Genomic Diversity of Mycobacterium tuberculosis Complex Strains in Cantabria (Spain), a Moderate TB Incidence Setting.

BACKGROUND: Tuberculosis (TB) control strategies are focused mainly on prevention, early diagnosis, compliance to treatment and contact tracing. The objectives of this study were to explore the frequency and risk factors of recent transmission of clinical isolates of Mycobacterium tuberculosis complex (MTBC) in Cantabria in Northern Spain from 2012 through 2013 and to analyze their clonal complexity for better understanding of the transmission dynamics in a moderate TB incidence setting. METHODS: DNA from 85 out of 87 isolates from bacteriologically confirmed cases of MTBC infection were extracted directly from frozen stocks and genotyped using the mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) method. The MIRU-VNTRplus database tool was used to identify clusters and lineages and to build a neighbor joining (NJ) phylogenetic tree. In addition, data were compared to the SITVIT2 database at the Pasteur Institute of Guadeloupe. RESULTS: The rate of recent transmission was calculated to 24%. Clustering was associated with being Spanish-born. A high prevalence of isolates of the Euro-American lineage was found. In addition, MIRU-VNTR profiles of the studied isolates corresponded to previously found MIRU-VNTR types in other countries, including Spain, Belgium, Great Britain, USA, Croatia, South Africa and The Netherlands. Six of the strains analyzed represented clonal variants. CONCLUSION: Transmission of MTBC is well controlled in Cantabria. The majority of TB patients were born in Spain. The population structure of MTBC in Cantabria has a low diversity of major clonal lineages with the Euro-American lineage predominating.

Antigen-induced cytokine and chemokine release test for tuberculosis infection using adsorption of stimulated whole blood on filter paper and multiplex analysis.

BACKGROUND: In vitro stimulation of whole blood or isolated blood cells with specific antigens is used for several purposes. Immediately following incubation with antigens, samples have to be centrifuged to stop the reactions by remaining cells and the supernatant refrigerated or analysed directly to preserve the analytes of interest, which makes samples difficult to prepare outside laboratories. We have tested whether spotting whole blood on filter paper after activation can be used in one of the tests for Mycobacterium tuberculosis infection (MTI), the QuantiFERON®-TB Gold In Tube test (QFT), where the spotting technique can make it suitable for use in locations without facilities like a centrifuge and a refrigerator. MATERIALS AND METHODS: Samples from 22 individuals undergoing screening for MTI and 10 healthy controls were incubated, centrifuged and IFN-γ measured by Enzyme-linked immunosorbent assay (ELISA), as described in the kit insert. In parallel, activated blood was spotted on filter paper (Schleicher & Schuell) and dried. The dried blood spot samples were analysed for 21 inflammatory markers with an in-house assay based on Luminex technology. RESULTS: Our multiplex measurements of inflammatory markers in samples from suspected MTI patients confirmed the IFN-γ findings in the QFT. IL-2, GM-CSF, IL-5, and IL-1ß were also found as useful markers for MTI. We were not able to distinguish between active tuberculosis and latent MTI. CONCLUSION: Applying blood on filter paper after incubation makes in vitro stimulation tests feasible in locations where heat and electricity is unavailable.