Luciana de Souza Nunes

High prevalence of Neisseria meningitidis hypervirulent lineages and emergence of W135:P1.5,2:ST-11 clone in Southern Brazil

OBJECTIVES The aim of this study was to characterize Neisseria meningitidis strains causing invasive disease in Rio Grande do Sul (RS), during 2003-2005, monitoring the occurrence of hypervirulent lineages, as well as to determine the diversity of PorA VR types for the corresponding isolates and clinical specimens. METHODS Isolates and clinical specimens were characterized by MLST and PorA VR typing. RESULTS This study demonstrated high prevalence of some hypervirulent lineages and emergence of new ones, including the emergence of lineages W135:P1.5,2:ST-11 complex, and C:P1.22,14-6:ST-103 complex. These lineages are probably responsible for the increasing incidence of serogroups C and W135, despite the overall decrease in serogroup B cases during the period. The most prevalent complex was serogroup B ST-32/ET-5 complex. The most prevalent PorA types found for serogroup B were P1.19,15, P1.7,16, and P1.18-1,3, representing a different distribution of PorA types compared to other states of Brazil. CONCLUSIONS This study highlights the importance of monitoring each population, even within the same country. The different distribution of PorA VR types in RS has implications in vaccine design and efficacy. Detailed and accurate meningococcal characterization is an important element in studies of meningococcal epidemiology, population biology, and evolution and provides information for the design of control strategies.

Multidrug-Resistant Nontuberculous Mycobacteria Isolated from Cystic Fibrosis Patients

ABSTRACT Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.

Performance of the GenoType MTBDRplus Assay Directly on Sputum Specimens from Brazilian Patients with Tuberculosis Treatment Failure or Relapse

ABSTRACT Rapid identification of drug resistance in clinical isolates of Mycobacterium tuberculosis is important in determining treatment for tuberculosis. The aim of this work was evaluate the performance of the GenoType MDRTBplus assay directly on sputum of patients who had treatment failure or relapse in a routine outpatient setting in southern Brazil.

Evaluation of respiratory syncytial virus group A and B genotypes among nosocomial and community-acquired pediatric infections in southern Brazil

BackgroundRespiratory syncytial virus (RSV) is the main cause of lower respiratory tract illness in children worldwide. Molecular analyses show two distinct RSV groups (A and B) that comprise different genotypes. This variability contributes to the capacity of RSV to cause yearly outbreaks. These RSV genotypes circulate within the community and within hospital wards. RSV is currently the leading cause of nosocomial respiratory tract infections in pediatric populations. The aim of this study was to evaluate the G protein gene diversity of RSV amplicons.MethodsNasopharyngeal aspirate samples were collected from children with nosocomial or community-acquired infections. Sixty-three RSV samples (21 nosocomial and 42 community-acquired) were evaluated and classified as RSV-A or RSV-B by real-time PCR. Sequencing of the second variable region of the G protein gene was performed to establish RSV phylogenetics.ResultsWe observed co-circulation of RSV-A and RSV-B, with RSV-A as the predominant group. All nosocomial and community-acquired RSV-A samples were from the same phylogenetic group, comprising the NA1 genotype, and all RSV-B samples (nosocomial and community-acquired) were of the BA4 genotype. Therefore, in both RSV groups (nosocomial and community-acquired), the isolates belonged to only one genotype in circulation.ConclusionsThis is the first study to describe circulation of the NA1 RSV genotype in Brazil. Furthermore, this study showed that the BA4 genotype remains in circulation. Deciphering worldwide RSV genetic variability will aid vaccine design and development.

Optimization of one-step duplex real-time RT-PCR for detection of influenza and respiratory syncytial virus in nasopharyngeal aspirates.

Viruses are major contributors to acute respiratory infection-related morbidity and mortality worldwide. The influenza (IF) viruses and human respiratory syncytial virus (RSV) play a particularly important role in the etiology of acute respiratory infections. This study sought to standardize a one-step duplex real-time RT-PCR technique to optimize diagnosis of IFA/IFB and RSVA/RSVB infection. Viral RNA was extracted with the commercially available QIAamp Mini Kit according to manufacturer instructions. RT-PCR was performed with primers to the matrix protein gene of IFA, the hemagglutinin gene of IFB and the N gene of RSVA and RSVB. The limits of detection were 1 copy/μL for IFA, 10 copies/μL for IFB, 5 copies/μL for RSVA, and 250 copies/μL for RSVB. The specificity of RT-PCR was determined by comparison against a panel of several respiratory pathogens. RT-PCR and indirect immunofluorescence (IIF) were compared in a sample of 250 nasopharyngeal aspirates (NPAs) collected during the year 2010. RT-PCR was more sensitive than IIF and able to detect viral co-infections. In summary, RT-PCR optimized for IFA/IFB and RSVA/RSVB is sensitive and specific for these viral agents and is therefore useful for assessment of the etiology of respiratory infections, whether for clinical or epidemiological purposes.

Letter to the editor: Escherichia coli harbouring mcr-1 gene isolated from poultry not exposed to polymyxins in Brazil

SA Lentz 1 2 , D de Lima-Morales 2 3 , VM Cuppertino 1 , LdS Nunes 3 , AS da Motta 1 , AP Zavascki 4 , AL Barth 3 , AF Martins 1 3 1. 1. ICBS – Instituto de Ciências Básicas da Saúde, UFRGS Univ. Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil 2. These authors contributed equally to this work. 3. LABRESIS – Lab. de Pesquisa em Resistência Bacteriana, HCPA Hosp. de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil 4. Infectious Diseases Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil

Antibiotic resistance and detection of the sul2 gene in urinary isolates of Escherichia coli in patients from Brazil

INTRODUCTION The present study aimed to assess the antibiotic resistance profiles and detect the presence of the sul2 gene in sulfamethoxazole-susceptible and resistant isolates of Escherichia coli obtained from outpatients and inpatients with urinary tract infections. METHODOLOGY The resistance profiles of 739 strains were assessed and the presence of the sul2 gene in 100 isolates was tested. RESULTS The antibiotics with the highest resistance rates were ampicillin (57.4%) and trimethoprim-sulfamethoxazole (44.7%). The presence of the gene sul2 was detected in 66.7% of outpatient samples and 67.9% of inpatient samples. CONCLUSIONS Our results demonstrate that E. coli isolates exhibit high resistance to various classes of antibiotics, highlighting the need for developing strategies to help in prescribing antibiotics.

Genetic Background of β-Lactamases in Enterobacteriaceae Isolates from Environmental Samples

The prevalence of β-lactamase-producing Enterobacteriaceae has increased worldwide. Although antibiotic-resistant bacteria are usually associated with hospitals, there are a growing number of reports of resistant bacteria in other environments. Concern about resistant microorganisms outside the hospital setting highlights the need to investigate mechanisms of antibiotic resistance in isolates collected from the environment. The present study evaluated the resistance mechanism to β-lactam antibiotics in 40 isolates from hospital sewage and surface water from the Dilúvio Stream, Porto Alegre City, Southern Brazil. The multiplex PCR technique was used to detect several resistance genes of β-lactamases: extended-spectrum β-lactamases (ESBLs), carbapenemases, and β-lactamase AmpC. After genes, detection amplicons were sequenced to confirm their identification. The clonal relationship was established by DNA macrorestriction using the XbaI enzyme, followed by pulsed-field gel electrophoresis (PFGE). The results indicated that resistance genes were present in 85% of the isolates. The most prevalent genes encoded narrow-spectrum β-lactamase, such as TEM-1 and SHV-1 with 70% of the strains, followed by carbapenemase KPC and GES (45%), ESBL types SHV-5 and CTX-M-8 (27.5%), and AmpC (ACT-1/MIR-1) (2.5%). Twelve isolates contained only one resistance gene, 14 contained two, and eight isolates had three resistance genes. PFGE indicated a clonal relationship among K. pneumoniae isolates. It was not possible to establish a clonal relationship between Enterobacter sp. isolates. The results highlight the potential of these resistance genes to spread in the polluted environment and to present a health risk to communities. This report is the first description of these resistance genes present in environmental samples other than a hospital in the city of Porto Alegre/RS.

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus.

BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Klebsiella pneumoniae carbapenemase-producing Serratia marcescens outbreak in a university hospital

Serratia marcescens is an important pathogen involved in hospitalacquired infections. Outbreaks have been reported and are difficult to eradicate. In neonates, the gastrointestinal tract represents an important reservoir for cross-contamination; however, in adult hospitalized patients, the respiratory tract is more important.1 To date, few reports of Klebsiella pneumoniae carbapenemase (KPC)-producing S marcescens have been described in Brazil. Recently, Da Silva et al2 described an outbreak of carbapenemresistant S marcescens with a focus on coproduction of KPC-2 and IMP-10 in a Brazilian university hospital. The outbreak occurred in an intensive care unit and all isolates were classified in the same clonal profile by pulsed-field gel electrophoresis (PFGE). We faced a simultaneous S marcescens outbreak in another Brazilian hospital, 1 of them by a KPC-producing clone. The outbreak occurred at Hospital Universitario Evangelico de Curitiba, a tertiary-care trauma reference hospital in southern Brazil with a total of 660 beds. All 17 isolates of S marcescens from 12 patients admitted to Hospital Universitario Evangelico de Curitiba September 11-October 22, 2015, were evaluated. Epidemiologic and microbiologic data of bacterial isolates are presented in Table 1. Bacterial isolates were identified and tested for antimicrobial susceptibility using the Vitek2 Compact System (bioMérieux, Durham, NC). Isolates were submitted to polymerase chain reaction for blaKPC using EasyQ KPC (bioMérieux, Marcy-lÉtoile, France) as previously described.3 One isolate from each patient (12 isolates) was genotyped by PFGE according to Kaufman et al.4 The first case of Serratia infection was identified September 11, 2015, in a blood culture in the neonatal intensive care unit (NICU). All patients in the NICU (19 beds) were placed under contact precautions, and surveillance cultures were collected from all patients. Two days later, S marcescens was identified in blood culture from a patient in an adult ICU. The units (NICU and adult ICU) are separated by 3 levels and health care workers are exclusive to each unit. Furthermore, the susceptibility profile of S marcescens was different between the patient in the NICU (carbapenem resistant) and the adult ICU (carbapenem susceptible). After the identification of these first cases, another 7 occurred in different units, and 5 were resistant to carbapenems. Carbapenemase-producing Enterobacteriaceae phenotipically identified by modified Hodge test was positive. All isolates of Serratia spp resistant to carbapenem were sent to molecular carbapenemase detection and presented the blaKPC-2 gene. Cultures from beds, furniture, water tap, sink, alcohol gel dispenser, ventilation circuits, and the nebulization system were taken. All cultures from both ICUs were negative for Serratia. After the last case, no more KPC-producing S marcescens organisms were identified in the institution at least 1 year after. According to PFGE, 8 isolates could be categorized into 2 profiles: type A (isolates 1, 2, 3, 6, 7) was the most prevalent and found in the adult ICU (all of them harboring blaKPC-2 gene) and the less prevalent type B (isolates 9, 10, 11) that showed a certain similarity with the isolates of the type A profile (79%) (Fig 1). All type B isolates were carbapenem-susceptible. This fact suggests that the isolates of both profiles may have the same common ancestor and