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Given the cost and unclear clinical impact of metagenomic next-generation sequencing (mNGS), laboratory stewardship may improve utilization. This retrospective observational study examines mNGS results from two academic medical centers employing different stewardship approaches. Eighty mNGS orders [54 cerebrospinal fluid (CSF) and 26 plasma] were identified from 2019 to 2021 at the University of Washington (UW), which requires director-level approval for mNGS orders, and the University of Utah (Utah), which does not restrict ordering. The impact of mNGS results and the relationship to traditional microbiology orders were evaluated. Nineteen percent (10/54) of CSF and 65% (17/26) of plasma studies detected at least one organism. Compared to CSF results, plasma results more frequently identified clinically significant organisms (31% vs 7%) and pathogens not detected by traditional methods (12% vs 0%). Antibiotic management was more frequently impacted by plasma versus CSF results (31% vs 4%). These outcome measures were not statistically different between study sites. The number and cumulative cost of traditional microbiology tests at UW were greater than Utah for CSF mNGS testing (UW: 46 tests, $6,237; Utah: 26 tests, $2,812; P < 0.05) but similar for plasma mNGS (UW: 31 tests, $3,975; Utah: 21 tests, $2,715; P = 0.14). mNGS testing accounted for 30%-50% of the total microbiology costs. Improving the diagnostic performance of mNGS by stewardship remains challenging due to low positivity rates and difficulties assessing clinical impact. From a fiscal perspective, stewardship efforts should focus on reducing testing in low-yield populations given the high costs of mNGS relative to overall microbiology testing expenditures. IMPORTANCE: Metagenomic next-generation sequencing (mNGS) stewardship practices remain poorly standardized. This study aims to provide actionable insights for institutions that seek to reduce the unnecessary usage of mNGS. Importantly, we highlight that clinical impact remains challenging to measure without standardized guidelines, and we provide an actual cost estimate of microbiology expenditures on individuals undergoing mNGS.
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BACKGROUND: Five organs (heart, right lung, liver, right, and left kidneys) from a deceased patient were transplanted into five recipients in four US states; the deceased patient was identified as part of a healthcare-associated fungal meningitis outbreak among patients who underwent epidural anesthesia in Matamoros, Mexico. METHODS: After transplant surgeries occurred, Fusarium solani species complex, a fungal pathogen with a high case-mortality rate, was identified in cerebrospinal fluid from the organ donor by metagenomic next-generation sequencing (mNGS) and fungal-specific polymerase chain reaction and in plasma by mNGS. RESULTS: Four of five transplant recipients received recommended voriconazole prophylaxis; four were monitored weekly by serum (1-3)-ß-d-glucan testing. All five were monitored for signs of infection for at least 3 months following transplantation. The liver recipient had graft failure, which was attributed to an etiology unrelated to fungal infection. No fungal DNA was identified in sections of the explanted liver, suggesting that F. solani species complex did not contribute to graft failure. The remaining recipients experienced no signs or symptoms suggestive of fusariosis. CONCLUSION: Antifungal prophylaxis may be useful in preventing donor-derived infections in recipients of organs from donors that are found to have Fusarium meningitis.
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We report an immunocompromised patient in Alabama, USA, 75 years of age, with relapsing fevers and pancytopenia who had spirochetemia after a tick bite. We identified Borrelia lonestari by using PCR, sequencing, and phylogenetic analysis. Increasing clinical availability of molecular diagnostics might identify B. lonestari as an emerging tickborne pathogen.
Assuntos
Borrelia , Febre Recorrente , Picadas de Carrapatos , Humanos , Febre Recorrente/diagnóstico , Alabama/epidemiologia , Picadas de Carrapatos/complicações , Filogenia , Borrelia/genéticaRESUMO
Molecular methods can enable rapid identification of Bartonella spp. infections, which are difficult to diagnose by using culture or serology. We analyzed clinical test results of PCR that targeted bacterial 16S rRNA hypervariable V1-V2 regions only or in parallel with PCR of Bartonella-specific ribC gene. We identified 430 clinical specimens infected with Bartonella spp. from 420 patients in the United States. Median patient age was 37 (range 1-79) years; 62% were male. We identified B. henselae in 77%, B. quintana in 13%, B. clarridgeiae in 1%, B. vinsonii in 1%, and B. washoensis in 1% of specimens. B. quintana was detected in 83% of cardiac specimens; B. henselae was detected in 34% of lymph node specimens. We detected novel or uncommon Bartonella spp. in 9 patients. Molecular diagnostic testing can identify Bartonella spp. infections, including uncommon and undescribed species, and might be particularly useful for patients who have culture-negative endocarditis or lymphadenitis.
Assuntos
Infecções por Bartonella , Bartonella henselae , Bartonella , Humanos , Masculino , Estados Unidos , Lactente , Pré-Escolar , Criança , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Feminino , RNA Ribossômico 16S/genética , Infecções por Bartonella/microbiologia , Reação em Cadeia da Polimerase/métodos , Técnicas de Amplificação de Ácido Nucleico , Bartonella henselae/genéticaRESUMO
Whole-genome sequencing (WGS) provides greater resolution than other molecular epidemiology strategies and is emerging as a new gold standard approach for microbial strain typing. The Bruker IR Biotyper is designed as a screening tool to identify bacterial isolates that require WGS to establish accurate relationships, but its performance and utility in nosocomial outbreak investigations have not been thoroughly investigated. Here, we evaluated the IR Biotyper by retrospectively examining isolates tested by WGS during investigations of potential nosocomial transmission events or outbreaks. Ninety-eight clinical isolates from 14 different outbreak investigations were examined: three collections of Acinetobacter baumannii (n = 2, n = 9, n = 5 isolates in each collection), one of Escherichia coli (n = 16), two of Pseudomonas aeruginosa (n = 2 and n = 5), two of Serratia marcescens (n = 9 and n = 7), five of Staphylococcus aureus (n = 8, n = 4, n = 3, n = 3, n = 17), and one of Stenotrophomonas maltophilia (n = 8). Linear regression demonstrated a weak, positive correlation between the number of pairwise genome-wide single-nucleotide polymorphisms (SNPs) and IR Biotyper spectral distance values for Gram-positive (r = 0.43, P ≤ 0.0001), Gram-negative (r = 0.1554, P = 0.0639), and all organisms combined (r = 0.342, P ≤ 0.0001). Overall, the IR Biotyper had a positive predictive value (PPV) of 55.81% for identifying strains that were closely related by genomic identity, but a negative predictive value (NPV) of 86.79% for identifying unrelated isolates. When experimentally adjusted cut-offs were applied to A. baumannii, P. aeruginosa, and E. coli, the PPV was 62% for identifying strains that were closely related and the NPV was 100% for identifying unrelated isolates. Implementation of the IR Biotyper as a screening tool in this cohort would have reduced the number of Gram-negative isolates requiring further WGS analysis by 50% and would reduce the number of S. aureus isolates needing WGS resolution by 48%.
Assuntos
Infecção Hospitalar , Escherichia coli , Humanos , Escherichia coli/genética , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Estudos Retrospectivos , Espectroscopia de Infravermelho com Transformada de Fourier , Análise de Fourier , Staphylococcus aureus/genética , Genoma Bacteriano/genética , Surtos de DoençasAssuntos
Antibacterianos , Azitromicina , Farmacorresistência Bacteriana , Macrolídeos , Sífilis , Treponema pallidum , Humanos , Antibacterianos/farmacologia , Antibacterianos/provisão & distribuição , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Macrolídeos/farmacologia , Macrolídeos/uso terapêutico , América do Norte , Sífilis/tratamento farmacológico , Sífilis/genética , Sífilis/microbiologia , Treponema pallidum/efeitos dos fármacos , Treponema pallidum/genética , Treponema pallidum/isolamento & purificação , Azitromicina/farmacologia , Azitromicina/uso terapêutico , Estados Unidos , Canadá , Penicilina G Benzatina/farmacologia , Penicilina G Benzatina/provisão & distribuição , Penicilina G Benzatina/uso terapêutico , Doxiciclina/farmacologia , Doxiciclina/provisão & distribuição , Doxiciclina/uso terapêuticoRESUMO
Due to the global SARS-CoV-2 pandemic, in-person laboratory medicine clerkships were converted to distance learning. The remote clerkship format provided advantages of allowing participation of students from more locations and greater scheduling flexibility but provided new challenges of maintaining learner engagement and providing experiential content of the laboratory environment. Gamification of educational content is one educational modality that has shown effectiveness in a multitude of different contexts to increase learner engagement and retention. Therefore, we created an interactive, educational 360° virtual reality walkthrough tour using off-the-shelf commercially available 360° cameras and software of the Transfusion service and Microbiology Laboratories. The process consists of taking multiple 360° still-images within the space, color-correction, blurring the faces of staff or sensitive information, adding navigation buttons, and other interactive elements. The virtual tours were used for both recruitment and education with further plans to integrate the learning modality into the curriculum. The clerkship is likely to remain as partially or fully as remote learning so such walkthrough tours will continue to remain relevant. This technology can be applied globally to other departments and institutions for education or recruitment.
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COVID-19 , Realidade Virtual , COVID-19/epidemiologia , Currículo , Humanos , Laboratórios , Pandemias , SARS-CoV-2RESUMO
Isolation of Cokeromyces recurvatus, a dimorphic mucormycete fungus, from clinical specimens poses a diagnostic challenge to physicians and laboratorians as this organism may represent a rare colonizer or true pathogen. Here, we report a case of Cokeromyces recurvatus present in a circumferential duodenal lesion. The patient is a 64-year-old with no past medical history, admitted with a three-week history of left upper quadrant abdominal pain. Computerized tomography scan identified duodenitis with significant gastric outlet obstruction, confirmed by the presence of a partially obstructing non-bleeding duodenal ulcer on upper endoscopy. Histology showed variably sized spherical structures without nuclei, reproductive tracts, or alimentary tracts. Small, clustered spherules representing putative endospores were observed within the larger structures and in the exudate. Based on the histology, the differential included Coccidioides spp, Emmonsia spp, or Chrysosporium spp. Additionally, gastric biopsies revealed concurrent Helicobacter pylori gastritis. The fungus was identified as C. recurvatus by broad-range fungal polymerase chain reaction performed on formalin-fixed paraffin-embedded biopsy tissue, as well as morphology and DNA sequencing of the cultured isolate. The fungus had low MICs to all major antifungal classes; however, in the context of the Helicobacter pylori infection, the patient was only treated with amoxicillin and clarithromycin with improvement in his symptoms before hospital discharge. Only three cases of Cokeromyces recurvatus isolated from the GI tract have been reported; this case highlights a unique clinical presentation in the small bowel in a patient without underlying medical conditions.
Assuntos
Obstrução da Saída Gástrica , Infecções por Helicobacter , Helicobacter pylori , Mucorales , Humanos , Pessoa de Meia-Idade , Obstrução da Saída Gástrica/diagnósticoRESUMO
Broad-range fungal PCR is a powerful tool for identifying pathogens directly from patient specimens; however, reported estimates of clinical utility vary and costs discourage universal testing. We investigated the diagnostic and clinical utility of broad-range fungal PCR by examining 9 years of results from sinonasal specimens, hypothesizing that this anatomic location would identify immunocompromised patients at high risk for invasive fungal disease. We retrospectively identified 644 PCRs and 1,446 fungal cultures from sinus sites. To determine the relative performance of each testing modality, we performed chart review on 52 patients having specimens submitted for culture and PCR on the same day. Positivity rates were significantly higher for PCR (37.1%) than culture (13.7%) but similar for formalin-fixed and fresh tissues (42.3% versus 34.6%). Relative to culture, PCR had significantly faster turnaround time to both preliminary (94.5 versus 108.8 h) and final positive (137.9 versus 278.5 h) results. Among chart-reviewed patients, 88% were immunocompromised, 65% had proven or probable fungal disease, and testing sensitivities for culture and PCR (67.5% and 85.0%) were not statistically different. Nevertheless, PCR identified pathogens not recovered by culture in 14.9% of cases and informed clinical decision-making in 16.7% of all reviewed cases, and sensitivity of PCR combined with culture (90.0%) was higher than that of culture alone. We conclude that broad-range fungal PCR is frequently informative for patients at risk of serious fungal disease and is complementary to and has faster turnaround time than culture. Formalin-fixed tissue does not adversely affect diagnostic yield, but anatomic site may impact assay positivity rates.
Assuntos
Micoses , Sinusite , DNA Fúngico/genética , Humanos , Micoses/diagnóstico , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Sensibilidade e Especificidade , Sinusite/diagnósticoRESUMO
The opportunistic pathogen Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF), a condition that predisposes patients to chronic lung infections. P. aeruginosa lung infections are difficult to treat because P. aeruginosa adapts to the CF lung, can develop multidrug resistance, and can form biofilms. Despite the clinical significance of P. aeruginosa, modeling P. aeruginosa infections in CF has been challenging. Here, we characterize Scnn1b-transgenic (Tg) BALB/c mice as P. aeruginosa lung infection models. Scnn1b-Tg mice overexpress the epithelial Na+ channel (ENaC) in their lungs, driving increased sodium absorption that causes lung pathology similar to CF. We intranasally infected Scnn1b-Tg mice and wild-type littermates with the laboratory P. aeruginosa strain PAO1 and CF clinical isolates and then assessed differences in bacterial clearance, cytokine responses, and histological features up to 12 days postinfection. Scnn1b-Tg mice carried higher bacterial burdens when infected with biofilm-grown rather than planktonic PAO1; Scnn1b-Tg mice also cleared infections more slowly than their wild-type littermates. Infection with PAO1 elicited significant increases in proinflammatory and Th17-linked cytokines on day 3. Scnn1b-Tg mice infected with nonmucoid early CF isolates maintained bacterial burdens and mounted immune responses similar to those of PAO1-infected Scnn1b-Tg mice. In contrast, Scnn1b-Tg mice infected with a mucoid CF isolate carried high bacterial burdens, produced significantly more interleukin 1ß (IL-1ß), IL-13, IL-17, IL-22, and KC, and showed severe immune cell infiltration into the bronchioles. Taken together, these results show the promise of Scnn1b-Tg mice as models of early P. aeruginosa colonization in the CF lung.
Assuntos
Fibrose Cística/genética , Modelos Animais de Doenças , Canais Epiteliais de Sódio/genética , Infecções Oportunistas/genética , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa/imunologia , Animais , Carga Bacteriana , Biofilmes/crescimento & desenvolvimento , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Canais Epiteliais de Sódio/imunologia , Feminino , Regulação da Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Interleucinas/genética , Interleucinas/imunologia , Transporte de Íons , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Infecções Oportunistas/imunologia , Infecções Oportunistas/microbiologia , Infecções Oportunistas/patologia , Plâncton/crescimento & desenvolvimento , Plâncton/imunologia , Plâncton/patogenicidade , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Sódio/metabolismo , Interleucina 22RESUMO
Universal screening of potential organ donors and recipients for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now recommended prior to transplantation in the United States during the coronavirus disease 19 (COVID-19) pandemic. Challenges have included limited testing capacity, short windows of organ viability, brief lead time for notification of potential organ recipients, and the need to test lower respiratory donor specimens to optimize sensitivity. In an early U.S. epicenter of the outbreak, we designed and implemented a system to expedite this testing and the results here from the first 3 weeks. The process included a Laboratory Medicine designee for communication with organ recovery and transplant clinical staff, specialized sample labeling and handoff, and priority processing. Thirty-two organs recovered from 14 of 17 screened donors were transplanted vs 70 recovered from 23 donors during the same period in 2019. No pretransplant or organ donors tested positive for SARS-CoV-2. Median turnaround time from specimen receipt was 6.8 hours (donors), 6.5 hours (recipients): 4.5 hours faster than daily inpatient median. No organ recoveries or transplantations were disrupted by a lack of SARS-CoV-2 testing. Waitlist inactivations for COVID-19 precautions were reduced in our region. Systems that include specialized ordering pathways and adequate testing capacity can support continued organ transplantation, even in a SARS-CoV-2 hyperendemic area.
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Teste para COVID-19/métodos , COVID-19/diagnóstico , Programas de Rastreamento/métodos , Transplante de Órgãos , Pandemias , SARS-CoV-2 , Transplantados , COVID-19/epidemiologia , Seguimentos , Humanos , Estudos Retrospectivos , Doadores de Tecidos/estatística & dados numéricosRESUMO
Multiple laboratory-developed tests (LDTs) and commercially available assays have emerged to meet diagnostic needs related to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. To date, there is limited comparison data for these different testing platforms. We compared the analytical performance of a LDT developed in our clinical laboratory based on CDC primer sets and four commercially available, FDA emergency use authorized assays for SARS-CoV-2 (Cepheid, DiaSorin, Hologic Panther, and Roche Cobas) on a total of 169 nasopharyngeal swabs. The LDT and Cepheid Xpert Xpress SARS-CoV-2 assays were the most sensitive assays for SARS-CoV-2 with 100% agreement across specimens. The Hologic Panther Fusion, DiaSorin Simplexa, and Roche Cobas 6800 failed to detect positive specimens only near the limit of detection of our CDC-based LDT assay. All assays were 100% specific, using our CDC-based LDT as the gold standard. Our results provide initial test performance characteristics for SARS-CoV-2 reverse transcription-PCR (RT-PCR) and highlight the importance of having multiple viral detection testing platforms available in a public health emergency.
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Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , COVID-19 , Teste para COVID-19 , Humanos , Nasofaringe/virologia , Pandemias , SARS-CoV-2 , Sensibilidade e EspecificidadeRESUMO
The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture ("16S Capture"). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 Staphylococcus aureus genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA.
Assuntos
Metagenômica , DNA Bacteriano/genética , Genes de RNAr , Humanos , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Mass spectrometry imaging (MSI) was used to elucidate host lipids involved in the inflammatory signaling pathway generated at the host-pathogen interface during a septic bacterial infection. Using Francisella novicida as a model organism, a bacterial lipid virulence factor (endotoxin) was imaged and identified along with host phospholipids involved in the splenic response in murine tissues. Here, we demonstrate detection and distribution of endotoxin in a lethal murine F. novicida infection model, in addition to determining the temporally and spatially resolved innate lipid inflammatory response in both 2D and 3D renderings using MSI. Further, we show that the cyclooxygenase-2-dependent lipid inflammatory pathway is responsible for lethality in F. novicida infection due to overproduction of proinflammatory effectors including prostaglandin E2. The results of this study emphasize that spatial determination of the host lipid components of the immune response is crucial to identifying novel strategies to effectively address highly pathogenic and lethal infections stemming from bacterial, fungal, and viral origins.
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Ciclo-Oxigenase 2/imunologia , Dinoprostona/imunologia , Francisella/patogenicidade , Infecções por Bactérias Gram-Negativas/imunologia , Interações Hospedeiro-Patógeno , Baço/imunologia , Animais , Ciclo-Oxigenase 2/deficiência , Ciclo-Oxigenase 2/genética , Dinoprostona/biossíntese , Eicosanoides/imunologia , Eicosanoides/metabolismo , Endotoxinas/biossíntese , Endotoxinas/toxicidade , Feminino , Francisella/fisiologia , Expressão Gênica , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/mortalidade , Infecções por Bactérias Gram-Negativas/patologia , Imunidade Inata , Inflamação , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Imagem Molecular , Fosfolipídeos/imunologia , Fosfolipídeos/metabolismo , Transdução de Sinais , Baço/metabolismo , Baço/patologia , Análise de SobrevidaAssuntos
Antifúngicos , DNA Fúngico , Farmacorresistência Fúngica , Análise de Sequência de DNA , Tinha , Trichophyton , Humanos , Tinha/microbiologia , Tinha/tratamento farmacológico , Tinha/diagnóstico , Trichophyton/genética , Trichophyton/efeitos dos fármacos , Trichophyton/isolamento & purificação , Trichophyton/classificação , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Farmacorresistência Fúngica/genética , DNA Fúngico/genética , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-IdadeRESUMO
The Group A Streptococcus remains a significant human pathogen causing a wide array of disease ranging from self-limiting to life-threatening invasive infections. Epithelium (skin or throat) colonization with progression to the subepithelial tissues is the common step in all GAS infections. Here, we used transposon-sequencing (Tn-seq) to define the GAS 5448 genetic requirements for in vivo fitness in subepithelial tissue. A near-saturation transposon library of the M1T1 GAS 5448 strain was injected subcutaneously into mice, producing suppurative inflammation at 24 h that progressed to prominent abscesses with tissue necrosis at 48 h. The library composition was monitored en masse by Tn-seq and ratios of mutant abundance comparing the output (12, 24 and 48 h) versus input (T0) mutant pools were calculated for each gene. We identified a total of 273 subcutaneous fitness (scf) genes with 147 genes (55 of unknown function) critical for the M1T1 GAS 5448 fitness in vivo; and 126 genes (53 of unknown function) potentially linked to in vivo fitness advantage. Selected scf genes were validated in competitive subcutaneous infection with parental 5448. Two uncharacterized genes, scfA and scfB, encoding putative membrane-associated proteins and conserved among Gram-positive pathogens, were further characterized. Defined scfAB mutants in GAS were outcompeted by wild type 5448 in vivo, attenuated for lesion formation in the soft tissue infection model and dissemination to the bloodstream. We hypothesize that scfAB play an integral role in enhancing adaptation and fitness of GAS during localized skin infection, and potentially in propagation to other deeper host environments.
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Genes Bacterianos/genética , Infecções dos Tecidos Moles/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Virulência/genética , Animais , Modelos Animais de Doenças , Aptidão Genética/genética , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Medicopsis species are rare fungal pathogens that frequently resist common antifungal therapies and are difficult to identify morphologically as conidia are produced in pycnidia, a key feature of coelomycetes. Immunocompromised patients are at risk of these infections, even after remote exposure, and typically present with phaeohyphomycoses without dissemination. We present the case of a renal transplant recipient 6.5 years post-transplant who developed a slowly progressive soft tissue infection mimicking a synovial cyst. A cultured isolate was identified as Medicopsis romeroi by sequencing of multiple ribosomal loci. The patient responded well to debridement and posaconazole therapy. Solid-organ transplant patients are at risk of opportunistic fungal infection long after transplant, and molecular methods are often required for definitive identification.
Assuntos
Ascomicetos/isolamento & purificação , Portador Sadio/diagnóstico , Portador Sadio/microbiologia , Hospedeiro Imunocomprometido , Feoifomicose/diagnóstico , Feoifomicose/microbiologia , Transplantados , Idoso , Ascomicetos/classificação , Ascomicetos/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Feminino , Humanos , Transplante de Rim , Análise de Sequência de DNA , Infecções dos Tecidos Moles/diagnóstico , Infecções dos Tecidos Moles/microbiologiaRESUMO
microRNAs (miRNAs) hold promise as biomarkers for a variety of disease processes and for determining cell differentiation. These short RNA species are robust, survive harsh treatment and storage conditions and may be extracted from blood and tissue. Pre-analytical variables are critical confounders in the analysis of miRNAs: we elucidate these and identify best practices for minimizing sample variation in blood and tissue specimens. Pre-analytical variables addressed include patient-intrinsic variation, time and temperature from sample collection to storage or processing, processing methods, contamination by cells and blood components, RNA extraction method, normalization, and storage time/conditions. For circulating miRNAs, hemolysis and blood cell contamination significantly affect profiles; samples should be processed within 2 h of collection; ethylene diamine tetraacetic acid (EDTA) is preferred while heparin should be avoided; samples should be "double spun" or filtered; room temperature or 4 °C storage for up to 24 h is preferred; miRNAs are stable for at least 1 year at -20 °C or -80 °C. For tissue-based analysis, warm ischemic time should be <1 h; cold ischemic time (4 °C) <24 h; common fixative used for all specimens; formalin fix up to 72 h prior to processing; enrich for cells of interest; validate candidate biomarkers with in situ visualization. Most importantly, all specimen types should have standard and common workflows with careful documentation of relevant pre-analytical variables.