Recurrent community-acquired pneumococcal meningitis in adults with and without identified predisposing factors.

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.

Integridade na pesquisa: ética profissional dos cientistas - O papel do Comitê de Integridade na Pesquisa do Instituto Adolfo Lutz / Research integrity: professional ethics of scientists - The role of the Research Integrity Committee at the Adolfo Lutz Institute

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)

Multiplex PCR to Streptococcus pneumoniae serotype identification directly in cerebrospinal fluid samples

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.

Implantação da rede laboratorial para realização do ensaio de liberação de interferon-gama (IGRA) para detecção de tuberculose latente no Estado de São Paulo: primeiros passos e desafios / Implementation of the laboratory network to carry out the interferon-gamma release assay (IGRA) to detect latent tuberculosis in the State of Sao Paulo: first steps and challenges

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.

Multiplex real-time PCR using SYBR Green: Unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid.

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.

Multiplex real-time PCR using SYBR Green: unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid

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)

Recent HIV infections: evaluation of a simple identification score for newly diagnosed patients.

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.

SARS-CoV-2 intralineage variation and temporal patterns of COVID-19 risk factors in three cities of southeastern Brazil: Age, sex, and race.

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.

Recent HIV infections: evaluation of a simple identification score for newly diagnosed patients

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.

Epidemiological and genetic aspects of the largest dengue outbreak recorded in 2015 in Southeastern Brazil.

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.

Field evaluation of COVID-19 antigen tests versus RNA based detection: Potential lower sensitivity compensated by immediate results, technical simplicity, and low cost.

One year into the coronavirus disease 2019 (COVID-19) pandemic, diagnostic strategies, although central for contact tracing and other preventive measures, are still limited. To meet the global demand, lower cost and faster antigen tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection are a convenient alternative to the gold standard reverse transcription-polymerase chain reaction (RT-PCR) assay. We tested laboratory-based RT-PCR RNA detection and two rapid antigen detection (RAD) tests, based on the immunochromatography test for nucleocapsid protein of SARS-CoV-2 (COVID-19 Ag ECO Test, ECO Diagnóstica, and Panbio COVID-19 Ag Rapid Test Abbott). Paired collection and testing were done in a small prospective open study in three clinical services in São Paulo, constituted of mostly symptomatic volunteers at collection (97%, 109/112) for a median of 4 days (interquartile range: 3-6), ranging from 1 to 30. Among the 108 paired RT-PCR/RAD tests, results were concordant in 96.4% (101/108). The test's performance was comparable, with an overall sensitivity of 87% and a specificity of 96%. These observations add to other data that suggest that antigen tests may provide reasonable sensitivity and specificity and deserve a role to improve testing strategies, especially in resource-limited settings.

Field evaluation of COVID­19 antigen tests versus RNA based detection: Potential lower sensitivity compensated by immediate results, technical simplicity, and low cost

One year into the coronavirus disease 2019 (COVID­19) pandemic, diagnosticstrategies, although central for contact tracing and other preventive measures, arestill limited. To meet the global demand, lower cost and faster antigen tests forsevere acute respiratory syndrome coronavirus 2 (SARS­CoV­2) detection are aconvenient alternative to the gold standard reverse transcription­polymerase chainreaction (RT­PCR) assay. We tested laboratory­based RT­PCR RNA detection andtwo rapid antigen detection (RAD) tests, based on the immunochromatography testfor nucleocapsid protein of SARS­CoV­2 (COVID­19 Ag ECO Test, ECO Diagnóstica,and Panbio COVID­19 Ag Rapid Test Abbott). Paired collection and testing weredone in a small prospective open study in three clinical services in São Paulo,constituted of mostly symptomatic volunteers at collection (97%, 109/112) for amedian of 4 days (interquartile range: 3­6), ranging from 1 to 30. Among the108 paired RT­PCR/RAD tests, results were concordant in 96.4% (101/108). Thetest's performance was comparable, with an overall sensitivity of 87% and aspecificity of 96%. These observations add to other data that suggest that antigentests may provide reasonable sensitivity and specificity and deserve a role toimprove testing strategies, especially in resource­limited settings.

Impacto do uso da reação em cadeia da polimerase para o diagnóstico de meningite bacteriana em substituição a contraimunoeletroforese / Impact of using polymerase chain reaction for the diagnosis of bacterial meningitis in substitution for counterimmunoelectrophoresis

Introdução: a meningite bacteriana é um grave problema de Saúde Pública mundial, tendo como principais agentes: Neisseria meningitidis, Streptococcus pneumoniae e Haemophilus influenzae. A metodologia de diagnóstico empregada no Instituto Adolfo Lutz ­ Centro de Laboratório Regional Santo André até o ano de 2011 era a contraimunoeletroforese (CIE), depois foi substituída pela reação em cadeia da polimerase em tempo real (qPCR), que apresenta maior sensibilidade. Objetivo: este trabalho objetivou comparar ambas as metodologias no período de 2009 a 2018, para avaliação do impacto da introdução da qPCR no diagnóstico das meningites bacterianas nos 7 municípios da região do ABC do Estado de São Paulo. Metodologia: foram avaliadas a quantidade total de exames realizados, a média mensal, a positividade no período, os municípios requisitantes e a prevalência das bactérias causadoras de meningite, no período de abril/2009 até dezembro/2018. Resultados: Foram 377 exames de CIE e 1305 de qPCR, com média anual de 230 exames em 2010-2013 e 130 exames em 2014-2018. Observou-se aumento da positividade entre as técnicas, 17,8% para CIE e 33,8% para qPCR. N. meningitidis foi responsável pela maioria dos casos entre 2011 e 2013, cerca de 61% dos casos positivos, enquanto que entre 2014 e 2018 foi S. pneumoniae, cerca de 53%. Conclusão: os resultados indicaram que a qPCR foi mais eficiente em detectar os agentes causadores de meningite bacteriana na região do que a técnica de CIE. Por fim, este trabalho suporta a implantação da metodologia de qPCR para diagnóstico de meningite em substituição de técnicas menos sensíveis.

Introduction: bacterial meningitis is still a serious worldwide public health problem, and the main etiological agents are: Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. The diagnostic methodology employed at the Adolfo Lutz Institute ­ Santo André Regional Laboratory Center until 2011 was the ounterimmunoelectrophoresis (CIE), then it was replaced by the real-time polymerase chain reaction (qPCR), which is more sensitivity. Objective: this study aimed to compare both methodologies from 2009 to 2018 to evaluate the impact of the introduction of qPCR in the diagnosis of bacterial meningitis in the 7 cities of the ABC region of São Paulo State. Methodology: the total number of tests performed, the month average, the positivity in the period, the requesting cities and the prevalence of bacteria causing meningitis were evaluated from April/2009 to December/2018. Results: there were 377 CIE exams and 1305 qPCR exams, with an annual average of 230 exams in 2010-2013 and 130 exams in 2014-2018. There was an increase in positivity between the performed techniques, 17.8% for CIE and 33.8% for qPCR. N. meningitidis accounted for most cases of bacterial meningitis between 2011 and 2013, about 61% of positive cases, whereas between 2014 and 2018 it was S. pneumoniae, with about 53%. Conclusion: the results indicated that qPCR was more efficient in detecting the agents that cause bacterial meningitis in the region than the CIE technique. Finally, this work supports the implementation of qPCR methodology for diagnosis of meningitis in replacement of less sensitive techniques.

Undiagnosed acute HIV infection identified through RNA testing of pooled serum samples obtained during a dengue outbreak in São Paulo, Brazil.

INTRODUCTION:: Improving HIV diagnostics and treatment is necessary to end the AIDS epidemic. Pooled plasma can be used to identify patients with acute HIV disease, even before serological tests. During dengue outbreaks, patients having symptoms common to other acute viral diseases might seek medical care. METHODS:: We evaluated HIV RNA in pooled seronegative dengue samples. RESULTS:: After excluding individuals with a known HIV diagnosis, an HIV-1 prevalence of 0.73% [95% confidence interval (CI) 0.23-1.76; 4/546 samples] was found. CONCLUSIONS:: Promoting strategies to diagnose these individuals and provide them with medical treatment might be instrumental for controlling the HIV epidemic.

Co-circulating serotypes in a dengue fever outbreak: Differential hematological profiles and phylogenetic relationships among viruses.

BACKGROUND: Dengue virus, represented by four distinct, genetically diverse serotypes, is the etiologic agent of asymptomatic to severe hemorrhagic diseases. The spatiotemporal dynamics of dengue serotypes and its association to specific diseases vary among the different regions worldwide. By 2007, and in São Paulo State, Brazil, dengue-case concentration in urban centers had changed to increased incidence in small- and medium-sized towns, the case of Marília. OBJECTIVES: The aim of this article was to distinguish dengue serotypes circulating during the 2007 Marília outbreak and define their association to demographic and hematological patient profiles, as well as the phylogenetic relationships among the different viruses. STUDY DESIGN: PCR amplicons corresponding to the junction of capsid and dengue pre-membrane encoding genes, obtained from dengue serologically positive patients, were sequenced. Hematological and demographic data of patients with different Dengue serotypes were evaluated by univariate and bivariate statistics. Dengue PCR sequences were used in phylogenetic relationships analyzed for maximum parsimony. RESULTS: Molecular typing confirmed co-circulation of the dengue serotypes 1 (DENV1) and 3 (DENV3), which presented divergent correlation patterns with regard to hematological descriptors. The increase in atypical lymphocytes, a likely indication of virus load, could be significantly associated to a decrease in leukocyte counts in the DENV3 group and platelet in the DENV1. Phylogenetic reconstitution revealed the introduction of DENV1 from northern Brazil and local divergence of DENV3 by either microevolution or viral introduction from other geographical regions or both. CONCLUSIONS: Dengue dynamics showed regional molecular-epidemiologic specificity, which has important implications for introduction of vaccines, disease management, and transmission control.

Undiagnosed acute HIV infection identified through RNA testing of pooled serum samples obtained during a dengue outbreak in São Paulo, Brazil

Abstract INTRODUCTION: Improving HIV diagnostics and treatment is necessary to end the AIDS epidemic. Pooled plasma can be used to identify patients with acute HIV disease, even before serological tests. During dengue outbreaks, patients having symptoms common to other acute viral diseases might seek medical care. METHODS: We evaluated HIV RNA in pooled seronegative dengue samples. RESULTS: After excluding individuals with a known HIV diagnosis, an HIV-1 prevalence of 0.73% [95% confidence interval (CI) 0.23-1.76; 4/546 samples] was found. CONCLUSIONS: Promoting strategies to diagnose these individuals and provide them with medical treatment might be instrumental for controlling the HIV epidemic.

Tuberculose pulmonar paucibacilar em Centros de Detenção Provisória / Paucibacillary pulmonary tuberculosis in ProvisionalDetention Centers

Cultura de micobactérias proporciona o crescimento de bacilos viáveis, mesmo presentes emescassa quantidade e não detectados pela baciloscopia. Neste estudo foram analisadas as amostrasde escarro que apresentaram baciloscopia negativa e cultura positiva. As amostras foram coletadasde 2008 a 2013, de indivíduos detidos em Centros de Detenção Provisória de Santo André,Mauá e Diadema, Estado de São Paulo. As metodologias utilizadas foram baciloscopia porcoloração Ziehl-Neelsen e cultura pelo Sistema BACTEC MGIT 960 e Ogawa-Kudoh. Dos11.529 exames realizados, 221 (1,9 %) apresentaram baciloscopias negativas e culturas positivas.Dos 221 isolados, 166 (75,1 %) pertenciam ao Complexo Mycobacterium tuberculosis, 21 (9,5 %)micobactérias não membros do Complexo Mycobacterium tuberculosis (MNT), 33 (14,9 %)Mycobaterium sp e uma cultura mista do Complexo M. tuberculosis e M. avium. MNT maisfrequentes foram M. avium (23,8 %) e M. fortuitum (19,0 %). A maioria dos isolados do ComplexoM. tuberculosis (155/166 - 93,4 %) foi sensível aos antimicrobianos. Sete amostras apresentaramresistência à isoniazida e uma apresentou multirresistência à isoniazida e rifampicina. Este estudomostra a importância da realização da cultura em escarros que apresentam baciloscopia negativano diagnóstico da TB e micobacteriose. O tratamento tardio causa a continuidade da transmissãoda doença e agravamento do quadro clínico.

Culture of mycobacteria induces the growth of viable bacillus occurring in small quantity,which are no detectable by bacilloscopy. This study aimed at identifying the mycobacteria isolatesfrom sputum presenting negative bacilloscopy and positive culture. The samples were collectedfrom 2008 to 2013 from criminals of Provisional Detention Centers in Santo André, Mauáand Diadema/SP. Smears were stained by Ziehl-Neelsen staining and the cultures were performedby the BACTEC MGIT 960 system and Ogawa-Kudoh culture medium. Of 11,529 isolates, 221(1.9 %) showed negative bacilloscopy and positive cultures. Of 221 isolates, 166 (75.1 %) belongedto Mycobacterium tuberculosis complex, 21 (9.5 %) were nontuberculous mycobacteria (NTM),33 (14.9 %) Mycobacterium sp, and one identified as a mixed culture of M. tuberculosis andM. avium complex. The most common NTM species were M. avium (23.8 %) and M. fortuitum(19.0 %). Most of the isolates (155/166-93.4 %) were susceptible to antimicrobial agents.Seven samples were resistant to isoniazid, and one presented multiresistance to isoniazid andrifampicin. This study shows the importance in performing sputum culture, when these samplesare negative on bacilloscopy in diagnosing TB and mycobacteriosis. The treatment delay resultsin the maintenance of disease transmission and worsening of clinical symptoms.

Undiagnosed acute HIV infection identified through RNA testing of pooled serum samples obtained during a gengue outbreak in São Paulo, Brazil

INTRODUCTION: Improving HIV diagnostics and treatment is necessary to end the AIDS epidemic. Pooled plasma can be used to identify patients with acute HIV disease, even before serological tests. During dengue outbreaks, patients having symptoms common to other acute viral diseases might seek medical care. METHODS: We evaluated HIV RNA in pooled seronegative dengue samples. RESULTS: After excluding individuals with a known HIV diagnosis, an HIV-1 prevalence of 0.73% [95% confidence interval (CI) 0.23-1.76; 4/546 samples] was found. CONCLUSIONS: Promoting strategies to diagnose these individuals and provide them with medical treatment might be instrumental for controlling the HIV epidemic. Keywords: HIV; Acute disease; Dengue

Tuberculose pulmonar paucibacilar em Centros de Detenção Provisória / Paucibacillary pulmonary tuberculosis in Provisional Detention Centers

Cultura de micobactérias proporciona o crescimento de bacilos viáveis, mesmo presentes em escassa quantidade e não detectados pela baciloscopia. Neste estudo foram analisadas as amostras de escarro que apresentaram baciloscopia negativa e cultura positiva. As amostras foram coletadas de 2008 a 2013, de indivíduos detidos em Centros de Detenção Provisória de Santo André, Mauá e Diadema, Estado de São Paulo. As metodologias utilizadas foram baciloscopia por coloração Ziehl-Neelsen e cultura pelo Sistema BACTEC MGIT 960 e Ogawa-Kudoh. Dos 11.529 exames realizados, 221 (1,9 %) apresentaram baciloscopias negativas e culturas positivas. Dos 221 isolados, 166 (75,1 %) pertenciam ao Complexo Mycobacterium tuberculosis, 21 (9,5 %) micobactérias não membros do Complexo Mycobacterium tuberculosis (MNT), 33 (14,9 %) Mycobaterium sp e uma cultura mista do Complexo M. tuberculosis e M. avium. MNT mais frequentes foram M. avium (23,8 %) e M. fortuitum (19,0 %). A maioria dos isolados do Complexo M. tuberculosis (155/166 - 93,4 %) foi sensível aos antimicrobianos. Sete amostras apresentaram resistência à isoniazida e uma apresentou multirresistência à isoniazida e rifampicina. Este estudo mostra a importância da realização da cultura em escarros que apresentam baciloscopia negativa no diagnóstico da TB e micobacteriose. O tratamento tardio causa a continuidade da transmissão da doença e agravamento do quadro clínico.

Culture of mycobacteria induces the growth of viable bacillus occurring in small quantity, which are no detectable by bacilloscopy. This study aimed at identifying the mycobacteria isolates from sputum presenting negative bacilloscopy and positive culture. The samples were collected from 2008 to 2013 from criminals of Provisional Detention Centers in Santo André, Mauá and Diadema/SP. Smears were stained by Ziehl-Neelsen staining and the cultures were performed by the BACTEC MGIT 960 system and Ogawa-Kudoh culture medium. Of 11,529 isolates, 221 (1.9 %) showed negative bacilloscopy and positive cultures. Of 221 isolates, 166 (75.1 %) belonged to Mycobacterium tuberculosis complex, 21 (9.5 %) were nontuberculous mycobacteria (NTM), 33 (14.9 %) Mycobacterium sp, and one identified as a mixed culture of M. tuberculosis and M. avium complex. The most common NTM species were M. avium (23.8 %) and M. fortuitum (19.0 %). Most of the isolates (155/166-93.4 %) were susceptible to antimicrobial agents. Seven samples were resistant to isoniazid, and one presented multiresistance to isoniazid and rifampicin. This study shows the importance in performing sputum culture, when these samples are negative on bacilloscopy in diagnosing TB and mycobacteriosis. The treatment delay results in the maintenance of disease transmission and worsening of clinical symptoms.