ReviewDNA markers for tuberculosis diagnosis
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)
- et al.
PolyTB: a genomic variation map for Mycobacterium tuberculosis
Tuberculosis
(2014) - et al.
Molecular diagnostics for tuberculosis
Pathology
(2015) - et al.
Commercial nucleic acid amplification tests in tuberculous meningitis-A meta-analysis
Diagn Microbiol Infect Dis
(2014) - et al.
Identification of nontuberculous mycobacteria by partial gene sequencing and public databases
Int J Mycobacteriol
(2014) - et al.
The role of IS6110 in the evolution of Mycobacterium tuberculosis
Tuberculosis (Edinb)
(2007) - et al.
Digital PCR assay detection of circulating Mycobacterium tuberculosis DNA in pulmonary tuberculosis patient plasma
Tuberculosis (Edinb)
(2016) - et al.
Use of digital droplet PCR to detect Mycobacterium tuberculosis DNA in whole blood-derived DNA samples from patients with pulmonary and extrapulmonary tuberculosis
Front Cell Infect Microbiol
(2017) - et al.
Detection of Mycobacterium tuberculosis-derived DNA in circulating cell-free DNA from a patient with disseminated infection using digital PCR
Int J Infect Dis
(2018) - et al.
IS6110 restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras, South India
Tuber Lung Dis
(1995) - et al.
Evaluation of LoopampTM MTBC detection kit for diagnosis of pulmonary tuberculosis at a peripheral laboratory in a high burden setting
Diagn Microbiol Infect Dis
(2018)
Mycobacterium species identification-A new approach via dnaJ gene sequencing
Syst Appl Microbiol
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
Evaluation of multiplex PCR using MPB64 and IS6110 primers for rapid diagnosis of tuberculous meningitis
Tuberculosis (Edinb)
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
Mycobacterium tuberculosis response regulators, DevR and NarL, interact in vivo and co-regulate gene expression during aerobic nitrate metabolism
J Biol Chem
Comparison of IS6110 and 'short fragment' devR (Rv3133c) gene targets with phenotypic methods for diagnosis of Mycobacterium tuberculosis
Med J Armed Forces India
Evaluation of 24 locus MIRU-VNTR genotyping of Mycobacterium tuberculosis isolates in Canada
Tuberculosis
Nontuberculous mycobacterial pulmonary infections
J Thorac Dis
General overview on nontuberculous mycobacteria, biofilms, and human infection
J Pathog
Rapid and simple approach for identification of Mycobacterium tuberculosis complex isolates by PCR-based genomic deletion analysis
J Clin Microbiol
Fact sheets: tuberculosis
Mycobacterium tuberculosis complex members adapted to wild and domestic animals
Adv Exp Med Biol
Stable association between strains of Mycobacterium tuberculosis and their human host populations
Proc Natl Acad Sci U S A
Robust, reliable and resilient: designing molecular tuberculosis tests for microscopy centers in developing countries
Expert Rev Mol Diagn
Selected culture and drug-susceptibility testing methods for drug-resistant Mycobacterium tuberculosis screening in resource-constrained settings
Expert Rev Mol Diagn
Comparison of culture and PCR methods for diagnosis of Mycobacterium tuberculosis in different clinical specimens
Jundishapur J Microbiol
Molecular diagnosis of mycobacteria
Clin Chem
Tuberculosis diagnostics: state of the art and future directions
Microbiol Spectr
Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic gram-negative bacilli
J Clin Microbiol
Genetic diversity in Mycobacterium tuberculosis
Curr Top Microbiol Immunol
Importance of differential identification of Mycobacterium tuberculosis strains for understanding differences in their prevalence, treatment efficacy, and vaccine development
J Microbiol
Identification of five novel Salmonella typhi-specific genes as markers for diagnosis of typhoid fever using single-gene target PCR assays
BioMed Res Int
Improved identification of rapidly growing mycobacteria by a 16S-23S internal transcribed spacer region PCR and capillary gel electrophoresis
PloS One
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
Multidrug-resistant tuberculosis (MDR-TB): 2013 update
Comparison of 14 molecular assays for detection of Mycobacterium tuberculosis complex in bronchoalveolar lavage fluid
J Clin Microbiol
Exploring alternative biomaterials for diagnosis of pulmonary tuberculosis in HIV-negative patients by use of the GeneXpert MTB/RIF assay
J Clin Microbiol
Detection of Mycobacterium tuberculosis DNA on the oral mucosa of tuberculosis patients
Sci Rep
Genotyping of Mycobacterium tuberculosis: application in epidemiologic studies
Future Microbiol
Diagnostic accuracy of nucleic acid amplification tests (NAATs) in urine for genitourinary tuberculosis: a systematic review and meta-analysis
BMC Infect Dis
Accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant tuberculosis: a systematic review and meta-analysis
Eur Respir J
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
Diagnostic accuracy of nucleic acid amplification based assays for tuberculous meningitis: a meta-analysis
J Infect
Impact of molecular diagnostics for tuberculosis on patient-important outcomes: a systematic review of study methodologies
PloS One
Diagnostic accuracy of nucleic acid amplification tests in urine for pulmonary tuberculosis: a meta-analysis
Int J Tubercul Lung Dis
Systematic review, meta-analysis and economic modelling of molecular diagnostic tests for antibiotic resistance in tuberculosis
Health Technol Assess
Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression
PloS One
In-house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum specimens: meta-analysis and meta-regression
BMC Microbiol
Strategies for differentiation, identification and typing of medically important species of mycobacteria by molecular methods
Vet Med
Differentiation of Mycobacterium species by direct sequencing of amplified DNA
J Gen Microbiol
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2020, TuberculosisCitation 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.
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2020, Journal of Microbiological MethodsCitation 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).
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2020, PET ClinicsCitation 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