Molecular detection, identification and drug resistance detection in Mycobacterium tuberculosis

This minireview presents recent developments in molecular methods for the diagnosis of tuberculosis, including detection, identification and determination of drug resistance of Mycobacterium tuberculosis . Tuberculosis remains one of the major causes of global death from a single infectious agent. This situation is worsened by the HIV/AIDS pandemic because one-third of HIV/AIDS patients are coinfected with M. tuberculosis . Also of great concern is the emergence of drug-resistant tuberculosis because there are almost no treatment options available for patients affected by highly resistant strains of M. tuberculosis . Advances in molecular biology techniques and a better knowledge of the molecular mechanisms of drug resistance have provided new tools for the rapid diagnosis of tuberculosis. Several nucleic acid amplification technologies have been developed and evaluated. New molecular approaches are being introduced continuously. This minireview will also comment on the future perspectives for the molecular diagnosis of tuberculosis and the feasibility for the implementation of these newer techniques in the clinical diagnostic laboratory.     

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTEC^TM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTEC^TM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTEC^TM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.     

Electrochemical-based biosensors for detection of Mycobacterium tuberculosis and tuberculosis biomarkers

Abstract

Early detection of tuberculosis (TB) reduces the interval between infection and the beginning of treatment. However, commercially available tests cannot discriminate between BCG-vaccinated healthy persons and patients. Also, they are not suitable to be used for immunocompromised persons. In recent years, biosensors have attracted great attention due to their simple utility, accessibility, and real-time outputs. These sensors are increasingly being considered as pioneering tools for point-of-care diagnostics in communities with a high burden of TB and limited accessibility to reference laboratories. Among other types of biosensors, the electrochemical sensors have the advantages of low-cost operation, fast processing, simultaneous multi-analyte analyzing, operating with turbid samples, comparable sensitivity and readily available miniaturization. Electrochemical biosensors are sub-divided into several categories including: amperometric, impedimetric, potentiometric, and conductometric biosensors. The biorecognition element in electrochemical biosensors is usually based on antibodies (immunosensors), DNAs or PNAs (genosensors), and aptamers (aptasensors). In either case, whether an interaction of the antigen–antibody/aptamer or the hybridization of probe with target mycobacterial DNA is detected, a change in the electrical current occurs that is recorded and displayed as a plot. Therefore, impedimetric-based methods evaluate resistance to electron transfer toward an electrode by a Nyquist plot and amperometric/voltammetric-based methods weigh the electrical current by means of cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Electrochemical biosensors provide a promising scope for the new era of diagnostics. As a consequence, they can improve detection of Mycobacterium tuberculosis traces even in attomolar scales.     

应用PCR技术检测结核分枝杆菌的临床分析

采用聚合酶链反应（Ｐｏｌｙｍｅｒａｎｓｅ Ｃｈｉａｎ Ｒｅａｔｉｏｎ,即ＰＣＲ）技术检测结核分枝杆菌Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｔｕｂｅｒｃｕｌｏｓｉｓ,一年多来共检测了１００例结核（肺、肾结核）患者的痰和尿液标本,结果ＰＣＲ检出阳性率为８１％,对照用储菌涂片抗酸染色法,阳性率为５８％,用常规培养法阳性率为２０％。而对５０例非结核患者的痰和尿液标本的检测,ＰＣＲ法仍有６％的阳性率,而用涂片或常规培     

IRF1 as a potential biomarker in Mycobacterium tuberculosis infection

Pulmonary tuberculosis (PTB) is a major global public health problem. The purpose of this study was to find biomarkers that can be used to diagnose tuberculosis. We used four NCBI GEO data sets to conduct analysis. Among the four data sets, GSE139825 is lung tissue microarray, and GSE83456 , GSE19491 and GSE50834 are blood microarray. The differential genes of GSE139825 and GSE83456 were 68 and 226, and intersection genes were 11. Gene ontology (GO) analyses of 11 intersection genes revealed that the changes were mostly enriched in regulation of leucocyte cell-cell adhesion and regulation of T-cell activation. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs revealed that the host response in TB strongly involves cytokine-cytokine receptor interactions and folate biosynthesis. In order to further narrow the range of biomarkers, we used protein-protein interaction to establish a hub gene network of two data sets and a network of 11 candidate genes. Eventually, IRF1 was selected as a biomarker. As validation, IRF1 levels were shown to be up-regulated in patients with TB relative to healthy controls in data sets GSE19491 and GSE50834 . Additionally, IRF1 levels were measured in the new patient samples using ELISA. IRF1 was seen to be significantly up-regulated in patients with TB compared with healthy controls with an AUC of 0.801. These results collectively indicate that IRF1 could serve as a new biomarker for the diagnosis of pulmonary tuberculosis.     

Small non‐coding RNA and colorectal cancer

Colorectal cancer (CRC) is the third most common malignance. Although great efforts have been made to understand the pathogenesis of CRC, the underlying mechanisms are still unclear. It is now clear that more than 90% of the total genome is actively transcribed, but lack of protein‐coding potential. The massive amount of RNA can be classified as housekeeping RNAs (such as ribosomal RNAs, transfer RNAs) and regulatory RNAs (such as microRNAs [miRNAs], PIWI‐interacting RNA [piRNAs], tRNA‐derived stress‐induced RNA, tRNA‐derived small RNA [tRFs] and long non‐coding RNAs [lncRNAs]). Small non‐coding RNAs are a group of ncRNAs with the length no more than 200 nt and they have been found to exert important regulatory functions under many pathological conditions. In this review, we summarize the biogenesis and functions of regulatory sncRNAs, such as miRNAs, piRNA and tRFs, and highlight their involvements in cancers, particularly in CRC.     

RNA polymerase II acts as an RNA‐dependent RNA polymerase to extend and destabilize a non‐coding RNA

RNA polymerase II (Pol II) is a well‐characterized DNA‐dependent RNA polymerase, which has also been reported to have RNA‐dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non‐coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3′‐end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α‐amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3′‐end.     

A novel genotypic test for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates by a multiplex probe array

AIMS: To develop and evaluate a novel genotypic test for rapid detection of rifampicin and isoniazid resistance of multidrug-resistant (MDR) Mycobacterium tuberculosis isolates by a multiplex probe array. METHODS AND RESULTS: A multiplex probe array was designed for genotypic test to simultaneously screen the mutations of rpoB, katG, inhA and ahpC genes, associated with rifampin and isoniazid resistance in M. tuberculosis, with a probe detecting one of the recently confirmed genetic markers of isoniazid resistance ahpC-6 and -9 locus added. By using the genotypic test developed, 52 MDR isolates were identified, among which 46 isolates had mutations in rpoB (88.5%) and 45 at codon 315 of katG, regulatory region of inhA and oxyR-ahpC intergenic region (86.5%), whereas all 35 susceptible isolates identified showed a wild-type hybridization pattern. The sensitivity and specificity were 88.5% and 100% for rifampicin resistance, and 86.5% and 100% for isoniazid resistance, respectively. CONCLUSION: A rapid and simultaneous detection of rifampicin and isoniazid resistance caused by the mutations of rpoB, katG, inhA and ahpC genes in M. tuberculosis isolates could be achieved by a multiplex probe array developed. SIGNIFICANCE AND IMPACT OF THE STUDY: This genotypic test protocol has the potential to be developed on clinical application for the rapid detection of drug resistant M. tuberculosis isolates before an efficient chemotherapy is initiated.     

Mass spectrometry applied to the identification of Mycobacterium tuberculosis and biomarker discovery

An adequate and effective tuberculosis (TB) diagnosis system has been identified by the World Health Organization as a priority in the fight against this disease. Over the years, several methods have been developed to identify the bacillus, but bacterial culture remains one of the most affordable methods for most countries. For rapid and accurate identification, however, it is more feasible to implement molecular techniques, taking advantage of the availability of public databases containing protein sequences. Mass spectrometry (MS) has become an interesting technique for the identification of TB. Here, we review some of the most widely employed methods for identifying Mycobacterium tuberculosis and present an update on MS applied for the identification of mycobacterial species.     

Cloning,expression, and functional characterization of the Mycobacterium tuberculosis secA gene

To better understand the protein secretion mechanisms involved in the growth and pathogenesis of Mycobacterium tuberculosis, we examined the secA gene from M. tuberculosis (tbsecA; cosmid sequence accession No. z95121.gb_ba). We generated plasmids containing the full-length tbsecA gene or a fusion containing the 5' sequence from the M. tuberculosis secA gene and the remainder from the Escherichia coli secA gene and evaluated the ability of each construct to complement the defective SecA protein in E. coli MM52ts when grown at the non-permissive temperature. The full-length tbsecA gene was unable to compensate for the temperature-sensitive defect, whereas E. coli MM52ts that has been transformed with plasmid pMF8TB226 containing a chimeric secA gene was able to grow at 42 degrees C. This work confirms that the topography of SecA and its ATP binding sites are highly conserved, whereas its membrane insertion domains are species specific.     

Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop‐mediated isothermal amplification assay

Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug‐resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing‐specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time‐consuming and technically demanding. In the present study, a loop‐mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain‐identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c‐targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207‐PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c‐multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains.     

Long non‐coding RNAs in non‐small cell lung cancer as biomarkers and therapeutic targets

Lung cancer‐associated mortality is the most common cause of cancer death worldwide. Non‐coding RNAs (ncRNAs), with no protein‐coding ability, have multiple biological roles. Long non‐coding RNAs (lncRNAs) are a recently characterized class of ncRNAs that are over 200 nucleotides in length. Many lncRNAs have the ability of facilitating or inhibiting the development and progression of tumours, including non‐small cell lung cancer (NSCLC). Because of their fundamental roles in regulating gene expression, along with their involvement in the biological mechanisms underlying tumourigenesis, they are a promising class of tissue‐ and/or blood‐based cancer biomarkers. In this review, we highlight the emerging roles of lncRNAs in NSCLC, and discuss their potential clinical applications as diagnostic and prognostic markers and as therapeutic targets.     

Identification and validation of l-asparaginase as a potential metabolic target against Mycobacterium tuberculosis

Multidrug-resistant Mycobacterium tuberculosis (Mtb) has emerged as a major health challenge, necessitating the search for new molecular targets. A secretory amidohydrolase, l -asparaginase of Mtb (MtA), originally implicated in nitrogen assimilation and neutralization of acidic microenvironment inside human alveolar macrophages, has been proposed as a crucial metabolic enzyme. To investigate whether this enzyme could serve as a potential drug target, it was studied for structural details and active site–specific inhibitors were tested on cultured Mycobacterial strain. The structural details of MtA obtained through comparative modeling and molecular dynamics simulations provided insights about the orchestration of an alternate reaction mechanism at the active site. This was contrary to the critical Tyr flipping mechanism reported in other asparaginases. We report the novel finding of Tyr to Val replacement in catalytic triad I along with the structural reorganization of a β-hairpin loop upon substrate binding in MtA active site. Further, 5 MtA-specific, active-site–based inhibitors were obtained by following a rigorous differential screening protocol. When tested on Mycobacterium culture, 3 of these, M3 (ZINC 4740895), M26 (ZINC 33535), and doxorubicin showed promising results with inhibitory concentrations (IC ₅₀) of 431, 100, and 56 µM, respectively. Based on our findings and considering stark differences with human asparaginase, we project MtA as a promising molecular target against which the selected inhibitors may be used to counteract Mtb infection effectively.