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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 1  |  Page : 76-80

Predictable repeatability issues with GeneXpert-Xpert MTB/RIF (version 4) derived rifampicin resistant tuberculosis results from South India: Appreciating the limits of a technological marvel!


1 Intermediate Reference Laboratory (TB), Trivandrum, Kerala, India
2 State TB Training and Demonstration Centre, Trivandrum, Kerala, India
3 Department of Community Medicine, Government Medical College, Palakkad, Kerala, India

Date of Web Publication24-Jul-2017

Correspondence Address:
Praveen Sanker
Intermediate Reference Lab (TB), State TB Cell, Red Cross Road, Vanchiyoor, Trivandrum, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_6_17

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  Abstract 

Background: GeneXpert MTB/RIF (Xpert), the fully automated cartridge-based nucleic acid amplification test for simultaneous identification of Mycobacterium tuberculosis complex and rifampicin resistance (RR), directly from samples is considered as a game changer for tuberculosis (TB) control programs worldwide. Methods: We are reporting serious issues with repeatability among a subgroup of Xpert (Version 4) identified RR results from South Indian state recently switched to Xpert by the National TB control program. Results: We have demonstrated that poor repeatability is frequently associated with those Xpert derived RR results, identified by detection of delayed amplification of any probe in the presence of positive analyte results for all probes. Another significant contributing factor was found to be lower bacterial loads in samples. The repeat tests were done by Xpert and/or by line probe assay depending on smear positivity. The finding is worrying as Xpert is recommended over other tests due to its reportedly better performance among low bacterial load samples such as pediatric, extra-pulmonary, HIV-TB co-infected, and smear negative pulmonary TB and the same samples, it seems are more likely to cause error prone RR results. Conclusions: We recommend for additional genotypic tests with specific mutant probes for detecting mutations at rpoB hot sites and growth based tests for all Xpert derived RR-TB cases identified by the above algorithm for confirmation of the presence of mutation, based on our available data.

Keywords: Delayed amplification, limits of Xpert, line probe assay, low bacterial load samples, negative probe-analyte result, positive probe-analyte result, repeatability, rifampicin false resistant, Xpert


How to cite this article:
Sanker P, Kottuthodi RP, Ambika AP, Santhosh VT, Balakrishnan R, Mrithunjayan SK, Moosan H. Predictable repeatability issues with GeneXpert-Xpert MTB/RIF (version 4) derived rifampicin resistant tuberculosis results from South India: Appreciating the limits of a technological marvel!. Biomed Biotechnol Res J 2017;1:76-80

How to cite this URL:
Sanker P, Kottuthodi RP, Ambika AP, Santhosh VT, Balakrishnan R, Mrithunjayan SK, Moosan H. Predictable repeatability issues with GeneXpert-Xpert MTB/RIF (version 4) derived rifampicin resistant tuberculosis results from South India: Appreciating the limits of a technological marvel!. Biomed Biotechnol Res J [serial online] 2017 [cited 2019 Oct 20];1:76-80. Available from: http://www.bmbtrj.org/text.asp?2017/1/1/76/211417


  Introduction Top


GeneXpert-Xpert MTB/RIF (Xpert-Cepheid, Sunnyvale, CA, USA) [Figure 1], the fully automated cartridge-based nucleic acid amplification test (NAAT) is considered as a game changer for tuberculosis (TB) control programs worldwide.[1],[2],[3] Fantastic automation, negligible bio-safety concerns, speed and considerably better sensitivity of the test even approaching that of cultures,[1],[2],[3] made it the preferred test for simultaneous identification of Mycobacterium tuberculosis complex (MTBC) with rifampicin (RIF) resistance (RR), especially among patients, likely to produce samples with low bacterial loads.[3] This real-time PCR based test identifies “RIF resistance associated mutations” by sensing a “significant” delay in amplification of the mutated segment/s compared to other normal segments, located in resistance determining region of rpoB gene of Mycobacterial genome. The “significant” delay ranges from just more than 4 cycles (positive analyte result present for all probes) [Figure 2] to complete drop out of the probe affected (negative analyte result for the affected probe) [Figure 3], depending on the type of mutation and the proportion of mutated population among a mixed population with normal wild type.[4]
Figure 1: GeneXpert system

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Figure 2: Rifampicin resistant result with positive analyte results for all probes

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Figure 3: Rifampicin resistant result with negative analyte result for any probe

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Kerala, South Indian state with 33 million population introduced Xpert, in all the 14 districts starting from March to June 2016 as part of the larger strategy of nationwide Xpert coverage by Revised National TB Control Program (RNTCP), India. During the following months till October, Xpert identified 84 RR pulmonary TB cases following which separate paired samples of sputa were sent to our laboratory – the State reference laboratory for further testing including susceptibility to second-line anti-TB drugs. As per the protocol followed at our laboratory, smear negative or scanty positive processed samples (light-emitting diode fluorescent microscopy) were subjected to a repeat Xpert, while the rest were subjected to line probe assay (LPA, genotype MTBDRplus, Hain Lifesciences GmbH, Nehren, Germany), before proceeding to culture and further susceptibility testing for the second-line drugs.


  Methods Top


We have examined the details submitted along with the samples received at our laboratory and collected the copies of Xpert results of all RNTCP Xpert identified RR-TB cases all over Kerala state (14 machines at 14 districts) from March to October 2016. Then, we compared that with the limited numbers of repeat Xpert results and/or LPA results of the corresponding samples available in our laboratory. The maximum usage among all the Xpert machines at the end of the study were < 200 tests per module and all the machines/kits were (understandably) Version 4 with sufficient period left for expiry of the kits which were meticulously stored below 22°C. Those results given by LPA as doubtful RR which necessitated confirmation by growth based tests (mutation present but of doubtful significance) were considered as resistant for a fair comparison. Hence, growth based DST was not taken for comparison.


  Results Top


Repeated Xpert test results were available for 12 out of the 71 RR sputum samples received at our laboratory from a total of 84 pulmonary RR cases identified all over the state by Xpert machines installed at districts [Figure 4]. Seven of them showed rifampicin sensitive (RS) results by the repeat Xpert, which were also, later found to be RS by LPA except in two cases, where LPA gave negative for MTBC result. A closer look revealed that all the seven cases were identified RR by district Xpert tests based on detection of “delayed amplification” (the difference of lowest and highest CT/cycle threshold >4 cycles) with positive analyte results for all probes.
Figure 4: Work flow chart

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After going through all the 84 RR Xpert results reported by district centers during the period, we have identified that 20 (24%) of them were identified based on “delayed amplification with all positive analyte results.”

Among 64 RR cases identified at districts by the presence of a negative analyte result for any probe, 54 samples were received at our laboratory and repeat Xpert results were available only for four [Figure 3]. All the four showed the same result with identical probe amplification on repeat testing thus showing full repeatability. Furthermore, out of the remaining fifty RR cases, LPA results were available for 45 cases and all except two cases were also found resistant by LPA with matching mutation sites. Due to the high agreement between Xpert and LPA among RR Xpert results identified by negative probe-analyte results (43/45, 96%) and when done among same sample sets at our laboratory (9/9, 100%) [Figure 4], we have combined both the data to get adequate numbers and then compared with the Xpert resistance determining algorithm [Table 1] and the Xpert bacterial load of samples [Table 2].
Table 1: Inconsistent* and consistent# results among rifampicin resistant cases identified by “delayed amplification” and “insignificant probe amplification” algorithm by Xpert

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Table 2: Inconsistent* and consistent# results among lower bacterial load and higher bacterial load rifampicin resistant samples by Xpert

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The comparison clearly shows that inconsistent results are highly likely to happen when the machine uses the “late amplification” criteria with positive analyte results for all probes. It also shows that low bacterial load results (>22 cycle threshold-CT) as another important contributing factor.


  Discussion Top


NAATs are currently the primary tool for identification of RR-TB and Xpert particularly is considered as a game changer for TB control programs in developing countries.[1],[2],[3] Initial studies have shown very high sensitivity and specificity for identification of RR by these tests.[3],[4],[5] Some of the issues with Xpert highlighted by other studies are false RR results related to existence of “disputed” and silent mutations,[6],[7],[8],[9],[10],[11] and significantly higher mutant proportions needed (65%–100% of the total bacterial population) for a positive identification,[3],[4] compared to the minimum 1% mutant population required for clinical resistance.[12] Solid media based proportion method and even liquid media based automated testing method (MGIT 960) to a certain extent, are capable of identifying such low proportions.[12]

Our data demonstrate that there is a serious possibility of inconsistency/possibly false RR results (10/15) on a subset of Xpert derived RR samples where “delayed CT” with positive analyte results for all probes are present. In contrast, almost all the RR (47/49) identified by negative analyte result of any probe (“complete drop out of any probe”) were shown consistent by subsequent LPA or Xpert. It is pertinent to remember here that the current version (Xpert MTB/RIF Version G4) is the product of changes in significant delta CT(The difference between the highest CT and the lowest CT) from 3.5 cycles (Xpert MTB/RIF Version G1) to 4.0 cycles (version G2) and subsequent fine tuning of other parameters in the later versions (G3 and G4).[13],[14] The WHO policy update 2013 for Xpert usage,[15] advises a repeat Xpert test for confirmation of Xpert derived RR results among groups with a lower RR probability for correcting “pre- and post-analytical” errors, which also points to an underlying consistency issue with the test among such groups.

Another significant factor in this study which seems to be related to inconsistent RR results is low/very low bacterial load (Xpert CT>22). A similar observation was made by Ocheretina et al. investigating a series of 22 TB cases.[16] We need to emphasize here that repeat testing in our study by Xpert and LPA were done on processed samples, whereas the first Xpert tests done by districts were on unprocessed sputum samples. Low bacterial load samples such as extra pulmonary samples, pediatric samples, sputum from HIV positive TB patients and smear negative pulmonary TB patients constitute the major subsets of patients targeted for Xpert in resource-limited settings like in India and indeed this is considered as one of the differentiating points favoring Xpert over other tests.[1],[2],[3] WHO has even recommended preference for Xpert in the case of extra-pulmonary samples with exclusivity in the case of certain samples.[15] In such a scenario, it is particularly worrying to find that Xpert is more likely to yield inconsistent RR results among the same type of samples, with which it is stretched to its limits of capability and where a repeat sample is very difficult as in extrapulmonary samples.

As per our data, 24% (20/84) of all RR identified by Xpert were identified by “delayed amplification” with positive analyte result for all probes and as to be expected, there are definite chances of regional variation and further studies with larger sample sizes are strongly suggested.

This laboratory data based study was conducted on RR samples from all patients selected systematically by the TB control program of India and identified by newly installed Xpert machines all over the state of Kerala for the study period. Our laboratory enjoys a monopoly as it is the only laboratory in the state certified for second-line anti-TB drugs susceptibility testing, which caters for the entire population of the state and hence receive samples from all RR-TB cases as soon as they get identified. This was a considerable strength of this study. However, the limited numbers of Xpert derived RR cases and repeat Xpert results are definite shortcomings and as stated above there are chances of regional variations.


  Conclusion Top


It seems like our limited data suggest that Xpert delta CT-based resistance determination, struggles in the absence of negative analyte result for any probe, more so among low bacterial load samples. Till this issue is cleared, it may be fair to recommend that all RR identified by Xpert machines based on the “delayed amplification” criteria with positive analyte results for all probes and perhaps those with a low bacterial load should be advised for a further genotypic test, which is capable of detecting well characterized mutant sequences. This should be followed by a growth based susceptibility testing, coupled with testing for other drugs, the results of which are anyway needed for ideal treatment options, more so if the sample is RR.

Acknowledgments

We appreciate the efforts of the staff members in Intermediate Reference Laboratory, Trivandrum for the completion of this article. We are thankful for the support provided by the State TB Officer/ADHS, Kerala.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Evans CA. GeneXpert – A game-changer for tuberculosis control? PLoS Med 2011;8:e1001064.  Back to cited text no. 1
    
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Small PM, Pai M. Tuberculosis diagnosis – Time for a game change. N Engl J Med 2010;363:1070-1.  Back to cited text no. 2
    
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Lawn SD, Mwaba P, Bates M, Piatek A, Alexander H, Marais BJ, et al. Advances in tuberculosis diagnostics: The Xpert MTB/RIF assay and future prospects for a point-of-care test. Lancet Infect Dis 2013;13:349-61.  Back to cited text no. 3
    
4.
Blakemore R, Story E, Helb D, Kop J, Banada P, Owens MR, et al. Evaluation of the analytical performance of the Xpert MTB/RIF assay. J Clin Microbiol 2010;48:2495-501.  Back to cited text no. 4
    
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Lawn SD, Nicol MP. Xpert® MTB/RIF assay: Development, evaluation and implementation of a new rapid molecular diagnostic for tuberculosis and rifampicin resistance. Future Microbiol 2011;6:1067-82.  Back to cited text no. 5
    
6.
Alonso M, Palacios JJ, Herranz M, Penedo A, Menéndez A, Bouza E, et al. Isolation of Mycobacterium tuberculosis strains with a silent mutation in rpoB leading to potential misassignment of resistance category. J Clin Microbiol 2011;49:2688-90.  Back to cited text no. 6
    
7.
Moure R, Martín R, Alcaide F. Silent mutation in rpoB detected from clinical samples with rifampin-susceptible Mycobacterium tuberculosis. J Clin Microbiol 2011;49:3722.  Back to cited text no. 7
    
8.
Yang B, Koga H, Ohno H, Ogawa K, Fukuda M, Hirakata Y, et al. Relationship between antimycobacterial activities of rifampicin, rifabutin and KRM-1648 and rpoB mutations of Mycobacterium tuberculosis. J Antimicrob Chemother 1998;42:621-8.  Back to cited text no. 8
    
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Jamieson FB, Guthrie JL, Neemuchwala A, Lastovetska O, Melano RG, Mehaffy C. Profiling of rpoB mutations and MICs for rifampin and rifabutin in Mycobacterium tuberculosis. J Clin Microbiol 2014;52:2157-62.  Back to cited text no. 9
    
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Taniguchi H, Aramaki H, Nikaido Y, Mizuguchi Y, Nakamura M, Koga T, et al. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis. FEMS Microbiol Lett 1996;144:103-8.  Back to cited text no. 10
    
11.
Sanker P, Ambika AP, Santhosh VT, Kottuthodi RP, Balakrishnan R, Mrithunjayan SK, et al. Are WHO approved nucleic acid amplification tests causing large-scale “false identification” of rifampicin-resistant tuberculosis? Programmatic experience from South India. Int J Mycobacteriol 2017;6:21-6.  Back to cited text no. 11
[PUBMED]  [Full text]  
12.
Canetti G, Froman S, Grosset J, Hauduroy P, Langerova M, Mahler HT, et al. Mycobacteria: Laboratory methods for testing drug sensitivity and resistance. Bull World Health Organ 1963;29:565-78.  Back to cited text no. 12
    
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Steingart KR, Schiller I, Horne DJ, Pai M, Boehme CC, Dendukuri N. Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev 2014:CD009593. Available from http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD009593.pub3/pdf. [Last accessed on 2017 May 24].  Back to cited text no. 13
    
14.
Foundation for Innovative New Diagnostics (FIND), Performance of Xpert MTB/RIF Version G4 Assay, Version 1.0; Dated 30th November, 2011. Available from: http://www.stoptb.org/wg/gli/assets/documents/map/findg4cartridge.pdf. [Last accessed on 2017 Feb 21].  Back to cited text no. 14
    
15.
WHO. Xpert MTB/RIF assay for the diagnosis of Pulmonary and Extrapulmonary TB in adults and children. Policy update: WHO; 2013. Available from: http://www.who.int/tb/publications/xpert-mtb-rif-assay-diagnosis-policy-update/en/. [Last accessed on 2017 May 24].  Back to cited text no. 15
    
16.
Ocheretina O, Byrt E, Mabou MM, Royal-Mardi G, Merveille YM, Rouzier V, et al. False-positive rifampin resistant results with Xpert MTB/RIF version 4 assay in clinical samples with a low bacterial load. Diagn Microbiol Infect Dis 2016;85:53-5.  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]


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