Tuberculosis (TB) remains a worldwide public health problem despite the fact that the organism was discovered more than a century ago. Infection with TB can result in two stages: asymptomatic latent tuberculosis infection (LTBI) or tuberculosis disease. About one-third of the world's population has latent TB. People infected with TB bacteria have a lifetime risk of falling ill with TB of 10%. However persons with compromised immune systems, such as people living with HIV/AIDS (PLHA), malnutrition or diabetes, or people who use tobacco, have a much higher risk of falling ill. TB is a leading killer of PLHA causing one quarter of all deaths.¹

Multidrug-resistant TB (MDR-TB) is a form of TB disease caused by a strain of M. tuberculosis complex that is resistant to rifampicin and isoniazid. The Global Project on Anti-Tuberculosis Drug Resistance Surveillance has been gathering data since 1994. The latest data indicates that every region of the world has reported multidrug-resistant tuberculosis (MDR-TB).² Detecting MDR-TB at an earlier stage is of utmost importance for both treatment and infection control. A critical mass of resistance seen in high burden countries has great potential not only to halt the progress of TB control but also to reverse it.³

Factors contributing to recent outbreaks and continued spread of MDR-TB include spread of HIV/AIDS, inefficient control procedures and laboratory delays in identification and susceptibility testing of Mycobacterium tuberculosis isolates. Multidrug- resistance occurring primarily as a consequence of poor treatment services could lead to a severe, stronger extensively drug-resistant tuberculosis (XDR-TB) if MDR-TB is not managed properly. Hence, early detection of MDR-TB cases are highly essential in order to curb the further spread of resistant strains.

Even though liquid-based indirect susceptibility tests have improved the turnaround time (TAT), they are still not rapid enough to allow timely decisions on patient management in case of MDR-TB. More rapid TB susceptibility tests are needed, particularly in TB high burden countries. Recently, some of the tests done directly from sputum specimens without the need for cuture are being studied with prospects for applicability in developing countries. One among them is the nitrate reductase assay (NRA), a calorimetric- based rapid culture technique.

The NRA test, initially introduced as an indirect assay is performed on solid medium as for the proportion method, though liquid-based assays have recently been studied.^4,5 The medium is supplemented with potassium or sodium nitrate at a concentration of 1000 mg/L to act as a growth indicator. Live M. tuberculosis organisms possess the nitro-reductase enzyme and will reduce nitrate to nitrite, which is then detected as a pink-purple colour when a detection reagent (Griess reagent) is added to the tube.⁶ This principle is used for DST where the drug containing media are tested and the colour change in these media indicate resistance to the particular drug being tested, while absence of any colour change indicates that the organism is susceptible to the drug.

Study settings: The study was a comparative-validation study carried out on patients of age greater than 18 years attending the Chest Clinic of Maulana Azad Medical College and from New Delhi Tuberculosis (NDTB) Centre, who were suspected to have multidrug-resistant (MDR) pulmonary tuberculosis (n =32). After proper counselling and obtaining their informed consent, these patients were included in the study.  All the relevant details were taken on a pre- designed proforma.

The following MDR- TB suspect definition was used to select patients:

1. Any TB patient who fails an RNTCP category I treatment regimen,e.

• an initially sputum smear positive patient who remains or becomes sputum positive after 5 months of taking the regimen or
• an initially smear negative patient who becomes smear positive at any time after starting the regimen

1. Any RNTCP Category II patient who is sputum smear positive at the end of 4 months of treatment or later, or
2. Close contacts of MDR-TB patients who are found to have smear positive pulmonary TB disease.

We excluded patients with negative sputum smear by direct microscopy and those not showing Mycobacterium tuberculosis in their culture.

Sample collection and transport: Two sputum specimens, five to ten ml each were collected in sterile leak proof containers from each MDR- pulmonary TB suspect attending the Chest Clinic of Lok Nayak Hospital and NDTB Centre (Intermediate Reference Laboratory). These samples were then transported quickly to the Mycobacteriology laboratory, Maulana Azad Medical College.

Laboratory Methods: The samples were decontaminated by N-Acetyl L-Cysteine and 1% Sodium hydroxide (NALC-NaOH) and used for conventional laboratory techniques (microscopy and Lowenstein Jensen (LJ) media culture) and the culture isolates were used for drug susceptibility testing (DST) using Proportion method and indirect nitrate reductase assay (NRA) on Blood Agar.

All LJ bottles were incubated at 37°C and examined daily for the first 5-7 days to detect the rapidly growing mycobacteria and contaminants, then weekly upto 8 weeks. Growth was confirmed by Ziehl- Neelson staining for AFB along with assessment of colony morphology, pigmentation and growth rate. Positive cultures were subcultured on LJ slopes for niacin and catalase test to confirm Mycobacterium tuberculosis.

Preparation of blood agar slants:

Blood agar-based medium (Himedia) was prepared according to the manufac­turer’s recommendations with few modifications. Five millilitres of glycerol per litre of medium and potassium nitrate (1000 μg/mL) were added to the medium later sterilised using an autoclave. After being cooled to 45ºC, the media was supplemented with 5% defibri­nated sheep blood.  The media was allowed to settle as slants. Media with antibiotics were prepared from stock antibiotic solutions. The critical antibiotic concentra­tions used in the blood agar medium were 0.2 μg/mL of Isoniazid and 1 μg/mL of Rifampicin.

Statistical analysis

The comparison of results of drug susceptibility testing by indirect NRA on blood agar was compared with that of proportion method (gold standard) and their percentage agreement was studied. NRA was validated against proportion method using sensitivity, specificity, positive predictive value and negative predictive value. Kappa value was calculated and a value of more than 0.75 represents excellent agreement beyond chance, a kappa below 0.40 represents poor agreement, and a kappa of 0.40 to 0.75 represents intermediate to good agreement. Data was entered in Microsoft Excel and for analysis SPSS latest version was used.

Clinico-social demographic data:

Table 1: Age and Gender wise distribution of cases in the study group (n=32)

From Table 1, we infer that the study participant’s age ranged from 18 years to 75 years. Maximum number of cases (n=16; 50%) were in the age group of 18-30 years. The mean age of the patients was 33.34 ± 2.34 years. It is clear that out of 32 total patients in the study, 68.8% (n=22) were males and 31.2% (n=10) were females. In the present study, common complaints by the patients in decreasing frequency were cough (n=26; 81.2%) and anorexia (n=26; 81.2%), followed by weight loss (n=25; 78.1%) and fever (n=25; 78.1%), breathlessness (n=16; 50%). Few participants also mentioned blood in sputum (n=6; 18.8%) and other complaints like chest pain (n=3; 9.4%). Crepitation on examination was present in 8 cases (25%).

Microbial data:

Of the entire sputum smear positive patients included, half of them (n=16) showed 2+ RNTCP grading, while a quarter (n=8) showed 3+ grading. About 15.6% of patients (n=5) had a 1+ grade, while 9.4% (n=3) had scanty bacilli per 100 oil immersion fields. All patients (n=32; 100%) had positive Lowenstein-Jensen medium growth of rough, tough and buff colonies that were AFB positive, showed canary yellow colour when tested for Niacin and produced less than 45 mm bubbles indicating weak catalase activity confirming Mycobacterium tuberculosis strain.

Drug Susceptibility data:

Table 2: Comparison of drug susceptibility testing (DST) results by indirect nitrate reductase assay (NRA) on blood agar Vs proportion method (PM) on LJ (n=31)

(Table 2) provides an overview of parameters like sensitivity and specificity of indirect blood agar NRA to detect resistance for isoniazid, which was 88.9% and 90.3% respectively while it was 89.5% and 100% respectively for rifampicin. It had an exemplary positive predictive value of 94.1% and 100% for INH and RIF respectively. An excellent agreement between the results of indirect NRA blood agar and proportion method for isoniazid and rifampicin resistance was found with kappa value of more than 0.8 for both.

Table 3: Number of MDR-TB cases detected

From Table.3, it is clear that the percentage of cases which came out to be MDR tuberculosis by proportion method (PM) on LJ medium and by indirect NRA on blood agar were 43.8% and 29% respectively.

Turnaround time (TAT):

The results were available by Day 7 for 48.4% of the strains by indirect NRA on blood agar, for 45.2% by Day 10 totalling to 93.6% and by 14 days for the remaining 6.4% strains. Thus by Day 14, the DST results were available for all (100%) samples.

Although since centuries before we had been diagnosing M. tuberculosis, its challenges still continue despite modern technological advancement. First, M. tuberculosis is a slow grower limiting its on-site patient care approach. Secondly, patients with pulmonary tuberculosis do not exhibit early symptoms leading to delay in diagnosis. Moreover, certain diagnostic tests cannot detect the bacteria if it is in low concentration.⁷

In this study, we provide an overview of the performance of a laboratory-based drug susceptibility method, nitrate reductase assay (NRA) using blood agar for Mycobacterium tuberculosis. The incidental growth of M.tuberculosis colonies on blood agar was first reported by Drancourt et al.,⁸ and termed this finding in the "end of dogma." This discovery led to further research on blood agar for cultivation of M.tuberculosis.^{9, 10, 11} It is preferred over conventional LJ medium owing to the special conditions required for media preparation of the latter and to the potential interactions between antibiotics and phospholipids in the LJ medium.

In the present study, comparison of indirect NRA on blood agar with proportional method (PM) on LJ media showed that there was an excellent agreement beyond chance in the DST results for isoniazid (90.3%) and rifampicin (93.5%) by the two methods with the kappa coefficient of 0.80 and 0.87 respectively. The sensitivity and specificity of indirect NRA on blood agar to detect resistance for isoniazid was 88.9% and 92.3% respectively and for rifampicin it was 89.5% and 100% respectively. The overall sensitivity and specificity of indirect NRA on blood agar for drug susceptibility testing was 89.2% and 96.2% respectively.

Various other studies have evaluated DST results on blood agar with gold standards. When the proportion method on blood agar was compared with radiometric proportion method, the sensitivity, specificity, positive and negative predictive value for INH and RIF was all 100% by Coban et al.¹² with a turnaround time of 14 days of incubation. Similarly, Yildiz et al.,¹³ compared the performance of proportion method on human and sheep blood agar with radiometric proportion method (BACTEC 460 TB) for INH drug susceptibility alone. The agreement was again 100% for INH.

However data on DST by NRA on blood agar are limited. In a comparable study by Coban et al.,¹⁴ agreement between indirect NRA on blood agar and BACTEC 460 TB was found to be 93.8% for both INH and RIF. The specificity of the NRA test for INH and RIF was found to be 94.2% and 96.4% and the sensitivity was found to be 92.8% and 90.4%, respectively correlating well with our study findings.

Drancourt and Raoult¹⁰ compared the Bactec 9000 MB system with blood agar and inferred that while the cost of blood agar was €1913 for the analysis of 1000 samples, it was increased to € 8990 with the Bactec 9000 MB system. Time of detection was 19±5 days for blood agar and 22±6 days for the Bactec 9000 MB system.  

Turnaround time in our study, for the indirect NRA on blood agar was Day 7 for 48.4% of the strains, 45.2% by Day 10 totalling to 93.6% and 14 days for the remaining 6.4% strains. In the study by Coban et al.,¹⁴ results were available between days 7-14 days, with the majority being available by the 10th day.

Limitations in the current study are Mycobacterium cannot be speciated based on the colony morphology on blood agar and further testing is required to confirm the species. Therefore it cannot be used for testing DST directly from specimen. The DST result of blood agar was not compared with MGIT, BACTEC or other automated system that can grow Mycobacterium early. Further studies with larger sample sizes are warranted to validate these findings.

Indirect nitrate reductase assay (NRA) on blood agar had an excellent agreement beyond chance with the proportion method for drug susceptibility testing (DST) of isoniazid and rifampicin. Being a basic medium used in most laboratories, blood agar can be a rapid and cost-effective alternative with an earlier turnaround time than most other conventional DST methods done on Lowenstein-Jensen medium, in a resource-limited setting. However further studies are required to compare DST of M .tuberculosis using the principle of NRA on blood agar. 

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