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Table of Contents
REVIEW ARTICLE
Year : 2015  |  Volume : 2  |  Issue : 2  |  Page : 118-129

WHO recommended tools to improve diagnosis of active and drug resistant tuberculosis


Center for Emerging Pathogens, Department of Medicine, New Jersey Medical School - Rutgers University, Newark, New Jersey, USA

Date of Web Publication5-Jul-2017

Correspondence Address:
Shubhada Shenai
Research Scientist/Research Teaching Specialist (RTS). Department of Medicine, Division of Infectious Diseases, Center for Emerging and Re-emerging Pathogens, Rutgers New Jersey Medical School, Rutgers Biomedical and Health Sciences, 185 South Orange Avenue, MSB A920G, Newark, NJ 07103
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.5530/ami.2015.4.3

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  Abstract 


The successful arrest or cure of tuberculosis (TB) depends largely on early case detection and proper treatment. Currently, several promising diagnostic tools with improved sensitivity and speed are available. Old microscopic and culture based technologies have been revisited and new molecular technologies have been developed. In recent years, World Health Organization (WHO) evaluated and endorsed different TB diagnostic tools and published various policy statements. These policy statements address improvements in smear microscopy, use of commercial and non-commercial culture based techniques and rapid molecular assays. This is the first comprehensive review compiling pros and cons of WHO recommended TB diagnostic tools for effective implementation in TB endemic regions.

Keywords: Tuberculosis, TB Diagnostic methods, Drug resistant TB


How to cite this article:
Shenai S. WHO recommended tools to improve diagnosis of active and drug resistant tuberculosis. Acta Med Int 2015;2:118-29

How to cite this URL:
Shenai S. WHO recommended tools to improve diagnosis of active and drug resistant tuberculosis. Acta Med Int [serial online] 2015 [cited 2019 May 19];2:118-29. Available from: http://www.actamedicainternational.com/text.asp?2015/2/2/118/209628




  Background Top


Tuberculosis (TB), a potentially fatal contagious disease, caused by Mycobacterium tuberculosis complex, is preventable and curable. TB is spread from person to person through the air by droplet nuclei that are produced when person with pulmonary or laryngeal TB cough. If not diagnosed and left untreated, each person with active pulmonary TB will infect on average between 10-15 people every year.[1] In 2013, 9.0 million people developed TB, and 1.5 million people died from the disease; 360 000 of whom were HIV positive individuals [Figure 1].[2] Nearly 3.5% of new and 20.5% of previously treated TB cases had multidrug-resistant TB (MDR-TB) [Figure 2], of which 9.0% were estimated to have extensively drug resistant TB (XDR-TB) [2]. Despite major efforts to increase case detection, one third of all TB cases are not still being diagnosed or notified to national TB programs each year[2] and less than 3% are tested to determine the pattern of drug resistance. A substantial percentage of patients with drug-susceptible TB (~35%) and a staggering (~85%) percentage of patients with MDR-TB remain undiagnosed.[3] Thus, prompt and accurate diagnosis of TB and drug resistance remains one of the biggest obstacles to global TB control.
Figure 1: Global Incidence of Tuberculosis. (Adapted from Reference 1). (a) shows the estimated global incidence of tuberculosis is 2013. Source: WHO report 2014, (b) shows estimated absolute numbers of TB cases and deaths (in millions per year), 1990-2013

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Figure 2: Percentage of new and previously treated TB cases that have MDR-TB (Adapted from Reference 1)

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In majority of high TB-burden countries, presumptive diagnosis of active TB is generally made on the basis of patient history, clinical presentation, radiological findings and smear microscopy. Although useful, radiological findings may not always provide absolute prediction of the disease. Smear microscopy is rapid, easy and cost-effective, but it lacks sensitivity (requires >104 bacilli/ml of sputum). Therefore, it has limited value for diagnosis of TB in children, human immune-deficiency virus (HIV) infected people and pauci- bacillary extra-pulmonary TB.[4],[5],[6] Further, microscopy cannot distinguish between viable and non-viable, drug susceptible and drug resistant M. tuberculosis complex or between different species of Mycobacteria. Culture-based methods are more reliable (detection limit: 10-100 CFU/mL sample). Culture also allows specific species identification and testing for recognition of drug susceptibility patterns. However, due to a slow growth rate of M. tuberculosis, conventional culture based techniques are time consuming and laborious.[7] Nucleic acid amplification tests (NAATs) can detect small quantities of genetic material (DNA or RNA) from M. tuberculosis complex and provide rapid results several weeks earlier than culture. NAATs are available as commercial kits or in-house tests. In-house polymerase chain reaction (PCR) is a molecular technique that allows sequence specific amplification of M. tuberculosis DNA and makes thousands of copies from very few DNA target molecules that can be detected by either electrophoresis on agarose gel or measuring the fluorescence intensity in Real Time PCR. In-house molecular assays are complex, labor-intensive and technically challenging. However, they are less expensive than commercial kits and hence widely used in developing countries. Although the specificity of NAATs is high, some NAATs have shown variable and low sensitivity, especially in extra-pulmonary and sputum smear-negative patients.[8],[9]

In this scenario, it is very essential to identify active and drug resistance TB cases as early as possible, so that the patient can be provided with the most effective treatment for rapid recovery and cure. In recent years, several promising TB diagnostic tools have become available and some are even being used in many countries. These tools must be adequately investigated and validated before they can be routinely used. WHO has evaluated selected TB diagnostic techniques and published various policy statements between 2007 and 2014. These policy statements address improvements in smear microscopy, use of commercial and non-commercial culture based assays and use of rapid molecular assays like line probe assay (LPA) and GeneXpert MTB/RIF (Xpert) for precise diagnosis of TB and DST.[10],[11],[12],[13] This review focuses on advantages and limitations of various WHO recommended TB diagnostic tests (as listed in [Table 1]).
Table 1: Tuberculosis diagnostic tests recommended of WHO for use in disease endemic region

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  Who Recommended TB Diagnostic Tools Top


LED Microscopy

Light- emitting diodes (LED) microscopy was developed for resource-limited countries to offer the benefits of fluorescence microscopy (FM) without the associated costs. As shown in [Figure 3], a small LED attachment can easily convert any compound light microscope into fluorescence microscope. Compared to mercury vapor fluorescent microscopes, LED microscopes have long life spans, lower maintenance requirements, require less power and are able to run on batteries.[14],[15],[16],[17] The bulbs have a very long half life, there is no UV light production and they are reported to perform equally well without a darkroom. These qualities make them 60% less expensive and feasible for use in low resource settings.[14],[15],[16],[17] LED microscopy allows a much larger area of the smear to be seen, resulting in up to 4 times faster examination of smear. Many studies have shown that LED-FM can play a key role in reaching WHO targets for TB detection, reducing laboratory workloads, and ensuring poor patients' access to TB diagnosis and prompt treatment.[11],[14],[15],[16],[17]
Figure 3: LED attachments to convert simple compound light microscope into LED Microscope. (a) LED attachment available at Zeiss Primo Star and LW Scientific Lumin, (b) LED attachment available at Zeiss Primo Star, LW Scientific Lumin and QBC ParaLens, (c) Commercial LED Microscopes available at Zeiss Primo Star, LW Scientific Lumin and QBC Diagnostic ParaLens, (d) LED illuminated auramine-O stained M. tuberculosis cells. Acid-fast organisms display a yellowish green fluorescence. (Figure adapted from Primo Star iLED webpage, http://www.zeiss.com/microscopy/en_de/products/light-microscopes/primo-star-iled.html)

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In 2009, the evidence for the efficacy of LED-FM was assessed by the WHO and as per results it was found to be 10% more sensitive[6] with no appreciable loss in specificity when compared with conventional Ziehl-Neelsen (ZN) microscopy and it had qualitative, operational and cost advantages over both conventional fluorescence and ZN microscopy. LED microscopy also showed 84% sensitivity (95% CI, 76-89%) and 98% specificity (95% CI, 85-97%) against culture as the reference standard.[11] When a microscopic reference standard was used, the overall sensitivity was 93% (95% CI, 85–97%), and the overall specificity was 99% (95% CI, 98–99%). A significant increase in sensitivity was reported when direct smears were used rather than concentrated smears (89% and 73%, respectively).[11],[14] Based on these results, in 2011, WHO endorsed replacement of conventional smear microscopy and FM with LED-FM in both high and low-volume laboratories.[11] In areas without high rates of MDR TB or HIV-associated TB, cost-effectiveness considerations favour the use of LED microscopy as the initial diagnostic tool, and thus the improved performance of direct smear microscopy. Despite the reduced specificity, LED-FM still has operational advantages that make it an attractive tool for TB laboratory diagnosis.

Limitations

Auramine stained smears fade quickly during storage, making subsequent rechecking of results unreliable.[18] The rate of fluorochrome fading (also called photo-bleaching) is affected by multiple factors including exposure to light, oxygen, temperature and pH. With the proposed wide-scale expansion of LED based FM to global TB laboratories there is concern that sensitivity of detection will decrease over time of storage, especially in pauci-bacillary smears, leading to the incorrect judgement. Therefore, appropriate external quality assurance procedures (EQA) are needed for TB laboratories planning to implement LED-FM for smear diagnostics.[18] National control programmes introducing new LED-FM services should monitor the performance of this method under operational conditions, as training needs of staff may be greater than anticipated.

Liquid Culture: Commercial Broth Based Culture Systems

Liquid cultures are faster, more accurate and more comprehensive than conventional solid media or smear microscopy. Although known for decades, the ability of a liquid medium to support a faster growth was heavily hampered by its susceptibility to contamination. The use of antimicrobial combinations suitable of inhibiting the growth of contaminants (Gram-positive, Gram-negative and fungi) represented a turning point. WHO issued policy recommendations on the use of the liquid culture and rapid species identification in 2007.[19] WHO approved automated liquid culture system include BacT/ALERT 3D (Biomerieux) and BACTEC MGIT 960 system (Becton, Dickinson).

BacT/ALERT 3D

BacT/Alert 3D (previously known as MB/BacT) is non-radiometric continuous monitoring system with a computerized database management. A modified Middlebrook 7H9 medium is used in which a supplement, a mixture of OADC enrichment and Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim, Vancomycin and Azlocillin is added at the moment of use. The system is based on detection of CO2. The bottles or medium have a CO2 sensor at the bottom and are inoculated through a rubber septum by means of a syringe. BacT/ALERT MB sterile, disposable culture bottles contain 29 ml of media and an internal sensor that detects carbon dioxide as an indicator of microbial growth. Many studies have evaluated the BacT/Alert D. The system turned out to be more rapid and more sensitive than conventional media while comparison with other automated and semi-automated systems did not reveal significant differences.[20],[21]

Mycobacterial Growth Indicator Tube (MGIT)

MGIT detects bacterial growth with the help of Oxygen (O2) sensitive fluorescent sensors, which are embedded in silicon and deposited on the bottom of the culture tube containing 7 ml of enriched modified Middle-brook 7H9 broth media. Contamination is controlled by the addition of a mixture of Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim and Azlocillin (PANTA) reconstituted with a poly-oxyethylene solution. The actively growing Mycobacteria deplete the dissolved O2 from the broth and allow the sensor to fluoresce when observed under UV light at 365 nm. It monitors the tubes at one-hour intervals, alters when they become positive and signals the end of the incubation period. On average diagnosis can be performed in 4-14 days (up to 42 days for negative results). Automated system has the capacity for up to 960 cultures at a time. It is also possible to use the MGIT tubes without instrumentation, by holding the tubes in a normal incubator and observing the fluorescence under a Wood's lamp. BACTEC MGIT 960 turned out to be clearly faster and more sensitive than solid media, while the comparison with other automated and semi-automated systems revealed substantially overlapping performance.[20],[21],[22]

Limitations of liquid cultures

The use of such a combination of antibiotics does not eliminate the decontamination step, which needs to be performed before inoculation of the samples. All decontamination methods are to some extent also harmful to mycobacteria, and culture is therefore not 100% sensitive. Bio-safety Laboratory (BSL)-2 facilities are required for processing specimens, inoculating cultures and handling positive cultures for species identification of DST. BSL-3 lab or Negative pressure room with Biosafety cabinet is required to open the MGIT positive tubes. Laboratory technicians require special training in BSL-3 safety precautions for specimens and cultures handling. Risk of contamination or carryover contamination due to the use of highly enriched growth media.

Species Identification of Mycobacteria from Positive Liquid cultures

Capilia TB assay

The Capilia TB (TAUNS, Numazu, Japan) assay is a quick, simple, lateral-flow immunochromatographic assay that detects the MPB64 antigen in M. tuberculosis complex culture isolates. It uses a monoclonal antibody to detect MPB64, one of the predominant proteins secreted by M. tuberculosis complex strains during cultures (from broth or from resuspended colonies from solid media). Results are available within 15 minutes. This assay does not require specialized equipment, cost effective and has been shown to be highly sensitive (>98%) and specific (>97%) for identification of M. tuberculosis complex from culture isolates.[23],[24],[25],[26],[27] New improved version of this test (Capilia TB-Neo) showed sensitivity and specificity of 99.2 and 96.4% in pure culture isolates and 89.3 and 100% in mixed culture isolates.[28]

Limitations

Mutations of the mpb64 gene result in false negative results. The results of M. bovis and M. bovis BCG cultures may vary since some BCG strains are known to lack mpb64 antigen productions. There is a requirement of BSL3 laboratory infrastructure with class I or class II microbiological safety cabinet to handle positive TB cultures.

Accuprobe

Accuprobe (Gen-Probe, San Diego, CA) a commercial isothermal method based on species specific DNA probes that hybridize to rRNA for the identification of limited number of important mycobacteria, including M. tuberculosis complex, M. avium, M. intracellulare, M. avium complex, M. kansasii and M. gordonae. The probe is a single stranded DNA oligonucleotide, complementary to a short species-specific sequence within a hyper variable region of the 16S ribosomal DNA. It is labelled with an acridinium ester, a chemiluminescent molecule, which gives light when properly excited on hybridization with complementary target. Thermal cyclers are not needed and the whole amplification step is carried out on a heating block at 42°C. The probes have been extensively evaluated in a clinical setting and have shown a very good sensitivity and specificity giving results in 2 hours. Overall, DNA probes show >99% sensitivity and specificity for MTB complex isolates and approximately 95% sensitivity and > 99% specificity for MAC isolates.[29]

Limitations

Although rare, some instances of cross-reaction between MTB complex probe and M. terrae and M. celatum have been reported.[30],[31] There is a requirement of BSL3 laboratory infrastructure with class I or class II microbiological safety cabinet to handle positive TB cultures.

Liquid culture DST

Methods for testing for drug susceptibility (DST) follow the same principle but use 2 culture samples; one with a drug added and one without the drug (a growth control). If the drug is active against the TB bacteria, it will inhibit growth and suppress fluorescence. Many studies have now been published on the application of the MGIT system for detection of resistance to first- and second-line anti- TB drugs.[32],[33],[34],[35] In all these studies, the MGIT system has shown very good results with a high correlation with the conventional methods on solid media and the BACTEC TB-460 system.

Limitations

Liquid culture DST is time consuming, as takes 7-14 additional days after the initial culture and identification. BSL-3 lab or Negative pressure room with level 2 Biosafety cabinet is required to handle M. tuberculosis positive cultures. Laboratory technicians require special training in BSL-3 safety precautions for cultures handling and DST.

Liquid Culture: Non-commercial culture and DST Methods

Several non commercial culture and DST methods have been developed specifically for settings with limited access to sophisticated laboratory infrastructure and technical expertise. In 2009, WHO assessed selected non-commercial culture and DST methods in a systematic, structured way and found sufficient evidence to recommend the use of colorimetric redox indicator (CRI) methods, microscopic observation drug susceptibility Method (MODS) in liquid culture and Nitrate Reductase Assay (NRA) for direct testing of sputum in reference laboratories.[13] The time to detection of MDR - TB DST may not be necessarily be faster with indirect testing and none of these methods can detect extensively drug-resistant TB.

CRI

These are indirect tests for rapid detection of drug resistance. Generally performed only on M. tuberculosis isolates grown from conventional cultures. They use redox indicators or tetrazolium salts to detect mycobacterial growth. In these tests the coloured redox indicator is added to the culture medium after M. tuberculosis has been exposed in vitro to different antibiotics.[36] Resistance is detected by a change in colour of the indicator, which is directly proportional to the number of viable M. tuberculosis cells in the medium. CRI methods are highly sensitive (pooled estimate, 98%; 95% confidence interval [CI], 96–99%) and specific (pooled estimate. 99%; 95% CI, 99-100%) for the detection of RIF resistance and also INH resistance (pooled sensitivity, 97%; 95% CI, 96–98%; pooled specificity, 98%; 95% CI, 97–99%).[13]

Limitations

The methods require manipulation of concentrated suspensions of mycobacteria, with a high risk for aerosol creation. CRI methods should therefore be performed under laboratory biosafety level 3 conditions at reference laboratory level. Laboratory technicians require special training in BSL-3 safety precautions for specimens and cultures handling.

MODS

MODS is recommended by the WHO as an interim solution for more timely diagnosis and DST in developing countries as it is rapid reliable and inexpensive. It is based on inoculation of specimen into drug free and drug containing media in a microtiter plates, followed by microscopic examination of early growth. Results are available generally in 9-9.5 days after inoculation.[37] MODS can be performed as a direct or an indirect test, by observing the characteristic cord formation of M. tuberculosis in a sealed micro-titre plates containing liquid culture medium through an inverted microscope. In 2006, study carried out by Moore et al in Peru, compared the performance of MODS to an automated liquid culture system and conventional Lowenstein-Jensen (L.J) proportional method for the detection of resistance to INH, RIF, Ethambutol (EMB) and Streptomycin (SM).[38] The agreement between MODS and the reference standard for DST in this study was 97% for INH, 100% for RIF, and 99% for INH and RIF combined (MDR). Lower values of agreement were obtained for EMB (95 %) and SM (92%).[38] Various studies on the accuracy of combined (direct and indirect) use showed that MODS is highly sensitive (pooled estimate, 98%; 95% CI, 95–99%) and specific (pooled estimate, 99%; 95% CI, 96–100%) for the detection of RIF resistance and slightly less so for INH (pooled sensitivity, 91%; 95% CI, 87–95%).[13] Recently Trollip et al published second-line drug susceptibility breakpoints for M. tuberculosis using the MODS assay and as per their results the second-line MODS assay yielded comparable results to MGIT second-line DST.[39]

Limitations

MODS is more challenging than smear microscopy, because it requires relatively high technical skill. Laboratory technicians may have difficulty distinguishing between the micro-colonies of M. tuberculosis complex and some nontuberculous mycobacteria (NTM). This could potentially have impact where NTM prevalence is high. As with all TB culture systems, BSL-2 facilities are required for processing specimens and inoculating cultures. BSL-3 facilities are required if tubes need to be opened and the cultured organisms manipulated, such as for speciation or further testing. Laboratory technicians require training in BSL-2 or BSL-3 safety precautions as appropriate and in the use of MODS

NRA

The NRA can be used as a direct test on smear-positive sputum specimens or as an indirect test on M. tuberculosis isolates. The nitrate reductase assay (NRA) is a quite simple and inexpensive technique based on the capacity of M. tuberculosis to reduce nitrate to nitrite, which is detected by adding a chemical reagent to the culture medium. M. tuberculosis is cultivated on L.J. medium in the presence of an antibiotic and its ability to reduce nitrate is measured after 10 days of incubation. Resistant strains will reduce the nitrate, which is revealed by a pink-red colour in the medium, while susceptible strains will lose this capacity as they are inhibited by the antibiotic. The assay has been evaluated in several studies for first-line drugs and ofloxacin with good results.[40],[41],[42],[43] Studies on combined (direct and indirect) use showed that NRA is highly sensitive (pooled estimate, 97%; 95% CI, 95–98%) and specific (pooled estimate, 100%; 95% CI, 99–100%) for the detection of RIF resistance and for INH resistance (pooled sensitivity, 97%; 95% CI, 95–98%; pooled specificity, 99%; 95% CI, 99–100%).[13]

Limitations

Indirect testing of clinical specimens with NRA is not faster than conventional phenotypic DST with liquid media. In comparison with the conventional indirect proportion DST method on L.J. medium, NRA requires similar staff skills and safety precautions. It should be performed inside an appropriate biosafety cabinet. Other NTM e.g. M. kansasii, M. sulgai, M. flavescens, M. terrae complex and most rapidly growing mycobacteria except M. cheloane also reduce nitrate and hence can be misdiagnosed especially when NRA is used as a direct test.[44] Species confirmation with additional supportive test would be helpful.

Genotypic Molecular Methods

WHO has recommended use of molecular-based tests that include Line Probe Assays (LPA) and GeneXpert MTB/RIF assay to diagnose RIF resistance TB considering RIF as a marker for MDR.[12] Both these tests are based on detection of mutations in the RIF Resistance-Determining Region (RRDR) of DNA-dependent RNA Polymerase gene (rpoB). Such mutations are found in 95–98% of M. tuberculosis strains determined to be RIF-resistant by the “gold standard” culture-based DST.

LPA for species identification and DST

In 2008 WHO endorsed the use of LPA for the rapid detection of Rif resistance TB (alone or in combination with INH).[12] The line probe assay uses the reverse line blot hybridization technology with differently specific DNA probes immobilized in parallel lines on a nitrocellulose strip or a nylon membrane.[45] The target DNA is PCR amplified using biotinylated primers and finally incubated with a strip or membrane coated with species specific probes. Once the hybridization has been carried out under highly stringent conditions and the unbound amplicons have been washed out, the hybridized probe is revealed as a coloured band, through an enhanced chemiluminiscence reaction, developed following the addition of a streptavidin-labled enzyme and a chromogenic substrate. 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, Zwijndrecht, Belgium) for the detection of resistance to RIF and the GenoType MTBDR assay (Hain Life sciences, GmbH, Germany) for the simultaneous detection of resistance to INH and RIF.

The Inno-LiPA Rif TB kit assay

The LiPA assay identifies the M. tuberculosis complex and simultaneously detect genetic mutations in the RRDR region of the rpoB gene RIF resistance.[46] Many studies evaluated the LiPA assay for detection of RIF resistance. Most of them were performed on M. tuberculosis isolates and just a few were applied directly to test sputum samples.[47],[48] It has been proposed as a good initial indicator of MDR resistance with a sensitivity of 98.5% for detecting RIF resistance.[49],[50] In a systematic review and meta-analysis of studies that applied the LiPA test, 12 of 14 studies performed in isolates had sensitivity greater than 95% and specificity of 100%. Four studies that applied LiPA directly to clinical specimens had 100 % specificity, and the sensitivity ranged from 80% to 100%.[51] In a large study conducted by Traore et al 2006, the utility of the LiPA test for detecting RIF resistance was assessed in 420 sputum samples originating from different countries. There was a 99.6% concordance between the RIF resistance obtained by culture and by the LiPA test, confirming that with an adequate DNA extraction method, the LiPA test allows rapid detection of resistance to RIF directly from sputum samples.[47]

GenoType MTBDRplus(R)

This assay is a DNA strip test that allows simultaneous molecular identification of TB and the most common genetic mutations in rpoB, katG and inhA genes causing resistance to RIF and INH.[52] In a study that evaluated the GenoType MTBDRplus assay in 143 M. tuberculosis isolates, 99 % of the MDR strains were found to have mutations in the rpoB gene and 88.4 % of strains with mutations in the codon 315 of the katG gene were also correctly identified.[49] The correlation with DNA sequencing was 100 %, and good sensitivity and specificity was obtained when compared to the conventional tests. As with other genotypic tests, there is interest in the application of these techniques directly to sputum samples. There are only two studies that address this issue. In the study by Hillemann et al., the GenoType MTBDR was tested directly in 42 smear positive sputum samples obtaining a concordance of 100 % when compared to conventional drug susceptibility testing.[53] In another study, the GenoType MTBDR was evaluated in 143 smear-positive sputum samples and it was able to correctly identify INH resistance in 48 (84.2 %) of the 57 specimens containing strains with resistance to high level of INH (0.4 μg/mL), and RIF resistance in 25 (96.2 %) of the 26 specimens containing RIF- resistant strains.[54] Various studies conducted at South Africa, Peru and Russia confirmed the consistent and excellent performance of the Genotype MTBDRplus assay on both M. tuberculosis culture isolates as well as smear positive sputum specimens.[12] Overall accuracy for RIF resistance and MDR detection was 99% and for INH resistance detection was 98%. In addition, the MTBDRplus assay provided interpretable results in 84% of specimens in which conventional DST results were unavailable due to contamination of the primary liquid culture.[12]

Hain Lifescience also released the GenoType MTBDRsl test in 2009, designed to test for resistance to second-line antiTB drugs (fluoroquinolones, ethambutol, aminoglycosides, and cyclic peptides). It can be used in combination with the MTBDRplus test to identify extensively drug-resistant TB.[55],[56] An updated version of the MTBDRplus was introduced by Hain in 2011 that has increased sensitivity for TB detection and may be performed directly on AFB smear-negative specimens. However this most recent Hain LPA version has not yet been endorsed by WHO for testing on smear-negative specimens. In an assessment of this new version of LPA in Moldova, the sensitivity and specificity for TB detection in 104 AFB smear-negative, culture-positive respiratory specimens were 80 and 99%, respectively, whereas those for RIF and INH resistance were 94 and 96%, respectively.[57]

Limitations

LPAs are as complex to perform as conventional culture and DST methods and require skilled and well-trained laboratory personnel, as well as adequate laboratory space and design to reduce the risk of false-positive results. Processing of smear-positive specimens for direct testing should be performed in a BSL-2 level laboratory, whereas performing LPAs on positive cultures requires BSL-3 laboratory infrastructure and equipment, including a class I or class II microbiological safety cabinet equipped with uninterrupted power supply (UPS). LPAs also require specific equipments such as a thermal cycler, shaking platform and water bath, heating block, hybridization oven etc. The laboratory must be designed to address the risk of amplicon contamination. Experience is also needed in result interpretation as unusual patterns of mutation and wild type bands can occur on occasion, especially in case of hetero-resistance (the presence of mixed population of resistant and wild type clinical isolates). Line probe assays do not eliminate the need for conventional culture and DST capability, as culture remains necessary for definitive diagnosis of TB in smear-negative patients, while conventional DST is required to diagnose XDR-TB.

The GeneXpert MTB/RIF Assay

In December 2010, WHO endorsed the use of GeneXpert MTB/RIF (Xpert; Cepheid, Sunnyvale, CA), a rapid, automated, cartridge-based nucleic acid amplification test that can simultaneously detect M. tuberculosis complex and resistance to RIF. Xpert assay amplifies a 192-bp segment of the M. tuberculosis rpoB gene in a heminested real time PCR and then uses molecular beacon technology. M. tuberculosis complex is detected using five overlapping molecular beacon probes (probes A to E) that are complementary to the entire 81-bp RIF resistance-determining “core” region of the wild-type rpoB gene.[58] The assay cartridge also contains lyophilized Bacillus globigii spores which serve as an internal sample processing and PCR control.[59] Unlike conventional nucleic acid test, Xpert is unique because the sample processing, PCR amplification and detection all these steps are integrated into a single self-enclosed Xpert cartridge [Figure 4]. Following sample loading, all steps in the assay are completely automated and the assay's sample reagent, used to liquefy sputum, has the ability to kill TB bacteria.[60],[61] These properties enable TB diagnosis and drug resistance testing almost anywhere without requiring the specialised laboratories and well trained technicians as needed for other rapid tests. As per WHO recommendations, Xpert should be used as the initial diagnostic test in individuals suspected of MDR-TB or HIV associated TB.[10]
Figure 4: GeneXpert MTB/RIF Assay Procedure [Ref. Boehme et al , NEJM 2010][62] Add two volumes of sample treatment reagent to each volume of sputum. Mix well, incubate at room temperature for 15 min. Transfer 2-3 mL to the GeneXpert Cartridge and then load the cartridge into the instrument. Once loaded inside the GeneXpert Instrument, DNA extraction, amplification and detection steps occur automatically and the user is provided with a printable test results

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During the evaluation of the Xpert assay to test unprocessed sputum specimens, Helb et al reported sensitivities of 100% for smear-positive specimens, 84.6% for smear negative solid culture-positive specimens, and 71.7% for smear-negative and solid-plus-liquid-culture-positive specimens.[58] The specificity was 100% for all samples. The assay correctly identified genomic DNA from 79 phylogenetically and geographically diverse strains of M. tuberculosis and no cross-reactivity occurred with a wide range of NTM or other organisms known to infect respiratory tract.[58],[59] The first multi-country evaluation done by the Foundation for Innovative and New Diagnostics (FIND) published in 2010 enrolled 1730 TB suspected patients at five study sites in South Africa, Peru, Azerbaijan and India. One direct test on sputum detected 98.2% patients with smear positive TB and 72.5% of patients with smear negative TB, with overall 99.2% specificity.[62] In patients with smear negative culture positive TB, processing one, two or three samples was associated with sensitivities of 72.5%, 85.1% and 97.6% respectively.[62] Since then there has been various studies published and all report a high Xpert sensitivity.[63],[64],[65],[67] In HIV-positive TB cases, the sensitivity appeared lower than in HIV-negative patients (76% compared with 89% pooled median sensitivity).[64],[65]

The overall pooled sensitivity of Xpert assay compared against culture as a reference standard in children presumed to have pulmonary TB was 66% in 10 studies where expectorated sputum or induced sputum was used (95% CI, 52–77%); the pooled sensitivity was 66% in 7 studies where samples from gastric lavage or aspiration were used (95% CI, 51–81%).[66],[67] The pooled specificity of Xpert against culture as the reference standard was at least 98%, with narrow confidence intervals. Moreover, Xpert assay was also found to be useful in a rapid detection of nearly 50 to 80% of extra-pulmonary TB cases in a variety of specimens with varied sensitivity depending on specimen type and processing method. Therefore, in late 2013, WHO expanded its recommendations on the use of Xpert MTB/RIF to include the diagnosis of TB in children and some forms of extra-pulmonary TB.[66],[67] Recently published systematic review by Maynard-Smith et al strongly supports WHO recommendation of the use of Xpert MTB/RIF for TB diagnosis in extra-pulmonary specimens like CSF, tissues and lymph node samples.[68]

Limitations

There are reports on false positive RIF resistance. False positive RIF's resistance is most likely because of mixed infections and mixture of drug-susceptible and drug-resistant populations.[69] In a study carried out by Rufai et al, the new G4 version cartridges did not detect a very rare mutation at P533L located at extreme end in the probe E region.[70] Therefore, RIF resistance results should be understood and interpreted carefully. Another important limitation of the MTB/RIF test is its cost, which may be prohibitive for a disease that principally affects poor people in poor communities. Although much more expensive than smear microscopy, affordability varies greatly between settings, and they appear to be comparable with the total costs of providing other rapid TB tests.

Summary and Future Perspectives

WHO approved methods for quality diagnosis of active and drug resistance TB; meet some but not all the requirements of an ideal rapid diagnostic test. It is important to realize that, at present, there is no stand alone test for the rapid detection of TB in all patients. Today, most of these techniques and equipments are increasingly complex and costly. Laboratory expertise and resources are required for these tests to become available in each and every local laboratory throughout the developing world and TB endemic region. However, as specimen delivery and communication of results can be rapidly and easily achieved, utilization of reference laboratories for rarely performed sophisticated tests could be a more practical approach. Laboratory strengthening with WHO recommended tools must be taken up by national TB programs and budgeted for the funding proposals. Quality diagnosis to reach the missing 3 million (or more) cases and provide them with proper treatment will be a crucial step towards the success of TB control programs.



 
  References Top

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