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IOSR Journal of Pharmacy

Vol. 2, Issue 1, Jan-Feb.2012, pp. 083-C

Gene Xpert MTB/RIF Assay: A New Hope for
Extrapulmonary Tuberculosis

Ankush Raj, Netrapal Singh, Promod K. Mehta*

Centre for Biotechnology, Maharslii Dayanand University, Rohtak 124001
^Corresponding author- Promod K Mehta (Associate professor)

Abstract

The diagnostic efforts for extrapulmonary tuberculosis (TB) have been severely hampered by the lack of diagnostic tests that
are accurate, simple to use and can be applied at the point of clinical care. This has been further enhanced by the widespread
inability to test for drug resistance. Gene Xpert® MTB/RIF test is a novel test for the diagnosis of extrapulmonary TB and
rifampicin resistance. This unique test can be used almost everywhere with minimal technical expertise, enabling diagnosis of
extrapulmonary TB and simultaneous assessment of rifampicin resistance to be completed within 2 h. In low-income
countries, however, its cost, environmental limitations and difficulties involved in supply and maintenance are major
obstacles. While it may be suitable for major reference hospitals, operational research is needed to evaluate the test and its
additional yield above high-quality smear microscopy. In high TB burden countries like India, WHO in 2010 endorsed some
guidelines for implantation of Gene Xpert® MTB/RIF test for diagnosis of TB and drug resistance.

Keywords- extrapulmonary tuberculosis, Gene Xpert® MTB/RIF, rifampicin, drug resistance, WHO.

1. Introduction

India has the world's largest burden of tuberculosis (TB), accounting for one-forth (24%) of the global TB incidence. The
global annual incidence estimate is 8.8 million cases, of which 1.5 million cases are from India [1]. According to Revised
National Tuberculosis Control Programme, 0.8 million new cases of extrapulmonary TB (EPTB) were observed in 2010 [1].
Less than 5% of new and previously treated TB patients were tested for MDR-TB in most countries in 2010 [1]. In India and
China, almost 50% of multidrug-resistant TB (MDR-TB) cases worldwide are estimated to occur [2]. In India, 15 to 20% of
TB cases are estimated to be cases of EPTB, which affects mainly the lymph nodes, meninges, kidney, spine, and growing
ends of the bones [3]. Since the causative agent Mycobacterium tuberculosis spreads from person to person, an efficient as
well as rapid diagnosis is a key objective of worldwide tuberculosis control programmes.

2. Present scenario of TB diagnosis

The major challenge in the diagnosis of EPTB is the frequently atypical clinical presentation simulating other inflammatory
and neoplastic conditions, which frequently results in a delay or deprivation of treatment. Therefore, a high degree of
suspicion is required for an early diagnosis and mostly, more than one technique is necessary for the confirmation of the
diagnosis. In lower-income countries, the lack of diagnostic infrastructure substantially aggravates the problem [4]. The
diagnosis of EPTB cases is challenging due to inadequate clinical sample volumes available and paucibacillary nature of the
biological samples. [5]. Several potential techniques have been employed for the diagnosis of EPTB specimens i.e. smear
microscopic examination, culture test (both manual and automated), serological assays (both antigen and antibody detection),
histological /cytological examination, Mantoux test, and PCR assays (conventional as well as Real-time PCR) [3].

The conventional Mycobacterium tuberculosis detection techniques based on microscopic examination of acid fast stained
specimens (by Ziehl-Neelsen method) are still highly used for diagnostic purpose especially in TB endemic countries,
although they fail to provide the required sensitivity and specificity and are unable to differentiate between M. tuberculosis
and non-tuberculous mycobacteria (NTM) [3]. Histological examination has limitations as it can not differentiate between TB
and non-TB infections caused by other related diseases e.g. sarcoidosis or NTM infections except for the demonstration of
stained tubercle bacilli [6]. Culture test has variable sensitivities of 3-80% in various clinical EPTB samples and it takes 4-8
weeks to get those results and also requires trained technicians [7]. The Mantoux test is widely employed world-wide but the
false-positive results are observed due to the previous bacillus Calmette-Guerin (BCG) immunization or exposure to atypical
mycobacteria and false-negative results also occur in aged individuals or immuno-suppressed individuals [71.

The reports on diagnostic tests such as enzyme-linked immunosorbent assay (ELISA), slide agglutination and PCR are
available for EPTB; however, the specificities and sensitivities of these tests are variable [4]. Also, these tests require a
number of manual steps, and some have a relatively long turnaround time. A series of meta-analyses has shown that nucleic
acid amplification tests (NAAT) have high specificity and positive predictive value with highly variable sensitivity in cases
of EPTB [8-10].

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Although the development of real-time PCR assays has improved the speeds, sensitivities, and specificities of these
molecular techniques due to its short turnaround time and ; iation of the procedure, the new real-time methods have still
not been widely adopted [11-12]. Cobas TaqMan MTB (Roche Molecular Systems, Branchburg, NJ) is a real-time PCR-
based kit using TaqMan hydrolysis probes and primers that bind to a specific, highly conserved region of the Mycobacterium
genome containing the gene for 16S rRNA [13].

3. Gene Xpert MTB/RIF Assay

Cephaid Gene Xpert MTB/RIF (Xpert) assay, which can detect M. tuberculosis complex and associated rifampin (RTF)
resistance directly from clinical samples using ultrasensitive hemi-nested PCR and molecular beacon technology that operate
in temperatures of 15-30°C, even in high-humidity environments [14]. This may represent the biggest advance in TB
diagnostics in past decades, as it allows the simultaneous detection of M. tuberculosis and RIF resistance in one assay,
requires minimum handling and training, and yields results within 2 h [14]. These characteristics also make it a potentially
attractive tool for extrapulmonary specimens.

4. Components of Xpert assay

The assay utilizes single-use plastic cartridges with multiple chambers that are preloaded with liquid buffers and lyophilized
reagent beads necessary for sample processing, DNA extraction and heminested real time PCR [15]. The different steps of
Xpert assay are following.

4.1 Cartridge

The assay utilizes single-use plastic cartridges with multiple chambers that are preloaded with liquid buffers and lyophilized
reagent beads necessary for sample processing, DNA extraction and heminested rt-PCR [15]. The cartridge incorporates a
syringe drive, a rotary drive and a filter upon which M. tuberculosis bacilli are deposited after being liberated from the
clinical material [16]. The test platform employs a sonic horn that inserts into the cartridge base to cause ultrasonic lysis of
the bacilli and release of the genetic material [16].

4.2 Hemi-nested real time PCR

The assay then amplifies a 192 bp segment of the rpoB gene using a hemi-nested rt-PCR reaction [17]. The assay also
contains lyophilized Bacillus globigii spores which serve as an internal sample processing and PCR control. The B. globigii
PCR assay is multiplexed with the M. tuberculosis assay [18].

4.3 Detection of RIF resistance.

RIF resistance is particularly amenable to rapid molecular detection since >95% of all rifampicin resistant strains contain
mutations localized within the 81 bp core region of the bacterial RNA polymerase rpoB gene, which encodes the active site
of the enzyme [18]. Moreover, mutations that produce in this region are highly detective for RIF resistance, whereas
susceptible isolates almost always have the same wild-type nucleotide sequence [16]. In addition, the rpoB core region is
flanked by M. tuberculosis-specific DNA sequences. Thus, by using PCR technology, it is possible to detect M. tuberculosis
and RIF resistance simultaneously, by targeting a single gene target. Thus, the rpoB gene represents a much better molecular
target for the simultaneous detection of TB and the key form of drug resistance because of higher sensitivity of rpoB gene
than kat G and inh A for isoniazid resistance [19].

4.4 Molecular becons

The Xpert assay utilizes molecular beacon technology to detect DNA sequences amplified in a hemi-nested real time PCR
assay [14]. Five different nucleic acid hybridization probes are used in the same multiplex reaction [18]. Each probe is
complementary to a different target sequence within the rpoB gene of RIF-susceptible M. tuberculosis and is labeled with a
differently colored fluorophore [16]. Together, these overlapping probes span the entire 81 bp core region of the rpoB gene
[18].

4.5 Detection of M. tuberculosis

M. tuberculosis is detected by the five overlapping molecular probes that collectively are complementary to the entire 81 bp
rpoB core region [18]. M. tuberculosis is identified when at least two of the five probes give positive signals with a cycle
threshold (C T ) of <38 cycles and that differ by no more than a prespecified number of cycles [18]. The B. globigii internal
control is positive when the single B. globigii specific probe produces a C T of <38 cycles. The standard user interface gives
the signal of presence or absence of M. tuberculosis and the presence or absence of rifampicin resistance, and a
semiquantitative estimate of the concentration of bacilli as defined by the C T range (high, <16; medium, 16-22; low, 22-28;
very low, >28). Assays that are negative for M. tuberculosis and for the B. globigii internal control are reported as invalid
assays [16].

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The basis for detection of RIF resistance is the difference between the first (early C T ) and the last (late C T ) M. tuberculosis-
specific beacon (AC T ). The system was originally configured such that resistance was reported when AC T was >3.5 cycles
and sensitive if <3.5 cycles. Since the assay terminates after 38 cycles, the assay was deemed indeterminate for RTF
resistance if the first probe C T is >34.5 cycles and the last probe has a C T of >38 cycles [18]. From May 2010 the automated
detection of RIF resistance was modified using a new AC T cut-off in order to improve the specificity for RIF resistance
detection [20].

5. Working of Xpert assay

Working of Xpert assay has been shown in fig. 1. Firstly clinical samples were treated with sodium hydroxide and
isopropanol containing sample reagent (SR) [16]. The SR is added to the sample at 2:1 ratio and incubated at room
temperature for 15 min [14]. This step is designed to reduce the viability of M. tuberculosis in sample at least 10 6 -fold to
reduce biohazard risk [21]. The treated sample is then manually transferred to the cartridge which is loaded into the
GeneXpert instrument. Subsequent processing is fully automated [16]. A schematic of the assay procedure is shown in Fig 1.

Fig.l. Various steps of Xpert Assay

6. Sensitivity and specificity

The specificity of the Xpert test in the diagnosis of pulmonary TB has been shown to be very high (97-100%) in
demonstration studies coordinated by the Foundation for Innovative New Diagnostics (FIND) in pulmonary samples [22].
Laboratory-based studies in Germany, France and Spain have assessed the utility of Xpert for TB diagnosis in nonrespiratory
samples, including pleural fluid, gastric fluid, tissue, cerebrospinal fluid, urine and stool [16]. 521 nonrespiratory samples
were detected with Xpert test having overall sensitivity of 77.3% and a specificity of 98.2%, using culture as gold standard
[23].

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Preliminary analysis demonstrated excellent performance of Xpert test on fine needle aspirates samples with high sensitivity
(100%) and specificity (93.8%) compared to MGIT culture [24]. Xpert test has the potential to significantly improve and
expedite the diagnosis of smear negative and extra-pulmonary TB at both hospital and point-of-care settings in regions with
overlapping HIV and TB epidemics [24].

Ligthelm et al. (2011) demonstrated the excellent diagnostic accuracy of the Xpert test in patients with tuberculous
lymphadenitis with sensitivity of 96.7% and specificity of 90% [25].

Recently, Tortoli et al. (2012) investigated a large number of consecutive extra-pulmonary clinical specimens (1,476)
including both paediatric (494) and adult samples with overall sensitivity (81%) and specificity (99.8%) using .combination
of culture and clinical diagnosis as gold standard [26].

Collectively, these results indicate substantial utility of Xpert for diagnosis of extra -pulmonary TB and further prospective
evaluations of clinical suspects are warranted to more precisely define the utility for different forms of extrapulmonary
disease.

7. Comparative study with other assays

From India, the Xpert was evaluated to test its utility in 547 patients with suspected extrapulmonary tuberculosis. For culture,
the sensitivity was low, 53% (150/283 specimens). Xpert sensitivity and specificity results were assessed in comparison to a
composite reference standard made up of smear and culture results and clinical, radiological, and histological findings [14].
The sensitivity of the Xpert assay was 81% (228/283 specimens) (64% [89/138] for smear-negative cases and 96% [139/145]
for smear -positive cases), with a specificity of 99.6% [14]. Causse et al. (201 1) tested a wide range of nonrespiratory samples
(n = 341) and reported a higher sensitivity of 95% and specificity of 100%, outperforming the commercially available Cobas
TaqMan® MTB assay [17]. In one previous study, Armand et al. (201 1) demonstrated the results from just 32 nonrespiratory
samples and found an overall sensitivity of 53% that was substantially lower than that obtained using an in-house IS6110-
based real-time PCR assay (78%) [27]. By contrast, the sensitivity of the Xpert assay was 75% while the IS6110 real time
assay's sensitivitiy was 0% in smear negative extrapulmonary specimens [28].

8. RIF resistance detection

The Xpert test has been evaluated for testing genomic DNA from M. tuberculosis isolates with 23 different commonly
occurring rpoB mutations with 100% sensitivity [29]. A total of 16 of the 23 mutations caused complete failure of detection
of at least one of the five probes and the remaining mutations produced a ACT of >3.5 cycles. All DNA samples from RIF
susceptible isolates were correctly identified as susceptible with an average ACT of 1.8 cycles [29]. Blakemore et al. (2010)
tested low concentrations of genomic DNA from 79 phylogenetically and geographically diverse strains of M. tuberculosis
that included 42 rifampicin susceptible and 37 resistant strains [18]. Further experiments were done in which DNA from
resistant and susceptible strains was mixed in varying ratios to assess how this impacted detection of rifampicin resistance
[18]. To enable detection, between 65% and 100% of the DNA from the rifampicin-resistant isolate had to be present,
depending on the mutation [18]. Whereas some mutations completely block probe hybridization, others only inhibit it. If only
partial inhibition occurs, then the presence of only a small concentration of wild -type amplicon would need to be present to
boost the probe signal into the normal range. Overall, this suggests that in patients with mixed infections, the Xpert assay
might only detect the resistant strain if it is the predominant one present. However, selection of resistant strains during the
course of standard TB treatment might lead to an apparent switch from a susceptible to a resistant phenotype when
comparing baseline testing with repeat testing during treatment [14].

9. Limitations of the Test

The Xpert test is a major advance in TB diagnostic testing, but has few limitations such as the limited shelf-life of the
diagnostic cartridges, some operating temperature and humidity restrictions, requirement for electricity supply, unknown
longterm robustness, and the need for annual servicing and calibration of each machine [15]. Laboratories in low-income
countries are littered with expensive equipment that no longer functions because it was inappropriate to the setting to which it
was donated. Ensuring sustainable systems for long-term provision of servicing and consumables may be more important and
challenging than initial implementation of the diagnostic equipment itself.

10. Impact in Low- and Middle-Income Settings

Despite numerous microbiological studies of improved TB diagnostic technologies, we remain remarkably ignorant of how
best to implement better tests to improve patient care, of who should receive the limited capacity for better tests to maximize
health impact, of how these tests may impact patient-relevant outcomes, and of how these issues vary between settings [15].
The impact of better diagnostic tests on the equity of care is largely unstudied and we don't know yet how this novel

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technology will affect the delays and costs faced by patients in their journey towards a cure for this archetypal disease of
poverty.

A major issue facing low- and middle-income countries wishing to implement this technology is the high cost compared to
smear microscopy. However, FIND has negotiated substantial cost reductions (relative to prices in the USA and Europe) for
use in the public sector in 1 16 high burden low- and middle-income countries [30]. The initial capital cost for the GeneXpert
instrument in these settings is considerably more than for microscopy but much lower than for conventional culture and drug
susceptibility testing, especially in view of the savings from dispensing with the need for expensive biosafety equipment [31].
Instruments require annual calibration, which is likely to prove difficult in remote settings, but the cost of this may be
minimized by the proposed development of a web-based system. The cost per test in early 201 1 (estimated at $18 per test) is
substantially greater than for microscopy, though similar to costs for performing culture and drug susceptibility testing [16].
In the short-term, implementation of the assay would represent a substantial financial increase in diagnostic costs for national
ministries of health but if the full benefits of the assay in terms of clinical outcomes and TB control are favorable, this may
prove to be a wise investment.

Although, findings of Vassall et al. (2011) suggested that Xpert is a cost-effective method of TB diagnosis, compared to a
base case of smear microscopy and clinical diagnosis of smear -negative TB in low- and middle-income settings where, with
its ability to substantially increase case finding, it has important potential for improving TB diagnosis and control [32]. The
extent of cost-effectiveness gain to TB programmes from deploying Xpert is primarily dependent on current TB diagnostic
practices.

11. WHO endorsement, costs & implementation (WHO 2010)

In the latter part of 2010, WHO reviewed all the available evidence regarding the Xpert assay, initially through an expert
group, then through the Strategic Technical Advisory Group for TB (STAG-TB) and finally within a wider WHO
consultation process. In December 2010 WHO endorsed the use of the assay [31] as follows:

• In December 2010 the WHO endorsed use of this assay as the initial diagnostic test in individuals suspected of having
MDR-TB or HTV associated TB (strong recommendation).

• WHO also endorsed use of the test as a follow-on to smear microscopy in settings where MDR-TB and HIV-associated
TB were less of a concern (conditional recommendation).

• Phased implementation from 2011 onwards is recommended, leading to full scale-up thereafter. The cost of cartridges
will decrease over time.

12. Conclusion

The Xpert technique has demonstrated a substantial capacity for the diagnosis of EPTB mostly from nonsterile fluids (gastric
aspirates and urine samples), lymph nodes or abscess aspirate specimens. The high cost of this novel technology is offset to
an extent by the rapid turnaround time as observed with the smear microscopic examination (less than two hours) with less
biohazard problems and only minimal training required. Hence, the Xpert test not only has good sensitivity and specificity for
the diagnosis of TB and detection of RIF resistance in EPTB but also perfectly fits the requirements of the Indian health care
setting.

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