Lab Diagnosis of MTB, MDR TB as per NTEP guidelines

Lab Diagnosis of MTB, MDRTB as per NTEP guidelines Basic concepts – Keep facts Primary (initial) resistance • TB patient’s initial Mycobacterium tuberculosis population resistant to drugs. Secondary (acquired) resistance • Drug resistant M. tuberculosis in initial population selected by inappropriate drug use (inadequate treatment or non-adherence). Definition of MDR-TB and XDR-TB MDR-TB • MDR-TB caused by strains of Mycobacterium tuberculosis resistant both Rifampicin and Isoniazid with or without resistance to other drugs. XDR-TB • Extensively drug-resistant TB (XDR-TB) is a subset of MDR-TB, where the bacilli, in addition to being resistant to R and H, are also resistant to fluoroquinolones and any one of the second-line injectable drugs (namely Kanamycin, Capreomycin, or Amikacin). Genesis of MDR TB • Resistance is a man-made amplification of a natural phenomenon. • Inadequate drug delivery is main cause of secondary drug resistance. • Secondary drug resistance is the main cause of primary drug resistance due to transmission of resistant strains. • MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti-TB are unlinked. When to suspect MDR-TB  Re-treatment patients who’s sputum smear remains positive after three months of intensive therapy.  Treatment failure and interruption cases.  Close contacts of MDR tuberculosis cases.  Positive diagnosis with; TB culture and susceptibility testing Factors Contributing to Development and Spread of MDR and XDR TB  Weak TB programs (DOTS)  Low completion/cure rates  Lack of treatment follow up and patient support  Unreliable drug supply  Diagnostic delay  Absent or inadequate infection control measures  Uncontrolled use of 2nd line drugs Laboratory Diagnosis of MDR-TB and XDR-TB  The laboratory is an essential component in TB control programs, and broader access to DST is a priority for most countries. Early choice of appropriate treatment is an essential determinant of favorable outcome, and rapid determination of drug resistance can allow a customized approach to treatment early in the course of the disease and can potentially reduce morbidity, mortality and infectiousness. Currently 3 technologies are available for diagnosis of MDR-TB viz.  The conventional solid egg-based LowensteinJensen (LJ) media. The liquid culture (Mycobacteria Growth Indicator Tube). The rapid molecular assays such as Line Probe Assay (LPA) and similar Nucleic Acid Amplification Tests like Xpert MTB/RIF. Fig: Diagnostic workflow CONVENTIONAL CULTURE-BASED METHODS • Using standard DST procedures with conventional methods, 8 to 12 weeks are required to identify drug-resistant microorganisms on solid media (e.g, Lowenstein-Jensen medium). In general such methods assess inhibition of M. tuberculosis growth in the presence of antibiotics to distinguish between susceptible and resistant strains. • Now Outdated - In addition, delayed identification of drug resistance results in adequate treatment, which may generate additional drug resistance and continued transmission in the community. LIQUID CULTURE-BASED METHODS  The BACTEC 460 TB radiometric system (Becton Dickinson, USA) was considered to be a major advancement when it was introduced, but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson, USA). Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs. Detection of drug resistance can be accomplished in days rather than weeks , although still constrained by high cost (equipment and consumables). Fig: Growth supplement and DST drugs Fig: BACTEC MGIT 960 machine Fig: MGIT Tube Diagnosis of MDR-TB and XDR-TB  The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity. Several approaches, phenotypic and molecular, have been explored to develop rapid, reliable and accurate methods for the rapid detection of drug resistance in M. tuberculosis. These methods should also be evaluated and applied in high-incidence areas. Novel, Rapid Molecular Methods The Xpert MTB/RIF • The Xpert MTB/RIF is a cartridge-based, automated diagnostic test that can identify Mycobacterium tuberculosis (MTB) DNA and resistance to rifampicin (RIF) by nucleic acid amplification technique (NAAT). • In December 2010, the World Health Organization (WHO) endorsed the Xpert MTB/RIF for use in TB endemic countries and declared it a major milestone for global TB diagnosis. Fig: Workflow of GeneXpert MTB/RIF assay LINE PROBE ASSAY  Line probe assays (LPAs) are another genetic or molecular test.  They can detect presence of MTB.  First-line LPAs can detect rifampicin and isoniazid resistance (MDR-TB)  Second-line LPAs can be used to detect resistance to the second-line injectable drugs and fluoroquinolones.  LPAs are very important for quickly guiding treatment decisions: 1. To detect resistance to isoniazid, which has worse health outcomes and is not detected by GeneXpert. 2. To determine which people with MDR-TB can take the shortened regimen. Fig: GenoType MTBDRplus probe strip Fig: GenoType MTBDRsl probe strip Advantages of molecular methods:  May be applied directly on clinical specimens.  Can also be used on specimens not suitable for growth-based assays.  Simplification and reduction of bio-safety requirements for both laboratory testing and specimen transport.  Large number of specimens can be processed at the same time.  Automation provides standardization & quality assurance of testing.  Able to detect mutations associated with lower levels of phenotypic resistance.  Detection of specific mutations allows to predict drug resistance to an entire family of drugs (e.g. Amino-glycosides). Advantages of Culture Methods:  Bacterial growth can be identified visually or by automated detection of its metabolism.  Provides a definitive diagnosis of TB.  Significantly increases the number of cases identified (30-50%) when compared to microscopy.  Provides the necessary isolates for conventional DST. Disadvantages of Culture Methods:  More complex and expensive.  Takes longer time.  Needs appropriate lab infrastructure & biosafety precautions.