RESUMO
Bacterial meningitis is still a significant public health concern, with high morbidity and mortality rates. Despite this, it is still a rare event that requires the bacterial invasion of the meninges. However, some predisposing factors can trigger recurrent episodes of meningitis. This study is aimed at determining the clinical characteristics and the molecular epidemiology of episodes of recurrent community-acquired meningitis with and without predisposing factors. For this purpose, we performed a retrospective study of our laboratory database during the period of 2010 to 2020. Additionally, using molecular tools developed in our previous works, the epidemiology of the pathogens causing these episodes was analyzed using cerebrospinal fluid samples, especially in the absence of isolated strains. We observed a total of 1,779 meningitis cases and 230 were caused by Streptococcus pneumoniae. Of those, 16 were recurrent meningitis episodes (16/1,779; 0.9%) from seven patients. Pneumococcus was the main agent responsible in these recurrent episodes and only two episodes were caused by Haemophilus influenzae. The mean age of these patients was 20 years old and three had predisposing factors which could have led to contracting meningitis. The samples presented different pneumococcal serotypes. Most of them were non-vaccine-covered serotypes and antibiotic susceptible strains. Therefore, it was demonstrated how the practical employment of molecular tools, developed for research, when applied in the routine of diagnosis, can provide important information for epidemiological surveillance. Furthermore, it was shown how pneumococcus was the leading cause of recurrent community-acquired meningitis without predisposing factors, suggesting that pneumococcal vaccination may be necessary, even in those groups of individuals considered to be less susceptible.
Assuntos
Infecções Comunitárias Adquiridas , Meningite Pneumocócica , Recidiva , Streptococcus pneumoniae , Humanos , Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/epidemiologia , Meningite Pneumocócica/epidemiologia , Meningite Pneumocócica/microbiologia , Adulto , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/classificação , Estudos Retrospectivos , Feminino , Masculino , Adulto Jovem , Pessoa de Meia-Idade , Adolescente , Fatores de Risco , Sorogrupo , Antibacterianos , Haemophilus influenzae/genética , Haemophilus influenzae/isolamento & purificação , Haemophilus influenzae/classificaçãoRESUMO
O Comitê de Integridade na Pesquisa do Instituto Adolfo Lutz (CIPIAL), com o objetivo de promover a cultura da integridade científica como um dos valores fundamentais defendidos pela instituição nas suas atividades de pesquisa, considera relevante compartilhar com a comunidade científica a sua implantação, destacando o seu papel no gerenciamento deste tema na instituição. Após a publicação de seu regimento, de acordo com as suas competências primordiais, o CIPIAL elaborou e publicou o Código de Boas Práticas Científicas do IAL com o objetivo de definir as políticas de integridade para orientar os profissionais envolvidos com a pesquisa. (AU)
The Research Integrity Committee of the Adolfo Lutz Institute (CIPIAL), with the aim of promoting the culture of scientific integrity as one of the fundamental values defended by the institution in its research activities, considers it relevant to share its implementation with the scientific community, highlighting its role in managing this issue at the institution. Following the publication of its rules and regulations, in accordance with its core competencies, CIPIAL drew up and published the IAL Code of Good Scientific Practice with the aim of defining integrity policies to guide professionals involved in research. (AU)
Assuntos
Pesquisadores , Má Conduta Científica , Revisão de Integridade Científica , Ética em PesquisaRESUMO
A tuberculose (TB) continua sendo um grande desafio para a saúde pública mundial e, para um controle eficiente, também é essencial identificar pessoas com tuberculose latente (ILTB). O ensaio de liberação de interferon-gama (IGRA), incorporado pelo SUS em 2021, permitirá ampliar o diagnóstico de ILTB, em complemento à prova tuberculínica. Para essa implantação, as coordenações do Programa Estadual e da Rede de Laboratórios de TB/SP iniciaram a identificação de executores do IGRA a partir da rede de laboratórios de TB e/ou CD4, para verificar possíveis barreiras para implantação do teste. Foram avaliados os insumos e os profissionais para execução do ensaio, a infraestrutura laboratorial e a disponibilidade de equipamentos. Dez laboratórios avaliaram amostras de sangue total com o kit QuantiFERON®-TB Gold Plus e relataram sua experiência quanto à logística de amostras, execução do ensaio e liberação de laudos. Para otimizar o exame, a coleta ocorreu em tubos heparinizados (sódio ou lítio). Foi sugerida a logística da rede de laboratórios de CD4, que foi utilizada por 20% dos laboratórios participantes, enquanto 50% optaram pelo agendamento. Não foram reportadas dificuldades na liberação de laudo. Dois laboratórios avaliaram o número de células T CD4+ prévio e no momento do IGRA, observando diferença em 10% dos pacientes, fator que pode ser relevante na análise do resultado. Ao todo, foram analisadas 383 amostras, 81 (21,1%) reagentes, 297 (77,5%) não reagentes e cinco (1,3%) indeterminados. Foi observada grande variação de positividade (3,6-50,0%) entre os laboratórios, provavelmente devido à população atendida. Apesar dos desafios encontrados, consideramos que a taxa média de positividade (~20%) sugere que a oferta do IGRA na rede pública possibilitará o aumento do diagnóstico de ILTB e melhor controle da TB.
RESUMO
Streptococcus pneumoniae causes invasive diseases of significant public health concern, such as meningitis. The culture of cerebrospinal fluid (CSF) samples, the standard technique for meningitis diagnoses, is not always positive. Consequently, meaningful information about the etiological agent is lost, which can compromise effective epidemiological surveillance and the improvement of immunization policies. This study aims to standardize a method to genotype pneumococcus in the CSF samples which could mitigate the absence of isolated strains, and also evaluate the prediction of this assay. We applied eight multiplex PCR (mPCR) assays to CSF samples paired with the Quellung reaction applied to the isolated strains. We also compared different master mix kits in the mPCR. Moreover, a retrospective study was conducted with CSF samples considered pneumococcus positive due to the presence of the lytA gene. Results showed that genotyping by the mPCR correlated 100% with the Quellung reaction, and genotyping was dependent on the master mix applied. In the retrospective study (2014–2020), 73.4% were successfully genotyped. The analyses of the receiver operating characteristic curve showed that the cycle threshold (Ct value) around 30 for the lytA gene had a 75% positive chance of successful genotyping, whereas with a Ct value > 35, the chance was 12.5%. Finally, we observed that genotype 19A was prevalent in the period (12%), information unknown until now due to the lack of isolated strains. Therefore, the mPCR of CSF samples can efficiently predict S. pneumoniae serotypes, especially in the absence of isolated strains, which can be a great tool for pneumococcal serotype surveillance.
RESUMO
Meningitis caused by Streptococcus pneumoniae is still a disease of great impact on Public health, which requires immediate diagnosis and treatment. However, the culture of clinical specimens is often negative and antibiotic susceptibility testing (AST) must be performed with isolated strains. Multiplex real-time polymerase chain reaction (qPCR) has high sensitivity and specificity, produces faster results to identify the pathogen, and it can also be an important tool to identify resistance antibiotic genes earlier than AST, especially in the absence of an isolated strain. This study developed a multiplex qPCR assay, using SYBR Green as a nonspecific dye, to detect antibiotic resistance genes to predict pneumococcal susceptibility/resistance in cerebrospinal fluid (CSF) samples from meningitis patients. From 2017 to 2020, CSF samples were cultured and analyzed by qPCR to detect the main three bacteria causing meningitis. Isolated and reference strains were applied in SYBR Green qPCR multiplex to detect pbp2b, ermB, and mef genes, and the results were compared with the AST. Pneumococcal-positive CSF samples (lytA-positive gene) without isolated strains were also tested to evaluate the antimicrobial susceptibility profile in the region from 2014 to 2020. From the received 873 CSF samples; 263 were cultivated, 149 were lytA-positive in the qPCR, and 25 produced viable isolated pneumococci strains, which were evaluated by AST. Melting temperature for each gene and the acceptance criteria were determined (pbp2b: 78.24-79.86; ermB: 80.88-82.56; mef: 74.85-76.34 ºC). A total of 48/51 strains presented a genetic profile in agreement with the AST results. Resistant strains to erythromycin and clindamycin were ermB-positive, and two were also mef-positive, indicating both resistance mechanisms were present. In the retrospective study of the genetic profile of resistance, 82 lytA-positive CSF samples plus 4 strains were applied in the SYBR Green qPCR multiplex: 51% of samples presented the wild genotype (pbp2b positive and ermB/mef negative); 15% were negative for all the three evaluated, indicating pneumococci resistant to penicillin; and 17% represented the multidrug-resistant pneumococci (pbp2b negative and ermB positive or pbp2b negative and ermB and mef positive). Therefore, SYBR Green qPCR multiplex proved to be a reliable tool to identify resistance genes in S. pneumoniae and would be less expensive than multiplex qPCR using specific probes. This could be easily introduced into the routine of diagnostic laboratories and provide a strong presumption of pneumococcal resistance, especially in the absence of isolated strains.
Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Antibacterianos/farmacologia , Benzotiazóis , Diaminas , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Quinolinas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos RetrospectivosRESUMO
Meningitis caused by Streptococcus pneumoniae is still a disease of great impact on Public health, which requires immediate diagnosis and treatment. However, the culture of clinical specimens is often negative and antibiotic susceptibility testing (AST) must be performed with isolated strains. Multiplex real-time polymerase chain reaction (qPCR) has high sensitivity and specificity, produces faster results to identify the pathogen, and it can also be an important tool to identify resistance antibiotic genes earlier than AST, especially in the absence of an isolated strain. This study developed a multiplex qPCR assay, using SYBR Green as a nonspecific dye, to detect antibiotic resistance genes to predict pneumococcal susceptibility/resistance in cerebrospinal fluid (CSF) samples from meningitis patients. From 2017 to 2020, CSF samples were cultured and analyzed by qPCR to detect the main three bacteria causing meningitis. Isolated and reference strains were applied in SYBR Green qPCR multiplex to detect pbp2b, ermB, and mef genes, and the results were compared with the AST. Pneumococcal-positive CSF samples (lytA-positive gene) without isolated strains were also tested to evaluate the antimicrobial susceptibility profile in the region from 2014 to 2020. From the received 873 CSF samples; 263 were cultivated, 149 were lytA-positive in the qPCR, and 25 produced viable isolated pneumococci strains, which were evaluated by AST. Melting temperature for each gene and the acceptance criteria were determined (pbp2b: 78.2479.86; ermB: 80.8882.56; mef: 74.8576.34 ºC). A total of 48/51 strains presented a genetic profile in agreement with the AST results. Resistant strains to erythromycin and clindamycin were ermB-positive, and two were also mef-positive, indicating both resistance mechanisms were present. In the retrospective study of the genetic profile of resistance, 82 lytA-positive CSF samples plus 4 strains were applied in the SYBR Green qPCR multiplex: 51% of samples presented the wild genotype (pbp2b positive and ermB/mef negative); 15% were negative for all the three evaluated, indicating pneumococci resistant to penicillin; and 17% represented the multidrug-resistant pneumococci (pbp2b negative and ermB positive or pbp2b negative and ermB and mef positive). Therefore, SYBR Green qPCR multiplex proved to be a reliable tool to identify resistance genes in S.pneumoniae and would be less expensive than multiplex qPCR using specific probes. This could be easily introduced into the routine of diagnostic laboratories and provide a strong presumption of pneumococcal resistance, especially in the absence of isolated strains. (AU)
Assuntos
Streptococcus pneumoniae , Resistência Microbiana a Medicamentos , Líquido Cefalorraquidiano , Corantes , Reação em Cadeia da Polimerase Multiplex , Substâncias Intercalantes , MeningiteRESUMO
OBJECTIVE: Recognize incident infection to better characterize the groups that fuel HIV epidemic. We propose a simple score to identify recent infections among newly diagnosed patients as a HIV surveillance tool. METHODS: Newly diagnosed patients were defined as recent infections when a negative serological test in the previous year was available. Laboratory tests, such as the avidity index (Bio-Rad, according to the CEPHIA protocol), chemiluminescent intensity (CMIA, architect, Abbott), and the nucleotide ambiguity index of partial pol sequences were used as proxies of recency. A simple score based on clinical symptoms of acute retroviral syndrome during the previous year, CD4+ T cell count, and viral load at admission was tested to assess the predictive power, using receiver operating characteristic (ROC) curves, to identify recent cases of infection. RESULTS: We evaluated 204 recently diagnosed patients who were admitted to the Ambulatório de Referência em Moléstias Infecciosas de Santo André (Santo André Reference Infectious Diseases Outpatient Clinic), in the metropolitan region of São Paulo, Brazil, recruited between 2011 and 2018. An HIV-negative test in the year prior to enrollment was documented in 37% of participants. The proportion of cases classified as recent infections (less than one year), according to the laboratory proxies were: 37% (67/181) for an avidity index < 40%, 22% (30/137) for a CMIA < 200, and 68% (124/181) for an ambiguity index < 0.5%. Using different combinations of recency definitions, our score showed an area under the ROC curve from 0.66 to 0.87 to predict recency. CONCLUSIONS: Using data from patients' interviews and routine laboratory tests at admission, a simple score may provide information on HIV recency and thus, a proxy for HIV incidence to guide public policies. This simple for the Brazilian public health system and other low- and middle-income countries.
Assuntos
Infecções por HIV , Brasil/epidemiologia , Contagem de Linfócito CD4 , Infecções por HIV/diagnóstico , Infecções por HIV/epidemiologia , Humanos , Incidência , Carga ViralRESUMO
The Santo André Regional Center from Adolfo Lutz Institute evaluated 91 537 samples by reverse transcription-polymerase chain reaction (RT-PCR) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from March 2020 to April 2021. The age, sex, and race of patients from three cities in southeastern Brazil, namely São Bernardo do Campo (SBC), Diadema, and Mauá were assessed in association to the rate of positive results using generalized linear models. Circulating lineages were obtained from GISAID and intralineage genetic variation was investigated employing Lasergene software. A declining number of reported cases around October to November 2020 separate two epidemic waves in the three cities. Mauá differed by the highest positive RT-PCR scores in January and February. GISAID classification of 38 SARS-CoV-2 complete genomic sequences showed the circulation of lineages P.1, B.1.1.28, P.2, B.1.1.332; P.1, P.2, B.1.1.28, B.1.1.33; and P.1, P.2 in SBC, Diadema and Mauá, respectively. Intralineage variation revealed a significant amino-acid substitution in the ORF3a encoding protein (A33S) present in four out of six (67%) P.1 Mauá isolates. As ORF3a encodes a nonselective Ca2+ permeable cation channel, supposed to interfere in airway homeostasis, specific mutations could increase its pathogenic effect resulting in a higher number of symptomatic individuals explaining why the second wave was more intense in Mauá city.
Assuntos
COVID-19 , SARS-CoV-2 , Brasil/epidemiologia , COVID-19/epidemiologia , Cidades/epidemiologia , Humanos , Fatores de Risco , SARS-CoV-2/genéticaRESUMO
ABSTRACT OBJECTIVE Recognize incident infection to better characterize the groups that fuel HIV epidemic. We propose a simple score to identify recent infections among newly diagnosed patients as a HIV surveillance tool. METHODS Newly diagnosed patients were defined as recent infections when a negative serological test in the previous year was available. Laboratory tests, such as the avidity index (Bio-Rad, according to the CEPHIA protocol), chemiluminescent intensity (CMIA, architect, Abbott), and the nucleotide ambiguity index of partial pol sequences were used as proxies of recency. A simple score based on clinical symptoms of acute retroviral syndrome during the previous year, CD4+ T cell count, and viral load at admission was tested to assess the predictive power, using receiver operating characteristic (ROC) curves, to identify recent cases of infection. RESULTS We evaluated 204 recently diagnosed patients who were admitted to the Ambulatório de Referência em Moléstias Infecciosas de Santo André (Santo André Reference Infectious Diseases Outpatient Clinic), in the metropolitan region of São Paulo, Brazil, recruited between 2011 and 2018. An HIV-negative test in the year prior to enrollment was documented in 37% of participants. The proportion of cases classified as recent infections (less than one year), according to the laboratory proxies were: 37% (67/181) for an avidity index < 40%, 22% (30/137) for a CMIA < 200, and 68% (124/181) for an ambiguity index < 0.5%. Using different combinations of recency definitions, our score showed an area under the ROC curve from 0.66 to 0.87 to predict recency. CONCLUSIONS Using data from patients' interviews and routine laboratory tests at admission, a simple score may provide information on HIV recency and thus, a proxy for HIV incidence to guide public policies. This simple for the Brazilian public health system and other low- and middle-income countries.
Assuntos
Humanos , Infecções por HIV/diagnóstico , Infecções por HIV/epidemiologia , Brasil/epidemiologia , Incidência , Contagem de Linfócito CD4 , Carga ViralRESUMO
Dengue virus, the etiological agent of dengue fever (DF) occurs in four genetically distinct serotypes (DENV1-4), being transmitted by female Aedes mosquitoes. DF incidence is increasing in Brazil, following vector dispersal, proliferation and DENV serotypes introduction, co-circulation and substitution. Medium- and small-sized cities in Sao Paulo State, such as Marilia (Midwest region), have been affected by huge epidemics. To understand the evolution of DENV epidemics in medium-sized cities, in this study a historical data on DENV incidence (2000-2015) in Marilia, was evaluated. Previous studies disclosed regional and specific DF outcomes associated with 2007 outbreak in that city, when co-circulating DENV1 and DENV3 presented different hematological profiles. In this study, characteristics of 2007 DENV epidemics were compared to the epidemiological, hematological and demographic outlines of the major outbreak of DENV1 in Marilia in 2015. DENV1 genetic diversity was assessed through capsid and pre-membrane junction encoding gene (CprM) sequencing. The results revealed circulation of DENV1 serotype from 2007 to 2015, with epidemics occurring every three-years until 2013 and then, increasing yearly. There were significant differences in hematological profiles of DENV1 patients between 2015 and 2007. CprM showed DENV1 genetic variability in 2015, contrasting with the unique sequence pattern in 2007. These results reinforce the regional and temporal characteristics of DENV epidemics that need local public health research to improve care for people and to limit the spread of new serotypes/genotypes to uninfected areas.