Comparative evaluation of nitrate reductase assay with conventional proportion method for isoniazid and rifampicin susceptibility testing in smear positive pulmonary tuberculosis patients

Introduction : With the surge of multidrug-resistant tuberculosis (MDR-TB) strains there is an escalating need for precise and cost-effective methods for a precipitous diagnostic and drug susceptibility testing (DST), chiefly in resource limited countries where tuberculosis is endemic. Aim : To find out the susceptibility pattern of Isoniazid (INH) and Rifampicin (RMP) in smear positive cases of pulmonary TB at MMIMSR Mullana and to evaluate the sensitivity and specificity of nitrate reductase assay (NRA) with standard proportion method for INH and RMP susceptibility testing. Material and Methods: A cross sectional test validation study was carried out at Mycobacteriology division in the Department of Microbiology on 100 smear positive sputum samples from pulmonary tuberculosis patients. Results: In comparison with L.J proportion method, NRA showed sensitivity, specificity, PPV, NPV, accuracy of 100%, 98.64%, 87.50%, 100%, and 98.76% respectively for INH. The sensitivity, specificity, PPV, NPV, accuracy of NRA compared to PM for RMP was 100%, 97.33%, 75.00%, 100%, 97.53%

Introduction : With the surge of multidrug-resistant tuberculosis (MDR-TB) strains there is an escalating need for precise and cost-effective methods for a precipitous diagnostic and drug susceptibility testing (DST), chiefly in resource limited countries where tuberculosis is endemic. Aim : To find out the susceptibility pattern of Isoniazid (INH) and Rifampicin (RMP) in smear positive cases of pulmonary TB at MMIMSR Mullana and to evaluate the sensitivity and specificity of nitrate reductase assay (NRA) with standard proportion method for INH and RMP susceptibility testing. Material and Methods: A cross sectional test validation study was carried out at Mycobacteriology division in the Department of Microbiology on 100 smear positive sputum samples from pulmonary tuberculosis patients. Results: In comparison with L.J proportion method, NRA showed sensitivity, specificity, PPV, NPV, accuracy of 100%, 98.64%, 87.50%, 100%, and 98.76% respectively for INH. The sensitivity, specificity, PPV, NPV, accuracy of NRA compared to PM for RMP was 100%, 97.33%, 75.00%, 100%, 97.53% respectively. Conclusion: Direct NRA is simple, easy to perform, prompt, reasonably less expensive, without requisite of expensive reagents and advanced instrumentation. It is highly specific and sensitive technique in the detection of drug resistant TB when tested in parallel with the proportion method. Therefore, NRA may be routinely employed in TB laboratories in developing countries for drug susceptibility testing of M. tuberculosis.

Introduction
Tuberculosis (TB) is a multi-systemic disease with a mutable presentation. indication of TB has been reported in human remains dated thousands of years. For a human pathogen with no known environmental reservoir, Mycobacterium tuberculosis has perfected the art of survival and has persisted in human populations from antiquity through modern time. 1 A total of 1.5 million people died from TB in 2018. Worldwide, TB is one of the top 10 causes of death and the leading cause from a single infectious agent. In 2018, an estimated 10 million people fell ill with TB worldwide. In 2018, the 30 high TB burden countries accounted for 87% of new TB cases. Eight countries account for two thirds of the total, with India leading the count. 2 The World Health Organisation (WHO) TB statistics for India for 2018 give an estimated incidence of 2.69 million cases. It is estimated that about 40% of the Indian population is infected with TB bacteria. 3 MDR and extensive drug resistant (XDR) in TB is a matter of great concern for TB control programs since there is no cure for some MDR-TB strains of Mycobacterium TB. 4 Regrettably, most of the available drug susceptibility testing (DST) methods are either very time-consuming or too expensive to be widely adopted in low and https://doi.org/10.18231/j.ijmmtd.2020.053 2454-4398/© 2020 Innovative Publication, All rights reserved. 237 middle-income countries. The proportion method (PM), the most commonly used method for determining drug susceptibility, requires up to four to six weeks for the results to be achieved. 5 In the last decade, fast, reproducible and low-cost phenotypic methods for determining the susceptibility to drugs have been described, such as the Microscopic Observation Broth-Drug Susceptibility assay 6 , the colorimetric redox-indicator 7 and the nitrate reductase assay (NRA). 8 Among the low-cost techniques, the NRA appears to be feasible as a rapid, specific, sensitive and easily implemented DST method, especially for low and middle-income countries. The procedure relies on the ability of metabolically active Mycobacterium TB to reduce nitrate to nitrite.

Study design
A cross sectional test validation study was carried out at Mycobacteriology division in the Department of Microbiology, MM Institute of Medical Sciences and Research Mullana, Ambala, Haryana. The study was conducted on 100 smear positive sputum samples from pulmonary tuberculosis patients according to RNTCP (Revised National Tuberculosis Control Programme), 9 the samples selected by random sampling method. The Patient inclusion criteria were all the smear positive sputum samples from pulmonary TB patients. All Smear negative cases of suspected pulmonary tuberculosis and Patients on anti-tuberculosis treatment with HIV-TB, co-infection, and other immune-compromised patients as evident from history were excluded. After taking written consent and noting the demographic details, The sputum specimens were digested, decontaminated by the standard NaOH-NALC procedure 10 and concentrated by centrifugation, the sputum samples of the patient were subjected to ZN (Ziehl-Neelsen) stain and the slides were examined under 100 X oil immersion (Nikon E100) and interpreted as per RNTCP guidelines.The sediment obtained after NaOH-NALC method was re-suspended in 3 ml of sterile distilled water. Out of 3 ml suspension, 1.5 ml was used for direct NRA and remaining 1.5 ml for the direct proportion method. The processed sample (0.2 ml) was inoculated on LJ media (LJ media was prepared in our laboratory) taking necessary aseptic precautions. The slopes were incubated at 37 o C for a maximum period of 8 weeks and inspected once a week for growth, 9 From the growth, a bacillary suspension was prepared by diluting the growth with sterile distilled water (adjusted the turbidity with McFarland standard number 1). The suspension was then inoculated on LJ media with INH (0.2µg/ml) and RMP (2µg/ml) and the slants were incubated at 37ºC. Another LJ slant with H37Rv (ATCC 27294) strain was inoculated with INH and RMP with same critical concentration (served as an internal quality control). The first reading was taken after 28 days and second on 40 th days of incubation respectively. The percentage resistance was calculated as the ratio of the number of colonies on the drug containing media to those on control media. If the ratio was greater than 1% then isolate was taken as resistant to these antibiotics. 11 For each specimen 0.5 ml of the undiluted decontaminated suspension was inoculated into 4.6 ml of 7H9-N medium (Middle Brook) containing RMP (Ranbaxy, Mumbai 2.0µg/ml) and another tube containing 4.6ml of 7H9-N medium with INH (Hi-media, Mumbai 0.2µg/ml). 0.5ml of the 1:10 dilution of sample in phosphate buffer saline (PBS) was inoculated into 4.6ml of drug free 7H9-N medium, which served as growth control. The control strain i.e. H37Rv was similarly inoculated on to the 7H9N medium with incorporation of the same antibiotics and potassium nitrate. All the tubes were then incubated at 37 0 C. After five days of incubation, 0.2ml of freshly prepared reagent mixture Griess reagent was added to 1 ml of drug free medium. The results were classified as negative (no color change) or positive (pink color) (Figure 1). If there was a change in color in the drug free tube, then the tubes with antibiotic (containing patient's sample) and the tube containing control strain were also tested. If there was no color change, then the tubes were re-incubated and the procedure repeated on the 7 th , 10 th , 14 th , 18th and 28 th day. For INH, results were interpreted as described by Angeby et al 6 An isolate was considered resistant if the color change in the INH tube was equal to or greater than that in 1:10 diluted growth control tube and was considered susceptible if there was no color change, or if the color change was less than that in 1:10 diluted growth control. For Rifampicin, an isolate was considered resistant if there was any color change in the RMP tube, and was considered susceptible if there was no color change in the RMP tube. Culture results were confirmed after examination of ZN smear prepared from the growth To rule out bacterial contamination two drops of each positive suspension was inoculated on to a blood agar plate, incubated at 37ºC, and examined after 24 hrs. 12 Internal quality control was done using the fully susceptible M.tuberculosis H37Rv (ATCC 27294) (Figures 2, 3 and 4)

Ethical considerations
The present study was approved by the Ethical Committee of Maharishi Markandeshwar Institute of Medical Sciences and Research. The approval was on the agreement that patient anonymity must be maintained, good laboratory practice, quality control ensured, and that every finding would be treated with utmost confidentiality and for the purpose of this research only. All work was performed according to the International Guidelines for Human Experimentation in Biomedical Research.Approval was obtained from the subjects by taking the informed consent.

Data management and statistical analysis
The data entry was carried using Microsoft Office Excel worksheet and then exported to statistical software and analyzed using appropriate statistical tests by using Statistical Package for Social Services (SPSS vs 21 for Mac IBM Inc Chicago).

Results
A total of 100 smear-positive sputum samples were included in the study.

Discussion
The "captain of all these men of death", TB has been a scourge of the human kind from time immemorial. Till date, no other disease in history matches the sheer magnitude of     the misery inflicted by TB on the human race in terms of morbidity and mortality. 13 The most worrisome drift during recent years is an upsurge in MDR-TB strains. An estimated 4.8 lakh cases of MDR-TB emerge annually worldwide with 1.3 lakh incident multi-drug resistant TB patient in India alone which includes 79000 MDR-TB Patients estimates among notified pulmonary cases. 3 This increase in MDR-TB is largely attributed to poor management of the DOTS Programme and the inability to rapidly diagnose drug resistance. Currently, drug-susceptibility testing is largely based on culture which is hampered by lengthy turn-around times, expensive equipment, high reagent costs and the need for skilled staff and extensive bio safety facilities. It is therefore indispensable that improved diagnostics are  12 Mishra et al, 15 Thapa et al. 16 In resource-constraint areas cost is an imperative aspect where most of the disease is prevalent. A cost by NRA as compared with PM and molecular assays accentuates the need of this colorimetric assay to be routinely used in laboratories in low-income countries. The supplementary equipment, media, and infrastructure required for NRA are routinely present in most microbiological laboratories. Moreover, NRA saves time and labour (avoids pre-isolation of MTB from sputum specimens) as this assay can be performed directly on smear positive sputum specimens. In the current study NRA was found to be a highly specific and sensitive technique in the detection of drug resistant MTB when tested in parallel with the PM. However, NRA posseses some limitations such as some strains (< 1%) of M. Tuberculosis lacks nitrate reductase activity rendering the test invalid. Considering this concern, the direct NRA DST could be performed in parallel with the reference standard (LJ PM) to confirm the presence of M.tuberculosis complex isolates.

Conclusion
NRA was found to be a highly specific and sensitive technique in the detection of drug resistant TB when tested in parallel with the PM. In addition, NRA was found to be rapid, inexpensive, easy to perform and does not require any special instrumentation. Therefore, NRA may be routinely employed in TB laboratories in developing countries for DST of M.tuberculosis. However, NRA possesses some limitations such as some strains (< 1%) of M. Tuberculosis lacks nitrate reductase activity rendering the test invalid. Considering this concern, the direct NRA DST could be performed in parallel with the reference standard (PM) to confirm the presence of M.tuberculosis complex isolates.

Conflicts of interest
All contributing authors declare no conflicts of interest.

Source of Funding
None.

Lubna Samad, Consultant
Cite this article: Mir RF, Peerzada BY, Samad L. Comparative evaluation of nitrate reductase assay with conventional proportion method for isoniazid and rifampicin susceptibility testing in smear positive pulmonary tuberculosis patients. IP Int J Med Microbiol Trop Dis 2020;6(4):237-242.