Characterisation and detection of Mycobacterium tuberculosis in pleural biopsies using Proteomics and Molecular techniques

Abstract

Tuberculosis (TB) remains a major global health problem. In 2012, an estimated 8.6 million people developed TB and 1.3 million died from the disease (including 320 000 deaths among HIV-positive people). All countries are affected, but most cases (85%) occur in Africa (30%) and Asia (55%), with India and China alone accounting for 35% of all cases. This high incidence is mainly due to high rates of human immunodefiency virus (HIV) and malnutrition that weaken the immune systems and speed up the spread of the disease. The management of TB has faced many challenges in the past, but the most important threats to global TB control are the HIV epidemic and the increasing prevalence of drug resistance. The threat of this disease has emphasised the need for basic science, translational and applied research, which target the development of improved TB diagnostics, drugs, treatment regimens, biomarkers of disease activity and vaccines. This disease often presents a diagnostic challenge because of its diverse clinical manifestations (pulmonary and extra-pulmonary) and low yield of acid-fast bacilli (AFB), especially in tissue specimens. According to WHO, inadequate case detection has now become the primary impediment to disease control. Although culture offers a better sensitivity, the incubation time may be up to six weeks, hence the need for rapid, affordable and sensitive methods for detection of M. tuberculosis, particularly in tissue specimens. Therefore, diagnostic tools that could improve case detection are therefore a high priority. Molecular techniques such as PrimeMix®Universal MTB detection assay and GenoType®MTBDRplus v2 assay have been evaluated and adopted for the diagnosis of TB in sputum samples, but their performance in tissue samples still needs to be explored. The purpose of this study was to characterise and detect M. tuberculosis in FFPE pleural biopsy specimens using Proteomics (nano-LC/MS/MS) and Molecular techniques (PrimeMix® Universal MTB ssay and GenoType®MTBDRplus v2 assay). Our results demonstrated that both molecular assays are able to detect M. tuberculosis complex in FFPE tissue; however PrimeMix®MTB Universal assay using StepOneTMplus real-time PCR instrument showed better sensitivity (59%) compared to GenoType®MTBDRplus v2 assay (32.1%). PrimeMix®Universal MTB ssay could be used to increase diagnostic accuracy in patients who have perplexing diagnostic problems associated with a granulomatous tissue response. This study showed that GenoType®MTBDRplus v2 assay was less sensitive, but highly specific when compared to PrimeMix®Universal MTB assay. However, PrimeMix®Universal MTB assay may be an efficient diagnostic adjunct to histology for diagnosis of TB, especially in samples where culture is deemed unsuitable. Therefore, PrimeMix®Universal MTB assay using StepOneTMplus real-time PCR instrument has the great potential for TB prevalence screening and GenoType®MTBDRplus v2 assay as the confirmatory test, which will aid in planning for TB diagnostics and treatment. This may lead to earlier treatment of TB patients resulting in decreased disease spread. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissue samples has enormous potential for the discovery and validation of disease biomarkers. This technique does not only provide the opportunity to determine the functional genome, but also facilitates the identification of proteins that have not been predicted by the genome analysis. An increasing number of proteomic studies are now being carried out to elucidate disease pathogenesis and identify potential disease-associated diagnostic, predictive and prognostic biomarkers. However, the conventional protein analysis approach is more difficult for the analysis of proteins using formalin-fixed paraffin embedded tissues because the formalin fixation process results in the cross-linking of proteins and thus intact proteins cannot be efficiently extracted. The present study highlights the optimisation of an efficient protocol, capable of extracting proteins for shotgun proteomics (nano-LC/MS/MS) using FFPE pleural tissue specimens. Protein extraction methods such as 2% SDS with long and short heating treatment and 6M Guanidine-HCL methods have been comparatively optimised and evaluated. It was found that incubation of tissue in a lysis buffer containing 2% SDS at high temperature led to the highest protein yield than the other two methods. However, with the use of optimised protein separation techniques, proteomics can unlock the proteome of the archived tissue for discovery and validation of biomarkers to improve disease diagnosis and therapy.