Use of GeneXpert Mycobacterium tuberculosis/rifampicin for rapid detection of rifampicin resistant Mycobacterium tuberculosis strains of clinically suspected multi-drug resistance tuberculosis cases

Introduction

Tuberculosis (TB) is the most common infectious disease worldwide caused by Mycobacterium tuberculosis (MTB).

In the global TB report [2014], WHO reported that in 2013, nine million people developed TB. At the same time, global burden of multidrug-resistant TB (MDR-TB) was estimated to be 480,000 cases leading to estimated 210,000 deaths (1). In Africa, 1.9% of new cases and 9.4% of diagnosed and treated patients are infected by an MDR strain (2). Multi-drug resistance (MDR) TB is defined as TB disease caused by a strain of M. tuberculosis that was resistant to at least isoniazid and rifampicin (RIF) (3). Emerging Multidrug-Resistant Tuberculosis-TB is one of the major concerns of health policy (4). Currently, less than 10% of multi-drug resistant tuberculosis (MDR-TB) cases in the world are detected (5). The rapid detection of M. tuberculosis in infected patients is essential for disease management (6). During the past few years, molecular methods have been developed to identify drug resistance causing gene muta-tions (7,8). One of the latest techniques is GeneXpert MTB/RIF, which can detect mutations in the rpoB gene only; due to close association of RIF resistance and MDR TB, this technique has been used to detect MDR TB cases (9). The technique has been thoroughly evaluated (10) and used in many countries (11). It has a sensitivity and specificity of 90.4% and 98.4%, respectively (12,13).

Culture is the “gold standard” for final determination, but it is time consuming and may take up 2 till 8 weeks (6). Molecular tests dramatically shorten diagnosis time from months to days (MTBDRplus) or even hours (GeneXpert MTB/RIF).The assay can generally be completed in less than 2 hours (14,15).

The objectives of this study were to use cartridge based nucleic acid amplification testing to evaluate patterns of RIF resistance in cases of sputum positive pulmonary TB and compare between phenotypic and genotypic testing of RIF resistance in MTB strains of clinically suspected MDR-TB isolated cases in western Algeria.

Methods

A prospective study was conducted at pneumophtisiology department (B) at Oran hospital (western Algeria) along the period of 2013 to 2014. The material of study came from different hospital centers and different public health sectors of western Algeria. Fifty clinically suspected MDR-TB cases were selected. An absolute confidentiality of the patients’ vital information was maintained for ethical purposes and an ethical approval was obtained from the institution in which the study was carried out.

The following variables were collected through an administered questionnaire during sputum collection: sex, age, treatment history (new or previously treated). Inclusion criteria used for MDR suspect consist of retreatment failure, retreatment cases sputum positive at 4 months, contact of known MDR-TB case, sputum positive retreatment case at diagnosis.

After identifying potential MDR-TB suspect cases, three sputum samples were collected from each patient. One specimen was used for direct microscopic examination by Ziehl-Neelsen method, one specimen was processed with N-acetyl-L-cysteine and sodium hydroxide before solid culture, and MTB/RIF test. The last specimen was used for direct testing with the Xpert MTB/RIF test. Xpert MTB/RIF assay was compared with conventional culture method for detecting TB and with conventional phenotypic drug susceptibility testing for detecting RIF’s resistance. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All authors declare that ‘written informed consent was obtained from the patient (or other approved parties) for publication of this case report and accompanying images.

Phenotypic drug susceptibility testing

Cultures obtained on Lowenstein-Jensen medium were collected and tested for drug susceptibility to RIF, isoniazid, ethambutol and streptomycin. Drug susceptibility testing was performed using the proportional method with Lowenstein-Jensen medium. The critical drug concentrations were 0.2 µg/mL for isoniazid, 40 µg/mL for RIF, 2 µg/mL for ethambutol and 4 µg/mL for streptomycin. The critical proportion of resistant bacillus necessary to define a resistant strain is 1% for the four tested drugs (15).

Genotypic drug susceptibility testing

For each of the samples; unscrew lid of sputum collection container; add Sample Reagent 2:1 (v/v) to the sample, replace the lid, and shake vigorously 10–20 times. Incubate for 15 minutes at room temperature. At one point between 5 and 10 minutes of the incubation again shake the specimen vigorously 10–20 times. Samples should be liquefied with no visible clumps of sputum. Particulate matter may exist that is not part of the sample. At least 2 mL of processed sample was taken with the plastic transfer pipette from the collection container to the single-use, disposable, self-contained GeneXpert cartridge. Then it was subjected to GeneXpert^® MTB/RIF to create a test. Results were noted after 2 hours.

Results

In this study, 50 clinically suspected MDR-TB cases were selected and their sputum samples were tested by phenotypic drug susceptibility methods and genotypic drug susceptibility methods using the test GeneXpert MTB/RIF.

Of total 50 cases included in this study, MTB was detected in all patients (100%) by GeneXpert MTB/RIF. However, RIF’s resistance was detected in only 21 cases (42%) by GeneXpert MTB/RIF. Gender distribution showed that 15 (71.43%) were male and 6 (28.57%) were female among RIF resistant cases (Table 1).The sex ratio was of 2.5. The distribution according to age showed that the majority of patients with RIF resistance belonged to age group of 31–40 years (n=10; 47.62%) followed by 21–30 and 41–50 years with (n=4; 19.04%) for each age group and 51–60 years with (n=3; 14.28%).

Table 1 Rifampicin sensitivity pattern
Full table

Table 1 shows that 15 (42.9%) male out of 35 and 6 (40.0%) female out of 15 were resistant to RIF, while 20 (57.1%) male out of 35 and 9 (60.0%) female out of 15 were sensitive to it.

42.85% of cases were retreatment failure cases, retreatment cases smear positive at 4 months were 23.82%. While 19.05% of cases were retreatment cases smear positive at diagnosis, and 14.28% patient had history of contact with MDR-TB (Table 2).

Table 2 Distribution of Rifampicin resistant cases according to patients’ category
Full table

Comparison of phenotypic and genotypic resistance drug susceptibility showed that all strains harboring mutations in rpoB were phenotypically resistant to RIF and isoniazid. Our results show that all RIF resistant strains detected by GeneXpert MTB/RIF were phenotypically confirmed as MDR strains (Table 3).

Table 3 Prevalence of multi-drug resistant tuberculosis (MDR-TB)
Full table

Twenty one cases were identified as being RIF resistant MTB by the conventional method. On comparing this with Xpert MTB/RIF; we noted a total of twenty one cases that are RIF resistant MTB. Sensitivity, specificity, positive predictive value and negative predictive value of Xpert MTB/RIF to detect RIF resistance in comparison to conventional phenotypic drug susceptibility technique were found equal to the rates of 100%, 100%, 100% and 100%, respectively (Table 4).

Table 4 Performance characteristics of the Xpert MTB/RIF assay compared to drug susceptibility testing for rifampicin (RIF)
Full table

Discussion

In this study our objectives were to use cartridge based nucleic acid amplification testing to evaluate patterns of RIF resistance in cases of sputum positive pulmonary TB and to compare between phenotypic and genotypic testing for resistance to RIF in MTB strains of clinically suspected MDR-TB cases.

Multidrug-resistant tuberculosis (MDR-TB) is defined as TB caused by strains of M. tuberculosis that are resistant to at least isoniazid and RIF (15). Mono-resistance to RIF is rare; however, 90% of RIF resistant isolates also exhibit resistance to isoniazid. Therefore, the detection of RIF resistance may serve as a surrogate marker for MDR M. tuberculosis (16). For RIF resistance detection, Xpert^® MTB/RIF provides accurate results and can allow rapid initiation of MDR-TB treatment (17).

In our study, 21 (42%) were RIF resistant, while 29 (58%) were RIF sensitive. This is similar to that reported by Trivedi (18) and Shah (19) where (37.3%) and (37.47%) were resistant to RIF respectively, but lower to the study of Chowgule who reported a very high incidence of RIF resistance of (66.8%) (20). This level of resistance was superior to the study of Rasaki et al. (21), Olusoji et al. (22), Lawson et al. (23), Ganguly et al. (6), where (7.2%), (8.6%), (19%), (29.87%) isolates were resistance to RIF respectively and Idigbe et al. (24) who reported only 2% of resistance to RIF in Lagos, Nigeria. However, no strain of RIF resistant was reported in the findings of Rasaki et al. (21).

There was male preponderance, 15 (71.43%) as against 6 (28.57%) female; this was in concord with the work of Ganguly et al. (6) where male subjects had prevalence of 85.71% as against 14.29% of females. Similarly, a European study by Faustini et al. (25) observed more drug resistant TB cases among men. This disparity could be due to the fact that male subjects were more exposed to risk factors of TB infection.

In the present study, the distribution according to age showed that the majority of patients with RIF resistance belonged to age group of 31–40 years (n=10; 47.62%) followed by 21–30 and 41–50 years with (n=4; 19.04%) for each age group. This was in concord with the study of Thomas et al. (26). In TRC, Chennai, 70% of the drug resistant patients were male and their mean age was 37. In a another study done by Robert et al. (27) the age and the sex distribution was similar to the study of Ganguly et al. (6) where maximum number of patients with RIF resistance were male and were in the age group of 21–30 years (26.53%) followed by 31–40 years (22.44%).

In our analyzed cohort, 42.85% of cases were retreatment failure cases, Retreatment (Previously CAT II) failure cases were found to be 41.83% among RIF resistant in the study of Ganguly et al. (6). In a study done by Sharma et al. (28) it was found that 34% of Cat- II failures were drug resistant which is similar to our study. A high prevalence of MDR-TB in Cat-II failure is not restricted to India and has been documented in Vietnam (29), Thailand (30) and Rowanda (31). Retreatment cases smear positive at 4 months were 23.82%, this level was superior to the study of Ganguly et al. (6) where retreatment cases smear positive at 4 months found to be 8.16% among RIF resistant cases. 19.05% of cases were retreatment cases smear positive at diagnosis. In studies by Sharma et al. (28) and Ganguly et al. (6) drug resistance was respectively found in 20% and 22.44% of Retreatment cases at diagnosis which is similar to our study. Ganguly et al. (6) found only one resistant case (1.02%) with history of contact with MDR TB. This is consistent with a study done by Singla et al. (32) in which only 0.66% of contacts developed MDR-TB. This was lower than what we found in our study where 14.28% patient presented history of contact with MDR-TB.

According to the World Health Organization (WHO) 650,000 people are infected worldwide and 12 million suffer from TB. In Africa, 1.9% of new cases and 9.4% of diagnosed and treated patients are infected by MDR strain (2). The results of this study showed that all strains harboring mutations in rpoB were phenotypically MDR-TB strains (resistant to RIF and isoniazid). This was comparable to 77.4% reported by Olusoji et al. (22). Few studies had documented the presence of cases infected by MDR strains in Nigeria, with prevalent rates ranging from 4–76.3% (33,34), but was much superior to the results found by Rasaki et al. (21) where forty four (31.4%) were positive and to another rates published in previous studies from India (18,35).

Compared to phenotypic DST, the MTB/RIF test correctly identified 21 of 21 patients (100% sensitive) with RIF-resistant bacteria and 29 of 29 (100% specific) with RIF-sensitive bacteria, this is similar to results showed in study conducted in Uganda where 64 smear-positive culture-positive sputa from patients previously treated for TB were tested and the Xpert MTB/RIF test detected nine of nine (100% sensitivity) cases of RIF resistance. RIF resistance was excluded in 55/55 susceptible cases—100% specificity (14). In a recent multicentre (Peru, Azerbaijan, South Africa and India) evaluation study of 1,730 patients with suspected drug-sensitive or multidrug-resistant pulmonary TB, the MTB/RIF test identified 200 of 205 patient (97.6% sensitive) with RIF-resistant bacteria and 504 of 514 (98.1% specific) with RIF-sensitive bacteria (36). In the study of Darwish et al. (37) the Xpert MTB/RIF revealed five out of the six cases as being RIF resistant MTB by the conventional method to be RIF resistant MTB with sensitivity 83% and specificity with 100%. Steingart et al. (17) emphasized that Xpert can be used as an initial diagnostic test for TB detection and RIF resistance in patients suspected of having TB, MDR-TB or HIV-associated TB.

Conclusions

The high sensitivity and specificity of Xpert MTB/RIF for RIF resistance detection support its use as an initial diagnostic test for RIF resistance. Therefore, implementation of molecular approaches for direct diagnosis of MDR TB, as a part of routine analysis in the laboratories of health care institutions, would be of great benefit in adapting treatment regimens, limiting dissemination of MDR TB strains.

Acknowledgements

The authors would like to thank the members of Pneumophtisiology Department (B) of Oran Hospital.

Footnote

Conflicts of Interest: The authors have no conflict of interest to declare.

Ethical Statement: This study was approved by the institutional ethic review board and informed consent was obtained from all patients.

References

1. Tuberculosis. WHO Global Tuberculosis Report 2014. Available online: http://www.who.int/tb/publications/global_report/gtbr14_executive_summary.pdf
2. WHO. Epidemiological fact sheets on HIV and AIDS, 2008 update: Core data on epidemiology and response. Djibouti. Who globalatlas.
3. WHO (2000). Anti-Tuberculosis drug resistance in the world: the WHO/IUATLD Global Project on Anti-tuberculosis drug Resistance Surveillance (Fourth global report) (WHO/HTM/TB/2008.394).
4. WHO (2011). Global Tuberculosis Control.
5. WHO. Multidrug and extensively drug-resistant TB (M/XDR-TB): 2010 Global Report on Surveillance and Response; 2010. Available online: http://whqlibdoc.who.int/publications/2010/9789241599191_eng.pdf
6. Ganguly J, Ray S, Nandi S, et al. A study to evaluate pattern of rifampicin resistance in cases of sputum positive pulmonary tuberculosis. J of Evolution of Med and Dent Sci 2015;4:4762-8. [Crossref]
7. Neonakis IK, Gitti Z, Krambovitis E, et al. Molecular diagnostic tools in mycobacteriology. J Microbiol Methods 2008;75:1-11. [Crossref] [PubMed]
8. García de Viedma D. Rapid detection of resistance in Mycobacterium tuberculosis: a review discussing molecular approaches. Clin Microbiol Infect 2003;9:349-59. [Crossref] [PubMed]
9. World Health Organization. Rapid implementation of the Xpert MTB/RIF diagnostic test (technical and operational ‘how-to’ practical con-siderations): WHO Library Cataloguing-in-Publication Data. Geneva, Switzerland: World Health Organization; 2011. Available online: , accessed August 23, 2013.http://whqlibdoc.who.int/publications/2011/9789241501569_eng.pdf
10. Marlowe EM, Novak-Weekley SM, Cumpio J, et al. Evaluation of the Cepheid Xpert MTB/RIF assay for direct detection of Mycobacterium tuberculosis complex in respiratory specimens. J Clin Microbiol 2011;49:1621-3. [Crossref] [PubMed]
11. Barnard M, Warren R, Gey Van Pittius N, et al. Genotype MTBDRsl line probe assay shortens time to diagnosis of extensively drug-resistant tuberculosis in a high-throughput diagnostic laboratory. Am J Respir Crit Care Med 2012;186:1298-305. [Crossref] [PubMed]
12. Chang K, Lu W, Wang J, et al. Rapid and effective diagnosis of tuberculosis and rifampicin resistance with Xpert MTB/RIF assay: a meta-analysis. J Infect 2012;64:580-8. [Crossref] [PubMed]
13. Zakham F, Chaoui I, Echchaoui AH, et al. Direct sequencing for rapid detection of multidrug resistant Mycobacterium tuberculosis strains in Morocco. Infect Drug Resist 2013;6:207-13. [PubMed]
14. Helb D, Jones M, Story E, et al. Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. J Clin Microbiol 2010;48:229-37. [Crossref] [PubMed]
15. David H, Lévy-Frébault V, Thorel MF. Méthodes de Laboratoire pour Mycobactériologie Clinique. Institute Pasteur, Paris, 1989:85.
16. Ioannidis P, Papaventsis D, Karabela S, et al. Cepheid GeneXpert MTB/RIF assay for Mycobacterium tuberculosis detection and rifampin resistance identification in patients with substantial clinical indications of tuberculosis and smear-negative microscopy results. J Clin Microbiol 2011;49:3068-70. [Crossref] [PubMed]
17. Steingart KR, Schiller I, Horne DJ, et al. Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database of Systematic Reviews 2014;1:CD009593. [PubMed]
18. Trivedi SS, Desai SG. Primary antituberculosis drug resistance and acquired rifampicin resistance in Gujarat, India. Tubercle 1988;69:37-42. [Crossref] [PubMed]
19. Shah AR, Agarwal SK, Shah KV. Study of drug resistance in previously treated tuberculosis patients in Gujarat, India. Int J Tuberc Lung Dis 2002;6:1098-101. [PubMed]
20. Chowgule RV, Deodhar L. Pattern of secondary acquired drug resistance to antituberculosis drug in Mumbai, India--1991-1995. Indian J Chest Dis Allied Sci 1998;40:23-31. [PubMed]
21. Rasaki SO. Rifampicin Resistant Tuberculosis in a Secondary Health Institution in Nigeria, West Africa. J Infect Dis Ther 2014;2:139. [Crossref]
22. Olusoji D, Eitayeb O, Olanrewaju O, et al. Global Advanced Research. J Microbiol 2013;2:022-025.
23. Lawson L, Habib AG, Okobi MI, et al. Pilot study on multidrug resistant tuberculosis in Nigeria. Ann Afr Med 2010;9:184-7. [Crossref] [PubMed]
24. Idigbe O, Sofola T, Akinosho R, et al. Initial drug resistance tuberculosis amongst HIV seropositive and seronegative prison inmates in Lagos, Nigeria. Int Conf AIDS 1998;12:137.
25. Faustini A, Hall AJ, Perucci CA. Risk factors for multidrug resistant tuberculosis in Europe: a systematic review. Thorax 2006;61:158-63. [Crossref] [PubMed]
26. Thomas A, Ramachandran R, Rehaman F, et al. Management of multi drug resistance tuberculosis in the field: Tuberculosis Research Centre experience. Indian J Tuberc 2007;54:117-24. [PubMed]
27. Robert J, Trystram D, Truffot-Pernot C, et al. Multidrug-resistant tuberculosis: eight years of surveillance in France. Eur Respir J 2003;22:833-7. [Crossref] [PubMed]
28. Sharma SK, Kumar S, Saha PK, et al. Prevalence of multidrug-resistant tuberculosis among category II pulmonary tuberculosis patients. Indian J Med Res 2011;133:312-5. [PubMed]
29. Quy HT, Lan NT, Borgdorff MW, et al. Drug resistance among failure and relapse cases of tuberculosis: is the standard re-treatment regimen adequate? Int J Tuberc Lung Dis 2003;7:631-6. [PubMed]
30. Yoshiyama T, Yanai H, Rhiengtong D, et al. Development of acquired drug resistance in recurrent tuberculosis patients with various previous treatment outcomes. Int J Tuberc Lung Dis 2004;8:31-8. [PubMed]
31. Rigouts L, Portaels F. DNA fingerprints of Mycobacterium tuberculosis do not change during the development of resistance to various antituberculous drugs. Tuber Lung Dis 1994;75:160. [Crossref] [PubMed]
32. Singla N, Singla R, Jain G, et al. Tuberculosis among household contacts of multidrug-resistant tuberculosis patients in Delhi, India. Int J Tuberc Lung Dis 2011;15:1326-30. [Crossref] [PubMed]
33. Dosumu EA, Osagie K, Shuaib A, et al. Multidrug resistant tuberculosis at the national hospital, Abuja Nigeria. Afr J Respir Med 2008;4:22-3.
34. Daniel O, Osman E. Prevalence and risk factors associated with drug resistant TB in South West, Nigeria. Asian Pac J Trop Med 2008;4:148-51. [Crossref] [PubMed]
35. Jain NK, Chopra KK, Prasad G. Initial and acquired isoniazid and rifampicin resistance to Mycobacterium tuberculosis and its implication for treatment. Indian J Tuberc 1992;39:121-4.
36. Boehme CC, Nabeta P, Hillemann D, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010;363:1005-15. [Crossref] [PubMed]
37. Darwish M, Abd El Wadood M. ALnagdi H. Diagnostic assessment of Xpert MTB/RIF in a sample of Mycobacterium tuberculosis Egyptian patients. Afr J Microbiol Res 2013;7:5107-13.