|Year : 2019 | Volume
| Issue : 3 | Page : 284-289
Detection of drug-resistant mycobacterium tuberculosis using Geno Type MTBDRplus assay in smear-positive cases
Aya M Abdel Dayem1, Samar H Sharkawy1, Rehab M Mohammed Fathy1, Omayma M Hassanin2, Asmaa Ali3
1 Department of Chest Diseases, Ain Shams University, Cairo, Egypt
2 Department of Cytogenetic & Molecular Biology, Ain Shams University, Cairo, Egypt
3 Department of Chest Directorates, Ministry of Health and Population, EL-Abassia Chest Hospital, Cairo, Egypt
|Date of Submission||29-Jan-2019|
|Date of Acceptance||07-May-2019|
|Date of Web Publication||4-Sep-2019|
MD, PhD Rehab M Mohammed Fathy
54 Abdel Rahman Elrafaey Street, El Hegaz Square, Heliopolis, Cairo, 11786
Source of Support: None, Conflict of Interest: None
Background Multidrug-resistance tuberculosis (MDR-TB) has become an annoying health problem with supreme concern in the developing countries. Several methods of detection of MDR-TB have developed over recent years.
Objective To investigate the diagnostic accuracy of GenoType MTBDRplus assay in detection of rifampicin (RIF)- and isoniazid (INH)-resistant isolates from sputum specimens of patients with active TB.
Patients and methods This is a cross-sectional study. Sputum samples from 50 cases with active TB were collected, and the drug sensitivity test was evaluated by two methods: the standard conventional culture method and the GenoType MTBDRplus assay as a newly developed genotypic (molecular) method.
Results The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of GenoType MTBDRplus assay in detection of RIF resistance were 100, 97.5, 90, 100, and 98%, respectively versus 100, 94.4, 87.5, 100, and 96%, respectively, for detection of INH resistance. The prevalence of MDR-TB by genotypic method was 12 versus 8% by conventional culture. On the contrary, drug resistance to either RIF or INH was detected in 28% of cases by the molecular method versus 30% of cases by using conventional culture.
Conclusion The diagnostic yield of GenoType MTBDRplus assay for detection of RIF and INH resistance was satisfactory enough to support its alternative use for rapid identification of MDR-TB isolates, facilitating early and proper anti-TB drug regimen.
Keywords: GenoType MTBDRplus assay, multidrug-resistant tuberculosis, rapid detection, mutation
|How to cite this article:|
Abdel Dayem AM, Sharkawy SH, Mohammed Fathy RM, Hassanin OM, Ali A. Detection of drug-resistant mycobacterium tuberculosis using Geno Type MTBDRplus assay in smear-positive cases. Egypt J Chest Dis Tuberc 2019;68:284-9
|How to cite this URL:|
Abdel Dayem AM, Sharkawy SH, Mohammed Fathy RM, Hassanin OM, Ali A. Detection of drug-resistant mycobacterium tuberculosis using Geno Type MTBDRplus assay in smear-positive cases. Egypt J Chest Dis Tuberc [serial online] 2019 [cited 2019 Oct 15];68:284-9. Available from: http://www.ejcdt.eg.net/text.asp?2019/68/3/284/266031
| Introduction|| |
Tuberculosis (TB) is considered a challenging global health problem especially in developing countries where the majority of TB morbidity and mortality cases occur . Greater attention is being paid to the increased emergence of multidrug-resistance tuberculosis (MDR-TB), defined as resistance to isoniazid (INH) and rifampicin (RIF), two of the first-line drugs in treatment regimens .
The WHO global TB report in 2018 stated that 3.5% of new TB cases and up to 18% of previously treated cases have MDR-TB , hindering efforts to control and manage this problem and threatening WHO’s target to eliminate TB by the year 2050 .
In Egypt, two recent studies researched the prevalence of MDR-TB among Egyptian population: the first was conducted in Alexandria as a representative part of Egypt between July 2008 and December 2012, reporting a 4.3% overall total prevalence of MDR-TB, and the second was a multicenter retrospective study, which included all patients with MDR-TB, admitted to El-Maamora Chest Hospital, Alexandria Governorate, El-Abassia Chest Hospital, Cairo Governorate, and El-Mansoura Chest Hospital, Dakahlia Governorate, from January 2006 to August 2015, concluding that acquired resistance was 96.4% and primary resistance was 3.6% ,. Moreover, the second national survey on drug resistance reported by the National Tuberculosis Control Program and conducted between 2010 and 2012 revealed an estimated MDR-TB prevalence of 3.4% among new cases compared with 32.4% among previously treated cases .
Diagnosis of active TB depends on detection of acid-fast bacilli (AFB)  followed by positive identification of Mycobacterium tuberculosis strains on solid or liquid culture (phenotypic), which still remains to be the “gold standard” reference for TB diagnosis as well as testing for drug sensitivity . However, this method takes 6–8 weeks, hence mandating the need for new methods for early detection of MDR-TB isolates to initiate proper treatment regimens without time delay .
Resistance to anti-TB drugs found in M. tuberculosis is mainly owing to presence of chromosomal mutations in genes encoding drug targets. MDR emerges owing to sequential accumulation of mutations in target genes .
Recently, several genotypic methods were developed for detection of mutations in rpo B and kat G genes causing resistance to INH and RIF , for examples, Cepheid Xpert MTB/RIF kit and the line probe assays . The aim of this study was to investigate the diagnostic accuracy of GenoType MTBDRplus assay in detecting MDR-TB strains from sputum of smear-positive TB cases.
| Patients and methods|| |
Study design and patients
This is a cross-sectional study, where 50 cases, diagnosed as positive for MTB by direct smear, were enrolled.
Exclusion criteria: patients who were smear negative for AFB even with clinical and radiological finding suggestive of TB infection were excluded.
A written consent before participation was provided by all participants, and the study protocol was given approval by the Institutional Research and Medical Ethics Committee of Ain Shams University.
All patients were subjected to the following: full history taking (including age, sex, smoking status, addiction habits, comorbidities especially diabetes mellitus and liver and renal disease, and contact with cases diagnosed with MDR-TB), clinical examination, chest radiography, and routine laboratory investigations, including liver function tests, kidney function tests, complete blood count and viral serology for hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV.
Microbiology assessment of sputum, including Ziehl–Neelsen staining and direct microscopy for AFB. Positive sputum specimens for AFB were subjected to the following:
- Sputum culture on Lowenstein–Jensen media (as gold standard test), where the culture time ranged from 6 to 8 weeks.
- Drug sensitivity test (DST) for RIF and INH by using the proportion method .
- Mutation detection including the following:
- Genomic DNA isolation of the MTB from sputum samples using Qiagen QIA amp DNA mini kit (Qiagen, Valencia, CA, USA) following manufacturer’s instructions.
- Concentration and purity of the extracted DNA was measured by Nano Drop ND-1000.
- Genotype analysis by real-time (RT) PCR strip technology using GenoType MTBDRplus assay (Hain Lifescience, Nehren, Germany) according to manufacturer’s instructions. RT-PCR had three steps: multiplex PCR, amplification, and reverse hybridization. The assay screens for the absence and/or presence of wild-type (WT) and/or mutant (MUT) DNA sequences within specific regions of three genes: the rpoB gene for RIF resistance, the katG gene for high-level INH resistance, and the inhA gene for low-level INH resistance. Each strip contains 27 reaction zones: six controls, including conjugate control (CC), amplification control (AC), M. tuberculosis complex, rpoB locus control, katG locus control, and inhA locus control, and the rest are 21 reaction zones for WT and MUT reaction zones, including eight rpoB WT, four MUT probes, one katG WT, two MUT probes, two inhA WT, and four MUT probes.
- Interpretation of results was as follows: the presence of CC bands indicates the efficiency of the conjugate and substrate. The presence of AC bands indicates the efficiency of DNA extraction and PCR procedures. The presence of the M. tuberculosis complex band indicates that the tested bacterium belongs to the M. tuberculosis complex. The three respective locus control bands (rpoB, katG, and inhA) indicate the presence of the specific gene region. The absence of the WT band is usually accompanied by the presence of MUT, which indicates resistance, and the presence of all WT bands without the MUT band indicates susceptible isolate. In rare cases, lack of WT band(s) without a corresponding MUT band could be observed owing to uncommon mutations in the probe region, and the presence of both WT and MUT bands in the same strip might be an indication for the presence of heteroresistance or mixed infection.
Results can be obtained within 24 h.
Data were managed using the Statistical Package for the Social Sciences (SPSS, SPSS Inc., Chicago, IL, USA) for Windows program, version 20.0. For all statistical analyses, P values less than 0.05 was considered statistically significant.
Descriptive statistics were represented as mean±SD for quantitative data and number (percentage) for qualitative data. The sensitivity, specificity, and positive and negative predictive values (PPV and NPV) of GenoType MTBDRplus assay in the diagnosis of MDR-TB against RIF, INH, or both were calculated.
Analytical statistics were performed between two groups using independent t test to compare between the two groups with normal continuous data distribution, Mann–Whitney test to compare between two groups with non-normal continuous data distribution, and χ2 test or Fisher’s exact test to compare between two or many categorical groups.
| Results|| |
The characteristics of the 50 enrolled patients are shown in ([Table 1]). Patients are grouped according to the susceptibility results of the GenoType MTBDRplus assay into group A (included 31 patients with susceptible TB isolates) and group B (included 19 patients with DR-TB isolates). The mean age was 37.54±11.06 years, and 70% (35/50) were males, with statistically significant association of male sex with group B (DR-TB) (P=0.01); however, there was no statistically significant association of age, smoking, addiction, or comorbidity with group B patients (DR-TB) (P>0.05)
From the 50 patients with TB, 22% (11/50) gave history of contact to MDR-TB cases, with statistically significant effect of positive contact history on DR-TB infection (P=0.008) ([Table 1]).
Overall, 66% (33/50) of the patients presented with unilateral lesion on chest radiography and 30% (15/50) of them had bilateral lesions, and only two (4%) cases had lesion-free chest radiographs, with no significant association of the radiographic pattern with DR-TB (P=0.7, 0.8, and 0.5, respectively) ([Table 1]).
The mean hemoglobin level was 9.30±2.13 g/dl, but it was significantly lower in patients with DR-TB (group B) (8.3±1.48 g/dl) (P=0.009) ([Table 1]).
Considering the virology profile of patients, HCV was positive in 14% (7/50) of patients, followed by HIV with 10% (5/50), and the least was HBV, with only two patients, comprising 4%; moreover, coinfection with HIV, HCV, and HBV had no statistically significant effect on DR-TB infection (P=0.3, 0.4, and 0.5, respectively) ([Table 1]).
The prevalence of MDR-TB was 12% (6/50) via genotypic (molecular) method versus 8% (4/50) by conventional culture method. Drug resistance to either RIF or INH was detected in 28% (14/50) of patients by molecular method versus 30% (15/50) by conventional culture. RIF resistance was detected in 8% (4/50) of patients by molecular method versus 10% (5/50) of patients by conventional culture, and also INH resistance was seen in 20% (20/50) of patients by both molecular and conventional methods ([Table 2]).
|Table 2 Drug susceptibility profile of the genotypic method (using GenoType MTBDRplus assay) versus conventional culture method|
Click here to view
The sensitivity, specificity, PPV, NPV, and accuracy of the genotypic (molecular) method in detection of RIF resistance were 100, 97.5, 90, 100, and 98%, respectively, versus 100, 94.4, 87.5, 100, and 96%, respectively, in detecting resistance to INH ([Table 3]).
|Table 3 Diagnostic accuracy of genotypic /molecular method in the detection of resistance of Mycobacterium tuberculosis strains to rifampicin and isoniazid|
Click here to view
| Discussion|| |
Conventional sputum culture and DST has long been essential to diagnose TB drug resistance . Unfortunately, this method is time consuming as it requires 6–8 weeks, with many problems encountered during standardization of tests, for example, the stability of the drugs in different culture media . Using this slow diagnostic procedure in diagnosis of TB resistance led to subsequent delay in initiating proper treatment regimens, giving a chance for development of MDR-TB strains; hence, developing rapid diagnostic assays would allow the control and enhance the outcome against this serious problem. This concept gave way to the development of newer molecular techniques for drug susceptibility testing for MTB. These techniques have gained strong research interest worldwide. The current work aims to investigate the diagnostic accuracy of the GenoType MTBDRplus assay (a commercialized molecular method) in detecting MDR-TB from smear-positive cases in comparison with the standard conventional culture method (phenotypic). We chose the GenoType MTBDRplus assay because the WHO has recently recommended it as a rapid screening test for patients who are suspected to be at risk of MDR-TB  and is now favored in different centers because of its shorter turnaround time and also the ability to use directly on clinical samples which is considered another key advantage, because it saves the time lost awaiting for the growth of colonies using the conventional culture method. In addition, MTBDRplus assay differs from INNO-LiPA and GeneXpert (other available rapid molecular tests) in its ability to detect both RIF and INH resistance at the same time. This addition of testing for mutations that cause INH resistance is very much recommended and appreciable because although some researchers regard resistance to RIF as a surrogate for MDR to a certain extent, there are still some TB strains that are resistant to RIF only, and therefore testing for both types of resistance separately and simultaneously at the same time would render more accurate results .
The current study is the first Egyptian study to use the GenoType MTBDRplus assay for early detection of DR-TB directly from patients’ sputum, and it was conducted on sputum specimens from 50 patients who were smear positive for M. tuberculosis. Results showed that overall prevalence of MDR-TB was 12% by molecular (genotypic) assay versus 8% by conventional (phenotypic) culture. On the contrary, drug resistance pattern was found in 28% of patients by molecular methods versus 30% by the conventional culture. RIF resistance was detected in 8% of patients by molecular method versus 10% by culture, whereas INH resistance was found in 20% of patients by both molecular and conventional methods. Susceptible strains were detected in 60% by the GenoType MTBDRplus assay versus 62% by conventional culture ([Table 2]). On comparing these results with those of Sharma et al.  who also studied the utility of Genotype MTBDRplus assay in detecting RIF and INH-resistant TB in Nepal, their results showed similar RIF resistance (11.1%), lower INH resistance (7.4%), and higher MDR (51.8%). Discordance of INH-resistance results between our study and theirs may be owing to the presence of genetic variations and mutations in other gene regions which are not targeted like ahpC gene encoding alkyl hydroperoxide reductase in INH-resistant strains .
The sensitivity, specificity, PPV, NPV, and accuracy of the molecular method in detection of RIF resistance were 100, 97.5, 90, 100, and 98%, respectively, whereas in detecting INH resistance, results were 100, 94.4, 87.5, 100, and 96%, respectively ([Table 3]). Our results are comparable to those of Khadka et al.  who evaluated the same assay and reported 93.3% sensitivity, 100% specificity, 100% PPV, and 97.4% NPV for RIF resistance. For INH resistance, the sensitivity, specificity, PPV, and NPV showed 93.3, 100, 100, and 97.4%, respectively. On the contrary, Omer et al.  found that the sensitivity and specificity of the GenoType MTBDRplus assay for the detection of RIF-resistant MTB isolates were 80.0 and 99.6%, respectively, versus 82.7 and 99.6%, respectively, for the detection of INH resistance.
Finally, a recent Chinese meta-analysis, including 40 studies, was conducted by Bai et al.  to comprehensively evaluate the overall diagnostic accuracy of the GenoType MTBDRplus assay in rapid detection of resistance of RIF, INH, and MDR, and concluded that pooled sensitivity and specificity for detection of resistance to RIF, INH, and MDR were 96 and 98%, respectively; 91 and 99%, respectively; and 91 and 99%, respectively. The pooled sensitivity was also found to be more variable and lower than specificity. They attributed this to the geographic and/or genetic variations in the distribution of drug-resistant strains of MTB and also to the fact that 5% of RIF-resistant MTB strains and 10–25% of low-level INH-resistant strains have no known resistance mutations.
The GenoType MTBDRplus test showed good accuracy in detecting resistance to RIF, INH as well as MDR, yet some important limitations remain to be addressed: first, the financial aspect especially in countries with limited resources, so further studies are needed to evaluate the cost-effectiveness of implementing it on a large scale as well as to study its effect on clinical outcomes. Second, there is still a great challenge in identifying MTB in smear-negative patients, especially in HIV coinfection. Third, there is a growing need for similar tests for extensively DR-TB. Finally, differences in geographical regions should be taken in consideration as well as different patient groups (HIV coinfection and pediatric).
| Conclusion|| |
The use of the GenoType MTBDRplus assay in early detection of MDR-TB directly from the sputum smear showed satisfactory diagnostic yield which may support its alternative use to conventional DST in daily clinical practice which would in turn enhance the proper MDR antituberculous drug regimen, increase the cure rate, decrease rate of transmission, avoid unnecessary use of medication, and improve overall clinical outcome.
Financial support and sponsorship
Conflicts of interest
There are no of conflicts of interest.
| References|| |
World Health Organization (WHO). Global tuberculosis report.The burden of disease caused by TB. Geneva: WHO; 2012:8–28.
Ahmad S, Mokaddas E. Recent advances in the diagnosis and treatment of multidrug-resistant tuberculosis. Respir Med 2009; 103:177–179.
World Health Organization (WHO). Global tuberculosis report. Geneva: WHO; 2018; 1–174.
Mohammad OI, Okab AA, Zaki ME. Situation of multidrug-resistant pulmonary tuberculosis in Alexandria governorate fromJuly 2008 to December 2012. Egypt J Bronchol 2016; 10:64–68.
Ibrahim E, Ibrahim A, Aly M, Messery A. Pattern of prevalence, risk factors and treatment outcomes among Egyptian patients with multidrug-resistant tuberculosis. Egypt J Chest Dis Tuberc 2017; 66:405–11.
National Tuberculosis Control Program. Tuberculosis control guidelines. Egypt: 2017; p. 13.
Mitchison DA. The diagnosis and therapy of tuberculosis during the past 100 years. Am J Respir Crit Care Med 2005; 171:699–706.
Sali M, De Maio F, Caccuri F, Campilongo F, Sanguinetti M, Fiorentini S et al.
Multicenter evaluation of the Anyplex Plus MTB/NTM MDR-TB assay for rapid detection of Mycobacterium tuberculosis complex and multidrug-resistant isolates in pulmonary and extrapulmonary specimens. J Clin Microbiol 2016; 54:59–63.
Mani V, Wang S, Inci F, De Libero G, Singhal A, Demirci U. Emerging technologies for monitoring drug-resistant tuberculosis at the point-of-care. Adv Drug Deliv Rev 2014; 78:105–117.
Boehme CC, Nabeta P, Hillemann Nicol MP, Shenai S, Krapp F, Jenny A et al.
Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010; 363:1005–1115.
World Health Organization (WHO). Molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis (MDR-TB). Policy statement. Geneva: WHO; 2008; 2–9.
Heifets L. Conventional methods for antimicrobial susceptibility testing of Mycobacterium tuberculosis
. In Bastian I, Portaels F, eds. Multidrug-resistant tuberculosis. 3rd edn. The Netherlands: Kluwer Academic Publisher; 2000; 133–143
Verma RK, Jain A. Antibodies to mycobacterial antigens for diagnosis of tuberculosis. FEMS Immunol Med Microbiol 2007; 51:453–461.
Victor TC, Warren R, Butt JL, Jordaan AM, Felix JV, Venter A et al.
Genome and MIC stability in Mycobacterium tuberculosis and indications for continuation of use of isoniazid in multidrug-resistant tuberculosis. J Med Microbiol 1997; 46:847–57.
Smith SE, Kurbatova EV, Cavanaugh JS, Cegielski JP. Global isoniazid resistance patterns in rifampin resistant and rifampin-susceptible tuberculosis. Int J Tuberc Lung Dis 2012; 16:203–205.
Sharma BK, Bhandari S, Maharjan B, Shrestha B, Banjara MR. Rapid detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis
using Genotype MTBDR plus assay in Nepal. International Scholarly Research Notices. 2014p. 6. Available at: http://dx.doi.org/10.1155/2014/648294
[Accessed 6 Dec 2018].
Ma X, Wang H, Deng Y, Liu Z, Xu Y, Pan X et al.
RPOB gene mutations and molecular characterization of rifampicin-resistant Mycobacterium tuberculosis isolates from Shandong Province, China. J Clin Microbiol 2006; 44:3409–3412.
Khadka JB, Maharjan B, Ghimire P. Early diagnosis of MDR-TB cases directly on sputum specimens by rapid molecular method. Int J Microbiol Res Rev 2013; 1: 92–96.
Omer ZB, Mekonnen Y, Worku A, Zewde A, Medhin G, Mohammed T et al.
Evaluation of the GenoType MTBDR plus assay for detection of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis
isolates in central. Int J Mycobacteriol 2016; 5:475–481. [Full text]
Bai Y, Wang Y, Shao C, Hao Y, Jin Y. GenoType MTBDR plus assay for rapid detection of multidrug resistance in Mycobacterium tuberculosis: a meta-analysis. PLoS One 2016; 11:1–20.
[Table 1], [Table 2], [Table 3]