RESUMEN
Using proteomics and complexome profiling, we evaluated in a year-long study longitudinal variations in the plasma proteome of kidney failure patients, prior to and after a kidney transplantation. The post-transplant period was complicated by bacterial infections, resulting in dramatic changes in the proteome, attributed to an acute phase response (APR). As positive acute phase proteins (APPs), being elevated upon inflammation, we observed the well-described C-reactive protein and Serum Amyloid A (SAA), but also Fibrinogen, Haptoglobin, Leucine-rich alpha-2-glycoprotein, Lipopolysaccharide-binding protein, Alpha-1-antitrypsin, Alpha-1-antichymotrypsin, S100, and CD14. As negative APPs, being downregulated upon inflammation, we identified the well-documented Serotransferrin and Transthyretin, but added Kallistatin, Heparin cofactor 2, and interalpha-trypsin inhibitor heavy chain H1 and H2 (ITIH1, ITIH2). For the patient with the most severe APR, we performed plasma complexome profiling by SEC-LC-MS on all longitudinal samples. We observed that several plasma proteins displaying alike concentration patterns coelute and form macromolecular complexes. By complexome profiling, we expose how SAA1 and SAA2 become incorporated into high-density lipid particles, replacing largely Apolipoprotein (APO)A1 and APOA4. Overall, our data highlight that the combination of in-depth longitudinal plasma proteome and complexome profiling can shed further light on correlated variations in the abundance of several plasma proteins upon inflammatory events.
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Proteínas Sanguíneas , Trasplante de Riñón , Proteoma , Humanos , Trasplante de Riñón/efectos adversos , Proteoma/análisis , Proteoma/metabolismo , Estudios Longitudinales , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/metabolismo , Proteínas de Fase Aguda/metabolismo , Persona de Mediana Edad , Masculino , Proteómica/métodos , Femenino , Insuficiencia Renal/sangre , Reacción de Fase Aguda/sangre , AdultoRESUMEN
Monoclonal gammopathy of undetermined significance (MGUS) is a plasma cell disorder characterized by the presence of a predominant monoclonal antibody (i.e., M-protein) in serum, without clinical symptoms. Here we present a case study in which we detect MGUS by liquid-chromatography coupled with mass spectrometry (LC-MS) profiling of IgG1 in human serum. We detected a Fab-glycosylated M-protein and determined the full heavy and light chain sequences by bottom-up proteomics techniques using multiple proteases, further validated by top-down LC-MS. Moreover, the composition and location of the Fab-glycan could be determined in CDR1 of the heavy chain. The outlined approach adds to an expanding mass spectrometry-based toolkit to characterize monoclonal gammopathies such as MGUS and multiple myeloma, with fine molecular detail. The ability to detect monoclonal gammopathies and determine M-protein sequences straight from blood samples by mass spectrometry provides new opportunities to understand the molecular mechanisms of such diseases.
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Gammopatía Monoclonal de Relevancia Indeterminada , Mieloma Múltiple , Paraproteinemias , Humanos , Gammopatía Monoclonal de Relevancia Indeterminada/diagnóstico , Paraproteinemias/diagnóstico , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/genética , Espectrometría de Masas , Inmunoglobulina GRESUMEN
BACKGROUND: Invasive aspergillosis (IA) by a triazole-resistant Aspergillus fumigatus is associated with high mortality. Real-time resistance detection will result in earlier initiation of appropriate therapy. METHODS: In a prospective study, we evaluated the clinical value of the AsperGenius polymerase chain reaction (PCR) assay in hematology patients from 12 centers. This PCR assay detects the most frequent cyp51A mutations in A. fumigatus conferring azole resistance. Patients were included when a computed tomography scan showed a pulmonary infiltrate and bronchoalveolar fluid (BALf) sampling was performed. The primary end point was antifungal treatment failure in patients with azole-resistant IA. RESULTS: Of 323 patients enrolled, complete mycological and radiological information was available for 276 (94%), and probable IA was diagnosed in 99/276 (36%). Sufficient BALf for PCR testing was available for 293/323 (91%). Aspergillus DNA was detected in 116/293 (40%) and A. fumigatus DNA in 89/293 (30%). The resistance PCR was conclusive in 58/89 (65%) and resistance detected in 8/58 (14%). Two had a mixed azole-susceptible/azole-resistant infection. In the 6 remaining patients, treatment failure was observed in 1. Galactomannan positivity was associated with mortality (P = .004) while an isolated positive Aspergillus PCR was not (P = .83). CONCLUSIONS: Real-time PCR-based resistance testing may help to limit the clinical impact of triazole resistance. In contrast, the clinical impact of an isolated positive Aspergillus PCR on BALf seems limited. The interpretation of the EORTC/MSGERC PCR criterion for BALf may need further specification (eg, minimum cycle threshold value and/or PCR positive on >1 BALf sample).
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Aspergilosis , Infecciones Fúngicas Invasoras , Aspergilosis Pulmonar Invasiva , Humanos , Estudios Prospectivos , Aspergilosis Pulmonar Invasiva/diagnóstico , Aspergilosis Pulmonar Invasiva/tratamiento farmacológico , Aspergilosis Pulmonar Invasiva/microbiología , Azoles/farmacología , Azoles/uso terapéutico , Aspergilosis/diagnóstico , Aspergilosis/tratamiento farmacológico , Aspergilosis/microbiología , Aspergillus , Aspergillus fumigatus , Infecciones Fúngicas Invasoras/diagnóstico , Infecciones Fúngicas Invasoras/tratamiento farmacológico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Triazoles/farmacología , Triazoles/uso terapéutico , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Farmacorresistencia FúngicaRESUMEN
Galactomannan (GM) testing of bronchoalveolar lavage (BAL) fluid samples has become an essential tool to diagnose invasive pulmonary aspergillosis (IPA) and is part of diagnostic guidelines. Enzyme-linked immunosorbent assays (ELISAs) (enzyme immunoassays [EIAs]) are commonly used, but they have a long turnaround time. In this study, we evaluated the performance of an automated chemiluminescence immunoassay (CLIA) with BAL fluid samples. This was a multicenter retrospective study in the Netherlands and Belgium. BAL fluid samples were collected from patients with underlying hematological diseases with a suspected invasive fungal infection. Diagnosis of IPA was based on the 2020 European Organisation for Research and Treatment of Cancer (EORTC)/Mycoses Study Group Education and Research Consortium (MSGERC) consensus definitions. GM results were reported as optical density index (ODI) values. ODI cutoff values for positive results that were evaluated were 0.5, 0.8, and 1.0 for the EIA and 0.16, 0.18, and 0.20 for the CLIA. Probable IPA cases were compared with two control groups, one with no evidence of IPA and another with no IPA or possible IPA. Qualitative agreement was analyzed using Cohen's κ, and quantitative agreement was analyzed by Spearman's correlation. We analyzed 141 BAL fluid samples from 141 patients; 66 patients (47%) had probable IPA, and 56 cases remained probable IPA when the EIA GM result was excluded as a criterion, because they also had positive culture and/or duplicate positive PCR results. Sixty-three patients (45%) had possible IPA and 12 (8%) had no IPA. The sensitivity and specificity of the two tests were quite comparable, and the overall qualitative agreement between EIA and CLIA results was 81 to 89%. The correlation of the actual CLIA and EIA values was strong at 0.72 (95% confidence interval, 0.63 to 0.80). CLIA has similar performance, compared to the gold-standard EIA, with the benefits of faster turnaround because batching is not required. Therefore, CLIA can be used as an alternative GM assay for BAL fluid samples.
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Enfermedades Hematológicas , Aspergilosis Pulmonar Invasiva , Aspergilosis Pulmonar , Humanos , Estudios Retrospectivos , Líquido del Lavado Bronquioalveolar/microbiología , Aspergilosis Pulmonar Invasiva/diagnóstico , Mananos/análisis , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: Although the Surviving Sepsis Campaign bundle recommends obtaining blood cultures within 1 hour of sepsis recognition, adherence is suboptimal in many settings. We, therefore, implemented routine blood culture collection for all nonelective ICU admissions (regardless of infection suspicion) and evaluated its diagnostic yield. DESIGN: A before-after analysis. SETTING: A mixed-ICU of a tertiary care hospital in the Netherlands. PATIENTS: Patients acutely admitted to the ICU between January 2015 and December 2018. MEASUREMENTS AND MAIN RESULTS: Automatic orders for collecting a single set of blood cultures immediately upon ICU admission were implemented on January 1, 2017. Blood culture results and the impact of contaminated blood cultures were compared for 2015-2016 (before period) and 2017-2018 (after period). Positive blood cultures were categorized as bloodstream infection or contamination. Blood cultures were obtained in 573 of 1,775 patients (32.3%) and in 1,582 of 1,871 patients (84.5%) in the before and after periods, respectively (p < 0.0001), and bloodstream infection was diagnosed in 95 patients (5.4%) and 154 patients (8.2%) in both study periods (relative risk 1.5; 95% CI 1.2-2.0; p = 0.0006). The estimated number needed to culture for one additional patient with bloodstream infection was 17. Blood culture contamination occurred in 40 patients (2.3%) and 180 patients (9.6%) in the before period and after period, respectively (relative risk 4.3; 95% CI 3.0-6.0; p < 0.0001). Rate of vancomycin use or presumed episodes of catheter-related bloodstream infections treated with antibiotics did not differ between both study periods. CONCLUSIONS: Implementation of routine blood cultures was associated with a 1.5-fold increase of detected bloodstream infection. The 4.3-fold increase in contaminated blood cultures was not associated with an increase in vancomycin use in the ICU.
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Cultivo de Sangre , Enfermedad Crítica/terapia , Sepsis/microbiología , Anciano , Cultivo de Sangre/métodos , Cultivo de Sangre/estadística & datos numéricos , Femenino , Humanos , Unidades de Cuidados Intensivos/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Admisión del Paciente , Estudios Prospectivos , Sepsis/sangre , Sepsis/diagnósticoRESUMEN
Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.
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Peroxidasa/antagonistas & inhibidores , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Humanos , Modelos Moleculares , Neutrófilos/fisiología , Fagocitosis , Unión Proteica , Conformación Proteica , Staphylococcus aureus/metabolismo , Regulación hacia ArribaRESUMEN
Staphylococcal superantigen-like (SSL) proteins, one of the major virulence factor families produced by Staphylococcus aureus, were previously demonstrated to be immune evasion molecules that interfere with a variety of innate immune defences. However, in contrast to characterised SSLs, which inhibit immune functions, we show that SSL13 is a strong activator of neutrophils via the formyl peptide receptor 2 (FPR2). Moreover, our data show that SSL13 acts as a chemoattractant and induces degranulation and oxidative burst in neutrophils. As with many other staphylococcal immune evasion proteins, SSL13 shows a high degree of human specificity. SSL13 is not able to efficiently activate mouse neutrophils, hampering in vivo experiments. In conclusion, SSL13 is a neutrophil chemoattractant and activator that acts via FPR2. Therefore, SSL13 is a unique SSL member that does not belong to the immune evasion class but is a pathogen alarming molecule. Our study provides a new concept of SSLs; SSLs not only inhibit host immune processes but also recruit human neutrophils to the site of infection. This new insight allows us to better understand complex interactions between host and S. aureus pathological processes.
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Proteínas Bacterianas/metabolismo , Neutrófilos/microbiología , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Staphylococcus aureus/patogenicidad , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Degranulación de la Célula , Factores Quimiotácticos/metabolismo , Femenino , Células HL-60 , Humanos , Evasión Inmune , Ratones Endogámicos , Activación Neutrófila , Neutrófilos/fisiología , Peritonitis/metabolismo , Peritonitis/microbiología , Estallido Respiratorio , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patologíaRESUMEN
Candida vulturna is a new member of the Candida haemulonii species complex that recently received much attention as it includes the emerging multidrug-resistant pathogen Candida auris. Here, we describe the high-quality genome sequence of C. vulturna type strain CBS 14366T to cover all genomes of pathogenic C. haemulonii species complex members.
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Candida/genética , Genoma Fúngico/genética , Candidiasis/microbiología , Humanos , Infecciones Oportunistas/microbiología , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Aspergillus fumigatus is a ubiquitous saprotrophic fungus and an opportunistic pathogen of humans and animals. Humans and animals can inhale hundreds of A. fumigatus spores daily. Normally this is harmless for humans, but in case of immunodeficiency, invasive pulmonary aspergillosis (IPA) can develop with a high mortality rate. A. fumigatus also causes non-invasive mycoses like sino-nasal aspergillosis (SNA) in dogs. RESULTS: In this study we compared A. fumigatus isolates from humans with suspected IPA, dogs with SNA, and a set of environmental isolates. Phylogenetic inference based on calmodulin (CaM) and beta-tubulin (benA) sequences did not reveal A. fumigatus sub-groups linked to the origin of the isolates. Genotyping and microsatellite analysis showed that each dog was infected by one A. fumigatus genotype, whereas human patients had mixed infections. Azole resistance was determined by antifungal susceptibility testing and sequencing of the cyp51A gene. A total of 12 out of 29 human isolates and 1 out of 27 environmental isolates were azole resistant. Of the azole resistant strains, 11 human isolates showed TR34/L98H (n = 6) or TR46/Y121F/T289A (n = 5). Phenotypically, isolates from dogs were more variable in growth speed and morphology when compared to those isolated from human and the environment. CONCLUSIONS: 1. A. fumigatus from dogs with SNA are phenotypically very diverse in contrast to their environmental and human counterparts. 2. Phenotypic variability can be induced during the chronic infection process in the sinus of the dogs. The basis of this heterogeneity might be due to genomic differences and/or epigenetic variations. 3. Differences in dogs is a could be a result of within-host adaption and might be triggered by environmental factors in the sinus, however this hypothesis still needs to be tested.
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Aspergilosis/microbiología , Aspergilosis/veterinaria , Aspergillus fumigatus/aislamiento & purificación , Enfermedades de los Perros/microbiología , Animales , Antifúngicos/farmacología , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Azoles/farmacología , Perros , Microbiología Ambiental , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Fenotipo , FilogeniaRESUMEN
Patients with haematological malignancies are at risk for invasive fungal diseases (IFD). A survey was conducted in all Dutch academic haematology centres on their current diagnostic, prophylactic and therapeutic approach towards IFD in the context of azole-resistance. In all 8 centres, a haematologist and microbiologist filled in the questionnaire that focused on different subgroups of haematology patients. Fungal prophylaxis during neutropaenia was directed against Candida and consisted of fluconazole and/or amphotericin B suspension. Mould-active prophylaxis was given to acute myeloid leukaemia patients during chemotherapy in 2 of 8 centres. All centres used azole prophylaxis in a subset of patients with graft-versus-host disease. A uniform approach towards the diagnosis and treatment of IFD and in particular azole-resistant Aspergillus fumigatus was lacking. In 2017, all centres agreed to implement a uniform diagnostic and treatment algorithm regarding invasive aspergillosis with a central role for comprehensive diagnostics and PCR-based detection of azole-resistance. This study (DB-MSG 002) will re-evaluate this algorithm when 280 patients have been treated. A heterogeneous approach towards antifungal prophylaxis, diagnosis and treatment was apparent in the Netherlands. Facing triazole-resistance, consensus was reached on the implementation of a uniform diagnostic approach in all 8 centres.
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Antifúngicos/administración & dosificación , Azoles/administración & dosificación , Manejo de la Enfermedad , Farmacorresistencia Fúngica , Neoplasias Hematológicas/complicaciones , Aspergilosis Pulmonar Invasiva/diagnóstico , Aspergilosis Pulmonar Invasiva/tratamiento farmacológico , Centros Médicos Académicos , Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Azoles/farmacología , Quimioprevención/métodos , Humanos , Aspergilosis Pulmonar Invasiva/prevención & control , Países Bajos , Prevalencia , Encuestas y CuestionariosRESUMEN
A survey of diagnosis and treatment of invasive aspergillosis was conducted in eight University Medical Centers (UMCs) and eight non-academic teaching hospitals in the Netherlands. Against a background of emerging azole resistance in Aspergillus fumigatus routine resistance screening of clinical isolates was performed primarily in the UMCs. Azole resistance rates at the hospital level varied between 5% and 10%, although rates up to 30% were reported in high-risk wards. Voriconazole remained first choice for invasive aspergillosis in 13 out of 16 hospitals. In documented azole resistance 14 out of 16 centres treated patients with liposomal amphotericin B.
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Anfotericina B/uso terapéutico , Aspergilosis/diagnóstico , Aspergilosis/tratamiento farmacológico , Aspergillus fumigatus/efectos de los fármacos , Voriconazol/uso terapéutico , Antifúngicos/uso terapéutico , Aspergilosis/epidemiología , Aspergillus fumigatus/aislamiento & purificación , Farmacorresistencia Fúngica , Humanos , Países Bajos/epidemiología , Encuestas y Cuestionarios , Voriconazol/farmacologíaRESUMEN
In order to cause colonization and invasive disease, pathogenic bacteria secrete proteins that modulate host immune defences. Identification and characterization of these proteins leads to a better understanding of the pathological processes underlying infectious and inflammatory diseases and is essential in the development of new strategies for their prevention and treatment. Current techniques to functionally characterize these proteins are laborious and inefficient. Here we describe a high-throughput functional selection strategy using phage display in order to identify immune evasion proteins. Using this technique we identified two previously uncharacterized proteins secreted by Staphylococcus aureus, SElX and SSL6 that bind to neutrophil surface receptors. SElX binds PSGL-1 on neutrophils and thereby inhibits the interaction between PSGL-1 and P-selectin, a crucial step in the recruitment of neutrophils to the site of infection. SSL6 is the first bacterial protein identified that binds CD47, a widely expressed cell surface protein recently described as an interesting target in anti-cancer therapy. Our findings provide new insights into the pathogenesis of S. aureus infections and support phage display as an efficient method to identify bacterial secretome proteins interacting with humoral or cellular immune components.
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Proteínas Bacterianas/metabolismo , Técnicas de Visualización de Superficie Celular/métodos , Interacciones Huésped-Patógeno , Proteínas de la Membrana/metabolismo , Neutrófilos/microbiología , Staphylococcus aureus/fisiología , Antígeno CD47 , Glicoproteínas de MembranaRESUMEN
Histoplasmosis is a frequent cause of infections in people living with HIV/AIDS (PLWHA). This study introduces the application of a Histoplasma capsulatum urine antigen lateral flow assay (LFA) for diagnosing disseminated histoplasmosis in PLWHA in Suriname. The LFA's diagnostic accuracy was compared with the current diagnostic approach, aiming to assess whether this test resulted in improved early detection and management. Additionally, the prevalence of histoplasmosis among advanced stage HIV patients without clinical suspicion of infection was evaluated using the same LFA. In total, 98 patients were included in the study, of which 58 were classified as "possible disseminated histoplasmosis (DH)" based on clinical criteria and 40 as "controls". Of these possible DH cases, only 19 (32.7%) had a positive LFA. During the study, decisions for treatment were made without the treating physician being aware of the LFA result. Only 55% of the patients who started treatment for histoplasmosis based on clinical criteria had a positive LFA, and 21% of untreated patients had a positive LFA. This study shows that combining clinical signs with LFA results enhances diagnostic accuracy and is cost effective, resulting in better treatment decisions.
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Infecciones por VIH , Histoplasma , Histoplasmosis , Humanos , Histoplasmosis/diagnóstico , Masculino , Femenino , Adulto , Suriname , Histoplasma/aislamiento & purificación , Infecciones por VIH/complicaciones , Persona de Mediana Edad , Antígenos Fúngicos/orina , Sensibilidad y Especificidad , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Infecciones Oportunistas Relacionadas con el SIDA/orina , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Inmunoensayo/métodosRESUMEN
Prokaryotes have evolved a multitude of defense systems to protect against phage predation. Some of these resemble eukaryotic genes involved in antiviral responses. Here, we set out to systematically project the current knowledge of eukaryotic-like antiviral defense systems onto prokaryotic genomes, using Pseudomonas aeruginosa as a model organism. Searching for phage defense systems related to innate antiviral genes from vertebrates and plants, we uncovered over 450 candidates. We validated six of these phage defense systems, including factors preventing viral attachment, R-loop-acting enzymes, the inflammasome, ubiquitin pathway, and pathogen recognition signaling. Collectively, these defense systems support the concept of deep evolutionary links and shared antiviral mechanisms between prokaryotes and eukaryotes.
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Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/virología , Inmunidad Innata , Bacteriófagos/genética , Bacteriófagos/fisiología , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética , Animales , Evolución Molecular , Inflamasomas/inmunología , Inflamasomas/genética , Eucariontes/virología , Eucariontes/genética , Eucariontes/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evolución Biológica , Plantas/inmunología , Plantas/virología , Plantas/microbiologíaRESUMEN
Prokaryotes encode multiple distinct anti-phage defense systems in their genomes. However, the impact of carrying a multitude of defense systems on phage resistance remains unclear, especially in a clinical context. Using a collection of antibiotic-resistant clinical strains of Pseudomonas aeruginosa and a broad panel of phages, we demonstrate that defense systems contribute substantially to defining phage host range and that overall phage resistance scales with the number of defense systems in the bacterial genome. We show that many individual defense systems target specific phage genera and that defense systems with complementary phage specificities co-occur in P. aeruginosa genomes likely to provide benefits in phage-diverse environments. Overall, we show that phage-resistant phenotypes of P. aeruginosa with at least 19 phage defense systems exist in the populations of clinical, antibiotic-resistant P. aeruginosa strains.
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Bacteriófagos , Infecciones por Pseudomonas , Fagos Pseudomonas , Humanos , Bacteriófagos/genética , Pseudomonas aeruginosa , Fagos Pseudomonas/genética , Infecciones por Pseudomonas/microbiología , AntibacterianosRESUMEN
BACKGROUND: Aspergillus tubingensis is a black Aspergillus belonging to the Aspergillus section Nigri, which includes species that morphologically resemble Aspergillus niger. Recent developments in species determination have resulted in clinical isolates presumed to be Aspergillus niger being reclassified as Aspergillus tubingensis by sequencing. We present a report of a patient with an osteomyelitis of the maxillary bone with a probable invasive Aspergillus tubingensis infection. CASE PRESENTATION: We describe an immune compromised patient suffering from osteomyelitis of the maxillary bone after tooth extraction. The osteomyelitis probably resulted in dentogenic pansinusitis presenting as an acute ethmoiditis. Histologic examination of biopsy samples showed osteomyelitis, and inflammation of the surrounding connective tissue. Cultures of the alveolar wound grew Aspergillus tubingensis. The patient was treated with liposomal amphoterocin B, which was changed to oral treatment with voriconazole based on susceptibility testing (MIC for voriconazole was 1 µg/ml). CONCLUSION: This case shows that Aspergillus tubingensis may have the potential to cause severe invasive infections in immunocompromised hosts. A larger proportion of Aspergillus tubingensis isolates are less susceptible to azoles compared to Aspergillus niger. Therefore, correct species identification and susceptibility testing is crucial for the choice of anti-fungal treatment, screening of azole resistance, and characterization of the pathogenic potential of the various species within Aspergillus section Nigri.
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Aspergilosis/microbiología , Aspergillus , Maxilar/microbiología , Infecciones Oportunistas , Osteomielitis/microbiología , Aspergilosis/diagnóstico , Aspergillus/clasificación , Aspergillus/genética , Humanos , Masculino , Maxilar/diagnóstico por imagen , Osteomielitis/diagnóstico , Filogenia , Radiografía , Tubulina (Proteína)/genética , Adulto JovenRESUMEN
Bacteriophages (phages) are viruses that specifically attack bacteria. Their use as therapeutics, which constitutes a promising alternative to antibiotics, heavily relies on selecting effective lytic phages against the pathogen of interest. Current selection techniques are laborious and do not allow for direct visualization of phage infection dynamics. Here, we present a method that circumvents these limitations. It can be scaled for high-throughput and permits monitoring of the phage infection in real time via a fluorescence signal readout. This is achieved through the use of a membrane-impermeant nucleic acid dye that stains the DNA of damaged or lysed bacteria and new phage progeny. We have tested the method on Pseudomonas aeruginosa and Klebsiella pneumoniae and show that an increase in fluorescence reflects phage-mediated killing. This is confirmed by other techniques including spot tests, colony plating, flow cytometry and metabolic activity measurements. Furthermore, we illustrate how our method may be used to compare the activity of different phages and to screen the susceptibility of clinical isolates to phage. Altogether, we present a fast, reliable way of selecting phages against Gram-negative bacteria, which may be valuable in optimizing the process of selecting phages for therapeutic use.