[Show abstract][Hide abstract] ABSTRACT: Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem. Early diagnosis of MDR-TB patients is essential for minimizing the risk of Mycobacterium tuberculosis (MTB) transmission. The conventional drug susceptibility testing (DST) methods for detection of drug-resistant M.tuberculosis are laborious and cannot provide the rapid detection for clinical practice.
The aim of this study was to develop a pyrosequencing approach for the simultaneous detection of resistance to rifampin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (SM), ofloxacin (OFL) and amikacin (AMK) in M. tuberculosis clinical isolates and sputum samples from re-treatment pulmonary tuberculosis ( PTB) patients. We identified the optimum conditions for detection mutation of rpoB, katG, rpsl, embB, gyrA and rrs gene by pyrosequencing. Then this approach was applied to detect 205 clinical isolates and 24 sputum samples of M. tuberculosis from re-treatment PTB patients.
The mutations of rpoB and gyrA gene were detected by pyrosequencig with the SQA mode, and the mutations of katG, rpsl, embB, gyrA and rrs gene were detected by pyrosequencing with SNP mode. Compared with the Bactec MGIT 960 mycobacterial detection system, the accuracy of pyrosequencing for the detection of RIF, INH, EMB, SM, AMK and OFL resistance in clinical isolates was 95.0%, 79.2%, 70.3%, 84.5%, 96.5% and 91.1%, respectively. In sputum samples the accuracy was 83.3%, 83.3%, 60.9%, 83.3%, 87.5% and 91.7%, respectively.
The newly established pyrosequencing assay is a rapid and high-throughput method for the detection of resistance to RIF, INH, SM, EMB, OFL and AMK in M.tuberculosis. Pyrosequencing can be used as a practical molecular diagnostic tool for screening and predicting the resistance of re-treatment pulmonary tuberculosis patients.
[Show abstract][Hide abstract] ABSTRACT: Accurate and early diagnosis of tuberculosis (TB) is of major importance in the control of TB. One of the most important technical advances in diagnosis of tuberculosis is the development of nucleic acid amplification (NAA) tests. However, the choice of the target sequence remains controversial in NAA tests. Recently, interesting alternatives have been found in hypothetical protein coding sequences from mycobacterial genome.
To obtain rational biomarker for TB diagnosis, the conservation of three hypothetical genes was firstly evaluated in 714 mycobacterial strains. The results showed that SCAR1 (Sequenced Characterized Amplified Region) based on Rv0264c coding gene showed the highest conservation (99.8%) and SCAR2 based on Rv1508c gene showed the secondary high conservation (99.7%) in M. tuberculosis (MTB) strains. SCAR3 based on Rv2135c gene (3.2%) and IS6110 (8%) showed relatively high deletion rate in MTB strains. Secondly, three SCAR markers were evaluated in 307 clinical sputum from patients in whom TB was suspected or patients with diseases other than TB. The amplification of IS6110 and 16SrRNA sequences together with both clinical and bacteriological identification was as a protocol to evaluate the efficacy of SCAR markers. The sensitivities and specificities, positive predictive value (PPV) and negative predictive value (NPV) of all NAA tests were higher than those of bacteriological detection. In four NAA tests, IS6110 and SCAR3 showed the highest PPV (100%) and low NPV (70% and 68.8%, respectively), and SCAR1 and SCAR2 showed the relatively high PPV and NPV (97% and 82.6%, 95.6% and 88.8%, respectively).
Our result indicated that SCAR1 and SCAR2 with a high degree of sequence conservation represent efficient and promising alternatives as NAA test targets in identification of MTB. Moreover, the targets developed from this study may provide more alternative targets for the development of a multisite system to effectively detect MTB in samples.
PLoS ONE 01/2013; 8(9):e73955. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 10-kDa culture filtrate protein (CFP10) and 6-kDa early-secreted target antigen (ESAT-6) play important roles in mycobacterial virulence and pathogenesis through a 1∶1 complex formation (CFP10/ESAT-6 protein, CE protein), which have been used in discriminating TB patients from BCG-vaccinated individuals. The B-cell epitopes of CFP10 and ESAT-6 separately have been analyzed before, however, the epitopes of the CE protein are unclear and the precise epitope in the positions 40 to 62 of ESAT-6 is still unknown.
In the present study, we searched for the B-cell epitopes of CE protein by using phage-display library biopanning with the anti-CE polyclonal antibodies. The epitopes were identified by sequence alignment, binding affinity and specificity detection, generation of polyclonal mouse sera and detection of TB patient sera.
One linear B-cell epitope (KWDAT) consistent with the 162(nd)-166(th) sequence of CE and the 57(th)-61(st) sequence of ESAT-6 protein was selected and identified. Significantly higher titers of E5 peptide-binding antibodies were found in the sera of TB patients compared with those of healthy individuals.
There was a B-cell epitope for CE and ESAT-6 protein in the position 40 to 62 of ESAT-6. E5 peptide may be useful in the serodiagnosis of tuberculosis, which need to be further confirmed by more sera samples.
PLoS ONE 01/2013; 8(1):e52848. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tuberculous pleurisy is one of the common extrapulmonary tuberculosis diseases. However, the diagnosis of tuberculous pleurisy still lacks a useful and effective tool, mainly due to paucity of Mycobacterium tuberculosis organisms in pleural effusion. Previous studies have confirmed that the MPT64 protein is highly specific and is secreted only by M. tuberculosis (MTB) complex. Therefore, in this study, we developed ELISA based on recombinantly expressed MPT64 in combination with rabbit polyclonal antibodies. The ELISA-MPT64 method was validated using MTB strains and tested against clinical samples. Nested PCR, Löwenstein-Jensen (L-J) culture and smear microscopy were employed as the comparative tools for assessing the performance of the assay. Our results demonstrate that the newly established ELISA-MPT64 technique is a rapid and useful tool for the diagnosis of tuberculous pleurisy.
Current Microbiology 06/2012; 65(3):313-8. · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Presently, tuberculosis (TB) poses a global threat to human health. The development of reliable laboratory tools is vital to the diagnosis and treatment of TB. MPT64, a protein secreted by Mycobacterium tuberculosis complex, is highly specific for TB, making antibody to MPT64 a reagent specific for the diagnosis of TB.
Antibody to MPT64 was obtained by a combination of genetic engineering and immunization by the system evolution of ligands by exponential enrichment. A high-affinity aptamer of antibody to MPT64 was selected from a random single-stranded DNA library, and a sandwich ELISA method based on this aptamer was developed. This ELISA method was used to detect TB in 328 serum samples, 160 from patients with pulmonary TB (PTB) and 168 from non-tuberculous controls.
The minimum limit of detection of the ELISA method was 2.5 mg/L, and its linear range varied from 10 mg/L to 800 mg/L. Its sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and area under the curve, with 95 % confidence intervals, were 64.4 % (56.7 %-71.4 %), 99.4 % (96.7 %-99.9 %), 108.2 (15.3-765.9), 0.350 (0.291-0.442) and 0.819 (0.770-0.868), respectively. No significant difference in sensitivity was observed between sputum smear positive (73/112, 65.2 %) and negative (30/48, 62.5 %) individuals.
This sandwich ELISA based on an MPT64 antibody aptamer may be useful for the serological diagnosis of PTB, both in sputum smear positive and negative patients.
[Show abstract][Hide abstract] ABSTRACT: The mycobacterial genome is inclined to polymerase slippage and a high mutation rate in microsatellite regions due to high GC content and absence of a mismatch repair system. However, the exact molecular mechanisms underlying microsatellite variation have not been fully elucidated. Here, we investigated mutation events in the hyper-variable trinucleotide microsatellite locus MML0050 located in the Rv0050 gene of W-Beijing and non-W-Beijing Mycobacterium tuberculosis strains in order to gain insight into the genomic structure and activity of repeated regions.
Size analysis indicated the presence of five alleles that differed in length by three base pairs. Moreover, nucleotide gains occurred more frequently than loses in this trinucleotide microsatellite. Mutation frequency was not completely related with the total length, though the relative frequency in the longest allele was remarkably higher than that in the shortest. Sequence analysis was able to detect seven alleles and revealed that point mutations enhanced the level of locus variation. Introduction of an interruptive motif correlated with the total allele length and genetic lineage, rather than the length of the longest stretch of perfect repeats. Finally, the level of locus variation was drastically different between the two genetic lineages.
The Rv0050 locus encodes the bifunctional penicillin-binding protein ponA1 and is essential to mycobacterial survival. Our investigations of this particularly dynamic genomic region provide insights into the overall mode of microsatellite evolution. Specifically, replication slippage was implicated in the mutational process of this microsatellite and a sequence-based genetic analysis was necessary to determine that point mutation events acted to maintain microsatellite size integrity while providing genomic diversity.
[Show abstract][Hide abstract] ABSTRACT: Objective
To evaluate the performance of phage amplified biologically assay (PhaB) for detecting tuberculosis (TB) in sputum in the pulmonary tuberculosis (PTB) patients.Methods
Shanghai Tuberculosis Key Laboratory of Shanghai Pulmonary Hospital participated in the project in collaboration with the laboratories of six hospitals and a total of 1660 eligible participants (1351 PTB patients and 309 non-TB patients) were included in the study. The sputum samples from the participants were detected by smear microscopy, PhaB, and Löwenstein-Jensen (L-J) culture method, respectively.ResultsThe overall sensitivity of PhaB were higher than that of L-J culture and smear microscopy (p
[Show abstract][Hide abstract] ABSTRACT: Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is an important receptor for Mycobacterium tuberculosis on human dendritic cells. Previous studies have shown that the variation, especially the -871A/G and -336A/G in DC-SIGN promoter influenced the susceptibility to tuberculosis. We therefore investigated whether polymorphisms in the DC-SIGN gene were associated with susceptibility to tuberculosis in an eastern Chinese population. A total of 237 culture-positive pulmonary tuberculosis case patients and 244 controls were genotyped for -871A/G and -336A/G by pyrosequencing. Our results suggested that the 2 promoter variants of DC-SIGN gene were not associated with susceptibility to tuberculosis in Chinese. Further analysis showed that the allele -336G was associated with a protective effect against fever in pulmonary tuberculosis patients, but not against cavity formation. In addition, we compared the allelic frequencies of -871A/G and -336A/G in African, Caucasian, and Asian groups. The results showed that the tw forms of allelic frequencies detected Chinese individuals in our study were similar to the reported frequencies in other Asian populations but differed significantly from those in the African and Caucasian groups studied.
Human immunology 11/2010; 72(2):183-6. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accurate and early diagnosis of tuberculosis (TB) is of major importance in the management and control of TB. Because the conventional bacteriological diagnosis of TB has several limitations, nucleic acid amplification (NAA) tests have emerged as promising alternatives. A potential problem with NAA tests is that some strains lack a target, which may be the one of main reasons for the much lower and highly variable accuracy in diagnosis. A possible solution may be to use more valid and applicable targets to increase detection accuracy.
In this paper, we designed a two-step program to obtain NAA test targets. Inter-simple sequence repeats (ISSR) based on oligonucleotide (GTG)(5) were first constructed to genotype Mycobacterium strains to obtain Mycobacterium tuberculosis (MTB)-specific fragment. Second, sequence characterized amplified region (SCAR) markers were developed from these species-specific sequences to identify MTB. Some 312 Mycobacterium strains were used to evaluate the efficacy of the SCAR markers, IS6110 element [specific identification of Mycobacterium tuberculosis complex (MTC)] and 16SrRNA gene (specific identification of Mycobacterium) amplification, together with traditional bacteriology testing was used as a control.
MTB-specific sequences located in a gene coding for Rv1508c, as a new NAA test target, were obtained using ISSR-PCR genotyping. Based on these sequences, the SCAR primer pairs MISP1 and MISP2 were designed. All 312 strains from Mycobacterium accurately produced the genus-specific 16SrRNA amplicon. 271 MTB strains and M. africanum were positive. However, all nontuberculous mycobacteria (NTM) strains and 1 MTB strain named 1143 were negative in both SCAR and IS6110 PCR amplification. M. bovis, bacille Calmette-Guérin (BCG) were IS6110-PCR positive, while SCAR-PCR was negative. Strain 1143 was defined as M. arupense with 99% identity by 16SrRNA gene sequencing identification, despite being diagnosed as MTB using traditional testing.
SCAR markers developed with this two-step program can be used as a new NAA test target to correctly detect MTB.
Clinical Chemistry and Laboratory Medicine 10/2010; 48(10):1501-5. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) remains a major health problem affecting millions of people worldwide. One-third of the world's population is infected with Mycobacterium tuberculosis, the etiologic agent of TB. A simple and rapid method to diagnose TB is urgently needed to be developed. The procedure of systematic evolution of ligands by exponential enrichment (SELEX) is a method in which single-stranded oligonucleotides (called aptamers) are selected from a wide variety of sequences, based on their interaction with a target molecule. Aptamers have been used in numerous investigations as therapeutic or diagnostic tools.
In this study, we apply a SELEX method to develop aptamers against MPT64 protein from M. tuberculosis. On this basis, a sandwich assay scheme with the complex of aptamer-MPT64 was designed and tested the feasibility of detecting M. tuberculosis by detecting MPT64 protein levels in the culture filtrates of 77 samples including M. tuberculosis and other Mycobacterium species.
There was a highly significant difference (p<0.01) between group A (non-TB Mycobacterium, bacille Calmette-Guérin) and group B (M. tuberculosis, M. bovis), when they were diagnosed with the sandwich assay scheme based on aptamer-protein complex to detect MPT64 protein levels in the culture filtrates of samples. When the cut-off point was at the optical density value of 0.58 (95%=0.764-0.946; Z=6.130, p=0.0001), the sandwich assay scheme based on aptamer-protein complex had a high sensitivity (negative ration, 24/27, 86.3%) and specificity (positive ration, 46/52, 88.5%).
Aptamer of MPT64 as a new detection tool, to a certain extent, is feasible to diagnose Mycobacterium tuberculosis.
Clinical Chemistry and Laboratory Medicine 02/2009; 47(4):405-11. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Influence of the recombinant culture filtered protein 10 (CFP-10) and early-secreted antigenic target 6kDa protein (ESAT-6) (r-CFP-10-ESAT-6, rCE) of Mycobacterium tuberculosis (Mtb) on human monocyte and macrophage activation was investigated using human monocyte, monocyte like THP-1 cell line and monocyte derived macrophage (MDM). rCE solely enhanced TNF-alpha release from human monocytes and THP-1 cells in a dose- and time-dependent manner. rCE enhanced expression of CD80 and CD40, it also synergized with IFN-gamma in induction of TNF-alpha production and HLA-DR expression. Pharmacological agents that selectively inhibit mitogen activated protein kinase activation markedly suppressed rCE-induced TNF- alpha release. However, continuous presence of rCE (>72h) during monocyte to macrophage differentiation inhibited macrophage response to LPS stimulation. Collectively, these data suggest that rCE might have differential influence on monocyte and macrophage activation, which might be correlated with Mtb immune evasion.
Biochemical and Biophysical Research Communications 01/2008; 365(3):534-40. · 2.28 Impact Factor