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REVIEW ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 3  |  Page : 323-331
 

Laboratory diagnosis of tuberculosis: Advances in technology and drug susceptibility testing


1 Department of Microbiology, Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla, Kerala, India
2 Department of Microbiology, Indira Gandhi Medical College and Research Institute, Puducherry, India

Date of Web Publication12-Oct-2017

Correspondence Address:
Seema Oommen
Department of Microbiology, Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla - 689 101, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_16_204

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 ~ Abstract 

There have been rapid technological advances in the detection of Mycobacterium tuberculosis and its drug susceptibility in clinical samples. These include advances in microscopic examination, in vitro culture and application of molecular techniques. The World Health Organization (WHO) has played a large role in evaluating these technologies for their efficacy and feasibility, especially in the developing countries. Amongst these, the Revised National Tuberculosis Control Programme (RNTCP), through its national network of designated microscopy centres and intermediate reference laboratories, has adopted certain technologies that are currently implemented in India. Advances in microscopy technology include fluorescent microscopy using light-emitting diode source, sodium hypochlorite microscopy and vital fluorescent staining of sputum smears. Automation of in vitro culture has markedly reduced the turnaround time (TAT), even in smear-negative samples, and permits simultaneous detection of resistant mutants. Molecular detection of drug resistance has further reduced the TAT, and the cartridge-based nucleic acid amplification test with its performance convenience and rapid results, appears poised to become the future of tuberculosis (TB) diagnosis in all settings, provided the cost of testing is reduced especially for use in private diagnostic settings. This article reviews technologies currently available for the diagnosis of TB, keeping in mind the WHO recommendations and the RNTCP practices. This is a thematic synthesis of data available on diagnosis in literature, preserving the conclusions of the primary studies.


Keywords: Culture, drug susceptibility, laboratory diagnosis, microscopy, molecular techniques, tuberculosis


How to cite this article:
Oommen S, Banaji N. Laboratory diagnosis of tuberculosis: Advances in technology and drug susceptibility testing. Indian J Med Microbiol 2017;35:323-31

How to cite this URL:
Oommen S, Banaji N. Laboratory diagnosis of tuberculosis: Advances in technology and drug susceptibility testing. Indian J Med Microbiol [serial online] 2017 [cited 2017 Dec 13];35:323-31. Available from: http://www.ijmm.org/text.asp?2017/35/3/323/216615



 ~ Introduction Top


In 2015, 10.4 million incident cases of tuberculosis (TB) were reported globally while the estimate of incidence (new TB cases per year) was 2.8 million cases in India.[1] The World Health Organization (WHO) Global TB report 2016 states that TB killed 1.4 million people (1.2 to 1.6 million) HIV-negative and 0.39 million (0.32 million to 0.46 million) HIV-positive individuals in 2015.[1] The 30 high TB burden countries accounted for 87% of all estimated incident cases worldwide. Of these, China, India and Indonesia alone accounted for 45% of global cases in 2015. An estimated 3.9% of new TB cases and 21% of previously treated cases have multidrug resistance (MDR). MDR-TB is defined as Mycobacterium tuberculosis complex (MTBC) isolates within vitro resistance to rifampicin (RIF) and isoniazid (INH) with or without resistance to other first-line anti-TB drugs.[2] MDR-TB isolates within vitro resistance to fluoroquinolone and one (or more) of the following injectable drugs: kanamycin (KM), amikacin (AMK) and capreomycin (CM), are defined as extensively drug-resistant TB (XDR-TB).[2]

In May 2016, WHO issued guidance that people with TB resistant to rifampicin, with or without resistance to other drugs, should be treated with an MDR-TB treatment regimen. This includes patients with MDR-TB as well as any other patient with TB resistant to rifampicin (MDR/RR-TB).[1] There were an estimated 580 000 incident cases of MDR/RR-TB in 2015, with cases of MDR-TB accounting for 83% of the total. In 2015, an estimated 250,000 people died of MDR TB.[1] The countries with the largest numbers of MDR/RR-TB cases are China, India and the Russian Federation[1] About 9.5 % of MDR-TB patients develop XDR-TB, which is even more difficult to treat.[2]

In addition to emergence of MDR, diagnosis of TB still remains a challenge especially in cases of extrapulmonary TB, non-mycobacterial infections and a growing population of immunosuppressed individuals including people with HIV. The choice of these tests also varies in case of high-burden countries wherein the focus of diagnosis is on detection of active cases, whereas in low-burden countries, the focus is to diagnose latent infection. The diagnostic tests currently available in most public health systems in high-burden countries today largely rely on age old microscopy. What was good in 1882 remains to be good in 2017 too. It is only in the last few years that public health programmes are looking beyond conventional microscopy for diagnosis of new cases. In 2015, there has been a paradigm shift in the WHO from stop TB to end TB strategy. To achieve this, gaps in detection and treatment have to be bridged by rolling out newer and faster techniques in diagnosis as well as detection of MDR with the availability of these facilities at the peripheral centres. Computer software has not been used. Currently available diagnostics can be classified as those that:

  • Directly detect the actively growing bacilli
  • Indirectly detect the immune response against the bacilli.


Direct detection of infection includes microscopy, culture, antigen detection and nucleic acid detection whereas indirect methods of detection include detection of immune response by tuberculin skin testing (TST) and interferon-gamma release assays (IGRAs). Other newer and experimental methods include volatile organic compounds (VOCs) detection and detection of beta-lactamase. This is a thematic synthesis of data available on diagnosis in literature, preserving the conclusions of primary studies referred to and provided in the references. The review is restricted to descriptive themes derived from the primary studies and does not generate analytical themes.


 ~ Direct Detection Methods Top


Microscopy

Sputum smear microscopy still remains the basis for diagnosis of TB in developing countries. The most regular practice is acid-fast staining using carbol fuschin and fluorochrome dye-auramine/rhodamine.

It is relatively fast, inexpensive and specific for TB in high incidence areas. Although highly specific, smear microscopy is insensitive – it detects roughly 50% of all the active cases of TB.[3] The higher load of bacilli (10,000 bacilli/ml) that need to be present leads to a varying sensitivity from 20% to 80% depending on various factors such as quality of the specimen and the training and motivation of laboratory personnel.[4] Sensitivity can be as low as 20% in children and HIV-infected people.[3] As per the current Revised National Tuberculosis Control Programme (RNTCP) guidelines, the patient should visit the clinic at least twice to submit a spot – early morning or spot-spot specimen. At least two ml specimen should be collected which should be mucopurulent. Sputum specimens should be examined within two days of collection. Studies have documented that the first sputum specimen detected 85.8% of TB cases with the second smear adding on to another 11.9%.[5] Relying on sputum microscopy alone may be dangerous from the public health point of view as 17% of smear-negative cases are known to transmit the disease.[6] Routine microscopy cannot differentiate between live and dead bacilli and hence cannot be used as a follow-up diagnostic test. It can neither be used to predict MDR nor the presence of non-tuberculous mycobacteria. Despite the multitude of disadvantages, in the absence of better alternatives, it is a useful tool in the basic laboratories common in developing countries. RNTCP has revised the diagnostic algorithm to allow for nucleic acid amplification detection based on cartridge based technologies (CBNAAT) for cases who are smear positive with presumptive MDR-TB/ or living in high MDR-TB areas (>5% and >20% among new and retreatment cases respectively). It also allows simultaneous testing of second specimen by the above-mentioned cartridge based technology for smear negative cases with radiological abnormalities and even those solely based on high clinical suspicion in spite of negative CXR and smear.

Fluorescent microscopy with light-emitting diodes

Conventional fluorescence microscopy (FM) using the quartz-halogen lamps or high-pressure mercury vapour lamps was expensive, vulnerable and required expert handling. Light-emitting diodes (LEDs) are more robust, sustainable and user-friendly, thus allowing advantages of FM at peripheral health-care systems. Royal blue colour LED lamps used in these newer generation fluorescent microscopes have an extremely long life expectancy (10,000 h vs. 200 for a conventional mercury lamp). Furthermore, they do not produce ultraviolet (UV) light, do not require darkened rooms and significantly decrease the instrument's power consumption, allowing longer lasting battery life and thus decreasing the cost incurred, unlike older lamps. FM increases the sensitivity of smear microscopy as it allows a much larger area of the smear to be seen, resulting in more rapid examination of the specimen (up to four times faster), allowing sixty slides to be screened per day as opposed to 25 using the Ziehl–Neelsen (ZN) method.[7] Being WHO recommended in 2009, the RNTCP has adopted LED microscopy to replace ZN method in its designated microscopy centres (DMCs) across India.

Instability of fluorescent stains under field conditions and instability of the stained smears for blinded rechecking have been reported.[8] Proper training of laboratory personnel is imperative as detection and interpretation of results may be affected.[9] Unlike Ziehl Neelsen, international guidance on quality assurance for FM does not currently exist and is under development.[10] The sensitivity, specificity, cost-effectiveness and cost-benefit of this approach have not yet been adequately established.

Front-loaded microscopy

Microscopy services usually require the patients to make repeated visits to the healthcare facility leading to increased drop-out patient rates. The WHO definition (2009) of a smear-positive case allows patients to be diagnosed as TB on a single smear. In front-loaded microscopy, the first two specimens are collected and examined on the same day a patient present to the clinic.[11] Patients with negative smears are asked to return with a morning specimen the next day, depending on whether routine services are based on two or three specimens being examined. Thus, TB patients can be offered treatment on the first day they present.

Sodium hypochlorite (bleach) microscopy

The digestion of sputum with house-hold bleach before sputum smear preparation and microscopy has been reported to be an effective, simple method to improve the yield of smear microscopy even in high HIV prevalence settings.[3] Progress on the development of a bleach microscopy method has been complicated by the wide heterogeneity and lack of standardisation in methods described. A standardised bleach method, the Mathare sodium hypochlorite (MaSH) method, has recently been developed and evaluated in Medecins Sans Frontieres sponsored studies in Mathare, Nairobi. The addition of standardised sodium hypochlorite solution to sputum followed by overnight sedimentation resulted in a 15% increase in TB cases detected.[3] The MaSH method is now being evaluated by the WHO's Tropical Disease Research initiative under operational conditions in large multi-country studies.

Vital fluorescent staining

Monitoring the response to TB treatment is essential for early detection of treatment failure or drug resistance. The most common monitoring tool available in resource-limited settings is sputum smear microscopy using ZN or auramine staining. Smear positivity at 3 months or later should be investigated with a culture and drug susceptibility testing (DST), while smear positivity at 5 months or later defines treatment failure. As smear microscopy cannot distinguish viable from dead bacilli, it is possible that a significant proportion of patients on treatment may continue to cough up dead bacilli from necrotic lung cavities and would be falsely labelled as 'smear positive'.[12] Culture is the only test that can identify viable bacilli, but it requires several weeks to report results and needs a high level of expertise and laboratory infrastructure. Therefore, these patients are at an increased risk of receiving an unnecessary prolonged or new treatment regimen. Recent studies proposed a simple and instant method for TB treatment monitoring, based on a common fluorescent viability marker, fluorescein diacetate (FDA),[13] in combination with smear microscopy. Unlike most fluorescent stains, FDA stains only living, cultivable bacteria thus guiding antimicrobial therapy before culture reports come in.

Newer microscopic technologies

Automated microscopic technology by TBDx (Signature Mapping Medical Sciences, USA) integrates robotic loading of stained slides and automated high-resolution digital image analysis to provide a result in minutes. The system has a 200-slide capacity reducing the burden on technicians. Early studies suggested improved sensitivity over the human eye, but specificity was reduced; thus, manual review of positive slides was necessary.[14] Performance studies are underway in Nigeria and South Africa, with further studies planned for Asia.

A second innovation to assist a microscopist is CellScope, a portable digital FM that provides enlarged digitalised images for review.[15]

Culture

Culture still remains the gold standard for diagnosis of TB, and it also permits the diagnosis of drug resistance, including the emerging mutations. Traditional egg-based (Lowenstein-Jensen) and agar-based (Middlebrook 7H10/11) methods are widely used. The limit of detection is 100 bacilli/ml, thus increasing the sensitivity compared to smear, but the growth in a conventional egg-based medium takes anywhere from 4 to 8 weeks with an additional 4 weeks for drug sensitivity by the conventional proportion method. Thus, it takes a median of 70 days to diagnose a case of MDR-TB by conventional culture methods.[16] Other limitations include requirement of biosafety facilities that are expensive to build and maintain and specially trained laboratory technicians to perform the procedure. Hence, TB cultures are performed only at national reference laboratories or in-hospital laboratories in large cities. Few developing countries have the capacity for good-quality DST for the first-line drugs, and even fewer have the capacity to test for the second-line drug resistance. In most countries, TB culture is reserved for treatment failure and drug-resistant cases and specimens often need to be sent to distant laboratories leading to delay in processing of specimens which in turn effect results.

The most common detection method that is WHO endorsed in 2007, and adopted by the RNTCP, is a liquid culture using Middlebrook 7H9 broth – the mycobacterial growth indicator tube (MGIT), a non-radiometric detection method which measures the consumption of oxygen by fluorescence.[3] As bacteria grow in the culture, the oxygen is utilised causing it to be fluorescent when placed under UV light. Methods for testing for drug susceptibility follow the same principle but use two culture samples: one with a drug added and one without the drug (a growth control) and is called the modified 1% proportion sensitivity testing method (PST). Usually an economic variant of proportion sensitivity testing using Lowenstein Jensen medium is incorporated as a backup. While CBNAAT is the first line option for testing for drug resistance, wherever possible, especially in cases of rifampicin resistance, DST results of all the drugs should be made available. If the test drug is active against the TB bacteria, it will inhibit growth and suppress fluorescence. In the manual system, a technician visually identifies fluorescence using a hand-held UV lamp containing apparatus. Automated systems have the capacity for up to 960 cultures at a time.

Such systems have been thoroughly evaluated in clinical settings and approved for the use in industrialised countries for years. Liquid culture systems provide results significantly faster than solid culture techniques: On an average, diagnosis can be performed in 9 days for a smear-positive case to 16 days for a smear-negative case.[17] A negative result is issued by 42 days and DST takes an average 7–14 days (range 4–14 days) after the initial culture.[3] Automated systems can benefit laboratories with a high workload and provide standardised reading of samples. Such systems have a sensitivity and specificity of nearly 100% (and marginally less for manual systems). Studies have shown that both automated and manual systems perform well in the detection of INH and RIF susceptibility but are not as effective for ethambutol (EMB) and streptomycin.[18],[19] Liquid media are more prone to contamination than solid media, leading to invalid results unless carefully controlled. Automated machines must be maintained, requiring on-site technical support from manufacturers or their agents. Internal quality control of culture involves testing of each batch for sterility and quality strain H37Rv for growth parameters. For DST; a sensitive strain (H37Rv) and mono-resistant strains are tested batch wise. For obtaining accreditation for DST random 10/100 isolates tested in the laboratory are sent to the national reference centre (NRL) in the first stage for real time assessment of the laboratory. This is followed by second stage for actual test of performance; 30 isolates are sent from the NRL annually to the laboratory. Concordance of >90% is required for isoniazid and rifampicin. Besides the MGIT, other culture methods are enumerated in [Table 1].
Table 1: Culture methods for Mycobacterium tuberculosis

Click here to view


Tools for rapidly identifying the species of culture isolates

Rapid speciation is necessary for the isolates grown in culture to differentiate between M. tuberculosis (MTB) complex and other species of Mycobacteria. Conventional biochemical methods of speciation using para-nitrobenzoic acid can take weeks. Strip speciation test can detect a TB-specific antigen (MPT 64) from positive liquid or solid cultures to confirm the presence of organisms belonging to M. tuberculosis complex. M. tuberculosis protein 64 (MPT-64) antigen is an M. tuberculosis complex (MTBC)-specific antigen secreted during bacterial growth. Studies have demonstrated that the rapid speciation tests compare favourably with other established phenotypic or genotypic methods. This test provides results within 15 min and is highly sensitive and specific. Sensitivity and specificity were found to be 98.6% and 97.9%, respectively, for the Capilia TB assay (Taunus, Japan), 99.0% and 100%, respectively, for the SD TB Ag MPT64 rapid test (Standard Diagnostics, South Korea) and 100% and 92.4% for the BD TBcID (Becton Dickinson, Sparks, MD).[25],[26],[27] Other than the equipment needed to culture TB, no additional equipment or consumables are needed to perform the test and it can detect M. tuberculosis complex even when mixed with non-tuberculous Mycobacteria. The results of Mycobacterium bovis and M. bovis Bacillus Calmette–Guérin (BCG) cultures may vary since some BCG strains are known to lack MPT 64 antigen production. The test detects organisms belonging to M. tuberculosis complex but does not specifically identify MTB strains. Most laboratories using liquid cultures including the intermediate reference laboratories (IRLs) in India utilize this antigen detection for rapid identification of M. tuberculosis complex in liquid culture as per the WHO recommendation in 2007–2008.

Antigen detection

Lipoarabinomannan (LAM) is a 17.5 kD glycolipid found in the outer cell wall of mycobacterial species.[28] LAM is one of the three major groups of interrelated lipopolysaccharides within the mycobacterial cell wall. All these molecules are non-covalently attached to the mycobacterial plasma membrane through the glycophospholipid anchor and extend to the surface of the cell wall. It is immunogenic and a major virulence factor promoting survival in human host. The test is available as an ELISA or dipstick method with a turnaround time (TAT) of 4–6 h or 20 min depending on the test kit used. The WHO recommends that it should not be used for the diagnosis of TB, except for HIV-positive in-patients with signs and symptoms of TB (pulmonary and/or extrapulmonary) who have a CD4 cell count ≤100 cells/μL or HIV-positive patients who are seriously ill, regardless of the CD4 count. For TB diagnosis among symptomatic patients, overall lateral flow-LAM pooled sensitivity was 44% and pooled specificity was 92%. Several hypotheses may explain the higher sensitivity of urine LAM detection in patients with HIV-related immunosuppression, including higher bacillary burden and antigen load, greater likelihood of TB in the genitourinary tract and greater glomerular permeability that allows increased antigen levels in urine.

Molecular detection: Nucleic acid amplification tests

Most molecular methods that are WHO endorsed detect the nucleic acid (DNA) of both live and dead bacilli. While molecular methods cannot as yet completely replace culture and phenotypic DST, implementation of these assays reduces the need for costly laboratory infrastructure and can detect MDR-TB relatively early, providing an attractive combination of speed and sensitivity. Nucleic acid amplification tests (NAATs) that were initially used widely and recommended by the United States Food and Drug Administration to assist the diagnosis of TB were polymerase chain reaction (PCR) (Amplicor PCR assay; Roche Molecular Systems, USA), transcription-mediated amplification (Amplified MTD assay [Gen-Probe Inc., USA], GenoType Mycobacteria Direct assay [Hain Lifescience, Germany]), strand displacement amplification (BD ProbeTec assay; Becton Dickinson, USA).[4] Since most of these tests involve amplification of target DNA, they are extremely susceptible to contamination from amplicons derived from positive specimens. Thus, strict infrastructure requirements must be observed, negative controls and inhibition controls must be included in each run to detect any potential amplicon contamination and inhibitors present, and wipe tests should be performed monthly to ensure that work areas remain clean. The advantage of these methods is that M. tuberculosis complex can be rapidly detected and identified; however, MTB cannot be ruled out due to issues related to sensitivity and inhibition. The pooled sensitivity and specificity of these tests were 85% (range, 36%–100%) and 97% (range, 54%–100%), respectively.[4] These techniques also have an important role in diagnosis of tuberculous meningitis.[4]

Currently, three methods exist; the first two being WHO recommended:

  1. Cartridge-based NAAT (CB-NAAT)
  2. Line probe assay (LPA)
  3. Loop-mediated amplification (LAMP).


Cartridge-based nucleic acid amplification test

The CB-NAAT is a semi-quantitative nested real-time PCR which detects both MTB and RIF resistance directly from clinical specimens. It is the WHO-recommended method in 2010 for the diagnosis of both pulmonary and extrapulmonary TB and for diagnosing paediatric TB. Under the current RNTCP guidelines, it is recommended for diagnosis of drug resistant-TB (DR-TB) in presumptive DR-TB and upfront diagnosis of TB in key population like paediatric tuberculosis, extra-pulmonary cases and people living with HIV. The analytical limit is 131 CFU/ml and the TAT is 2–3 h. Results can be ideally available while patient waits in the clinic. Because the cartridges are self-contained, the problem of cross-contamination between samples is eliminated. Sputum is liquefied and inactivated with a sample reagent which kills over 99.9% of TB bacilli in the specimen, and 2 ml of the material is transferred into a cartridge and this is inserted in the MTB-RIF test platform. Inside the cartridge, the sample is automatically filled, washed, filtered, by ultrasonic lysis of the filter captured organisms to release the DNA. It uses three specific primers and five unique molecular probes to ensure high degree of specificity. The primers amplify a portion of the rpoB gene 81 bp RIF resistance determining region. The probes are capable to differentiate between wild-type (WT) and conserved sequence and mutations in the core region.

The sensitivity was 99.8% for smear- and culture-positive cases and 90.2% for smear-negative, culture-positive cases.[29] The sensitivity of a single direct MTB/RIF test for culture-confirmed TB was 92.2% and rose to 96.0% with the additional testing of a pelleted sample. For the detection of smear-negative, culture-positive TB, the sensitivity of the assay was 72.5% for one test, 85.1% for two tests and 90.2% for three tests. The estimated specificity was 99.2% for a single direct MTB/RIF test, 98.6% for two MTB/RIF tests and 98.1% for three MTB/RIF tests. The MTB/RIF test correctly detected RIF resistance with a sensitivity of 99.1% and 100% specificity.[29] Thus, the test detects TB in essentially all smear-positive samples and the majority of smear-negative samples. The presence of non-tuberculous Mycobacteria does not confound testing. The cartridges are stable at room temperature. In future, the same technology may be also used for HIV viral load detection.

Issue to be considered while using CB-NAAT is the presence of mono-resistance to INH which is not detected in this test. INH mono-resistance is documented to be 7%–11% in the first-line treatment failures and newly diagnosed and previously untreated patients, respectively.[30] Loss of therapeutic efficacy of this important anti-TB drug has considerable implications for treatment and control strategies. Both live and dead bacilli are picked up by the CB-NAAT thus making this test in the current format useless to assess post-therapy efficacy. Concerns exist regarding false-positive RIF resistance results; hence, samples found to be resistant must be confirmed by a second Xpert MTB/RIF test or an LPA and phenotypic culture testing. In case an indeterminate result is obtained on the first specimen, a repeat testing of a new specimen by CBNAAT is required, if the result of this is also indeterminate, testing by culture and DST or Line Probe assay is mandated. Each cartridge has its internal quality control viz sample processing control and Probe Check control. If Probe check control fails the test is stopped and an error is generated (>5% errors need to be investigated). The sample processing control must be positive when MTB is NOT detected but may be positive or negative is MTB is detected. The test requires a trained and computer-literate operator, a stable supply of electricity and air-conditioned settings.[31],[32]

Line probe assay

This strip test detects TB DNA and genetic mutations associated with drug resistance from smear-positive sputum specimens or culture isolates after DNA extraction and PCR amplification. This is a hybridisation assay that allows differentiation between Mycobacterium species. Each strip consists of 27 reaction zones (bands), including six controls (conjugate, amplification, M. tuberculosis complex, rpoB, katG and inhA controls), eight rpoB WT and four mutants (MUT) probes, one katG WT and two MUT probes and two inhA WT and four MUT probes. Theoretically the TAT is 5–6 h but the entire procedure usually takes upto 72 hours. It has a good sensitivity and specificity when performed on smear-positive and on culture isolates. Sensitivity, specificity and positive and negative predictive values were 98.9%, 99.4%, 97.9% and 99.7% for the detection of RIF resistance; while it was 94.2%, 99.7%, 99.1% and 97.9%, respectively, for the detection of INH resistance and 98.8%, 100%, 100% and 99.7%, respectively, for the detection of MDR compared to conventional results, thus confirming their value in rapid screening of patients suspected of MDR-TB.[33] WHO has endorsed LPA for MDR-TB in 2009. Two commercially available products are (1) InnoLiPA assay-Innogenetics, Belgium, and (2) Hain Lifescience GenoType® MTBDRplus.

Geographical variation in the prevalence of mutations associated with RIF and in particular with INH resistance may result in varying performance of LPAs in different epidemiological settings. Introduction of LPAs should preferably be preceded by an assessment of sensitivity and specificity of these assays in a representative collection of MDR and non-MDR isolates at country or at regional level. LPAs are as complex to perform as conventional culture and DST and require skilled and well-trained laboratory personnel, as well as adequate laboratory space and design (BSL-2/3 level laboratory with Class II Biological Safety Cabinet) to reduce the risk of false-positive results. LPAs 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 DST is required to confirm if not diagnose MDR/XDR TB.

Detection of resistance to the second-line drugs: At present, the 'gold standard' DST method measures phenotypic resistance by the culture-based indirect proportion method, which has been standardised for solid and liquid media for the detection of ofloxacin (OFX) and AMK resistance.[34],[35] Using these platforms, a DST may take anywhere between 10 days and 6 weeks. Excluding second-line drug resistance is a critical prerequisite for identifying patients who can be placed on the shorter MDR-TB regimen. Hain's MTBDRsl version was developed with this in mind to shorten the TAT of second-line DST testing to 1–2 days. These assays detect mutations in the gyrA gene (fluoroquinolone resistance), rrs gene (KM, AMK and CM resistance) and embB gene (EMB resistance). Any smear-positive or MDR culture-positive patient (adult or children) who has a positive Hain MDRplus assay may undergo the MTBDRsl version. This enables a rapid diagnosis of pre-XDR or XDR-TB in a smear-positive patient or if an MDR isolate is used. The sensitivity for detecting OFX, AMK and extensive drug resistance directly from clinical samples was 90.7%, 100% and 92.3%, respectively, and the specificity for detection was 98.1%, 99.4% and 99.6%, respectively.[36] Inclusion of moxifloxacin in an RIF-resistant or MDR-TB regimen is best guided by phenotypic testing.[2] This test is the first and only currently WHO-recommended rapid test in May 2016 for the detection of additional resistance in MDR-TB patients as well as XDR-TB.[2] Detection of second-line resistance by the second LPA means that MDR-TB patients should not be given the shorter treatment regimen as this leads to the development of XDR-TB and treatment failures. These recommendations do not eliminate the need for phenotypic DST to confirm resistance to other drugs and to monitor the emergence of additional drug resistance during treatment. The NRL retests 20/50 duplicate strains from a participating laboratory. This is followed by annual proficiency testing. Internal and external concordance should be >95%. Both, CB-NAAT and LPA are WHO-recommended technologies adopted by the RNTCP at IRLs in India.

Loop-mediated amplification

Molecular amplification methods are proven technologies for the detection of TB but have not been widely used in remote settings because of the cost and complexity. LAMP is a simple, rapid, specific and cost-effective nucleic acid amplification method solely developed by Eiken Chemical Co., Ltd, Japan.[37] It is characterised by the use of four different primers specifically designed to recognise six distinct regions on the target gene and the reaction process proceeds at a constant temperature using auto-cycling strand displacement reaction targeting the six regions of the gyrB and 16S rRNA genes. Amplification and detection of gene can be completed in a single step, by incubating the mixture of samples, primers, DNA polymerase with strand displacement activity and substrates at a constant temperature (about 65°C). It provides high amplification efficiency, with DNA being amplified 109–1010 times in 15–60 min. Because of its high specificity, the presence of amplified product can indicate the presence of target gene. LAMP is a simple isothermal DNA amplification method that does not require a thermocycler or detection system and allows visual detection of amplification, possibly allowing it to be used at lower levels of the health system. It detects M. tuberculosis complex but does not detect resistance. The assay had a detection limit of 5–50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of M. tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare from a solid medium culture. This LAMP-based assay is simple, rapid and sensitive; a result is available in 35 min for a solid medium culture and in 60 min for a liquid medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.

Various other isothermal NAAT includes:[38]

  • Recombinase polymerase amplification (RRA, Twist Dx, UK)
  • Cross-priming amplification (CPA-China)
  • Helicase-dependent amplification (HAD-USA)
  • Nicking enzyme amplification reaction (NEAR-USA)
  • Epistem (UK) and Xceleris (India) are working on providing a rapid TB diagnostic test using gene drive technology. This is a hand-held device which incorporates a rapid nucleic acid amplification specific to M. tuberculosis complex as well as RIF resistance.



 ~ Indirect Method of Detection Top


The lack of a distinctive antibody response and inability to differentiate latent infection from active disease compromised immunoassay technologies to such an extent that in 2012, the WHO issued a negative endorsement, urging practitioners not to use serological tests for diagnosis of TB.[39]

Exposure to MTB may result in latent TB infection. A person with latent TB infection usually leads a healthy life without developing active TB disease. Two billion people have latent TB infection, but only a fraction (<10 million a year) fall sick with active TB disease.[40] TST and IGRA cannot differentiate between latent TB and active TB.

Tuberculin skin testing

Popularly known as Mantoux test involves injecting the purified protein derivative (PPD) of MTB intradermally in the forearm and the resulting reaction is read after 48–72 h.[41] It was developed by Koch in 1890, but the intradermal technique currently in use was described in 1912 by Charles Mantoux, a French physician who developed on the work of Koch and Clemens von Pirquetto to create his test in 1907.[42] While using tuberculin test, it should be remembered that, in general, it detects only presence or absence of infection, i.e., exposure to MTB or latent TB. At present, only two tuberculins have been accepted as standard tuberculins by WHO, i.e., PPD-S PPD, prepared according to the method described by Siebert, from MTB and PPD RT 23. The International Standard Tuberculins is in the custody of the Laboratory of Biological Standards, Staten, Serum Institute, Copenhagen, Denmark.

PPD-RT 23 with Tween 80 of strength 1 TU and 2 TU is standardised tuberculin available in India supplied by BCG Vaccine Laboratory, Guindy, Chennai. Other tuberculins available in the market are not standardised. Tween 80 is a detergent added to tuberculin to prevent their adsorption on glass or plastic surface. The optimal strength of PPD is 2 TU. A positive skin test is indicated by a skin reaction at the point of the injection. In our country, it is used in high-risk groups, especially children, household contacts and HIV-infected patients. A positive test is considered when 10 mm or more induration is present. However, vaccination with the BCG vaccine can also lead to a reaction at the TST site (as can repeat TST testing), which limits the test's usefulness in vaccinated children or people repeatedly tested because of high risks of exposure (such as healthcare workers).

Interferon-gamma release assay

This is anin vitro assay wherein T-cells sensitised with MTB on encountering mycobacterial antigen (early secretory antigenic target 6 [ESAT-6] and culture filtrate protein 10 [CFP-10] and TB7.7), release interferon-gamma (a TH1 cytokine).[43] Advantages of this test are that the antigen used is recognised by T-cells of TB patients and not by BCG-vaccinated or healthy unvaccinated individuals. It has a very high specificity and much less likely than the TST to be confounded by exposure to environmental mycobacteria or by prior BCG vaccination. It does not boost responses that will be measured by subsequent tests as happens with TST. IGRAs do not require a second visit to the clinic to evaluate the test result, thus potentially reducing costs to the patient. Results can be available within 24 h. Disadvantages the sample drawn should be incubated within 16 h for older IGRAs and 8 h for the T-SPOT of collection which may require the use of portable incubators or establishment of systems enabling transportation to properly equipped laboratories for testing. Commercially available tests are QuantiFERON-TB Gold (QFT-G) and QuantiFERON-TB Gold in Tube (QFT-GIT) (Cellestis, Australia) and T-SPOT TB (Immunotec, UK).

There are insufficient data and low-quality evidence on the performance of IGRAs in low- and middle-income countries, typically those with a high TB and/or HIV burden. In 2011, WHO has recommended against the use of IGRAs in these scenarios. IGRAs and the TST cannot accurately predict the risk of infected individuals developing active TB disease. Neither IGRAs nor the TST should be used for the diagnosis of active TB disease. IGRAs are costlier and technically complex to do than the TST. Given comparable performance but increased cost, replacing TST by IGRAs as a public health intervention in resource-constrained settings, including India, is not recommended.[40] RNTCP advises against use of IGRAs for diagnosis in adults in high burden countries.


 ~ Other Indirect Methods Top


Volatile organic compounds

'In persons affected with phthisis, if the sputa which they cough up have a heavy smell when poured upon coals, and if the hairs of the head fall off, the case will prove fatal,' Hippocrates, Greek physician 460–410 BC.[44] These VOCs can be detected in urine, sputum and breath using a mass spectrophotometer and a gas chromatography.

Giant African pouched rats (Cricetomys gambianus) are trained to detect TB in sputum specimens by smell alone. In the study conducted in Sub-Saharan African countries by rats trained to sniff out TB-positive or TB-negative sputum specimens. The sensitivity was found to be 73% and specificity 93%.[45],[46]

A breath analyser test was developed as a point of care test by Menssana Inc. to be used in the community to screen high-risk groups. Patients cough into a disposable device which is placed in the instrument for detection of VOCs. The instrument is fully portable and runs off rechargeable AA batteries. The test is performed and readout obtained in under 10 min. Limited performance data are available and further evaluation studies are required.[47]

Beta-lactamase detection

A promising alternative to smear microscopy is a detection of a metabolic signature of MTB. Recently, a panel of fluorogenic substrates metabolised by BlaC, a TB-specific β-lactamase, was developed.[48] A prototype under development by Global Biodiagnostics, Temple, TX, USA, demonstrated high specificity and sensitivity (10 CFU in unprocessed sputum) and enabled detection in a homemade box containing a LED, filters and a mobile phone camera.


 ~ Conclusion Top


At DMCs, LED microscopy to detect MTB has been adopted by the RNTCP to replace conventional ZN staining. In IRLs, commercial automated liquid culture-MGIT is the current gold standard for detecting MDR-TB and XDR-TB, including in smear-negative cases. The LPA is suitable to detect MDR-TB only in smear-positive cases. Genotypic automated NAATs have considerable advantage in programme management and surveillance of MDR-TB. The WHO has worldwide banned the use of commercial sero-diagnostic tests (gazette notification, vide 433E, 7th June 2012) and the RNTCP discourages the use of the indirect test IGRA in India for adults.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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2004 - Indian Journal of Medical Microbiology
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