Role of Xpert MTB/RIF Assay in the Diagnosis of Pulmonary Tuberculosis and Rifampicin Resistance Taher Abdel -Aziz 1, Naglaa Azab 2, Eman AboElabass 3 & I.M El-deen 4 1 Ph.D of Biochemistry, Faculty of Science, Port Said University. 2 Professor of Biochemistry, Faculty of Medecine, Benha University. 3 Bsc of Science Faculty, Port Said University. 4 Professor of Chemistry, Faculty of Science, Port Said University. ABSTRACT Objective: The aim of this study is to evaluate the role of Genexpert (Xpert MTB/RIF) in the detection of the (MTB) complex DNA and mutations associated with rifampicin (RIF) resistance directly from sputum and comparing it with the conventional methods. Subjects and methods: one hundred patients referred for clinical and/ or radiological suspicion of TB. Early morning two consecutive morning sputum samples were collected. One of them was tested directly by Ziehl-Neelsen staining method and Xpert MTB/RIF assay. The other was cultured on LJ media for isolation of TB bacilli. Results: The GeneXpert sensitivity was found to be is 97.4%, specificity 65.2 %. The sensitivity of GeneXpert in evaluation of the rifampicin sensitivity in this study was found to be 96.97% and the specificity found to be 63.63%. Conclusion: GeneXpert is a single test that can detect both M. tuberculosis complex and rifampicin resistance within two hours. GeneXpert is a good negative test but not a good positive. Keywords: TB, GeneXpert, RIF resistance. 1. Introduction Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis (MTB). Tuberculosis generally affects the lungs, but can also affect other parts of the body [1]. Mycobacterium tuberculosis continues to cause significant morbidity and mortality in low and middle income countries. The World Health Organization (WHO) estimates that one-third of the world s population is infected with tuberculosis, with approximately 9 million new tuberculosis cases and 1.4 million deaths worldwide each year (WHO, 2016). The latest value for incidence of tuberculosis (per 100,000 people) in Egypt was 14 at 2016 [2]. Rapid diagnosis of mycobacterial disease is critical, and attempts to shorten the time to detection of such organisms deserve attention. Smear examination is a rapid method for detection of mycobacteria in a clinical specimen, especially sputum but the limitations are low sensitivity and inability to diagnose rifampicin sensitivity pattern [3]. Culture is the gold standard method and drug resistance test (DST) can be done but it is costly and therefore unavailable in most sites. There is also the risk of contamination and prolonged turnaround time due to the slow growth rate of the TB bacilli [4]. In order to overcome conventional methods low sensitivity and diagnostic delays, Xpert MTB/RIF, a fully automated molecular test, has been introduced. It can detect the presence of Mycobacterium tuberculosis (MTB) complex DNA and mutations associated with rifampicin (RIF) resistance directly from sputum in less than 2 hours, and it minimizes staff manipulation and biosafety risk [5]. The aim of this study is to evaluate the role of Genexpert (Xpert MTB/RIF) in the detection of the presence of mycobacterium tuberculosis (MTB) complex DNA and mutations associated with rifampicin (RIF) resistance directly from sputum and comparing it with the conventional methods. 2. Subjects and Methods This study was conducted at Benha Chest hospital and the laboratory of Benha Faculty of Medicine. It was carried out on 100 patients during Page 90
the period from July 2017 to July 2018. They were referred for clinical and/ or radiological suspicion of TB. All the procedures used in this study were approved by the research ethics committee of faculty of Science, Port Said university, Egypt. An informed consent was obtained from all patients included in this study. All patients under study were subjected to: full history taking, clinical examination including general and chest examination, chest X-ray and laboratory investigation including complete Blood Count (CBC), liver function tests, renal function tests and sputum examination for MTB detection. Sputum specimen collection Early morning two consecutive expectorated morning sputum samples were collected on the same day by asking the patient to cough deeply in a dry sterile wide screw capped container. Three successive early morning sputum samples can be collected if the first sample show negative staining results. The minimum acceptable volume of sputum was 2 ml in each container. One of the sputum specimens were tested directly by Ziehl-Neelsen staining method [6], and Xpert MTB/RIF assay [7]. The other sputum specimen were cultured on Lowenstein-Jensen media for isolation of TB bacilli [8]. 3. Statistical analysis All statistical analyses were carried out in STATA/SE version 11.2 for Windows (STATA Corporation, College Station, Texas). Statistical significance was accepted at p value <0.05 (S), while a p value >0.05 was considered non-significant. The Student s t-test (t) was used to test differences between two groups regarding parametric data.the agreement tests were done using kappa statistics. 4. Results The age of all cases ranged from 23 to 75 years old, with age median of 49 years. Of the study patients, 67 were males and 33 were females. Table (1): Direct smear examination results of the 100 patients by Ziehl Neelsen (ZN) stain. ZN Number Percentage Positive 73 73% Negative 27 27% Table (2): Lowenstein-Jensen medium results of the 100 patients. LJ Positive Negative Number 77 23 Percentage 77% 23% Table (3): GeneXpert test results of the 100 patients: GeneXpert Positive Negative Number 83 17 Percentage 83% 17% Table (4): Comparison between GeneXpert (GX) test & Lowenstein Jensen medium (LJ) of the 100 patients. LJ Positive Negative Kappa Positive 75(75%) 8 (8%) GX 0.689 Negative 2 (2%) 15 (15) Table (5): Comparison between Rifampicin sensitivity(rs) according to GeneXpert test & according to LJ medium of the 77 culture positive patients. RS according to LJ Kappa R S RS according to GX R 7(9.1%) 2(2.6%) S 4(5.2%) 64 (83.1%) 0.656 The GeneXpert sensitivity was found to be is 97.4%, specificity 65.2 %. The sensitivity of GeneXpert in evaluation of the rifampicin sensitivity in this study was found to be 96.97% and the specificity found to be 63.63%. 5. Discussion This study was carried on 100 patients, who were referred for clinical and/ or radiological suspicion of TB. Male patients accounted for 67% of the study subjects and 33% were females. The age of all cases ranged from 23 to 75 years old with mean age of 40. 5 + 14.243 years. The age median in this study found to be 49 years. This study is in agreement of the study of Khalil and Butt, who included 93 patients in their study with suspected pulmonary tuberculosis and found that the mean age of the patients was 38.56 ± 19.045 years [9]. On the other hand, the study of Fernandes et al, showed age median obviously less than concluded by this study, as they found the age median of their study to be 35.7 years [10]. Page 91
This study is coinciding with previous extensively documented results which concluded that the global TB epidemic is characterized by significant differences in prevalence between men and women, that rates of TB are much higher among men than women in large areas of the world [11]. Controversly, the study of Khalil and Butt showed that about 36 patients (38.7%) were males while the rest were female patients, which is in disagreement with this study [9]. This disparity could be due to the fact that male subjects were more exposed to risk factors of TB infection. However, some scholars believe that these differences are in part, if not wholly, due to the effects of confounding variables such as differential access to care, which would bias case reporting [11]. In this study, by direct smear examination (using Ziehl Neelsen stain) in the 100 patients, 73% of the patients were Ziehl Neelsen positive and 23% were negative, by Lowenstein-Jensen medium culture, 77 % of patients were, positive and 23% were negative, regarding to GeneXpert test results, 83% of the patients were positive and 17% were negative. Using the Lowenstein-Jensen medium culture as the gold standard for the diagnosis of MTB, The GeneXpert test had superior performance for rapid diagnosis of Mycobacterium tuberculosis over existing AFB smear microscopy, due to the fact that GeneXpert can detect as low as 131 colony forming units per ml in M. tuberculosis spiked sputum but identification by ZN requires at least 10,000 bacilli per ml of sputum [12]. These results are in agreement with the previous studies tested the performance of both methods [13, 14 & 15]. Increasing the number of MTB positive patients in the GeneXpert test by 6% in comparison to the gold standard, LJ medium is comparable with other studies results [13, 14, 15 & 16]. This is could be explained, as the GeneXpert is working by PCR amplification of DNA of live and dead bacilli but LJ works on live bacilli only [17]. Regarding to the agreement test between the mentioned diagnostic tools, Ziehl Neelsen, GeneXpert test & Lowenstein Jensen medium. In this study, there was good agreement between GeneXpert and Lowenstein Jensen medium with kappa value of 0.689, fair agreement between GeneXpert and Ziehl Neelsen with kappa value of 0.311 and finally moderate agreement between Lowenstein Jensen medium and Ziehl Neelsen with kappa value of 0.521 in the diagnosis of pulmonary TB. These results are relatively comparable with the results of Orina et al who reported similar results regarding the agreement between GeneXpert and Ziehl Neelsen with fair agreement and kappa value of 0.318 but rather different results regarding the agreement between Lowenstein Jensen medium and Ziehl Neelsen to be fair also with kappa value of 0.3099 and the agreement between GeneXpert and Lowenstein Jensen medium to be very good with kappa value of 0.988 [18]. The study of Hasan et al is in agreement with this study regarding the agreement between GeneXpert and Lowenstein Jensen medium to be good [19]. In this study, rifampicin sensitivity testing using LJ medium in the 77 MTB culture positive patients resulted in: 85.7% of the patients were sensitive and 14.3% were resistant. While 89.2% of the 83 GeneXpert positive patients were sensitive and 10.8 % of them were resistant according to GeneXpert test. There is significant agreement concerning the rifampicin sensitivity testing according to GeneXpert test and LJ medium in studied subjects. The degree of agreement is considered good agreement, with kappa value of 0.656. Rifampicin resistant cases have been reported from various studies. The Rifampicin resistance prevalence rate of 14.3% found in this study is comparable with 16.7% rate reported in Okonkwo et al [4] and much higher than reported by Khalil and Butt (6.45%) [9], and 5.8% recorded by Oluwaseun et al [20]. Regarding to the agreement between the LJ medium and GeneXpert method in the detection of rifampicin sensitivity, previous studies recorded agreement with the results of this study as Jing et al, who found that the degree of agreement were good as that of this study, with kappa value of 0.746 [21]. In this study, the GeneXpert sensitivity was found to be is 97.4%, specificity 65.2 %. In semi-agreement with this study, a study was done in Abassia chest hospital by Hussain et al suggested that GeneXpert is a powerful tool for PTB diagnosis with good sensitivity (93%) [22]. However, the specificity was higher than revealed in this study (98.3%). The specificity in this study (65.2 %) was relatively low compared to some previous studies, as 8 false positive results were recorded, likely owing to the previously mentioned data that GeneXpert is working by PCR amplification of DNA of live and dead bacilli but LJ works on live bacilli only [17]. Walters et al shows specificity Page 92
comparable to ours (60%) [23]. High rates of sensitivity and specificity have been reported for different clinical specimens (Blakemore et al., 2010; Boehme et al., 2011; Kim et al., 2012). 6. Conclusion GeneXpert is a single test that can detect both M. tuberculosis complex and rifampicin resistance within two hours after starting the test, with minimal hands-on technical time. GeneXpert is a good negative test but not a good positive as it cannot differentiate between live and dead bacilli, so we cannot use it in the patients follow up. 7. References [1] World Health Organization (2015): Global Tuberculosis Report 20th edition. [2] World Health Organization (2017): Global Tuberculosis Report 2017. [3] Laskar O., Akram H., Jannatul F., Mominur R. et al (2017): GeneXpert MTB/RIF Assay for Rapid Identification of Mycobacterium Tuberculosis and Rifampicin Resistance Directly from Sputum Sample. Journal of Enam Medical College; 7 (2):86-86. [4] Okonkwo C., Onwunzo C., Chukwuka P. et al (2017): The Use of the Gene Xpert Mycobacterium tuberculosis/rifampicin (MTB/Rif) Assay in detection of multi drug Resistant Tuberculosis (MDRTB) in Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria. J HIV Retrovirus, 3:1. [5] Piatek S., Cleeff V., Alexander H. et al (2013): GeneXpert for TB diagnosis: planned and purposeful implementation. Global Health: Science and Practice ; 1(1):18 23. [6] Acharya T.(2016): Ziehl-Neelsen technique (AFB Staining): Principle, Procedure and reporting. laboratory diagnosis of Bacterial Disease, Staining techniques in Microbiology microbeonline. [7] Miotto P., Bigoni S., Migliori B. et al (2012): Early tuberculosis treatment monitoring by Xpert (R) MTB/RIF. European Respiratory Journal ; 39( 5)1269 1271. [8] Pfyffer G., Murray R., Baron J.(2007): Mycobacterium: general characteristics, laboratory detection, and staining procedures, p. 543 572In In Murray P. R., Baron E. J., Jorgensen J. H., Landry M. L., Pfaller M. A., editors. (ed.), Manual of clinical microbiology, 9th ed ASM Press, Washington, DC. [9] Khalil F and Butt T. (2015): Diagnostic Yield of Bronchoalveolar Lavage Gene Xpert in Smear Negative and Sputum-Scarce Pulmonary Tuberculosis. Journal of the College of Physicians and Surgeons Pakistan; 25 (2): 115-118. [10] Fernandes P., Yog M., Gaeddert M. et al (2018): Sex and age differences in Mycobacterium tuberculosis infection in Brazil 146,(12)1503-1510. Epidemiology and Infection, [11] Rhines A. (2013): Tuberculosis The role of sex differences in the prevalence and transmission of tuberculosis; 93 (2013) 104-107. [12] Steingart R., Schiller I., Horne J. et al(2014): Xpert MTB/RIF assay for pulmonary tu-berculosis and rifampicin resistance in adults. Cochrane Database of Systematic Reviews ;1(1): 21. CD009593. [13] Bajrami B., Mulliqi G, Kurti A. et al (2018): Assessment of diagnostic accuracy of GeneXpert Mycobacterium tuberculosis/rifampicin in diagnosis of pulmonary tuberculosis in Kosovo Biomedical and Biotechnology Research Journal (BBRJ); 2(3): 191-195. [14] Bajrami R., Mulliqi D., Kurti A. et al (2016): Comparison of GeneXpert MTB/RIF and conventional methods for the diagnosis of tuberculosis in Kosovo. J Infect Dev Ctries; 10(4):418-422. [15] Badal-Faesen S., Firnhaber C., Kendall M. et al (2017): Impact of Larger Sputum Volume on Xpert MTB/RIF Assay Detection of Mycobacterium tuberculosis in Smear-Negative Individuals with Suspected Tuberculosis J Clin Med.; 6(8): 78. [16] Agrawal M., Ashish Bajaj A., Bhatia V. et al (2016): Comparative Study of GeneXpert with ZN Stain and Culture in Samples of Suspected Pulmonary Tuberculosis J Clin Diagn Res; 10(5):09 12. [17] Barnard A., Irusen M., Bruwer W. et al (2015): Koegelenberg The utility of Xpert MTB/RIF performed on bronchial washings obtained in patients with suspected pulmonary tuberculosis in a high prevalence setting. BMC Pulm Med. ;15:103. [18] Orina G., Ong'wen S., Amolo A. and Orindi O. et al (2017): Comparative Study of Smear Microscopy, Gene Xpert and Culture and Sensitivity Assays in Detection of Mycobacterium tuberculosis on Sputum Samples among Tuberculosis Suspected Cases in Nyamira County Referral Hospital, Mycobacterial Diseases ; 7:3. [19] Hasan Z., Shakoor S., Arif F.et al (2017): Evaluation of Xpert MTB/RIF testing for rapid diagnosis of childhood pulmonary tuberculosis in children by Xpert MTB/RIF testing of stool samples in a low resource setting;bmc Research Notes;10:(1) 473. [20] Oluwaseun E., Akaniyi P., Onabanjo O. et al (2013): Primary multi drug resistant tuberculosis among HIV SeroPositive and Sero Negative patients in Abeokuta, Southwest Nigeria. American Journal of Research Communication;1:(2)224-225. [21] Jing H., Lu Z., Deng Y. et al (2017): Evaluation of Xpert MTB/RIF in detection of pulmonary and extrapulmonary tuberculosis cases in China, Int J Clin Exp Pathol ;10(4):4847-4851. [22] Husseiny S., Moussa A., Faten S. et al (2016): Evaluation of GeneXpert MTB/RIF assay for direct diagnosis of pulmonary tuberculosis. PMCID; 37(10): 1076 1081. Page 93
[23] Walters E., Gie P., Hesseling C. et al (2012): Rapid diagnosis of pediatric intrathoracic tuberculosis from stool samples using the Xpert MTB/RIF Assay: A pilot study. Pediatr Infect Dis J;31:13-16. [24] Blakemore R., Story E., Helb D. et al (2010): Evaluation of the analytical performance of the Xpert MTB/RIF assay. Journal of Clinical Microbiology, 48(7): 2495 2501. Page 94