Aspergillus sensitisation detection using point-of-care lateral flow assay in moderate to severe asthma.

Allergic fungal airway diseases are associated with asthma exacerbations and poor control. However, the early identification of allergic Aspergillus airway diseases can be challenging, especially in resource-poor countries. We aimed to evaluate the clinical utility of the point-of-care Aspergillus IgG-IgM lateral flow assay in diagnosing Aspergillus airway diseases in patients with moderate-severe asthma. Patients with moderate-severe asthma, severe asthma with fungal sensitisation (SAFS) and allergic bronchopulmonary aspergillosis (ABPA) were recruited. Clinical information was extracted from clinical records. Blood samples were collected for serological tests. Serum samples were evaluated using Aspergillus immunochromatographic test (ICT). A total of 65 patients were recruited into the study, of whom 23.1% had clinical diagnosis of ABPA, 18.5% had SAFS and 58.5% had moderate-to-severe asthma who did not fit ABPA or SAFS criteria. The ICT test gave a sensitivity of 69 [95% confidence interval: 51-88]% and a specificity of 77 [60-88]% in predicting a positive Aspergillus IgG test. The sensitivity and specificity for a positive Aspergillus IgE were 77 [59-88]% and 86 [71-94]%, respectively. The majority (sensitivity: 87 [62-96]%) of patients with ABPA had positive ICT results, with a specificity of 70%. The negative predictive value was high (95 [82-99]%) with a low negative likelihood ratio (< 0.2), making it potentially useful in ruling out ABPA. The ICT assay may be valuable in ruling out ABPA in resource-limited countries where serological investigations are less feasible. The ICT assay may be particularly useful in ruling out ABPA and warrants further validation.

Allergic Aspergillus diseases can make patients with asthma more unwell, but this is difficult to diagnose, especially in developing countries where tests are not widely available. We have found that a cheap, easy-to-use and fast test may be useful in diagnosing allergic Aspergillus diseases.

Direct Whole-Genome Sequencing of Sputum Accurately Identifies Drug-Resistant Mycobacterium tuberculosis Faster than MGIT Culture Sequencing.

The current methods available to diagnose antimicrobial-resistant Mycobacterium tuberculosis infections require a positive culture or only test a limited number of resistance-associated mutations. A rapid accurate identification of antimicrobial resistance enables the prompt initiation of effective treatment. Here, we determine the utility of whole-genome sequencing (WGS) of M. tuberculosis directly from routinely obtained diagnostic sputum samples to provide a comprehensive resistance profile compared to that from mycobacterial growth indicator tube (MGIT) WGS. We sequenced M. tuberculosis from 43 sputum samples by targeted DNA enrichment using the Agilent SureSelectXT kit, and 43 MGIT positive samples from each participant. Thirty two (74%) sputum samples and 43 (100%) MGIT samples generated whole genomes. The times to antimicrobial resistance profiles and concordance were compared with Xpert MTB/RIF and phenotypic resistance testing from cultures of the same samples. Antibiotic susceptibility could be predicted from WGS of sputum within 5 days of sample receipt and up to 24 days earlier than WGS from MGIT culture and up to 31 days earlier than phenotypic testing. Direct sputum results could be reduced to 3 days with faster hybridization and if only regions encoding drug resistance are sequenced. We show that direct sputum sequencing has the potential to provide comprehensive resistance detection significantly faster than MGIT whole-genome sequencing or phenotypic testing of resistance from cultures in a clinical setting. This improved turnaround time enables prompt appropriate treatment with associated patient and health service benefits. Improvements in sample preparation are necessary to ensure comparable sensitivities and complete resistance profile predictions in all cases.

Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections.

Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens.

Characterization of the interaction between human respiratory syncytial virus and the cell cycle in continuous cell culture and primary human airway epithelial cells.

Viruses can modify conditions inside cells to make them more favorable for replication and progeny virus production. One way of doing this is through manipulation of the cell cycle, a process that describes the ordered growth and division of cells. Analysis of model cell lines, such as A549 cells and primary airway epithelial cells, infected with human respiratory syncytial virus (HRSV) has shown alteration of the cell cycle during infection, although the signaling events were not clearly understood. In this study, targeted transcriptomic analysis of HRSV-infected primary airway epithelial cells revealed alterations in the abundances of many mRNAs encoding cell cycle-regulatory molecules, including decreases in the D-type cyclins and corresponding cyclin-dependent kinases (CDK4 and CDK6 [CDK4/6]). These alterations were reflected in changes in protein abundance and/or relocalization in HRSV-infected cells; taken together, they were predicted to result in G(0)/G(1) phase arrest. In contrast, there was no change in the abundances of D-type cyclins in A549 cells infected with HRSV. However, the abundance of the G(1)/S phase progression inhibitor p21(WAF1/CIP1) was increased over that in mock-treated cells, and this, again, was predicted to result in G(0)/G(1) phase arrest. The G(0)/G(1) phase arrest in both HRSV-infected primary cells and A549 cells was confirmed using dual-label flow cytometry that accurately measured the different stages of the cell cycle. Comparison of progeny virus production in primary and A549 cells enriched in G(0)/G(1) using a specific CDK4/6 kinase inhibitor with asynchronously replicating cells indicated that this phase of the cell cycle was more efficient for virus production.

Prospective observational study of SARS-CoV-2 infection, transmission and immunity in a cohort of households in Liverpool City Region, UK (COVID-LIV): a study protocol.

INTRODUCTION: The emergence and rapid spread of COVID-19 have caused widespread and catastrophic public health and economic impact, requiring governments to restrict societal activity to reduce the spread of the disease. The role of household transmission in the population spread of SARS-CoV-2, and of host immunity in limiting transmission, is poorly understood. This paper describes a protocol for a prospective observational study of a cohort of households in Liverpool City Region, UK, which addresses the transmission of SARS-CoV-2 between household members and how immunological response to the infection changes over time. METHODS AND ANALYSIS: Households in the Liverpool City Region, in which members have not previously tested positive for SARS-CoV-2 with a nucleic acid amplification test, are followed up for an initial period of 12 weeks. Participants are asked to provide weekly self-throat and nasal swabs and record their activity and presence of symptoms. Incidence of infection and household secondary attack rates of COVID-19 are measured. Transmission of SARS-CoV-2 will be investigated against a range of demographic and behavioural variables. Blood and faecal samples are collected at several time points to evaluate immune responses to SARS-CoV-2 infection and prevalence and risk factors for faecal shedding of SARS-CoV-2, respectively. ETHICS AND DISSEMINATION: The study has received approval from the National Health Service Research Ethics Committee; REC Reference: 20/HRA/2297, IRAS Number: 283 464. Results will be disseminated through scientific conferences and peer-reviewed open access publications. A report of the findings will also be shared with participants. The study will quantify the scale and determinants of household transmission of SARS-CoV-2. Additionally, immunological responses before and during the different stages of infection will be analysed, adding to the understanding of the range of immunological response by infection severity.

Detection of carbapenemases, AmpC and ESBL genes in Acinetobacter isolates from ICUs by DNA microarray.

The accumulation of multiple inherent and acquired resistance mechanisms in Acinetobacter spp. results in emergence of "pandrug resistant" strains which is one of the major concerns in healthcare sectors worldwide. Surveillance of the carbapenemase/ extended-spectrum ß-lactamases (ESBLs) genes in A. baumannii by phenotypic methods is challenging especially in developing countries, like Pakistan. In this context, a novel microarray (CT 103XL Check-MDR) assay was used for simultaneous detection of genes encoding clinically important carbapenemases and ESBLs. The results were compared with the phenotypic methods including MHT, Rapidec Carba NP, EDTA+DDST and Rosco (KPC/MBL). The results of the microarray were also confirmed by PCR. All of the strains of A. baumannii (47) were resistant to imipenem and meropenem. Microarray and PCR results showed presence of OXA-23 in all the isolates of A. baumannii while 36.17% also harbored PER. Rosco kit test showed 100% sensitivity to detect carbapenemases but exhibited low specificity to classify them. Rapidec Carba NP test has 100% sensitivity and specificity to detect the carbapenemases when compared with microarray. Sensitivity and specificity of microarray assay were 100% for bla-genes in comparison to PCR. This reveals that Check-MDR CT103 XL assay is an accurate method for the identification of ESBLs and carbapenemase genes in A. baumannii in comparison to the other methods.