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1.
Nucleic Acids Res ; 52(2): 921-939, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38033324

RESUMO

An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications.


Assuntos
RNA Guia de Sistemas CRISPR-Cas , RNA , RNA/genética , Sistemas CRISPR-Cas
2.
J Clin Microbiol ; 61(5): e0004623, 2023 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-37129508

RESUMO

Campylobacter ureolyticus is an emerging pathogen increasingly appreciated as a common cause of gastroenteritis and extra-intestinal infections in humans. Outside the setting of gastroenteritis, little work has been done to describe the genomic content and relatedness of the species, especially regarding clinical isolates. We reviewed the epidemiology of clinical C. ureolyticus cultured by our institution over the past 10 years. Fifty-one unique C. ureolyticus isolates were identified between January 2010 and August 2022, mostly originating from abscesses and blood cultures. To clarify the taxonomic relationships between isolates and to attribute specific genes with different clinical manifestations, we sequenced 19 available isolates from a variety of clinical specimen types and conducted a pangenomic analysis with publicly available C. ureolyticus genomes. Digital DNA:DNA hybridization suggested that these C. ureolyticus comprised a species complex of 10 species clusters (SCs) and several subspecies clusters. Although some orthologous genes or gene functions were enriched in isolates found in different SCs and clinical specimens, no association was significant. Nearly a third of the isolates possessed antimicrobial resistance genes, including the ermA resistance gene, potentially conferring resistance to macrolides, the treatment of choice for severe human campylobacteriosis. This work effectively doubles the number of publicly available C. ureolyticus genomes, provides further clarification of taxonomic relationships within this bacterial complex, and identifies target SCs for future analysis.


Assuntos
Infecções por Campylobacter , Campylobacter jejuni , Campylobacter , Gastroenterite , Humanos , Infecções por Campylobacter/microbiologia , Genômica , Antibacterianos , Gastroenterite/microbiologia , DNA , Campylobacter jejuni/genética
3.
J Clin Microbiol ; 59(3)2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33328178

RESUMO

Bronchoalveolar lavage (BAL) culture is a standard, though time-consuming, approach for identifying microorganisms in patients with severe lower respiratory tract (LRT) infections. The sensitivity of BAL culture is relatively low, and prior antimicrobial therapy decreases the sensitivity further, leading to overuse of empirical antibiotics. The Unyvero LRT BAL Application (Curetis GmbH, Germany) is a multiplex molecular panel that detects 19 bacteria, 10 antibiotic resistance markers, and a fungus, Pneumocystis jirovecii, in BAL fluid in ∼4.5 h. Its performance was evaluated using 1,016 prospectively collected and 392 archived specimens from 11 clinical trial sites in the United States. Overall positive and negative percent agreements with culture results for identification of bacteria that grow in routine cultures were 93.4% and 98.3%, respectively, with additional potential pathogens identified by Unyvero in 21.7% of prospectively collected specimens. For detection of P. jirovecii, the positive percent agreement with standard testing was 87.5%. Antibiotic resistance marker results were compared to standard antibiotic susceptibility test results to determine positive predictive values (PPVs). PPVs ranged from 80 to 100%, based on the microorganism and specific resistance marker(s). The Unyvero LRT BAL Application provides accurate detection of common agents of bacterial pneumonia and of P. jirovecii The sensitivity and rapidity of this panel suggest significant clinical value for choosing appropriate antibiotics and for antibiotic stewardship.


Assuntos
Reação em Cadeia da Polimerase Multiplex , Pneumonia Bacteriana , Líquido da Lavagem Broncoalveolar , Resistência Microbiana a Medicamentos , Alemanha , Humanos , Pneumonia Bacteriana/diagnóstico , Sensibilidade e Especificidade
4.
J Clin Microbiol ; 59(2)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33139422

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the challenges inherent to the serological detection of a novel pathogen such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Serological tests can be used diagnostically and for surveillance, but their usefulness depends on their throughput, sensitivity, and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three major SARS-CoV-2 antigens-spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP). Specificity was assessed using 213 prepandemic samples. Sensitivity was measured and compared to that of the Abbott Architect SARS-CoV-2 IgG assay using serum samples from 125 unique patients equally binned (n = 25) into 5 time intervals (≤5, 6 to 10, 11 to 15, 16 to 20, and ≥21 days from symptom onset). With samples obtained at ≤5 days from symptom onset, the 3Flex assay was more sensitive (48.0% versus 32.0%), but the two assays performed comparably using serum obtained ≥21 days from symptom onset. A larger collection (n = 534) of discarded sera was profiled from patients (n = 140) whose COVID-19 course was characterized through chart review. This revealed the relative rise, peak (S, 23.8; RBD, 23.6; NP, 16.7 [in days from symptom onset]), and decline of the antibody response. Considerable interperson variation was observed with a subset of extensively sampled intensive care unit (ICU) patients. Using soluble ACE2, inhibition of antibody binding was demonstrated for S and RBD, and not for NP. Taking the data together, this study described the performance of an assay built on a flexible and high-throughput serological platform that proved adaptable to the emergence of a novel infectious agent.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Microesferas , SARS-CoV-2/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/sangue , COVID-19/patologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Feminino , Fluorimunoensaio , Humanos , Imunoglobulina G/sangue , Cinética , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
5.
Clin Infect Dis ; 70(1): 19-25, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30801635

RESUMO

BACKGROUND: Previous reports suggested that US methicillin-resistant Staphylococcus aureus (MRSA) strain epidemiology has changed since the rise of USA300 MRSA. We describe invasive MRSA trends by strain type. METHODS: Data came from 5 Centers for Disease Control and Prevention Emerging Infections Program sites conducting population-based surveillance and collecting isolates for invasive MRSA (ie, from normally sterile body sites), 2005-2013. MRSA bloodstream infection (BSI) incidence per 100 000 population was stratified by strain type and epidemiologic classification of healthcare exposures. Invasive USA100 vs USA300 case characteristics from 2013 were compared through logistic regression. RESULTS: From 2005 to 2013, USA100 incidence decreased most notably for hospital-onset (6.1 vs 0.9/100 000 persons, P < .0001) and healthcare-associated, community-onset (10.7 vs 4.9/100 000 persons, P < .0001) BSIs. USA300 incidence for hospital-onset BSIs also decreased (1.5 vs 0.6/100 000 persons, P < .0001). However, USA300 incidence did not significantly change for healthcare-associated, community-onset (3.9 vs 3.3/100 000 persons, P = .05) or community-associated BSIs (2.5 vs 2.4/100 000 persons, P = .19). Invasive MRSA was less likely to be USA300 in patients who were older (adjusted odds ratio [aOR], 0.97 per year [95% confidence interval {CI}, .96-.98]), previously hospitalized (aOR, 0.36 [95% CI, .24-.54]), or had central lines (aOR, 0.44 [95% CI, .27-.74]), and associated with USA300 in people who inject drugs (aOR, 4.58 [95% CI, 1.16-17.95]). CONCLUSIONS: Most of the decline in MRSA BSIs was from decreases in USA100 BSI incidence. Prevention of USA300 MRSA BSIs in the community will be needed to further reduce burden from MRSA BSIs.


Assuntos
Bacteriemia/epidemiologia , Infecções Comunitárias Adquiridas/epidemiologia , Infecção Hospitalar/epidemiologia , Staphylococcus aureus Resistente à Meticilina , Vigilância da População , Infecções Estafilocócicas/epidemiologia , Adulto , Idoso , Bacteriemia/microbiologia , Infecções Comunitárias Adquiridas/microbiologia , Infecção Hospitalar/microbiologia , Feminino , Humanos , Incidência , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Infecções Estafilocócicas/microbiologia , Estados Unidos , Adulto Jovem
6.
Emerg Infect Dis ; 25(7): 1281-1288, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31211681

RESUMO

Pseudomonas aeruginosa is intrinsically resistant to many antimicrobial drugs, making carbapenems crucial in clinical management. During July-October 2015 in the United States, we piloted laboratory-based surveillance for carbapenem-resistant P. aeruginosa (CRPA) at sentinel facilities in Georgia, New Mexico, Oregon, and Tennessee, and population-based surveillance in Monroe County, NY. An incident case was the first P. aeruginosa isolate resistant to antipseudomonal carbapenems from a patient in a 30-day period from any source except the nares, rectum or perirectal area, or feces. We found 294 incident cases among 274 patients. Cases were most commonly identified from respiratory sites (120/294; 40.8%) and urine (111/294; 37.8%); most (223/280; 79.6%) occurred in patients with healthcare facility inpatient stays in the prior year. Genes encoding carbapenemases were identified in 3 (2.3%) of 129 isolates tested. The burden of CRPA was high at facilities under surveillance, but carbapenemase-producing CRPA were rare.


Assuntos
Carbapenêmicos/farmacologia , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/microbiologia , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Resistência beta-Lactâmica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carbapenêmicos/uso terapêutico , Criança , Pré-Escolar , Doenças Transmissíveis Emergentes/história , Comorbidade , Feminino , História do Século XXI , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Infecções por Pseudomonas/história , Vigilância em Saúde Pública , Estados Unidos/epidemiologia , Adulto Jovem
7.
Artigo em Inglês | MEDLINE | ID: mdl-30803966

RESUMO

The convergence of hypervirulence and multidrug resistance in Klebsiella pneumoniae is a significant concern. Here, we report the first screen for hypermucoviscosity, a trait associated with increased virulence, using a U.S. surveillance collection of carbapenem-resistant (CR) K. pneumoniae isolates. We identified one hypermucoviscous isolate, which carried a gene encoding the KPC-3 carbapenemase, among numerous resistance genes. The strain further exhibited colistin heteroresistance undetected by diagnostics. This convergence of diverse resistance mechanisms and increased virulence underscores the need for enhanced K. pneumoniae surveillance.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Carbapenêmicos/farmacologia , Colistina/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Genótipo , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Virulência
8.
Emerg Med J ; 35(6): 357-360, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29523721

RESUMO

OBJECTIVES: Skin and soft tissue infections (SSTI) caused by methicillin-resistant Staphylococcus aureus (MRSA) are prevalent in the emergency department (ED). We determined whether MRSA nasal carriage better identifies patients with MRSA wound infection than clinical risk factors or emergency medicine (EM) provider's choice of discharge prescriptions. METHODS: Adult patients presenting to a large academic medical centre ED in the USA with SSTI between May 2010 and November 2011 were screened. Research assistants administered a questionnaire regarding MRSA risk factors, and MRSA nares swab PCR testing, wound culture results and information on antibiotics prescribed at discharge were collected. Measures of classification accuracy for nares swab, individual risk factors and physician's prescription for MRSA coverage were compared with gold standard wound culture. RESULTS: During the study period, 116 patients with SSTI had both wound cultures and nares swabs for MRSA. S. aureus was isolated in 59.5%, most often MRSA (75.4%). Thirty patients (25.9%) had a positive MRSA nares swab and culture for a sensitivity of 57.7% and specificity of 92.2%. Positive predictive value (PPV) for MRSA nares swab was 85.7% and positive likelihood ratio was 7.4, while negative predictive value was 72.8% and negative likelihood ratio 0.5. None of the individual risk factors nor EM provider's prescription for MRSA coverage had a PPV or positive likelihood ratio higher than nares swabs. CONCLUSIONS: MRSA nares swab is a more accurate predictor of MRSA wound infection compared with clinical risk factors or EM provider's choice of antibiotics. MRSA nares swab may be a useful tool in the ED.


Assuntos
Cavidade Nasal/microbiologia , Infecções Cutâneas Estafilocócicas/diagnóstico , Adulto , Antibacterianos/uso terapêutico , Técnicas Bacteriológicas/métodos , Feminino , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Testes de Sensibilidade Microbiana/métodos , Pessoa de Meia-Idade , New York , Prevalência , Estudos Prospectivos , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Infecções Cutâneas Estafilocócicas/epidemiologia , Staphylococcus aureus/patogenicidade , Inquéritos e Questionários , Infecção dos Ferimentos/diagnóstico
9.
Antimicrob Agents Chemother ; 60(12): 7043-7045, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27620481

RESUMO

There has been an increase in the number of pertussis cases reported since the introduction of the acellular pertussis vaccine. While children that present with pertussis have a characteristic whooping cough, adults can simply have a persistent, nonspecific cough and remain undiagnosed. Macrolide antibiotics, such as azithromycin, are the currently recommended treatment for pertussis. Solithromycin is a new macrolide and the first fluoroketolide with broad activity against a wide spectrum of bacterial pathogens and has completed clinical development for community-acquired bacterial pneumonia. This study reports the potent in vitro activity of solithromycin against a collection of recent isolates of Bordetella pertussis.


Assuntos
Antibacterianos/farmacologia , Bordetella pertussis/efeitos dos fármacos , Macrolídeos/farmacologia , Triazóis/farmacologia , Bordetella pertussis/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana
10.
Microbiol Spectr ; 12(9): e0381523, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39041813

RESUMO

Reliable detection of mecA and mecC-mediated beta-lactam resistance using automated antimicrobial susceptibility test systems is critical for patient care. The aim of this study was to compare the performance of the new cefoxitin screen test (oxsf02n) on the Vitek 2 card (Vitek 2) and BD Phoenix PMC-100 Gram-Positive AST Panel (Phoenix) against the reference method for the detection of mecA (and mecC)-mediated beta-lactam resistance. Two hundred fifty clinical fresh and stock Staphylococcus spp. isolates were included. There were 120 mecA-positive, 10 mecC-positive, and 120 mecA and mecC-negative isolates. Cefoxitin screen and oxacillin tests were performed on Vitek 2 and Phoenix and by their respective reference methods (disk diffusion for the cefoxitin screen test and broth microdilution for oxacillin) for all isolates. PCR testing was also performed to confirm the presence of mecA and/or mecC genes. Results from each system were compared to the reference methods. Statistical hypotheses were evaluated both for Vitek 2 compared to the reference methods and Vitek 2 compared to the Phoenix. Compared to the reference method, the Vitek 2 cefoxitin screen test had 100% sensitivity/98% specificity and the Phoenix cefoxitin screen test had 84% sensitivity/100% specificity for the detection of mecA (and mecC)-mediated beta-lactam resistance. When the oxacillin test was combined with the cefoxitin screen for Vitek 2, the sensitivity and specificity were unchanged. However, when the oxacillin and cefoxitin screen tests were combined for the Phoenix, the sensitivity increased to 100% and the specificity remained unchanged (100%). When considering cefoxitin alone, the Vitek 2 screen test showed a higher sensitivity than the Phoenix for the detection of mecA and mecC-mediated beta-lactam resistance. However, currently, both systems use a combination of the cefoxitin and oxacillin tests to interpret the final result, and both reached a high level of performance when cefoxitin and oxacillin results were combined.IMPORTANCEThis research marks the inaugural evaluation of the revamped cefoxitin screen test version in Vitek 2, juxtaposing it against reference methods and a primary competitor BD Phoenix.


Assuntos
Antibacterianos , Cefoxitina , Testes de Sensibilidade Microbiana , Cefoxitina/farmacologia , Testes de Sensibilidade Microbiana/métodos , Antibacterianos/farmacologia , Humanos , Proteínas de Bactérias/genética , Oxacilina/farmacologia , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Resistência beta-Lactâmica , Infecções Estafilocócicas/microbiologia , Proteínas de Ligação às Penicilinas/genética
11.
Antimicrob Agents Chemother ; 57(6): 2443-50, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23507280

RESUMO

The semen-derived enhancer of viral infection (SEVI) is a positively charged amyloid fibril that is derived from a self-assembling proteolytic cleavage fragment of prostatic acid phosphatase (PAP(248-286)). SEVI efficiently facilitates HIV-1 infection in vitro, but its normal physiologic function remains unknown. In light of the fact that other amyloidogenic peptides have been shown to possess direct antibacterial activity, we investigated whether SEVI could inhibit bacterial growth. Neither SEVI fibrils nor the unassembled PAP(248-286) peptide had significant direct antibacterial activity in vitro. However, SEVI fibrils bound to both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Neisseria gonorrhoeae) bacteria, in a charge-dependent fashion. Furthermore, SEVI fibrils but not the monomeric PAP(248-286) peptide promoted bacterial aggregation and enhanced the phagocytosis of bacteria by primary human macrophages. SEVI also enhanced binding of bacteria to macrophages and the subsequent release of bacterially induced proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-1ß). Finally, SEVI fibrils inhibited murine vaginal colonization with Neisseria gonorrhoeae. These findings demonstrate that SEVI has indirect antimicrobial activity and that this activity is dependent on both the cationic charge and the fibrillar nature of SEVI.


Assuntos
Amiloide/metabolismo , Amiloide/farmacologia , Antibacterianos/farmacologia , Macrófagos/microbiologia , Fagocitose/efeitos dos fármacos , Proteínas Tirosina Fosfatases/química , Sêmen/química , Staphylococcus aureus/efeitos dos fármacos , Vaginose Bacteriana/prevenção & controle , Fosfatase Ácida , Amiloide/química , Animais , Antibacterianos/metabolismo , Citocinas/biossíntese , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Tirosina Fosfatases/metabolismo , Sêmen/metabolismo , Staphylococcus aureus/metabolismo
12.
Microbiol Resour Announc ; 12(9): e0029323, 2023 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-37650621

RESUMO

The complete genome of Kalamiella piersonii clinical isolate URMC-2103A041 from human bacteremia was determined using the hybrid assembly of short- and long-read sequencing chemistry. The genome contains a 3.93 Mb chromosome, three circular plasmids, and one linear plasmid.

13.
Artigo em Inglês | MEDLINE | ID: mdl-37124477

RESUMO

Objective: To evaluate the demographic, maternal, and community-level predictors of pediatric respiratory syncytial virus (RSV) and influenza diagnosis among an urban population of children residing in Rochester, NY. Study design: A test-negative case-control design was used to investigate various non-clinical determinants of RSV and influenza diagnosis among 1,808 children aged 0-14 years who presented to the University of Rochester Medical Center (URMC) or an affiliated health clinic in Rochester, NY between 2012-2019. These children were all tested for RSV and influenza via polymerase-chain-reaction (PCR) method, including RSV and influenza diagnosis of all severity types. Test results were linked to medical records, birth certificates, questionnaires administered through the Statewide Perinatal Data System, and the US census by census tracts to obtain information on child, maternal, demographic, and socio-economic characteristics. Results: Overall the strongest predictor of RSV and influenza diagnosis was child's age, with every year increase in child's age, risk for RSV decreased (OR: 0.75; 95% CI: 0.71, 0.79) and risk for influenza increased (OR: 1.20; 95%: 1.16, 1.24). In addition to age, non-private insurance type was positively associated with influenza diagnosis. When considering the proportion of positive cases for RSV and influenza over all PCR tests by respiratory season, a spike in influenza cases was observed in 2018-2019. Conclusions: Age was a strong predictor of RSV and influenza diagnosis among this urban sample of children.

14.
Microbiol Resour Announc ; 11(12): e0084622, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36374079

RESUMO

The genomes of two human monkeypox virus strains from recently reported cases in our local region that were associated with the 2022 global outbreak were sequenced. Genomes from clinical isolates provide valuable information for epidemiological tracking and analysis of strain evolution and can be especially important during the early phases of outbreaks.

15.
mBio ; 12(1)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500343

RESUMO

Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States.IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.


Assuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Estados Unidos
16.
J Clin Microbiol ; 48(7): 2469-75, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20444971

RESUMO

Mupirocin susceptibility testing of Staphylococcus aureus has become more important as mupirocin is used more widely to suppress or eliminate S. aureus colonization and prevent subsequent health care- and community-associated infections. The present multicenter study evaluated two susceptibility testing screening methods to detect mupirocin high-level resistance (HLR), broth microdilution (BMD) MICs of >or=512 microg/ml, and a 6-mm zone diameter for a disk diffusion (DD) test with a 200-microg disk. Initial testing indicated that with Clinical and Laboratory Standards Institute methods for BMD and DD testing, the optimal conditions for the detection of mupirocin HLR were 24 h of incubation and reading of the DD zone diameters with transmitted light. Using the presence or absence of mupA as the "gold standard" for HLR, the sensitivity and specificity of a single-well 256 microg/ml BMD test were 97 and 99%, respectively, and those for the 200-microg disk test were 98 and 99%, respectively. Testing with two disks, 200 microg and 5 microg, was evaluated for its ability to distinguish HLR isolates (MICs >or= 512 microg/ml), low-level-resistant (LLR) isolates (MICs = 8 to 256 microg/ml), and susceptible isolates (MICs

Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Mupirocina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação
17.
Clin Lab Med ; 40(4): 421-431, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33121612

RESUMO

Planning for a new laboratory is exciting and daunting. The project's success starts with an agreed on vision and scope as defined by key stakeholders. In addition to the work of architects and building professionals, such projects require major investment in upfront thought, time, and commitment from laboratory directors, supervisors, and technologists who will use the space. Incorporating design features critical to efficient and flexible workflow as required to meet growing and changing needs will extend the lifetime of the space. Open floor plans may challenge some sensibilities and biosafety concerns but are the vogue for BSL-2 laboratories.


Assuntos
Arquitetura de Instituições de Saúde , Laboratórios , Microbiologia , Contenção de Riscos Biológicos , Humanos
18.
Front Microbiol ; 11: 1747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32849376

RESUMO

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae pose significant treatment and infection prevention challenges. Escherichia coli sequence type (ST) 131 associated with the bla CTX-M-15 gene has been the dominant lineage of ESBL-producing E. coli in the US and worldwide. In this study, our objective was to determine the ß-lactamase profile, means of dissemination, prevalence, and the clonal identity of ESBL-producing E. coli in our region of Western New York. Whole-genome SNP-based phylogenomics was used to assess 89 ceftriaxone-resistant (CTR) E. coli. Isolates were collected from both inpatients and outpatients and from urine and sterile-sites over a 2 month period in 2017 or throughout the year, respectively. ST131 was the predominant ST (46.0%), followed by ST38 (15.7%). The bla CTX-M-15 gene was commonly found in 53.7% of ST131 isolates, whereas the bla CTX-M-27 gene was found in 26.8% of ST131, though was significantly associated with ST38, and was found in 71.4% of those strains. When compared to ST131, ST38 E. coli exhibited increased frequency of resistance to nitrofurantoin and decreased frequency of resistance to ciprofloxacin and ampicillin-sulbactam. Using Nanopore long-read sequencing technology, an analysis of the ESBL genetic context showed that the bla CTX-M-15 gene was chromosomal in 68.2% of ST131, whereas the bla CTX-M-27 gene was plasmid-borne in all ST131 and 90% of ST38 isolates. Notably, the bla CTX-M-27 gene in ST38 resided on highly-related (99.0-100.0% identity and 65.0-98.0% query coverage) conjugative IncF plasmids of distinct plasmid multi-locus sequence types (pMLSTs) from those in ST131. Furthermore, ST131 and ST38 were found to harbor different antibiotic resistance gene and virulence factor profiles. These findings raise the possibility of an emerging ESBL-producing E. coli lineage in our region.

19.
Artigo em Inglês | MEDLINE | ID: mdl-33086756

RESUMO

Experimental and epidemiological evidence suggests that environmental toxicants may influence susceptibility to influenza and respiratory syncytial virus (RSV). The objective of the present study was to estimate the association between blood lead concentrations and the odds of child influenza or RSV infection. A test-negative, case-control study was conducted among 617 children, <4 years of age, tested for influenza/RSV from 2012-2017 in Rochester, NY. There were 49 influenza cases (568 controls) and 123 RSV cases (494 controls). Blood lead concentrations reported in children's medical records were linked with influenza/RSV lab test results. Covariables were collected from medical records, birth certificates, and U.S. census data. In this sample, evidence of an association between blood lead levels and RSV or influenza diagnosis was not observed. Children with a lead level ≥1 µg/dL vs. <1 µg/dL had an adjusted odds ratio (aOR) and 95% confidence limit of 0.95 (0.60, 1.49) for RSV and 1.34 (0.65, 2.75) for influenza. In sex-specific analyses, boys with lead concentrations ≥1 µg/dL vs. <1 µg/dL had an aOR = 1.89 (1.25, 2.86) for influenza diagnosis, while the estimates were inconsistent for girls. These results are suggestive of sex-specific associations between blood lead levels and the risk of influenza, although the sample size was small.


Assuntos
Influenza Humana , Chumbo , Infecções por Vírus Respiratório Sincicial , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Influenza Humana/epidemiologia , Chumbo/sangue , Chumbo/toxicidade , Masculino , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sinciciais Respiratórios
20.
medRxiv ; 2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33052354

RESUMO

The COVID-19 pandemic has highlighted challenges inherent to serological detection of a novel pathogen like SARS-CoV-2. Serological tests can be used diagnostically and for surveillance, but their usefulness depends on throughput, sensitivity and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three SARS-CoV-2 antigens-spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP). Specificity was assessed using 213 pre-pandemic samples. Sensitivity was measured and compared to the Abbott™ ARCHITECT™ SARS-CoV-2 IgG assay using serum from 125 unique patients equally binned ( n = 25) into 5 time intervals (≤5, 6 to 10, 11 to 15, 16 to 20, and ≥21 days from symptom onset). With samples obtained at ≤5 days from symptom onset, the 3Flex assay was more sensitive (48.0% vs. 32.0%), but the two assays performed comparably using serum obtained ≥21 days from symptom onset. A larger collection ( n = 534) of discarded sera was profiled from patients ( n = 140) whose COVID-19 course was characterized through chart review. This revealed the relative rise, peak (S, 23.8; RBD, 23.6; NP, 16.7; in days from symptom onset), and decline of the antibody response. Considerable interperson variation was observed with a subset of extensively sampled ICU patients. Using soluble ACE2, inhibition of antibody binding was demonstrated for S and RBD, and not for NP. Taken together, this study described the performance of an assay built on a flexible and high-throughput serological platform that proved adaptable to the emergence of a novel infectious agent.

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