Elsevier

Tuberculosis

Volume 113, December 2018, Pages 139-152
Tuberculosis

Review
DNA markers for tuberculosis diagnosis

https://doi.org/10.1016/j.tube.2018.09.008Get rights and content

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is an infectious disease with more than 10.4 million cases and 1.7 million deaths reported worldwide in 2016. The classical methods for detection and differentiation of mycobacteria are: acid-fast microscopy (Ziehl-Neelsen staining), culture, and biochemical methods. However, the microbial phenotypic characterization is time-consuming and laborious. Thus, fast, easy, and sensitive nucleic acid amplification tests (NAATs) have been developed based on specific DNA markers, which are commercially available for TB diagnosis. Despite these developments, the disease remains uncontrollable. The identification and differentiation among MTBC members with the use of NAATs remains challenging due, among other factors, to the high degree of homology within the members and mutations, which hinders the identification of specific target sequences in the genome with potential impact in the diagnosis and treatment outcomes. In silico methods provide predictive identification of many new target genes/fragments/regions that can specifically be used to identify species/strains, which have not been fully explored. This review focused on DNA markers useful for MTBC detection, species identification and antibiotic resistance determination. The use of DNA targets with new technological approaches will help to develop NAATs applicable to all levels of the health system, mainly in low resource areas, which urgently need customized methods to their specific conditions.

Introduction

Mycobacterial infections in humans remain a serious health problem worldwide due to the failure of the specific immune response to eradicate the pathogens. Currently, there are more than 150 species in the genus Mycobacterium [1], and they can be classified into three main groups based on clinical importance for diagnosis and treatment purposes: A) Mycobacterium tuberculosis complex (MTBC), which causes tuberculosis (TB), B) Mycobacterium leprae, which cause leprosy, and C) non-tuberculous mycobacteria (NTM), which are usually opportunistic pathogens that cause diseases generally in immunocompromised patients, mainly as pulmonary (more frequent), skin, soft tissue and disseminated infections (Fig. 1) [2]. Rapid and reliable identification of mycobacterial infections is critical in guiding public health and primary care decisions due to the species-dependent differences in epidemiology, host spectrum, geographic range, pathogenicity, and drug susceptibility [3].

TB is a worldwide infectious disease with more than 10.4 million cases and 1.7 million deaths reported in 2016 [4]. It is commonly caused by 1) Mycobacterium tuberculosis (Mtb), frequently caused by 2) M. bovis and 3) M. africanum, and occasionally caused by 4) M. microti, 5) M. caprae, 6) M. canettii, 7) M. pinnipedii, 8) Dassie bacillus, 9) M. mungi, 10) M. orygis, and 11) M. bovis BCG (Fig. 1) [5].

Human TB may occur due to human-to-human or animal-to-human (zoonotic) transmission. The natural host for Mtb, M. africanum and M. canettii is human, while the others are transmitted from animals, such as bovidae (M. bovis), rodents (M. microti), goats (M. caprae), marine mammals (M. pinnipedii), hyrax (Dassie bacillus), mongooses (M. mungi), and oryxes (M. orygis) [6]. They are clonal populations evolved from a single progenitor species that has diversified by the acquisition of spontaneous mutations rather than by horizontal gene transfer (HGT) [7].

Direct acid-fast microscopy using Ziehl-Neelsen staining is the most widely used method in low-resource settings to diagnose TB [8]. However, it has low sensitivity (requires approximately 5,000–10,000 bacilli per 1 mL of sputum for detection) and specificity [9]. The classical gold standard for differentiation and identification of mycobacteria are culture and biochemical methods [10]. The use of traditional phenotypic microbiological techniques, such as culturing in Lӧwenstein-Jensen medium are time-consuming (MTBC are slow-growers that require more than three weeks for positive cultures), laborious (MTBC need fastidious culture requirements), and unstable (MTBC demonstrate variable gene expression based on cultivation conditions) [11]. A more advanced development using liquid culture incorporating a Mycobacterial Growth Indicator Tube (MGIT™) (Becton Dikinson, USA) allows faster (8–14 days), more sensitive (10% increase in detection cases) and convenient detection (automation in detecting growth) [12]. The MGIT™ system also enables drug-sensitivity testing to be carried out to detect drug-resistant strains [12].

Molecular identification has emerged as an alternative or complement to traditional microbiological identification as it is faster, easier, and more sensitive. Nucleic acid amplification tests (NAATs) provide accurate and quick methods for identification of microorganisms without the need for cumbersome biochemical tests, and are particularly useful to detect slow-growing and fastidious microorganisms [13]. Also, molecular characterization is more sensitive compared to culture methods, thus preventing misdiagnosis or false negative results. Classical genotyping methods for MTBC such as IS6110 restriction fragment length polymorphism (RFLP), spoligotyping, and mycobacterial interspersed repeat units-variable number of tandem repeats (MIRU-VNTR) have enabled strain typing [14]. It is important to study strain variation to understand the disease prevalence because genetic diversity in MTBC strains have translated into phenotypic variation (i.e., virulence and immunogenicity), which will affect the efficacy of treatment [14,15].

With the development of NAATs, a single pair of primers can be used as diagnostic markers to detect pathogens at a single gene target resolution, which could lead to a simple, cost-effective, and more effective DNA-based detection methods [16]. Mycobacterium-specific genes enabled fast detection of all mycobacterial strains and differentiation from non-mycobacterial strains. This is extremely important for diagnosis of mycobacterial infections because even a fast-growing Mycobacterium needs at least three to seven days to grow [17]. MTBC-specific genes are important to detect all the causative agents for TB and to prevent the risk of misdiagnosis. With the advancement in DNA molecular techniques, even though these target genes are categorized under Mycobacterium genus- or MTBC-specific genes, they can be used for subsequent species differentiation. Differentiation among MTBC members is important to improve clinical and therapeutic management of TB and for epidemiology purposes, i.e., exclusion of pyrazinamide (PZA) treatment in M. bovis infection, detection of transmission between animal products and humans, and the study of species distribution in different geographical areas [18]. The emergence of multidrug resistant-TB (MDR-TB) and extremely drug resistant-TB (XDR-TB) strains have led to high disease burden and mortality. It is estimated that annually, approximately 3.7% of new TB cases are MDR-TB, and 9% of them are in fact XDR-TB [19]. The increasing presence of TB cases harboring drug resistant strains need rapid, sensitive, and specific approaches, which is being addressed with the use of NAATs [12].

Having a specific target gene does promise high positive predictive values and low false negative results. However, in terms of analytical sensitivity, a gene with high copy numbers, i.e., IS6110 (up to 25 copies in Mtb genome), plays an important role in determining the limit of detection of an assay, and thus contributes to higher sensitivity diagnostic tests [20].

Also, the biological samples used to detect the microorganisms affect the overall outcome of the diagnosis. Since mycobacteria colonize human lungs and cause pulmonary diseases, respiratory specimens, such as sputum, is commonly used to detect the microorganisms via sputum smear microscopy [8].

Regarding the impact of the type of sample, using NAATs, Shenai et al. (2013) showed that sputum remains the best biological fluid, in terms of sensitivity, either without processing (raw) (100%) or processed (decontaminated with N-acetyl-l-cysteine and sodium hydroxide) (100%), compared to saliva (38.5%), blood (8.3%), urine (3.8%) and exhaled breath concentrate (0%) to detect Mtb using Xpert MTB/RIF [21]. Oral swabbing is an alternative to invasive procedures or coughing, which, using IS6110 PCR, showed high sensitivity (90%), when at least 2 swabs per patient were analyzed [22].

This review is focused on the application of reported DNA markers for: A) detection of MTBC, B) MTBC species identification, and C) determination of antibiotic resistance (Fig. 2). It also highlights the importance of the use of bioinformatics tools for new marker identification and the urgent need of point-of-care (POC) NAAT-based development for low-resource areas.

Some excellent publications available in this area are recommended for additional information [12,[23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]].

Section snippets

Detection of MTBC

In the TB diagnostic process the specific detection of MTBC with a suitable differentiation from NTM, is of paramount importance. In this section, some of the main reported DNA markers used for this objective are presented.

MTBC species identification

The diversity among MTBC members is associated with differences in bacterial virulence, pathogenicity, and drug susceptibility, which highlight the importance of the availability of markers with capacity of species differentiation. In this section, some DNA targets useful for differentiation among the members of the complex will be presented.

Determination of antibiotic resistance

MDR-TB strains are resistant to rifampicin (RIF) and isoniazid (INH), the two most powerful first-line antibiotic drugs used for the treatment of TB, but susceptible to second-line anti-TB drugs [4]. XDR-TB strains are resistant to at least one fluoroquinolone (FQ) and at least one of the three second-line injectable agents, amikacin (AMK), capreomycin (CAP), and kanamycin (KAN), in addition to first-line antibiotics [138]. The use of MDR-TB- and XDR-TB-specific genes serve dual functions, i.e,

Mining new target DNA markers for TB diagnosis

The development of WGS and bioinformatics tools enable massive amounts of data to be analyzed together. This top-down approach using the genomics data has successfully identified specific target genes, such as mtss90, for diagnosis of TB [124]. Using in silico methods, recent studies have also reported new genetic markers with potential for TB diagnosis or treatment. Calero et al. (2013), have identified specific Mtb genes codifying antigens that have high potential for presentation by MHC

Transforming NAATs into POC diagnostic tests for low resource settings

Despite all the efforts in developing new, sensitive and specific tests, many of them already endorsed by the WHO, one of the main challenges is the application of these NAATs in high-burden countries, which are low-resource settings and meeting the ASSURED criteria recommended by the WHO (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users) [207].

In high-incidence areas, microscopy centers represent the most important place in the TB

Conclusion

Genus, species and strain differentiation in addition to drug resistance detection can be achieved using DNA-based methods. The advancement in computational genomic approaches has enabled many potential new target genes and anti-tuberculous drug targets to be identified, to be explored. The integration to new technological platforms with bioinformatics will allow to improve the current diagnostic methods and to develop new POCD suitable for application in low-resource high endemic areas.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

This work was supported by LRGS Grant (203.PPSK.67212001), Ministry of Education (Higher Education), Malaysia.

References (219)

  • M. Yamada-Noda et al.

    Mycobacterium species identification-A new approach via dnaJ gene sequencing

    Syst Appl Microbiol

    (2007)
  • B.W. Lee et al.

    DNA amplification by the polymerase chain reaction for the rapid diagnosis of tuberculous meningitis. Comparison of protocols involving three mycobacterial DNA sequences, IS6110, 65 kDa antigen, and MPB64

    J Neurol Sci

    (1994)
  • S.P. Lekhak et al.

    Evaluation of multiplex PCR using MPB64 and IS6110 primers for rapid diagnosis of tuberculous meningitis

    Tuberculosis (Edinb)

    (2016)
  • A. Martin et al.

    Evaluation of the BD MGIT TBc Identification Test (TBc ID), a rapid chromatographic immunoassay for the detection of Mycobacterium tuberculosis complex from liquid culture

    J Microbiol Methods

    (2011)
  • V. Malhotra et al.

    Mycobacterium tuberculosis response regulators, DevR and NarL, interact in vivo and co-regulate gene expression during aerobic nitrate metabolism

    J Biol Chem

    (2015)
  • A.K. Sahni et al.

    Comparison of IS6110 and 'short fragment' devR (Rv3133c) gene targets with phenotypic methods for diagnosis of Mycobacterium tuberculosis

    Med J Armed Forces India

    (2013)
  • S. Christianson et al.

    Evaluation of 24 locus MIRU-VNTR genotyping of Mycobacterium tuberculosis isolates in Canada

    Tuberculosis

    (2010)
  • M.M. Johnson et al.

    Nontuberculous mycobacterial pulmonary infections

    J Thorac Dis

    (2014)
  • S. Faria et al.

    General overview on nontuberculous mycobacteria, biofilms, and human infection

    J Pathog

    (2015)
  • L.M. Parsons et al.

    Rapid and simple approach for identification of Mycobacterium tuberculosis complex isolates by PCR-based genomic deletion analysis

    J Clin Microbiol

    (2002)
  • Fact sheets: tuberculosis

    (2018)
  • K.M. Malone et al.

    Mycobacterium tuberculosis complex members adapted to wild and domestic animals

    Adv Exp Med Biol

    (2017)
  • A.E. Hirsh et al.

    Stable association between strains of Mycobacterium tuberculosis and their human host populations

    Proc Natl Acad Sci U S A

    (2004)
  • C.M. Denkinger et al.

    Robust, reliable and resilient: designing molecular tuberculosis tests for microscopy centers in developing countries

    Expert Rev Mol Diagn

    (2013)
  • V. Ausina Ruiz et al.

    Selected culture and drug-susceptibility testing methods for drug-resistant Mycobacterium tuberculosis screening in resource-constrained settings

    Expert Rev Mol Diagn

    (2013)
  • A. Gholoobi et al.

    Comparison of culture and PCR methods for diagnosis of Mycobacterium tuberculosis in different clinical specimens

    Jundishapur J Microbiol

    (2014)
  • H. Soini et al.

    Molecular diagnosis of mycobacteria

    Clin Chem

    (2001)
  • M. Pai et al.

    Tuberculosis diagnostics: state of the art and future directions

    Microbiol Spectr

    (2016)
  • Y.W. Tang et al.

    Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic gram-negative bacilli

    J Clin Microbiol

    (1998)
  • S. Gagneux

    Genetic diversity in Mycobacterium tuberculosis

    Curr Top Microbiol Immunol

    (2013)
  • H. Chae et al.

    Importance of differential identification of Mycobacterium tuberculosis strains for understanding differences in their prevalence, treatment efficacy, and vaccine development

    J Microbiol

    (2018)
  • Y.X. Goay et al.

    Identification of five novel Salmonella typhi-specific genes as markers for diagnosis of typhoid fever using single-gene target PCR assays

    BioMed Res Int

    (2016)
  • T.J. Gray et al.

    Improved identification of rapidly growing mycobacteria by a 16S-23S internal transcribed spacer region PCR and capillary gel electrophoresis

    PloS One

    (2014)
  • A. Somoskovi et al.

    Direct comparison of the genotype MTBC and genomic deletion assays in terms of ability to distinguish between members of the Mycobacterium tuberculosis complex in clinical isolates and in clinical specimens

    J Clin Microbiol

    (2008)
  • Multidrug-resistant tuberculosis (MDR-TB): 2013 update

    (2013)
  • O.W. Akkerman et al.

    Comparison of 14 molecular assays for detection of Mycobacterium tuberculosis complex in bronchoalveolar lavage fluid

    J Clin Microbiol

    (2013)
  • S. Shenai et al.

    Exploring alternative biomaterials for diagnosis of pulmonary tuberculosis in HIV-negative patients by use of the GeneXpert MTB/RIF assay

    J Clin Microbiol

    (2013)
  • R.C. Wood et al.

    Detection of Mycobacterium tuberculosis DNA on the oral mucosa of tuberculosis patients

    Sci Rep

    (2015)
  • M. Kato-Maeda et al.

    Genotyping of Mycobacterium tuberculosis: application in epidemiologic studies

    Future Microbiol

    (2011)
  • C. Altez-Fernandez et al.

    Diagnostic accuracy of nucleic acid amplification tests (NAATs) in urine for genitourinary tuberculosis: a systematic review and meta-analysis

    BMC Infect Dis

    (2017)
  • R.R. Nathavitharana et al.

    Accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant tuberculosis: a systematic review and meta-analysis

    Eur Respir J

    (2017)
  • E.O. Babafemi et al.

    Effectiveness of real-time polymerase chain reaction assay for the detection of Mycobacterium tuberculosis in pathological samples: a systematic review and meta-analysis

    Syst Rev

    (2017)
  • R. Gupta et al.

    Diagnostic accuracy of nucleic acid amplification based assays for tuberculous meningitis: a meta-analysis

    J Infect

    (2018)
  • S.G. Schumacher et al.

    Impact of molecular diagnostics for tuberculosis on patient-important outcomes: a systematic review of study methodologies

    PloS One

    (2016)
  • D. Marangu et al.

    Diagnostic accuracy of nucleic acid amplification tests in urine for pulmonary tuberculosis: a meta-analysis

    Int J Tubercul Lung Dis

    (2015)
  • F. Drobniewski et al.

    Systematic review, meta-analysis and economic modelling of molecular diagnostic tests for antibiotic resistance in tuberculosis

    Health Technol Assess

    (2015)
  • D.I. Ling et al.

    Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression

    PloS One

    (2008)
  • L.L. Flores et al.

    In-house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum specimens: meta-analysis and meta-regression

    BMC Microbiol

    (2005)
  • L. Dvorska et al.

    Strategies for differentiation, identification and typing of medically important species of mycobacteria by molecular methods

    Vet Med

    (2001)
  • T. Rogall et al.

    Differentiation of Mycobacterium species by direct sequencing of amplified DNA

    J Gen Microbiol

    (1990)
  • Cited by (15)

    • CRISPR-based biosensing is prospective for rapid and sensitive diagnosis of pediatric tuberculosis

      2020, International Journal of Infectious Diseases
      Citation Excerpt :

      Second, the target selection of CRISPR platforms is essential. For diagnosis of TB, gene targets that are specific and conserved for TB are advantageous to make assay more ideal (Chin et al., 2018) (e.g. IS6110 may be more suitable than easily mutated rpoB). At the same time, the target sequence should be long enough for choosing a suitable candidate near the PAM sequence.

    • Identification of a Mycobacterium tuberculosis-specific gene marker for diagnosis of tuberculosis using semi-nested melt-MAMA qPCR (lprM-MAMA)

      2020, Tuberculosis
      Citation Excerpt :

      However, bacterial culture is a time-consuming method as MTBC members are slow-growers, requiring at least a week to grow in liquid culture or three weeks in solid medium [5]. Several nucleic acid amplification tests (NAATs), some of them endorsed by World Health Organization (WHO), have been developed for TB diagnosis supported by their fast, sensitive and specific detection capacity [6], which are based on the detection of MTBC such as real-time PCR assays [Xpert® MTB/RIF (Xpert) (Cepheid, USA), Abbott RealTime MTB (Abbott Laboratories, USA) and Roche Cobas® MTB (Roche Diagnostics, Switzerland)], line probe assays (LiPA) [GenoType MTBC (Hain Lifescience GmbH, Germany), GenoType MTBDRplus (Hain Lifescience GmbH, Germany) and Nipro NTM + MDRTB detection kit 2 (Nipro, Japan)], and loop-mediated isothermal amplification assays [Loopamp™ MTBC detection kit (TB-LAMP) (Eiken, Japan)] [7]. Target genes used in the development of these assays such as rpoB gene in Xpert, IS6110 gene in TB-LAMP and 23S rRNA in GenoType MTBDRplus [8–10] are highly homologous among the MTBC members and cannot be used for species differentiation.

    • Development of an immunochromatographic lateral flow dipstick for the detection of Mycobacterium tuberculosis 16 kDa antigen (Mtb-strip)

      2020, Journal of Microbiological Methods
      Citation Excerpt :

      Chest radiography is sensitive in the diagnosis of pulmonary TB, but with low specificity, especially in people with HIV infection (WHO, 2006). Despite simple molecular-based diagnostic tests to detect Mtb such as Xpert MTB/RIF and TB-LAMP have been developed, their implementation in low resource high TB burden countries is still difficult due to many factors such as precarious infrastructure and lack of skilled personnel among others (Niemz and Boyle, 2012; Denkinger et al., 2013; Chin et al., 2018; Monedero-Recuero, 2018). Since TB remains a major global health problem and is responsible for millions of incident cases and deaths each year (Organization WH, 2019), new diagnostic tools for widespread use in resource-constrained settings are needed urgently, which could be a paramount contribution to TB control (Pai et al., 2017).

    • Novel Quantitative PET Imaging Techniques in Tuberculosis

      2020, PET Clinics
      Citation Excerpt :

      Globally, an estimated 10 million new patients developed tuberculosis (TB) in 2018, of which around 1.2 million people (among human immunodeficiency virus [HIV] negative) and 251,000 (among HIV positive), respectively, died.1,2

    View all citing articles on Scopus
    View full text