|Year : 2014 | Volume
| Issue : 1 | Page : 23-27
Genotype MTBDRplus line probe assay for rapid and direct detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex from sputum samples
Nitin Mohan1, Pappala Bala Chandrasekhar2, Indugula Jyothi Padmaja1, Neeraj Raizada3, Polimati Subba Rao4, Bandi Sasidhar Kumar5
1 Department of Microbiology, Andhra Medical College, Visakhapatnam, Andhra Pradesh, India
2 Microbiologist, C and DST Laboratory G.H.C.C.D, Andhra Medical College, Visakhapatnam, Andhra Pradesh, India
3 Medical Officer, Foundation for Innovative New Diagnostics (FIND, India), New Delhi, India
4 Department of Pulmonary Medicine, G.H.C.C.D, Andhra Medical College, Visakhapatnam, Andhra Pradesh, India
5 District T.B Control Officer, District T.B Control Centre, G.H.C.C.D, Visakhapatnam, Andhra Pradesh, India
|Date of Web Publication||10-Mar-2014|
S/O Dr. P Mohan Rao Subudhi, Gandhinagar First line, Berhampur - 760 001, Odisha
Source of Support: The study was conducted at C & DST laboratory (IRL),
Andhra Medical College, Visakhapatnam. FIND provides technical support
to the laboratory under RNTCP., Conflict of Interest: None
Context: Rapid identification of resistant strains is crucial for the early administration of appropriate therapy and to curtail their spread. Genotype MTBDRplus is an assay for rapid detection of gene mutations in sputum samples.
Aims: i) To detect the presence of rifampicin and isoniazid associated mutations directly in smear positive sputum samples. ii) Analysis of mutations in the region of North Coastal Andhra Pradesh.
Materials and Methods: A total of 151 sputum samples were processed by N-acetyl L-cysteine - sodium hydroxide method. The sediment obtained was used for smear preparation, culture and nucleic acid extraction. Multiplex amplification and reverse hybridization were then performed according to manufacturer's instructions. The presence of a mutation is indicated by the lack of hybridization on one or more of wild-type probes with or without hybridization on the mutation probes.
Results: Hundred percent readable results were achieved from sputum samples smear grade of 2+ and 3+. All strips had bands indicating Mycobacterium tuberculosis complex and were also confirmed by culture and biochemical tests. The strains resistant to rifampicin only, isoniazid only and multidrug resistant were 55(36.7%), 42(28%) and 40(26.7%) respectively. The most frequent type of mutation for rifampicin resistance was Serine-531-Leucine. The rate of mutations detected by negative hybridization results with wild-type probes was 17.5%.
Conclusions: Potential for testing large number of isolates rapidly and directly from sputum samples makes Genotype MTBDRplus suitable as a first line screening assay for multidrug resistant tuberculosis. Most frequent mutation causing resistance is Serine-531-Leucine.
Keywords: Genotype MTBDRplus, inhA gene (fatty-acid enoyl-acyl carrier protein reductase), katG gene (catalase-peroxidase), rpoB gene (b-subunit of RNA polymerase)
|How to cite this article:|
Mohan N, Chandrasekhar PB, Padmaja IJ, Raizada N, Rao PS, Kumar BS. Genotype MTBDRplus line probe assay for rapid and direct detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex from sputum samples. J NTR Univ Health Sci 2014;3:23-7
|How to cite this URL:|
Mohan N, Chandrasekhar PB, Padmaja IJ, Raizada N, Rao PS, Kumar BS. Genotype MTBDRplus line probe assay for rapid and direct detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex from sputum samples. J NTR Univ Health Sci [serial online] 2014 [cited 2021 Oct 28];3:23-7. Available from: https://www.jdrntruhs.org/text.asp?2014/3/1/23/128426
| Introduction|| |
In June 2008, the World Health Organization endorsed the use of molecular line-probe assays for multidrug resistant tuberculosis (MDR-TB) screening, and the Genotype MTBDRplus assay has since been introduced for routine practice in various countries.  However, owing to the geographical diversity of Mycobacterium tuberculosis clinical isolates across the world, there is a possibility of varying efficacy of genotyping based methods which may have diagnostic implications. 
The aims of the study are i) to detect the presence of rifampicin and isoniazid associated mutations directly in smear positive sputum samples and ii) analysis of mutations in the region of North Coastal Andhra Pradesh.
| Materials and Methods|| |
The study was conducted from June to July 2011. A total of 151 acid-fast bacilli (AFB) smear positive sputum samples were analyzed. The specimens were initially digested and decontaminated by N-acetyl L-cysteine - sodium hydroxide method. After centrifugation and supernatant removal, the sediment was resuspended in one to two milliliter of phosphate buffer and used for smear preparation (Ziehl-Neelsen's staining), culture and nucleic acid extraction.
The Genotype MTBDRplus line probe assay (HAIN Lifescience GmbH, Nehren, Germany) was carried out according to the manufacturer's instructions. About 500 μL of the decontaminated and concentrated specimen was centrifuged at 10000 g for 15 min, the supernatant was discarded, and the pellet was resuspended in 100 μL of molecular grade water. The specimen was then heat killed at 95°C (hot oven) for 20 min. This was followed by a 15 min sonication step. After sonication, 5 μl of the supernatant was used immediately for polymerase chain reaction (PCR), while the remainder was stored at –20°C. Amplification was done according to the protocol for sputum material (Multiplex PCR): denaturation at 95°C for 15 min; ten cycles of denaturation at 95°C for 30 s and elongation at 58°C for 120 s; an additional 30 cycles of denaturation at 95°C for 25 s, annealing at 53°C for 40 s, and elongation at 70°C for 40 s; and a final extension step at 70°C for 8 min.
The biotin-labeled PCR product was denatured and hybridized to a strip with specific oligonucleotide probes. One probe is complementary with an Mycobacterium tuberculosis complex (MTBC) specific region of the 23S rRNA gene (TUB), and one probe is specific for the rpoB gene (b-subunit of RNA polymerase), i.e. rpoB-Uni and should be always positive for all MTBC strains, while eight wild-type (WT) probes (WT1 to WT8) encompass the region of the rpoB gene encoding amino acids 505 to 533. Four other probes are specific for the most common mutations (MUT): D516V, H526Y, H526D, and S531L (probes rpoB MUT1, rpoB MUT2A, rpoB MUT2B, and rpoB MUT3, respectively). One probe detects a katG-specific gene (catalase-peroxidase) region and should always be positive for all MTBC strains. Three other probes are specific for the S315 region of katG. One is the wild-type probe (katG WT), while two others (katG MUT1 and MUT2) are designed to detect the AGC-to-ACC (S315T) and the AGC-to-ACA (S315T) mutations. Along with locus control for inhA gene (fatty-acid enoyl-acyl carrier protein reductase) there are two wild type probes and four mutation probes (inhA MUT1, inhA MUT2, inhA MUT3A and inhA MUT 3B) to detect mutations in inhA promoter region. rpoB predicts rifampicin (RIF) resistance, katG predicts high level isoniazid (INH) resistance and inhA predicts low level INH resistance. After hybridization, membrane strips were attached to evaluation sheet, read and interpreted.
In conclusion, when all wild-type probes gave a positive signal and all mutation probes reacted negatively, the isolate was considered susceptible.
- When at least one negative signal was obtained with wild type probe, the isolate was considered resistant.
- When the resistance was due to mutations described above, a positive reaction was obtained with one of the mutation probe and was always accompanied by a negative reaction with corresponding wild type probe.
Mycobacterium tuberculosis H 37 RV was used as control. Both positive and negative controls were maintained for each run.
Samples were also inoculated onto Lowenstein Jensen (LJ) media and the colonies obtained were subjected to biochemical tests for identification of Mycobacterium tuberculosis.
| Results|| |
A total of 151 sputum samples were studied. All sputum samples were graded depending on the AFB count. Forty-three samples (28.4%) were graded 1+; 54 samples (35.8%) 2+; 54 samples (35.8%) 3+ [Table 1]. Readable Genotype MTBDRplus assay results [Figure 1] were obtained for 150 DNA extracts obtained from sputum samples comprising 99.3% of all extracts available for testing. Of these all strips had TUB bands indicating presence of Mycobacterium tuberculosis complex. Colonies obtained on Lowenstein-Jensen medium from same samples were subjected to biochemical tests for identification, and all were positive for Mycobacterium tuberculosis. One sample (AFB count 1+) gave invalid result (no bands at all). Better results (100% readable results) were achieved with sputum samples with higher grades, i.e. 2+ and 3+.
|Table 1: Performance of genotype MTBDRplus assay depending on concentration of AFB in sputum specimens (n = 151)|
Click here to view
|Figure 1: Genotype MTBDRplus kit hybridization results. Every run had Negative control (NC), Master Mix negative control (MMXC) and Positive control (PC), i.e. H37RV|
Click here to view
All wild-type probes gave a positive signal and all mutation probes reacted negatively in 93 (62%) DNA samples extracted from sputum, i.e. sensitive to rifampicin and isoniazid. Mutations conferring resistance to rifampicin and isoniazid were detected in 55 (36.7%) and 42 (28%), respectively, in DNA samples extracted from sputum, [Table 2] and [Table 3]. In 40 (26.7%) DNA samples where rifampicin resistance was detected, mutations in katG and/or inhA genes were also detected indicating they were isoniazid resistant also, i.e. MDR.
|Table 2: Variety of mutations associated with rifampicin resistance (rpoB gene)|
Click here to view
|Table 3: Variety of mutations associated with isoniazid resistance (katG and inhA gene)|
Click here to view
In a vast majority of rifampicin resistant isolates (N = 45; 81.8%) codon 531 was affected. The most frequent type of mutation responsible for rifampicin resistance is Ser-531-Leu mutation (74.5%). In the present study, four mutations (S531L, H526Y, H526D, and D516V) accounting for resistance in 47(85.4%) of 55 rifampicin resistant isolates were identified by the kit mutation (MUT) probes specific for four mutations (rpoB MUT3, rpoB MUT2A, rpoB MUT2B and rpoB MUT1 respectively). The amino acid modifications detected in other rifampicin resistant strains (N = 8 or 14.5%) correspond to mutations that were not targeted by an oligonucleotide probe on strip. They were therefore detected by negative hybridization result with one of the eight wild-type-specific probes (WT1 to WT8).
Mutations associated with isoniazid resistance were more diverse: 30 strains (71.4%) had mutations in katG (codon 315) gene only, 11 strains (26.2%) had mutations in the inhA gene only and 1 strain had mutations both in katG and inhA genes. Percentage of mutations in isoniazid resistant isolates revealed by negative hybridization results with wild type probes is 21.4%.
The rate of identified specific mutations in the present study was 82.5%.
| Discussion|| |
Andhra Pradesh (AP) is one of the largest states in India with an approximate population of 80 million. The state records 1,14,624 (TB India, 2009) tuberculosis (TB) patients annually with approximately 6% of them suspected to be harboring MDR. AP state has recently initiated the second line anti TB treatment under the Directly Observed Treatment Short course plus program of Revised National Tuberculosis Control Program The State TB control program, also proposes to use the molecular diagnostic tests for MDR TB in near future. In the current study we used the molecular assay (HAIN Lifescience GmbH Genotype MTBDRplus) for rapid detection of resistance to the most important anti-TB drugs (RIF and INH) on sputum samples in North Coastal AP. Attempt to record the baseline information on the mutations prevailing in this part of the country was made.
The assay is simple to perform and can be completed within 24 h. The test does not require viable organisms and hence is safe to perform. Interpretation of the test strips was easy. The intensities of the different hybridization bands varied. The proportion of interpretable Genotype MTBDRplus assay results in our study (99.3%) was similar to that reported by Hillemann et al. (98.6%).  According to their study MTBDRplus is reliable for prediction of resistance directly in smear positive sputum samples and made a similar observation that 40 amplification cycles was adequate. An increase in PCR cycles would increase the sensitivity of the assay to amplicon contamination.
The present study demonstrated multidrug resistance levels of 26.7%. A study by Lingala et al.,  on samples from Hyderabad and Koraput revealed multidrug resistance levels of 34.7% by culture and drug susceptibility testing on Lowenstein-Jensen medium and sequencing. Among mutations associated with the resistance to rifampicin and isoniazid, the dominance of mutations in the codon 531 of the rpoB gene and codon 315 of the katG gene was similar or close to previously reported studies worldwide and in India [Table 4].
|Table 4: Dominant mutations of rifampicin and isoniazid resistance in different studies|
Click here to view
Ser-531-Leu is the most frequent type of mutation responsible for RMP resistance. Similar finding was seen in study by Mai NT Huyen et al.  The rate of identified specific mutations (82.5%) in the present study was lower than that reported by Russia (93%).  It suggests that set of MUT probes used does not cover all mutations prevailing in North Coastal A.P. It may hence be apt to customize the detection kits so as to make them suitable to detect the locally prevalent mutations.
The percentage of mutations revealed by negative hybridization result with wild type probe in the present study is 17.5%. It indicates that the MTBDR DNA strip assay is also capable of recording the presence of rarely found mutations that can be encountered with a higher prevalence in certain countries. 
Molecular tests are more popular and are being tried in conjunction with the conventional tests in several settings, as they are expected to yield faster results. Currently, available molecular tests are designed to detect known mutations commonly occurring in Mycobacterium tuberculosis isolates. Uncommon mutations not covered by the standard molecular diagnostic kits could have missed the diagnosis had they been tested with molecular tests alone. A meta-analysis of direct susceptibility testing for MDR-TB by Freddie Bwanga et al. suggested that direct Genotype MTBDRplus assay would be 50% cheaper than conventional testing. 
| Conclusions|| |
Potential for testing large number of isolates rapidly and directly from sputum samples makes the MTBDRplus assay a suitable first line screening test. The assay can identify the most frequent mutations involved in resistance to RIF and INH and can reveal the presence of additional mutations by negative hybridization results with the wild-type probes. The performance of the assay may also be affected by selection of probes and target population. Most frequent mutation causing resistance is Ser-531-Lue. It can also be used for epidemiological analyses of the types of mutation.
| References|| |
|1.||Huyen MN, Tiemersma EW, Lan NT, Cobelens FG, Dung NH, Sy DN, et al. Validation of the GenoType MTBDRplus assay for diagnosis of multidrug resistant tuberculosis in South Vietnam. BMC Infect Dis 2010;10:149-57. |
|2.||Lingala MA, Srikantam A, Jain S, Rao KV, Rao PV. Clinical and geographical profiles of rpoB gene mutations in Mycobacterium tuberculosis isolates from Hyderabad and Koraput in India. J Microbiol Antimicrob 2010;2:13-8. |
|3.||Hillemann D, Rüsch-Gerdes S, Richter E. Evaluation of Genotype for MTBDRplus assay rifampicin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol 2007;45:2635-40. |
|4.||Brossier F, Veziris N, Truffot-Pernot C, Jarlier V, Sougakoff W. Performance of Genotype MTBDR line probe assay for detection of resistance to rifampicin and isoniazid in strains of Mycobacterium tuberculosis with Low- and High-Level resistance. J Clin Microbiol 2006;44:3659-64. |
|5.||Somoskovi A, Dormandy J, Mitsani D, Rivenburg J, Salfinger M. Use of smear-positive samples to assess the PCR-based Genotype MTBDR assay for rapid, direct detection of Mycobac teriumtuberculosis Complex as well as its resistance to isoniazid and rifampicin. J Clin Microbiol 2006;44:4459-63. |
|6.||Nikolayevskyy V, Balabanova Y, Simak T, Malomanova N, Fedorin I, Drobniewski F. Performance of Genotype MTBDRplus assay in the diagnosis of tuberculosis and drug resistance in Samara, Russian Federation. BMC Clin Pathol 2009;9:2. |
|7.||Negi SS, Singh U, Gupta S, Khare S, Rai A, Lal S. Characterisation of rpoB gene for detection of rifampicin drug resistance by SCCP and Sequence analysis. Indian J Med Microbiol 2011;27:226-30. |
|8.||Bwanga F, Hoffner S, Haile M, Joloba ML. Direct susceptibility testing for multi drug resistant tuberculosis: A meta-analysis. BMC Infect Dis 2009;9:67-82. |
[Table 1], [Table 2], [Table 3], [Table 4]
|This article has been cited by|
||Phenotypic and genotypic drug sensitivity profiles of Mycobacterium tuberculosis infection and associated factors in northeastern Ethiopia
| ||Fikru Gashaw,Berhanu Erko,Yalemtsehay Mekonnen,Bazezew Yenew,Misikir Amare,Balako Gumi,Gobena Ameni |
| ||BMC Infectious Diseases. 2021; 21(1) |
|[Pubmed] | [DOI]|
||Analysis of drug resistance mutations in pulmonary Mycobacterium tuberculosis isolates in the Southern coastal region of Andhra Pradesh, India
| ||Polu Giri Prasad,Mohammad Shaik Jasmine,Kota Neela Mani kanta,Karumanchi Deepthi,Uday Sankar Allam |
| ||The Brazilian Journal of Infectious Diseases. 2019; |
|[Pubmed] | [DOI]|