Identification of host gene expression biomarkers for tuberculosis

The presence of disease, including infectious disease, has been observed to give rise to specific patterns of gene expression in peripheral whole blood, regardless of disease site. These gene expression signatures allow for distinction between diseases and have the potential to reform diagnostics, particularly in diseases and patient groups for whom current diagnostics are unreliable, like Tuberculosis (TB). Although TB is a treatable infectious disease, it has high morbidity and mortality, especially in low resource countries and HIV infected patients. In this thesis, I propose a bioinformatics toolbox that derives minimal transcriptomic signatures from microarray datasets acquired from heterogeneous groups regardless of underlying co-infections and geographic locations. The transcripts’ expression values are then aggregated into a single value disease risk score (DRS) for every patient, that allows for classification between the disease groups in a binary manner. The toolbox was employed to analyse an adult and a paediatric TB transcriptomic study, comprising HIV infected and uninfected patients from sub-Saharan Africa. In the adult study, the DRS based on a 27-transcript signature distinguished culture confirmed TB from latent TB infection (LTBI), while 44 transcripts distinguished TB from other diseases phenotypically similar to TB (OD), with high sensitivity and specificity. Out-of-sample validation was performed using a publicly available dataset. In the paediatric study, a 51-transcript signature distinguished TB from OD and a 42-transcript signature from LTBI. The signatures were validated out-of-sample using an independent cohort and benchmarked against culture-negative TB patients and Xpert® MTB/RIF, currently used for detection of M. tuberculosis. This thesis provides proof of principle that minimal host blood transcriptional signatures are able to distinguish TB from LTBI and OD regardless of HIV infection. The subsequent transformation of the signatures into a score for every patient may facilitate disease categorisation and potentially development of diagnostic tools.Open Acces

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

Bioinformatics: living on the edge

A report on the 11th European Conference on Computational Biology (ECCB), Basel, Switzerland, September 9-12, 2012

Distinct effects of HIV protease inhibitors and ERAD inhibitors on zygote to ookinete transition of the malaria parasite

In an effort to eradicate malaria, new interventions are proposed to include compound/vaccine development against pre-erythrocytic, erythrocytic and mosquito stages of Plasmodium. Drug repurposing might be an alternative approach to new antimalarials reducing the cost and the time required for drug development. Previous in vitro studies have examined the effects of protease inhibitors on different stages of the Plasmodium parasite, although the clinical relevance of this remains unclear. In this study we tested the putative effect of three HIV protease inhibitors, two general aspartyl protease inhibitors and three AAA-p97 ATPase inhibitors on the zygote to ookinete transition of the Plasmodium parasite. Apart from the two general aspartyl inhibitors, all other compounds had a profound effect on the development of the parasites. HIVPIs inhibited zygote to ookinete conversion by 75%–90%, while the three AAA-p97 ATPase inhibitors blocked conversion by 50%–90% at similar concentrations, while electron microscopy highlighted nuclear and structural abnormalities. Our results highlight a potential of HIV protease inhibitors and p97 inhibitors as transmission blocking agents for the eradication of malaria

Genome-wide host RNA signatures of infectious diseases: discovery and clinical translation

The use of whole blood gene expression to derive diagnostic biomarkers capable of distinguishing between phenotypically similar diseases holds great promise but remains a challenge. Differential gene expression analysis is used to identify the key genes that undergo changes in expression relative to healthy individuals, as well as to patients with other diseases. These key genes can act as diagnostic, prognostic and predictive markers of disease. Gene expression 'signatures' in the blood hold potential to be used for the diagnosis of infectious diseases, where current diagnostics are unreliable, ineffective or of limited potential. For diagnostic tests based on RNA signatures to be useful clinically, the first step is to identify the minimum set of gene transcripts that accurately identify the disease in question. The second requirement is rapid and cost effective detection of the gene expression levels. Whilst signatures have been described for a number of infectious diseases, 'clinic-ready' technologies for RNA detection from clinical samples are limited, though existing methods such as reverse transcription-polymerase chain reaction (RT-PCR) are likely to be superseded by a number of emerging technologies, which may form the basis of the translation of gene expression signatures into routine diagnostic tests for a range of disease states

Transcriptomic Profiling in Childhood H1N1/09 Influenza Reveals Reduced Expression of Protein Synthesis Genes

We compared the blood RNA transcriptome of children hospitalized with influenza A H1N1/09, respiratory syncytial virus (RSV) or bacterial infection, and healthy controls. Compared to controls, H1N1/09 patients showed increased expression of inflammatory pathway genes and reduced expression of adaptive immune pathway genes. This was validated on an independent cohort. The most significant function distinguishing H1N1/09 patients from controls was protein synthesis, with reduced gene expression. Reduced expression of protein synthesis genes also characterized the H1N1/09 expression profile compared to children with RSV and bacterial infection, suggesting that this is a key component of the pathophysiological response in children hospitalized with H1N1/09 infection

Diagnosis of childhood tuberculosis and host RNA expression in Africa

Improved diagnostic tests for tuberculosis in children are needed. We hypothesized that transcriptional signatures of host blood could be used to distinguish tuberculosis from other diseases in African children who either were or were not infected with the human immunodeficiency virus (HIV

Detection of tuberculosis in HIV-infected and-uninfected African adults using whole blood RNA expression signatures: a case-control study

Background: A major impediment to tuberculosis control in Africa is the difficulty in diagnosing active tuberculosis (TB), particularly in the context of HIV infection. We hypothesized that a unique host blood RNA transcriptional signature would distinguish TB from other diseases (OD) in HIV-infected and -uninfected patients, and that this could be the basis of a simple diagnostic test. Methods and Findings: Adult case-control cohorts were established in South Africa and Malawi of HIV-infected or -uninfected individuals consisting of 584 patients with either TB (confirmed by culture of Mycobacterium tuberculosis [M.TB] from sputum or tissue sample in a patient under investigation for TB), OD (i.e., TB was considered in the differential diagnosis but then excluded), or healthy individuals with latent TB infection (LTBI). Individuals were randomized into training (80%) and test (20%) cohorts. Blood transcriptional profiles were assessed and minimal sets of significantly differentially expressed transcripts distinguishing TB from LTBI and OD were identified in the training cohort. A 27 transcript signature distinguished TB from LTBI and a 44 transcript signature distinguished TB from OD. To evaluate our signatures, we used a novel computational method to calculate a disease risk score (DRS) for each patient. The classification based on this score was first evaluated in the test cohort, and then validated in an independent publically available dataset (GSE19491). In our test cohort, the DRS classified TB from LTBI (sensitivity 95%, 95% CI [87–100]; specificity 90%, 95% CI [80–97]) and TB from OD (sensitivity 93%, 95% CI [83–100]; specificity 88%, 95% CI [74–97]). In the independent validation cohort, TB patients were distinguished both from LTBI individuals (sensitivity 95%, 95% CI [85–100]; specificity 94%, 95% CI [84–100]) and OD patients (sensitivity 100%, 95% CI [100–100]; specificity 96%, 95% CI [93–100]). Limitations of our study include the use of only culture confirmed TB patients, and the potential that TB may have been misdiagnosed in a small proportion of OD patients despite the extensive clinical investigation used to assign each patient to their diagnostic group. Conclusions: In our study, blood transcriptional signatures distinguished TB from other conditions prevalent in HIV-infected and -uninfected African adults. Our DRS, based on these signatures, could be developed as a test for TB suitable for use in HIV endemic countries. Further evaluation of the performance of the signatures and DRS in prospective populations of patients with symptoms consistent with TB will be needed to define their clinical value under operational conditions