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1.
Sci Immunol ; 6(56)2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33622974

RESUMEN

Hyperinflammation contributes to lung injury and subsequent acute respiratory distress syndrome (ARDS) with high mortality in patients with severe coronavirus disease 2019 (COVID-19). To understand the underlying mechanisms involved in lung pathology, we investigated the role of the lung-specific immune response. We profiled immune cells in bronchoalveolar lavage fluid and blood collected from COVID-19 patients with severe disease and bacterial pneumonia patients not associated with viral infection. By tracking T cell clones across tissues, we identified clonally expanded tissue-resident memory-like Th17 cells (Trm17 cells) in the lungs even after viral clearance. These Trm17 cells were characterized by a a potentially pathogenic cytokine expression profile of IL17A and CSF2 (GM-CSF). Interactome analysis suggests that Trm17 cells can interact with lung macrophages and cytotoxic CD8+ T cells, which have been associated with disease severity and lung damage. High IL-17A and GM-CSF protein levels in the serum of COVID-19 patients were associated with a more severe clinical course. Collectively, our study suggests that pulmonary Trm17 cells are one potential orchestrator of the hyperinflammation in severe COVID-19.

2.
J Virol Methods ; 290: 114093, 2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33549574

RESUMEN

BACKGROUND: In immunocompromised patients, BK Virus (BKV) reactivation may cause serious disease with high morbidity. Particularly for patient management after solid organ transplantation, monitoring of viral load in different clinical specimens is crucial to ensure early diagnosis and response to reactivation. In this study, we evaluated the clinical performance of a custom designed primer /probe set for detection of BKV on the cobas® 6800, a high-throughput platform, employing the open channel of the system for integration of a lab-developed test (LDT). MATERIALS/METHODS: A primer/probe set was optimized for the use on a high-throughput platform. Clinical performance was assessed in EDTA-plasma, serum and urine samples. Limit-of-detection (LOD) was determined by using a dilution series of BKV WHO standard. A CE-labeled PCR test (Altona Diagnostics) was used as a comparison to the assay. RESULTS: The LOD for the LDT BKV assay was 6.7 IU/mL. Inter-and intra-run variability (at 5 x LOD) was low (<1.5 Ct in all specimens). All quality control panel specimens (Instand Germany n = 19) were correctly identified. Of 290 clinical samples tested, results were concordant for 280 samples. Sensitivity and specificity of the assay were 96 % and 98 % respectively. The quantitative analysis revealed a strong correlation (linear regression) between the CE-labelled comparator assay and the new BKV LDT assay with r2 = 0.96 for n = 123 urine samples and r2 = 0.98 for n = 167 plasma/serum samples. CONCLUSION: Compared to a CE-IVD assay, the adapted LDT showed good analytical and clinical sensitivity and specificity for the detection and quantification of BKV in different clinical specimens. It represents a convenient solution to automate the LDT workflow with low hands-on time and thus facilitates high-throughput screening for BKV reactivation in immunocompromised patients.

3.
JAMA Ophthalmol ; 2021 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-33475692

RESUMEN

Importance: Current recommendations are to avoid tissue for corneal transplant from donors with coronavirus disease 2019 (COVID-19) or those who were recently exposed to COVID-19 owing to the lack of knowledge about the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in corneal tissues. Evidence of SARS-CoV-2 in corneal tissue would seem to have clinical relevance for corneal transplant. Objectives: To investigate the presence of viral SARS-CoV-2 RNA in corneal discs of deceased patients with confirmed COVID-19 and assess viral genomic and subgenomic RNA load, possible infectivity, and histologic abnormalities. Design, Setting, and Participants: A case series was conducted of 11 deceased patients with COVID-19 who underwent autopsy between March 20 and May 14, 2020. Eleven corneal discs (1 corneal disc per patient) were harvested for molecular detection of viral genomic and subgenomic RNA, virus isolation, and immunohistochemistry. The SARS-CoV-2 RNA loads were compared with RNA loads in the conjunctival and throat swab samples and aqueous humor, vitreous humor, and blood samples. Main Outcomes and Measures: Evidence of SARS-CoV-2 RNA in human corneas. Results: This study comprised 11 patients (6 women [55%]; mean [SD] age, 68.5 [18.8] years). In 6 of 11 eyes (55%), SARS-CoV-2 genomic RNA was detected in the cornea; subgenomic RNA was present in 4 of these 6 eyes (67%). Infectivity or the presence of viral structural proteins could not be confirmed in any eye. However, patients whose corneal disc was positive for SARS-CoV-2 RNA also had positive results for SARS-CoV-2 RNA in 4 of 6 conjunctival swab samples, 1 of 3 aqueous humor samples, 3 of 5 vitreous humor samples, and 4 of 5 blood samples. Overall, conjunctival swab samples had positive results for SARS-CoV-2 RNA in 5 of 11 cases. Postmortem SARS-CoV-2 viremia was detected in 5 of 9 patients. Conclusions and Relevance: Viral genomic and subgenomic RNA of SARS-CoV-2 was detected in the cornea of patients with COVID-19 viremia. The risk of COVID-19 infection via corneal transplant is low even in donors with SARS-CoV-2 viremia, but further research is necessary to assess the rate of SARS-CoV-2 transmission via corneal transplant.

4.
J Med Microbiol ; 70(2)2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33404401

RESUMEN

Introduction. Laboratories worldwide are facing high demand for molecular testing during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, which might be further aggravated by the upcoming influenza season in the northern hemisphere.Gap Statement. Given that the symptoms of influenza are largely indistinguishable from those of coronavirus disease 2019 (COVID-19), both SARS-CoV-2 and the influenza viruses require concurrent testing by RT-PCR in patients presenting with symptoms of respiratory tract infection.Aim. We adapted and evaluated a laboratory-developed multiplex RT-PCR assay for simultaneous detection of SARS-CoV-2 (dual target), influenza A and influenza B (SC2/InflA/InflB-UCT) on a fully automated high-throughput system (cobas6800).Methodology. Analytical performance was assessed by serial dilution of quantified reference material and cell culture stocks in transport medium, including pretreatment for chemical inactivation. For clinical evaluation, residual portions of 164 predetermined patient samples containing SARS-CoV-2 (n=52), influenza A (n=43) or influenza B (n=19), as well as a set of negative samples, were subjected to the novel multiplex assay.Results. The assay demonstrated comparable analytical performance to currently available commercial tests, with limits of detection of 94.9 cp ml-1 for SARS-CoV-2, 14.6 cp ml-1 for influenza A and 422.3 cp ml-1 for influenza B. Clinical evaluation showed excellent agreement with the comparator assays (sensitivity of 98.1, 97.7 and 100 % for Sars-CoV-2 and influenza A and B, respectively).Conclusion. The SC2/InflA/InflB-UCT allows for efficient high-throughput testing for all three pathogens and thus provides streamlined diagnostics while conserving resources during the influenza season.

5.
Emerg Infect Dis ; 27(1)2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33327991

RESUMEN

Analyses of infection chains have demonstrated that severe acute respiratory syndrome coronavirus 2 is highly transmissive. However, data on postmortem stability and infectivity are lacking. Our finding of nasopharyngeal viral RNA stability in 79 corpses showed no time-dependent decrease. Maintained infectivity is supported by virus isolation up to 35 hours postmortem.


Asunto(s)
/virología , Nasofaringe/virología , ARN Viral/aislamiento & purificación , Mucosa Respiratoria/virología , /aislamiento & purificación , Cadáver , Humanos
8.
Artículo en Inglés | MEDLINE | ID: mdl-33007476

RESUMEN

OBJECTIVES: Investigation whether in depth characterization of virus variant patterns can be used for epidemiological analysis of the first SARS-CoV-2 infection clusters in Hamburg, Germany. METHODS: Metagenomic RNA- and amplicon-sequencing and subsequent variant calling in 25 respiratory samples from SARS-CoV-2 infected patients involved in the earliest infection clusters in Hamburg. RESULTS: Amplikon sequencing and cluster analyses of these SARS-CoV-2 sequences allowed the identification of the first infection cluster and five non-related infection clusters occurring at the beginning of the viral entry of SARS-CoV-2 in the Hamburg metropolitan region. Viral genomics together with epidemiological analyses revealed that the index patient acquired the infection in Northern Italy and transmitted it to two out of 134 contacts. Single nucleotide polymorphisms clearly distinguished the virus variants of the index and other clusters and allowed to track in which sequences worldwide these mutations were first described. Minor variant analyses identified the transmission of intra-host variants in the index cluster and household clusters. CONCLUSIONS: SARS-CoV-2 variant tracing allows the identification of infection clusters and the follow up of infection chains occurring in the population. Furthermore, the follow up of minor viral variants in infection cluster can provide further resolution on transmission events indistinguishable on consensus sequence level.

9.
Lancet Neurol ; 19(11): 919-929, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33031735

RESUMEN

BACKGROUND: Prominent clinical symptoms of COVID-19 include CNS manifestations. However, it is unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, gains access to the CNS and whether it causes neuropathological changes. We investigated the brain tissue of patients who died from COVID-19 for glial responses, inflammatory changes, and the presence of SARS-CoV-2 in the CNS. METHODS: In this post-mortem case series, we investigated the neuropathological features in the brains of patients who died between March 13 and April 24, 2020, in Hamburg, Germany. Inclusion criteria comprised a positive test for SARS-CoV-2 by quantitative RT-PCR (qRT-PCR) and availability of adequate samples. We did a neuropathological workup including histological staining and immunohistochemical staining for activated astrocytes, activated microglia, and cytotoxic T lymphocytes in the olfactory bulb, basal ganglia, brainstem, and cerebellum. Additionally, we investigated the presence and localisation of SARS-CoV-2 by qRT-PCR and by immunohistochemistry in selected patients and brain regions. FINDINGS: 43 patients were included in our study. Patients died in hospitals, nursing homes, or at home, and were aged between 51 years and 94 years (median 76 years [IQR 70-86]). We detected fresh territorial ischaemic lesions in six (14%) patients. 37 (86%) patients had astrogliosis in all assessed regions. Activation of microglia and infiltration by cytotoxic T lymphocytes was most pronounced in the brainstem and cerebellum, and meningeal cytotoxic T lymphocyte infiltration was seen in 34 (79%) patients. SARS-CoV-2 could be detected in the brains of 21 (53%) of 40 examined patients, with SARS-CoV-2 viral proteins found in cranial nerves originating from the lower brainstem and in isolated cells of the brainstem. The presence of SARS-CoV-2 in the CNS was not associated with the severity of neuropathological changes. INTERPRETATION: In general, neuropathological changes in patients with COVID-19 seem to be mild, with pronounced neuroinflammatory changes in the brainstem being the most common finding. There was no evidence for CNS damage directly caused by SARS-CoV-2. The generalisability of these findings needs to be validated in future studies as the number of cases and availability of clinical data were low and no age-matched and sex-matched controls were included. FUNDING: German Research Foundation, Federal State of Hamburg, EU (eRARE), German Center for Infection Research (DZIF).


Asunto(s)
Betacoronavirus/aislamiento & purificación , Encéfalo/patología , Encéfalo/virología , Infecciones por Coronavirus/patología , Neumonía Viral/patología , Anciano , Anciano de 80 o más Años , Autopsia/métodos , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/genética , Femenino , Alemania/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Neuropatología , Pandemias , Neumonía Viral/epidemiología , Neumonía Viral/genética , Transcriptoma/genética
10.
J Clin Virol ; 132: 104650, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33011643

RESUMEN

BACKGROUND: The ongoing SARS-CoV-2 pandemic presents a unique challenge to diagnostic laboratories. There are preliminary studies correlating qRT-PCR results from different materials to clinical outcomes, yet, comparability is limited due to the plethora of different assays used for diagnostics. In this study we evaluate clinical performance and linear range for the SARS-CoV-2 IVD (cobas6800/8800 system, a fully automated sample-to-result platform) in different clinically relevant matrix materials outside official specifications. METHODS: Assay performance was assessed in human plasma, BAL/BL and transport medium following chemical inactivation. For analytical evaluation, respective matrix materials were spiked with SARS-CoV-2 RNA in ten-fold dilution series. The efficacy of chemical inactivation by guanidine hydrochloride solution was confirmed in cell culture infectivity experiments. For correlation, a total of 289 predetermined clinical samples including respiratory swabs, plasma and lower respiratory tract specimens were subjected to the SARS-CoV-2 IVD test and results were compared. RESULTS: The SARS-CoV-2 IVD showed excellent linearity over four to six log steps depending on matrix material. Chemical inactivation resulted in a reduction in plaque forming units of at least 3.5 log steps, while having no significant impact on assay performance. Inter-run consistency from three different testing sites demonstrated excellent comparability of RT-PCR results (maximum deviation was 1.53 CT). Clinical evaluation for respiratory swabs showed very good agreement with the comparator assay (Positive agreement 95.7 %, negative agreement 98.9 %). CONCLUSION: The SARS-CoV-2 IVD test for the cobas6800/8800 systems offers excellent linear range and inter-run consistency for quantification of SARS-CoV-2 RNA in different matrices outside official specifications.

11.
J Clin Virol ; 130: 104549, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32763809

RESUMEN

BACKGROUND: The global market for SARS-CoV-2-immunoassays is becoming ever more crowded with antibody-tests of various formats, targets and technologies, careful evaluation is crucial for understanding the implications of individual test results. Here, we evaluate the clinical performance of five automated immunoassays on a set of clinical samples. METHODS: Serum/plasma samples of 75 confirmed COVID-19 patients and 320 pre-pandemic serum samples of healthy blood donors were subjected to two IgG and three total antibody SARS-CoV-2-immunoassays. All test setups were automated workflows. RESULTS: Positivity of assays (onset of symptoms > 10 days) ranged between 68.4 % and 81.6 % (Diasorin 68.4 %, Euroimmun 70.3 %, Siemens 73.7 %, Roche 79.0 % and Wantai 81.6 %). All examined assays demonstrated high specificity of >99 % (Euroimmun, Diasorin: 99.1 %, Wantai: 99.4 %) but only two reached levels above 99.5 % (Roche: 99.7 %, Siemens 100 %). Interestingly, there was no overlap in false positive results between the assays. The strongest correlation of quantitative results was observed between the Diasorin and Euroimmun IgG tests (r2 = 0.76). Overall, we observed no difference in the distribution of test results between female and male patients (p-values: 0.18-0.87). A significant difference between severely versus critically ill patients was demonstrated for the Euroimmun, Diasorin, Wantai and Siemens assays (p-values:0.041). CONCLUSION: All assays showed good clinical performance. Our data confirm that orthogonal test strategies as recommended by the CDC can enhance clinical specificity. However, the suboptimal rates of test positivity found at time of hospitalization in this cohort underline the importance of molecular diagnostics to rule out/confirm active infection with SARS-CoV-2.


Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Pruebas Serológicas/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Automatización de Laboratorios , Betacoronavirus , Técnicas de Laboratorio Clínico , Estudios de Cohortes , Infecciones por Coronavirus/inmunología , Reacciones Falso Positivas , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/inmunología , Sensibilidad y Especificidad , Adulto Joven
13.
Microbiol Resour Announc ; 9(23)2020 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-32499358

RESUMEN

Here, we describe the complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab sample from a female patient with COVID-19 who was infected in Hamburg, northern Germany.

14.
Dtsch Med Wochenschr ; 145(17): e96-e100, 2020 08.
Artículo en Alemán | MEDLINE | ID: mdl-32572869

RESUMEN

BACKGROUND: In children, the infection with SARS-CoV-2, the cause of COVID-19, tends to be clinically inapparent more often or less severe than in adults. The spread of this infection from children poses a danger to vulnerable adults. Therefore, child care institutions and schools currently are widely closed. METHODS: Since the status of infection tends to be congruent in mothers and their children, we tested 401 mothers of children between 1 and 10 years in the city of Rostock (State of Mecklenburg-Westpomerania, northeast of Germany), for the presence of RNA of SARS-CoV-2 in throat swabs, and of antibodies against SARS-CoV-2 in serum. RESULTS: In none of the mothers tested, RNA of this virus was detected in the throat swab. In the ELISA test, IgG antibodies were positive in one serum sample, IgA antibodies were positive in 11, and borderline in 3 serum samples. All 401 sera were negative in the indirect immunofluorescence test (IIFT) with FITC-labeled IgG, IgA, und IgM antibodies. CONCLUSION: At the time of this study, neither SARS-CoV-2 RNA, nor specific antibodies against SARS-CoV-2 were detectable in the mothers tested in Rostock.


Asunto(s)
Anticuerpos Antivirales/sangre , Betacoronavirus/inmunología , Infecciones por Coronavirus/epidemiología , Neumonía Viral/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Betacoronavirus/genética , Betacoronavirus/aislamiento & purificación , Niño , Preescolar , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Alemania/epidemiología , Humanos , Lactante , Masculino , Tamizaje Masivo/métodos , Persona de Mediana Edad , Orofaringe/virología , Pandemias , Neumonía Viral/diagnóstico , Neumonía Viral/inmunología , Reacción en Cadena de la Polimerasa , Prevalencia , ARN Viral/análisis , Adulto Joven
16.
Ann Intern Med ; 173(4): 268-277, 2020 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-32374815

RESUMEN

BACKGROUND: The new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 210 000 deaths worldwide. However, little is known about the causes of death and the virus's pathologic features. OBJECTIVE: To validate and compare clinical findings with data from medical autopsy, virtual autopsy, and virologic tests. DESIGN: Prospective cohort study. SETTING: Autopsies performed at a single academic medical center, as mandated by the German federal state of Hamburg for patients dying with a polymerase chain reaction-confirmed diagnosis of COVID-19. PATIENTS: The first 12 consecutive COVID-19-positive deaths. MEASUREMENTS: Complete autopsy, including postmortem computed tomography and histopathologic and virologic analysis, was performed. Clinical data and medical course were evaluated. RESULTS: Median patient age was 73 years (range, 52 to 87 years), 75% of patients were male, and death occurred in the hospital (n = 10) or outpatient sector (n = 2). Coronary heart disease and asthma or chronic obstructive pulmonary disease were the most common comorbid conditions (50% and 25%, respectively). Autopsy revealed deep venous thrombosis in 7 of 12 patients (58%) in whom venous thromboembolism was not suspected before death; pulmonary embolism was the direct cause of death in 4 patients. Postmortem computed tomography revealed reticular infiltration of the lungs with severe bilateral, dense consolidation, whereas histomorphologically diffuse alveolar damage was seen in 8 patients. In all patients, SARS-CoV-2 RNA was detected in the lung at high concentrations; viremia in 6 of 10 and 5 of 12 patients demonstrated high viral RNA titers in the liver, kidney, or heart. LIMITATION: Limited sample size. CONCLUSION: The high incidence of thromboembolic events suggests an important role of COVID-19-induced coagulopathy. Further studies are needed to investigate the molecular mechanism and overall clinical incidence of COVID-19-related death, as well as possible therapeutic interventions to reduce it. PRIMARY FUNDING SOURCE: University Medical Center Hamburg-Eppendorf.


Asunto(s)
Autopsia/métodos , Infecciones por Coronavirus/mortalidad , Neumonía Viral/mortalidad , Embolia Pulmonar/mortalidad , Tromboembolia Venosa/mortalidad , Anciano , Anciano de 80 o más Años , Betacoronavirus , Causas de Muerte , Femenino , Alemania/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Estudios Prospectivos , Tomografía Computarizada por Rayos X
17.
J Clin Virol ; 128: 104390, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32388471

RESUMEN

BACKGROUND: The ongoing SARS-CoV-2 pandemic presents a unique challenge for diagnostic laboratories around the world. Automation of workflows in molecular diagnostics is instrumental for coping with the large number of tests ordered by clinicians, as well as providing fast-tracked rapid testing for highly urgent cases. In this study we evaluated a SARS-CoV-2 LDT for the NeuMoDx 96 system, a fully automated device performing extraction and real-time PCR. METHODS: A publicly available SARS-CoV-2 RT-PCR assay was adapted for the automated system. Analytical performance was evaluated using in-vitro transcribed RNA and clinical performance was compared to the cobas 6800-based reference assay within the lab. RESULTS: The Envelope (E) Gene-LDT displayed good analytical performance with an LoD of 95.55 cp/mL and no false positives during evaluation of cross-reactivity. A total of 176 patient samples were tested with both the E-Gene-LDT and the reference assay. Positive and negative agreement were 100 % and 99.2 % respectively. Invalid-rate was 6.3 %. CONCLUSION: The E-Gene-LDT showed analytical and clinical performance comparable to the cobas6800-based reference assay. Due to its random-access workflow concept and rapid time-to-result of about 80 min, the system is very well suited for providing fast-tracked SARS-CoV-2 diagnostics for urgent clinical samples in the hospital setting.


Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Pandemias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Betacoronavirus , Técnicas de Laboratorio Clínico/instrumentación , Infecciones por Coronavirus/diagnóstico , Hospitales , Humanos , Neumonía Viral , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Factores de Tiempo , Flujo de Trabajo
18.
Euro Surveill ; 25(9)2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32156329

RESUMEN

Facing the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), high-volume respiratory testing is demanded in laboratories worldwide. We evaluated the performance of a molecular assay for the detection of SARS-CoV-2 on a high-throughput platform, the cobas 6800, using the 'open channel' for integration of a laboratory-developed assay. We observed good analytical performance in clinical specimens. The fully automated workflow enables high-throughput testing with minimal hands-on time, while offering fast and reliable results.


Asunto(s)
Técnicas de Laboratorio Clínico , Coronavirus , Virus del SRAS , Síndrome Respiratorio Agudo Grave , Betacoronavirus , Infecciones por Coronavirus/diagnóstico , Humanos , Pandemias , Neumonía Viral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad
19.
J Microbiol Methods ; 172: 105882, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32119956

RESUMEN

BACKGROUND: The cobas® omni Utility Channel enables users to integrate lab-developed tests (LDTs) on the cobas® 6800 System to perform molecular diagnostics with high-throughput capacity and full automation. At present, there are no CE- or FDA-approved tests for stool pathogens on this system. To assess the performance of stool as a matrix, we evaluated the analytical and clinical performance of an LDT for detection of Clostridioides difficile (C. difficile) toxin B using the Utility Channel (C.diff_UTC). METHODS: A 10% stool suspension prepared from liquid stool samples diluted in phosphate buffered saline was used for analysis. Limit of detection (LoD) was determined in six dilutions with 126 replicates/dilution. Clinical evaluation was performed using 514 predetermined patient stool samples from two study sites in Germany. The C.diff_UTC was compared with LC 480 amplification and an LDT or the R-BioPharm C. difficile assay. Discrepant results were further analyzed using the GeneXpert C. difficile assay. RESULTS: Limit of detection was 23.48 cfu/mL (95% Confidence Interval [CI]: 19.14-31.01) with inter-run variation of <2 cycle thresholds at 3 × and 10 × LoD. No cross-reactivity was observed with a panel of fecal organisms and pathogens. Bioinformatic analysis showed coverage of the major C. difficile toxinotypes by the primer/probe set. Clinical evaluation revealed sensitivity of 96.7% (95% CI: 88.7-99.6) and specificity of 99.3% (95% CI: 98.0-99.9) compared with the reference method; inhibition rate was 3.5% (18/514). CONCLUSION: Using a predesigned primer/probe set, the C.diff_UTC assay features analytical performance and clinical sensitivity and specificity comparable to currently available nucleic acid amplification tests (NAATs) and is suitable for high-throughput testing. This was a proof-of-concept study, indicating the cobas Utility Channel could likely be adapted for other clinically relevant stool pathogens in outbreak scenarios.

20.
BMC Infect Dis ; 20(1): 170, 2020 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-32087681

RESUMEN

BACKGROUND: Infectious meningitis is a serious disease and patient outcome relies on fast and reliable diagnostics. A syndromic panel testing approach like the FilmArray ME can accelerate diagnosis and therefore decrease the time to pathogen specific therapy. Yet, its clinical utility is controversial, mainly because of a remaining uncertainty in correct interpretation of results, limited data on its performance on clinical specimens and its relatively high costs. The aim of this study was to analyze clinical performance of the assay in a real life setting at a tertiary university hospital using a pragmatic and simple sample selection strategy to reduce the overall cost burden. METHODS: Over a period of 18 months we received 4623 CSF samples (2338 hospitalizations, 1601 individuals). FilmArray ME analysis was restricted to CSF-samples with a high pretest probability of infectious meningitis, e.g. positive Gram-stain, samples in which leukocytes and/or bacteria were evident or urgent suspicion of infection was communicated by clinicians. N = 171 samples matched to our risk criteria and were subjected to FilmArray ME analysis. Those samples were also analyzed by reference methods: culture only (n = 45), PCR only (n = 20) or both methods (n = 106). RESULTS: 56/171 (32.75%) were FilmArray ME positive. Bacterial pathogens were detected in 30/56 (53.57%), viral pathogens were detected in 27/56 (48.21%) and yeast DNA was detected in 1/56 (1.79%) of positive samples. Double detection occurred in 2/56 samples. In 52/56 (92.86%) FilmArray ME positive samples, results could be confirmed by the reference assays (sensitivity = 96.30%, specificity =96.58%). CONCLUSION: The FilmArray ME assay is a fast and reliable diagnostic tool for the management of infectious meningitis and can easily be implemented in routine diagnostic workflows. However, correlation of test results and underlying clinical symptoms requires experienced users and the awareness of potentially false negative or false positive results. Moreover, considering the need for antimicrobial susceptibility testing, the use of molecular tests as a stand-alone diagnostic cannot be recommended.


Asunto(s)
Pruebas Diagnósticas de Rutina/métodos , Encefalitis/diagnóstico , Meningitis/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Coloración y Etiquetado/métodos , Pruebas Diagnósticas de Rutina/economía , Encefalitis/líquido cefalorraquídeo , Encefalitis/microbiología , Encefalitis/virología , Violeta de Genciana , Alemania , Hospitales Universitarios , Humanos , Laboratorios , Meningitis/líquido cefalorraquídeo , Meningitis/microbiología , Meningitis/virología , Técnicas de Diagnóstico Molecular/economía , Reacción en Cadena de la Polimerasa Multiplex/economía , Fenazinas , Estudios Prospectivos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Coloración y Etiquetado/economía , Centros de Atención Terciaria
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