scholarly journals COVID-19-Associated Invasive Aspergillosis: Data from the UK National Mycology Reference Laboratory

2020 ◽  
Vol 59 (1) ◽  
pp. e02136-20 ◽  
Author(s):  
Andrew M. Borman ◽  
Michael D. Palmer ◽  
Mark Fraser ◽  
Zoe Patterson ◽  
Ciara Mann ◽  
...  
Keyword(s):  
Critically Ill ◽  
Incidence Rates ◽  
Clinical Samples ◽  
Reference Laboratory ◽  
Serum Samples ◽  
Pulmonary Aspergillosis ◽  
Content Type ◽  
Specific Pcr ◽  
The Uk ◽  
Respiratory Specimens

ABSTRACTCOVID-19-associated pulmonary aspergillosis (CAPA) was recently reported as a potential infective complication affecting critically ill patients with acute respiratory distress syndrome following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, with incidence rates varying from 8 to 33% depending on the study. However, definitive diagnosis of CAPA is challenging. Standardized diagnostic algorithms and definitions are lacking, clinicians are reticent to perform aerosol-generating bronchoalveolar lavages for galactomannan testing and microscopic and cultural examination, and questions surround the diagnostic sensitivity of different serum biomarkers. Between 11 March and 14 July 2020, the UK National Mycology Reference Laboratory received 1,267 serum and respiratory samples from 719 critically ill UK patients with COVID-19 and suspected pulmonary aspergillosis. The laboratory also received 46 isolates of Aspergillus fumigatus from COVID-19 patients (including three that exhibited environmental triazole resistance). Diagnostic tests performed included 1,000 (1-3)-β-d-glucan and 516 galactomannan tests on serum samples. The results of this extensive testing are presented here. For a subset of 61 patients, respiratory specimens (bronchoalveolar lavage specimens, tracheal aspirates, and sputum samples) in addition to serum samples were submitted and subjected to galactomannan testing, Aspergillus-specific PCR, and microscopy and culture. The incidence of probable/proven and possible CAPA in this subset of patients was approximately 5% and 15%, respectively. Overall, our results highlight the challenges in biomarker-driven diagnosis of CAPA, especially when only limited clinical samples are available for testing, and the importance of a multimodal diagnostic approach involving regular and repeat testing of both serum and respiratory samples.

scholarly journals Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

2015 ◽  
Vol 53 (4) ◽  
pp. 1137-1143 ◽  
Author(s):  
Antonina A. Votintseva ◽  
Louise J. Pankhurst ◽  
Luke W. Anson ◽  
Marcus R. Morgan ◽  
Deborah Gascoyne-Binzi ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Solid Phase ◽  
Clinical Samples ◽  
Reference Laboratory ◽  
Whole Genome ◽  
Mycobacterial Species ◽  
Accurate Identification ◽  
Content Type

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) asMycobacterium tuberculosiswere successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

scholarly journals Molecular Diagnosis of Invasive Aspergillosis and Detection of Azole Resistance by a Newly Commercialized PCR Kit

2017 ◽  
Vol 55 (11) ◽  
pp. 3210-3218 ◽  
Author(s):  
Eric Dannaoui ◽  
Frédéric Gabriel ◽  
Manuel Gaboyard ◽  
Gaëlle Lagardere ◽  
Lucile Audebert ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Azole Resistance ◽  
Rrna Gene ◽  
28S Rrna ◽  
Serum Samples ◽  
Content Type ◽  
28S Rrna Gene

ABSTRACTAspergillus fumigatusis the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance inA. fumigatusis worrisome. The aim of this study was to validate the new MycoGENIEA. fumigatusreal-time PCR kit and to evaluate its performance on clinical samples for the detection ofA. fumigatusand its azole resistance. This multiplex assay detects DNA from theA. fumigatusspecies complex by targeting the multicopy 28S rRNA gene and specific TR34and L98H mutations in the single-copy-numbercyp51Agene ofA. fumigatus. The specificity ofcyp51Amutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinicalA. fumigatusisolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for theAspergillus28S rRNA gene and 6 copies for thecyp51Agene harboring the TR34and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR34and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection ofA. fumigatusDNA and azole resistance due to TR34and L98H mutations in clinical samples.

scholarly journals Microsphere-Based IgM and IgG Avidity Assays for Human Parvovirus B19, Human Cytomegalovirus, and Toxoplasma gondii

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Yilin Wang ◽  
Lea Hedman ◽  
Visa Nurmi ◽  
Inga Ziemele ◽  
Maria F. Perdomo ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Human Cytomegalovirus ◽  
Parvovirus B19 ◽  
Clinical Samples ◽  
Human Parvovirus B19 ◽  
Serum Samples ◽  
Content Type ◽  
Reference Methods ◽  
Igg Avidity ◽  
Highly Sensitive

ABSTRACT Human parvovirus B19 (here B19), human cytomegalovirus (HCMV), and Toxoplasma gondii infections during pregnancy can lead to severe complications. While traditional diagnosis of infections is mostly confined to one pathogen at a time, a multiplex array is a feasible alternative to improve diagnostic management and cost-efficiency. In the present study, for these three pathogens, we developed microsphere-based suspension immunoassays (SIAs) in multiplex and monoplex formats for the detection of antimicrobial IgM antibodies as well as corresponding chaotrope-based IgG avidity SIAs. We determined the diagnostic performances of the SIAs versus in-house and commercial reference assays using a panel of 318 serum samples from well-characterized clinical cohorts. All the newly developed assays exhibited excellent performance compared to the corresponding high-quality reference methods. The positive and negative percent agreements of the IgM SIAs in comparison with reference methods were 95 to 100% and 98 to 100%, and those of the IgG avidity SIAs were 92 to 100% and 95 to 100%, respectively. Kappa efficiency values between the SIAs and the corresponding reference assays were 0.91 to 1. Furthermore, with another panel comprising 391 clinical samples from individuals with primary infection by B19, HCMV, or T. gondii, the IgM SIAs were highly sensitive for the detection of acute infections, and the IgG avidity SIAs were highly specific for the separation of primary infections from past immunity. Altogether, the strategy of IgM multiplex screening followed by IgG avidity reflex testing can provide high-throughput and accurate means for the detection and stage determination of B19, HCMV, and T. gondii infections. IMPORTANCE Human parvovirus B19, human cytomegalovirus, and Toxoplasma gondii are ubiquitous pathogens. Their infections are often asymptomatic or mild in the general population yet may be transmitted from mother to fetus during pregnancy. Maternal infections by these pathogens can cause severe complications to the fetus or congenital abnormalities. As a rule, the risk of maternal transmission is critically related to the infection time; hence, it is important to determine when a pregnant woman has acquired the infection. In this study, we developed new diagnostic approaches for the timing of infections by three pathogens. All the new assays appeared to be highly sensitive and specific, providing powerful tools for medical diagnosis.

scholarly journals Prospective Evaluation of a New Aspergillus IgG Enzyme Immunoassay Kit for Diagnosis of Chronic and Allergic Pulmonary Aspergillosis

2016 ◽  
Vol 54 (5) ◽  
pp. 1236-1242 ◽  
Author(s):  
C. Dumollard ◽  
S. Bailly ◽  
S. Perriot ◽  
M. P. Brenier-Pinchart ◽  
C. Saint-Raymond ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Operating Characteristic ◽  
Allergic Bronchopulmonary Aspergillosis ◽  
Roc Curves ◽  
Serum Samples ◽  
Prospective Multicenter Study ◽  
Pulmonary Aspergillosis ◽  
Content Type ◽  
Chronic Pulmonary Aspergillosis ◽  
Commercial Enzyme Immunoassay

Anti-AspergillusIgG antibodies are important biomarkers for the diagnosis of chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA). We compared the performance of a new commercial enzyme immunoassay (EIA) (Bordier Affinity Products) with that of the Bio-Rad and Virion\Serion EIAs. This assay is novel in its association of two recombinant antigens with somatic and metabolic antigens ofAspergillus fumigatus. In a prospective multicenter study, 436 serum samples from 147 patients diagnosed with CPA (136 samples/104 patients) or ABPA (94 samples/43 patients) and from 205 controls (206 samples) were tested. We obtained sensitivities of 97%, 91.7%, and 86.1%, and specificities of 90.3%, 91.3%, and 81.5% for the Bordier, Bio-Rad, and Virion\Serion tests, respectively. The Bordier kit was more sensitive than the Bio-Rad kit (P< 0.01), which was itself more sensitive than the Virion\Serion kit (P= 0.04). The Bordier and Bio-Rad kits had similar specificity (P= 0.8), both higher than that of the Virion\Serion kit (P= 0.02). The area under the receiver operating characteristic (ROC) curves confirmed the superiority of the Bordier kit over the Bio-Rad and the Virion\Serion kits (0.977, 0.951, and 0.897, respectively;P< 0.01 for each comparison). In a subset analysis of 279 serum samples tested with the Bordier and Bio-Rad kits and an in-house immunoprecipitin assay (IPD), the Bordier kit had the highest sensitivity (97.7%), but the IPD tended to be more specific (71.2 and 84.7%, respectively;P= 0.10). The use of recombinant, somatic, and metabolic antigens in a single EIA improved the balance of sensitivity and specificity, resulting in an assay highly suitable for use in the diagnosis of chronic and allergic aspergillosis.

scholarly journals Streptococcus pneumoniae Serotype 1 Burden in the African Meningitis Belt: Exploration of Functionality in Specific Antibodies

2015 ◽  
Vol 22 (4) ◽  
pp. 404-412 ◽  
Author(s):  
S. Blumental ◽  
J. C. Moïsi ◽  
L. Roalfe ◽  
M. Zancolli ◽  
M. Johnson ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Age Groups ◽  
Serum Samples ◽  
Content Type ◽  
The United Kingdom ◽  
Meningitis Belt ◽  
Serotype 1 ◽  
Functional Immunity ◽  
Specific Igg ◽  
The Uk

ABSTRACTStreptococcus pneumoniaeserotype 1 (Sp1) constitutes an important cause of seasonal endemic meningitis in all age groups in the African meningitis belt. Despite a higher meningitis incidence, the Burkinabé population has an Sp1-specific antibody seroprevalence similar to that reported in the United Kingdom (UK). We aimed to establish whether the opsonophagocytic activity (OPA) of pneumococcal IgG naturally present in Burkina Faso differs from that seen in individuals in the UK and to compare the OPAs generated by natural and vaccine-induced immunity. Samples collected from pneumococcal vaccine-naive Burkinabé and UK subjects were matched for age (1 to 39 years) and anti-Sp1 IgG level, analyzed for OPA to 3S. pneumoniaeserotypes (1, 5, and 19A), and compared to postvaccine samples. Furthermore, the Burkinabé samples were assessed for IgG avidity and serotype-specific IgM concentrations. One hundred sixty-nine matched serum samples from both populations were selected. A greater proportion of Burkinabé subjects aged 1 to 19 years had functional Sp1 activity (OPA ≥ 8) compared to UK subjects (12% versus 2%,P< 0.001); however, the proportions were similar among adults (9%). The correlation between Sp1 IgG concentration and OPA was good (P< 0.001), but many individuals had nonfunctional IgG, which was not related to avidity. While the Sp1 IgM concentrations correlated with OPA, not all of the function in serum samples with low IgG could be attributed to IgM. Finally, vaccine-induced Sp1-specific IgG was more functional than equivalent amounts of naturally occurring IgG. In conclusion, despite a substantially higher pneumococcal meningitis incidence, no decreased functional immunity to Sp1 could be evidenced in the Burkinabé population compared to that in the population from the UK. Furthermore, the naturally induced antibodies were less functional than vaccine-induced antibodies.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Computer-supported Detection of M-Components and Evaluation of Immunoglobulins after Capillary Electrophoresis

2001 ◽  
Vol 47 (1) ◽  
pp. 110-117 ◽  
Author(s):  
Magnus Jonsson ◽  
Joyce Carlson ◽  
Jan-Olof Jeppsson ◽  
Per Simonsson
Keyword(s):  
Capillary Electrophoresis ◽  
Decision Support ◽  
Protein Separation ◽  
Serum Proteins ◽  
Cost Effective ◽  
Clinical Samples ◽  
Serum Samples ◽  
Computerized Decision Support ◽  
Cost Effective Method ◽  
Mathematical Algorithms

Abstract Background: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenström macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system. Methods: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the γ- and β-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested. Results: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma. Conclusions: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.

scholarly journals Population (Antibody) Testing for COVID-19—Technical Challenges, Application and Relevance, an English Perspective

Vaccines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 550
Author(s):  
Peter A. C. Maple
Keyword(s):  
National Level ◽  
Herd Immunity ◽  
Population Based ◽  
Biological Properties ◽  
Limited Extent ◽  
Antibody Testing ◽  
Serum Samples ◽  
Oral Fluids ◽  
Antibody Assays ◽  
The Uk

In the UK, population virus or antibody testing using virus swabs, serum samples, blood spots or oral fluids has been performed to a limited extent for several diseases including measles, mumps, rubella and hepatitis and HIV. The collection of population-based infection and immunity data is key to the monitoring of disease prevalence and assessing the effectiveness of interventions such as behavioural modifications and vaccination. In particular, the biological properties of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its interaction with the human host have presented several challenges towards the development of population-based immunity testing. Measuring SARS-CoV-2 immunity requires the development of antibody assays of acceptable sensitivity and specificity which are capable of accurately detecting seroprevalence and differentiating protection from non-protective responses. Now that anti-COVID-19 vaccines are becoming available there is a pressing need to measure vaccine efficacy and the development of herd immunity. The unprecedented impact of the SARS-CoV-2 pandemic in the UK in terms of morbidity, mortality, and economic and social disruption has mobilized a national scientific effort to learn more about this virus. In this article, the challenges of testing for SARS-CoV-2 infection, particularly in relation to population-based immunity testing, will be considered and examples given of relevant national level studies.

scholarly journals Sex differences in the association between major cardiovascular risk factors in midlife and dementia: a cohort study using data from the UK Biobank

BMC Medicine ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jessica Gong ◽  
Katie Harris ◽  
Sanne A. E. Peters ◽  
Mark Woodward
Keyword(s):  
Risk Factors ◽  
Blood Pressure ◽  
Sex Differences ◽  
Cardiovascular Risk ◽  
Cardiovascular Risk Factors ◽  
Sex Difference ◽  
Incidence Rates ◽  
Cox Proportional Hazards ◽  
Uk Biobank ◽  
The Uk

Abstract Background Sex differences in major cardiovascular risk factors for incident (fatal or non-fatal) all-cause dementia were assessed in the UK Biobank. The effects of these risk factors on all-cause dementia were explored by age and socioeconomic status (SES). Methods Cox proportional hazards models were used to estimate hazard ratios (HRs) and women-to-men ratio of HRs (RHR) with 95% confidence intervals (CIs) for systolic blood pressure (SBP) and diastolic blood pressure (DBP), smoking, diabetes, adiposity, stroke, SES and lipids with dementia. Poisson regression was used to estimate the sex-specific incidence rate of dementia for these risk factors. Results 502,226 individuals in midlife (54.4% women, mean age 56.5 years) with no prevalent dementia were included in the analyses. Over 11.8 years (median), 4068 participants (45.9% women) developed dementia. The crude incidence rates were 5.88 [95% CI 5.62–6.16] for women and 8.42 [8.07–8.78] for men, per 10,000 person-years. Sex was associated with the risk of dementia, where the risk was lower in women than men (HR = 0.83 [0.77–0.89]). Current smoking, diabetes, high adiposity, prior stroke and low SES were associated with a greater risk of dementia, similarly in women and men. The relationship between blood pressure (BP) and dementia was U-shaped in men but had a dose-response relationship in women: the HR for SBP per 20 mmHg was 1.08 [1.02–1.13] in women and 0.98 [0.93–1.03] in men. This sex difference was not affected by the use of antihypertensive medication at baseline. The sex difference in the effect of raised BP was consistent for dementia subtypes (vascular dementia and Alzheimer’s disease). Conclusions Several mid-life cardiovascular risk factors were associated with dementia similarly in women and men, but not raised BP. Future bespoke BP-lowering trials are necessary to understand its role in restricting cognitive decline and to clarify any sex difference.

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