India tops the global list for Drug resistant Tuberculosis, but inadequate and expensive laboratory culture techniques have led to delay in the diagnosis and treatment. We studied the potential of an alternative method which could be cost-effective by combining the drugs in the same tube for identification of drug resistance. Drug Susceptibility Test (DST) results of 1000 sputum samples are got from suspected TB patients against INH (isoniazid) and Rifampicin by two techniques: a) a modified technique with both drugs in the same MGIT tube and b) a standard technique with the antibiotics in separate MGIT tubes for the diagnosis of MDR-TB (Multidrug Resistant). 39 samples were contaminated and were excluded from final analysis. 198 were smear positives by the concentrated Ziehl-Neelsen’s staining method. 219 were found to be culture positive out of which 195 were identified as M. tuberculosis complex. 40 (20.5%) strains were identified as MDR-TB by the conventional method and 39 were picked up by the modified DST. INH and Rifampicin mono-resistance accounted for 32 (16.4%) and 4 (2%) respectively. 99% concordance was observed between the two tests in categorizing MDR-TB. Similarly modified technique with combination of the second line Antibiotics-Ofloxacin, Kanamycin and Capreomycin was applied on the identified MDR strains in a stepwise manner. 6 (15%) were identified as Pre-XDR strains and 2 (5%) were found to be XDR-TB strains. This study implies that combining drugs in the same tube may be an equivalent and possibly a cost-effective alternative which needs to be explored further.
WHO declared TB (Tuberculosis) as the number one killer among the Infectious diseases in the world in 2015 [
Drug resistance has complicated the Tuberculosis epidemic, making early diagnosis and treatment even more difficult. Multi-Drug Resistance (MDR-TB) is defined as resistance of Mycobacterium tuberculosis to the first-line drugs, Isoniazid and Rifampicin. Further progression of resistance across the drug categories led to the isolation of Extensively Drug Resistant Tuberculosis (XDR-TB) strains in 2006 [
WHO in 1994 launched the Global Project on anti-tuberculosis drug resistance surveillance which advised for routine DST of representative samples for categorization into MDR and XDR Tuberculosis [
The principle of Anti Tuberculosis Therapy (ATT) is based on combination therapy where multiple drugs are given in combination simultaneously for effective eradication of the bacteria in vivo. This concept was the basis for our study, and we wanted to check the applicability of simultaneous combination of drugs in the same tube for Drug Susceptibility Testing in vitro. There are various ways to detect Multi-Drug Resistant Tuberculosis. Techniques like Line Probe Assay are very expensive and require technical expertise [
The study design is a comparative study of diagnostic accuracy. The modified test was the novel test with combination of antibiotics―INH and Rifampicin in the same MGIT tube. The Reference standard was the conventional test with INH and Rifampicin in separate MGIT tubes. The modified test and Reference standard tests were done simultaneously. The study involved 1000 patients with suspected tuberculosis-both in-patients and out-patients who presented themselves to a tertiary-care teaching hospital in urban south India. Inclusion Criteria involved Sputum samples of patients in the age group-4 years and above with suspected tuberculosis, who were registered for sputum AFB smear examination in the RNTCP Clinic. The samples were collected from the RNTCP (Revised National Tuberculosis Control Program) clinic, between January 2013 to December 2015, purely based on the inclusion in registry and convenience sampling, after the patient’s informed consent. Sputum samples which were less than 1 ml were excluded. The study was approved by the St. John’s Medical College & Hospital Institutional Ethical Review Board. Bangalore, India and funded by the St John’s Research Institute- Infectious Diseases Unit. As this was a prospective analysis, written informed consent was obtained from the study participants. Patient information was anonymized prior to analysis. The write up was done following the Standards for the Reporting of Diagnostic Accuracy Studies―STARD guidelines. This was a Non-superiority study.
In order to examine a 90% sensitivity in identifying drug resistance using the new modified Drug susceptibility test (DST) as compared to the conventional DST, with 10% precision and 95% Confidence Interval, at least 35 Multi drug resistant strains need to be isolated. The Statistics of prevalence of MDR Tuberculosis in India is around 2% - 3% for newly diagnosed TB and around 20% for previously treated TB. Since we were not categorizing patients according to the treatment status, we took an in-between value of 10% for which 350 culture positive samples were to be obtained to get 35 Drug resistant strains. Taking an arbitrary value of 20% of all samples would be culture positive, final number of samples required was calculated as 1750. We arrived at a final sample size of 1000 for convenience and financial reasons. (During sample collection and pro- cessing we reached 40 drug resistant cases within the 1000 sample size).
These samples were subjected to decontamination and homogenization using NaLC-NaOH method for liquid culture and solid culture. Smears made from concentrated specimens were stained by the Ziehl-Neelsen’s technique. All the smears were read within 48 hours of preparation using a light microscope (magnification 1000×). The slides were categorized as AFB positive or negative depending on the presence or absence of acid-fast bacilli (AFB) using the WHO/ IUATLD and RNTCP scale, with a positive result corresponding to ≥1 AFB per 100 high-power fields (HPFs).
Clinical samples after digestion and concentration were inoculated in liquid culture (BACTEC MGIT, Beckton Dickinson) and solid culture media (commercial Lowenstein Jenson Medium (LJ)) and were incubated at 37˚C. MGIT was incubated for six weeks and LJ media up to eight weeks. The culture positive samples were confirmed to be AFB by Ziehl-Neelsen’s staining. Culture positive for Mycobacteria either from liquid or solid media, were used for the study. ATCC H37Rv strain and Mycobacterium fortuitum were used as the standard Positive and negative control respectively [
TB Antigen MPT64 rapid ICT kit, manufactured by Standard Diagnostics, Seoul, South Korea, was used as per the manufacturer’s instructions for differentiation between M. tuberculosis complex and MOTT (Mycobacteria Other Than Tuberculosis). Initial 450 samples were differentiated using both the MPT64 Antigen test and the PNBA (p-Nitro Benzoic Acid) test, which is the Gold Standard. Both the tests showed a 100% concordance in our study and the data on 450 samples was published earlier [
Reference Standard-Mycobacterial Growth Indicator Tube-Antibiotic Susceptibility Test (MGIT-AST):
MGIT-AST (Becton Dickinson, Sparks, MD, ABD) was performed as recommended by the manufacturer. MGIT tubes were marked as per the drug and 0.8 mL of oleic acid-albumin-dextrose-catalase was added in each of the 3 tubes (GC-Growth Control, INH, RIF) that together constituted a MGIT-AST kit: growth control. Antibiotics were added at a volume of 100 µl from the stock solutions. Final concentrations were 0.1 μg/mL for INH, 1 μg/mL for RIF. A 1:5 sterile saline concentration of McFarland 0.5 standard suspension was inoculated into each tube at a volume of 0.5 mL. Drug susceptibility testing sets were incubated at 37˚C and continuously monitored until a susceptible or resistant result was obtained. The drug susceptibility test results were reported by the instrument (micro MGIT machine), once the GC became positive [
The powdered drug Isoniazid and Rifampicin were procured from Sigma Aldrich and the dilutions were made using the suitable solvents. All stock solutions were sterilized using a 0.2 µm polycarbonate filter membrane, and the first 20% of the initial filtrate was discarded. Stock solutions were stored at −80˚C in small aliquots. A tube containing INH + RIF with concentration 0.1 μg/mL for INH, 1 μg/mL for RIF with 50 µl of each drug was added into a single tube with the combined volume of 100 µl and was incubated at 37˚C and continuously monitored until a susceptible or resistant result was obtained. The drug susceptibility testing set results was reported by the instrument (micro MGIT machine), once the GC became positive [
The MDR strains detected from the first DST subsequently underwent second line DST in a stepwise manner. All the MDR strains were first put through a DST with Ofloxacin alone. If found to be Ofloxacin resistant, they had to undergo DST against injectable drugs. Conventional method was done with single antibiotic per tube with either Ofloxacin or Kanamycin or Capreomycin at concentration of 2.0 μg/mL for Ofloxacin, and concentration of 2.5 μg/mL for Kanamycin and Capreomycin. 100 µl of each drug from the stock solutions was added per tube [
The hypothesis was that if the strain is XDR it should show growth in the tubes with either combinations, whereas a Pre-XDR strain would not grow in the combination tube due to the presence of a sensitive single drug. Validation of this test was done against the standard test with 4 strains (details included in the supplementary material).
We did an additional observation of Clinical Outcomes for the Drug resistant TB patients and it was collected via phone calls and Medical Records. Details collected included possible risk factors for developing drug resistant tuberculosis, clinical outcome including death/cure/ still on treatment, also past history of tuberculosis/exposure to drugs.
The Sensitivity, Specificity, Positive and Negative Predictive Values were calculated for comparison between the tests―DST-Modified test Versus Conventional method and Liquid Culture Versus Solid culture results. Prevalence adjusted Kappa value and concordance were also calculated. McNemar’s test was used to assess the significance of the difference between the groups.
All of the 1000 patients with presumptive TB included in the study underwent smear, culture (both solid and liquid) and once isolated, drug susceptibility testing (DST) in the period between 2013 and 2015. The sample processing with numbers at each step is depicted in detail in
Out of the 219 samples positive by culture, there were 151 samples positive by the LJ method-solid media and 218 samples positive by MGIT-liquid media.
Culture positives | Culture negative | |
---|---|---|
N = 219 | N = 742 | |
smear positives | 168 | 30 |
N = 198* | ||
smear negatives | 51 | 712 |
N = 763* |
Note *35 smear negative samples were contaminated and were excluded from analysis. 4 smear positive samples was contaminated with AFB was excluded from analysis. Culture positive means total yield of both solid and liquid cultures and excluding the contaminants, making a total number of 961.
Out of the 218 samples which were culture positive, sixty seven samples were culture positive only by means of liquid media. One sample which was negative in Liquid media was positive in solid. The sensitivity and specificity of liquid as compared with the solid media (gold standard) was found to be 99% and 91% with Positive Predictive Value and Negative Predictive Value being 68.8% & 99.86% respectively (
Culture positives by solid medium N = 151 | Culture negative by solid medium N = 799 | |
---|---|---|
Culture positives by liquid medium N = 218 | 150 | 68 |
Culture negatives by liquid medium N = 743* | 1 | 731 |
Specificity and Sensitivity for the culture for mycobacteria was found to be 91.4% and 99.3%. The PPV was 68.8% and NPV was 99.8%.
with solid media in our study compared to the liquid media, contrary to what has been observed. The overall contamination rate for LJ and MGIT was 5% and 3.9%, respectively in our study, which was statistically significant (p = 0.039).
The 219 culture positive samples were differentiated into 195 (89%) Mycobacterium tuberculosis complex (MTBC) and 24 (11%) MOTT―Mycobacte- rium Other Than Tuberculosis by the MPT64 Antigen Test.
Drug Susceptibility Testing done on all the 195 MTBC strains revealed 119 (61%) susceptible (INH & RIF) strains and the remaining 76 (38.9%) strains were found to be with varying drug susceptibility profiles. Among those 76 strains, forty (20.5%) were found to be MDR TB isolates, 32 isolates were resistant to INH alone and 4 isolates were resistant to RIF alone by the conventional DST method. Among the mono-resistant cases, INH mono-resistance was the commonest (16.4%).
All 195 culture positive strains underwent both conventional DST and the modified DST. Comparing the 1st line DSTs, there were 40 detected to be MDR by the Conventional technique, compared to 39 by the Modified DST (
The 40 MDR strains underwent 2nd line DST further in a stepwise manner, as depicted in
We did an additional observation of interest in our study by looking at the Clinical Outcomes/previous history for the patients with Drug Resistant Tuberculosis. Clinical outcome defined as death or cure could be obtained for only 19 patients out of the 40 MDR cases. There were 3 deaths and 16 were either cured or doing well on ongoing treatment. Among the six Pre XDR cases, 5 were lost to follow up, and one died. Among the 2 XDR cases, one died and the second patient was lost to follow up. Among the 4 Rifampicin mono-resistant cases, data was obtained for 2 patients and they were cured by first line ATT. We also tried to get data on possible risk factors for developing drug resistance among the patients. Diabetes mellitus, Smoking and HIV (Human Immunodeficiency Virus) were found to be present in 10, 5 and 1 patient respectively for whom the information were available. The history of prior ATT use was available for a very few MDR patients, there were 9 relapse, 9 default and 7 failure cases.
In a high burden country like India, it is ideal to do a Culture and Drug Susceptibility testing for every patient with suspected TB. Though MGIT/liquid culture is slightly more expensive, the fact that the results are available in a fraction of time compared to solid cultures makes it a more reasonable choice in order to avoid patients being lost to follow up. The need of the hour is a rapid and a reliable test, with the sensitivity and specificity comparable to the gold standard which is the solid media culture. DST again is more time consuming in the solid media than in the liquid media. In our study we investigated a DST technique with MGIT/liquid media, with multiple drugs in the same tube, compatible with automation, standardization and rapid time to result. Our study, though had limited numbers of Drug Resistant strains, showed 99% concordance between the modified technique and the conventional technique for the first line drugs and almost 100% concordance for the second line drugs. This translates to a reduction in the number of MGIT tubes involved in doing DST, which could mean a reduction in expenditure for diagnosis of drug resistance, especially in a high burden setting like India. This kind of a concept of combining antibiotics is fairly unexplored, as evident by the dearth of literature. Further research with formal sample size calculation needs to be undertaken to assess the validity of the test, since the numbers in our study were small.
There was only one sample which was not detected as MDR by the modified technique. The same sample which was positive for MDR strain by the standard technique was later re-cultured and the repeat DST showed the same strain MDR by the conventional technique and Sensitive by the Modified method. Since the conventional technique is the gold standard, it was decided to categorise that strain into MDR. On follow up of that particular patient by telephone, we found that the patient had responded to first line Anti Tuberculosis treatment and was declared cured.
The contamination rates were found to be higher in LJ media-5% compared to 3.9% with the liquid media. This has been observed elsewhere too [
This study also highlighted the trends of culture positivity prevailing in a tertiary care centre in urban Bangalore. 19.5% of our sputum samples (195 out of the 1000) were culture positive. This was comparable to a large scale retrospective analysis done in a tertiary care centre in Delhi, where 18.9% of the sputum samples (1240 out of 6569 samples) showed positive mycobacterial culture [
Looking at the Drug-resistant trends, our study showed an overall prevalence of 20.5% MDR (40 out of 195 culture positive) cases among the culture positive patients in our centre. Similar rates were quoted from studies from Delhi which showed a prevalence of 28.2% of cultures with MDR strains in a retrospective sample analysis from 2009-2012 [
These rates reiterate the importance of doing culture and drug susceptibility in all the tuberculosis cases, especially in a high burden country like India. This is possible only with a larger network of laboratories doing drug susceptibility to 1st and 2nd line drugs in India, as well as more cost effective measures of doing DST.
Clinical outcomes of the patients with MDR were also observed in our study through chart reviews and telephone calls, but only a few could be followed up. Among those patients, the presence of high risk factors- diabetes mellitus, HIV, smoking, and prior treatment with ATT were noted [
The main limitation of this study was that the numbers of MDR strains were very limited to ascertain the concordance between the modified DST technique and conventional method. In addition, the cost effectiveness of the test was not analysed using formal methods. Also, when we look at the applicability of this novel method of DST, technical experience is needed when doing combination of antibiotics in the same tube on a large scale, which is another potential limitation.
In general, better rapid diagnostic tests are needed for effective detection and early commencement of treatment of drug resistant TB. In a country like India with limited budget allocated to TB control, more focus needs to be given to reduce costs on drug susceptibility testing and allocate funds in a promising potential alternative for DST without compromising on the sensitivity and specificity. Our study though small in sample size shows that combination of antibiotics in the same tube could be a potential cost-effective alternative for DST.
Further studies with a formal sample size need to be planned to look into concordance and validate the possibility of making the modified method as a potential alternative and also look into the cost-effectiveness since there is a reduction in the number of MGIT tubes involved in this DST.
We would like to acknowledge Dr. Tony Raj, Dr. George D’Souza, Dr. Prem K Mony, Dr. Tinku Thomas and Sumithra Selvam for their valuable time and contribution, and also acknowledge St John’s Medical College hospital, especially the Tuberculosis/RNTCP clinic, as well as the Department of Microbiology for their valuable support in recruitment and data. We would also like to acknowledge the St John’s Research Institute for giving us the laboratory support and funding for the tests and material involved.
This particular project was funded exclusively by the Infectious Diseases Unit of the St John’s Research Institute. This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
The authors assert that all procedures contributing to this research work comply with the ethical standards of the National and Institution Ethics Committee at St. John’s National Academy of Health Sciences on the use of patient samples. It also complies with Helsinki Declaration of 1975, as revised in 2008.
Kumar, J.A.J.U., Srinivasa, H. and Chiramal, J.A. (2017) A Comparative Study of Drug Susceptibility Testing Techniques for Identification of Drug Resistant TB in a Tertiary Care Centre, South India. Journal of Tuberculosis Research, 5, 44-57. https://doi.org/10.4236/jtr.2017.51005