COMPARISON OF A DNA BASED PCR APPROACH WITH CONVENTIONAL METHODS FOR THE DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN MOROCCO

<p><strong>Background:</strong> Worldwide, tuberculosis (TB) is a major public health problem and the rapid diagnosis and appropriate chemotherapy become the first priority and a serious challenge to improve TB treatment.</p><p>In the objective of early TB diagnosis and rapid detection of <em>Mycobacterium tuberculosis </em>(MTB) in the clinical specimens, the utility of the Polymerase Chain Reaction (PCR) using the Insertion Sequence 6110 (IS6110) as target was compared to conventional methods.</p><p><strong>Methods:</strong> Out of 305 patients with different clinical manifestations: suspected, new, drug relapse, drug failure and chronic cases were enrolled in this study and tested by mycobacteriological and PCR techniques for the investigation about the tubercle bacilli.</p><p><strong>Results:</strong> The results of the in house IS6110 PCR showed a good sensitivity (92, 42%) and high specificity (98%), the positive and negative predictive values were 96.4 % and 95.3 % respectively.</p><p><strong>Conclusion:</strong> This study showed clearly that the PCR testing using the IS6110 in the routine analysis is a potential tool for the rapid TB diagnosis, especially for critical cases and would be of great interest to help the clinician in the misdiagnosed critical cases by the traditional radiology.</p&gt

Immune activation and regulatory T cells in Mycobacterium tuberculosis infected lymph nodes

Abstract Background Lymph node tuberculosis (LNTB) is the most frequent extrapulmonary form of tuberculosis (TB). Studies of human tuberculosis at sites of disease are limited. LNTB provides a unique opportunity to compare local in situ and peripheral blood immune response in active Mycobacterium tuberculosis (Mtb) disease. The present study analysed T regulatory cells (Treg) frequency and activation along with CD4+ T cell function in lymph nodes from LNTB patients. Results Lymph node mononuclear cells (LNMC) were compared to autologous peripheral blood mononuclear cells (PBMC). LNMC were enriched for CD4+ T cells with a late differentiated effector memory phenotype. No differences were noted in the frequency and mutifunctional profile of memory CD4+ T cells specific for Mtb. The proportion of activated CD4+ and Tregs in LNMC was increased compared to PBMC. The correlation between Tregs and activated CD4+ T cells was stronger in LNMC than PBMC. Tregs in LNMC showed a strong positive correlation with Th1 cytokine production (IL2, IFNγ and TNFα) as well as MIP-1α after Mtb antigen stimulation. A subset of Tregs in LNMC co-expressed HLA-DR and CD38, markers of activation. Conclusion Further research will determine the functional relationship between Treg and activated CD4+ T cells at lymph node sites of Mtb infection

Molecular Typing of Mycobacterium Tuberculosis Complex by 24-Locus Based MIRU-VNTR Typing in Conjunction with Spoligotyping to Assess Genetic Diversity of Strains Circulating in Morocco.

Standard 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing allows to get an improved resolution power for tracing TB transmission and predicting different strain (sub) lineages in a community.During 2010-2012, a total of 168 Mycobacterium tuberculosis Complex (MTBC) isolates were collected by cluster sampling from 10 different Moroccan cities, and centralized by the National Reference Laboratory of Tuberculosis over the study period. All isolates were genotyped using spoligotyping, and a subset of 75 was genotyped using 24-locus based MIRU-VNTR typing, followed by first line drug susceptibility testing. Corresponding strain lineages were predicted using MIRU-VNTRplus database.Spoligotyping resulted in 137 isolates in 18 clusters (2-50 isolates per cluster: clustering rate of 81.54%) corresponding to a SIT number in the SITVIT database, while 31(18.45%) patterns were unique of which 10 were labelled as "unknown" according to the same database. The most prevalent spoligotype family was LAM; (n = 81 or 48.24% of isolates, dominated by SIT42, n = 49), followed by Haarlem (23.80%), T superfamily (15.47%), >Beijing (2.97%), > U clade (2.38%) and S clade (1.19%). Subsequent 24-Locus MIRU-VNTR typing identified 64 unique types and 11 isolates in 5 clusters (2 to 3isolates per cluster), substantially reducing clusters defined by spoligotyping only. The single cluster of three isolates corresponded to two previously treated MDR-TB cases and one new MDR-TB case known to be contact a same index case and belonging to a same family, albeit residing in 3 different administrative regions. MIRU-VNTR loci 4052, 802, 2996, 2163b, 3690, 1955, 424, 2531, 2401 and 960 were highly discriminative in our setting (HGDI >0.6).24-locus MIRU-VNTR typing can substantially improve the resolution of large clusters initially defined by spoligotyping alone and predominating in Morocco, and could therefore be used to better study tuberculosis transmission in a population-based, multi-year sample context

Detailed Results obtained including demographic, drug-resistance and genotyping information on 5 Clusters and 69 unique patterns defined by identical spoligotyping and 24-loci MIRU from 75 <i>M</i>.<i>tuberculosis</i> strain isolated in Morocco.

<p>ID: Identifying number</p><p>DST: Drug SusceptibilityTesting</p><p>MLVA-MtbC15-9: Multi Locus Variant Allele-Mycobacterium tuberculosis complex15-9</p><p>Detailed Results obtained including demographic, drug-resistance and genotyping information on 5 Clusters and 69 unique patterns defined by identical spoligotyping and 24-loci MIRU from 75 <i>M</i>.<i>tuberculosis</i> strain isolated in Morocco.</p

Genetic tree based on spoligotyping and 24-locus MIRU-VNTR data of 75 <i>M</i>. <i>tuberculosis</i> isolates from10 Moroccan cities.

<p>A dendogram was generated using the UPGMA algorithm using tools available from the MIRU-VNTR<i>plus</i> identification database (see text). Isolates are identified according to their corresponding spoligotype international type (SIT; boxed), according to the SITVIT database.</p

Detailed Results obtained including demographic, drug-resistance and genotyping information on 5 Clusters and 69 unique patterns defined by identical spoligotyping and 24-loci MIRU from 75 <i>M</i>.<i>tuberculosis</i> strain isolated in Morocco.

<p>ID: Identifying number</p><p>DST: Drug SusceptibilityTesting</p><p>MLVA-MtbC15-9: Multi Locus Variant Allele-Mycobacterium tuberculosis complex15-9</p><p>Detailed Results obtained including demographic, drug-resistance and genotyping information on 5 Clusters and 69 unique patterns defined by identical spoligotyping and 24-loci MIRU from 75 <i>M</i>.<i>tuberculosis</i> strain isolated in Morocco.</p

Detailed Results obtained including demoraphic, drug-resistance and genotyping information on 5 clusters including 11 isolates defined by 24-loci MIRU patterns from 75 <i>M</i>.<i>tuberculosis</i> strain isolated in Morocco.

<p>ID: Identifying number</p><p>MLVA-MtbC15-9: Multi Locus Variant Allele-Mycobacterium tuberculosis complex15-9</p><p>Detailed Results obtained including demoraphic, drug-resistance and genotyping information on 5 clusters including 11 isolates defined by 24-loci MIRU patterns from 75 <i>M</i>.<i>tuberculosis</i> strain isolated in Morocco.</p