Outcomes from a national screening program for Ukrainian refugees at risk of drug resistant tuberculosis in Wales.

High rates of drug-resistant tuberculosis in Ukraine suggest screening is necessary to mitigate public health hazards for host populations. A pathway was implemented in Wales and data prospectively collected Between 8 April and 21 December 2022. Of 5425 Ukrainian arrivals, notifications were received by TB teams on 2395 (44%) of whom 1955 (82%) were screened. The refugees were young (median age 30, IQR 14-41), and predominantly female (66.1%). Interferon- gamma release assay (IGRA) tests were positive in 112 (6.5%). One Case of active tuberculosis was identified (0.05%). Our data supports European guidelines that routine screening of this population is not recommended, but we remain uncertain as to the risks of this population going forwards.

Burden of nosocomial COVID-19 in Wales: results from a multicentre retrospective observational study of 2508 hospitalised adults.

The burden of nosocomial SARS-CoV-2 infection remains poorly defined. We report on the outcomes of 2508 adults with molecularly-confirmed SARS-CoV-2 admitted across 18 major hospitals, representing over 60% of those hospitalised across Wales between 1 March and 1 July 2020. Inpatient mortality for nosocomial infection ranged from 38% to 42%, consistently higher than participants with community-acquired infection (31%-35%) across a range of case definitions. Those with hospital-acquired infection were older and frailer than those infected within the community. Nosocomial diagnosis occurred a median of 30 days following admission (IQR 21-63), suggesting a window for prophylactic or postexposure interventions, alongside enhanced infection control measures.

COVID-19 managed on respiratory wards and intensive care units: Results from the national COVID-19 outcome report in Wales from March 2020 to December 2021.

BACKGROUND: A COVID-19 hospital guideline was implemented across all 18 acute hospitals in Wales in March 2020, promoting ward management of COVID pneumonitis and data collected across the first 3 Waves of the pandemic (Wave 1 March 1st 2020 to November 1st 2020, Wave 2 November 2st 2020 to February 21st 2021 and Wave 3 June 1st 2021 to December 14th 2021). The aim of this paper is to compare outcomes for patients by admission setting and type of ventilatory support given, with a particular focus on CPAP therapy. METHODS: This is a retrospective observational study of those aged over 18 admitted to hospital with community acquired COVID-19 between March 2020 and December 2021. The outcome of interest was in-hospital mortality. Univariate logistic regression models were used to compare crude outcomes across the waves. Multivariable logistic regression models were used to assess outcomes by different settings and treatments after adjusting for Wave, age, sex, co-morbidity and deprivation. RESULTS: Of the 7,803 records collected, 5,887 (75.4%) met the inclusion criteria. Analysis of those cases identified statistically significant outcome improvements across the waves for all patients combined (Waves 1 to 3: 31.5% to 18.8%, p<0.01), all ward patients (28.9% to 17.7%, p<0.01), and all ICU patients (44.3% to 32.2%, p = 0.03). Sub group analyses identified outcome improvements in ward patients without any oxygen therapy (Waves 1 to 3: 22.2% to 12.7%, p<0.01), with oxygen therapy only (34.0% to 12.9%, p<0.01) and with CPAP only (63.5% to 39.2%, p<0.01). The outcome improvements for ICU patients receiving CPAP only (35.7% to 24.6%, p = 0.31) or invasive ventilation (61.6% to 54.6%, p = 0.43) were not statistically significant though the numbers being admitted to ICU were small. The logistic regression models identified important age and comorbidity effects on outcomes. The multivariable model that took these into account suggested no statistically significantly greater risk of death for those receiving CPAP on the ward compared to those receiving CPAP in ICU (OR 0.89, 95% CI: 0.49 to 1.60). CONCLUSIONS: There were successive reductions in mortality in inpatients over the three Waves reflecting new treatments and better management of complications. Mortality for those requiring CPAP was similar in respiratory wards and ICUs after adjusting for differences in their respective patient populations.

A Systematic Review and Meta-Analysis of Inpatient Mortality Associated With Nosocomial and Community COVID-19 Exposes the Vulnerability of Immunosuppressed Adults.

Background: Little is known about the mortality of hospital-acquired (nosocomial) COVID-19 infection globally. We investigated the risk of mortality and critical care admission in hospitalised adults with nosocomial COVID-19, relative to adults requiring hospitalisation due to community-acquired infection. Methods: We systematically reviewed the peer-reviewed and pre-print literature from 1/1/2020 to 9/2/2021 without language restriction for studies reporting outcomes of nosocomial and community-acquired COVID-19. We performed a random effects meta-analysis (MA) to estimate the 1) relative risk of death and 2) critical care admission, stratifying studies by patient cohort characteristics and nosocomial case definition. Results: 21 studies were included in the primary MA, describing 8,251 admissions across 8 countries during the first wave, comprising 1513 probable or definite nosocomial COVID-19, and 6738 community-acquired cases. Across all studies, the risk of mortality was 1.3 times greater in patients with nosocomial infection, compared to community-acquired (95% CI: 1.005 to 1.683). Rates of critical care admission were similar between groups (Relative Risk, RR=0.74, 95% CI: 0.50 to 1.08). Immunosuppressed patients diagnosed with nosocomial COVID-19 were twice as likely to die in hospital as those admitted with community-acquired infection (RR=2.14, 95% CI: 1.76 to 2.61). Conclusions: Adults who acquire SARS-CoV-2 whilst already hospitalised are at greater risk of mortality compared to patients admitted following community-acquired infection; this finding is largely driven by a substantially increased risk of death in individuals with malignancy or who had undergone transplantation. These findings inform public health and infection control policy and argue for individualised clinical interventions to combat the threat of nosocomial COVID-19, particularly for immunosuppressed groups. Systematic Review Registration: PROSPERO CRD42021249023.

Detection of mycobacterial antigen responses in lung but not blood in HIV-tuberculosis co-infected subjects.

Forty-seven HIV-infected adults had broncho-alveolar lavage stimulated with purified protein derivate of Mycobacterium tuberculosis. Eighteen of 19 (95%) with tuberculosis co-infection had interferon-gamma synthetic CD4 lymphocyte responses > 1% versus three of 28 (11%) without (P < 0.0001). Lung response was unrelated to blood CD4 cell count. BAL HIV tuberculosis responses were similar in 25 HIV-uninfected tuberculosis patients. Responses in matched blood samples were often undetectable. Therefore, immunological tuberculosis assays seem less affected by HIV co-infection when lung-based.

Optimal gating strategies for determining bronchoalveolar lavage CD4/CD8 lymphocyte ratios by flow cytometry.

Alterations in bronchoalveolar lavage (BAL) CD4/CD8 T cell subset ratios have been demonstrated in a variety of different respiratory disorders and the measurement of these changes may be diagnostically helpful. Flow cytometry (FCM) is a precise technology that offers many advantages over conventional cytospin techniques to determine T cell subset ratios in tissue fluids such as BAL. However, the optimum gating strategies for evaluating these parameters by FCM have not been evaluated. Here, the CD4/CD8 ratios in 33 BAL samples were compared using three different methods by FCM with two different flow cytometers. Bland Altman analysis demonstrated clinically insignificant differences between two simplified staining and gating strategies and a more complex "gold standard" method. These findings confirm the precision of FCM for BAL T cell subset ratio analysis and suggest that the optimal gating strategy may be a simple panel using only CD45, CD4 and CD8.

Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.

BACKGROUND: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. METHODS: Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. RESULTS: Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. CONCLUSIONS: The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells.

The role of flow cytometry in the interferon-gamma-based diagnosis of active tuberculosis and its coinfection with HIV-1--A technically oriented review.

TB remains uncontrolled. In resource-rich countries, only approximately 60% of diagnoses are confirmed by culture. The number is lower in resource-poor environments. Huge scope therefore exists for alternative diagnostic strategies. Counting antigen-specific lymphocytes by virtue of cytokine production following 8-16 h stimulation with tuberculosis antigens is currently the strategy of choice. Several methods exist, including ELISA, ELISpots, and flow cytometry. Although it is clear that blood samples stimulated by ESAT-6 and CFP-10 antigens discriminate between TB infection and BCG vaccination, it is flow-cytometry that seems to be able to distinguish active TB disease from mere TB exposure. Of the various flow-protocols including four-color tests (CD45-CD3-CD4-IFNgamma), three-color tests (CD3-CD4-IFNgamma) and two-color tests (CD4-IFNgamma), even the simplest is performing well, provided that the results are expressed as percentage of IFN-gamma+ cells per CD4+ lymphocytes (%IFNgamma/CD4+). Studies using broncho-alveloar lavage (BAL) and Induced-Sputum (ISp) show that TB-specific CD4+IFN-gamma+ T cells accumulate in the lung in pulmonary and extra-pulmonary TB at frequencies >5-20-fold more frequent than in blood. This pulmonary homing is absent following BCG immunization. The use of PPD to stimulate CD4+IFN-gamma+ cells in the lung in active TB leads to >3-12-fold greater responses than seen with CFP-10 or ESAT-6, and any interference from BCG vaccination is absent. This method is unaffected by HIV coinfection, which has always been the problem for other immune-based diagnostics. Further, lung-based samples provide material for rapid tests of both the IFN-gamma assay and bacteriology, and importantly, these tests are amenable for future simplification with automated fluorescence-image cytometers.Another development of the multiparameter analytical power of flow-cytometry is to use markers for "lung-seeking" populations of CD4+ T cells in blood, obviating lung sampling. In active TB, but not in BCG vaccinees, TB-specific memory CD4+ T cells can be found in blood that are dominantly CD27-negative and probably lung seeking and can be diagnostically useful.

Increased proportions of activated and proliferating memory CD8+ T lymphocytes in both blood and lung are associated with blood HIV viral load.

One mechanism of HIV pathogenesis has been proposed to be the activation of T lymphocytes, resulting in proliferation and decreased survival of these activated cells. Others have suggested that co-infections may exacerbate this process. These hypotheses were tested by examining the relationship between HIV blood viral load with the proportion of activated and proliferating CD8+ lymphocytes in lung and blood. In the lung these responses were compared in patients with or without respiratory pathogens. Thirty-five HIV-positive patients and five controls underwent bronchoalveolar lavage (BAL). BAL and blood samples were fixed and permeabilized and the proportions of memory CD8+ lymphocytes that expressed the activation marker CD38 and the cell cycling marker Ki67 were measured by flow cytometry. CD38bright CD8+ lymphocytes from both sites increased with increasing blood viral load. In BAL there was no significant difference between the CD38 activation status in those with respiratory pathogens compared with those without. More CD38bright CD8+ lymphocytes expressed Ki67 when compared with the CD38dim lymphocytes. These findings provide evidence that HIV is the primary factor in stimulating CD8+ cell activation.

Determination of bronchoalveolar lavage leukocyte populations by flow cytometry in patients investigated for respiratory disease.

BACKGROUND: Characteristic changes in the proportions of leukocyte populations in bronchoalveolar lavage (BAL) reflect different disease states in the lung. The standard method for examination of BAL leukocytes is by microscopy of cytospin preparations. This method may not be the optimum technique due to difficulties in distinguishing cell types morphologically and due to the low number of cells routinely counted. We hypothesized that flow cytometry (FCM) may be a more precise tool for investigating BAL. METHODS: 100 BALs were performed on 92 patients. All samples were stained using the pan-leukocyte marker (CD45) in combination with a granulocyte marker (CD15) and a cell viability marker (7-aminoactinomycin D). Selected samples were also stained with an eosinophil marker (CD23). These samples were run on an FCM and the results compared with leukocyte differentials obtained by light microscopy of parallel cytospin preparations. RESULTS: Close correlations between the two methods were demonstrated for the enumeration of all leukocyte subsets, but the coefficient of variation was considerably lower by FCM than by cytospin. CONCLUSIONS: These findings, combined with the speed of FCM and the ability to perform simple lymphocyte phenotyping, argue in favor of this becoming the method of choice for investigating BAL.

Purified protein derivative-activated type 1 cytokine-producing CD4+ T lymphocytes in the lung: a characteristic feature of active pulmonary and nonpulmonary tuberculosis.

Because tuberculosis (TB) is primarily a pulmonary disease, we examined the cytokine responses of CD4(+) T lymphocytes in bronchoalveolar lavage (BAL) fluid after incubation with purified protein derivative (PPD) in human immunodeficiency virus-negative patients with TB and control subjects with nontuberculous respiratory disease. Parallel blood and BAL fluid samples from each subject were incubated with or without PPD, and the proportions of CD4(+) T lymphocytes producing interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha were measured by flow cytometry. The proportions of PPD-activated IFN-gamma- and TNF-alpha-producing CD4(+) cells were low among control subjects (median, 0.33% and 0.78%, respectively). By contrast, among patients with TB, strong IFN-gamma and TNF-alpha responses were demonstrated (median, 24.0% and 32.4%, respectively), regardless of whether the TB was pulmonary or nonpulmonary. Measurement of type 1 cytokine production by CD4(+) T lymphocytes in response to PPD in BAL fluid is a promising new diagnostic test for active TB in immunocompetent individuals.