Lessons learned: Retrospective assessment of outcomes and management of patients with advanced HIV disease in a semi-urban polyclinic in Epworth, Zimbabwe.

INTRODUCTION: HIV continues to be one of the leading causes of infectious death worldwide and presentation with advanced HIV disease is associated with increased morbidity and mortality. Recommendations for the management of advanced HIV disease include prompt screening and treatment of opportunistic infections, rapid initiation of ART and intensified adherence support. We present treatment outcomes of a cohort of patients presenting with advanced HIV disease in a semi-urban Zimbabwean polyclinic. METHODS: Retrospective cohort analysis of adult patients enrolled for care at Epworth polyclinic, Zimbabwe between 2007 and end June 2016. Treatment outcomes at 6 and 12 months were recorded. Multivariate logistical regression analysis was undertaken to identify risk factors for presentation with advanced HIV Disease (CD4 count less than 200 cells/mm3 or WHO stage 3 or 4) and risks for attrition at 12 months. RESULTS: 16,007 anti-retroviral therapy naive adult patients were included in the final analysis, 47.4% of whom presented with advanced HIV disease. Patients presenting with advanced HIV disease had a higher mortality rate at 12 months following enrollment compared to early stage patients (5.11% vs 0.45%). Introduction of a package of differentiated care for patients with a CD4 count of less than 100 cells/mm3 resulted in diagnosis of cryptococcal antigenaemia in 7% of patients and a significant increase in the diagnosis of TB, although there was no significant difference in attrition at 6 or 12 months for these patients compared to those enrolled prior to the introduction of the differentiated care. CONCLUSIONS: The burden of advanced HIV disease remained high over the study period in this semi-urban polyclinic in Zimbabwe. Introduction of a package of differentiated care for those with advanced HIV disease increased the diagnosis of opportunistic infections and represents a model of care which can be replicated in other polyclinics in the resource constrained Zimbabwean context.

Author Correction: Progression of whole-blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in severe influenza.

In the version of this article initially published, a source of funding was not included in the Acknowledgements section. That section should include the following: P.J.M.O. was supported by EU FP7 PREPARE project 602525. The error has been corrected in the HTML and PDF version of the article.

Progression of whole-blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in severe influenza.

Transcriptional profiles and host-response biomarkers are used increasingly to investigate the severity, subtype and pathogenesis of disease. We now describe whole-blood mRNA signatures and concentrations of local and systemic immunological mediators in 131 adults hospitalized with influenza, from whom extensive clinical and investigational data were obtained by MOSAIC investigators. Signatures reflective of interferon-related antiviral pathways were common up to day 4 of symptoms in patients who did not require mechanical ventilator support; in those who needed mechanical ventilation, an inflammatory, activated-neutrophil and cell-stress or death ('bacterial') pattern was seen, even early in disease. Identifiable bacterial co-infection was not necessary for this 'bacterial' signature but was able to enhance its development while attenuating the early 'viral' signature. Our findings emphasize the importance of timing and severity in the interpretation of host responses to acute viral infection and identify specific patterns of immune-system activation that might enable the development of novel diagnostic and therapeutic tools for severe influenza.

The Transcriptional Signature of Active Tuberculosis Reflects Symptom Status in Extra-Pulmonary and Pulmonary Tuberculosis.

BACKGROUND: Mycobacterium tuberculosis infection is a leading cause of infectious death worldwide. Gene-expression microarray studies profiling the blood transcriptional response of tuberculosis (TB) patients have been undertaken in order to better understand the host immune response as well as to identify potential biomarkers of disease. To date most of these studies have focused on pulmonary TB patients with gene-expression profiles of extra-pulmonary TB patients yet to be compared to those of patients with pulmonary TB or sarcoidosis. METHODS: A novel cohort of patients with extra-pulmonary TB and sarcoidosis was recruited and the transcriptional response of these patients compared to those with pulmonary TB using a variety of transcriptomic approaches including testing a previously defined 380 gene meta-signature of active TB. RESULTS: The 380 meta-signature broadly differentiated active TB from healthy controls in this new dataset consisting of pulmonary and extra-pulmonary TB. The top 15 genes from this meta-signature had a lower sensitivity for differentiating extra-pulmonary TB from healthy controls as compared to pulmonary TB. We found the blood transcriptional responses in pulmonary and extra-pulmonary TB to be heterogeneous and to reflect the extent of symptoms of disease. CONCLUSIONS: The transcriptional signature in extra-pulmonary TB demonstrated heterogeneity of gene expression reflective of symptom status, while the signature of pulmonary TB was distinct, based on a higher proportion of symptomatic individuals. These findings are of importance for the rational design and implementation of mRNA based TB diagnostics.

Differential Production of Type I IFN Determines the Reciprocal Levels of IL-10 and Proinflammatory Cytokines Produced by C57BL/6 and BALB/c Macrophages.

Pattern recognition receptors detect microbial products and induce cytokines, which shape the immunological response. IL-12, TNF-α, and IL-1ß are proinflammatory cytokines, which are essential for resistance against infection, but when produced at high levels they may contribute to immunopathology. In contrast, IL-10 is an immunosuppressive cytokine, which dampens proinflammatory responses, but it can also lead to defective pathogen clearance. The regulation of these cytokines is therefore central to the generation of an effective but balanced immune response. In this study, we show that macrophages derived from C57BL/6 mice produce low levels of IL-12, TNF-α, and IL-1ß, but high levels of IL-10, in response to TLR4 and TLR2 ligands LPS and Pam3CSK4, as well as Burkholderia pseudomallei, a Gram-negative bacterium that activates TLR2/4. In contrast, macrophages derived from BALB/c mice show a reciprocal pattern of cytokine production. Differential production of IL-10 in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages was due to a type I IFN and ERK1/2-dependent, but IL-27-independent, mechanism. Enhanced type I IFN expression in LPS-stimulated C57BL/6 macrophages was accompanied by increased STAT1 and IFN regulatory factor 3 activation. Furthermore, type I IFN contributed to differential IL-1ß and IL-12 production in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages via both IL-10-dependent and -independent mechanisms. These findings highlight key pathways responsible for the regulation of pro- and anti-inflammatory cytokines in macrophages and reveal how they may differ according to the genetic background of the host.

Analysis of Transcriptional Signatures in Response to Listeria monocytogenes Infection Reveals Temporal Changes That Result from Type I Interferon Signaling.

Analysis of the mouse transcriptional response to Listeria monocytogenes infection reveals that a large set of genes are perturbed in both blood and tissue and that these transcriptional responses are enriched for pathways of the immune response. Further we identified enrichment for both type I and type II interferon (IFN) signaling molecules in the blood and tissues upon infection. Since type I IFN signaling has been reported widely to impair bacterial clearance we examined gene expression from blood and tissues of wild type (WT) and type I IFNαß receptor-deficient (Ifnar1-/-) mice at the basal level and upon infection with L. monocytogenes. Measurement of the fold change response upon infection in the absence of type I IFN signaling demonstrated an upregulation of specific genes at day 1 post infection. A less marked reduction of the global gene expression signature in blood or tissues from infected Ifnar1-/- as compared to WT mice was observed at days 2 and 3 after infection, with marked reduction in key genes such as Oasg1 and Stat2. Moreover, on in depth analysis, changes in gene expression in uninfected mice of key IFN regulatory genes including Irf9, Irf7, Stat1 and others were identified, and although induced by an equivalent degree upon infection this resulted in significantly lower final gene expression levels upon infection of Ifnar1-/- mice. These data highlight how dysregulation of this network in the steady state and temporally upon infection may determine the outcome of this bacterial infection and how basal levels of type I IFN-inducible genes may perturb an optimal host immune response to control intracellular bacterial infections such as L. monocytogenes.

The Blood Transcriptome of Experimental Melioidosis Reflects Disease Severity and Shows Considerable Similarity with the Human Disease.

Melioidosis, a severe human disease caused by the bacterium Burkholderia pseudomallei, has a wide spectrum of clinical manifestations ranging from acute septicemia to chronic localized illness or latent infection. Murine models have been widely used to study the pathogenesis of infection and to evaluate novel therapies or vaccines, but how faithfully they recapitulate the biology of human melioidosis at a molecular level is not known. In this study, mice were intranasally infected with either high or low doses of B. pseudomallei to generate either acute, chronic, or latent infection and host blood and tissue transcriptional profiles were generated. Acute infection was accompanied by a homogeneous signature associated with induction of multiple innate immune response pathways, such as IL-10, TREM1, and IFN signaling, largely found in both blood and tissue. The transcriptional profile in blood reflected the heterogeneity of chronic infection and quantitatively reflected the severity of disease. Genes associated with fibrosis and tissue remodeling, including matrix metalloproteases and collagen, were upregulated in chronically infected mice with severe disease. Transcriptional signatures of both acute and chronic melioidosis revealed upregulation of iNOS in tissue, consistent with the expression of IFN-γ, but also Arginase-1, a functional antagonist of the iNOS pathway, and was confirmed by immunohistochemistry. Comparison of these mouse blood datasets by pathway and modular analysis with the blood transcriptional signature of patients with melioidosis showed that many genes were similarly perturbed, including Arginase-1, IL-10, TREM1, and IFN signaling, revealing the common immune response occurring in both mice and humans.

The application of transcriptional blood signatures to enhance our understanding of the host response to infection: the example of tuberculosis.

Despite advances in antimicrobials, vaccination and public health measures, infectious diseases remain a leading cause of morbidity and mortality worldwide. With the increase in antimicrobial resistance and the emergence of new pathogens, there remains a need for new and more accurate diagnostics, the ability to monitor adequate treatment response as well as the ability to predict prognosis for an individual. Transcriptional approaches using blood signatures have enabled a better understanding of the host response to diseases, leading not only to new avenues of basic research, but also to the identification of potential biomarkers for use in diagnosis, prognosis and treatment monitoring.

Identification of the key differential transcriptional responses of human whole blood following TLR2 or TLR4 ligation in-vitro.

The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS), human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that in vitro stimulated human whole blood can be used to interrogate the early transcriptional kinetic response of innate cells to TLR ligands. Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation.

Systems approaches to studying the immune response in tuberculosis.

Tuberculosis (TB) remains a disease of considerable mortality and morbidity. Studies employing microarrays to derive transcriptional profiles of the host response during TB, which combined with data from experimental systems have highlighted a potentially detrimental role for type I interferons during infection, with important implications for vaccine and therapeutic development. In addition, these studies have provided candidate biomarkers which may advance diagnosis and treatment monitoring. These studies thus exemplify the promise of a systems biology approach to study complex infectious disease such as TB.

Vaccination against tuberculosis: how can we better BCG?

Tuberculosis remains one of the most significant human diseases of the developing world, accounting for 3800 worldwide deaths per day. Although we currently have a vaccine for tuberculosis, BCG, this is insufficient at protecting from adult pulmonary tuberculosis in the parts of the world where a good vaccine is most needed. This has prompted the search for new vaccination strategies that can protect better than BCG, or can boost BCG-induced immunity. We discuss these subjects in line with what is known of the immune responses to BCG and Mycobacterium tuberculosis - the etiological agent of the disease, as well as the particular difficulties facing development of new vaccines against tuberculosis. A greater understanding of the factors constituting optimal protection against Mycobacterium tuberculosis infection, as well as which pathogenic factors facilitate active disease, will accelerate the delivery of safe vaccines able to restrict active tuberculosis and thus impede contagion.

Socio-demographic determinants and prevalence of Tuberculosis knowledge in three slum populations of Uganda.

BACKGROUND: Knowledge of tuberculosis has been shown to influence health seeking behaviour; and urban slum dwellers are at a higher risk of acquiring tuberculosis than the general population. The study aim was to assess knowledge of tuberculosis and identify the associated socio-demographic determinants, in order to inform tailored interventions for advocacy, communication and social mobilisation in three urban-slum communities of Uganda. METHODS: A cross-sectional survey of 1361 adults between April and October 2011. Data was analyzed by descriptive statistics. Adjusted odds ratios (aOR) and 95% confidence intervals (95% CI) of potential determinants of tuberculosis (TB) knowledge were estimated by multivariable ordinal logistic regression using Stata 11.2 software. RESULTS: We found low knowledge of TB cause (26.7%); symptoms (46.8%), transmission (54.3%), prevention (34%) and free treatment (35%). Knowledge about TB treatment (69.4) and cure (85.1) was relatively high. Independent determinants of poor knowledge of TB in the multivariable analysis included (aOR, 95% CI) lack of formal education (0.56; 0.38 - 0.83, P = 0.004), unemployment (0.67; 0.49 - 0.90, P = 0.010) and never testing for HIV (0.69; 0.51 - 0.92, P < 0.012). Whilst, older age (1.73; 1.30 - 2.29, P < 0.001) and residing in Lira (2.02; 1.50 - 2.72, P < 0.001) were independent determinants of higher knowledge of TB. CONCLUSION: This study revealed deficiencies in the public health knowledge about TB symptoms, diagnosis and treatment among urban-slum dwellers in Uganda. Tuberculosis control programmes in similar settings should consider innovative strategies for TB education, advocacy, communication and social mobilisation to reach the youth, unemployed and less-educated; as well as those who have never tested for HIV.