Polymyxin Resistance in Salmonella: Exploring Mutations and Genetic Determinants of Non-Human Isolates.

Until 2015, polymyxin resistance was primarily attributed to chromosomal mutations. However, with the first report of mobile colistin resistance (mcr-1) in commensal Escherichia coli from food animals in China, the landscape has changed. To evaluate the presence of polymyxin resistance in Salmonella spp., a drop screening test for colistin and polymyxin B was carried out on 1156 isolates of non-human origin (animals, food, and the environment), received in Brazil, between 2016 and 2021. Subsequently, 210 isolates with resistant results in the drop test were subjected to the gold-standard test (broth microdilution) for both colistin and polymyxin B. Whole-genome sequencing (WGS) of 102 resistant isolates was performed for a comprehensive analysis of associated genes. Surprisingly, none of the isolates resistant to colistin in the drop test harbored any of the mcr variants (mcr-1 to mcr-10). WGS identified that the most common mutations were found in pmrA (n= 22; T89S) and pmrB (n = 24; M15T, G73S, V74I, I83A, A111V). Other resistance determinants were also detected, such as the aac(6')-Iaa gene in 72 isolates, while others carried beta-lactamase genes (blaTEM-1blaCTX-M-2, blaCMY-2). Additionally, genes associated with fluoroquinolone resistance (qnrB19, qnrS1, oqxA/B) were detected in 11 isolates. Colistin and polymyxin B resistance were identified among Salmonella from non-human sources, but not associated with the mcr genes. Furthermore, the already-described mutations associated with polymyxin resistance were detected in only a small number of isolates, underscoring the need to explore and characterize unknown genes that contribute to resistance.

Molecular characterization and whole-genome sequencing of Corynebacterium diphtheriae causing skin lesion.

We present a case of skin lesion caused by nontoxigenic Corynebacterium diphtheriae. Genomic taxonomy analyses corroborated the preliminary identification provided by mass spectrometry. The strain showed a susceptible phenotype with increased exposure to penicillin, the first drug of choice for the treatment. An empty type 1 class integron carrying only the sul1 gene, which encodes sulfonamide resistance, was found flanked by transposases. Virulence factors involved in adherence and iron uptake, as well as the CRISPR-Cas system, were predicted. MLST analysis revealed the ST-681, previously reported in French Guiana, a European territory.

Molecular characterization and phylogenetic analysis of the first Corynebacterium rouxii strains isolated in Brazil: a recent member of Corynebacterium diphtheriae complex.

BACKGROUND: Corynebacterium diphtheriae complex was formed by the species C. diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in the recent past. In addition to C. diphtheriae, C. ulcerans and C. pseudotuberculosis species can carry the tox gene, which encodes diphtheria toxin. Currently, three new species have been included in the complex: Corynebacterium rouxii, Corynebacterium silvaticum, and Corynebacterium belfantii. C. rouxii is derived from the ancient Belfanti biovar of C. diptheriae. We provide the complete genome sequences of two non-toxigenic strains C. rouxii isolated from a cat with a purulent infection in Brazil. The taxonomic status and sequence type, as well as the presence of resistance and virulence genes, and CRISPR-Cas system were additionally defined. RESULTS: The genomes showed an average size of 2.4 Mb and 53.2% GC content, similar to the type strain of the species deposited in Genbank/NCBI. Strains were identified as C. rouxii by the rMLST database, with 95% identity. ANI and DDH in silico were consistent with values above the proposed cut-off points for species limit, corroborating the identification of the strains as C. rouxii. MLST analyses revealed a new ST, which differs from ST-537 only by the fusA allele. No horizontal transfer resistance gene was predicted in both genomes and no mutation was detected in the constitutive genes gyrA and rpoB. Some mutations were found in the seven penicillin-binding proteins (PBPs) detected. The tox gene was not found, but its regulatory gene dtxR was present. Among the predicted virulence genes are those involved in iron uptake and adherence, in addition to the DIP0733 protein involved in epithelial cell adhesion and invasion. The CRISPR-Cas type I-E system was detected in both genomes, with 16 spacer sequences each. Of them, half are unknown according to the databases used, indicating that there is an unexplored reservoir of corynebacteriophages and plasmids. CONCLUSIONS: This is the first genomic study of C. rouxii reported in Brazil. Here we performed taxonomic analysis and the prediction of virulence factors. The genomic analyses performed in this study may help to understand the potential pathogenesis of non-toxigenic C. rouxii strains.

Genomic analysis and antimicrobial activity of ß-lactam/ß-lactamase inhibitors and other agents against KPC-producing Klebsiella pneumoniae clinical isolates from Brazilian hospitals.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are highly disseminated worldwide, and isolates co-resistant to other antimicrobial agents pose a threat to effective antimicrobial therapy. Therefore, evaluation of novel antimicrobial drugs is needed to identify potential treatments with better outcomes. We evaluated the in vitro activity of novel antimicrobial drugs/combinations against 97 KPC-producing Klebsiella pneumoniae isolates recovered from different hospitals in Brazil during 2021-2022. Clonality, resistance and virulence genes were detected by whole-genome sequencing. The majority of the isolates (54.6%) were classified as extensively drug resistant or multidrug resistant (44.3%); one isolate showed a pandrug resistance phenotype. The most active antimicrobial agents were meropenem-vaborbactam, cefiderocol, and ceftazidime-avibactam, with sensitivities higher than 90%; resistance to ceftazidime-avibactam was associated with KPC-33 or KPC-44 variants. Colistin and polymyxin B were active against 58.6% of the isolates. The 97 isolates were distributed into 17 different sequence types, with a predominance of ST11 (37.4%). Although high in vitro susceptibility rates were detected for meropenem-vaborbactam and cefiderocol, only ceftazidime-avibactam is currently available in Brazil. Our findings showed limited susceptibility to antimicrobial drugs employed for infection treatment of carbapenem-resistant K. pneumoniae, underscoring the urgent need for stringent policies for antimicrobial stewardship to preserve the activity of such drugs.

Technical comparison of MinIon and Illumina technologies for genotyping Chikungunya virus in clinical samples.

New-generation sequencing (NGS) techniques have brought the opportunity for genomic monitoring of several microorganisms potentially relevant to public health. The establishment of different methods with different mechanisms provides a wide choice, taking into account several aspects. With that in mind, the present aim of the study was to compare basic genomic sequencing metrics that could potentially impact genotyping by nanopores from Oxford Nanopore Technologies and by synthesis from Illumina in clinical samples positive for Chikungunya (CHIKV). Among the metrics studied, running time, read production, and Q score were better represented in Illumina sequencing, while the MinIOn platform showed better response time and greater diversity of generated files. That said, it was possible to establish differences between the studied metrics in addition to verifying that the distinctions in the methods did not impact the identification of the CHIKV virus genotype.

Phenotypic and molecular characterization and complete genome sequence of a Corynebacterium diphtheriae strain isolated from cutaneous infection in an immunized individual.

Diphtheria is an infectious disease potentially fatal that constitutes a threat to global health security, with possible local and systemic manifestations that result mainly from the production of diphtheria toxin (DT). In the present work, we report a case of infection by Corynebacterium diphtheriae in a cutaneous lesion of a fully immunized individual and provided an analysis of the complete genome of the isolate. The clinical isolate was first identified by MALDI-TOF Mass Spectrometry. The commercial strip system and mPCR performed phenotypic and genotypic characterization, respectively. The antimicrobial susceptibility profile was determined by the disk diffusion method. Additionally, genomic DNA was sequenced and analyzed for species confirmation and sequence type (ST) determination. Detection of resistance and virulence genes was performed by comparisons against ResFinder and VFDB databases. The isolate was identified as a nontoxigenic C. diphtheriae biovar Gravis strain. Its genome presented a size of 2.46 Mbp and a G + C content of 53.5%. Ribosomal Multilocus Sequence Typing (rMLST) allowed the confirmation of species as C. diphtheriae with 100% identity. DDH in silico corroborated this identification. Moreover, MLST analyses revealed that the isolate belongs to ST-536. No resistance genes were predicted or mutations detected in antimicrobial-related genes. On the other hand, virulence genes, mostly involved in iron uptake and adherence, were found. Presently, we provided sufficient clinical data regarding the C. diphtheriae cutaneous infection in addition to the phenotypic and genomic data of the isolate. Our results indicate a possible circulation of ST-536 in Brazil, causing cutaneous infection. Considering that cases of C. diphtheriae infections, as well as diphtheria outbreaks, have still been reported in several regions of the world, studies focusing on taxonomic analyzes and predictions of resistance genes may help to improve the diagnosis and to monitor the propagation of resistant clones. In addition, they can contribute to understanding the association between variation in genetic factors and resistance to antimicrobials.

Whole genome sequencing of Bacillus anthracis isolated from animal in the 1960s, Brazil, belonging to the South America subclade.

Bacillus anthracis causes anthrax disease and can affect humans and other animals. This zoonotic disease has an impact on the economic and health aspects. B. anthracis population is divided into three major clades: A (with worldwide distribution), B, and C (restricted to specific regions). Anthrax is most common in agricultural regions of central and southwestern Asia, sub-Saharan Africa, Southern and Eastern Europe, the Caribbean, and Central and South America. Here, we sequenced by short and long reads technologies to generate a hybrid assembly of a lineage of B. anthracis recovered from animal source in the 1960s in Brazil. Isolate identification was confirmed by phenotypic/biochemical tests and MALDI-TOF MS. Antimicrobial susceptibility was performed by in-house broth microdilution. B. anthracis IAL52 was susceptible to penicillin, amoxicillin, doxycycline, levofloxacin, and tetracycline but non-susceptible to ciprofloxacin. IAL52 was classified as sequence type ST2, clade A.Br.069 (V770 group). Sequencing lineages of B. anthracis, especially from underrepresented regions, can help determine the evolution of this critical zoonotic and virulent pathogen.

Genomics and Antimicrobial Susceptibility of Clinical Pseudomonas aeruginosa Isolates from Hospitals in Brazil.

Pseudomonas aeruginosa, an opportunistic pathogen causing infections in immunocompromised patients, usually shows pronounced antimicrobial resistance. In recent years, the frequency of carbapenemases in P. aeruginosa has decreased, which allows use of new beta-lactams/combinations in antimicrobial therapy. Therefore, the in vitro evaluation of these drugs in contemporary isolates is warranted. We evaluated the antimicrobial susceptibility and genomic aspects of 119 clinical P. aeruginosa isolates from 24 different hospitals in Brazil in 2021-2022. Identification was performed via MALDI-TOF-MS, and antimicrobial susceptibility was identified through broth microdilution, gradient tests, or disk diffusion. Whole-genome sequencing was carried out using NextSeq equipment. The most active drug was cefiderocol (100%), followed by ceftazidime-avibactam (94.1%), ceftolozane-tazobactam (92.4%), and imipenem-relebactam (81.5%). Imipenem susceptibility was detected in 59 isolates (49.6%), and the most active aminoglycoside was tobramycin, to which 99 (83.2%) isolates were susceptible. Seventy-one different sequence types (STs) were detected, including twelve new STs described herein. The acquired resistance genes blaCTX-M-2 and blaKPC-2 were identified in ten (8.4%) and two (1.7%) isolates, respectively. Several virulence genes (exoSTUY, toxA, aprA, lasA/B, plcH) were also identified. We found that new antimicrobials are effective against the diverse P. aeruginosa population that has been circulating in Brazilian hospitals in recent years.

Performance of the tetra-primer PCR technique compared to PCR-RFLP in the search for rs12979860 (C/T) and rs8099917 (T/G) single nucleotide polymorphisms (SNPs) in the IFNL4 gene / Desempenho da técnica tetra-primer PCR em relação a PCR-RFLP na pesquisa de polimorfismos de nucleotídeos únicos (SNPs) rs12979860 (C/T) e rs8099917 (T/G) no gene IFNL4

Single nucleotide polymorphisms (SNPs, rs12979860 e rs8099917) in the Interferon Lambda 4 gene (IFNL4, formerly IFNL3and/or IL28B) has been associated with failure in the innate immune response, sustained virological response in hepatitis C, and HTLV-1-associated myelopathy (HAM) development. To search for these polymorphisms several methodologies can be employed, such as sequencing, real-time or quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism analysis in PCR products (PCR-RFLP), and tetra-primer PCR. The present study compared the performance of the tetra-primer PCR in relation to the PCR-RFLP, both optimized in the Research HTLV Laboratory of the Center of Immunology of Instituto Adolfo Lutz in São Paulo. One hundred DNA samples obtained from patients of STD/Aids Reference Centre in São Paulo, previously analyzed for IL28B SNPs by PCR-RFLP were selected for analysis, after confirming that they represent all IL28B SNPs patterns described in the literature. The results obtained showed concordance between the PCR-RFLP and the tetra-primer PCR SNPs results, and because of the low cost, easy to perform, and minor employment of biological specimen and reagents, the tetra-primer PCR is of choice to be used in routine. (AU)

Polimorfismos de nucleotídeos únicos (single nucleotide polymorphisms, SNPs rs12979860 e rs8099917) no gene que codifica o Interferon Lambda 4 (IFNL4, antigamente IFNL3 e/ou IL28B) têm sido associados às falhas na resposta imune inata e resposta virológica sustentada na hepatite C, e a mielopatia associada ao HTLV-1 (HTLV-1-associated myelopathy, HAM). A pesquisa destes polimorfismos pode empregar diversas metodologias: sequenciamento, reação em cadeia da polimerase em tempo real ou quantitativa (quantitative polymerase chain reaction, qPCR), análise de fragmentos de restrição enzimática em produtos de PCR (restriction fragment length polymorphism in PCR products, PCR-RFLP) e a tetra-primer PCR. Este estudo comparou o desempenho da tetra-primer PCR em relação a PCR-RFLP, ambas otimizadas no Laboratório de Pesquisa em HTLV do Centro de Imunologia do Instituto Adolfo Lutz de São Paulo. Foram selecionadas 100 amostras de DNA obtidas de pacientes do Centro de Referência e Treinamento em DST/Aids de São Paulo cujos SNPs na IL28B foram anteriormente determinados por PCR-RFLP e representaram todos os perfis descritos em literatura. Os resultados obtidos mostraram concordância entre elas, e pelo fato da tetra-primer PCR ter menor custo, ser de fácil execução, empregar menos tempo, insumos e material biológico, é a técnica de escolha para uso em rotina. (AU)

Co-production of Classes A and B Carbapenemases BKC-1 and VIM-2 in a Clinical Pseudomonas Putida Group Isolate from Brazil.

Emergence of resistance to classical antimicrobial agents is a public health issue, especially in countries with high antimicrobial consumption rates. Carbapenems have been employed as first-choice option for empirical treatment complicated infections. However, in the last decades, frequency of carbapenemase-producing Gram-negative bacteria has rising, demanding the use of alternative antimicrobial agents. By sequencing the entire genomes with short and long reads technologies, we report the isolation and genomic characterization of a carbapenem-resistant Pseudomonas clinical isolate. The identification based on average nucleotide identity indicates a putative new species into the Pseudomonas putida Group, which carries both the blaBKC-1 and blaVIM-2 carbapenemase genes. The blaBKC-1 was found to be on a transferable IncQ plasmid backbone, whereas blaVIM-2 was found in a new integron, In2126 (intl1∆-blaVIM-2-aacA7-blaVIM-2∆-aacA27-3'CS), described in this study. Our findings indicate that co-occurrence of classes A and B carbapenemase enzymes underscores the evolving emergence of more complex antimicrobial resistance in opportunistic pathogens.

Development and Validation of Multiplex Quantitative Real-Time PCR Assays for Simultaneous Detection and Differentiation of HTLV-1 and HTLV-2, Using Different PCR Platforms and Reagent Brands.

Brazil currently has the highest number of individuals infected with human T-lymphotropic virus 1- and 2- (HTLV-1 and HTLV-2) globally. At present, neither molecular protocols nor commercial assays are available for HTLV-1/-2 diagnosis or validated by the Brazilian Ministry of Health regulatory agency (ANVISA). We developed and validated two in-house multiplex quantitative real-time PCR for HTLV-1/-2 (mqPCR_HTLV) assays, targeting the pol and tax genes, for the simultaneous identification of HTLV-1, HTLV-2, and the albumin reference gene. The robustness of the assays was evaluated on two platforms using seven commercial master mix formulations. The reactions employed double plasmids (pHTLV1-Alb and pHTLV2-Alb) for the standard curve's construction and for expressing the detection limit of the assays. They were able to detect 10 and 10 copies of HTLV-1 and 10 and 70 copies of HTLV-2 for the tax and pol targets, respectively. High efficiency was obtained using both the platforms and all the reagents evaluated and were successfully reproduced by other analysts. DNA samples from HTLV-1/-2-infected and non-infected patients and from HIV/HTLV-coinfected patients were evaluated to determine the feasibility of their use in routine diagnosis. The mqPCR_HTLV (pol and tax) assays demonstrated an overall specificity of 100% and a sensitivity of 97.4% when testing samples from patients without HIV infection, and sensitivities of 77.1% (pol) and 74.6% (tax) in samples from HIV/HTLV-coinfected patients. In addition, they resolved the issue of HTLV western blotting (WB) indeterminate and WB-untyped results in 45.5 and 66.7% of cases, respectively. The developed mqPCR_HTLV (pol and tax) assays indicated their feasibility for efficient and reliable HTLV diagnosis in various core facility laboratories under different conditions and supplies.

Genomic characterization of a multi-drug resistant, CTX-M-65-producing clinical isolate of Salmonella Infantis isolated in Brazil.

A multi-drug resistant, CTX-M-65 producing Salmonella Infantis was identified from a patient in Brazil. Whole genome sequencing followed by hybrid assembly (short and long reads) indicated the presence of blaCTX-M-65 in a pESI-like megaplasmid in this ST32 isolate and phylogenetic analysis showed high similarity with IncFIB S. Infantis isolates from food and poultry in the USA.

Development and Validation of Multiplex Quantitative Real-Time PCR Assays for Simultaneous Detection and Differentiation of HTLV-1 and HTLV-2, Using Different PCR Platforms and Reagent Brands

Brazil currently has the highest number of individuals infected with human T-lymphotropic virus 1- and 2- (HTLV-1 and HTLV-2) globally. At present, neither molecular protocols nor commercial assays are available for HTLV-1/-2 diagnosis or validated by the Brazilian Ministry of Health regulatory agency (ANVISA). We developed and validated two in-house multiplex quantitative real-time PCR for HTLV1/-2 (mqPCR_HTLV) assays, targeting the pol and tax genes, for the simultaneous identification of HTLV-1, HTLV-2, and the albumin reference gene. The robustness of the assays was evaluated on two platforms using seven commercial master mix formulations. The reactions employed double plasmids (pHTLV1-Alb and pHTLV2-Alb) for the standard curve's construction and for expressing the detection limit of the assays. They were able to detect 10 and 10 copies of HTLV-1 and 10 and 70 copies of HTLV-2 for the tax and pol targets, respectively. High efficiency was obtained using both the platforms and all the reagents evaluated and were successfully reproduced by other analysts. DNA samples from HTLV-1/-2-infected and non-infected patients and from HIV/HTLV-coinfected patients were evaluated to determine the feasibility of their use in routine diagnosis. The mqPCR_HTLV (pol and tax) assays demonstrated an overall specificity of 100% and a sensitivity of 97.4% when testing samples from patients without HIV infection, and sensitivities of 77.1% (pol) and 74.6% (tax) in samples from HIV/HTLV-coinfected patients. In addition, they resolved the issue of HTLV western blotting (WB) indeterminate and WB-untyped results in 45.5 and 66.7% of cases, respectively. The developed mqPCR_HTLV (pol and tax) assays indicated their feasibility for efficient and reliable HTLV diagnosis in various core facility laboratories under different conditions and supplies. (AU)

HTLV-1 and HTLV-2 infections in patients with endemic mycoses in São Paulo, Brazil: A cross-sectional, observational study.

Background: Brazil is a country endemic for human T-lymphotropic virus 1 and 2 (HTLV-1 and HTLV-2), systemic mycoses such as paracoccidioidomycosis (PCM) and histoplasmosis (HP), and aspergillosis (AP). The prevalence of HTLV-1/-2 infections in individuals with endemic mycoses in Latin America is unknown; however, an association between HTLV-1 and severe PCM and HP has been observed in Peru. Addressing this knowledge gap, we searched for HTLV-1/-2 antibodies in serum samples sent to the Instituto Adolfo Lutz, São Paulo, Brazil, for systemic mycosis diagnosis. Methods: We used 387 sera from a biorepository that had seropositive results for Paracoccidioides spp. (G1, n=212), Histoplasma capsulatum (G2, n=95), Aspergillus spp. (G3, n=61), and at least two of these fungi (G4, n=19). We searched for the presence of HTLV-1/-2 antibodies using commercial immunoassays: enzyme immunoassay (HTLV-I+II Murex, Diasorin), western blotting (HTLV Blot 2.4, MP Biomedicals), and line immunoassay (INNO-LIA HTLV I/II, Fujirebio). Demographic characteristics were evaluated in each group. Findings: Different regions in São Paulo were sampled. Most samples were from males (76.2%; p=0.001), except for G3, in which no sex bias was detected. Mean age differences were observed between groups: patients with PCM and HP had a similar mean age (42.8 and 42.0 years, respectively), while those with AP and co-fungal infection were older (55.1 and 52.8 years, respectively, (p<0.001). Noteworthy, males were older than females in G1 (p=0.005). Screening detected HTLV-1/2 antibodies in five samples (1.30%; 95% CI: 0.8-1.8%), with two borderline results. HTLV-1/2 was confirmed in two samples: 2/387 (0.52%; 0.063-1.85%): one HTLV-2, male, 42 years, from G1: 1/212 (0.47%; 0.012-2.60%), and one HTLV-1, male, 51 years, from G3: 1/61 (1.64%; 0.042-8.80%). Interpretation: In the state of São Paulo, HTLV-1 and HTLV-2 seem to circulate in male patients with systemic mycoses, and since HTLV-1 could impact fungal disease severity, the identification of co-infection is important regardless of prevalence. Funding: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), and Instituto Adolfo Lutz.

HTLV-1 and HTLV-2 infections in patients with endemic mycoses in São Paulo, Brazil: a cross-sectional, observational study

Background Brazil is a country endemic for human T-lymphotropic virus 1 and 2 (HTLV-1 and HTLV-2), systemic mycoses such as paracoccidioidomycosis (PCM) and histoplasmosis (HP), and aspergillosis (AP). The prevalence of HTLV-1/-2 infections in individuals with endemic mycoses in Latin America is unknown; however, an association between HTLV-1 and severe PCM and HP has been observed in Peru. Addressing this knowledge gap, we searched for HTLV-1/-2 antibodies in serum samples sent to the Instituto Adolfo Lutz, São Paulo, Brazil, for systemic mycosis diagnosis. Methods We used 387 sera from a biorepository that had seropositive results for Paracoccidioides spp. (G1, n=212), Histoplasma capsulatum (G2, n=95), Aspergillus spp. (G3, n=61), and at least two of these fungi (G4, n=19). We searched for the presence of HTLV-1/-2 antibodies using commercial immunoassays: enzyme immunoassay (HTLV-I+II Murex, Diasorin), western blotting (HTLV Blot 2.4, MP Biomedicals), and line immunoassay (INNOLIA HTLV I/II, Fujirebio). Demographic characteristics were evaluated in each group. Findings Different regions in São Paulo were sampled. Most samples were from males (76.2%; p=0.001), except for G3, in which no sex bias was detected. Mean age differences were observed between groups: patients with PCM and HP had a similar mean age (42.8 and 42.0 years, respectively), while those with AP and co-fungal infection were older (55.1 and 52.8 years, respectively, (p<0.001). Noteworthy, males were older than females in G1 (p=0.005). Screening detected HTLV-1/2 antibodies in five samples (1.30%; 95% CI: 0.8−1.8%), with two borderline results. HTLV-1/2 was confirmed in two samples: 2/387 (0.52%; 0.063−1.85%): one HTLV-2, male, 42 years, from G1: 1/212 (0.47%; 0.012−2.60%), and one HTLV-1, male, 51 years, from G3: 1/61 (1.64%; 0.042−8.80%). Interpretation In the state of São Paulo, HTLV-1 and HTLV-2 seem to circulate in male patients with systemic mycoses, and since HTLV-1 could impact fungal disease severity, the identification of co-infection is important regardless of prevalence.(AU)

A clinical Pseudomonas chlororaphis isolate harboring a new blaVIM-2 borne integron, In2088, isolated from bloodstream infection in a newborn patient.

We report the isolation and genomic characterization of a VIM-2 producing Pseudomonas chlororaphis causing bloodstream infection in a newborn in Brazil. A new integron, In2088 (intI1-blaVIM-2-aacA7-aacA27-gcu241), was identified and the first P. chlororaphis genome from a clinical isolate was deposited in public databases.

SARS-CoV-2 variants in severely symptomatic and deceased persons who had been vaccinated against COVID-19 in São Paulo, Brazil.

COVID-19 vaccination began in São Paulo, Brazil in January 2021, first targeting healthcare workers (HCWs) and the elderly, using the CoronaVac vaccine (Sinovac/Butantan) and subsequently the Oxford/AstraZeneca (ChAdOx1) vaccine (AstraZeneca/FIOCRUZ-RJ). Studies on such vaccines have shown efficacy in preventing severe cases and deaths, but there is a lack of information regarding their effectiveness. This manuscript presents data from the Instituto Adolfo Lutz (IAL), a public health laboratory located in São Paulo City that receives samples from 17 Regional Health Departments under the Secretary of Health of São Paulo, for SARS-CoV-2 genomic surveillance. Through May 15, 2021 IAL received 20 samples for analysis from COVID-19 vaccinated individuals who needed hospitalization and/or died from COVID-19. Next-generation sequencing was performed on an Ion Torrent S5 platform using the AmpliSeq™ SARS-CoV-2 kit. Almost all cases were vaccinated with CoronaVac and presented the gamma variant of concern (VOC). Cases of death were observed mostly in the elderly in nursing homes, and severe cases in younger frontline HCWs. This data confirmed that the SARS-CoV-2 gamma variant is highly transmissible, severe, and lethal for COVID-19 in these groups of individuals, thereby highlighting the importance of continuous vaccination and non-pharmacological prevention measures to avoid virus dissemination and the emergence of new VOCs.

La vacunación contra la COVID-19 empezó en São Paulo (Brasil) en enero del 2021 con los trabajadores de atención de salud (personal de salud) y las personas mayores, empleando la vacuna de CoronaVac (Sinovac/Butantan) y posteriormente la vacuna de Oxford/AstraZeneca (ChAdOx1) (AstraZeneca/FIOCRUZ-RJ). Los estudios sobre estas vacunas han mostrado su eficacia en la prevención de los casos graves y las muertes, pero existe falta de información con respecto a su efectividad. En este artículo se presentan datos del Instituto Adolfo Lutz (IAL), un laboratorio de salud pública ubicado en la ciudad de São Paulo que recibe muestras de 17 departamentos regionales de salud bajo la Secretaría de Salud de São Paulo, relativos a la vigilancia genómica del SARS-CoV-2. Hasta el 15 de mayo del 2021, el IAL había recibido 20 muestras para su análisis de personas vacunadas contra la COVID-19 que necesitaron hospitalización o murieron a causa de esta enfermedad. Se realizó una secuenciación de nueva generación en una plataforma Ion Torrent S5 mediante el kit para el SARS-CoV-2 AmpliSeq™. Casi todos los pacientes se habían vacunado con CoronaVac y presentaban la variante de preocupación gamma. Se observaron muertes principalmente de personas mayores en residencias y casos graves en personal de salud más joven de primera línea. Estos datos confirmaron que la variante gamma del SARS-CoV-2 es sumamente transmisible, grave y letal para la COVID-19 entre estos grupos y destacan la importancia de continuar con la vacunación y las medidas preventivas no farmacológicas para evitar la propagación del virus y la aparición de nuevas variantes de preocupación.

A vacinação contra a COVID-19 começou em São Paulo, Brasil, em janeiro de 2021, primeiramente dirigida a profissionais da saúde e idosos, utilizando a vacina CoronaVac (Sinovac/Butantan), e posteriormente a vacina Oxford/AstraZeneca (ChAdOx1) (AstraZeneca/Fiocruz-RJ). Os estudos sobre tais vacinas revelaram eficácia na prevenção de casos graves e mortes, mas há falta de informação em relação à sua efetividade. Este manuscrito apresenta dados do Instituto Adolfo Lutz (IAL), um laboratório de saúde pública localizado no município de São Paulo, que recebe amostras de 17 Departamentos Regionais de Saúde da Secretaria Estadual de Saúde de São Paulo para vigilância genômica do SARS-CoV-2. Até 15 de maio de 2021, o IAL recebeu 20 amostras para análise de indivíduos vacinados contra a COVID-19 que necessitaram de hospitalização e/ou morreram por COVID-19. O sequenciamento de nova geração foi realizado em plataforma Torrente de íon S5, utilizando o kit AmpliSeq™ SARS-CoV-2. Quase todos os casos foram vacinados com CoronaVac e apresentaram a variante de preocupação (VOC) gama. Os óbitos foram observados principalmente nos idosos de casas de repouso, e os casos graves em profissionais de saúde mais jovens da linha de frente. Esses dados confirmaram que a variante SARS-CoV-2 gama é altamente transmissível, grave e letal para COVID-19 nesses grupos de indivíduos, destacando, assim, a importância da vacinação contínua e de medidas preventivas não farmacológicas para evitar a disseminação viral e o surgimento de novas VOC.

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 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).