Diagnosis of extrapulmonary tuberculosis by GeneXpert MTB/RIF Ultra assay

ABSTRACT

Introduction

Diagnosis of extrapulmonary tuberculosis (EPTB) is an arduous task owing to different anatomical locations, unusual clinical presentations, and sparse bacillary load in clinical specimens. Although GeneXpert® MTB/RIF is a windfall in TB diagnostics including EPTB, it yields low sensitivities but high specificities in many EPTB specimens. To further improve the sensitivity of GeneXpert®, GeneXpert® Ultra, a fully nested real-time PCR targeting IS6110, IS1081 and rpoB (Rv0664) has been endorsed by the WHO (2017), wherein melt curve analysis is utilized to detect rifampicin-resistance (RIF-R).

Area covered

We described the assay chemistry/work design of Xpert Ultra and evaluated its performance in several EPTB types, that is, TB lymphadenitis, TB pleuritis, TB meningitis, and so on, against the microbiological reference standard or composite reference standard. Notably, Xpert Ultra exhibited better sensitivities than Xpert, but mostly at the compensation of specificity values. Moreover, Xpert Ultra exhibited low false-negative and false-positive RIF-R results, compared with Xpert. We also detailed other molecular tests, that is, Truenat MTB^TM/TruPlus, commercial real-time PCR, line probe assay, and so on, for EPTB diagnosis.

Expert opinion

A combination of clinical features, imaging, histopathological findings, and Xpert Ultra are adequate for definite EPTB diagnosis so as to initiate an early anti-tubercular therapy.

Plain Language Summary

We discussed the assay chemistry and evaluated performance of GeneXpert®MTB/RIF Ultra to identify the TB germs and resistance to one of the potent bactericidal drugs, that is, rifampicin in different types of extrapulmonary TB (EPTB, TB in organs other than lungs) and compared its performance with its ancestor, that is, GeneXpert. We briefly outlined the other molecular tests, such as Truenat MTB^TM/Truenat MTB^TM Plus, commercial real-time PCR, line probe assay, and so on for EPTB diagnosis. While evaluating Xpert Ultra results, the ‘trace call’ has been introduced, whose interpretation is important. By and large, Xpert Ultra assay outperformed in TB lymphadenitis, TB pleuritis, and TB meningitis when compared with Xpert, though limited information is available on other EPTB forms. Overall, the sensitivity of Xpert Ultra has been increased in most of EPTB cases, but mostly at the cost of specificity values.

SUMMARY

We detailed the assay chemistry and efficiency of GeneXpert® MTB/RIF Ultra to detect Mtb and RIF-R in different clinical forms of EPTB, against culture or composite reference standard. We compared the pros and cons of Xpert Ultra with its predecessor, that is, Xpert and also outlined the other molecular tests, that is, Truenat MTB^TM/TruenatMTB^TM Plus, commercial real-time PCR (e.g. Cobas TaqMan Mtb), line probe assay, and so on, to diagnose EPTB. While interpreting Xpert Ultra results, the ‘trace call’ has been incorporated, its interpretation is crucial that needs consideration of previous TB history, HIV status and whether the individual has childhood/adult TB. Overall, Xpert Ultra performed well in TB lymphadenitis, TB pleuritis, and TB meningitis, while limited information is available on other EPTB types, for which more studies are needed to ensure the authenticity of assay.

KEYWORDS:

• Extrapulmonary tuberculosis
• diagnosis
• GeneXpert® MTB/RIF ultra
• RIF-R
• trace calls
• Truenat MTB^TM
• LPA
• GeneXpert® MTB/RIF

Article highlights

• Diagnosis of extrapulmonary TB (EPTB) is quite challenging owing to atypical clinical presentation, deep inaccessible lesions in most of EPTB cases and low bacillary load in clinical specimens.

• Timely and accurate EPTB diagnosis as well as detection of drug resistance (DR) is crucial to initiate anti-tubercular medication so as to prevent morbidity/mortality.

• GeneXpert® MTB/RIF Ultra is a fully nested real-time PCR targeting IS6110, IS1081 and rpoB, which is utilized for detecting Mtb and rifampicin resistance (RIF-R) within 80 min.

• In this article, we detailed the assay chemistry/work flow of Xpert Ultra and evaluated the diagnostic performance of this assay in various EPTB types, against culture (on LJ/MGIT-960) or composite reference standard.

• Xpert Ultra results are semi-quantitatively categorized as ‘high, medium, low, very low, trace call, and not detected.’ The interpretation of ‘trace call’ shall be done carefully, especially in HIV-TB co-infected individuals, patients with a previous TB history/ATT follow-up and if the individuals suffer from childhood or adult TB.

• However, other parameters, that is, clinical presentation, detailed imaging (i.e. ultrasound, CT scan, MRI, etc.) and histopathological examination need to be considered to confirm the EPTB diagnosis in individuals with ‘trace results’ attained by Xpert Ultra.

• Notably, Xpert Ultra exhibited better sensitivities than its predecessor Xpert in most of EPTB specimens but at the compensation of some specificity values. Among different EPTB types, Xpert Ultra performed well in TB lymphadenitis, pleural TB and TB meningitis, though limited information is available on other EPTB types.

• We also detailed other molecular assays for the detection of Mtb and DR (including RIF-R), that is, line probe assay (LPA), Truenat MTB/Truenat MTB Plus and Truenat MTB-RIF Dx as well as whole genome sequencing (WGS), whereas commercial real-time PCR, loop-mediated isothermal amplification, and immuno-PCR (I-PCR)/nano-based I-PCR assays detect Mtb only.

• The main disadvantage of Xpert Ultra (similar to Xpert) is that it only detects RIF-R among a variety of anti-TB drugs. To overcome this, LPA or WGS may be utilized to detect MDR/XDR-TB cases.

Acknowledgments

We sincerely thank Michael Loeffelholz, Cepheid, Sunnyvale, CA, USA for critically reading the manuscript and providing valuable suggestions/comments that help us to improve this manuscript.

Declaration of interest

PK Mehta and B Dahiya acknowledge the Council of Scientific and Industrial Research (21(1068)/18/EMR-II), New Delhi for awarding the Emeritus Scientist Fellowship and Research Associate Fellowship, respectively. A Soni acknowledges the University Grants Commission, New Delhi for awarding the Junior Research Fellowship. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Cepheid provided a scientific accuracy review at the request of the journal editor.

Reviewers Disclosure

Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose.

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This paper was not funded.