|Year : 2017 | Volume
| Issue : 4 | Page : 568-574
Evaluation of genotype MTBDRplus line probe assay in detection of rifampicin and isoniazid resistance in comparison to solid culture drug susceptibility testing in a tertiary care centre of western Uttar Pradesh
Shariq Ahmed1, Indu Shukla1, Nazish Fatima1, Sumit K Varshney1, Mohammad Shameem2
1 Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
2 Department of TB and Respiratory Diseases, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
|Date of Web Publication||1-Feb-2018|
Dr. Shariq Ahmed
Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh - 202 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Isoniazid (INH) and rifampicin (Rif) are the key first-line antituberculosis drugs, and resistance to these drugs i.e., multi-drug-resistant tuberculosis (MDR-TB), is likely to result in treatment failure and poor clinical outcomes. India has the highest burden of TB and MDR-TB in the world, disproportionately high even for India's population. The GenoType® MTBDRplus molecular method allows rapid detection of Rif and INH resistance. Aim: The present study was done to compare the performance of line probe assay test (GenoType® MTBDRplus) with solid culture method for an early diagnosis of MDR-TB. Methods: Totally 1503 sputum samples of MDR-TB suspects were subjected to fluorescent microscopy. Decontamination was done by N-acetyl-L-cysteine and sodium hydroxide method. Fluorescent microscopy-positive samples were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay. Sixty-two random samples were compared with phenotypic drug susceptibility testing (DST) (1% proportion method) using solid culture method by Lowenstein–Jensen media. Results: The sensitivity, specificity, positive predictive value and negative predictive value for detection of resistance to Rif were 94.74%, 95.35%, 90% and 97.62% and to INH were 92.00%, 91.89%, 88.46% and 94.44%, respectively, in comparison with the phenotypic DST. Conclusion: GenoType® MTBDRplus has good sensitivity and specificity in detecting MDR-TB cases with a significantly lesser turnaround time as compared to conventional DST method and simultaneous detection of Rif and INH resistance. This technique saves several weeks of time required for culture and DST.
Keywords: Drug resistance, GenoType® MTBDRplus assay, line probe assay, rapid diagnosis of multidrug-resistant tuberculosis, tuberculosis
|How to cite this article:|
Ahmed S, Shukla I, Fatima N, Varshney SK, Shameem M. Evaluation of genotype MTBDRplus line probe assay in detection of rifampicin and isoniazid resistance in comparison to solid culture drug susceptibility testing in a tertiary care centre of western Uttar Pradesh. Indian J Med Microbiol 2017;35:568-74
|How to cite this URL:|
Ahmed S, Shukla I, Fatima N, Varshney SK, Shameem M. Evaluation of genotype MTBDRplus line probe assay in detection of rifampicin and isoniazid resistance in comparison to solid culture drug susceptibility testing in a tertiary care centre of western Uttar Pradesh. Indian J Med Microbiol [serial online] 2017 [cited 2020 Jan 25];35:568-74. Available from: http://www.ijmm.org/text.asp?2017/35/4/568/224435
| ~ Introduction|| |
Tuberculosis (TB) remains a major global health problem. In 2012, an estimated 86 million people developed TB and 1.3 million died from the disease. With a population of around 1.24 billion, India is the largest country in the South East Asian region. It ranks first among the high TB burden countries which contributed 26% of the estimated global incident TB cases in 2012 and also among the 27 multidrug-resistant TB (MDR-TB) high-burden countries worldwide, contributing to 21% of all MDR-TB cases estimated among notified cases. MDR-TB is caused by strains of Mycobacterium tuberculosis (MTB) that are resistant to at least rifampicin (Rif) and isoniazid (INH), the two key drugs in the treatment of the disease. Since 2006, the presence of even more resistant strains of MTB labelled as extensively drug resistant (XDR)-TB has been recognised.,,
The global threat of MDR-TB to control TB underscores the importance of prompt and rapid identification of such resistant MTB strains. INH and Rif are the key first-line anti-TB drugs, and resistance to these drugs i.e., MDR-TB, is likely to result in treatment failure and poor clinical outcomes., India has the highest burden of TB and MDR-TB in the world, disproportionately high even for India's population.
As in vitro confirmation of resistance to INH and Rif is essential for the diagnosis of MDR-TB, a major reason for the delay in starting MDR-TB treatment is the prolonged time taken by the laboratory to make the diagnosis of drug resistance.,,
The mean time to detect drug resistance on egg-based Lowenstein–Jenson (L–J) media is around 3–4 months. Even using the more modern broth-based liquid culture systems, culture and drug susceptibility test (DST) results from sputum specimens still take several weeks.
Liquid media-based tests, such as the BACTEC ®, MB/BacT ®, ESP ® and MGIT ® systems, are more rapid, but also more costly and require sophisticated laboratories and trained personnel. However, newly developed molecular-based methods have advantages over conventional phenotypic methods in terms of both accuracy and turnaround time.
The line probe assay (LPA) (MTBDRplus) detects mutations associated with the rpoB gene for Rif resistance, katG genes for high-level INH resistance and the inhA regulatory region gene for low-level INH resistance. In June 2008, the World Health Organisation (WHO) recommended the use of molecular LPA for the diagnosis of MDR-TB.
The present study was done to compare the performance of LPA test (GenoType ® MTBDRplus assay) with solid culture method among MDR-TB suspects for an early diagnosis of MDR-TB at a tertiary care centre in western Uttar Pradesh.
| ~ Methods|| |
The study was approved by the Institutional Ethics and Research Advisory Committee, Faculty of Medicine, Aligarh Muslim University (AMU), Aligarh. Informed consent was not required in this study since it was a part of routine investigations done in the department.
The present study was conducted in Culture and DST Laboratory (Revised National TB Control Programme [RNTCP] certified), Department of Microbiology, J.N. Medical College, AMU, Aligarh, on the sputum samples received of the MDR-TB-suspected patients (according to Programmatic Management of Drug Resistant Tuberculosis guidelines) from the out- and inpatient departments of the hospital and from various TB units in and around Aligarh region from January 2015 to August 2016.
Specimen collection and processing
The patients were advised to collect 4–5 ml of early-morning sputum in a sterilised 50 ml Falcon tube. They were instructed to rinse their mouth with pure water and clean their teeth before collection to avoid contamination with food and other particles. The 2 consecutive days' sputum samples were collected as per the RNTCP criteria.
- One spot specimen when the patient first attends the hospital
- One next early-morning specimen.
Both the sputum samples were subjected to fluorescent microscopy and cultured on solid L–J medium. The LPA test was done in one of the samples having a higher bacillary load. The sputum specimens were handled in Class II biosafety cabinet and were decontaminated by N-acetyl-L-cysteine and sodium hydroxide (NALC-NaOH) method., Subsequently, the sediments were suspended in 1–1.5 ml sterile phosphate buffer (pH: 6.8), and two bottles of L–J medium were inoculated with each sample. A volume of 500 ml of the processed sample was used for DNA isolation in a screw-capped tube.
Conventional drug susceptibility testing
The DST was carried out in L–J solid media by economic variant of 1% proportion method according to the standard operating procedure of RNTCP. Rif and INH were tested with concentrations of 40 and 0.2 mg/ml, respectively. All isolates were identified as MTB by their slow growth rate, colony morphology, inability to grow on L–J media containing p-nitrobenzoic acid (500 mg/ml) and niacin-positive and catalase-negative tests. Any strain with 1% (the critical proportion) of bacilli resistant to any of the two drugs – Rif and INH – was classified as resistant to that drug.
Line probe assay
The GenoType ® MTBDRplus LPA was performed according to the manufacturer's (Hain Lifescience, Nehren, Germany) instructions. Three steps for LPA test included DNA extraction, multiplex polymerase chain reaction (PCR) amplification and reverse hybridisation. These steps were carried out in three separate rooms with restricted access and unidirectional workflow., Sputum specimens were decontaminated using NALC-NaOH. Following suspension of the pellet in phosphate buffer, 500 μl of decontaminated sample was pipetted to a 1.5 ml microcentrifuge tube with screw cap. It was centrifuged for 15 min at 10,000 ×g in a centrifuge tube with aerosol-tight rotor. Supernatant was discarded and suspended in 100 μl lysis buffer (A-LYS) using a vortex mixer and then incubated for 5 min at 95°C in a hot air oven or water bath. Then, 100 μl neutralisation buffer (A-NB) was added and vortexed briefly. It was centrifuged for 5 min at 10,000 ×g. Multiplex PCR was performed using 45 μl amplification mix consisting of 10 μl AM-A and 35 μl AM-B. 5 μl DNA template was added in each tube in a separate room and amplification was performed with the final volume of 50 μl using a thermal cycler and amplification protocol provided by Hain Lifescience. Hybridisation was performed in TwinCubator as per instructions provided by the manufacturers. After completion of hybridisation, strips were washed, removed and fixed to GenoType ® MTBDRplus assay worksheet for interpretation.,
Each strip of LPA had 27 reaction zones (bands), including six controls (conjugate, amplification, TUB, rpoB, katG and inhA controls), eight rpoB wild-type (WT1–WT8), four mutant probes (rpoB MUT D516V, rpoB MUT H526Y, rpoB MUT H526D and rpoB MUT S531L), one katG wild-type and two mutant probes (katG MUT S315T1 and katG MUT S315T2) and two inhA wild-type and four mutant probes (inhA MUT1 C15T, inhA MUT2 A16G, inhA MUT3A T8C and inhA MUT3B T8A) [Figure 1]. Either missing of wild-type band or the presence of mutant band was taken as an indication of a resistant strain. To give a valid result, all six expected control bands should appear correctly. Otherwise, the result is considered invalid. The turnaround time (hours) was calculated from the collection of samples to the availability of results.
Data were analysed using MedCalc developed by MedCalc Software (acacialaan 22, 8400 ostend, Belgium). Data were presented as frequency (percentage). Sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) with 95% confidence intervals were calculated. P < 0.05 was considered statistically significant.
| ~ Results|| |
A total of 1503 sputum samples of MDR-TB-suspected patients (1088 males and 415 females) were enrolled in the study. All sputum samples were subjected to fluorescent microscopy (Auramine O) and culture. Nine hundred and four fluorescent-positive samples were subjected to LPA. Eight hundred and fifty-six samples showed presence of TUB band but was absent in 48 samples [Table 1].
|Table 1: Detection of Mycobacterium tuberculosis complex band by line probe assay done on fluorescent microscopy positive samples (n=904)|
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Testing of all the 1503 samples by culture yielded at least one positive growth for MTB complex (MTBC) in 843 (50.09%) patients. Samples from 611 (40.65%) patients had no growth and in 49 (3.26%) patients the cultures were contaminated [Table 2]. Of the 904 cultures, 800 (88.5%) were MTB, 52 (5.8%) were culture negative, 3 (0.3%) demonstrated were nontubercular mycobacteria (NTM) and the remaining 49 (5.4%) were contaminated [Table 3].
|Table 2: Mycobacterial culture examination of multi-drug resistant tuberculosis suspected patients on Lowenstein–Jenson medium (n=1503)|
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|Table 3: Mycobacterium tuberculosis detection by line probe assay versus solid culture in 904 smear positive sputum samples (fluorescent microscopy)|
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The LPA correctly identified MTB in 796 of 800 MTB culture-positive samples (sensitivity: 99.5%). Four samples were MTB negative (absence of TUB bands). All three sputum specimens having NTM also demonstrated absence of TUB bands on LPA tests (sensitivity: 100%) [Table 3].
The smear examination yielded the following bacillary load: 73 (8.1%) smears were graded as scanty, 440 (48.7%) as 1+, 214 (23.67%) as 2+ and 177 (19.58%) as 3+. There was a significantly higher likelihood of obtaining an interpretable Genotype ® MTBDRplus result from a specimen with a positive smear grade compared to those specimens with a scanty smear grading [Table 4].
|Table 4: Association between smear positivity grades and line probe assay test result|
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Of the 856 samples that were TUB band positive on LPA, 180 (21.03%) samples were found to be resistant to both Rif and INH (MDR Rif + INH). LPA was performed on smear-positive samples. Monoresistance to Rif was detected in 50 (5.84%) and monoresistance to INH was detected in 80 (9.25%) cases. Five hundred and forty-six (63.79%) samples were sensitive to both Rif and INH (Rif + INH) [Table 5].
|Table 5: Anti-mycobacterial sensitivity profile of multi-drug resistant tuberculosis suspected patients as detected by line probe assay (n=856)|
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Of these 856 samples, 62 random cultures were subjected to solid culture DST on L–J medium. Nineteen samples came out to be resistant and 25 were resistant to isoniazid. Seventeen samples were resistant to both Rif and INH. Keeping L–J DST as gold standard test, sensitivity and specificity of Genotype ® MTBDRplus assay for the detection of Rif resistance came out to be 94.74% and 95.35%, respectively. Similarly, PPV and NPV of Genotype ® MTBDRplus assay for the detection of Rif resistance came out to be 90% and 97.62%, respectively [Table 6]. Moreover, for INH resistance, sensitivity, specificity, PPV and NPV of Genotype ® MTBDRplus assay came out to be 92.00%, 91.89%, 88.46% and 94.44%, respectively [Table 7].
|Table 6: Correlation of rifampicin resistance on Lowenstein–Jenson drug susceptibility testing with line probe assay|
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|Table 7: Correlation of Isoniazid resistance on Lowenstein–Jenson drug susceptibility testing with line probe assay|
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| ~ Discussion|| |
Timely diagnosis and prompt treatment of infectious cases are the only key elements in reducing the spread of MDR-TB. Accurate and early diagnosis of MDR-TB is highly desirable as it interrupts further transmission of the disease and avoids empirical addition of lifesaving drugs and thus amplification of drug resistance and creation of extensively drug-resistant TB. It also avoids unnecessary cost of administration and occurrence of serious side effects of second-line anti-TB drugs in case one is dealing with drug-sensitive MTB strains.
Conventional culture and drug susceptibility testing (DST) on solid media is a time-consuming process and these systems have been supplemented with automated liquid culture systems in many diagnostic laboratories with decreased time to detection and greater sensitivity. However, the time for testing resistance is still about 7–10 days, beginning from the time that a positive culture is obtained., The most rapid results could be achieved by molecular methods including commercial or in-house DNA hybridisation or amplification methods which allow detection of MTB as well as drug resistance in clinical samples within 1–2 days., Early diagnosis of Rif and INH drug-resistant MTB is essential for efficient treatment and control of MDR-TB.
The present study was conducted in a Culture and DST Laboratory (RNTCP certified), and accredited for carrying out LPA in a tertiary care centre in Western Uttar Pradesh. We evaluated the performance of LPA test directly on sputum samples obtained from MDR-TB suspects. Some of these samples were subsequently tested by phenotypic (L–J proportion method) DST and the results were compared. A good concordance between LPA and conventional solid culture DST was observed in our study. LPA showed the advantage of shorter turnaround time as compared to solid DST.
Since LPA can be done directly on the smear-positive samples, and provided valid sensitivity results in contaminated (48/49) and or negative culture result (12/52), it overcomes these limitations of the conventional solid culture methods, hence surpassing the limitations of solid culture DST such as reduced recovery of viable Mycobacteria and contamination translated into better and timely diagnosis of MDR-TB, thus leading to prompt treatment and reduced transmission of MDR-TB.
In the present study, it was seen that 19/73 (26%) invalid results were obtained when the sputum bacillary load was <1+. Hence, it can be said that efficiency of LPA decreases when sputum bacillary load is <1+.
In our study, out of the 856 MTBC-positive samples, MDR were 180 (21.03%), monoresistant Rif were 50 (5.84%), monoresistant INH were 80 (9.35%) and 546 (63.79%) were sensitive to both Rif and INH. findings have now been presented in [Table 8],[Table 9],[Table 10] form mentioning relevant citations. Similar findings were depicted by various authors as shown in [Table 8].
|Table 8: Studies showing percentages of MDR, Rif monoresistant, INH resistant, Rif and INH sensitive detected|
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|Table 9: Studies showing sensitivity, specificity, PPV and NPV for the detection of rifampicin resistance|
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|Table 10: Studies showing sensitivity, specificity, PPV and NPV for the detection of isoniazid resistance|
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In the present study, sensitivity, specificity, PPV and NPV for the detection of rifampicin resistance came out to be 94.74%, 95.35%, 90% and 97.62%. These values are within the range of the observations made by the various studies done across India and around the world as shown in [Table 9].
However, for detection of INH resistance, in comparison to the conventional DST, the sensitivity, specificity, PPV and NPV of the MTBDRplus assay were 92%, 91.89%, 88.46% and 94.44%, respectively. Similar observations were seen to be made from various studies across India and around the world as shown in [Table 10].
The results in our study are in concordance with that of other recent studies, which have reported the sensitivity of MTBDRplus assay in detecting Rif resistance varying between 92% and 99% and INH resistance between 73% and 92%.,,, Our results were in agreement with the meta-analysis done by Ling et al.
The study findings suggest that LPA is suitable for routine use in settings where a standardised second-line anti-TB drug regimen is provided to MDR-TB cases and where Rif resistance is also treated with the same standardised MDR-TB regimen.
It is well known that DST of anti-tubercular drugs by conventional methods is difficult due to various technical reasons., In addition, it may take upto 6 weeks to get a phenotypic DST result and during this period the patient may infect his/her contacts. Recent studies have demonstrated the feasibility of the Genotype ® MTBDRplus assay, which was utilised as an effective tool for MDR-TB screening in countries with a high burden of drug-resistant TB and showed a good concordance with phenotypic DST results.,, However, rapid DST has some drawbacks, generally related to the low concentration of bacilli and possible presence of different type of Mycobacteria (i.e., a mixture of sensitive and resistance clones) in sputum specimen.
A major limitation of molecule genetic detection of drug resistance remains that not all mutations conferring resistance to anti-TB drugs are known. This is especially true in detecting INH resistance and explains the comparatively low sensitivity. Other limitations of Genotype ® MTBDRplus assay include need for appropriate infrastructure, adequately trained and skilled laboratory personnel.
| ~ Conclusion|| |
The present study conducted in the geographic region with a high prevalence of TB shows that GenoType ® MTBDRplus has good sensitivity and specificity in detecting MDR-TB cases with a significantly lesser turnaround time as compared to conventional DST method and simultaneous detection of Rif and INH resistance. This technique saves several weeks of time required for culture and DST. Hence, a routine use of LPA can substantially reduce the time to diagnose RIF and INH resistance and can potentially enable earlier commencement of appropriate drug therapy, thereby facilitating prevention of further transmission of drug-resistant strains. In the background of rising drug resistance in MTB, the MTBDRplus assay may be a good tool in the diagnosis and management of MDR-TB. LPAs do not carry the biohazard risk associated with the cultivation of MTB in conventional methods. However, LPAs are not a complete replacement for conventional culture and DST, as MTB culture is still required for smear-negative specimens and bacillary count <1+ on smear examination.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]