Genomic characterization of invasive meningococcal X isolates from Brazil, 1992–2022 / Caracterización genómica de aislamientos invasivos de meningococo X de Brasil, 1992–2022

Introduction: Invasive meningococcal disease (IMD) is a major health problem. Given the post-COVID-19 pandemic scenario with the loosening of the non-pharmacological measures to control the virus transmission and considering the observed global reduction of meningococcal vaccination coverage, an increase in IMD cases can be expected. Methodology: Using whole-genome sequencing, we characterized six Neisseria meningitidis serogroup X (MenX) isolates recovered from IMD cases in Brazil in the last 30 years. Results: The predominance (66.6%, 4/6) of ST2888 presenting fHbp 160, NHBA 129, NadA 21, and PorA 19,15 was found on isolates. Two novel STs, 15458 and 15477, were described. Conclusion: This study describes the circulation of MenX lineage ST2888 in Brazil, previously reported only in Europe. Continuous universal surveillance is crucial to implement prompt public health measures aiming to prevent and control non-vaccine preventable serogroup X IMD cases.(AU)

Genomic characterization of invasive meningococcal X isolates from Brazil, 1992-2022.

INTRODUCTION: Invasive meningococcal disease (IMD) is a major health problem. Given the post-COVID-19 pandemic scenario with the loosening of the non-pharmacological measures to control the virus transmission and considering the observed global reduction of meningococcal vaccination coverage, an increase in IMD cases can be expected. METHODOLOGY: Using whole-genome sequencing, we characterized six Neisseria meningitidis serogroup X (MenX) isolates recovered from IMD cases in Brazil in the last 30 years. RESULTS: The predominance (66.6%, 4/6) of ST2888 presenting fHbp 160, NHBA 129, NadA 21, and PorA 19,15 was found on isolates. Two novel STs, 15458 and 15477, were described. CONCLUSION: This study describes the circulation of MenX lineage ST2888 in Brazil, previously reported only in Europe. Continuous universal surveillance is crucial to implement prompt public health measures aiming to prevent and control non-vaccine preventable serogroup X IMD cases.

SARS-CoV-2 reinfection in a healthcare professional in inner Sao Paulo during the first wave of COVID-19 in Brazil.

Coronavirus Disease 2019 pandemic remains a threat to public health. We report 2 cases of Coronavirus Disease 2019 infection in the same healthcare professional in Brazil. Genomic analysis identified that primoinfection was caused by the endemic lineage B.1.1.33 while reinfection by the lineage B.1.1.44, a lineage with an additional V1176F mutation in S protein.

Two Novel Mutations Associated with Polymyxin-B Resistance in a Pandemic Lineage of Uropathogenic Escherichia coli of the Sequence Type 69.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) are frequent pathogens worldwide, impacting on the morbidity and economic costs associated with antimicrobial treatment. OBJECTIVES: We report two novel mutations associated with polymyxin-B resistance in an UPEC isolate collected in 2019. METHODS: Isolate was submitted to antimicrobial susceptibility testing including broth microdilution for polymyxin B. Whole genome was sequenced and analyzed. RESULTS: Polymyxin-B total inhibition occurred at 16 mg/L (resistant). UPEC isolate was assigned to the phylogroup D, serotype O117:H4, and Sequence Type 69. mcr genes were not detected, but two novel mutations in the pmrA/basS (A80S) and pmrB/basR (D149N) genes were identified. CONCLUSIONS: The occurrence of non-mcr polymyxin resistance in E. coli from extraintestinal infections underscores the need of a continuous surveillance of this evolving pathogen.

COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols.

The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS-CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity and quality of the RNA input. This study compared the performance and cost-effectiveness of three different kits/reagents for RNA extraction used in COVID-19 diagnosis in Sao Paulo, Brazil. A total of 300 NP/OP samples belonging to suspected cases of COVID-19 stored in a biorepository were randomly selected, and RNA was extracted using (i) automated extraction (Loccus, Extracta Kit FAST), (ii) manual extraction (BioGene Kit, Bioclin, Quibasa), and (iii) quick extraction methods (Lucigen, Quick DNA Extract Kit). Next, the samples were tested using RT-qPCR for SARS-CoV-2 with the Allplex 2019-nCoV modified assay and the Charité-Berlin protocol. All assays/kits were used according to the manufacturer's instructions. For the Allplex kit, the sensitivity in detecting SARS-CoV-2 with previously extracted RNA by different procedures was 100.0% for Loccus, 100.0% for BioGene and 91.9% for Quick. Using the Charité-Berlin protocol, the sensitivities were 81.4% for Loccus, 81.2% for BioGene and 60.7% for Quick. The least sensitive target gene and the gene most affected by RNA extraction procedures was the RNA-dependent RNA polymerase gene (Charité-Berlin protocol). No false-positive SARS-CoV-2 results were detected using RNA obtained from any of the different protocols. In conclusion, Loccus and BioGene RNA extractions were efficient for RT-qPCR assays, and although the BioGene procedure is less expensive, Loccus is the best choice because it allows the rapid handling of hundreds or thousands of samples, a desirable feature during pandemics. Although less sensitive, the Quick extraction is useful during outbreaks coupled with the Allplex amplification kit for SARS-CoV-2 diagnosis (κ = 0.925).

SARS-CoV-2 reinfection in a healthcare professional in inner Sao Paulo during the first wave of COVID-19 in Brazil

Coronavirus Disease 2019 pandemic remains a threat to public health. We report 2 cases of Coronavirus Disease 2019 infection in the same healthcare professional in Brazil. Genomic analysis identified that primoinfection was caused by the endemic lineage B.1.1.33 while reinfection by the lineage B.1.1.44, a lineage with an additional V1176F mutation in S protein.

COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols

The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS­CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity and quality of the RNA input. This study compared the performance and cost-effectiveness of three different kits/reagents for RNA extraction used in COVID-19 diagnosis in Sao Paulo, Brazil. A total of 300 NP/OP samples belonging to suspected cases of COVID-19 stored in a biorepository were randomly selected, and RNA was extracted using (i) automated extraction (Loccus, Extracta Kit FAST), (ii) manual extraction (BioGene Kit, Bioclin, Quibasa), and (iii) quick extraction methods (Lucigen, Quick DNA Extract Kit). Next, the samples were tested using RT-qPCR for SARS-CoV-2 with the Allplex 2019-nCoV modified assay and the Charité-Berlin protocol. All assays/kits were used according to the manufacturer's instructions. For the Allplex kit, the sensitivity in detecting SARS-CoV-2 with previously extracted RNA by different procedures was 100.0% for Loccus, 100.0% for BioGene and 91.9% for Quick. Using the Charité-Berlin protocol, the sensitivities were 81.4% for Loccus, 81.2% for BioGene and 60.7% for Quick. The least sensitive target gene and the gene most affected by RNA extraction procedures was the RNA-dependent RNA polymerase gene (Charité-Berlin protocol). No false-positive SARS-CoV-2 results were detected using RNA obtained from any of the different protocols. In conclusion, Loccus and BioGene RNA extractions were efficient for RT-qPCR assays, and although the BioGene procedure is less expensive, Loccus is the best choice because it allows the rapid handling of hundreds or thousands of samples, a desirable feature during pandemics. Although less sensitive, the Quick extraction is useful during outbreaks coupled with the Allplex amplification kit for SARS-CoV-2 diagnosis (κ = 0.925).

Genomic surveillance of Neisseria meningitidis serogroup B invasive strains: Diversity of vaccine antigen types, Brazil, 2016-2018.

BACKGROUND: Neisseria meningitidis serogroup B remains a prominent cause of invasive meningococcal disease (IMD) in Brazil. Because two novel protein-based vaccines against serogroup B are available, the main purpose of this study was to provide data on the diversity and distribution of meningococcal vaccine antigen types circulating in Brazil. METHODOLOGY: Genetic lineages, vaccine antigen types, and allele types of antimicrobial-associated resistance genes based on whole-genome sequencing of a collection of 145 Neisseria meningitidis serogroup B invasive strains recovered in Brazil from 2016 to 2018 were collected. RESULTS: A total of 11 clonal complexes (ccs) were identified among the 145 isolates, four of which were predominant, namely, cc461, cc35, cc32, and cc213, accounting for 72.0% of isolates. The most prevalent fHbp peptides were 24 (subfamily A/variant 2), 47 (subfamily A/variant 3), 1 (subfamily B/variant 1) and 45 (subfamily A/variant 3), which were predominantly associated with cc35, cc461, cc32, and cc213, respectively. The NadA peptide was detected in only 26.2% of the isolates. The most frequent NadA peptide 1 was found almost exclusively in cc32. We found seven NHBA peptides that accounted for 74.5% of isolates, and the newly described peptide 1390 was the most prevalent peptide exclusively associated with cc461. Mutated penA alleles were detected in 56.5% of the isolates, whereas no rpoB and gyrA mutant alleles were found. CONCLUSION: During the study period, changes in the clonal structure of circulating strains were observed, without a predominance of a single hyperinvasive lineage, indicating that an epidemiologic shift has occurred that led to a diversity of vaccine antigen types in recent years in Brazil.

Hypervirulent Klebsiella pneumoniae as Unexpected Cause of Fatal Outbreak in Captive Marmosets, Brazil.

After the sudden death of captive marmosets in São Paulo, Brazil, we conducted a histologic and microbiologic study. We found hyperacute septicemia caused by hypermucoviscous sequence type 86 K2 Klebsiella pneumoniae. We implemented prophylactic antimicrobial therapy, selected dedicated staff for marmoset interactions, and sanitized the animals' fruit to successfully control this outbreak.

Importation and early local transmission of COVID-19 in Brazil, 2020.

We conducted the genome sequencing and analysis of the first confirmed COVID-19 infections in Brazil. Rapid sequencing coupled with phylogenetic analyses in the context of travel history corroborate multiple independent importations from Italy and local spread during the initial stage of COVID-19 transmission in Brazil.

Genomic and Clinical Characterization of IMP-1-Producing Multidrug-Resistant Acinetobacter bereziniae Isolates from Bloodstream Infections in a Brazilian Tertiary Hospital.

Acinetobacter baumannii is the main species of the Acinetobacter genus; however, non-baumannii Acinetobacter (NBA) species causing infections have been described for the past years, as well as antimicrobial resistance. In this study, we describe the occurrence of two multidrug-resistant (MDR) IMP-1-producing Acinetobacter bereziniae isolates recovered from bloodstream infections in different patients but in the same intensive care unit among 134 carbapenem-resistant Acinetobacter screened. Antimicrobial susceptibility testing revealed resistance to carbapenems, extended spectrum, and antipseudomonad cephalosporins, amikacin, and trimethoprim-sulfamethoxazole. Both A. bereziniae isolates shared the same ApaI-pulsed-field gel electrophoresis (PFGE) pattern. Whole-genome sequencing of both isolates revealed that blaIMP-1 was embedded into an In86 Class I integron carrying also sul1, aac(6')-31, and aadA genes. A new sequence type (ST1309 Pasteur) was deposited. The virulence genes lpxC and ompA, seen in A. baumannii, were detected in the A. bereziniae strains. Recognition of A. bereziniae causing invasive MDR infection underscores the role of NBA species as human pathogens especially in at-risk patients.

Importation and early local transmission of COVID-19 in Brazil, 2020

We conducted the genome sequencing and analysis of the first confirmed COVID-19 infections in Brazil. Rapid sequencing coupled with phylogenetic analyses in the context of travel history corroborate multiple independent importations from Italy and local spread during the initial stage of COVID-19 transmission in Brazil. (AU)

Molecular analysis of clonally related Salmonella Typhi recovered from epidemiologically unrelated cases of typhoid fever, Brazil.

BACKGROUND: The primary method of molecular subtyping for the identification and investigation of outbreaks has been pulsed-field gel electrophoresis (PFGE). In some cases, this technique has not been able to show discrimination between the unrelated strains that can be achieved by whole genome sequencing (WGS). METHODS: The aim of this study was to determine the strengths and drawbacks of WGS using different analytic approaches compared to traditional typing method, PFGE, for retrospectively typing clusters cases of 28 S. Typhi. RESULTS: We evaluated three analytical approaches on the WGS data set (Nucleotide Difference (ND), (SNPs) and Whole genome multi locus sequence typing (wgMLST) that identically classified the clusters-related strains into two clusters, cluster A (with strains from 2017), and Cluster B (with strains from 2007). CONCLUSIONS: In this study WGS based typing, was able to compete with PFGE for differentiation of the clusters of S. Typhi strains.

Outbreak of G2P[4] rotavirus gastroenteritis in a retirement community, Brazil, 2015: An important public health risk?

The present study described a group A rotavirus (RVA) outbreak in an age-care facility in Brazil, using epidemiologic and molecular diagnostic methods. A descriptive clinical, epidemiological and environmental investigation was conducted. Stool samples were collected and screened for RVA, Norovirus (NoV), Enteric Adenovirus 40/41 (AdV 40/41) and Astrovirus (AstV) using ELISA, RT-PCR, qRT-PCR, electron microscopy and sequencing methods. Outbreak occurred during 26th-29th October, 2015; 28 individuals affected (22 residents; 6 staff). The attack rate was 25.9% and 8.5% among residents (median-age: 85.5 years) and staff (median-age: 28 years), respectively. Female staff was identified as the index case. RVA G2P[4] genotype was detected in 87.5% (7/8). Genetic analysis demonstrated that the outbreak involved one single strain, suggesting a common-source infection. RVA should be considered during outbreaks investigations in residential facilities, and raise the question if the current licensed RVA vaccines for children could also be helpful for the elderly.

O impacto do último enxágue na citotoxicidade de produtos críticos passíveis de processamento / Impacto del último enjuague en la citotoxicidad de productos críticos pasibles de procesamiento / The impact of the final rinse on the cytoxicity of critical products submitted for processing

RESUMO Objetivo Avaliar a citotoxicidade de produtos submetidos à contaminação desafio, limpeza baseada em procedimento operacional padrão (POP) validado e enxágue final em diferentes tipos de água: de torneira, deionizada, destilada, tratada por osmose reversa e ultrapurificada. Método Estudo experimental e laboratorial. Foram utilizadas como amostras 130 cânulas de hidrodissecção, 26 por grupo experimental, caracterizados, de acordo com a água utilizada no último enxágue. As amostras foram submetidas à contaminação desafio interna e externamente por uma solução contendo 20% sangue de carneiro desfibrinado e 80% de Cloreto de Sódio a 0,9%. Em seguida, tiveram o lúmen preenchido por solução viscoelástica, permanecendo em contato com o contaminante por 50 minutos, sendo então, processadas, de acordo com um POP validado. A citotoxicidade foi avaliada pela captura do corante vital vermelho neutro. Resultados Ausência de citotoxicidade nos extratos das amostras. Conclusão As amostras não demonstraram citotoxicidade, independentemente da qualidade de água utilizada no último enxágue. Os resultados apresentados puderam ser alcançados unicamente por meio do uso de um procedimento operacional padrão de limpeza validado, baseado em literatura científica, em recomendações oficiais e na legislação relacionada.

RESUMEN Objetivo Evaluar la citotoxicidad de productos sometidos a contaminación desafío, limpieza siguiendo procedimiento validado y enjuague en diferentes tipos de agua: de caño, desionizada, destilada, tratada por ósmosis inversa y ultrapurificada. Método fueron utilizados 130 canulas de hidrosección, 26 por grupo experimental, caracterizadas por el tipo agua utilizada en el último enjuague. La muestras fueron contaminadas interna y externamente por una solución con 20% sangre de carnero desfibrinado y 80% de Cloruro de Sodio a 0,9%. Luego el lumen fue cubierto por la solución viscoelastica, permaneciendo en contacto con el contaminante 50 minutos y posteriormente procesados, siguiendo orientaciones del procedimiento padrón validado. El ensayo de citotoxicidad se realizó mediante la incorporación del colorante vital rojo neutro. Resultados ausencia de toxicidad en extractos de las muestras. Conclusión no hubo toxicidad en las muestras, independiente del agua utilizada en el último enjuague. Los resultados fueron alcanzados gracias al uso del procedimiento operacional padrón de limpieza validado, embasado en literatura científica, recomendaciones oficiales y en legislación relacionada.

ABSTRACT Objective To assess the cytotoxicity of products subsequent to a cleaning process based on a validated standard operating procedure (SOP), and a final rinse with different types of water: tap, deionized, distilled, treated by reverse osmosis and ultra-purified. Method This was an experimental and laboratory study. The sample consisted of 130 hydrodissection cannulas, 26 per experimental group, characterized according to type of water used in the final rinse. The samples were submitted to internal and external contamination challenge with a solution containing 20% defibrinated sheep blood and 80% of sodium chloride 0.9%. Next, the lumens were filled with a ophthalmic viscosurgical device, remaining exposed for 50 minutes, and then were processed according to the validated SOP. Cytotoxicity was assessed using neutral red uptake assay. Results No cytoxicity was detected in the sample extracts. Conclusion The samples did not display signs of cytotoxicity, regardless of final rinse quality. The results obtained were reached by using only a validated cleaning operating procedure, based on the scientific literature, and on official recommendations and related regulation.

The impact of the final rinse on the cytoxicity of critical products submitted for processing. / O impacto do último enxágue na citotoxicidade de produtos críticos passíveis de processamento.

Objective To assess the cytotoxicity of products subsequent to a cleaning process based on a validated standard operating procedure (SOP), and a final rinse with different types of water: tap, deionized, distilled, treated by reverse osmosis and ultra-purified. Method This was an experimental and laboratory study. The sample consisted of 130 hydrodissection cannulas, 26 per experimental group, characterized according to type of water used in the final rinse. The samples were submitted to internal and external contamination challenge with a solution containing 20% defibrinated sheep blood and 80% of sodium chloride 0.9%. Next, the lumens were filled with a ophthalmic viscosurgical device, remaining exposed for 50 minutes, and then were processed according to the validated SOP. Cytotoxicity was assessed using neutral red uptake assay. Results No cytoxicity was detected in the sample extracts. Conclusion The samples did not display signs of cytotoxicity, regardless of final rinse quality. The results obtained were reached by using only a validated cleaning operating procedure, based on the scientific literature, and on official recommendations and related regulation.

[Cytotoxicity of PVC tubes sterilized in ethylene oxide after gamma radiation exposure]. / Citotoxicidade de tubos de PVC esterilizados em óxido de etileno após exposição à radiação gama.

Do materials sterilized using gamma rays become toxic when re-sterilized in ethylene oxide? This question guided the objective of this study, which was to investigate the potential cytotoxic effect of PVC sterilized by gamma radiation and re-sterilized with EO by the agar diffusion method in cell cultures. Nine PVC tubes were subjected to gamma radiation sterilization and were re-sterilized in EO. The tubes were divided into a total of 81 units of analysis that were tested so as to represent the internal and external surfaces and mass of each tube. It was concluded that the PVC materials sterilized in gamma radiation and re-sterilized in EO are not cytotoxic.

Citotoxicidade de tubos de PVC esterilizados em óxido de etileno após exposição à radiação gama / Cytotoxicity of PVC tubes sterilized in ethylene oxide after gamma radiation exposure / Citotoxicidad de tubos de PVC esterilizados en óxido de etileno luego de exposición a la radiación gamma

Materiais esterilizados em raios gama, ao serem re-esterilizados em óxido de etileno (EO), formam substâncias tóxicas? Esta questão norteou o objetivo deste estudo, que foi investigar o potencial efeito citotóxico do PVC esterilizado em radiação gama e re-esterilizado em EO pelo método da difusão em ágar em culturas celulares. Nove tubos de PVC foram submetidos à esterilização em radiação gama e re-esterilizados em EO. Os tubos foram divididos em um total de 81 unidades de análise, que foram testadas de forma a representar as superfícies internas, externas e massa de cada tubo. Concluiu-se que os materiais de PVC esterilizados em Radiação Gama e consecutivamente re-esterilizados em EO não são citotóxicos.

Do materials sterilized using gamma rays become toxic when re-sterilized in ethylene oxide? This question guided the objective of this study, which was to investigate the potential cytotoxic effect of PVC sterilized by gamma radiation and re-sterilized with EO by the agar diffusion method in cell cultures. Nine PVC tubes were subjected to gamma radiation sterilization and were re-sterilized in EO. The tubes were divided into a total of 81 units of analysis that were tested so as to represent the internal and external surfaces and mass of each tube. It was concluded that the PVC materials sterilized in gamma radiation and re-sterilized in EO are not cytotoxic.

Los materiales esterilizados con rayos gama, al ser re-esterilizados en óxido de etileno (EO), ¿forman substancias tóxicas? Esta pregunta orientó el objetivo del presente estudio, que fue investigar el potencial efecto citotóxico del PVC esterilizado en radiación gamma y re-esterilizado en EO por el método de difusión en agar en cultivos celulares. Nueve tubos de PVC fueron sometidos a esterilización por radiación gamma y re-esterilizados en EO. Se les aplicaron en total 81 unidades de análisis, las cuales fueron testeadas de manera tal de representar las superficies internas, externas y la masa de cada tubo. Se concluyó en que los materiales de PVC esterilizados con Radiación Gamma y, posteriormente, con EO, no son citotóxicos.