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Background: Tuberculosis is a challenging infectious disease in India. Early diagnosis and drug susceptibility test (DST) are needed for programmatic management of tuberculosis. Conventional DST may take upto three and half months by solid culture method. Line probe assay (GenoType MTBDRplus) is a rapid molecular test for detection of rifampicin and isoniazid resistance directly from smear positive sputum samples which give result within one and half day.
Objectives: The aim of this study was to determine the performance of line probe assay (GenoType MTBDRplus) as a rapid detection technique for timely diagnosis of multi drug resistant tuberculosis ( MDR-TB) and mutation associated with rpoB, katG and inhA gene in north India at tertiary care centre.
Methods: we have performed line probe assay, GenoTypeMTBDRplus in 177 MDR suspected patients directly from smear positive sputum samples in a accredited laboratory at Department of Medicine, All India institute of medical science New Delhi. Results were compared with conventional drug susceptibility test on solid media by 1 % proportion method.
Results: Sensitivity and specificity were 100 % and 99% for detection of rifampicin resistance; 90% and 99% for detection of isoniazid resistance; 97% and 100% for detection of MDR-TB. Overall concordance of line probe assay and conventional drug susceptibility testing (DST) was 95.7 %. Prevalence of katG gene mutation conferring INH resistance was 89.5% (43/48) that was higher in MDR-TB (80% , 28/35). As compared to KatG mutation inhA mutation was very low (12.5%) in our study. The rpoB mutations are found in all 39 rifampicin resistant samples. The prevalence of rpoB S531L and KatG S315T1 mutation in MDR-TB were 80% and 94.2% respectively. Line probe assay also works well on culture isolates. This assay shown superior performance over samples which were culture contaminated and culture negative but smear positive on solid media. Turnaround time of line probe assay was one and half day (from sample collection to result available in the laboratory) whereas it was 28 days for conventional culture growth and another 42 days for DST.
Line probe assay is highly sensitive and specific for rifampicin resistant and MDR having very less turnaround time therefore it has the potential to help in early treatment of MDR-TB patients and prevention of transmission of drug resistant strains of Mycobacterium tuberculosis.
Multi-drug resistant tuberculosis (MDR-TB) caused by Mycobacterium tuberculosis is defined as resistant to both isoniazid and rifampicin with or without resistance to other drugs. it is a phenomenon that is threating to destabilize global tuberculosis (TB) control (1). It was estimated that current rates of multidrug resistant TB in new and previously treated cases are globally at 2.9 % and 15.3 % respectively. India and China covered almost 50 % MDR-TB in the world (2).
The prevalence of MDR TB has previously been reported to be low at less than 3% among new cases and 12-17 % in re-treatment cases based on study conducted at Gujarat, Maharashtra and Andhra Pradesh (3). Timely diagnosis and accurate detection of drug resistant Mycobacterium tuberculosis is necessary for efficient treatment and control of MDR-TB.
Solid or liquid culture method for drug susceptibility testing is a time consuming method as availability of their results vary from week to months (4). The World Health Organization and collaborating partners like Foundation of Innovative New Diagnostics have endorsed the use of the molecular test line probe assay (GenoType MTBDRplus, Hain Lifescience GmbH, Nehren, Germany) for rapid detection of MDR-TB patients directly from sputum specimen and culture of Mycobacterium tuberculosis (8 ). The assay detects mutations associated with the rpoB gene for RIF resistance, katG genes for high level INH resistance, and in the inhA regulatory region gene for low-level INH resistance (9).
Rifampicin (RIF) resistance is most frequently associated with mutation in relatively small fragment (81bp) of the rpoB gene encoding for the áµ-subunit of the RNA polymerase where as isoniazid (INH) resistance is mainly caused by mutations in one of several regions of the katG gene, the inhA regulatory and coding region, and the ahpC-oxyR, ndh, and kasA genes (9-12).
The aim of this study was to determine the performance of line probe assay (the GenoType MTBDRplus assay) as rapid detection technique for timely diagnosis and better management of MDR-TB and mutations associated with rpoB, katG and inhA gene in north India.
Patients from six districts and chest clinics of All india institute of medical sciences , New Delhi are participated in the study. This study was conducted at the Tuberculosis Laboratory at Department of Medicine, All India Institute of Medical Science (AIIMS), New Delhi. The laboratory is accredited by the National Mycobacteriology Accreditation System of central TB Division ministry of Health, Govt. of India. Study was approved by ethics committee of AIIMS, New Delhi. Previously treated pulmonary tuberculosis patients were subjected to sputum smear examination (graded according to revised national tuberculosis programme) and those having bacillary load 1+ or more, were enrolled for the study during August 2011 to April 2012.
Specimen collection and processing
Two sputum samples (spot and morning) were collected per patients in 50 ml sterile falcon tubes. Both samples were subjected to smear and culture. LPA was done in one of the both sample having higher bacillary load.
All specimens were performed in a class II biosafety cabinet in a BSL-3 laboratory. Sputum specimens were decontaminated with N-acetyl-L-cystein-sodium hydroxide (Nalc-NaOH) method. After decontamination, the concentrated sediments were suspended in 1-1.5 ml sterile phosphate buffer (pH 6.8) and two L-J slopes were inoculated for each samples. Smears were prepared for Ziehl-Neelsen staining. 500 µl of processed sample was taken in screw caped tube for DNA isolation. Remaining processed sample was stored at -20áµ’C for retesting of discrepant or invalid results. Culture was performed by solid media (Lowenstein- Jensen media).
Conventional drug susceptibility testing
DST was carried out by using Lowenstein-Jensen solid media by economic variant of 1 % proportion method according to the standard operating procedure of Revised National Tuberculosis Control Programme. Two drugs rifampicin and isoniazid were tested with their concentrations 40 µg/ml and 0.2 µg/ml respectively. All isolates were identified as Mycobacterium tuberculosis by their slow growth rate, colony morphology, inability to grow on L-J media containing p-nitrobenzoic acid (500 µg/ml), niacin and catalase test. Any strain with 1% (the critical proportion) of bacilli resistant to any of the both drugs - Rifampicin and Isoniazid were classified as resistant to that drug.
Rapid drug susceptibility testing
The GenoType MTBDRplus line probe assay was performed according to the manufacturer's (Hain Life science GmbH Nehren, Germany) instructions. The test has three steps: DNA extraction, multiplex polymerase chain reaction (PCR) amplification and reverse hybridization. These steps were carried out in separate rooms with restricted access and unidirectional workflow. DNA extraction was performed in BSL-3 laboratory. A 500 µl of decontaminated sediment was used for DNA extraction that included heat killing (95áµ’C/20min), sonication (15 min) and centrifugation (13000g/8 min). 5 µl of supernatant (DNA) was used for the PCR. Master mixture for amplification consisted of 35 µl primer nucleotide mixture (provided with kit), 5µl of 10X PCR buffer with 15 mM Mgcl2, 2µl of 25 mM MgCl2, o.2µl (1U) of HotStar Taq DNA polymerase (provided by Hain life science), 3 µl nuclease free molecular grade water and 5µl of supernatentant (DNA) in a final volume of 50µl. The amplification protocol consisted of 15 min of denaturation at 95áµ’C followed by 10 cycles comprising denaturation at 95áµ’C for 30 sec and elongation at 58áµ’C for 120 sec. An additional 20 cycles comprising 95áµ’C for 25 sec, 53áµ’C for 40 sec and 70áµ’C for 40 sec and a final extension at 70áµ’C for 8 min. Hybridization was performed with the automatic machine (twincubator). After hybridization and washing, strips were removed, fixed on paper and results were interpreted. Each strips of LPA has 27 reaction zones (bands), including six controls (conjugate, amplification, M.tuberculosis complex (TUB), rpoB, katG, and inhA controls), eight rpoB wild-type (WT1-WT8) and four mutant probes (rpoB MUT D516V, rpoB MUT H526Y, rpoBMUT H526D, and rpoB MUT S531L) one katG wild-type and two mutant probes (katG MUT T1S315T1 and MUT T2S315T2), and two inhA wild type and four mutant probes (inhA MUT1 C15T, inhA MUT2 A16G, inhA MUT3A T8C, inhA MUT3B T8A (Figure 1). Either missing of wild-type band or the presence of mutant probe was taken as an indication of resistant strain. LPA was performed independent of culture and DST. Turnaround time was calculated from collection of sample to result reporting.
177 previously treated smear positive patients were enrolled for the study where 109 were male and 68 were female. The mean age of patients was 3 4. 4 + 14.09. Samples from all the patients were subjected to smear, culture, DST and LPA. Smear examination shown that 46 (25.98%) were 1+ AFB, 72 (40.67%) were 2+ and 59 (33.33%) were 3+. Of these 177 patients, 168 (94.91%) were culture positive, 5 (2.82%) were culture negative and 4 were contaminated. All 168 positive cultures were subjected to conventional DST of which 37 (22.02%) were MDR, 16 (9.52%) were INH monoresistant , 2 (1.19%) were rifampicin monoresistant, 111 (66.07%) were susceptible to both isoniazid and rifampicin and two were confirmed as a NTM which had also missing of TUB band in LPA.
Among all 177 sputum samples LPA gave complete interpretable results in 171 (97.17%) samples including two NTM as stated above, samples from four patients were culture negative and 3 were contaminated. Therefore 163 had both conventional DST and interpretable LPA results.
Of the total 37 MDR conventional DST results, LPA gave 36 interpretable results where 35 were also MDR but one was rifampicin mono-resistant. All two rifampicin monoresistant were detected by line probe assay. Among 16 isoniazid mono-resistant conventional DST results, only 12 were shown similar results by LPA. Overall concordance of line probe assay and conventional DST was 95.70 % (156/163).
Considering the conventional Lowenstein - Jensen 1 % proportion method as a gold standard, Performance parameter for detection of rifampicin, isoniazid, and multidrug resistance by LPA were calculated (Table 1) from specimens for which both results were available
Mutation pattern of LPA
In all 39 rifampicin resistant strains detected by line probe assay, S531L mutations (MUT3 band) were found in 30 (76.92%). Higher proportion of this mutation were found in MDR strains (28/35= 80%) as compared to rifampicin monoresistant (2/4 =50%) strains. Other mutations associated with rifampicin resistant in MDR strains were H526D (4/35 ), D516V (3/35) and H526Y(1/35) but we were not found these three mutations in rifampicin monoresistant (0/4). One MDR strains had missing of WT8 and MUT3 band, but MUT2B band (H526D mutation) was present. Other one MDR strains had missing of WT8 band and no any mutation band was present. One rifampicin monoresistant had missing of WT2 band and other one had missing of WT7 band. Rest two rifampicin monoresistant strains had common mutation that was detected by absence of WT8 band and presence of MUT3 band that patterns were more common in MDR strains (Table 2).
The most common mutations found in isoniazid resistant strains were KatG mutation (43/48=89.5%) which are more common in MDR (33/35=94.28%) as compared to isoniazid monoresistant strains (10/13=76.9%). The prevalence of inhA mutation were only 12.5% (6/48) which are more common in isoniazid monoresistant (3/13=23%) as compared to MDR-TB (3/35=8.5%). Only one (MDR) strain in our study had both katG and inhA mutation. Non of the single inhA MUT2 (A16G mutation), inhA MUT3A (T8C mutation) and inhA MUT3B (T8A mutation) band were identified in our study population.
Turnaround time of line probe assay was less than 2 days whereas it was 28 days for conventional culture growth and another 42 days for DST
Line probe assay from culture isolates.
We have perform LPA on 52 culture isolates of sputum samples as another part of study of which 30 isolates were of known DST pattern. Among these 30 DST results, 5 were each of rifampicin and isoniazid monoresistant, 8 were MDR, 12 were sensitive to both drug. We found 100% (30/30) similarities in isoniazid resistant pattern. Discordant results were found in 2 rifampicin susceptibility test where in one isolates LPA gave sensitive result in place of rifampicin monoresistant, indicates that in this isolates mutation may present out side of rpoB gene that is not targeted by LPA. In other, we got MDR in place of Isoniazid monoresistance. The higher concordance results of isoniazid shows that all mutation in these isolates conferring to INH resistance are present within the genomic region that were incorporated in LPA. Of rest 22 cultures isolates, 20 were from from smear negative and culture positive sputum samples and two were extra pulmonary culture isolates. Conventional DST results were not available in these 20 isolates but we got 100 % (20/20) interpretable results by LPA. It shows the advantage of LPA in case of smear negative and culture positive sample. Of the two extrapulmonary samples, one was smear positive (2+) lymp node aspirate and other was smear negative BAL sample of HIV positive patient. Both were routine samples of the laboratory . There was no TUB in LPA test of lymph node aspirate, DST result shown resistant to rifampicin and isoniazid but PNB test was also positive later it confirmed as NTM. There was sensitive (rifampicin and isoniazid) result in BAL sample by both method.
We have evaluated the line probe assay (GenoType MTBDRplus assay) for rapid detection of rifampicin, isoniazid and MDR-TB. The performance of line probe assay was very similar to conventional culture and DST by Lowenstein-Jensen proportion method. In our study MDR prevalence in sauth delhi like badarpur, okhla region is 27.05 % and it is 19.35 % in west delhi like sagarpur, mahavir inclave, dwarika constituency. The sensitivity for detection of rifampicin resistance, isoniazid resistance and multi drug resistance was 100 , 90.30 and 97.22. The specificity for detection of rifampicin, isoniazid and multidrug resistance tuberculosis was 99.20, 99.09 and 97.22 respectively which shows comparable to other studies (4-6). Apart from that, we found valid results by line probe assay in majority of cases where there was culture contamination (4/3=75%) or no culture growth (4/6=66.6%) demonstrating this assay may offer superior patient care service over conventional DST. As reported previously, rifampicin resistance was highly associated with mutation in the 81 base pair region of the rpoB gene, mostly 500-533. Major mutation for all rifampicin resistant in our study was S531L (76.92%) which is more common in MDR (80%) is consistent with those found in previous studies (5,6). We got one false rifampicin resistance by line probe assay which shown WT2 missing band which is rare mutation, this is an agreement with previous report (5). Major discrepancy had come from isoniazid. Line probe assay was failed to detect isoniazid resistant in 5 specimen (in which one was MDR), it indicates that mutation was present at other genomic regions that were not targeted by recent line probe assay.
Distribution of katG and inhA gene mutation differs in different geographical regions (8-12). We found that the most common mutation were located in the katG gene (89.5%) Which was more common in MDR (94.2%). KatG S315T1 was the predominant mutation with 88.5% in MDR and 69.2% in INH monoresistant. Mutations in inhA gene (12.24%) was very less as compared to katG gene. This inhA gene mutation was slightly high in INH monoresistant as compared to MDR. Only one strain of our study had both katG and inhA mutation. Twenty three percent of isoniazid resistant (3/13) and 5.71% (2/35) of MDR strains were detected as isoniazid resistant by a mutation in the inhA gene only which reflects that recent version of line probe assay is more effective than previous which did not incorporated with inhA gene.
Line probe assay is rapid molecular method for detection of rifampicin, isoniazid from smear positive sputum samples and culture isolates of M. tuberculosis. It is highly sensitive and specific to rifampicin and MDR having very less turnaround time therefore it has the potential to help in management and treatment of MDR-TB patients and prevention of transmission drug resistant strains of Mycobacterium tuberculosis.