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 Table of Contents  
THE EVOLUTION - LABORATORY DIAGNOSIS OF TUBERCULOSIS
Year : 2017  |  Volume : 4  |  Issue : 1  |  Page : 34-44

The expanding canvas of rapid molecular tests in detection of tuberculosis and drug resistance


Department of Microbiology, P D Hinduja National Hospital, Mumbai, Maharashtra, India

Date of Web Publication6-Nov-2017

Correspondence Address:
Camilla Rodrigues
Department of Microbiology, P D Hinduja National Hospital, Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/astrocyte.astrocyte_63_17

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  Abstract 


In many developed countries, tuberculosis (TB) is considered a disease of the past. However, the impact of this disease can be devastating even today, especially in resource poor countries suffering from high burdens of both TB and human immunodeficiency virus (HIV). One of the greatest threats to global TB control is the growing prevalence of drug-resistant bacilli. Correctly diagnosing drug-resistant TB patients is more problematic in resource-limited settings as there is no or limited infrastructure for drug susceptibility testing (DST) of TB bacilli. The conventional phenotypic DST method for TB takes weeks before declaring the results and initiating proper anti-TB treatment. The evolution of molecular diagnostic methods has revolutionized the TB diagnostics. These methods are accurate, rapid, easy to perform, and can solve controversial issues related to TB diagnosis as well as drug susceptibility. It is important to link these rapid molecular techniques with the conventional methods to determine the impact of the disease.

Keywords: Drug resistance, drug susceptibility tests, point-of-care tests, TB


How to cite this article:
Shah A, Rodrigues C. The expanding canvas of rapid molecular tests in detection of tuberculosis and drug resistance. Astrocyte 2017;4:34-44

How to cite this URL:
Shah A, Rodrigues C. The expanding canvas of rapid molecular tests in detection of tuberculosis and drug resistance. Astrocyte [serial online] 2017 [cited 2020 Mar 28];4:34-44. Available from: http://www.astrocyte.in/text.asp?2017/4/1/34/217660




  Introduction Top


Tuberculosis (TB) has afflicted mankind through the millennia, not sparing anyone; be it the poor or the rich, the common man, or the ruler. The global burden of TB has been mountainous throughout the past century and continues to remain even so today. It is estimated that more than 14 lakh persons die from this infectious disease around the globe, which includes 4.8 lakh in India alone.[1] The problems confronting us now is due to decades of neglect partly fostered by misconception that we had drugs to control the disease. With increasing prevalence of drug resistance cases, smear negative pulmonary, and extrapulmonary TB cases as well as the unholy alliance between human immunodeficiency virus (HIV) and TB, the need for highly sensitive and rapid diagnostic tools are becoming a vital aspect of management for early initiation of appropriate anti-tubercular treatment.[1]

TB diagnosis nowadays comprises microscopy, liquid culture (MGIT), as well as rapid molecular diagnostic techniques such as Xpert MTB/RIF and Line Probe Assay.


  Microscopy Top


Given the poor specificity of tuberculin skin test (TST), the importance of smear and culture confirmation cannot be overemphasized. Smear examination by Ziehl–Neelson (Z/N) stain has the advantage of simplicity, availability, and rapidity, however, the sensitivity is affected by the skill and experience of the microscopist, the number of specimens examined, and the concentration of organisms in the sputum. Microscopy can detect up to 70% of culture positive samples with a lower limit of detection of 5 × 103 organisms/ml.[2],[3] Optimizing microscopy includes methods such as bleach and guanidinium hydrochloride, as well as using more sensitive methods as fluorescent microscopy. Mycobacterial cells appear bright yellow or orange against a greenish background. This method can be used to enhance the detection of mycobacteria directly in patients' specimens. The advantage of this method is that lower magnification can be used, enabling rapid screening over a wider area.[4]

A systematic review evaluating the diagnostic yield of each of the 3 sputum specimens inferred that microscopy analysis of the third specimen provided a low incremental diagnostic yield of 2–5%. Based on these findings, the World Health Organization (WHO) recommends the implementation of “same-day-diagnosis” approach, emphasizing that all countries using the three-specimen case-finding strategy to switch to two-specimen case-finding strategy.[5]


  Culture Techniques Top


Traditionally, culture is the gold standard and solid culture was the method used for confirmatory diagnosis of TB. Although having higher sensitivity compared to smear microscopy, it has a long turnaround time (TAT) of approximately 4–8 weeks. In addition, drug susceptibility testing (DST) takes 2–3 weeks, thus resulting in further delay in initiation of appropriate anti-tubercular treatment. Broth-based liquid cultures have profound advantages over solid culture, capable of detecting as low a level as 10 to 103 viable bacilli per millilitre of the specimen, thereby increasing the case yield by 10%.[6]

Conventional tests

Agar-based methods: Mycobacterial culture is a far more sensitive test than smear examination and allows biochemical identification of the species, considerably enhancing the specificity. Agar-based media allow detection of colonies in 10–12 days, whereas the most commonly used egg-based Lowenstein–Jensen Medium (LJ) usually takes 18–24 days.[7],[8] Susceptibility testing can also be performed on LJ but the turnaround time is approximately 3 months for culture and susceptibility.

Automated liquid culture methods:

  1. Mycobacterial Growth Indicator Tube (MGIT) 960 TB: The test employs a new state of the art fluorescent technology that enables result towards positivity as rapidly as 7–10 days.[9] MGIT 960 TB is used for the isolation and accurate identification of Mycobacteria
  2. MB Bact is based on a colorimetric CO2 sensor that is altered by bacterial metabolism.[10]


These liquid culture systems, however, have limitations: (1) They are more prone to contamination by other nonmycobacterial organisms (8.6%)[11] or nontuberculous mycobacteria (NTM); even in experienced laboratories, approximately 5–7% of specimens do not yield results because of contamination. (2) Cross-contamination between samples during culture inoculation, that is, carryover of bacilli from positive to negative specimens, is possible (4%). A recent study reported that liquid culture systems are more accurate and cost-effective than solid cultures for the diagnosis of TB in HIV-positive patients in a resource-limited setting.[12]

Liquid culture systems are capable of producing positive results in 2 weeks for the vast majority of sputum smear-positive specimens and within 3 weeks for smear-negative specimens.


  Drug Susceptibility Tests Top


Detecting drug resistance in TB is done by either phenotypic methods or genotypic methods.

Phenotypic methods

Phenotypic methods are generally based on the growth of M. tuberculosis in the culture media. Until and unless culture does not grow, there is absolute no role of phenotypic methods.

Phenotypic methods include:

  • Growth observation
  • Detection of metabolic activity or products
  • Phage based technologies


Conventional methods

The in-vitro DST is a universally accepted way of selecting appropriate drugs for chemotherapy for TB patients. These tests also provide useful epidemiological data for planning of large scale treatment. Three methods described by WHO are absolute concentration, resistance ratio and proportion method.[13],[14]

The BACTEC MGIT 960 1% proportion methods has been approved by the US FDA and is also considered to be the “gold standard” for the drug susceptibility testing to anti-TB drugs. The principle of this assay is similar to that explained before for liquid culture using BACTEC MGIT 960.

Nonconventional methods

These methods include Microscopic Observation Direct Susceptibility (MODS), Thin Layer Agar (TLA), phage-based methods, colorimetric redox indicator methods, and nitrate reductase assays (NRAs); they are optimized to facilitate rapid determination of drug-susceptibility pattern of MTB strains.

Genotypic methods

TB drug resistance is associated with mutation (s) in the genes relevant to responses to each drug. Genotypic methods for detecting mutations in these genes have been proposed to overcome the limitation of phenotypic DST, which is, without the culture growing, phenotyping methods cannot be used.

Genotypic methods include molecular methods which 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.

Molecular methods

Majority of molecular tests have been focused on detection of nucleic acid, both DNA and RNA, that are specific to both M. tuberculosis, by amplification techniques such as polymerase chain reaction (PCR) and detection of mutation in the genes that are associated with resistance to anti-tubercular drugs by sequencing or nucleic acid hybridization.

Advantages of molecular methods over phenotypic methods:

  • Molecular tests can be done directly from the sample
  • High specificity as well as sensitivity
  • More accurate and reproducible, giving diagnosis within 48–72 hours
  • Less biohazard risk


Molecular diagnostics for TB have evolved because the long turn-around-time for culture methods. These molecular diagnostics include both detection of organism directly from the sample as well as we can have idea about the drug resistance pattern.

  • Direct detection of TB from clinical samples
  • Additional genotypic methods for detection of drug resistance.


i) Direct detection by nucleic acid amplification directly from clinical samples allows for initiation of treatment if the clinical picture is consistent. Various in house PCR assays include amplification of genes encoding mycobacterial antigens, repetitive sequences, ribosomal RNA, etc., Commercial FDA approved systems include Amplicor (Roche Diagnostics) and AMTD (GenProbe) Nucleic acid amplification.

a. Polymerase chain reaction (PCR): PCR is an in-vitro method for amplifying specific DNA sequence. Starting with extremely minute amounts of a particular nucleic acid sequence from any source, PCR enzymatically generates millions or billions of exact copies, thereby making genetic analysis of tiny samples a relatively simple process. Nested PCR certainly enhances the sensitivity of PCR.

TB PCR test amplifies IS6110 region which is present in MTB complex. Results of PCR should be carefully interpreted as it is DNA-based technology and cannot differentiate between dead and viable organisms. False positive results may be seen in old TB cases. False negative results may be seen in extremely paucibacillary cases and needs to be correlated clinically. Few Indian strains lack IS6110, in such cases this test is of no use. The detection limit of this assay is up to 10 copies/ml.

Application of TB-PCR in routine practice is of very limited value because of high rate of false positive cases as well as contamination issues.

b. Transcription mediated amplification (TMA): TMA uses a species-specific sequence of ribosomal RNA (rRNA) as the target for reverse transcriptase. The advantage of this technology is that dead cells have no transcription machinery, hence, only viable cells are picked up and amplified.

DNA amplification technology can amplify minute quantities of DNA to levels that can be readily seen following routine agarose gel electrophoresis. However, amplification can amplify even minute quantities of contaminating DNA. False positive results are the major concern. Moreover, the presence of an organism in a clinical specimen does not necessarily indicate disease. Various target antigens have been used IS6110, MPB64, 16S rRNA gene, 65kd, 38kD rpo B, etc.

In theory, nucleic acid amplification (NAA) tests are capable of amplifying a single copy of the target genomic sequence. In practice, although molecular tests hold promise in diagnosis, they have a modest sensitivity in extrapulmonary specimens.

c. Real-time PCR techniques: This same principle of amplification is employed in real-time PCR. However, instead of looking at bands on a gel at the end of the reaction, the process is monitored in “real time.” Literally, the reaction is placed in to a real-time PCR machine that watches the reaction occur with a camera or detector.

There are several different techniques that are used to allow the progress of a PCR reaction to be monitored but they all have one thing in common. They all link the amplification of DNA to the generation of fluorescence, which can simply be detected with a camera during each PCR cycle. Hence, as the number of gene copies increases during the reaction, so the fluorescence increases.

The main advantages of real-time PCR techniques are the speed of the test and a lower risk of contamination. Real-time PCR techniques have been applied to M. tuberculosis strains and, more recently, directly to clinical samples.

Molecular beacon assays, which work on the principle of real-time PCR, are based on a stem and loop structure with the loop in the probe. Easier to perform, real time formats such as GeneXpert are being currently evaluated directly from samples. These assays have huge potential as they are rapid and can be performed in a real world setting out of the molecular lab.

ii) Molecular methods for detection of drug resistance: broadly classified as

Probe-based methods/DNA-chip based methods

  • CB-NAAT or Xpert MTB/RIF
  • Line Probe Assays
  • Microarray
  • Sequencing-based methods
  • Pyrosequencing
  • Whole Genome Sequencing


Genotypic methods for drug resistance

Genetic studies have determined that, in M. tuberculosis, resistance to anti-tubercular drugs is the consequence of spontaneous mutations in genes encoding either the drug target or enzymes involved in drug activation. Resistance-associated random chromosomal mutations have been described for all first-line drugs; however, no single genetic alteration has yet been found that results in the polyresistant or MDR phenotype.[15] Rather, polyresistance or multidrug resistance develops through the sequential acquisition of mutations at multiple loci. Unfortunately, the multiple locations of the mutations for each drug and the fact that resistance to a particular drug can involve a mutation in more than one gene or in several distinct loci of the same gene have made the molecular detection of drug resistance a challenging task.

More than 95% of rifampicin-resistant isolates possess mutations in the Rifampicin Resistance Determining Region (RRDR: 81-bp region, codon 507–533) of the rpoB gene, whereas 70–80% of isoniazid-resistant isolates harbour mutations in the katG and inhA genes. Subsequent genetic studies have also revealed the presence of certain mutations to be associated with conferring resistance to clinically important second-line anti-tubercular drugs. For example, mutations in the gyrA (codon 90–94) and gyrB (codon 510) region have been known to confer resistance in approximately 85% of the fluoroquinolone-resistant isolates, whereas approximately 80% of aminoglycoside resistant isolates are known to possess mutations at codon 1401 and 1484 of the rrs gene. Using these molecular data, several commercial and in-house assays have been developed to facilitate rapid determination of MDR as well as XDR-TB with a turn-around-time of approximately 2–3 days.


  Line Probe Assays (Solid Phase Hybridization Technique) Top


There are currently two commercially available solid-phase hybridization techniques for the rapid detection of drug resistance in TB – The Line Probe Assay (INNO-LiPA Rif TB Assay, Innogenetics, Belgium) for the detection of resistance to RIF and the GenoType MTBDR assay (Hain Lifesciences, Germany) for the simultaneous detection of resistance to isoniazid and rifampicin. INNO-LiPA Assay is not available in India.

A systematic review evaluated the use of Genotype MTBDRplus assays and reported higher diagnostic accuracy for determination of rifampicin resistance (pooled sensitivity, 98.1%; pooled specificity, 98.7%) than isoniazid resistance (pooled sensitivity, 84.3%; pooled specificity, 99.5%) for both clinical isolates and direct clinical specimens.[16],[17] Overall sensitivity and specificity for Genotype MTBDplus in smear positive samples is 96.4% and 100%, respectively, compared to smear-negative samples where it is 77.8% and 97.2%, respectively.[16],[17]

GenoType MTBDRsl (MTBDRsl) [Figure 1] is a rapid DNA-based test for detecting specific mutations associated with resistance to fluoroquinolones and second-line injectable drugs (SLIDs) in MTB complex. MTBDRsl version 2.0 (released in 2015) identifies the mutations detected by version 1.0, as well as additional mutations. The test may be performed on a culture isolate or a patient specimen, which eliminates delays associated with culture. Version 1.0 requires a smear-positive specimen, whereas version 2.0 may use a smear-positive or -negative specimen.[18]
Figure 1: Templates for Genotype MTBDRsl ver 1.0 and ver 2.0.

Click here to view


The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance (96%) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93%). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4%, respectively. Now a days, GT blot, an automated reverse hybridisation instrument, is used for the performing Line Probe Assay [Figure 2].
Figure 2: GT Blot- automated reverse hybridisation (Line Probe Assay).

Click here to view


Advantages of line probe assay

  • Useful for both smear-positive as well as smear-negative pulmonary samples.
  • According to various mutation bands, this test is helpful to indicate the level of resistance (high or low) to particular drug, such that treatment can be changed accordingly.
  • Early diagnosis of both MDR as well as XDR-TB as results are available within 48 hours


Disadvantages of line probe assay

  • Extrapulmonary smear-negative samples show very low sensitivity and specificity, and hence, are not useful.
  • Silent mutation may result in false prediction of resistance.
  • It requires proper training of the staff and technical excellence so cannot be done at periphery level


CB-NAAT or Xpert MTB/RIF – Cartridge- Based Nucleic Acid Amplification Test

The most recent commercial diagnostic tool developed by Cepheid Inc. in collaboration with Foundation for Innovative New Diagnostics [Figure 3].
Figure 3: Xpert MTB/RIF

Click here to view


The test is based on a real-time hemi-nested PCR test which detects the presence of M. tuberculosis complex bacilli.[19] Using 5 molecular beacons which span the rpoB gene 81-bp rifampin resistance-determining region (RRDR), the test simultaneously determines susceptibility to rifampicin, which can be used as a surrogate marker for MDR.[19] The turn-around-time is <2 h, so a very useful point-of-care test in a routine practice.

The closed-cartridge system makes it possible for the assay to be used outside the laboratory environment, and studies assessing biosafety have suggested that the use of Xpert MTB/RIF carries a smaller biohazard risk than smear microscopy.[20] The risk of cross-contamination is also reduced with the closed cartridge system.[21]

The test has shown a sensitivity above 90% for culture-positive tuberculosis, with high specificity in sputum samples. Sensitivity in individuals with HIV coinfection is over 80%.[22],[23],[24] A recent Cochrane review concluded that the Xpert MTB/RIF as an initial replacement for sputum smear showed a pooled sensitivity of 88% [95% credible interval (CrI), 83–92%] and a pooled specificity of 98% (95% CrI, 97–99%).[25]

Several studies have reported successful use of the Xpert MTB/RIF test on extrapulmonary samples, with overall sensitivities of over 80% and specificity reaching 100%.[26],[27],[28],[29],[30] The Xpert test has true diagnostic potential with good sensitivity (86–100%) for specimens such as synovial, pericardial, and peritoneal fluids; pus; and fine-needle aspirates and moderate sensitivity (63 to 73%) for tissues, lymph nodes, and pleural fluid but poor sensitivity (29%) in the case of CSF.[30] A preprocessing step (concentrating the specimen by centrifuging it at high speed and then using the pellet for processing) might be required to increase the sensitivity for paucibacillary specimen types such as CSF.[30] The Xpert MTB/RIF has no role in follow-up cases. Thus, once the treatment is initiated based on the Xpert MTB/RIF test, on subsequent follow-up, there is no need to repeat the test again as it is of no use.

The Xpert MTB/RIF (Xpert) assay, detects Mycobacterium tuberculosis (Mtb) with a limit of detection (LOD) of 133 CFU/ml sputum, and detects mutations in the MtbrpoB gene which cause rifampicin resistance (RIF-R).

Advantages of Xpert MTB/RIF

  • Less dependent on the user's skills, and routine staff with minimal training can use the test
  • It has a short turn-around-time and simultaneously detects M. tuberculosis and RIF resistance in less than 3 h
  • Closed cartridge-based system so less chances of biohazard


Disadvantages of Xpert MTB/RIF

  • Sensitivity is only 60–80% in smear-negative TB
  • Sensitivity is very low (around 30–40%) for sterile body fluids
  • Silent mutation or abnormal real-time PCR curves may result in false prediction of resistance


Microarrays, also known as biochips or DNA chips, have been proposed as genotypic methods [31] for detecting drug resistance in M. tuberculosis. They are based on the hybridization of DNA obtained from clinical samples to high density oligonucleotides immobilized on a solid support such as miniaturized glass slides.

Pyrosequencing (PSQ) is a DNA sequencing technique based on the detection of the pyrophosphate released during DNA synthesis,[32],[33],[34] and is well suitable for large-scale screening for a short length DNA fragment.

PSQ can detect mutations associated with first- and second-line anti-TB drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations.

The molecular targets included katG, the inhA promoter, and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance.

PSQ data were compared to phenotypic MGIT 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP.

PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.[35],[36]

PSQ is of immense help to resolve the issue when there is discordant results between phenotypic DST by MGIT and any molecular techniques (Xpert MTB/RIF or LiPA).

[Table 1] showing comparison between Line Probe Assay and Xpert MTB/RIf.
Table 1: Comparison between LPA and Xpert

Click here to view



  Future of Tuberculosis Diagnosis Top


Xpert Ultra: 24 March 2017, On World TB Day, Cepheid, Rutgers New Jersey Medical School and FIND announced a new version of the Xpert MTB/RIF test, the Xpert® MTB/RIF Ultra (Ultra), for the diagnosis of TB and rifampicin resistance. WHO also issued a recommendation that Ultra can be used as an alternative to the existing Xpert MTB/RIF test for the diagnosis of TB and detection of rifampicin resistance in all settings.

A new molecular TB test with a limit of detection (LOD) equivalent to the 10–100 CFU/ml LOD of liquid culture and improved RIF-R detection. The new Ultra assay is much more sensitive than Xpert, and is as sensitive as liquid TB culture. Ultra detects RIF-R as efficiently as Xpert; however, the specificity of Ultra RIF-R is likely to be higher due to improvements in assay design. The Ultra assay should significantly increase TB detection in smear-negative patients and provide more reliable RIF-R detection.

Xpert Omni: A new device called the GeneXpert Omni is intended for point-of-care testing for TB and rifampicin resistance, using the same cartridges as those used in the current Genexpert machine. It is smaller, lighter, and less expensive than the current Genexpert, with a built-in 4-hour battery. Because of above mentioned characteristics, it is very useful particularly in the remote settings where very limited infrastructure is available for rapid diagnosis of TB.

Truenat MTB: A chip-based nucleic acid amplification test involve sputum processing using Trueprep-MAG™ (nanoparticle-based protocol run on a battery-operated device) and real-time PCR performed on the Truelab Uno™ analyzer (handheld, battery-operated thermal cycler). The preliminary study shows that the Truenat MTB test allows detection of TB in approximately 1 hour and can be utilized in near-care settings to provide quick and accurate diagnosis. It has good sensitivity and specificity for the diagnosis of TB but also fits the requirements of the resource-limited health care settings. Large studies are required to obtain better estimates of the Truenat MTB performance.

Genome sequencing

Whole-genome sequencing (WGS) of bacterial genomes allows simultaneous identification of all known resistance mutations as well as markers, with which transmission can be monitored.[37]

However, with the mean time to a positive MGIT culture being 14 days, most WGS results are not available for more than 2 weeks, which is too long a delay before starting therapy.[38]

Recently, a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement ofculture. This oligonucleotide enrichment technology [SureSelectXT (Agilent) method] has been developed to obtain the first M. tuberculosis genome sequences directly from both smear-positive and smear-negative sputum [Figure 4].[39],[40],[41],[42]
Figure 4: WGS workflow for Mycobacterium tuberculosis.[39],[40],[41],[42]

Click here to view


Algorithms for integrated use of conventional and molecular diagnostic assays for rapid TB diagnosis and determination of drug resistance

Despite a pool of available diagnostic assays, there is still no single test completely reliable for the laboratory diagnosis of TB. Diagnostic algorithms in developed countries with a low incidence of TB focus on the screening process so as not to miss any patient with TB, whereas those in resource-limited settings with a higher incidence of HIV-associated and DR TB focus primarily on identifying patients at risk of MDR TB infection to facilitate initiation of appropriate anti-tubercular treatment and thereby interrupt further transmission.

To effectively curb the rise and spread of MDR-TB throughout the globe, there is an urgent need to quickly and accurately determine clinical drug susceptibility profiles of DR isolates. The correlation of genotypic test results, including line probe assays and PSQ, with a specific range of MIC values for each drug, presents an invaluable tool for quickly guiding treatment decisions in clinical laboratories worldwide. Because effective MDR-TB treatment relies upon our knowledge of quantitative susceptibility testing results, the correlation of genotypic test results with phenotypic resistance levels is a critical component of TB diagnosis that should be further evaluated to prevent the spread of drug resistance and promote the optimum use of the few drugs available for TB treatment.

For RIF, mutations within the 81bp region of the rpoB gene, encoding the B subunit of a DNA-dependent RNA polymerase, are responsible for conferring RIF resistance. Canonical mutations in this gene, including 516Val, 526Asp/Tyr and 531Leu, are well documented, whereas amino acid changes at codons 511, 515, 516, 518, 521, 522, and 533 have not been thoroughly evaluated for their association with phenotypic RIF-resistance. Notably, high-level RIF-resistance is reported more frequently than lower levels of resistance, supporting the analysis of these other, potentially RIF-resistance associated mutations for diagnostic purposes [Table 2].[43]
Table 2: Important mutations associated with RIF and INH

Click here to view


For INH, the accumulation of mutations in the katG gene and inhA promoter leads to development of resistance. Mutations within katG prevent the activation of the gene's respective prodrug, resulting in high-level INH resistance [Table 2].

Mutations in the inhA promoter gene are known to increase the level of protein expression, and are generally correlated with low-level INH resistance,[43] is a point of clinical relevance, as isolates with this mutation might be treated with higher doses of INH [Table 2].

When patients with MDR-TB are compared to patients with MDR-TB that have additional fluoroquinolones resistance, those with fluoroquinolones resistance appear to have a more serious form of disease, in that treatment success becomes less common and the risk of developing XDR-TB increases.[44] Cross-resistance to the fluoroquinolones is frequent and fluoroquinolones resistance in MTB is usually associated with mutations in the conserved quinolone resistance-determining region (QRDR) of gyrA, particularly at codons 90 and 94.[44]

Phenotypically, gyrA mutations with Ala90Val, Ser91Pro, or Asp94Ala showed a low level of resistance to fluoroquinolones, have the potential to be rapidly diagnosed and treated with standard or increased dose of fluoroquinolones, whereas Asp94Asn/Tyr, Asp94Gly, or Asp94His mutations demonstrated a high level of resistance [Table 3].
Table 3: Important mutations associated with FQs and AGs

Click here to view


Amikacin (AMK)/kanamycin (KAN), and capreomycin (CAP) are known to effect protein synthesis in MTB, and resistance to these compounds is primarily conveyed by changes in the 16S rRNA (encoded by the rrs gene).[45] The rrs mutation, A1401G, can cause high-level AMK/KAN resistance and lower-level CAP resistance. Eis, an aminoglycoside acetyltransferase, catalyzes the transfer of an acetyl group from acetyl-coenzyme A to an amine group of aminoglycoside. It has been reported that Eis of M. tuberculosis shows a multiacetylation capability at the 2'-, 3'-, or 6'- positions of aminoglycoside antibiotics.[45]

The rrs canonical mutation, A1401G, was found to confer a high level of AMK and KAN resistance to MTB isolates, however, a moderate level of CAP resistance. eis promoter mutations were generally determined to confer lower levels of AMK and CAP resistance and low-to-moderate levels of KAN resistance [Table 3].

Hence, it is very important that all clinicians follow an algorithm to maintain a balance in the number of requests for certain diagnostic procedures; otherwise, laboratory services will be overburdened with unnecessary testing and will also increase the cost burden on the patient. Thus, effective planning [Figure 5] would facilitate the successful integration of conventional and molecular methodologies for rapid diagnosis of MDR TB in a reference laboratory setting in a middle incidence country.
Figure 5: Cost effective algorithm using WHO approved tests for rapid TB diagnosis and determination of drug resistance.

Click here to view


Despite the clear advantages that molecular methods offer for drug susceptibility testing in terms of turn-around-time, the cost implications should be borne in mind for resource-constrained settings as ours. Associated laboratory infrastructure including proper design and quality control issues to avoid cross-contamination and amplicon generation is paramount. Lastly, wherever the genetic basis of resistance is not fully characterized, drug resistance should be confirmed by well standardized phenotypic methods.


  Conclusions Top


Diagnosis of MTB poses a major challenge to health care facilities and the research community in resource-constrained settings. The more sophisticated molecular tools for diagnosis and DST are not widely available as in developed countries. Ideally, a diagnostic test for MTB should be accurate, fast, easy-to-implement, sustainable, and affordable. Acknowledging the need to facilitate a timely transition from the research laboratories to programmatic level implementation, WHO has sped-up the intake of new technologies that are evidence based. The evidence is provided by diagnostic accuracy evaluations, where accuracy performance has been used as a surrogate of patient-important outcomes.

All the above mentioned molecular tests have better performance characteristics in terms of increased sensitivity and better accuracy for detecting MDR as well as XDR-TB, and thus represent a better diagnostic tools for the rapid detection of resistance to first-line as well as second-line drugs; this should be recommended in countries like India with a high burden of MDR/XDR-TB or in retreatment case. Based on the report of these tests, clinicians can start proper treatment very early in the context of the disease progression as well as helpful in prevention of drug resistance incidences because of improper empirical therapy.

Rapid molecular diagnosis has a definite edge over conventional phenotypic methods being highly sensitive and specific, as well as results are available in 48–72 hours. 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 hours. Line probe assays detect point mutations in rifampicin, isoniazid, fluroquinolones, as well as aminoglycosides. The canonical mutations are useful in deciding treatment options. With smear-positive samples, line probe assays show higher sensitivity and specificity compared to smear-negative ones. PSQ and other sequencing methods are useful, particularly in cases of discrepancies between phenotypic and genotypic methods. Emphasis should be placed on developing rapid and accurate sequencing techniques directly from the sample. The landscape of TB diagnostics offer newer technologies with point-of-care assays enabling TB diagnosis to be available easily from bench to bedside.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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