Transcriptomics for child and adolescent tuberculosis.

Tuberculosis (TB) in humans is caused by Mycobacterium tuberculosis (Mtb). It is estimated that 70 million children (<15 years) are currently infected with Mtb, with 1.2 million each year progressing to disease. Of these, a quarter die. The risk of progression from Mtb infection to disease and from disease to death is dependent on multiple pathogen and host factors. Age is a central component in all these transitions. The natural history of TB in children and adolescents is different to adults, leading to unique challenges in the development of diagnostics, therapeutics, and vaccines. The quantification of RNA transcripts in specific cells or in the peripheral blood, using high-throughput methods, such as microarray analysis or RNA-Sequencing, can shed light into the host immune response to Mtb during infection and disease, as well as understanding treatment response, disease severity, and vaccination, in a global hypothesis-free manner. Additionally, gene expression profiling can be used for biomarker discovery, to diagnose disease, predict future disease progression and to monitor response to treatment. Here, we review the role of transcriptomics in children and adolescents, focused mainly on work done in blood, to understand disease biology, and to discriminate disease states to assist clinical decision-making. In recent years, studies with a specific pediatric and adolescent focus have identified blood gene expression markers with diagnostic or prognostic potential that meet or exceed the current sensitivity and specificity targets for diagnostic tools. Diagnostic and prognostic gene expression signatures identified through high-throughput methods are currently being translated into diagnostic tests.

Treatment of Multisystem Inflammatory Syndrome in Children.

BACKGROUND: Evidence is urgently needed to support treatment decisions for children with multisystem inflammatory syndrome (MIS-C) associated with severe acute respiratory syndrome coronavirus 2. METHODS: We performed an international observational cohort study of clinical and outcome data regarding suspected MIS-C that had been uploaded by physicians onto a Web-based database. We used inverse-probability weighting and generalized linear models to evaluate intravenous immune globulin (IVIG) as a reference, as compared with IVIG plus glucocorticoids and glucocorticoids alone. There were two primary outcomes: the first was a composite of inotropic support or mechanical ventilation by day 2 or later or death; the second was a reduction in disease severity on an ordinal scale by day 2. Secondary outcomes included treatment escalation and the time until a reduction in organ failure and inflammation. RESULTS: Data were available regarding the course of treatment for 614 children from 32 countries from June 2020 through February 2021; 490 met the World Health Organization criteria for MIS-C. Of the 614 children with suspected MIS-C, 246 received primary treatment with IVIG alone, 208 with IVIG plus glucocorticoids, and 99 with glucocorticoids alone; 22 children received other treatment combinations, including biologic agents, and 39 received no immunomodulatory therapy. Receipt of inotropic or ventilatory support or death occurred in 56 patients who received IVIG plus glucocorticoids (adjusted odds ratio for the comparison with IVIG alone, 0.77; 95% confidence interval [CI], 0.33 to 1.82) and in 17 patients who received glucocorticoids alone (adjusted odds ratio, 0.54; 95% CI, 0.22 to 1.33). The adjusted odds ratios for a reduction in disease severity were similar in the two groups, as compared with IVIG alone (0.90 for IVIG plus glucocorticoids and 0.93 for glucocorticoids alone). The time until a reduction in disease severity was similar in the three groups. CONCLUSIONS: We found no evidence that recovery from MIS-C differed after primary treatment with IVIG alone, IVIG plus glucocorticoids, or glucocorticoids alone, although significant differences may emerge as more data accrue. (Funded by the European Union's Horizon 2020 Program and others; BATS ISRCTN number, ISRCTN69546370.).

Geographical and temporal trends and seasonal relapse in Plasmodium ovale spp. and Plasmodium malariae infections imported to the UK between 1987 and 2015.

BACKGROUND: Plasmodium ovale spp. and P. malariae cause illness in endemic regions and returning travellers. Far less is known about these species than P. falciparum and P. vivax. METHODS: The UK national surveillance data, collected 1987 to 2015, were collated with the International Passenger Survey and climatic data to determine geographical, temporal and seasonal trends of imported P. ovale spp. and P. malariae infection. RESULTS: Of 52,242 notified cases of malaria, 6.04% (3157) were caused by P. ovale spp. and 1.61% (841) by P. malariae; mortality was 0.03% (1) and 0.12% (1), respectively. Almost all travellers acquired infection in West or East Africa. Infection rate per travel episode fell fivefold during the study period. The median latency of P. malariae and P. ovale spp. was 18 and 76 days, respectively; delayed presentation occurred with both species. The latency of P. ovale spp. infection imported from West Africa was significantly shorter in those arriving in the UK during the West African peak malarial season compared to those arriving outside it (44 days vs 94 days, p < 0.0001), implying that relapse synchronises with the period of high malarial transmission. This trend was not seen in P. ovale spp. imported from East Africa nor in P. malariae. CONCLUSION: In West Africa, where malaria transmission is highly seasonal, P. ovale spp. may have evolved to relapse during the malarial high transmission season. This has public health implications. Deaths are very rare, supporting current guidelines emphasising outpatient treatment. However, late presentations do occur.

Subcutaneous ivermectin use in the treatment of severe Strongyloides stercoralis infection: two case reports and a discussion of the literature.

BACKGROUND: Strongyloides stercoralis infection presents with varying degrees of severity, but it often primarily involves the small bowel. In severe infection and cases of hyperinfection, ileus and small-bowel obstruction may prevent enteral absorption of anthelminthics such as ivermectin. At present there are no parenteral anthelminthics licensed for use in humans. METHODS: Here, we describe two cases of severe S. stercoralis infection treated with an unlicensed veterinary preparation of subcutaneous ivermectin, and we discuss the published reports of the use of this treatment elsewhere. RESULTS: Both patients were successfully treated with subcutaneous ivermectin, and both recovered completely. CONCLUSIONS: Despite the limited published experience of parenteral ivermectin use, there is evidence that it may be a safe and effective treatment for severe strongyloidiasis. However, more data are needed to guide dosing schedules and monitoring for toxicity.

A health integrated platform for pharmacy clinical intervention data management and intelligent visual analytics and reporting.

Objective: Our initiative aimed to improve the system used to capture pharmacist clinical interventions to better support staff to document, manage and identify trends in medication-related problems (MRPs). The aim of the study was to develop an electronic tool which is easily accessible by most electronic devices with secure data storage and access. Methods: A REDCap® database was designed for documentation of pharmacy clinical interventions. Information documented can be retrieved in real-time and can be integrated to Microsoft Power BI® for real-time data visualisation. The dashboards were customised to display useful information including pharmacy clinical intervention details, common MRPs, common medications involved available to users at real time. Results: A total of 4343 interventions were documented from July 2022 to March 2023. The most common MRPs were omission of regular medications 876 (20.17%), condition untreated 722(16.62%), and contraindications apparent 451 (10.38%). The most common medications involved include iron 244 (5.62%), enoxaparin 231 (5.32%), macrogol laxatives 208 (4.79%), multivitamin 206 (4.74%), colecalciferol 179(4.12%), tramadol 156 (3.59%). Conclusion: This study demonstrated the significance of integration of health application tools of REDcap and Power BI in the data management and intelligent visual analytics and reporting.

Immunoglobulin, glucocorticoid, or combination therapy for multisystem inflammatory syndrome in children: a propensity-weighted cohort study.

Background: Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments. Methods: The Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370. Findings: We enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2-11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75-1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58-1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91-1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70-1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11-0·20]; p<0·0001) and glucocorticoids alone (0·68 [0·50-0·93]; p=0·014) versus intravenous immunoglobulin alone. Persistent fever (from day 2 onward) was less common with intravenous immunoglobulin plus glucocorticoids compared with either intravenous immunoglobulin alone (OR 0·50 [95% CI 0·38-0·67]; p<0·0001) or glucocorticoids alone (0·63 [0·45-0·88]; p=0·0058). Coronary artery aneurysm occurrence and resolution did not differ significantly between treatment groups. Interpretation: Recovery rates, including occurrence and resolution of coronary artery aneurysms, were similar for primary treatment with intravenous immunoglobulin when compared to glucocorticoids or intravenous immunoglobulin plus glucocorticoids. Initial treatment with glucocorticoids appears to be a safe alternative to immunoglobulin or combined therapy, and might be advantageous in view of the cost and limited availability of intravenous immunoglobulin in many countries. Funding: Imperial College London, the European Union's Horizon 2020, Wellcome Trust, the Medical Research Foundation, UK National Institute for Health and Care Research, and National Institutes of Health.

Relationship between molecular pathogen detection and clinical disease in febrile children across Europe: a multicentre, prospective observational study.

Background: The PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice. Methods: Febrile children and controls were recruited on presentation to hospital in 9 European countries 2016-2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed. Findings: Of 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92-5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07-7.59), Group A streptococcus (OR 2.73, 95% CI 1.13-6.09) and E. coli (OR 2.7, 95% CI 1.02-6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11-0.46), influenza B (OR 0.12, 95% CI 0.02-0.37) and RSV (OR 0.16, 95% CI: 0.06-0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23-0.72) and EBV (OR 0.71, 95% CI 0.56-0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively. Interpretation: Most febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics. Funding: EU Horizon 2020 grant 668303.

Comparative transcriptomic analysis of whole blood mycobacterial growth assays and tuberculosis patients' blood RNA profiles.

In vitro whole blood infection models are used for elucidating the immune response to Mycobacterium tuberculosis (Mtb). They exhibit commonalities but also differences, to the in vivo blood transcriptional response during natural human Mtb disease. Here, we present a description of concordant and discordant components of the immune response in blood, quantified through transcriptional profiling in an in vitro whole blood infection model compared to whole blood from patients with tuberculosis disease. We identified concordantly and discordantly expressed gene modules and performed in silico cell deconvolution. A high degree of concordance of gene expression between both adult and paediatric in vivo-in vitro tuberculosis infection was identified. Concordance in paediatric in vivo vs in vitro comparison is largely characterised by immune suppression, while in adults the comparison is marked by concordant immune activation, particularly that of inflammation, chemokine, and interferon signalling. Discordance between in vitro and in vivo increases over time and is driven by T-cell regulation and monocyte-related gene expression, likely due to apoptotic depletion of monocytes and increasing relative fraction of longer-lived cell types, such as T and B cells. Our approach facilitates a more informed use of the whole blood in vitro model, while also accounting for its limitations.

Characterisation of the blood RNA host response underpinning severity in COVID-19 patients.

Infection with SARS-CoV-2 has highly variable clinical manifestations, ranging from asymptomatic infection through to life-threatening disease. Host whole blood transcriptomics can offer unique insights into the biological processes underpinning infection and disease, as well as severity. We performed whole blood RNA Sequencing of individuals with varying degrees of COVID-19 severity. We used differential expression analysis and pathway enrichment analysis to explore how the blood transcriptome differs between individuals with mild, moderate, and severe COVID-19, performing pairwise comparisons between groups. Increasing COVID-19 severity was characterised by an abundance of inflammatory immune response genes and pathways, including many related to neutrophils and macrophages, in addition to an upregulation of immunoglobulin genes. In this study, for the first time, we show how immunomodulatory treatments commonly administered to COVID-19 patients greatly alter the transcriptome. Our insights into COVID-19 severity reveal the role of immune dysregulation in the progression to severe disease and highlight the need for further research exploring the interplay between SARS-CoV-2 and the inflammatory immune response.

Host transcriptional response to TB preventive therapy differentiates two sub-groups of IGRA-positive individuals.

We hypothesised that individuals with immunological sensitisation to Mycobacterium tuberculosis (Mtb), conventionally regarded as evidence of latent tuberculosis infection (LTBI), would demonstrate binary responses to preventive therapy (PT), reflecting the differential immunological consequences of the sterilisation of viable infection in those with active Mtb infection versus no Mtb killing in those who did not harbour viable bacilli. We investigated longitudinal whole blood transcriptional profile responses to PT of Interferon gamma release assay (IGRA)-positive tuberculosis contacts and IGRA-negative, tuberculosis-unexposed controls. Longitudinal unsupervised clustering analysis with a subset of 474 most variable genes in antigen-stimulated blood separated the IGRA-positive participants into two distinct subgroups, one of which clustered with the IGRA-negative controls. 117 probes were differentially expressed over time between the two cluster groups, many of them associated with immunological pathways important in mycobacterial control. We contend that the differential host RNA response reflects lack of Mtb viability in the group that clustered with the IGRA-negative unexposed controls, and Mtb viability in the group (1/3 of IGRA-positives) that clustered away. Gene expression patterns in the blood of IGRA-positive individuals emerging during the course of PT, which reflect Mtb viability, could have major implications in the identification of risk of progression, treatment stratification and biomarker development.

Examining the Complex Relationship Between Tuberculosis and Other Infectious Diseases in Children.

Millions of children are exposed to tuberculosis (TB) each year, many of which become infected with Mycobacterium tuberculosis. Most children can immunologically contain or eradicate the organism without pathology developing. However, in a minority, the organism overcomes the immunological constraints, proliferates and causes TB disease. Each year a million children develop TB disease, with a quarter dying. While it is known that young children and those with immunodeficiencies are at increased risk of progression from TB infection to TB disease, our understanding of risk factors for this transition is limited. The most immunologically disruptive process that can happen during childhood is infection with another pathogen and yet the impact of co-infections on TB risk is poorly investigated. Many diseases have overlapping geographical distributions to TB and affect similar patient populations. It is therefore likely that infection with viruses, bacteria, fungi and protozoa may impact on the risk of developing TB disease following exposure and infection, although disentangling correlation and causation is challenging. As vaccinations also disrupt immunological pathways, these may also impact on TB risk. In this article we describe the pediatric immune response to M. tuberculosis and then review the existing evidence of the impact of co-infection with other pathogens, as well as vaccination, on the host response to M. tuberculosis. We focus on the impact of other organisms on the risk of TB disease in children, in particularly evaluating if co-infections drive host immune responses in an age-dependent way. We finally propose priorities for future research in this field. An improved understanding of the impact of co-infections on TB could assist in TB control strategies, vaccine development (for TB vaccines or vaccines for other organisms), TB treatment approaches and TB diagnostics.

Clinical, geographical, and temporal risk factors associated with presentation and outcome of vivax malaria imported into the United Kingdom over 27 years: observational study.

OBJECTIVE: To examine temporal and geographical trends, risk factors, and seasonality of imported vivax malaria in the United Kingdom to inform clinical advice and policy. DESIGN: Observational study. SETTING: National surveillance data from the UK Public Health England Malaria Reference Laboratory, data from the International Passenger Survey, and international climactic data. PARTICIPANTS: All confirmed and notified cases of malaria in the UK (n=50,187) from 1987 to 2013, focusing on 12,769 cases of vivax malaria. MAIN OUTCOME MEASURES: Mortality, sociodemographic details (age, UK region, country of birth and residence, and purpose of travel), destination, and latency (time between arrival in the UK and onset of symptoms). RESULTS: Of the malaria cases notified, 25.4% (n=12,769) were due to Plasmodium vivax, of which 78.6% were imported from India and Pakistan. Most affected patients (53.5%) had travelled to visit friends and relatives, and 11.1% occurred in tourists. Imported P vivax is concentrated in areas with large communities of south Asian heritage. Overall mortality was 7/12,725 (0.05%), but with no deaths in 9927 patients aged under 50 years. Restricting the analysis to those aged more than 50 years, mortality was 7/2798 (0.25%), increasing to 4/526 (0.76%) (adjusted odds ratio 32.0, 95% confidence interval 7.1 to 144.0, P<0.001) in those aged 70 years or older. Annual notifications decreased sharply over the period, while traveller numbers between the UK and South Asia increased. The risk of acquiring P vivax from South Asia was year round but was twice as high from June to September (40 per 100,000 trips) compared with the rest of the year. There was strong seasonality in the latency from arrival in the UK to presentation, significantly longer in those arriving in the UK from South Asia from October to March (median 143 days) versus those arriving from April to September (37 days, P<0.001). CONCLUSIONS: Travellers visiting friends and family in India and Pakistan are most at risk of acquiring P vivax, and older patients (especially those >70 years) are most at risk of dying; these groups should be targeted for advice before travelling. The risk of acquiring vivax malaria is year round but higher during summer monsoons, masked by latency. The latency of time to clinical presentation of imported vivax malaria in the UK is highly seasonal; seasonal latency has implications for pretravel advice but also for the control of malaria in India and Pakistan. A reduced incidence of vivax malaria in travellers may mean further areas of South Asia can be considered not to need malaria chemoprophylaxis.

Geographical concentration of falciparum malaria treated in the UK and delay to treatment with artesunate in severe cases: an observational study.

OBJECTIVES: To quantify geographical concentration of falciparum malaria cases in the UK at a hospital level. To assess potential delay-to-treatment associated with hub-and-spoke distribution of artesunate in severe cases. DESIGN: Observational study using national and hospital data. SETTING AND PARTICIPANTS: 3520 patients notified to the Malaria Reference Laboratory 2008-2010, 34 patients treated with intravenous artesunate from a tropical diseases centre 2002-2010. MAIN OUTCOME MEASURES: Geographical location of falciparum cases notified in the UK. Diagnosis-to-treatment times for intravenous artesunate. RESULTS: Eight centres accounted for 43.9% of the UK's total cases; notifications from 107 centres accounted for 10.2% of cases; 51.5% of hospitals seeing malaria notified 5 or fewer cases in 3 years. Centres that saw <10 cases/year treat 26.3% of malaria cases; 6.1% of cases are treated in hospitals seeing <2 cases/year. Concentration of falciparum malaria was highest in Greater London (1925, 54.7%), South East (515, 14.6%), East of England (402, 11.4%) and North West (192, 5.4%). The North East and Northern Ireland each notified 5 or fewer cases per year. Median diagnosis-to-treatment time was 1 h (range 0.5-5) for patients receiving artesunate in the specialist centre; 7.5 h (range 4-26) for patients receiving it in referring hospitals via the hub-and-spoke system (p=0.02); 25 h (range 9-45) for patients receiving it on transfer to the regional centre from a referring hospital (p=0.002). CONCLUSIONS: Most UK hospitals see few cases of falciparum malaria and geographical distances are significant. Over 25% of cases are seen in hospitals where malaria is rare, although 60% are seen in hospitals seeing over 50 cases over 3 years. A hub-and-spoke system minimises drug wastage and ensures availability in centres seeing most cases but is associated with treatment delays elsewhere. As with all observational studies, there are limitations, which are discussed.