Decreased neutrophil-associated miRNA and increased B-cell associated miRNA expression during tuberculosis.

MicroRNAs are short non-coding RNAs that regulate gene expression by binding to, and suppressing the expression of genes. Research show that microRNAs have potential to be used as biomarkers for diagnosis, treatment response and can be used for therapeutic interventions. Furthermore, microRNA expression has effects on immune cell functions, which may lead to disease. Considering the important protective role of neutrophils and B-cells during M.tb infection, we evaluated the expression of microRNAs, known to alter function of these cells, in the context of human TB. We utilised real-time PCR to evaluate the levels of microRNA transcripts in the peripheral blood of TB cases and healthy controls. We found that neutrophil-associated miR-197-3p, miR-99b-5p and miR-191-5p transcript levels were significantly lower in TB cases. Additionally, B-cell-associated miR-320a, miR-204-5p, miR331-3p and other transcript levels were higher in TB cases. The miRNAs differentially expressed in neutrophils are predominantly implicated in signalling pathways leading to cytokine productions. Here, the decreased expression in TB cases may imply a lack of suppression on signalling pathways, which may lead to increased production of pro-inflammatory cytokines such as interferon-gamma. Furthermore, the miRNAs differentially expressed in B-cells are mostly involved in the induction/suppression of apoptosis. Further functional studies are however required to elucidate the significance and functional effects of changes in the expression of these microRNAs.

Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo).

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON® -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions.

Differential gene expression of activating Fcγ receptor classifies active tuberculosis regardless of human immunodeficiency virus status or ethnicity.

New diagnostics and vaccines for tuberculosis (TB) are urgently needed, but require an understanding of the requirements for protection from/susceptibility to TB. Previous studies have used unbiased approaches to determine gene signatures in single-site populations. The present study utilized a targeted approach, reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), to validate these genes in a multisite study. We analysed ex vivo whole blood RNA from a total of 523 participants across four sub-Saharan countries (Ethiopia, Malawi, South Africa, and The Gambia) with differences in TB and human immunodeficiency virus (HIV) status. We found a number of genes that were expressed at significantly lower levels in participants with active disease than in those with latent TB infection (LTBI), with restoration following successful TB treatment. The most consistent classifier of active disease was FCGR1A (high-affinity IgG Fc receptor 1 (CD64)), which was the only marker expressed at significantly higher levels in participants with active TB than in those with LTBI before treatment regardless of HIV status or genetic background. This is the first study to identify a biomarker for TB that is not affected by HIV status or geo-genetic differences. These data provide valuable clues for understanding TB pathogenesis, and also provide a proof-of-concept for the use of RT-MLPA in rapid and inexpensive validation of unbiased gene expression findings.

Emergence and diversity of different HIV-1 subtypes in South Africa, 2000-2001.

HIV-1 is a major health problem in South Africa with an average prevalence rate of 29.1% in pregnant women and between 4.9 and 6.1 million people infected. Using env gp120 V3 serotyping and genotyping techniques 410 patient samples were investigated. Most of the samples were obtained from different clinics in the greater Cape Town area of the Western Cape Province in South Africa. These included an academic hospital, state and private clinics, an informal settlement, sex worker cohorts, and the blood transfusion services. RNA was extracted from plasma samples followed by RT-PCR and sequencing of the env gp120 V3 region. Sequence fragments were assembled using Sequencher V4.7 and subsequently codon aligned. Distance calculation, tree construction methods, and bootstrap analysis were implemented using MEGA version 4.0. Viral load measurements indicated that HIV-1 RNA levels from 74 samples were below the assay detection limit. Three hundred thirty-six samples were used for env PCR and sequencing and 320 were assigned to subtypes. The majority of the sequences were subtyped as C (n = 285, 89.0%). Other subtypes detected were subtype A (n = 10, 3.1%); subtype B (n = 22, 6.8%); one each of subtypes F1, G, U, and a CH recombinant. Whether this diversity will have major implications for HIV-1 evolution and vaccine development in this region remains undetermined.