, penA, ponA and porB) and performed RNA sequencing to evaluate resistance mechanisms. To perform the transcriptome analysis, the NG isolate (MIC CRO 0.125mg/L) (R) was cultured in both GC medium plates and GC medium plates with CRO subinhibitory concentration (0.06mg/L) (RC) and a isolate with MIC CRO 0.0005mg/L was culture in GC medium plates (S). Illumina RNA sequencing was performed (Novaseq 100bp paired-end) in biological triplicates. RNA-Seq data analysis was performed in Rockhopper system. The SNP patterns found for MIC CRO isolates (0.03 - 0.125mg / L) were as follows: mtrR: -35A del, ponA: L421P, penA: mosaic, porB: G120K / A121D. The isolates with MIC <0.004mg /L did not present mutations mostly. The mutation patterns found in isolates with reduced sensitivity are identical to those of isolates that have resistance to CRO (MIC = 0.250mg/L), showing the lack of genotypic differentiation in strains with unequal susceptibility to CRO. By analyzing RNA-Seq data, it was possible to identify 244 genes differentially expressed between isolate S and isolate R. Among the 20 genes with major difference in expression, 7 are related to phage-associated proteins, 5 are antisense RNAs and 4 are related to the type III modification-restriction system. In the comparison between the RC isolate and the R isolate, it was possible to observe 116 differentially expressed genes, and among the 20 genes with the greatest difference in expression, we found 10 antisense RNAs and 3 hypothetical protein genes. It was possible to observe that the type III restriction-modification system may have relevance in the CRO resistance phenotype, as well as the transcription of genes associated with the phage, present in the transcriptome of the 3 experiments, which lacks experimental validation. In addition, the presence of CRO appears to induce a response to DNA damage in NG, mediated by DinB and RecA. Furthermore, we found that genomic resistance components are not exclusively responsible for determining resistance to CRO. We observed numerous hypothetical proteins and the presence of antisense RNAs in the experiments suggesting transcriptional regulations. Due to the lack of revised information on the NG genome, it was not possible to enrich data on the set of differentially expressed genes, highlighting fragile point of in silico analysis

Genomic epidemiology of Mycobacterium tuberculosis in Santa Catarina, Southern Brazil (Scientific Reports, (2020), 10, 1, (12891), 10.1038/s41598-020-69755-9)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.publishersversionpublishe