Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the top 10 causes of death worldwide. Drug-resistant tuberculosis (DR-TB) poses a major threat to the World Health Organization's “End TB” strategy which has defined its target as the year 2035. In 2019, there were close to 0.5 million cases of DRTB, of which 78% were resistant to multiple TB drugs. The traditional culture-based drug susceptibility test (DST - the current gold standard) often takes multiple weeks and the necessary laboratory facilities are not readily available in low-income countries. Whole genome sequencing (WGS) technology is rapidly becoming an important tool in clinical and research applications including transmission detection or prediction of DR-TB. For the latter, many tools have recently been developed using curated database(s) of known resistance conferring mutations. However, documenting all the mutations and their effect is a time-taking and a continuous process and therefore Machine Learning (ML) techniques can be useful for predicting the presence of DR-TB based on WGS data. This can pave the way to an earlier detection of drug resistance and consequently more efficient treatment when compared to the traditional DST.

Abstract Background: Surveillance of multidrug resistant/extensively drug-resistant tuberculosis (MDR/XDR-TB) is essential to guide disease dissemination control measures. Brazil contributes to a significant fraction of tuberculosis (TB) cases worldwide, but only few reports addressed MDR/XDR-TB in the country. Methods: This cross-sectional, laboratory-based study describes the phenotypic resistance profiles of isolates obtained between January 2008 and December 2011 in Bahia, Brazil, and sociodemographic, epidemiological, and clinical characteristics (obtained from mandatory national registries) of the corresponding 204 MDR/XDR-TB patients. We analyzed the mycobacterial spoligotyping and variable number of tandem repeats of mycobacterial interspersed repetitive units in 12-loci profiles obtained from Salvador. Results: MDR/XDR-TB patients were predominantly male, had a median age of 43 years, belonged to black ethnicity, and failed treatment before MDR-TB diagnosis. Nearly one-third of the isolates had phenotypic resistance (evaluated by mycobacteria growth indicator tube assay) to second-line anti-TB drugs (64/204, 31%), of which 22% cases (14/64) were diagnosed as XDR-TB. Death was a frequent outcome among these individuals and was associated with resistance to second-line anti-TB drugs. Most isolates successfully genotyped belonged to the Latin-American Mediterranean (LAM) Family, with an unprecedented high proportion of LAM10-Cameroon subfamily bacilli. More than half of these isolates were assigned to a unique cluster by the genotyping methods performed. Large clusters of identical genotypes were also observed among LAM SIT42 and SIT376 strains. Conclusions: We highlight the need for strengthening local and national efforts to perform early detection of TB drug resistance and to prevent treatment discontinuation to limit the emergence of drug-resistant strains.

Introduction: COVID-19 broke out in Wuhan in China in December 2019, soon reached pandemic level causing thousands of deaths worldwide. It presents mostly mild flu cases; however, it can evolve into a severe respiratory syndrome that is responsible for hospitalizations and deaths. Objective: To describe the epidemiological and spatial profile of confirmed cases of influenza-like syndrome (GB) by COVID-19 in the state of Pará. Method: Descriptive and ecological study, referring to cases of COVID-19 reported in the E-SUS VE. From the variables of the epidemiological profile, spatial distribution and temporal trend of the disease. Results: The spatial distribution showed that the concentration of cases is located in the southern municipalities of the state. In the profile variables: female gender (53.16%), age group (61.14%) 30 and 59 years, chronic heart disease represented (41.99%), diabetes (31.22%) and (16.30%) chronic respiratory diseases Health professionals infected (8%). Clinical features: fever (71.99%), cough (64.57%), and sore throat (46.78%). Confirmation criteria: rapid antibody test (84.39%), rapid antigen test (7.61%), and RT-PCR (7.48%). The temporal trend of the disease showed the peak of cases in the 20th epidemiological week. Conclusion: Mostly women, most frequent heart diseases and diabetes, confirmed by rapid test and classical symptoms of GS. A flattening of the curve was observed after the 21st week, possibly due to the lockdown intervention. However, it was shown the high number of cases on 07/07/2020 in several regions, specifically in municipalities of the southern region of the state.

Introdução: A COVID-19 surgiu em Wuhan na China em dezembro de 2019, logo alcançou o nível de pandemia causando milhares de mortes no mundo. Se apresenta em sua maioria casos gripais leves, entretanto pode evoluir para uma síndrome respiratória grave que é responsável pelas hospitalizações e óbitos. Objetivo: Descrever o perfil epidemiológico e espacial dos casos confirmados de Síndrome Gripal (SG) por COVID-19 no estado do Pará. Método: Estudo descritivo e ecológico, referente aos casos de COVID-19 notificados no E-SUS VE. A partir das variáveis do perfil epidemiológico, distribuição espacial e tendência temporal da doença. Resultados: A distribuição espacial mostrou que a concentração dos casos localiza-se nos municípios do Sul do estado. Nas viariáveis do perfil: sexo feminino (53,16%), faixa etária (61,14%) 30 e 59 anos, doenças cardíacas crônicas representou (41,99%), diabetes (31,22%) e (16,30%) doenças respiratórias crônicas Profissionais da saúde infectados (8%). Características clínicas: febre (71,99%),tosse (64,57%) e dor de garganta (46,78%). Critério de confirmação: teste rápido- anticorpo (84.39%), teste rápido antígeno (7.61%) e RT-PCR (7.48%). Na tendência temporal da doença evidenciou-se o pico de casos na 20º semana epidemiológica. Conclusão: A maioria mulheres, doenças cardíacas e diabetes mais evidentes, confirmados a maioria por teste rápido e sintomatologia clássica da SG. Observou-se o achatamento da curva após a 21º semana, possivelmente, por conta da intervenção lockdown. Entretanto mostrou-se a alta de casos na data de 07/07/2020 em diversas regiões, especificamente em municípios da região sul do estado.

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

BACKGROUND The evaluation of procedures for drug susceptibility prediction of Mycobacterium tuberculosis based on genomic data against the conventional reference method test based on culture is realistic considering the scenario of growing number of tools proposals based on whole-genome sequences (WGS). OBJECTIVES This study aimed to evaluate drug susceptibility testing (DST) outcome based on WGS tools and the phenotypic methods performed on isolates of M. tuberculosis Lineage 1 from the state of Pará, Brazil, generally associated with low levels of drug resistance. METHODOLOGY Culture based DST was performed using the Proportion Method in Löwenstein-Jensen medium on 71 isolates that had been submitted to WGS. We analysed the seven main genome sequence-based tools for resistance and lineage prediction applied to M. tuberculosis and for comparison evaluation we have used the Kappa concordance test. FINDINGS When comparing the WGS-based tools against the DST, we observed the highest level of agreement using TB-profiler. Among the tools, TB-profiler, KvarQ and Mykrobe were those which identified the largest number of TB-MDR cases. Comparing the four most sensitive tools regarding resistance prediction, agreement was observed for 43 genomes. MAIN CONCLUSIONS Drug resistance profiling using next-generation sequencing offers rapid assessment of resistance-associated mutations, therefore facilitating rapid access to effective treatment.

ABSTRACT In Fortaleza, the capital of Ceara State, Brazil, the detection rate of tuberculosis (TB) in 2018 was 65.5/100,000 inhabitants with a cure rate of 59.1%, which is higher than the country average. This study investigated the risk factors associated with drug-resistant tuberculosis (DR-TB) and identified the drug-resistance phenotype and resistance-conferring mutations. The geographic distribution of DR-TB in Fortaleza, Brazil, was also determined. From March 2017 to February 2018, 41 DR-TB isolates and 69 drug-susceptible pulmonary TB isolates were obtained from patients seen at a referral hospital in Fortaleza, Brazil. Samples were subjected to phenotypic and genetic analysis of resistance; the spatial distribution of the participants was also analyzed. Primary resistance was high (50.9%) among participants. The following risk factors for DR were identified: being female ( p = 0.03), having diabetes ( p < 0.01), history of previous TB disease ( p < 0.01), and the number of intra-domiciliary contacts ( p < 0.01). Analysis by multiplex allele-specific polymerase chain reaction detected mutations in the genes katG (65.8%) , rpoB (43.9%), inhA promoter (14.6%), and gyrA (9.8%). Sequencing identified mutations in the the genes katG (75.6%), inhA promoter (19.5%), rpoB (85.4%), and gyrA (100%). There was no mutation in the rrs gene. Spatial analysis showed DR-TB isolates distributed in areas of low socioeconomic status in the city of Fortaleza. Our results emphasized the importance of detecting resistance to TB drugs. The resistance found in the gene gyrA is of concern due to the high number of pre-extensive DR-TB cases in Fortaleza.

Abstract Pseudomonas aeruginosa is the leading cause of nosocomial infections with high mortality rates owing to the limited therapeutic options for multidrug-resistant Pseudomonas aeruginosa (MDRPA) and metallo-beta-lactamase (MBL)-producing strains. Herein, we present a meta-analysis exploring the association between MDRPA and São Paulo MBL-1 (SPM-1)-producing strains vs. mortality. Online databases were screened to identify studies published between 2006 and 2016. A total of 15 studies, comprising 3,201 cases of P. aeruginosa infection, were included. Our results demonstrated a higher mortality rate among patients infected with MDRPA (44.6%, 363/813) than those with non-MDRPA infection (24.8%, 593/2,388) [odds ratio (OR) 2.39, 95% confidence interval (CI) 1.70-3.36, p <0.00001]. The risk of mortality in patients with non-SPM-1 strains was four times higher than that observed in the patients of the SPM-1 group; however, no statistically significant difference was observed (p = 0.43). In conclusion, the results of our study demonstrated that patients infected with MDRPA had a significantly higher mortality rate than that of patients infected with non-MDRPA strains, especially patients with bloodstream infection (BSI), immunosuppression, and inadequate antimicrobial therapy. The absence of studies on the molecular aspects of blaSPM-1 and its association with mortality limited the analysis; therefore, our results should be interpreted with caution. Our findings also highlight the need for more studies on the molecular aspects of resistance and the peculiarities of different nosocomial settings.

RESUMO Objetivo: Descrever as manifestações clínicas de pacientes com infecção pulmonar por micobactérias do complexo Mycobacterium abscessus (CMA) e compará-las com as daqueles infectados com outras micobactérias não tuberculosas (MNT). Métodos: Estudo de coorte retrospectivo envolvendo 43 pacientes divididos em dois grupos: grupo CMA, com pacientes com infecção pulmonar por CMA (n = 17); e grupo MNT, com pacientes com infecção pulmonar por MNT que não CMA (n = 26). Os pacientes foram previamente tratados com o esquema rifampicina, isoniazida, pirazinamida e etambutol antes de o diagnóstico de MNT ser confirmado com culturas positivas em duas amostras de escarro diferentes. As sequências nucleotídicas dos genes hsp65, RNAr 16S e/ou rpoB foram analisadas para a identificação das micobactérias. Foram coletadas características demográficas, clínicas e radiológicas, assim como respostas terapêuticas e desfechos. Resultados: A única manifestação clínica significativamente mais frequente no grupo CMA que no grupo MNT foi hiporexia (p = 0,0306). A chance de haver a necessidade de utilização de um segundo esquema terapêutico foi quase 12 vezes maior no grupo CMA que no grupo MNT. O sucesso terapêutico foi significativamente maior no grupo MNT que no grupo CMA (83,2% vs. 17,6%; p < 0,0001). A chance de recidiva no grupo CMA foi aproximadamente 37 vezes maior que no grupo MNT. Conclusões: Na amostra estudada, a resposta terapêutica da doença pulmonar causada por CMA evoluiu de forma menos favorável do que naquela causada pelas demais MNT.

ABSTRACT Objective: To describe the clinical manifestations of patients with pulmonary infection caused by mycobacteria of the Mycobacterium abscessus complex (MABSC), and to compare these manifestations with those of patients infected with other nontuberculous mycobacteria (NTM). Methods: This was a retrospective cohort study involving 43 patients divided into two groups: the MABSC group, consisting of patients with pulmonary infection caused by MABSC (n = 17); and the NTM group, consisting of patients with pulmonary infection caused by NTM other than MABSC (n = 26). Patients were previously treated with a regimen of rifampin, isoniazid, pyrazinamide, and ethambutol before the diagnosis of NTM was confirmed by two culture-positive sputum samples. The nucleotide sequences of the hsp65, 16S rRNA, and/or rpoB genes were analyzed to identify the mycobacteria. Data were collected on demographic, clinical, and radiological characteristics, as well as on treatment responses and outcomes. Results: Loss of appetite was the only clinical manifestation that was significantly more common in the MABSC group than in the NTM group (p = 0.0306). The chance of having to use a second treatment regimen was almost 12 times higher in the MABSC group than in the NTM group. Treatment success was significantly higher in the NTM group than in the MABSC group (83.2% vs. 17.6%; p < 0.0001). The chance of recurrence was approximately 37 times higher in the MABSC group than in the NTM group. Conclusions: In the study sample, treatment response of pulmonary disease caused by MABSC was less favorable than that of pulmonary disease caused by other NTM.

Abstract: INTRODUCTION: The spread of multidrug-resistant Pseudomonas aeruginosa in Brazilian hospitals has greatly impacted upon the morbidity and mortality of individuals in intensive care units. Given the lack of information regarding the dynamics of multidrug resistance in northern Brazil, we analyzed the clinical and microbiological features of nosocomial infections caused by P. aeruginosa. METHODS Between January 2010 and March 2012, we conducted a retrospective cohort study of P. aeruginosa isolates from 54 patients who were hospitalized in intensive care units. The clinical and epidemiologic variables were analyzed, including the patients' demographic data and comorbidities, and the lengths of the intensive care unit stays, the classification of the infections as nosocomial, the use of invasive procedures, antimicrobial therapy, and the patients' outcomes. We undertook susceptibility tests, molecular detection of the metallo-β-lactamase genes, and genotypic analyses of the isolates using the repetitive element-polymerase chain reaction. RESULTS: Multidrug resistance occurred most frequently among isolates from adults who had been hospitalized for an average of 87.1 days. The use of mechanical ventilation and urinary catheters were risk factors for infection. The four isolates that harbored the blaSPM-1-like gene showed >95% genetic similarity. CONCLUSIONS This study's findings show that P. aeruginosa has a high death rate, and that inadequate treatment and invasive procedures are risk factors for infection. This is the first report describing the detection of the blaSPM-1-like gene in northern Brazil. These results highlight the need for better monitoring and a greater understanding of nosocomial infections and their public health impacts.

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

INTRODUÇÃO: O surto ocorreu entre fevereiro a junho de 2006 e incluiu identificação de casos, análise dos prontuários, culturas ambientais, análise de resistência e genotipagem dos isolados de Serratia marcescens. MÉTODOS: Os cultivos foram compostos de 13 isolados de sangue e 17 swabs de reto e mãos e amostras do ar. RESULTADOS: Os dados obtidos por eletroforese de campo pulsado evidenciaram três cepas que contaminaram 24 pacientes. Infecção sistêmica foi mais comum em neonatos com menor peso, longo tempo de internação, nascimento prematuro e uso de respiração mecânica. CONCLUSÕES: Foi evidenciada a natureza multifatorial do surto. Foi encontrado um clone endêmico de S. marcescens.

INTRODUCTION: The outbreak occurred between February and June 2006 and included identification of the cases, analysis of medical records, cultures from environmental sources, resistance analyses and genotyping profile of Serratia marcescens. METHODS: The cultures were composed of 13 blood isolates, 17 rectal and hand swabs and air sampling. RESULTS: The data obtained by pulsed-field gel electrophoresis exhibited three strains that contaminated 24 patients. Systemic infection was the most common in neonates with lower weight, long periods of hospitalization, premature delivery and the use of mechanical ventilation. CONCLUSIONS: This investigation revealed the multifactorial nature of the outbreak. An endemic clone of S. marcescens was detected.

INTRODUÇÃO: Salmonella Typhi é o agente da febre tifóide (doença caracterizada por febre, cefaléia, mialgia, artralgia, diarréia ou constipação), cujo quadro pode se complicar e levar o paciente a óbito. No Brasil, a febre tifóide é endêmica nas regiões Norte e Nordeste, com surtos ocorridos nos meses de intenso calor. OBJETIVO: Analisar e comparar a variabilidade genética de S. Typhi isoladas de surto e casos esporádicos de febre tifóide ocorridos em determinado período na cidade de Belém (PA). MATERIAL E MÉTODOS: Foram analisadas 20 amostras de S. Typhi: 10 isoladas de um surto ocorrido no bairro do Guamá, Belém, entre os meses de dezembro/2005 e março/2006, e 10 de casos esporádicos ocorridos em diferentes localidades da mesma cidade e no mesmo período do surto. A caracterização genética foi realizada pela análise do perfil de macrorrestrição obtido pela enzima XbaI e definido por eletroforese em gel de campo pulsado (PFGE). RESULTADOS: A análise de XbaI-PFGE das amostras estudadas demonstrou uma similaridade genética de 83% a 100%. CONCLUSÃO: Este estudo pôde demonstrar a relação clonal das amostras S. Typhi causadoras de surto e de casos esporádicos de febre tifóide ocorridos na cidade de Belém no período de dezembro/2005 a março/2006.

BACKGROUND: Salmonella Typhi is the causative agent of typhoid fever, illness characterized by fever, migraine, myalgia, arthralgia, diarrhea or constipation, which may have complications and cause death. In Brazil, the typhoid fever is endemic in the Northern and Northeastern regions, with outbreaks occurring in scorching months. OBJECTIVE: To analyse and compare the genetic variability of S. Typhi strains isolated from outbreaks and sporadic cases of typhoid fever occurred in the city of Belém (PA) between December 2005 and March 2006. MATERIAL AND METHODS: Twenty samples of S. Typhi were analyzed: 10 of them were isolated from an outbreak occurred in Guamá neighborhood in Belém, between December 2005 and March 2006, and the other 10 were isolated from sporadic cases in different neighborhoods of the same city in the same outbreak period. The genetic characterization was performed by macrorestriction analysis of genomic DNA with XbaI enzyme defined by pulsed-field gel electrophoresis (PFGE). RESULTS: The Xbal-PFGE analysis of the studied samples revealed a genetic similarity of 83% to 100%. CONCLUSION: This study demonstrated the clonal relation between the S. Typhi samples from the outbreak and from the sporadic cases of typhoid fever occurred in the city of Belém between December 2005 and March 2006.

A reação em cadeia da polimerase usada para amplificação de uma seqüência interna de um fragmento previamente amplificado (nested-PCR) foi investigada como uma alternativa complementar a pesquisa de bacilos álcool ácido resistentes e a cultura do Mycobacterium tuberculosis em meio de Lowenstein-Jensen. Foram investigadas 144 amostras de escarro de pacientes suspeitos de tuberculose encaminhados ao Laboratório de Tuberculose do Instituto Evandro Chagas em Belém, no período de junho de 2002 a dezembro de 2003. Das 144 amostras, 121 foram caracterizadas como tuberculose, 119 foram positivas na cultura, 95 na baciloscopia e 128 na nested-PCR. A sensibilidade da nested-PCR foi 96% (116/121), enquanto a especificidade foi 48% (11/23). A nested-PCR poderá ser uma ferramenta complementar para o diagnóstico da tuberculose, pois apresenta sensibilidade equivalente à cultura, no entanto, necessita de maiores avaliações visando minimizar o número de resultados falso-positivos.

The polymerase chain reaction used for amplifying an internal sequence of a previously amplified fragment (nested-PCR) was investigated as a complementary alternative for searching for alcohol-acid resistant bacilli and Mycobacterium tuberculosis cultures in Lowenstein-Jensen medium. 144 sputum samples were investigated from patients with suspected tuberculosis that were sent to the Tuberculosis Laboratory of the Evandro Chagas Institute in Belém, between June 2002 and December 2003. From the 144 samples, 121 were characterized as tuberculosis: 119 were positive in cultures, 95 under bacilloscopy and 128 using nested-PCR. The sensibility of the nested-PCR was 96% (116/121), while the specificity was 48% (11/23). Nested-PCR may be a complementary tool for diagnosing tuberculosis, since it presents sensitivity equivalent to that of cultures. However, further evaluations are needed with the aim of minimizing the number of false-positive results.

A reação em cadeia da polimerase (PCR) e suas variações, como a nested-PCR, têm sido destacadas como técnicas moleculares promissoras para o diagnóstico rápido da tuberculose (TB). No presente estudo avaliou-se a nested-PCR utilizando-se como marcadores moleculares a seqüência IS6110 e o antígeno b aplicados ao diagnóstico da TB. Foram submetidas a baciloscopia, cultura e nested-PCR 136 amostras clínicas de pacientes com suspeita de TB. O diagnóstico de tuberculose pulmonar foi atribuído a 116 pacientes e, desses, 97 foram multibacilares e 111 apresentaram cultura positiva para M. tuberculosis. As reações de nested-PCR identificaram 70% (antígeno b) e 94% (IS6110) dos casos paucibacilares. Os valores de sensibilidade determinados para cultura, nested-PCR do IS6110 e antígeno b foram 95%, 98% e 86%, respectivamente. A especificidade foi de 100%, 15% e 45% para cultura, nested-PCR do IS6110 e antígeno b, respectivamente. O diagnóstico molecular da tuberculose deve estar fundamentado na análise conjunta de vários parâmetros, como baciloscopia, cultura, manifestações clínicas, prova terapêutica e história prévia de tuberculose.

Chain reaction of polimerase (PCR) and its variations such as the nested-PCR have been outstanding as promising molecular techniques for the fast diagnosis of the tuberculosis (TB). In the present study the nested-PCR was evaluated using as molecular markers the sequence IS6110 and the antigen b applied to the diagnosis of TB. One hundred and thirty six clinical samples of patients with suspicion of TB were submitted to the baciloscopia, culture and nested-PCR. The diagnosis of pulmonary tuberculosis was attributed to 116 patients, of these, 97 were multi-bacilli and 111 presented positive culture for M. tuberculosis. The nested-PCR reactions identified 70% (antigen b) and 94% (IS6110) of the acid-fast smear negative cases of tuberculosis. The sensibility values determined for culture, and nested-PCR of IS6110 and antigen b were 95%, 98% and 86%, respectively. The specificity was 100%, 15% and 45% for culture, nested-PCR of IS6110 and antigen b, respectively. The molecular diagnosis of the tuberculosis should be based in the combined analysis of several parameters, as the baciloscopia, culture, clinical manifestations, therapeutic proves and previous history of tuberculosis.