|Year : 2020 | Volume
| Issue : 4 | Page : 93-99
Molecular diagnosis of tuberculosis with emphasis on Xpert Mycobacterium tuberculosis assay – Clinical review
Gajanan S Gaude, Samskruti Vishwanath
Department of Pulmonary Medicine, J. N. Medical College, KAHER's, Belgaum, Karnataka, India
|Date of Submission||14-Jul-2019|
|Date of Acceptance||26-May-2020|
|Date of Web Publication||19-Oct-2020|
Dr. Gajanan S Gaude
Department of Pulmonary Medicine, J. N. Medical College, KAHER's, Belgaum - 590 010, Karnataka
Source of Support: None, Conflict of Interest: None
Tuberculosis (TB), due to Mycobacterium tuberculosis(MTB), remains a major public health issue. It causes ill health for approximately 10 million people each year and is one of the top ten causes of death worldwide. For the past 5 years, it has been the leading cause of death from a single infectious agent, ranking above HIV/AIDS. Effective diagnosis of pulmonary TB requires the availability – on a global scale – of standardized, easy-to-use, and robust diagnostic tool that would allow the direct detection of both the MTB complex and the resistance to key drugs, such as rifampicin. The latter result can serve as a marker for multidrug-resistant (MDR) MTB and has been reported in >95% of the MDR-TB isolates. Here, we review some of the recent molecular methods in the diagnosis of TB.
Keywords: GeneXpert test, line probe assay, molecular methods, tuberculosis
|How to cite this article:|
Gaude GS, Vishwanath S. Molecular diagnosis of tuberculosis with emphasis on Xpert Mycobacterium tuberculosis assay – Clinical review. J Clin Sci 2020;17:93-9
|How to cite this URL:|
Gaude GS, Vishwanath S. Molecular diagnosis of tuberculosis with emphasis on Xpert Mycobacterium tuberculosis assay – Clinical review. J Clin Sci [serial online] 2020 [cited 2020 Nov 26];17:93-9. Available from: https://www.jcsjournal.org/text.asp?2020/17/4/93/298456
| Introduction|| |
Tuberculosis (TB), due to Mycobacterium tuberculosis (MTB), remains a major public health issue. It causes ill health for approximately 10 million people each year and is one of the top ten causes of death worldwide. For the past 5 years, it has been the leading cause of death from a single infectious agent, ranking above HIV/AIDS. India is one among the six countries that account for 60% of all new TB cases, worldwide. In 2016, there were an estimated 1.3 million TB deaths among HIV-negative people and an additional 374,000 deaths among HIV-positive people, and drug-resistant TB is a persistent threat, with 490,000 million cases of multidrug-resistant TB (MDR-TB) emerging in 2016. The rapid detection of MTB in the respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopic methods. As much as 50%–60% of acid-fast bacilli (AFB) culture-positive clinical specimens may fail to reveal AFB on the smear made from the specimen. This leads to ineffective TB detection and the emergence and transmission of drug-resistant MTB strains, thus jeopardizing global TB control activities. Effective diagnosis of pulmonary TB (PTB) requires the availability – on a global scale – of standardized, easy-to-use, and robust diagnostic tool that would allow the direct detection of both the MTB complex and the resistance to anti-TB drugs. The rapid availability of reliable test will result into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community. The objective of the present review is to assess the availability of various genetic-based tests in the diagnosis of MTB and to assess their utility in day-to-day management of the TB cases, including drug-resistant MTB.
Genetic basis of diagnosis of tuberculosis
The greatest breakthrough, as for the entire field of infectious diseases, came from biotechnology, with the introduction of nucleic acid amplification techniques (NATs)., Gene amplification can achieve the goal of reducing the generation time of microorganisms to minutes and the goal of replacing biological growth on artificial media by the enzymatic reproduction of nucleic acids in vitro. The importance of nucleic acid amplification methods lies in their wide applicability in the life sciences and their potential to revolutionize the practice of medicine. Examples are nucleic acid sequence analysis and genetic fingerprinting. To understand NATs, they are subdivided into three categories: (i) target amplification systems; (ii) probe or primer amplification systems, which exploit hybridization of short probes (primers) to the target and various enzyme activities to modify or synthesize deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); and (iii) signal amplification, in which the signal generated from probes is enhanced by means of compound probes or branched-probe technology, without the aid of the above-mentioned enzymes. The above technologies forin vitro amplification of mycobacterial nucleic acids are used mainly to reduce the time necessary to detect the pathogen in clinical specimens; to increase the sensitivity and specificity; and to simplify the test by automation and incorporation of nonisotopic detection formats. Each method has certain advantages, but the impact of any single method on clinical sensitivity has not yet been convincingly demonstrated. Applications that target RNA, including NASBA, TMA, or Q-Beta, are expected to be more sensitive, because RNA already occurs in high copy numbers in the bacterial cells. However, it is often the case that a higher analytical sensitivity does not necessarily improve clinical sensitivity. Rather, the performance of NATs in the detection of MTB depends largely on factors such as collection, volume, and preparation of samples. Higher analytical sensitivity may even translate into a loss of specificity. Nested polymerase chain reaction (PCR), a popular modification of PCR using nested sets of primers and amplification of the amplified nucleic acids from a first round of PCR, is not only very sensitive but also extremely prone to carry-over contamination. Genotypic methods include molecular methods that detect the genes associated with mutation. Unlike phenotypic methods, genotypic methods do not depend on the culture to grow. These can be done directly from the suspected sample.
The basic inherent step necessary for all of these methods to work is the hybridization of nucleic acid probes to a specific target in the genome or RNA of MTB. Nucleic acid probes are selected segments of DNA or RNA sequences that are chemically easy to synthesize and that have been labeled with enzymes, antigenic substrates, chemiluminescent moieties, or radioisotopes available as commercially prepared kits. Under stringent conditions, they bind (hybridize) rapidly and specifically to target nucleotide complementary sequences. A current example of how efficient hybridization can be presented by the probe technology used to identify the species of mycobacteria from cultures (Gen-Probe Accuprobe method). This method is now considered the state of the art for the rapid culture confirmation of MTB or the Mycobacterium avium complex. The target used by this method is the 16S ribosomal RNA (rRNA). Therefore, it was straightforward to use this molecule or the gene coding for the 16S rRNA, namely the 16S ribosomal DNA (rDNA), as a powerful target for amplification of mycobacteria both on the genus and the species level., Similarly, this strategy is applicable to other targets in the RNA operon, e.g., 23S RNA, or the more variable 23S-16S rDNA spacer, which is common to all mycobacteria. Species identification in these assays is performed by probing, restriction enzyme analysis, or direct sequencing.
IS6110: A repetitive target
The only repetitive target useful for an NAT in TB, which is so far available, is an insertion sequence (IS) designated IS6110. The latter is specific for the MTB complex, generally occurs in 1–20 copies per cell, making it an ideal target for amplification, and is generally used for all protocols.
The specimens used for the molecular testing in TB are usually taken from the lower respiratory tract such as sputum or bronchoalveolar lavage (BAL), biopsies, and cerebrospinal fluid (CSF). Some of the genetic tests such as Xpert MTB/rifampin (RIF) assay has been cleared for the application to extrapulmonary specimens. Due to problems with inhibitors, analysis of stool and blood samples is not yet recommended. It is worth noting that molecular techniques detect bacteria, or particles of the bacteria, after centrifugation. Therefore, plasma, serum, or swabs are inadequate specimens. Moreover, it is important to know that culture is always performed in parallel to the molecular method. All the available molecular tests are summarized in [Table 1].
|Table 1: Phenotypic and genotypic tests for the diagnosis of tuberculosis|
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Xpert Mycobacterium tuberculosis/rifampin assay
In December 2010, the WHO recommended the use of the Xpert MTB/RIF assay for the early diagnosis of TB. It utilizes real-time PCR (rt-PCR) technology to both diagnose TB and detect rifampicin resistance concurrently using unprocessed clinical specimens, regardless of their smear status. Nucleic acid amplification tests have long held great promise for TB diagnosis and the rapid detection of drug resistance. However, sensitivity for TB diagnosis has been modest and variable in the sputum smear-negative and extra-PTB (EPTB). Rifampicin resistance is particularly amenable to rapid molecular detection since >95% of all rifampicin-resistant strains contain mutations localized within the 81 bp core region of the bacterial RNA polymerase rpoB gene, which encodes the active site of the enzyme. Thus, the rpoB gene represents a much better molecular target for the simultaneous detection of TB and the key form of drug resistance.
Molecular beacon technology
The Xpert MTB/RIF assay utilizes molecular beacon technology to detect DNA sequences amplified in a hemi-nested rt-PCR assay. Five different nucleic acid hybridization probes are used in the same multiplex reaction. Each probe is complementary to a different target sequence within the rpoB gene of rifampicin-susceptible MTB and is labeled with a differently colored fluorophore. A mutation within these sequences interferes with hybridization such that the conformational integrity of the probe may be retained in the nonfluorescing state. Thus, a mutation anywhere in the core region of the rpoB gene results in either delayed onset or complete suppression of fluorescence of the corresponding molecular beacon. This prototype assay was found to have high sensitivity and specificity for the detection of rifampicin resistance.
GeneXpert® diagnostic platform
The GeneXpert diagnostic system was originally developed by Cepheid Inc. for the detection of anthrax. The GeneXpert system has been successful in combining on-board sample preparation with fully-automated rt-PCR amplification and detection functions. The cartridge-based system incorporates microfluidic technology and fully automated nucleic acid analysis to purify, concentrate, detect, and identify targeted nucleic acid sequences from unprocessed clinical samples. An expanding range of different organisms may be detected using pathogen-specific cartridges within the same GeneXpert test platform. The test platform is modular, with each module independently processing one cartridge at a time [Figure 1]. Machines with 1, 4, 16, and 48 modules are available, permitting multiple assays to be run concurrently and independently. Xpert MTB/RIF assay utilizes single-use plastic cartridges with multiple chambers that are preloaded with liquid buffers and lyophilized reagent beads necessary for sample processing, DNA extraction, and hemi-nested rt-PCR. When performed on unprocessed sputum samples, the assay can generate results within 2 h with <15 min of hands-on time.
Utility of Xpert Mycobacterium tuberculosis/rifampin assay in different clinical settings
In October 2013, the WHO issued the updated Policy Guidance 2, providing revised recommendations on using of Xpert MTB/RIF to diagnose PTB, pediatric TB, EPTB, and rifampicin resistance. The test has a pooled sensitivity of 88% and a pooled specificity of 99% which included 22 studies. In Smear negative PTB patients, Xpert MTB/RIF yielded a pooled sensitivity of 68% and pooled specificity of 99%. In HIV patients with PTB, the pooled sensitivity of Xpert MTB/RIF assay was 79%. One study showed that GeneXpert helps in increased early case detection to diagnose PTB in people living with HIV as compared to fluorescent microscopy and also to detect rifampicin resistance with high specificity. A cross-sectional study conducted by Atehortúa et al. observed that with the Xpert MTB/RIF test, overall performance was similar to the one achieved under ideal conditions. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 87%, 91%, 68%, and 97%, respectively. Mbelele et al. studied the performance of Xpert MTB/RIF assay on the sputum sediment samples, which included 262 patients, and observed that the sensitivity, specificity, PPV, and NPV were 92%, 85%, 74%, and 96%, respectively in diagnosing TB. Meyer et al. studied the efficacy of Xpert on different kinds of sputum and found that patients with salivary sputum showed a trend toward a substantially higher proportion of samples that were Xpert-positive as compared with those with all other sputum sample types. Blood-stained sputum produced the lowest sensitivity and salivary sputum the highest (66%).
Xpert Mycobacterium tuberculosis/rifampin assay on bronchoalveolar lavage fluid
BAL and aspirates can be subjected to Xpert assay when the sputum smear status is negative. Several studies done on the BALF to know the sensitivity and specificity of Xpert assay with one of the studies showed sensitivity of 92.3% and specificity of 87.7%. In a retrospective study done by Le Palud et al., BAL Xpert MTB/RIF assay was 60% sensitive and culture was 66% sensitive, but culture took longer time for diagnosis as compared to Xpert MTB/RIF assay. A study conducted by Khalil and Butt in Pakistan with high prevalence of TB showed that the Xpert assay helped in diagnosing the disease in early stage had a sensitivity of 91.86% and a specificity of 71.42%. The study conducted by Lu et al. in China observed that the sensitivity and specificity of the Xpert MTB/RIF assay were 84.5% and 98.9%, respectively, and those for smear microscopy and AFB cultures were 36.2% and 100%, respectively. Cochrane systematic review has observed that this test is highly accurate. As compared to culture, Xpert has about 88% sensitivity and 98% specificity for PTB in adults. In smear-negative patients with TB, Xpert had a sensitivity of 67%. For rapid detection of rifampicin resistance, the sensitivity is 94% and specificity is 98%. Hence, BALF can be used for the earlier diagnosis of sputum smear-negative PTB cases.
Xpert Mycobacterium tuberculosis/rifampin assay in diagnosing extrapulmonary tuberculosis
The role of Xpert MTB/RIF assay in diagnosing EPTB with using culture as the reference standard, the pooled sensitivity of Xpert MTB/RIF in the lymph node tissues or aspirates is 84.9%. The pooled sensitivity in the gastric fluid is 83.8% and in other tissue specimens is 81.2%. In CSF, the pooled sensitivity of Xpert MTB/RIF compared against culture as a reference standard is 79.5%. In the pleural fluid, the pooled sensitivity of Xpert MTB/RIF as compared to culture is 43.7%. Pleural fluid is a suboptimal sample for the bacterial confirmation of pleural TB, regardless of the method used. The sensitivity of Xpert MTB/RIF in testing samples of pleural fluid is very low. Xpert MTB/RIF for diagnosing EPTB is still comparatively weak. Scott et al. showed that when culture was used as the reference, Xpert MTB/RIF's overall sensitivity was 59% and specificity was 92% for pus, sensitivity for lymph node aspirates was 80% and for other fluids sensitivity was 51% (ascitis - 59% and pleural - 47%). A systemic review including 21 studies with a total of 6026 nonrespiratory samples showed that comparing Xpert and culture done on the same samples, the sensitivity was very heterogeneous with a median sensitivity of 0.83, whereas specificities were typically very high (median, 0.98). Pooled summary estimates of sensitivity varied substantially between sample types: lymph node tissue - 0.96, tissue samples of all types - 0.88, pleural fluid - 0.34, and gastric aspirates - 0.78. Median sensitivities for CSF testing and nonpleural serous fluid samples were 0.85 and 0.67, respectively., An another systemic review, which included 18 studies involving 4461 samples, showed that the Xpert MTB test had sensitivity differed substantially between sample types. In lymph node tissues or aspirates, Xpert pooled sensitivity was 83.1% versus culture. In the CSF, Xpert pooled sensitivity was 80.5%; in the pleural fluid, the sensitivity was 21.4%. Xpert pooled specificity was consistently 98.7% across different sample types. The WHO now recommends Xpert over conventional tests for the diagnosis of TB in the lymph nodes and other tissues and as the preferred initial test for the diagnosis of TB meningitis. The data for other of specimens such as ascitic fluid, pericardial fluid, urine, blood, and stool were limited and therefore were not considered for analysis by subgroup.
Truenat Mycobacterium tuberculosis
A chip-based nucleic acid amplification test involves sputum processing using Trueprep-MAG™ (nanoparticle-based protocol run on a battery-operated device) and rtPCR performed on the Truelab Uno™ analyzer. The preliminary study shows that the Truenat MTB test allows detection of TB in approximately 1 h and can be utilized in near-care settings to provide quick and accurate diagnosis. It not only has good sensitivity and specificity for the diagnosis of TB but also fits the requirements of the resource-limited healthcare settings. Large studies are required to obtain better estimates of the Truenat MTB performance.
Line probe assay
Line probe assays (LPAs) are tests that use PCR and reverse hybridization methods for the rapid detection of mutations associated with drug resistance. LPA are designed to identify MTB complex and simultaneously detect mutations associated with drug resistance. One of the disadvantages with these assays is that they have an open-tube format, which can lead to cross-contamination and an increased risk of false-positive results. LPA technology is suitable for use at national/central reference laboratories or at laboratories where there is proven capacity to conduct molecular testing. There must be adequate and appropriate laboratory infrastructure and equipment. This must also include the necessary biosafety precautions and the prevention of contamination. Appropriate laboratory staff also needs to be trained to conduct LPA procedures. LPAs are the WHO-approved tests for rapid detection of drug resistance to first- and second-line agents. In LPA, mutations are detected by (i) the binding of amplicons to probes targeting the most commonly occurring mutations (MUT probes) or (ii) inferred by the lack of hybridization (i.e., lack of binding) of the amplicons to the corresponding WT probes. The posthybridization reaction leads to the development of colored bands on the test strip detecting probe binding. Although LPA can detect the mutations that are most frequently identified in resistant strains, some mutations that confer resistance are outside the regions covered by the test, and therefore, resistance cannot be completely excluded even in the presence of all WT probes. Some mutations are identified specifically by MUT probes, whereas others are only inferred by the absence of binding of the amplicons to WT probes. The lack of binding of a WT probe without simultaneous binding of a mutant probe is likely caused by the presence of a resistance mutation. There are three types of LPA available:
- Inno-LiPA Mycobacteria v2 is an LPA for the simultaneous detection and identification of the genus Mycobacterium and 16 different mycobacterial species. The test is based on nucleotide differences in the 16S–23S rRNA spacer region
- LPA for the identification of the MTB and rifampicin resistance: The LiPA rpoB PCR can be performed on all respiratory specimens and other specimens where the detection of rifampicin resistance in MTB is the primary purpose of the investigation
- LPA for identifying species from Mycobacterium genus and detecting potential MDR-TB and extensively MDR-TB. The GenoType MTBDR plus test allows for the detection of MTB complex and simultaneously its resistance to rifampicin and/or isoniazid by mutations in the rpo B and kat G/inh A (high/low isoniazid resistance) genes, respectively.
Tuberculosis peptide nucleic acid fluorescence in situ hybridization
Fluorescence in situ hybridization using peptide nucleic acid (PNA) probes allows differentiation between tuberculous and nontuberculous mycobacteria in the smears of mycobacterial cultures. PNA molecules are pseudopeptides with DNA-binding capacity, in which the sugar phosphate backbone of DNA has been replaced by a polyamide backbone.
New PCR-based genotyping techniques include spacer oligonucleotide typing (spoligotyping), IS6110-based restriction fragment length polymorphism (RFLP), and mycobacterial interspersed repetitive unit (MIRU) typing. Genotyping is useful in analyzing suspected outbreaks of TB in institutions such as hospitals, schools, and prisons.
- Spoligotyping: This test can be used for both detection and typing of MTB, through PCR amplification of a highly polymorphic direct repeat locus in the genome of MTB. Prior culturing of the bacteria is needed and the results are available from culture within 1 day, with a sensitivity of 96% and a specificity of 98%
- DNA fingerprinting (RFLP): This type of test, using IS6110-based RFLP, has proven useful in phylogenetic studies of TB bacilli, particularly since IS6110 is unique for the MTB complex. RFLP DNA fingerprinting is the gold standard for strain typing in mycobacteriology, and this method of genotyping has been standardized in order to increase the inter- and intra-laboratory comparability so that it could be used for subspeciation of MTB. The disadvantages of RFLP genotyping are that a large cell mass are required and that comparison is difficult since the results are band patterns, hard to convert into digital formats
- MIRU typing: This test is a technique based on variable numbers of tandem repeat at 12 loci in the genome of MTB. This is still under investigation and requires further testing in clinical settings.
Interferon-gamma release assays
This test detects the latent tuberculous infection in the body. It is cytokine detection assay which measures the cell-mediated immune response elicited against MTB. Interferon-gamma release assays (IGRAS) measure the interferon (IFN)-gamma released by sensitized white blood cells. Four IGRAs which use different antigens to stimulate IFN-gamma release and different methods of measurement have been approved in the USA. QuantiFERON-TB was approved as an aid for diagnosing latent TB infection and is no longer commercially available since it was replaced by QuantiFERON-TB Gold, which is approved as an aid for diagnosing both latent infection and active disease. Newer tests include QuantiFERON-TB Gold In-Tube test and the T-SPOT.TB test. QuantiFERON-TB Gold is an ELISA test which detects the release of IFN-gamma in fresh heparinized whole blood from sensitized persons upon incubation with synthetic peptides simulating ESAT-6 and culture filtrate protein-10. The patient only needs to visit once, for specimen collection, and results can be obtained in 48 h. QuantiFERON-TB Gold In-Tube test was developed to overcome the limitation of QuantiFERON-TB Gold, which could only be used in facilities where blood testing could begin within a few hours of its collection. This test uses a mixture of 14 peptides representing ESAT-6, CFP-10, and a part of TB. T-SPOT.TB test incubates peripheral blood mononuclear cells with mixtures of peptides and uses an enzyme-linked immunospot assay to detect increases in the number of T-cells that secrete IFN-gamma.
Monokine-amplified interferon-gamma release assays
This is a newer test for the diagnosis of latent tubercular infection and is still under investigations. Given that IFN release leads to subsequent release of IFN-responsive chemokines such as MIG and IP-10, the identification of these chemokines might provide a sensitive tool for the detection of mycobacterial infection and antigen-specific T-cell responses.
Whole-genome sequencing (WGS) is becoming an affordable and accessible method that can identify microevolution within MTB lineages as they are transmitted between hosts. There are two classes of sequencers that exist: the first-generation sequencer and the second-generation (widely known as the next-generation sequencer). The first-generation sequencer is relatively slow but has a high-throughput and low cost. The second-generation has a lower throughput, higher cost, and is able to sequence multiple genomes in less than a day. The WGS can detect various types of mutations better than the Xpert MTB assay. Moreover, WGS could avoid false positives when a polymorphism in the rifampicin-resistance determining region of rpoB is detected. WGS has not been used as a routine diagnostic tool for TB, partly because of the need to culture MTB for several weeks until an adequate amount of DNA can be extracted. WGS data can be obtained several weeks before the drug susceptibility test data is available. DNA sequencing could also be used to confirm RIF resistance from Xpert MTB/RIF. However, the method is very costly and requires further clinical research to be used in clinical practice.
| Summary|| |
Newer molecular tests have better performance characteristics in terms of increased sensitivity and better accuracy for early diagnosis of MTB as well as for detecting MDR and extensively drug-resistant TB and thus represent better diagnostic tools for the rapid detection of resistance to first-line as well as second-line drugs. These molecular diagnostic tests has a definite edge over conventional phenotypic methods being highly sensitive and specific, as well as the results are available in 48–72 h. Xpert MTB/RIF is very easy to perform, robust, and rapid system to detect rifampicin resistance directly from the samples, irrespective of smear status within 2 h. The landscape of TB diagnostics offer newer technologies with point-of-care assays enabling TB diagnosis to be available easily from bench to bedside, and this will help in early diagnosis as well as to prevent the transmission of TB in the community.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
World Health Organization. Global Tuberculosis Report 2017. WHO Report 2017. Available from: http://www.who.int
. [Last accessed on 2019 Jun 20].
World Health Organization. The Global Plan to Stop TB, 2006-2015. Geneva: World Health Organization; 2015. Available from: http://www.stoptb.org
. [Last accessed on 2019 Jun 20].
World Health Organization. Global Tuberculosis Control: Epidemiology, Strategy, Financing. WHO Report. World Health Organization; 2016. Available from: http://www.who.int
. [Last accessed on 2019 Jun 20].
Morris S, Bai GH, Suffys P, Gomez PL, Fairchok M, Rouse D. Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis
. J Infect Dis 1995;171:954-60.
Nurwidya F, Handayani D, Burhan E, Yunus F. Molecular diagnosis of tuberculosis. Chonnam Med J 2018;54:1-9.
Castan P, de Pablo A, Fernández-Romero N, Rubio JM, Cobb BD, Mingorance J, et al
. Point-of-care system for detection of Mycobacterium tuberculosis
and rifampin resistance in sputum samples. J Clin Microbiol 2014;52:502-7.
Centers for Disease Control and Prevention (CDC). Availability of an assay for detecting Mycobacterium tuberculosis
, including rifampin-resistant strains, and considerations for its use – United States, 2013. MMWR Morb Mortal Wkly Rep 2013;62:821-7.
Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al
. Rapid molecular detection of tuberculosis and rifampin resistance. N
Engl J Med 2010;363:1005-15.
Sohn H, Pai M, Dendukuri N, Kloda LA, Boehme CC, Steingart KR. Xpert MTB/RIF test for detection of pulmonary tuberculosis and rifampicin resistance. Cochrane Database of Systematic Reviews 2012, Issue 1. Art. No.: CD009593. DOI: 10.1002/14651858.CD009593.
Persing DH. In vitro
nucleic acid amplification techniques. In: Persing DH, Smith TF, Tenover FC, White TJ, editors. Diagnostic Molecular Microbiology, Principles and Applications. Washington DC: ASM Press; 1993. p. 51-87.
Pfyffer GE, Kissling P, Jahn EM, Welscher H, Salfinger M, Weber R, Diagnostic performance of amplified Mycobacterium tuberculosis
direct test with cerobrospinal fluid, other non-respiratory, and respiratory specimens. J Clin Microbiol 1996;34:834-41.
Miyazaki Y, Koga H, Kohno S, Kaku M. Nested polymerase chain reaction for detection of Mycobacterium tuberculosis
in clinical samples. J Clin Microbiol 1993;31:2228-32.
Tenover CF. Diagnostic deoxyribonucleic acid probes for infectious disease. Clin Microbiol Rev 1988;1:82-101.
Tevere VJ, Hewitt PL, Dare A, Hocknell P, Keen A, Spadoro JP, et al
. Detection of Mycobacterium tuberculosis
by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis
-specific probe. J Clin Microbiol 1996;34:918-23.
Kox LF, van Leeuwen J, Knijper S, Jansen HM, Kolk AH. PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples. J Clin Microbiol 1995;33:3225-33.
Takewaki S, Okuzumi K, Ishiko H, Nakahara K, Ohkubo A, Nagai R. Genus-specific polymerase chain reaction for the mycobacterial dnaJ gene and species-specific oligonucleotide probes. J Clin Microbiol 1993;31:446-50.
Clarridge JE 3rd
, Shawar RM, Shinnick TM, Plikaytis BB. Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis
in a routine mycobacteriology laboratory. J Clin Microbiol 1993;31:2049-56.
Kirschner P, Rosenau J, Springer B, Teschner K, Feldmann K, Böttger EC. Diagnosis of mycobacterial infections by nucleic acid amplification: 18-month prospective study. J Clin Microbiol 1996;34:304-12.
Lawn SD, Nicol MP. Xpert® MTB/RIF assay: Development, evaluation and implementation of a new rapid molecular diagnostic for tuberculosis and rifampicin resistance. Future Microbiol 2011;6:1067-82.
Sumangala V, Venkatesha DT, Chennaveerappa PK, Gayathree LJ. Role of GeneXpert® MTB/RIF assay for early diagnosis of pulmonary tuberculosis in people living with HIV. Int Med Dent 2017;4:56-60.
Atehortúa S, Ramírez F, Echeverri LM, Peñata A, Ospina S. Xpert MTB/RIF test performance assay in respiratory samples at real work settings in a developing country. Biomedica 2015;35:125-30.
Mbelele PM, Aboud S, Mpagama SG, Matee MI. Improved performance of Xpert MTB/RIF assay on sputum sediment samples obtained from presumptive pulmonary tuberculosis cases at Kibong'oto infectious diseases hospital in Tanzania. BMC Infect Dis 2017;17:808.
Meyer AJ, Atuheire C, Worodria W, Kizito S, Katamba A, Sanyu I. Sputum quality and diagnostic performance of GeneXpert MTB/RIF among smear-negative adults with presumed tuberculosis in Uganda. PLoS One 2017;12:E0180572. Available from: https://doi.org/10.1371/journal.pone.0180572
. [Last accessed on 2019 Jun 20].
Barnard DA, Irusen EM, Bruwer JW, Plekker D, Whitelaw AC, Deetlefs JD, et al
. The utility of Xpert MTB/RIF performed on bronchial washings obtained in patients with suspected pulmonary tuberculosis in a high prevalence setting. BMC Pulm Med 2015;15:103.
Le Palud P, Cattoir V, Malbruny B, Magnier R, Campbell K, Oulkhouir Y, et al
. Retrospective observational study of diagnostic accuracy of the Xpert® MTB/RIF assay on fiberoptic bronchoscopy sampling for early diagnosis of smear-negative or sputum-scarce patients with suspected tuberculosis. BMC Pulm Med 2014;14:137.
Khalil KF, Butt T. Diagnostic yield of Bronchoalveolar Lavage gene Xpert in smear-negative and sputum-scarce pulmonary tuberculosis. J Coll Physicians Surg Pak 2015;25:115-8.
Lu Y, Zhu Y, Shen N, Tian L, Sun Z. Evaluating the diagnostic accuracy of the Xpert MTB/RIF assay on bronchoalveolar lavage fluid: A retrospective study. Int J Infect Dis 2018;71:14-9.
Scott LE, Beylis N, Nicol M, Nkuna G, Molapo S, Berrie L, et al
. Diagnostic accuracy of Xpert MTB/RIF for extra-pulmonary tuberculosis specimens: Establishing a laboratory testing algorithm for South Africa. J Clin Microbiol 2014;52:1818-23.
Maynard-Smith L, Larke N, Peters JA, Lawn SD. Diagnostic accuracy of the Xpert MTB/RIF assay for extrapulmonary and pulmonary tuberculosis when testing non-respiratory samples: A systematic review. BMC Infect Dis 2014;14:709.
Iram S, Zeenat A, Hussain S, Wasim Yusuf N, Aslam M. Rapid diagnosis of tuberculosis using Xpert MTB/RIF assay-Report from a developing country. Pak J Med Sci 2015;31:105-10.
Denkinger CM, Schumacher SG, Boehme CC, Dendukuri N, Pai M, Steingart KR. Xpert MTB/RIF assay for the diagnosis of extra-pulmonary tuberculosis: A systematic review and meta-analysis. Eur Respir J 2014;44:435-46.
World Health Organization. The Use of Molecular Line Probe Assays for the Detection for the Detection of Resistance to Second-Line anti-Tubercular Drugs: Policy Guidance. WHO Publication. World Health Organization; 2016. p. 1-52.
Scarparo C, Piccoli P, Rigon A, Ruggiero G, Nista D, Piersimoni C. Direct identification of mycobacteria from MB/BacT alert 3D bottles: Comparative evaluation of two commercial probe assays. J Clin Microbiol 2001;39:3222-7.
Tortoli E, Mariottini A, Mazzarelli G. Evaluation of INNO-LiPA MYCOBACTERIA v2: Improved reverse hybridisation multiple DNA probe assay for mycobacterial identification. J Clin Microbiol 2003;41:4418-20.
Rossau R, Traore H, De Beenhouwer H, Mijs W, Jannes G, De Rijk P, et al
. Evaluation of the INNO-LiPA Rif. TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis
complex and its resistance to rifampin. Antimicrob Agents Chemother 1997;41:2093-8.
Sam IC, Drobniewski F, More P, Kemp M, Brown T. Mycobacterium tuberculosis
and rifampin resistance, United Kingdom. Emerg Infect Dis 2006;12:752-9.
Anochie PI, Onyeneke EC, Ogu AC, Onyeozirila AC, Aluru S, Onyejepu N, et al
. Recent advances in the diagnosis of Mycobacterium tuberculosis
. Germs 2012;2:110-20.
Pai M, Denkinger CM, Kik SV, Rangaka MX, Zwerling A, Oxlade O, et al
. Gamma interferon release assays for detection of Mycobacterium tuberculosis
infection. Clin Microbiol Rev 2014;27:3-20.
Metcalfe JZ, Everett CK, Steingart KR, Cattamanch A, Huang L, Hopewell PC, et al
. Ineterferon-gamma release assay for active pulmonary tuberculosis diagnosis in adults in low and middle income countires: Systematic review and meta-analysis. J Infect Dis 2011;204 Suppl 4:S1120-9.
Köser CU, Ellington MJ, Peacock SJ. Whole-genome sequencing to control antimicrobial resistance. Trends Genet 2014;30:401-7.
Witney AA, Cosgrove CA, Arnold A, Hinds J, Stoker NG, Butcher PD. Clinical use of whole genome sequencing for Mycobacterium tuberculosis
. BMC Med 2016;14:46.
Witney AA, Gould KA, Arnold A, Coleman D, Delgado R, Dhillon J, et al
. Clinical application of whole-genome sequencing to inform treatment for multidrug-resistant tuberculosis cases. J Clin Microbiol 2015;53:1473-83.
McAlister AJ, Driscoll J, Metchock B. DNA sequencing for confirmation of rifampin resistance detected by Cepheid Xpert MTB/RIF assay. J Clin Microbiol 2015;53:1752-3.