Rapid diagnosis of drug resistant tuberculosis: current perspectives and challenges

Rapid diagnosis of drug resistant tuberculosis: current perspectives and challengesMuktikesh Dash

Review Article

Department of Microbiology, MKCG Medical College, Berhampur-760004, Odisha. India.E-mail: [email protected]: 21-06-2012 | Accepted: 05-08-2012 | Published Online: 20-08-2012This is an Open Access article distributed under the terms of the Creative Commons Attribution License (creativecommons.org/licenses/by/3.0)Conflict of interest: None declared Source of funding: Nil

Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis complex remains one of the major public health problems, especially in developing countries. The emergence of drug resistant tuberculosis (both multi-drug resistant and extensively drug resistant tuberculosis) is widely considered a serious threat to global TB control. Rapid diagnosis of drug resistant tuberculosis is one of the cornerstones for global TB control as it allows early epidemiological and therapeutic interventions. The present article provides an overview of the various diagnostic options available for drug resistant tuberculosis, including rapid conventional tools and newer molecular methods. Newly developed rapid phenotypic tests include automated liquid based culture and susceptibility tests, thin layer agar cultures, TK medium, microscopic-observation drug susceptibility assay and phage-based assay. Among newly developed molecular methods, real-time polymerase chain reaction (RT-PCR) and line probe assays (LPAs) have been commercialised and widely used in clinical laboratories. To effectively address the threats of drug resistant tuberculosis, global initiatives are required to scale-up culture and drug susceptibility testing capacities. In parallel efforts are needed to expand the use of novel and emerging molecular technologies for rapid diagnosis of drug resistance.

Key words: Tuberculosis; diagnostic tests; drug resistance; PCR; mycobacterium.

Introduction

The impact of tuberculosis (TB) can be devastating even today, especially in developing countries suffering from high burdens of both TB and human immunodeficiency virus (HIV) infections. In 2009 there were 9.4 million new cases of TB globally, causing 1.7 million deaths [1]. Tuberculosis is a major public health problem in India which accounts for one-fifth of the global tuberculosis incident cases. Each year nearly 2 million people in India develop tuberculosis, of which around 0.87 million are infectious cases. It is estimated that annually around 330,000 Indians die due to tuberculosis [2]. Drug resistance has enabled it to spread with a vengeance. The prevalence of multi-drug resistant tuberculosis (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB) are increasing throughout the world both among new tuberculosis cases as well as among previously treated ones [3]. Fortunately, the past few years have seen an

unprecedented level of funding and activity focused on the development of new tools for diagnosis of drug resistant tuberculosis. This should go a long way in helping us arrest the spread of the disease.

Drug resistant tuberculosis

MDR-TB is a form of TB caused by a strain of M. tuberculosis resistant to the most potent first line anti-tuberculous drugs, i.e. isoniazid (INH) and rifampicin (RIF). It has been estimated that India and China account for nearly 50% of the global burden of MDR-TB cases. Approximately 5% of all pulmonary TB cases in India may be MDR. MDR rates are low in new, untreated cases. The incidence in such cases ranges from 1 to 5% (mostly <3%) in different parts of India [4-6]. However, during the last decade, there has been an increase in reported incidences of drug resistance in Category II cases, particularly among those treated irregularly, or with incorrect regimens and doses. In such cases, the incidence of

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MDR-TB varies from 12-17% [6].

XDR-TB, is defined as TB caused by a strain of M. tuberculosis that is resistant to RIF and INH, as well as to any member of the quinolone family and at least one of the second-line anti-tuberculous injectable drugs i.e., Kanamycin, Capreomycin, or Amikacin. XDR-TB was first described in 2006. Since then, there have been documented cases in six continents and 55 countries [7]. The global prevalence of XDR-TB has been difficult to assess. The prevalence of XDR-TB has been reported from India, which varies between low i.e., 2.4% to as high as 33.3% among HIV infected persons suffering from MDR-TB [8,9]. Treatment outcomes are significantly worse for patients with XDR-TB, compared to patients with drug-susceptible TB or MDR TB [10,11]. In the first recognised outbreak of XDR-TB, 53 patients in KwaZulu-Natal, South Africa, who were co-infected with XDR TB and human immunodeficiency virus (HIV), survived for an average of 16 days, with a mortality of 98% [12]. XDR-TB raises concerns of a future tuberculosis epidemic with restricted treatment options, and jeopardises the major gains made in tuberculosis control.

Totally drug resistant tuberculosis (TDR-TB) or extremely drug resistant tuberculosis (XXDR TB) is resistant to all first line and second line anti-tubercular drugs. Four cases were detected in Mumbai who were resistant to all first line and second line drugs [13]. This kind of rapid progression of drug resistance from MDR, to XDR and TDR-TB underlines the need for rapid and accurate diagnosis of drug resistant tuberculosis.

Conventional methods for diagnosis of drug resistant tuberculosis

Mycobacterium tuberculosis is an extremely slow growing organism. Using standardised drug susceptibility testing (DST) with conventional methods, 8 to 12 weeks are required to identify drug resistant tuberculosis on solid media (i.e., Lowenstein-Jensen medium). In general, these methods assess inhibition of M. tuberculosis growth in presence of antibiotics to distinguish between susceptible and resistant strains. As the results usually take weeks, inappropriate choice of treatment regimen may result in death such as in case of XDR-TB (especially in HIV co-infected

patients). In addition, delayed diagnosis of drug resistance results in inadequate treatment, which may generate additional drug resistance and continued transmission in community.

Rapid phenotypic methods for diagnosis of drug resistant tuberculosis

Rapid automated liquid based culture and sensitivity tests- Automated liquid culture systems such as BACTEC radiometric system (Bactec 460TB;Becton Dickinson, USA), non radiometric systems MB/BacT ALERT (BioMerieux, France), Versa TREK (Trek Diagnostic System, USA) and Mycobacteria Growth Indicator Tube (MGIT 960; Becton Dickinson, USA) are more sensitive and have a shorter turn-around time than solid media cultures. These are also less labour intensive and therefore, less vulnerable to manual errors. But automated systems still require few weeks to obtain final results [14]. Also, these instruments are costly, require maintenance and can be extremely difficult for most public health laboratories in developing countries.

Thin layer agar (TLA) cultures and TK Medium- Thin layers of 7H11 middle brook solid agar medium are used to detect microcolonies by conventional microscopy. As it can be adapted for the rapid detection of drug resistance directly from sputum samples, it has an average turn-around time of 11 days [15]. Newly developed test such as TK medium (Salubris Inc., USA) is a colorimetric system that indicates growth of mycobacteria by changing the colour of the growth medium. Metabolic activity of growing mycobacteria changes the colour of the culture medium, and this allows for an early positive identification before bacterial colonies appear. Unfortunately, there is insufficient published evidence on the field performance of these tests in developing countries [16].

Microscopic observation drug susceptibility assay (MODS)- The MODS assay is based on characteristic cord formation of M. tuberculosis that can be visualised microscopically (‘strings and tangles’ appearance) in liquid medium with or without antimicrobial drugs (for DST) [17]. The test sensitivity is better than traditional methods using LJ media with a turnaround time of 7 days for culture and drug sensitivity. Besides, it is cheap, simple

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and fairly accurate [18]. One minor disadvantage of MODS assay is the requirement for an inverted microscope for observation of the mycobacterial growth.

Phage based assay- Phage amplification-based test (FAST Plaque-Response, Biotech Laboratories Ltd., UK), has been developed for direct use on sputum specimens. Drug resistance is diagnosed when M. tuberculosis is detected in samples that contain the drug (i.e., RIF). When these assays were performed on M. tuberculosis culture isolates, they have shown high sensitivity and variable specificity, but evidence is lacking about the accuracy when they are directly applied to sputum specimens [19]. It also requires high standards of bio-safety and quality control.

Rapid molecular methods for diagnosis of drug resistant tuberculosis

Since publication of genome details (M. tuberculosis H37Rv strain) in 1998, these have been utilised in development of nucleic acid amplification (NAA) tests for diagnosis of drug resistant tuberculosis. A number of NAA tests are now available, manual and automated, commercial and in-house, with varying performance characteristics. Real-time polymerase chain reaction (RT-PCR) and line probe assays (LPA) have been commercialised and widely used in clinical laboratories.

Real-time polymerase chain reaction (RT-PCR)- Molecular tools are based on identification of specific mutations responsible for drug resistance, which are detected by the process of nucleic acid amplification in conjunction with electrophoresis, sequencing or hybridisation. Direct sequencing techniques such as real-time polymerase chain reaction (RT-PCR) uses wild-type primer sequences to amplify genes and enables the use of specific probes to identify mutations. Among these is a recently introduced semi-quantitative nested RT-PCR, i.e., GeneXpert MTB/RIF (Cepheid, USA and FIND Diagnostics, Geneva, Switzerland). It integrates and automates sample processing and simultaneously detects M. tuberculosis and rifampicin resistance within single-use disposable cartridges. A study examined 1730 patients with suspected drug-sensitive or multidrug-resistant pulmonary tuberculosis across Peru, Azerbaijan, South Africa and India. There was sensitive detection of M. tuberculosis and rifampicin

resistance directly from untreated sputum in less than two hours with minimal hands-on time [20]. The W.H.O. has recently supported the use of this system as an initial diagnostic test in respiratory specimens of patients with high clinical suspicion of having tuberculosis or who could be multidrug resistant [21]. These tests are expensive and complicated, even if highly sensitive and specific.

Line probe assays (LPAs)- Line probe assays are a family of novel DNA strip tests that use both PCR and reverse hybridisation methods. In these assays, a specific target sequence is amplified, and applied on nitrocellulose membranes. Specific DNA probes on the membrane hybridise with the amplified sequence applied on it. Colour conjugates make the amplified target sequences appear as coloured bands. These tests have been designed to identify M. tuberculosis and simultaneously detect genetic mutations related to drug resistance both from clinical samples as well as culture isolates.

Commercially available kits include the INNO-LiPA Rif.TB (Innogenetics, Belgium), the GenoType MTBDR, MTBDRplus, MTBDRsl assay (Hain Lifescience, Germany). INNO-LiPA Rif.TB test is able to identify M. tuberculosis complex and simultaneously detect genetic mutations in the rpoB gene region related to rifampicin resistance. The GenoType MTBDR assay, introduced in 2004, identifies M. tuberculosis complex and simultaneously detects mutations in the rpoB gene as well as mutations in the katG gene for high-level isoniazid resistance. The second generation MTBDRplus and MTBDRsl assays also detect mutations in the inhA gene for low-level isoniazid resistance and mutations in the gyrA, rrs and embB genes, respectively. The new MTBDRsl assay may represent a reliable tool for detection of floroquinolone, amikacin, capreomycin and ethambutol resistance. A recent laboratory evaluation study from South Africa estimated the accuracy of the GenoType MTBDRplus assay performed directly on AFB smear-positive sputum specimens. It showed high sensitivity, specificity, positive and negative predictive values for detection of rifampicin and INH resistance. However, a meta-analysis on this assay found that sensitivity estimates for INH resistance were comparatively modest [22,23].



Revised National Tuberculosis Control Programme (RNTCP) and drug resistant tuberculosis

The Revised National Tuberculosis Control Programme (RNTCP) plans to strengthen laboratory capacity for M. tuberculosis culture, drug sensitivity testing (C-DST) and Line probe assay (LPA) across India. To date, 35 RNTCP accredited laboratories including 14 LPA and 4 liquid culture laboratories in public and private sectors are serving patients while another 30 laboratories are under the process of up-gradation and accreditation under RNTCP, most of them include LPA and Liquid Culture for first and second line drugs [24]. In a policy statement released in June 2008, the WHO endorsed the use of LPA for rapid screening of patients at risk of MDRTB and recommended the use of line probe assays only on culture isolates and smear-positive sputum specimens. It is not recommended as a complete replacement for conventional culture and drug susceptibility testing [25]. As of January 2012, diagnosis of XDRTB can only be confirmed at three laboratories in India, which are quality assured for second-line anti-tuberculosis drug susceptibility testing of flouroquinolones and injectables. These are the National Reference Laboratories (NRL) of TRC/NIRT Chennai, NTI Bangalore and LRS Institute, New Delhi. Routine fluoroquinolone and injectable DST (i.e. XDR TB diagnosis) on all MDRTB patients at the beginning of treatment has been recommended by the RNTCP National Laboratory Committee in 2011, but the capacity to conduct that testing is not yet present in most culture and DST laboratories used by RNTCP. Capacity building for second line DST is being undertaken through these NRLs [24].

Conclusion

Effective control of drug resistant tuberculosis will require massive scaling-up of culture and DST capacity, and simultaneous use of rapid molecular assays. Furthermore, all molecular tests require DNA extraction, gene amplification and detection of mutations and are, therefore, relatively expensive, demand resources and skills. These are usually unavailable in developing countries where rates of drug resistant tuberculosis are high. The challenge, therefore, is to not only develop new tools, but to also make sure that benefits of promising new tools actually reach the populations that need it most, but can least afford them.

Key Points

• The emergence of drug resistant tuberculosis is a serious threat to control of tuberculosis. For initiating early treatment and prevention, rapid diagnosis of drug resistant tuberculosis is essential.

• Conventional methods take weeks for detection and result in delayed diagnosis resulting in deterioration of patient’s condition and inadequate treatment, which may generate additional drug resistance and continued transmission in community.

• Newly developed rapid phenotypic tests including automated liquid based culture, thin layer agar cultures, TK medium, microscopic-observation drug susceptibility assay and phage-based assay are expensive and not suitable for field testing in developing countries.

• The WHO has supported the use of RT-PCR system as an initial diagnostic test in respiratory specimens of patients with high clinical suspicion of having tuberculosis or who could be multidrug resistant.

• WHO endorsed the use of LPA for rapid screening of patients at risk of MDRTB and recommended its use only on culture isolates and smear-positive sputum specimens. It is not recommended as a complete replacement for conventional culture and drug susceptibility testing.

• Global initiatives are required to scale-up culture and drug susceptibility testing capacities and expand the use of novel and emerging molecular technologies for rapid diagnosis of drug resistance.

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Health & Medicine

Rapid diagnosis of drug resistant tuberculosis: current perspectives and challenges