Detection and Quantification of HspX Antigen in Sputum Samples Using Plasmonic Biosensing: Toward a Real Point-of-Care (POC) for Tuberculosis Diagnosis.

Advancements that occurred during the last years in the diagnosis of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis infection, have prompted increased survival rates of patients. However, limitations related to the inefficiency of an early detection still remain; some techniques and laboratory methods do not have enough specificity and most instruments are expensive and require handling by trained staff. In order to contribute to a prompt and effective diagnosis of tuberculosis, we report the development of a portable, user-friendly, and low-cost biosensor device for its early detection. By using a label-free surface plasmon resonance (SPR) biosensor, we have established a direct immunoassay for the direct detection and quantification of the heat shock protein X (HspX) of Mtb, a well-established biomarker of this pathogen, directly in pretreated sputum samples. The method relies on highly specific monoclonal antibodies that are previously immobilized on the plasmonic sensor surface. This technology allows for the direct detection of the biomarker without amplification steps, showing a limit of detection (LOD) of 0.63 ng mL-1 and a limit of quantification (LOQ) of 2.12 ng mL-1. The direct analysis in pretreated sputum shows significant differences in the HspX concentration in patients with tuberculosis (with concentration levels in the order of 116-175 ng mL-1) compared with non-tuberculosis infected patients (values below the LOQ of the assay).

Genome analysis shows a common evolutionary origin for the dominant strains of Mycobacterium tuberculosis in a UK South Asian community.

We have investigated the Mycobacterium tuberculosis strain types present in the South Asian population of the UK, in which tuberculosis is particularly prevalent. In contrast to the widespread Beijing strains which have the variable number tandem repeats (VNTR) profile 42435, isolates with the VNTR profile 42235, jointly with 02335 or 42234 profiles, appear more frequently in tuberculosis patients of South Asian ethnic origin (SA-strains) in the UK than in any other ethnic group. Using microarray-based comparative genomics to distinguish total or partially deleted genes, we found that three of the common deleted regions in the SA-strains were identical to some deleted genes in the strain CH, which caused an outbreak among South Asian patients in Leicester in 2001 but were different from genomic deletions found in Beijing/W strains. Analysis of some of the deleted regions revealed differences in comparison to the strain CH including the polymorphism in some of the PE/PPE and Esat-6 genes, which may be responsible for the diversity of antigenic variation or differences in the activation of the host immune response. Interrupted genes or the replacement by insertion elements was confirmed in some of the deleted genomic regions. Our results are consistent with the hypothesis that the SA-strains may present common features, implying a common origin for this group of strains.

Molecular features of Mycobacterium avium human isolates carrying a single copy of IS1245 and IS1311 per genome.

Human clinical isolates of the Mycobacterium avium complex, from hospitals in Bogotá, were studied using a wide range of molecular tests including PCR restriction-enzyme analysis (PRA) of the hsp65 gene. Up to 21 of the isolates were identified as M. avium PRA variant III (Mav III), a variant obtained only from isolates on the American continent. In contrast to previous reports, restriction fragment length polymorphism analysis using IS1245 and IS1311 showed a single copy for each insertion sequence (IS) in the majority (19/21) of the Colombian Mav III isolates under study. In order to analyse whether the ISs were inserted in a relevant genomic region, experimental conditions were established to determine the insertion loci of each single copy of both ISs in the genome. Analysis of genomic insertion loci indicated that both IS1245 and IS1311 were present in areas containing putatively truncated integrases and/or transposases, which may have an influence on the mobility of the inserted IS. In addition, a conserved genomic region was identified for the insertion of IS1311; this region could be part of the IS1311 itself.

Alignment of multiple complete genomes suggests that gene rearrangements may contribute towards the speciation of Mycobacteria.

To more accurately define the taxonomic relationships among species belonging to the genus Mycobacterium we have applied and compared three complete genome sequence comparison procedures to existing systems. These included a nucleotide sequence comparison including both coding and no-coding regions of the genome and two genomic-order comparisons using MAUVE and M-GCAT software to provide comparative gene synteny. These methods clearly differentiated a panel of genomes from reference mycobacterial species. Overall, the speciation of bacteria through determination of gene rearrangements were consistent with the gold standard method for species definition in bacteria, DNA-DNA hybridization however within the context of this system, individual components of the Mycobacterium tuberculosis complex (MTBC) did not show sufficient diversity to classify them as a separate species. The high number of gene rearrangements observed between the species tested suggests that gene reorganization of the genome represents an important contributor to speciation within the genus Mycobacterium and other related genera. The absence of rearrangements amongst MTBC supports their consideration as a single genospecies. Some gene rearrangements provided clear internal synteny between genomes of mycobacterial strains belonging to a same species and we suggest these could be used to classify subspecies.

Mycobacterium colombiense sp. nov., a novel member of the Mycobacterium avium complex and description of MAC-X as a new ITS genetic variant.

Forty-five mycobacterial strains isolated from 23 Colombian HIV-positive patients were identified as members of the Mycobacterium avium complex (MAC) and were characterized using different molecular approaches. Seven of the isolates showed characteristic features that allowed them to be differentiated from other members of the complex. The isolates had a novel 16S-23S rRNA internal transcribed spacer (ITS 1) gene sequence which is described as a new sequevar, MAC-X. All of the seven novel isolates gave a positive result with the MAC-specific AccuProbe (Gen-Probe), but tested negative for Mycobacterium avium and Mycobacterium intracellulare species-specific probes (64 and 100 % of the isolates, respectively). The novel isolates could be differentiated phenotypically from other members of the MAC on the basis of the production of urease and by a consistent mycolic acid pattern. The novel isolates shared some characteristics with M. avium, such as the avium variant I (av-I) pattern of the hsp65 gene as determined by PCR restriction analysis and a positive PCR result for the mig (macrophage-induced) gene. However, the novel isolates showed a unique 16S rRNA gene sequence. DNA-DNA relatedness values, from 24 to 44 %, confirmed the distinction of the novel isolates from other members of the MAC at the genetic level and their status as members of a separate species. The novel isolates are proposed as representatives of a novel species, Mycobacterium colombiense sp. nov., that is closely related to M. avium within the MAC. The type strain is 10B(T) (=CIP 108962(T)=CECT 3035(T)).

A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor.

Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrow-derived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.

IS6110 mediates increased transcription of the phoP virulence gene in a multidrug-resistant clinical isolate responsible for tuberculosis outbreaks.

Drug resistance in Mycobacterium tuberculosis complex strains is solely due to chromosomal mutations that could affect bacterial virulence. Molecular epidemiology studies have shown that resistant strains are less likely to be clustered than susceptible strains. However, a few multidrug-resistant (MDR) M. tuberculosis complex strains have been described as causing outbreaks, suggesting that they have restored virulence or increased transmission. One of the biggest MDR tuberculosis outbreaks documented to date was caused by the B strain of M. bovis. Restriction fragment length polymorphism fingerprinting revealed that the B strain contains two copies of IS6110. Here, we mapped and sequenced the regions flanking the two copies of IS6110 in the B strain. Ligation-mediated PCR showed that one of these IS6110 copies is located within the promoter region of phoP, a transcriptional regulator that is essential for M. tuberculosis virulence. We used PCR to screen 219 MDR M. tuberculosis complex strains (90.4% of all MDR isolates) isolated in Spain between 1998 and 2002 and found that the B strain was the only strain that contained a copy of IS6110 in the phoP promoter. To determine whether IS6110 affects phoP promoter activity in the B strain, we individually cloned the phoP gene and its promoter region (including IS6110 from the B strain and the equivalent region from M. tuberculosis without IS6110 as a control) into a mycobacterial replicative plasmid and transformed M. smegmatis with the resulting plasmid. Primer extension analysis showed that phoP transcription was strongly upregulated when the promoter region contained IS6110, as in the case of the B strain.