Development and validation of a quick, automated, and reproducible ATR FT-IR spectroscopy machine-learning model for Klebsiella pneumoniae typing.

The reliability of Fourier-transform infrared (FT-IR) spectroscopy for Klebsiella pneumoniae typing and outbreak control has been previously assessed, but issues remain in standardization and reproducibility. We developed and validated a reproducible FT-IR with attenuated total reflectance (ATR) workflow for the identification of K. pneumoniae lineages. We used 293 isolates representing multidrug-resistant K. pneumoniae lineages causing outbreaks worldwide (2002-2021) to train a random forest classification (RF) model based on capsular (KL)-type discrimination. This model was validated with 280 contemporaneous isolates (2021-2022), using wzi sequencing and whole-genome sequencing as references. Repeatability and reproducibility were tested in different culture media and instruments throughout time. Our RF model allowed the classification of 33 capsular (KL)-types and up to 36 clinically relevant K. pneumoniae lineages based on the discrimination of specific KL- and O-type combinations. We obtained high rates of accuracy (89%), sensitivity (88%), and specificity (92%), including from cultures obtained directly from the clinical sample, allowing to obtain typing information the same day bacteria are identified. The workflow was reproducible in different instruments throughout time (>98% correct predictions). Direct colony application, spectral acquisition, and automated KL prediction through Clover MS Data analysis software allow a short time-to-result (5 min/isolate). We demonstrated that FT-IR ATR spectroscopy provides meaningful, reproducible, and accurate information at a very early stage (as soon as bacterial identification) to support infection control and public health surveillance. The high robustness together with automated and flexible workflows for data analysis provide opportunities to consolidate real-time applications at a global level. IMPORTANCE We created and validated an automated and simple workflow for the identification of clinically relevant Klebsiella pneumoniae lineages by FT-IR spectroscopy and machine-learning, a method that can be extremely useful to provide quick and reliable typing information to support real-time decisions of outbreak management and infection control. This method and workflow is of interest to support clinical microbiology diagnostics and to aid public health surveillance.

Distinction between Enterococcus faecium and Enterococcus lactis by a gluP PCR-Based Assay for Accurate Identification and Diagnostics.

It was recently proposed that Enterococcus faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis. Our goals were to develop a PCR assay to rapidly differentiate these species and to discuss the main phenotypic and genotypic differences from a clinical perspective. The pan-genome of 512 genomes of E. faecium and E. lactis strains was analyzed to assess diversity in genes between the two species. Sequences were aligned to find the best candidate gene for designing species-specific primers, and their accuracy was tested with a collection of 382 enterococci. E. lactis isolates from clinical origins were further characterized by whole-genome sequencing (Illumina). Pan-genome analysis resulted in 12 gene variants, with gene gluP (rhomboid protease) being selected as the candidate for species differentiation. The nucleotide sequence of gluP diverged by 90 to 92% between sets, which allowed species identification through PCR with 100% specificity and no cross-reactivity. E. lactis strains were greatly pan-susceptible and not host specific. Hospital E. lactis isolates were susceptible to clinically relevant antibiotics, lacked infection-associated virulence markers, and were associated with patients presenting risk factors for enhanced bacterial translocation. Here, we propose a PCR-based assay using gluP for easy routine differentiation between E. faecium and E. lactis that could be implemented in different public health contexts. We further suggest that E. lactis, a dominant human gut species, can cross the gut barrier in severely ill, immunodeficient, and surgical patients. Knowing that bacterial translocation may be a sepsis promoter, the relevance of infections caused by E. lactis strains, even if they are pan-susceptible, should be explored. IMPORTANCE Enterococcus faecium is a WHO priority pathogen that causes severe and hard-to-treat human infections. It was recently proposed that E. faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis; therefore, some of the human infections occurring globally are being misidentified. In this work, we developed a PCR-based rapid identification method for the differentiation of E. faecium and E. lactis and discussed the main phenotypic and genotypic differences of these species from a clinical perspective. We identified the gluP gene as the best candidate, based on the phylogenomic analysis of 512 published pan-genomes, and validated the PCR assay with a comprehensive collection of 382 enterococci obtained from different sources. Further detailed analysis of clinical E. lactis strains showed that they are highly susceptible to antibiotics and lack the typical virulence markers of E. faecium but are able to cause severe human infections in immunosuppressed patients, possibly in part due to gut barrier translocation.

In vitro activity of imipenem/relebactam against Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal (SUPERIOR and STEP studies).

OBJECTIVES: To study the in vitro activity of imipenem/relebactam and comparators and the imipenem/relebactam resistance mechanisms in a Pseudomonas aeruginosa collection from Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17) surveillance studies. METHODS: P. aeruginosa isolates (n = 474) were prospectively recovered from complicated urinary tract (cUTI), complicated intra-abdominal (cIAI) and lower respiratory tract (LRTI) infections in 11 Portuguese and 8 Spanish ICUs. MICs were determined (ISO broth microdilution). All imipenem/relebactam-resistant P. aeruginosa isolates (n = 30) and a subset of imipenem/relebactam-susceptible strains (n = 32) were characterized by WGS. RESULTS: Imipenem/relebactam (93.7% susceptible), ceftazidime/avibactam (93.5% susceptible) and ceftolozane/tazobactam (93.2% susceptible) displayed comparable activity. The imipenem/relebactam resistance rate was 6.3% (Portugal 5.8%; Spain 8.9%). Relebactam restored imipenem susceptibility to 76.9% (103/134) of imipenem-resistant isolates, including MDR (82.1%; 32/39), XDR (68.8%; 53/77) and difficult-to-treat (DTR) isolates (67.2%; 45/67). Among sequenced strains, differences in population structure were detected depending on the country: clonal complex (CC)175 and CC309 in Spain and CC235, CC244, CC348 and CC253 in Portugal. Different carbapenemase gene distributions were also found: VIM-20 (n = 3), VIM-1 (n = 2), VIM-2 (n = 1) and VIM-36 (n = 1) in Spain and GES-13 (n = 13), VIM-2 (n = 3) and KPC-3 (n = 2) in Portugal. GES-13-CC235 (n = 13) and VIM type-CC175 (n = 5) associations were predominant in Portugal and Spain, respectively. Imipenem/relebactam showed activity against KPC-3 strains (2/2), but was inactive against all GES-13 producers and most of the VIM producers (8/10). Mutations in genes affecting porin inactivation, efflux pump overexpression and LPS modification might also be involved in imipenem/relebactam resistance. CONCLUSIONS: Microbiological results reinforce imipenem/relebactam as a potential option to treat cUTI, cIAI and LRTI caused by MDR/XDR P. aeruginosa isolates, except for GES-13 and VIM producers.

Imipenem-Relebactam Susceptibility in Enterobacterales Isolates Recovered from ICU Patients from Spain and Portugal (SUPERIOR and STEP Studies).

Imipenem-relebactam is a novel ß-lactam-ß-lactamase inhibitor combination. We evaluated the in vitro activity of imipenem-relebactam and comparators against Enterobacterales clinical isolates recovered in 8 Spanish and 11 Portuguese intensive care units (ICUs) (SUPERIOR, 2016-2017; STEP, 2017-2018). Overall, 747 Enterobacterales isolates (378 Escherichia coli, 252 Klebsiella spp., 64 Enterobacter spp., and 53 other species) were prospectively collected from ICU patients with complicated intraabdominal (cIAI), complicated urinary tract (cUTI), and lower respiratory tract (LRTI) infections. MICs were determined (ISO-broth microdilution), and whole-genome sequencing (WGS) was performed in a subset of isolates displaying susceptible and resistant imipenem-relebactam MICs. Imipenem-relebactam (98.7% susceptible) showed similar activity to ceftazidime-avibactam (99.5% susceptible) and higher than ceftolozane-tazobactam (86.9% susceptible). Imipenem-relebactam was inactive against 1.3% (10/747) isolates, all of them due to carbapenemase production (9 K. pneumoniae and 1 E. cloacae). Imipenem-relebactam was active against 100% of extended-spectrum ß-lactamase (ESBL)-E. coli and ESBL-Klebsiella spp. isolates and 80.4% of carbapenemase-Klebsiella spp. producers. Carbapenemase genes were confirmed by WGS in 41 Klebsiella spp.: OXA-48 (20/41), KPC-3 (14/41), OXA-181 (4/41), NDM-1 (1/41), OXA-48 + VIM-2 (1/41), and KPC-3 + VIM-2 (1/41). In Klebsiella spp. isolates, relebactam restored imipenem susceptibility in all KPC-3 producers, and resistant isolates (7/41) were mostly OXA-48 + CTX-M-15-K. pneumoniae high-risk clones (7/9). Intercountry differences were detected as follows: OXA-48 (17/21) was dominant in Spain, unlike KPC-3 (14/15) in Portugal. Imipenem-relebactam was 100% active against CTX-M-15-ST131-H30Rx-E. coli high-risk clone, predominant in both countries. Our results depict the potential role of imipenem-relebactam in ICU patients with cIAIs, cUTIs, and LRTIs due to wild-type ESBL- and carbapenemase-producing Enterobacterales, particularly KPC producers. IMPORTANCE We comparatively evaluate the in vitro activity of a drug combination consisting of a carbapenem (imipenem) and a novel inhibitor of beta-lactamases (relebactam), a mechanism that destroys beta-lactam antibiotics. We assess the activity against a collection of Enterobacterales clinical isolates recovered from difficult-to-treat infections in patients admitted to different intensive care units in Portugal and Spain. Imipenem-relebactam shows excellent activity in avoiding common resistance mechanisms in this setting, such as extended-spectrum beta-lactamases and carbapenemases widely distributed, including KPCs. We show few resistant isolates (<2%). Molecular characterization by whole-genome sequencing shows that most of the resistant isolates produced specific carbapenemase, such as OXA-48 or metalo-betalactamases. Our study updates the activity of imipenem-relebactam in light of current epidemiology in a hospital setting in which the use of this combination is needed due to the presence of infections due to multidrug-resistant isolates.

Confronting Ceftolozane-Tazobactam Susceptibility in Multidrug-Resistant Enterobacterales Isolates and Whole-Genome Sequencing Results (STEP Study).

Ceftolozane-tazobactam (C/T) is frequently used for infections caused by multidrug-resistant (MDR)-Enterobacterales isolates. Whole-genome sequencing (WGS, Illumina-Hiseq 4000/NovaSeq 6000, OGC, UK) was used to study the population structure, the resistome and the virulome of C/T-susceptible and -resistant MDR Escherichia spp. (n=30) and Klebsiella spp. (n=78) isolates, recovered from lower respiratory, intra-abdominal and urinary tract infections of ICU patients from 11 Portuguese Hospitals (STEP study, 2017-2018). Minimum inhibitory concentrations (MICs) were determined (ISO-broth microdilution, breakpoints EUCAST-2020). In Escherichia spp., a weak concordance between the phenotypic and the WGS method (P=0.051) was observed in the carbapenemase detection (3/30) [blaVIM-2 (2/3), blaKPC-3 (1/3)]; VIM-2-Escherichia coli isolates were C/T-susceptible and only the KPC-3-Escherichia marmotae producer showed C/T-resistance. Overall, CTX-M-15-E. coli-ST131-O25:H4-H30-Rx (11/30) was the most frequent subclone, followed by CTX-M-27-E. coli-ST131-O25:H4-H30 (4/4). Moreover, a wide resistome and virulome were detected in all E. coli isolates. Among Klebsiella spp. isolates [K. pneumoniae (67/78), K. aerogenes (7/78), K. oxytoca (2/78), K. variicola (2/78)], concordance (P<0.001) was observed between the phenotypic and the genomic carbapenemase detection (21/78) [blaKPC-3 (14/21), blaOXA-48 (3/21), blaOXA-181 (3/21)]. A high correlation between C/T-resistance and carbapenemase detection was established (P<0.05). Overall, a high clonal diversity was observed, mainly in KPC-3-producing K. pneumoniae isolates. An extensive resistome was detected in Klebsiella spp. isolates, whereas virulence determinants were mostly identified in carbapenemase producers (P<0.001). WGS is a powerful tool for typing characterization and microbiological study of MDR-Enterobacterales pathogens. Furthermore, carbapenemase genes are associated with C/T-resistance in Klebsiella spp., but other mechanisms might also be involved.

Distinct epidemiology and resistance mechanisms affecting ceftolozane/tazobactam in Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal depicted by WGS.

OBJECTIVES: To analyse the epidemiology, the resistome and the virulome of ceftolozane/tazobactam-susceptible or -resistant Pseudomonas aeruginosa clinical isolates recovered from surveillance studies in Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17). METHODS: P. aeruginosa isolates were recovered from intra-abdominal, urinary tract and lower respiratory tract infections in ICU patients admitted to 11 Portuguese and 8 Spanish hospitals. MICs were determined (ISO-standard broth microdilution, EUCAST 2020 breakpoints). A subset of 28 ceftolozane/tazobactam-resistant P. aeruginosa isolates were analysed and compared with 28 ceftolozane/tazobactam-susceptible P. aeruginosa strains by WGS. RESULTS: Clonal complex (CC) 235 (27%) and CC175 (18%) were the most frequent, followed by CC244 (13%), CC348 (9%), CC253 (5%) and CC309 (5%). Inter-hospital clonal dissemination was observed, limited to a geographical region (CC235, CC244, CC348 and CC253 in Portugal and CC175 and CC309 in Spain). Carbapenemases were detected in 25 isolates (45%): GES-13 (13/25); VIM type (10/25) [VIM-2 (4/10), VIM-20 (3/10), VIM-1 (2/10) and VIM-36 (1/10)]; and KPC-3 (2/25). GES-13-CC235 (13/15) and VIM type-CC175 (5/10) associations were observed. Interestingly, KPC-3 and VIM-36 producers showed ceftolozane/tazobactam-susceptible phenotypes. However, ceftolozane/tazobactam resistance was significantly associated with GES-13 and VIM-type carbapenemase production. Six non-carbapenemase producers also displayed ceftolozane/tazobactam resistance, three of them showing known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene, ftsI (R504C and F533L). Overall, an extensive virulome was identified in all P. aeruginosa isolates, particularly in carbapenemase-producing strains. CONCLUSIONS: GES-13-CC235 and VIM type-CC175 were the most frequent MDR/XDR P. aeruginosa clones causing infections in Portuguese and Spanish ICU patients, respectively. Ceftolozane/tazobactam resistance was mainly due to carbapenemase production, although mutations in PBP-encoding genes may additionally be involved.

Impact of urinary selective antibiogram in primary care / Impacto do antibiograma seletivo nas uroculturas em cuidados primários

ABSTRACT INTRODUCTION: Selective reporting of antibiotic susceptibility test results (selective antibiogram), is increasingly recognized as one of the key strategies of antibiotic stewardship programs. The objective of this study is to determine the impact of selective susceptibility reporting on ciprofloxacin utilization and Escherichia coli susceptibility to ciprofloxacin in the outpatient setting. MATERIAL AND METHODS: A selective reporting policy was created and implemented in 2011. The policy involves the non-reporting of ciprofloxacin susceptibility to Enterobacteriaceae isolated in a urine sample when there was susceptibility to other agents with narrow spectrum. The outcomes evaluated were outpatient ciprofloxacin utilization and E. coli susceptibility to ciprofloxacin between January 2011 and December 2018 RESULTS: Between 2011 and 2018 we detected an increased susceptibility rate of E. coli to ciprofloxacin from 79% to 87% (p < 0.001) and a decreased incidence rate of E. coli resistant to ciprofloxacin from 2.52 to 0.87 (p < 0.001). The ciprofloxacin dropped from 0.75 defined daily doses (DHD) to 0.36 DHD and there was a compensatory increase in nitrofurantoin and fosfomycin utilization. DISCUSSION: Our study showed that selective reporting can influence prescribing practice in a community level and encourages clinicians to select more narrow-spectrum and cost-effective antimicrobial agents in UTIs. CONCLUSION: Our results suggest that selective antibiogram should be considered an effective prevention strategy to reduce targeted antimicrobial utilization.

RESUMEN INTRODUCCIÓN: La notificación selectiva de resultados de la prueba de susceptibilidad bacteriana (antibiograma selectivo) es conocida como una de las estrategias clave de los programas de optimización de antimicrobianos. El objetivo de este estudio fue determinar el impacto del antibiograma selectivo en el consumo de ciprofloxacino y la sensibilidad de la bacteria Escherichia coli al ciprofloxacino en la atención básica. MATERIAL Y MÉTODOS: La política de informe selectivo fue creada e implementada en 2011 e incluyó la no trasmisión, en el antibiograma, de sensibilidad de Enterobacteriaceae al ciprofloxacino en muestras urinarias cuando había sensibilidad a otros agentes de espectro reducido. Los desenlaces evaluados fueron el consumo de ciprofloxacino y la evolución de sensibilidad de E. coli al ciprofloxacino entre enero de 2011 y diciembre de 2018. RESULTADOS En esse período, se detectó un aumento en la tasa de sensibilidad de E. coli al ciprofloxacino, del 79% al 87% (p < 0,001). La tasa de incidencia de E. coli resistente al ciprofloxacino descendió de 2,52 a 0,87 (p < 0,001). El consumo de ciprofloxacino tuvo un descenso de 0,75 doses por mil habitantes día (DHD) a 0,36 DHD. Al mismo tiempo, un aumento compensatorio se observó en el consumo de nitrofurantoína y fosfomicina. DISCUSIÓN: Nuestro estudio demostró que el uso del antibiograma selectivo influyó en la práctica de prescripción de antimicrobianos y animó a los médicos generales a elegir antimicrobianos de espectro más reducido y con mejor relación de costo-beneficio. CONCLUSIÓN: Nuestros resultados sugieren que la utilización de antibiogramas selectivos debe ser considerada una estrategia efectiva en la reducción del consumo de determinados antimicrobianos.

RESUMO INTRODUÇÃO: A notificação seletiva dos resultados do teste de suscetibilidade aos antimicrobianos (antibiograma seletivo) é conhecida como uma das estratégias-chave dos programas de apoio à prescrição de antibióticos. O objetivo deste estudo foi determinar o impacto do antibiograma seletivo no consumo de ciprofloxacino e a suscetibilidade da bactéria Escherichia coli ao ciprofloxacino nos cuidados primários. MATERIAL E MÉTODOS: A política de notificação seletiva foi criada e implementada em 2011 e envolveu a não transmissão, no antibiograma, da suscetibilidade da família Enterobacteriaceae ao ciprofloxacino em amostras urinárias quando existia suscetibilidade a outros agentes com menor espectro. Os desfechos avaliados foram consumo de ciprofloxacino e evolução da suscetibilidade de E. coli ao ciprofloxacino entre janeiro de 2011 e dezembro de 2018. RESULTADOS: Nesse período, um aumento foi detectado na taxa de suscetibilidade de E. coli ao ciprofloxacino, de 79% a 87% (p < 0,001). A taxa de incidência de E. coli resistente ao ciprofloxacino diminuiu de 2,52 para 0,87 (p < 0,001). O consumo de ciprofloxacino teve uma queda de 0,75 doses diárias definidas (DHD) para 0,36 DHD. Simultaneamente, um aumento compensatório foi observado no consumo de nitrofurantoína e fosfomicina. DISCUSSÃO: Nosso estudo demonstrou que a utilização do antibiograma seletivo influenciou a prática de prescrição dos antimicrobianos e incentivou os clínicos gerais a selecionar antimicrobianos de espectro de ação mais reduzido e com melhor relação custo-benefício. CONCLUSÃO: Nossos resultados sugerem que a utilização de antibiogramas seletivos deve ser considerada uma estratégia eficaz na redução do consumo de determinados antimicrobianos.

Evidence for the Dissemination to Humans of Methicillin-Resistant Staphylococcus aureus ST398 through the Pork Production Chain: A Study in a Portuguese Slaughterhouse.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 was recovered from infections in humans exposed to animals, raising public health concerns. However, contact with food producing chain as a means of transmission of LA-MRSA to humans remains poorly understood. We aimed to assess if pork production chain is a source of MRSA ST398 for human colonization and infection. MRSA from live pigs, meat, the environment, and slaughterhouse workers were analyzed by Pulsed-Field Gel Electrophoresis (PFGE), spa, MLST typing, SNPs and for antibiotic resistance and virulence gene profiles. We compared core and accessory genomes of MRSA ST398 isolated from slaughterhouse and hospital. We detected MRSA ST398 (t011, t108, t1451) along the entire pork production chain (live pigs: 60%; equipment: 38%; meat: 23%) and in workers (40%). All MRSA ST398 were multidrug resistant, and the majority carried genes encoding biocide resistance and enterotoxins. We found 23 cross-transmission events between live pigs, meat, and workers (6-55 SNPs). MRSA ST398 from infection and slaughterhouse environment belonged to the same clonal type (ST398, t011, SCCmec V), but differed in 321-378 SNPs. Pork production chain can be a source of MRSA ST398 for colonization of human slaughterhouse workers, which can represent a risk of subsequent meat contamination and human infection.

Pulmonary nocardiosis: A Single Center Study.

OBJECTIVES: Nocardiosis is a rare infection caused by Nocardia spp., a gram-positive bacteria non-commensal of the human flora. Nocardiosis usually presents with lung infection but may disseminate to other organs, most frequently the brain. The major risk factor is immunosuppression, but lung diseases also increase the risk of infection. Treatment with antibiotics is usually prolonged. In this study, we made a retrospective analysis of pulmonary nocardiosis cases and a review of the available literature. METHODS: We made a retrospective analysis of all pulmonary nocardiosis cases from 13 years (January 2005 to December 2017) in our institution, selecting patients from pulmonology and infectious diseases consultation. RESULTS: We found four patients diagnosed with pulmonary nocardiosis, three males (patients 1, 2 and 3) and one female (patient 4). Median age was 71 ± 15 years old. Different specimens were identified (N. cyriacigeorgica, Nocardia spp., N. nova, and N. wallacei/transvalensis). Bronchofibroscopy with bronchoalveolar lavage culture was the most frequent diagnostic procedure (patients 1 and 4). Only patient 2 presented an unfavorable response to treatment and died from septic shock. CONCLUSIONS: Pulmonary nocardiosis has a good prognosis if diagnosed early and treated adequately. It should always be considered in the differential diagnosis of pulmonary infections concomitant with brain or other soft tissue lesion, especially in immunocompromised patients.

Linezolid-resistant (Tn6246::fexB-poxtA) Enterococcus faecium strains colonizing humans and bovines on different continents: similarity without epidemiological link.

OBJECTIVES: poxtA is the most recently described gene conferring acquired resistance to linezolid, a relevant antibiotic for treating enterococcal infections. We retrospectively screened for poxtA in diverse enterococci and aimed to characterize its genetic/genomic contexts. METHODS: poxtA was screened by PCR in 812 enterococci from 458 samples (hospitals/healthy humans/wastewater/animals/retail food) obtained in Portugal/Angola/Tunisia (1996-2019). Antimicrobial susceptibility testing was performed for 13 antibiotics (EUCAST/CLSI). poxtA stability (∼500 generations), transfer (filter mating), clonality (SmaI-PFGE) and location (S1-PFGE/hybridization) were tested. WGS (Illumina-HiSeq) was performed for clonal representatives. RESULTS: poxtA was detected in Enterococcus faecium from six samples (1.3%): a healthy human (rectal swab) in Porto, Portugal (ST32/2001); four farm cows (milk) in Mateur, Tunisia (ST1058/2015); and a hospitalized patient (faeces) in Matosinhos, Portugal (ST1058/2015). All expressed resistance to linezolid (MIC = 8 mg/L), chloramphenicol, tetracycline and erythromycin, with variable resistance to ciprofloxacin and streptomycin. ST1058-poxtA-carrying isolates from Tunisia and Portugal differed by two SNPs and had similar plasmid content. poxtA, located in an IS1216-flanked Tn6246-like element, co-hybridized with fexB on one or more plasmids per isolate (one to three plasmids of 30-100 kb), was stable after several generations and transferred only from ST1058. ST1058 strains carried resistance/virulence genes (Efmqnr/acm) possibly induced under selective quinolone treatment. CONCLUSIONS: poxtA has been circulating in Portugal since at least 2001, corresponding to the oldest description worldwide to date. We also extend the reservoir of poxtA to bovines. The similar linezolid-resistant poxtA-carrying strains colonizing humans and livestock on different continents, and without a noticeable relationship, suggests a recent transmission event or convergent evolution of E. faecium populations in different hosts and geographic regions.

In vitro activity of ceftolozane-tazobactam against Enterobacterales and Pseudomonas aeruginosa causing urinary, intra-abdominal and lower respiratory tract infections in intensive care units in Portugal: The STEP multicenter study.

The STEP surveillance study was designed to increase knowledge about distribution of multidrug-resistant (MDR) Enterobacterales and Pseudomonas aeruginosa in Portugal, focusing on the intensive care unit (ICU). Antimicrobial susceptibility of common agents was also evaluated and compared with that of one of the latest therapeutic introductions, ceftolozane-tazobactam (C/T). Clinical isolates of Enterobacterales (n=426) and P. aeruginosa (n=396) from patients admitted in Portuguese ICUs were included. Activity of C/T and comparators was investigated using standard broth microdilution. Isolates were recovered from urinary tract (UTI, 36.9%), intra-abdominal (IAI, 24.2%) and lower respiratory tract (LRTI, 38.9%) infections. In P. aeruginosa, overall distribution of MDR/extremely-drug resistant (XDR)/pan-drug resistant (PDR) isolates accounted for 21.2%, 23.2% and 0.8%, respectively. C/T was the most potent agent tested against P. aeruginosa and MDR/XDR/PDR phenotypes. In Escherichia coli, extended-spectrum beta-lactamases (ESBL) and carbapenemase (CP) phenotypes accounted for 16.6% and 1.7%, respectively, whereas in Klebsiella spp., ESBL and CP-phenotypes represented 28.5% and 17.9%, respectively. Overall, susceptibility of C/T against Enterobacterales was 86.9%. C/T was the least affected agent in E. coli (99.4% susceptibility), whereas its activity was moderate in Klebsiella spp. (71.5%) and Enterobacter spp. (70.4%), due in part to a high rate of ESBL and CP-phenotypes. In Enterobacterales, blaKPC was the most prevalent CP gene (63.0%), followed by blaOXA-48 (33.3%) and blaVIM (3.7%). These microbiological results reinforce C/T as a therapeutic option in ICU patients with UTI, IAI or LRTI due to P. aeruginosa or Enterobacterales isolates, but not for CP producers.

Self-sampling coupled to the detection of HPV 16 and 18 E6 protein: A promising option for detection of cervical malignancies in remote areas.

OBJECTIVE: To evaluate both the performance and acceptability of a method coupling self-sampling with detection of cervical malignancy via elevated HPV 16 and 18 E6 oncoproteins (OncoE6™ Cervical Test) in remote areas in Brazil. METHODS: Women living in rural villages in proximity to Coari city, Amazonas, Brazil were invited to participate in a cervical cancer screening study. 412 subjects were enrolled; there were no refusals. In addition to E6 protein detection, DNA was extracted from the brushes and evaluated for HPV genotypes by PCR (PGMY09/11), followed by typing by the Papillocheck™ if positive. Subjects who were found to be positive for OncoE6 or HPV-DNA were referred for colposcopy. RESULTS: For 110 subjects (27%) this was the first cervical cancer exam. Overall the HPV-DNA prevalence was 19.1% (n = 79); 1.4% (n = 6) were positive by the OncoE6 Test. Fifty-six women attended the invitation for colposcopy where nine had an abnormal cervix and were subsequently biopsied. Histopathological analysis revealed 2 CIN3, 2 carcinomas and 5 CIN1. OncoE6 called two out of the three HPV 16 or 18 associated CIN3+ lesions. CONCLUSIONS: The findings suggest that self-administered sample collection in combination with OncoE6 Test is feasible in this population. This could enable expanded screening coverage while ensuring a high specificity which is imperative given the remote geographic location, since women bearing abnormal test results would necessitate travel and logistical burden to access colposcopy and treatment.

Incidence and serotype characterisation of Streptococcus agalactiae in a Portuguese hospital.

AIMS: Streptococcus agalactiae, commonly known as group B Streptococcus (GBS), has been recognised as a worldwide causative pathogenic agent of neonatal sepsis, meningitis and pneumonia. To better understand the behaviour of S. agalactiae in pregnant women from a hospital from the North of Portugal, retrospective analyses were performed to describe epidemiological, clinical and microbiological characteristics of the isolates obtained. METHODS: Based on laboratorial records and the hospital's patient files, a 6-year retrospective study was performed to analyse S. agalactiae isolates from screened pregnant women between 35 and 37 weeks of gestation and hospitalised neonates from pregnant women between 24 and 41 weeks of gestation admitted in Hospital Pedro Hispano. Serotype characterisation was also performed in 67 GBS strains. RESULTS: In 6692 pregnant women between 35 and 37 weeks of gestation screened between 2011 and 2016, a total of 1377 S. agalactiae isolates (21%) were found. A high percentage (40%) of unknown colonisation status among hospitalised neonates from pregnant women between 24 and 41 weeks of gestations was also found. The incidence of neonatal sepsis was 8.7 (95% CI 7.0 to 10.8) cases per 1000 live births. Regarding serotype characterisation, serotype III (22.4%) was the most frequent, followed by serotype Ia (19.4%) and serotypes Ib and V (both with 17.9%). CONCLUSION: High epidemiological values of GBS colonisation and incidence were found in this study. In Portugal studies on the epidemiology and behaviour of S. agalactiae remain limited, reinforcing the importance and need for S. agalactiae screening across the country.

Arboviral diseases in the Western Brazilian Amazon: a perspective and analysis from a tertiary health & research center in Manaus, State of Amazonas.

The Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), located in Manaus, the capital of the State of Amazonas (Western Brazilian Amazon), is a pioneering institution in this region regarding the syndromic surveillance of acute febrile illness, including arboviral infections. Based on the data from patients at the FMT-HVD, we have detected recurrent outbreaks in Manaus by the four dengue serotypes in the past 15 years, with increasing severity of the disease. This endemicity has culminated in the simultaneous circulation of all four serotypes in 2011, the first time this has been reported in Brazil. Between 1996 and 2009, 42 cases of yellow fever (YF) were registered in the State of Amazonas, and 71.4% (30/42) were fatal. Since 2010, no cases have been reported. Because the introduction of the yellow fever virus into a large city such as Manaus, which is widely infested by Aedes mosquitoes, may pose a real risk of a yellow fever outbreak, efforts to maintain an appropriate immunization policy for the populace are critical. Manaus has also suffered silent outbreaks of Mayaro and Oropouche fevers lately, most of which were misdiagnosed as dengue fever. The tropical conditions of the State of Amazonas favor the existence of other arboviruses capable of producing human disease. Under this real threat, represented by at least 4 arboviruses producing human infections in Manaus and in other neighboring countries, it is important to develop an efficient public health surveillance strategy, including laboratories that are able to make proper diagnoses of arboviruses.

Arboviral diseases in the Western Brazilian Amazon: a perspective and analysis from a tertiary health & research center in Manaus, State of Amazonas

The Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), located in Manaus, the capital of the State of Amazonas (Western Brazilian Amazon), is a pioneering institution in this region regarding the syndromic surveillance of acute febrile illness, including arboviral infections. Based on the data from patients at the FMT-HVD, we have detected recurrent outbreaks in Manaus by the four dengue serotypes in the past 15 years, with increasing severity of the disease. This endemicity has culminated in the simultaneous circulation of all four serotypes in 2011, the first time this has been reported in Brazil. Between 1996 and 2009, 42 cases of yellow fever (YF) were registered in the State of Amazonas, and 71.4% (30/42) were fatal. Since 2010, no cases have been reported. Because the introduction of the yellow fever virus into a large city such as Manaus, which is widely infested by Aedes mosquitoes, may pose a real risk of a yellow fever outbreak, efforts to maintain an appropriate immunization policy for the populace are critical. Manaus has also suffered silent outbreaks of Mayaro and Oropouche fevers lately, most of which were misdiagnosed as dengue fever. The tropical conditions of the State of Amazonas favor the existence of other arboviruses capable of producing human disease. Under this real threat, represented by at least 4 arboviruses producing human infections in Manaus and in other neighboring countries, it is important to develop an efficient public health surveillance strategy, including laboratories that are able to make proper diagnoses of arboviruses.

Pediatric atrophic rhinosinusitis: what can we do?

A 5-year-old female had history of chronic foul smelling nasal discharge. Rhinoscopy showed greenish crusts lining the nasal cavities and inferior turbinates were shriveled significantly. Nasal cavity cultures of crusts by swab revealed Klebsiella ozaenae making the diagnosis of primary atrophic rhinosinusitis. After several unsuccessful treatment, we have decided to try sulfamethoxazole-trimethoprim prophylaxis and 1 year later there was a complete clinical improvement. There are many medical therapies and surgical options described, but none of them showed effective at long term. We present antibiotic prophylaxis as a viable alternative for long term control of the disease.