We evaluated the upgraded Amplified Mycobacterium Tuberculosis Direct Test kit (AMTD) (Gen-Probe Inc.) for the direct detection of
Mycobacterium tuberculosis in respiratory and non-respiratory ...specimens, and compared the results between the traditional 30,000 RLUs cutoff criteria (C) and three equivocal ranges (30,000–100,000, R1; 30,000–500,000, R2; and 30,000–1,000,000, R3). We tested 663 respiratory and 238 non-respiratory samples from 464 patients. The gold standard was considered to be the combination of culture and clinical data. One hundred and nineteen samples were from 56 patients with pulmonary tuberculosis, and 36 samples were from 19 patients with extrapulmonary tuberculosis. When C criteria was applied, the sensitivity and specificity values were 90.8 and 93.0% for respiratory specimens, while they were 88.9 and 92.1% for non-respiratory specimens (
p = NS). The sensitivity was significantly higher in smear-positive specimens (96.7%) than in smear-negative ones (81.0%) (
p < 0.05). When compared with C criteria, the overall sensitivity was maintained at 90.3% for R1 criteria, and slightly decreased to 89.7% for R2 and R3 criteria (
p = NS). Overall, specificity increased significantly from 92.9% (C) to 97.5% (R1), 99.1% (R2), and 99.2% (R3). Application of R2 or R3 criteria improved significantly the specificity of the test with little decrease in sensitivity.
RepA is the replication initiator protein of the Pseudomonas plasmid pPS10 and is also able to autoregulate its own synthesis. Here we report a genetic and functional analysis of a leucine ...zipper‐like (LZ) motif located at the N‐terminus of RepA. It is shown that the LZ motif modulates the equilibrium between monomeric and dimeric forms of the protein and that monomers of RepA interact with sequences at the origin of replication, oriV, while dimers are required for interactions of RepA at the repA promoter. Further, different residues of the LZ motif are seen to have different functional roles. Leucines at the d positions of the putative alpha‐helix are relevant in the formation of RepA dimers required for transcriptional autoregulation. They also modulate other RepA‐RepA interactions that result in cooperative binding of protein monomers to the origin of replication. The residues at the b/f positions of the putative helix play no relevant role in RepA‐RepA interactions. These residues do not affect RepA autoregulation but do influence replication, as demonstrated by mutants that, without affecting binding to oriV, either increase the host range of the plasmid or are inactive in replication. It is proposed that residues in b/f positions play a relevant role in interactions between RepA and host replication factors.
The initiator protein of the plasmid pPS10, RepA, has a putative hellx-turn-hellx (HTH) motif at its C-terminal end. RepA dimers bind to an inverted repeat at the repA promoter (repAP) to ...autoregulate RepA synthesis. 7lsqb;D. Garcia de viedma, etal. (1996) EMBOJ. in press. RepA monomers bind to four direct repeats at the origin of replication (oriV) to initiate pPS10 replication This report shows that randomly generated mutations In RepA, associated with deficiencies in autoregulation, map either at the putative HTH motif or In its vicinity. These mutant proteins do not promote pPS10 replication and are severely affected in binding to both the repAP and ortV regions in vitro. Revertats of a mutant that map in the vicinity of the HTH motif have been obtained and correspond to a second amino acid substitution far upstream of the motif. However, reversion of mutants that map in the helices of the motif occurs less frequently, at least by an order of magnitude. All these data indicate that the helices of the HTH motif play an essential role in specific RepA-DNA interactions, although additional regions also seem to be Involved in DNA binding activity. Some mutations have slightly different effects in replication and autoregulation, suggesting that the role of the HTH motif in the interaction of RepA dimers or monomers with their respective DNA targets (IR or DR) is not the same.
ABSTRACT
The Latin American-Mediterranean (LAM) family of
Mycobacterium tuberculosis
is believed to be the cause of ∼15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage ...of the LAM family in Brazil by a single characteristic genomic deletion designated RD
Rio
. Using the Brazilian strains, we pinpoint an Ag85C
103
single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism RFLP analysis) that correctly identified all LAM family strains. Importantly, all RD
Rio
strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between “wild-type” and RD
Rio
strains, were then used to analyze an international collection of
M. tuberculosis
strains. RD
Rio
M. tuberculosis
was identified from four continents involving 11 countries. Phylogenetic analysis of the IS
6110
-RFLP patterns from representative RD
Rio
and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C
103
SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS
6110
element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RD
Rio
strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RD
Rio
sublineage.
The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of similar to 15% of tuberculosis cases worldwide. Previously, we defined a prevalent ...sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RD super(Rio). Using the Brazilian strains, we pinpoint an Ag85C super(103) single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism RFLP analysis) that correctly identified all LAM family strains. Importantly, all RD super(Rio) strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between "wild-type" and RD super(Rio) strains, were then used to analyze an international collection of M. tuberculosis strains. RD super(Rio) M. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RD super(Rio) and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C super(103) SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RD super(Rio) strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RD super(Rio) sublineage.
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