|Year : 2013 | Volume
| Issue : 2 | Page : 130-137
A rapid and low-cost microscopic observation drug susceptibility assay for detecting TB and MDR-TB among individuals infected by HIV in South India
S Solomon1, P Balakrishnan1, R Vignesh1, G Waldrop1, SS Solomon2, KG Murugavel1, N Kumarasamy3, T Yepthomi3, S Poongulali3, CR Swathirajan1, V Sreenivasan3, C Chandrasekar4, J Suriakumar4, A Mahilmaran4, G Manoharan5, DAJ Moore6
1 Infectious Diseases Laboratory, YRG Centre for AIDS Research and Education (YRG CARE), Chennai, India
2 Medical Centre, YRG Centre for AIDS Research and Education (YRG CARE); Department of Chest Diseases, International Training and Education Center for Health India (I-TECH), Chennai, India
3 Medical Centre, YRG Centre for AIDS Research and Education (YRG CARE), Chennai, India
4 Department of Chest Diseases, Balaji Medical College and Hospital, Chennai, India
5 Department of Chest Diseases, Government Hospital of Thoracic Medicine, Chennai, India
6 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, USA and TB Centre and Department of Clinical Research, London School of Hygiene and Tropical Medicine, London, UK
|Date of Submission||06-Aug-2012|
|Date of Acceptance||30-Oct-2012|
|Date of Web Publication||19-Jul-2013|
Infectious Diseases Laboratory, YRG Centre for AIDS Research and Education (YRG CARE), Chennai, India
Source of Support: The World Bank – Development Marketplace Grant, Conflict of Interest: None
Background: The converging epidemics of HIV and tuberculosis (TB) pose one of the greatest public health challenges of our time. Rapid diagnosis of TB is essential in view of its infectious nature, high burden of cases, and emergence of drug resistance. Objective: The purpose of this present study was to evaluate the feasibility of implementing the microscopic observation drug susceptibility (MODS) assay, a novel assay for the diagnosis of TB and multi-drug-resistant tuberculosis (MDR-TB) directly from sputum specimens, in the Indian setting. Materials and Methods: This study involved a cross-sectional, blinded assessment of the MODS assay on 1036 suspected cases of pulmonary TB in HIV-positive and HIV-negative patients against the radiometric method, BD-BACTEC TB 460 system. Results: Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of the MODS assay in detecting MTB among TB suspected patients were 89.1%, 99.1%, 94.2%, 95.8%, respectively. In addition, in the diagnosis of drug-resistant TB, the MODS assay was 84.2% sensitive for those specimens reporting MDR, 87% sensitivity for those specimens reporting INH mono-resistance, and 100% sensitive for specimens reporting RIF mono-resistance. The median time to detection of TB in the MODS assay versus BACTEC was 9 versus 21 days (P < 0.001). Conclusion: Costing 5 to 10 times lesser than the automated culture methods, the MODS assay has the potential clinical utility as a simple and rapid method. It could be effectively used as an alternative method for diagnosing TB and detection of MDR-TB in a timely and affordable way in resource-limited settings.
Keywords: HIV/TB, Low-cost, MDR-TB, Microscopic Observation Drug Susceptibility assay, Tuberculosis, TB diagnosis
|How to cite this article:|
Solomon S, Balakrishnan P, Vignesh R, Waldrop G, Solomon S S, Murugavel K G, Kumarasamy N, Yepthomi T, Poongulali S, Swathirajan C R, Sreenivasan V, Chandrasekar C, Suriakumar J, Mahilmaran A, Manoharan G, Moore D. A rapid and low-cost microscopic observation drug susceptibility assay for detecting TB and MDR-TB among individuals infected by HIV in South India. Indian J Med Microbiol 2013;31:130-7
|How to cite this URL:|
Solomon S, Balakrishnan P, Vignesh R, Waldrop G, Solomon S S, Murugavel K G, Kumarasamy N, Yepthomi T, Poongulali S, Swathirajan C R, Sreenivasan V, Chandrasekar C, Suriakumar J, Mahilmaran A, Manoharan G, Moore D. A rapid and low-cost microscopic observation drug susceptibility assay for detecting TB and MDR-TB among individuals infected by HIV in South India. Indian J Med Microbiol [serial online] 2013 [cited 2018 May 20];31:130-7. Available from: http://www.ijmm.org/text.asp?2013/31/2/130/115225
| ~ Introduction|| |
Tuberculosis (TB) continues to be a leading cause of morbidity and mortality in developing countries. In 2009, there were an estimated 9.4 million incident cases of TB globally.  India has the highest TB burden globally, accounting for 21% of the global incidence and two- thirds of all cases in Southeast Asia. HIV and TB co-infection form a lethal combination, each speeding the other's progress. The global HIV pandemic has presented an immense challenge to the diagnosis, treatment, and prevention of TB, and also it is one of the most important public health problems in India.
The urgent need for improved diagnostic tools for detection of TB and multi-drug-resistant tuberculosis (MDR-TB) has been repeatedly highlighted. ,, Microscopic examination for acid-fast bacilli (AFB) remains the cornerstone of TB diagnosis despite reports of eroded sensitivity especially among HIV patients.  Conventional culture method on solid medium for detection of TB and MDR-TB is time consuming and takes several months to report the results. Commercially available liquid culture systems, although having short turn around time (TAT), are not accessible in settings where the need is greatest  and are prohibitively expensive and require laboratories with advanced infrastructure. , Given the context of the emerging MDR-TB and extensively drug-resistant TB (XDR-TB) strains, there is an urgent need in resource-limited settings for a new, high-performing, inexpensive, and rapid diagnostic method for effective detection of TB and drug-resistant TB. 
The simple microscopic observation drug susceptibility (MODS) assay has been developed and validated in Lima, Peru. ,, The assay uses two well-known properties of Mycobacterium tuberculosis: (i) the rate of growth in liquid medium is considerably quicker than that in solid medium, and (ii) the morphology of M. tuberculosis in liquid culture is characteristic and recognizable, consisting of "cords" and "tangles." The MODS assay has been studied on both smear-positive and smear-negative sputum specimens with good results.  Furthermore, the possibility for low cross-contamination in resource-limited laboratories has also been illustrated.  The objective of this present study was to evaluate for the suitability of an MODS assay in detecting TB and MDR-TB in sputum specimens from both HIV-positive and HIV-negative cases.
| ~ Materials and Methods|| |
Following a technology transfer from Peru, where the methodology was developed ( http://www.modsperu.org/ ), and successful standardization of the MODS assay in a tertiary HIV care clinic in south India, a study was designed to compare the performance of the MODS assay against BACTEC TB460 system (Becton Dickinson, CA, USA). The study protocol and informed consent forms (Tamil, Telugu, and English) were approved by the Institutional Ethical Committee.
Patients and specimens
The sputum specimens were collected from patients attending an HIV tertiary care clinic and other hospitals in south India. Patients with clinical symptoms suggestive of TB were included and those who had received anti-TB treatment and medication such as amikacin, capreomycin, ciprofloxacin, cycloserine, ethionamide, kanamycin, moxifloxacin, and ofloxacin in the 30 days prior to screening were excluded from the study. The HIV status of the individuals was ascertained according to National AIDS Control Organization (NACO, 2007) guidelines. The specimens collected from the tertiary HIV care clinic were kept in the refrigerator until processing and all the specimens were processed within 48 h of collection. The specimens transported from other regions were at ambient temperature during the transit (ranging from 3 to 24 days, 7 median days) and then stored in the refrigerator on receipt of the specimens until processing at the testing laboratory.
Detection of TB and MDR-TB
All sputum specimens were digested and decontaminated of other bacteria by the standard N-acetyl-L-cysteine (NALC)-NaOH-sodium citrate method.  An aliquot of the specimen was used for microscopical examination of Ziehl-Neelsen stained smears and the remainder was used for parallel testing with BACTEC TB 460 culture system and MODS culture assay after decontamination and concentration. The results of MODS were blinded until completion of the test with BACTEC TB 460 system.
BACTEC TB460 system
The BACTEC TB460 culture was performed as per the manufacturer's instructions. The culture-positive strains were differentiated into two groups - M. tuberculosis complex (MTB complex) and Mycobacterium other than Tuberculosis bacilli (MOTT bacilli) - by the NAP differentiation test. The strains of MTB complex were subjected to the drug susceptibility testing for rifampicin (RIF) and isoniazid (INH) drugs. The MOTT bacilli were inoculated into Lowenstein-Jenson (LJ) medium for future identification of the species.
Following a two-week laboratory training and technology transfer from Lima, Peru, MODS assay was performed as described previously.  Wells containing decontaminated specimens, 7H9 broth (Difco, Becton Dickinson, MD, USA), OADC (Becton Dickinson, MD, USA), and MGIT-PANTA (Becton Dickinson, MD, USA), with or without drug, were examined from third day of the inoculation of the specimen under an inverted light microscope (Nikon, Tokyo, Japan) at ×10 and ×40 magnifications, and after that on every alternate day until 21 days for the presence of characteristic tangled growth [Figure 1] of M. tuberculosis in the drug-free wells. The drug concentration of isoniazid and rifampicin were 0.4 μL and 1.0 μL, respectively. Negative controls were run in each plate and, on a separate plate, one drug-susceptible control strain of M. tuberculosis and an MDR-TB control strain were included every day the assay was run.
|Figure 1: Characteristic "tangles" and "cord" formation by M. tuberculosis in MODS assay (Magnifi cation ×10)|
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Positive cultures were identified by cord formation, characteristic of M. tuberculosis growth, in liquid medium in drug-free control wells. The organism in a specimen was considered susceptible if growth was observed in the drug-free control wells but not in the wells containing INH and RIF drugs. To minimize cross-contamination and occupational exposure, the culture plates were permanently sealed in a clear, transparent polythene cover using the thermo-sealer [Figure 2]a and b after inoculation and were subsequently examined with the sealed polythene cover. Fungal or bacterial contamination was recognized by rapid overgrowth and clouding in wells. If contamination was detected, the fresh cultures were set up with a backup processed specimens stored.
|Figure 2: (a) Alternative method of sealing the MODS plate using a thermo-sealer, instead of zip-loc covers. (b) Sealed MODS plate|
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Moreover, when faster growth (usually within 3 to 5 days) is observed without any characteristic tangled cord appearance of MTB and not resembling mat-like bacterial or cottony fungal contamination, the wells were followed up until 21 days and then the lids were removed under Class II biosafety cabinet and the culture fluids were aspirated for making smears and were ZN stained. The presence of AFB with the absence of characteristic tangled growth was recorded as MOTT bacilli.
All data analyses were performed using SAS (version 9.0, SAS Institute Inc., Cary, NC, USA) and R for Mac OS X GUI 1.35 (R Foundation for statistical computing, Vienna, Austria) statistical software. Concordance between the two diagnostic methods and resistance profiles was determined by sensitivity, specificity, predictive values, and Kappa statistics (with 95% confidence intervals). BACTEC TB460 was used as the reference standard for all analyses, although recognizing its limited accuracy as a gold standard and hence the denominator for the sensitivity of detection by each method (MODS and TB-BACTEC 460 System) was culture positivity by either method. The Wilcoxon signed-rank test was used to compare the time of diagnosis and the time to resistance profile between the two methods. A P value of less than 0.05 was considered statistical significance.
| ~ Results|| |
Of the 1036 specimens processed in our study, 366 (35%) were collected from HIV-positive patients, 185 (18%) specimens were collected from HIV-negative patients, and 485 (47%) were collected from patients whose HIV status was unknown. Overall, the median age of the patients was 36, with 67% of the specimens collected from male patients. A total of 303 sputum specimens (29%) were processed within 48 h of collection while 733 (71%) of the specimens experienced delayed processing. Delayed processing occurs as consequence of the aforementioned travel time and storage protocol for specimens collected in other cities. A total of 261 (25%) specimens reported a positive MTB culture result according to the BACTEC TB460 System. Additionally, a total of 41 (4%) specimens reported a positive MOTT culture result. Contamination was observed in either MODS or BACTEC systems in 65 specimens. Owing to the reprocessing time, these specimens were subsequently excluded from days until detection analyses.
Concordance of detection
MTB Detection by MODS when compared with BACTEC
Overall MTB sensitivity reached 89.1%, with highest sensitivity observed among HIV-negative patients (93.9%) followed by HIV-positive patients (89.2%) and HIV status unknown (84.2%). Overall specificity reached 99.1%; the highest specificity observed was 98.7% in patients with unknown HIV statuses. The MODS assay detected 14 cases of MTB that were culture negative by BACTEC TB 460, and of which 5 were AFB smear positive. The sensitivities, specificities, predictive, and Kappa values of MTB detection in different patient groups are given in the [Table 1].
|Table 1: Performance characteristics of MODS assay to detect MTB and MOTT across the study populations as compared against BACTEC TB 460|
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MOTT Detection by MODS when compared with BACTEC
There was decreased overall sensitivity for MOTT detection by MODS than for MTB, with an MOTT overall sensitivity of 62.1%. High MOTT sensitivity was observed in specimens from HIV-negative patients (83.3%). The lowest MOTT sensitivity (50%) was observed in specimens from patients with unknown HIV statuses. The highest specificity for MOTT detection was observed in both HIV- positive patients and patients with unknown HIV statuses (99.1%), with the overall specificity also reaching 99.1%. The sensitivities, specificities, predictive, and Kappa values of MOTT detection in different patient groups are given in the [Table 1]. Four specimens were tested positive for MOTT by MODS while being culture negative by BACTEC, and of these 2 were AFB smear positive [Table 2].
|Table 2: Additional cases of MTB and MOTT detected by MODS assay that were culture negative by BACTEC TB 460|
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Detection of TB with an AFB smear negative result
Among AFB smear negative specimens (n = 186), which grew 15 MTB and 7 MOTT isolates, the overall sensitivity, specificity, PPV, NPV, and Kappa values are 87.9%, 99.3%, 93.5%, 98.5% and 0.89, respectively for detection of MTB. Interestingly, there was increased overall sensitivity of MODS in smear negative specimens for detection of MOTT, with an overall sensitivity of 100%. The overall specificity, PPV, NPV, and Kappa values are 99%, 75%, 100%, and 0.85, respectively, for detection of MOTT.
Concordance of resistance
Overall, MODS reported 84.2% sensitivity for those specimens reporting MDR (n = 16), 87% sensitivity for those specimens reporting INH mono-resistance (n = 20), and 100% sensitivity for those specimens reporting RIF mono-resistance (n = 1). On stratification by HIV status, MODS achieved 100% sensitivity, specificity, PPV, and NPV for detection of INH mono-resistance and MDR among HIV-negative patients. The sensitivities, specificities, predictive, and Kappa values for detection of drug resistance strains are given in the [Table 3].
|Table 3: Performance characteristics of MODS assay in INH and RIF susceptibility assay across the study populations as compared against BACTEC TB 460|
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Days to detection
Across the board, MODS reported a significantly (P < 0.001) shorter time to detection of TB and resistance detection than BACTEC. The median day for TB detection and drug-resistance detection for MODS was 9 days for the total population and also for specimens from HIV-positive and HIV-negative patients. The median day of MODS TB detection and drug-resistance detection for specimens from HIV unknown patients increased to 11 days. In contrast, the BACTEC method reported a median of 21 days for detection of TB, regardless of HIV status, and 29 days to detect drug resistance profile, also regardless of HIV status. The contamination rates were higher in stored specimens (6.4%) than in fresh specimens (2.6%) processed by MODS method, whereas the rate of contamination remained the same in both groups (1.6%) processed by BACTEC. However, there were no significant differences between these groups in contamination rates. The days to detection of TB and resistance from different patient groups are shown in the [Figure 3], [Figure 4] and [Figure 5]. In addition, the performance characteristics of MODS were not found to be affected by storing the specimens for more than 48 h, which implies the practical feasibility of using it in field studies permitting batch processing of specimens in resource-limited settings.
|Figure 3: HIV status had no signifi cant effect on time to culture positivity in MODS or BACTEC|
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|Figure 4: Time to diagnosis of TB (a) and MDR-TB (b) by MODS is signifi cantly shorter than time to TB and MDR-TB diagnosis by BACTEC|
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|Figure 5: Sputum sample storage had no signifi cant effect on time to culture positivity in MODS or BACTEC|
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| ~ Discussion|| |
Smear microscopy is the mainstay in the diagnosis of TB infection in resource-limited settings. Although cheap, simple, and rapid, smear microscopy suffers from low specificity and variable sensitivity and can neither differentiate between live and dead bacilli nor identify MOTT.  The advantages of culture-based diagnosis over sputum smear include an increased sensitivity of diagnosis and also the availability of susceptibility data. However, available conventional tests such as MGIT and BACTEC are simply not feasible for developing nations because of their high costs and equipment requirements.
The present study with a large sample size with different groups of patients from south India has demonstrated that MODS is a reliable method for detecting and determining drug susceptibility of MTB as reported in other regions. ,,,,,,, The sensitivity of the MODS assay in detecting TB slightly varied between HIV-positive (89.2%) and -negative (93.9%) patients; the difference could have been due to the low shedding of MTB in the sputum of the HIV patients as reported, , alteration of the normal host immune response to MTB in persons with HIV, cavitation, and transfer of TB bacilli into respiratory secretions is markedly reduced, thereby hampering diagnosis by sputum microscopy and culture. In the present study, the median detection time for culture of Mycobacteria (both M. tuberculosis and MOTT) and for detecting drug-resistant strains by the MODS was 9 days as reported earlier. ,,, Importantly, in the present study, processing stored sputum specimens (that were not processed within 48 h) had not influenced the results. Hence, the specimens can be stored at 2-8°C until sufficient numbers and this batch processing of the specimens could bring down the cost and labor.
The study also revealed that detection of MOTT could be possible by MODS and the sensitivity for detecting MOTT bacilli is 62.1% among overall TB suspects and 68.8% among HIV-positive patients. Although the sensitivity in detection of MOTT is comparatively low, still it is helpful to the settings, where the MOTT is not uncommon like India. The increasing number of MOTT as opportunistic pathogens in HIV disease is being reported , and the detection of MOTT could be an additional advantage where MTB culture is negative. However, this is the first study to report the detection of MOTT using the MODS assay. However, the biosafety procedures should be very stringent while handling the culture plates for the detection of MOTT by AFB smear.
TB presents atypically in many cases, with some of the classic signs and symptoms being absent and a high rate of smear-negative pulmonary TB and more particularly in areas with high HIV prevalence. , Hence, the performance of MODS assay with smear-negative TB cases is very critical. The MODS assay has been studied on both smear-positive and smear-negative (reported the sensitivity as high as 94%) sputum samples with good results. ,, The present study with smear-negative cases revealed overall sensitivity (87.9%) and specificity (99.3%) of MODS in detection of TB and, in the case of MOTT, the sensitivity is 100%.
The performance results for MODS in the detection of MDR-TB are similar to those obtained in other studies. ,,,,,, The combined results for INH and RIF (in MDR-TB detection) had good agreement rates (84.2% sensitivity and Kappa value 0.85). In HIV-negative patients, excellent agreement rates (100% sensitivity and Kappa value 1) were observed. Accurate detection of INH and RIF resistance is clinically significant since resistance to these two agents defines per se MDR TB, which requires substantially different treatment than non-MDR TB. Therefore, the MODS assay could be an efficient tool in screening HIV patients for TB/MDR TB infection, and this screening could be substantially useful in better delivery of clinical management services to HIV patients. The MODS assay has been successfully used recently as a screening assay to rule out active TB disease before administration of INH as preventive therapy in HIV patients in Lima, Peru. 
In addition, MODS assay culture is safe, since culture and direct drug-susceptibility testing occur within a closed system in sealed cover. The original protocol uses a Zip-loc bag to seal the MODS plate, and, in the current study, an electric thermo-sealer was used to seal the end twice adjacently [Figure 2]a and b to ensure that there is no leakage. Complete sealing is likely to provide better safety than a Zip-loc bag. Moreover, an electric thermo-sealer is not expensive (10-15 USD). The present study additionally has shown that MOTT also could be detected using the MODS assay; but it requires opening the MODS culture plate to confirm it by AFB staining and which is likely to be associated with increased biohazard issues. The working cost of the MODS is approximately 2 USD  and in contrast to other commercially available culture assays which cost approximately 10-20 USD.
To conclude, given its simplicity, low-cost, low-tech, and accuracy, MODS assay could be easily be adapted as routine TB testing in resource-limited settings such as India, where TB is highly prevalent and highest quality diagnostic tools are least available. However, as there are very limited reports available on smear-negative cases, further studies with larger sample size could throw light on the performance of the MODS assay in detecting smear-negative cases, which is very common in HIV patients.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]