Multi Drug Resistant Tuberculosis Detection by Phenotypic Method inTreatment Naïve Pulmonary Tuberculosis Patients

Introduction

Tuberculosis (TB), a curable and preventable communicable disease caused by the bacilli, Mycobacterium tuberculosis continues as the leading cause of death due to a single infectious agent above HIV/AIDS. Currently, TB is the thirteenth leading cause of death globally and might rank as the second leading cause of death from a single infectious agent after COVID- 19. India ranks first among the eight countries that share two-thirds of the global total of TB. The year 2020, witnessed a large global drop in the reporting of newly diagnosed TB patients due to the ongoing COVID-19 pandemic, which has also upturned the years of progress achieved in the global elimination of TB.¹

The emergence of drug resistance (DR) in TB, observed from the time anti-TB drugs were available for their initial use in the late 1940s, is hindering the nation’s national strategic plan (NSP) target of ending TB by 2025.2,3The proportion of multi drug-resistant tuberculosis (MDRTB), defined as resistance to Rifampicin and Isoniazid – the two potent first-line drugs, in treatment naïve and previously treated patients is maintained at 3-4% and 18-21% respectively, globally, for a decade.^1,4

Drug susceptibility tests in TB are done for the following reasons: a) To identify relapse or re-treatment cases, b) To change the drug regimen when drug resistance is suspected and c) For conducting drug resistance surveillance studies in a region/country. Three methods are generally available for determining phenotypic drug susceptibility of mycobacteria on LJ (LowensteinJensen) media. They are the proportion method, absolute concentration method (MIC method) and the resistance ratio method. All three methods are equally satisfactory, when properly standardized and performed. In India, large numbers of laboratories and laboratories under the National Tuberculosis Elimination Program (NTEP) have standardized proportion methods of drug susceptibility testing (DST). Hence, the proportion method is advised as the routine and commonly used DST method.⁵

Considering the higher chances of transmission of resistant strains to treatment-naive patients, drug resistance testing (DRT) for MTB is recommended for all confirmed cases of TB. Phenotypic DST in TB is still considered a reliable approach to detect DR in TB due to its ability to quantitatively predict both resistance and susceptibility, in addition to its excellent clinical correlation and quality control networks.³ Hence the present study was undertaken to identify MDR -TB in treatment-naive pulmonary TB patients by phenotypic DST.

Methods

The current study was a hospital-based observational, cross-sectional study conducted on treatment-naive pulmonary TB patients visiting the Respiratory Medicine Department from September 2018- to June 2021. The study was approved by the Institutional Ethics Committee (VIEC). No. VIEC/2019/APP/001.

Culture-positive isolates from newly diagnosed pulmonary tuberculosis patients of all age groups, both male and female, with no previous treatment history of anti-tubercular therapy (or less than one-month history) were included. Culture positive isolates from extra-pulmonary tuberculosis patients and those who have taken anti-tubercular drugs for more than 28 days were excluded from the study. Culture positive isolates obtained from treatment-naive or newly diagnosed pulmonary tuberculosis was confirmed as MTB by their slow growth rate, colony morphology on LJ media (rough, tough, buff-coloured colonies), smear microscopy from cultures and SD Bioline TB Ag MPT64 Rapid test (Figure 1).⁶

Isolates were then subjected to Indirect Drug susceptibility testing using an economic variant of the 1% proportion method on Lowenstein Jensen (LJ) media slants for five first-line anti-tuberculosis drugs. The antitubercular drugs used include: Streptomycin (STR) (4 µg/ml), Isoniazid (INH) (0.2 µg/ml), Rifampicin (RIF) (40 µg /ml), Ethambutol (EMB) (2 µg /ml) and pyrazinamide (PZA) (200 µg/ml).

The proportion method estimates the proportion of mutants resistant to a given drug by calculating the number of colonies on drug-free media and drugcontaining media. The proportion of bacilli resistant to a given drug is then determined by expressing the resistant portion as a percentage of the total population.⁵

Approximately 4-5 mg growth from the primary culture was taken with a standard wire loop and placed in a McCartney bottle containing 1ml sterile distilled water (SDW) and 3 mm size, 6 glass beads to prepare bacterial inoculum for proportion method. Under continuous shaking, 4-5 ml of distilled water was added gradually and the bottle was vortexed for 20-30 seconds. Allow the coarse particles to settle down. Decant the suspension carefully into another clear, sterile McCartney bottle. The final concentration of 1 mg/ml of tubercle bacilli, (S1- neat suspension) was made by matching with McFarland standard No.1.

1 ml SDW with six 3 mm glass beads + 1 loopful

(3 mm internal diameter) of culture

                          ↓

Vortexed for 20 – 30 seconds

                          ↓

To the above add 4 ml of SDW

Adjusted turbidity with McFarland 0.05 with SDW

               (neat suspension)

                          ↓

2 loopful of neat + 2 ml of SDW (S2 -10 –2 dilution)

                          ↓

2 loopful of S2 + 2 ml of SDW (S4 -10-4 dilution)

One standard loopful of the 10–2 and 10–4 dilution of the bacterial suspension was inoculated onto drug-free as well as drug-containing LJ slopes. The slopes were incubated at 37°C. The first reading of proportion tests was read on the 28th day and the final result was on the 42nd day of inoculation.

The following criteria were used for the interpretation of the test:

• The first reading was taken on the 28th day after inoculation

• Colonies were counted first on the neat, followed by the 10-2 and 10-4 dilutions (both drug-free and drugcontaining slopes)

• The number of resistant bacilli contained in the inoculum was obtained by the average number of colonies grown on the drug-containing slopes

• Any strain with 1% (the critical proportion) of bacilli resistant to any of the antibiotics, was classified as resistant to that drug. The highest count obtained on the drug-free and the drug-containing medium should be taken for calculating the proportion of resistant bacilli (regardless of whether both counts were obtained on the 28th day, or both on the 42nd day of reporting). MDR results if available to be given on the 28th day.

The following formula was used for identifying drug-resistant populations

Highest no. of growth on drug-containing medium/ Growth on drug-free medium× 100

More than or equal to 1% = Resistant

Less than or equal to 1% = Sensitive

• If the result on the 28th day is resistant, no further reading of the test for that drug is required: the strain is classified as resistant for that drug.

• If a strain is reported as negative on the 28th day, it should be read on the 42nd day too, before releasing the result.

• If the strain is resistant to all the drugs on the 28th day, then the report is given on the same day.

• Repeat the test, if growth on the control media is poor even after six weeks.

H37RV procured from National Tuberculosis Institute (NTI), Bengaluru was used as the internal quality control strain

Data was entered in MS Excel and analysed using SPSS version 19. Frequencies and proportions, which are descriptive statistics were used to summarize the data

Results

A total of 240 culture-positive isolates obtained from treatment naïve pulmonary tuberculosis patients were enrolled in the study, which included 161 (67%) from male patients and 79 (33%) from female patients (Table 1). Of the 240 isolates subjected to phenotypic drug susceptibility testing by 1% proportion method, 127 (52.9%) isolates were susceptible to all the first-line anti-tubercular drugs (Figure 2). Of the 127 susceptible strains, 45 (35.4%) were from females and 82 (64.6%) were from males. Resistance to any drug was observed in 113 (47 %) isolates. Resistance to any drugs exhibited by female and male patients were 34 (30%) and 79 (70%) of the culture-positive isolates respectively (Table 2).

Any rifampicin resistance was observed in 53 (22%) of the isolates, any isoniazid resistance was observed in 45 (18.8%) of the isolates, any streptomycin resistance was observed in 47 (19.6%) of the isolates and any pyrazinamide resistance was observed in 63 (26.25%) of the isolates. No resistance was observed for the first-line antibiotic, Ethambutol, by any of the culture-positive isolates (Table 3).

Mono resistance to Rifampicin, Isoniazid, Streptomycin and Pyrazinamide was observed in 11 (4.6%), 8 (3.3%), 15 (6.3%) and 14 (5.8%) of isolates respectively (Table 4).

MDR pattern, (resistance to RIF+INH combination, with or without resistance to other first-line drugs) (Figure 3) were observed in 30 isolates out of the 240 culture positives subjected to DST by 1% proportion method, making a proportion of 12.5% (30/240). The different MDR patterns observed in the study were: RIF+INH12 (40%), RIF+INH+STR- 2 (7%), RIF+INH+PYR- 9 (30%) & RIF+INH+STR+PYR- 7 (23%) (Table 5).

Discussion

Drug-resistant TB is a growing concern, posing a great threat to public health and a major challenge in TB control programmes globally compared to drugsusceptible TB, due to the requirement of complex and prolonged therapy with highly expensive and toxic drugs, with severe side effects and lower success rates. Hence, prompt diagnosis of TB and surveillance of drug-resistant patterns in MTB strains is highly essential for understanding the burden of drug-resistant TB and developing measures for preventing transmission of drug resistance in the community, especially in countries with low-resource settings, where facilities to diagnose MDR-TB is not readily available.⁷ The current study was intended to assess the level of drug resistance to any of the first-line anti-TB drugs and the incidence of MDR-TB in MTB strains isolated from treatment-naive pulmonary TB patients.

Culture positive isolates obtained from treatment-naive males and females in our study was 67% and 33% respectively (Table1), which was concomitant with the similar study by Banik et al., which yielded 65% and 35% pure isolates from men and women population respectively.⁸

Resistances to any of the first-line drugs was 47% and pan sensitivity was 53% in the current study (Table 2). Garrido et al., reported pan susceptibility and any resistance to first-line drugs in 85.7% and 14.3% of isolates respectively.⁹ However, Banik et al., reported pan sensitivity in only 30% and any resistance in 70% isolates.⁸

The resistance rates (%) observed to various firstline drugs in the current study were Rifampicin 22, Isoniazid 18.8, Streptomycin 19.6, Pyrazinamide 26.3, and Ethambutol 0 (Table 3). The resistance rates to individual anti-tubercular drugs considerably varied among different studies.^8-15 None of our isolates exhibited resistance to Ethambutol antibiotic in the present study. The rate of initial resistance to Ethambutol was low ranging from 0 - 4.2 percent globally.¹³ At the global level, a Tanzanian study did not report any resistance to the Ethambutol drug, similar to our findings.¹⁵

The prevalence of mono-resistance to Rifampicin, Isoniazid, Streptomycin and Pyrazinamide observed in the current study was 4.6%, 3.3%, 6.3% and 5.8% respectively (Table 4). Mono resistance, is defined as the resistance exhibited to a single first-line anti-TB drug only.¹⁶ Different surveys done by WHO using standardized guidelines showed that the levels of primary resistance to isoniazid as a single-agent ranged from 0 - 16.9% for streptomycin; 0.1 - 23.5% for rifampicin; 0 – 3%.¹³ Mono resistance was noted mostly to Isoniazid (30%) and pyrazinamide (10%) in a study in Meghalaya.⁸ Mathuria et al., reported a lower rate of resistance to a single drug, ranging from 0.0 to 6.3% in new cases in a multi-centre study from North India.¹⁴

The proportion of MDR-TB (12.5%) was found to be high in the study population in the present study (Table 5). Drug resistance rates to first-line drugs among new TB patients varies across the globe due to the differences in performances of TB control programme, sample size, study area, bias in patient selection, geographical settings, differences in the methodology adopted for the studies etc.⁸ Sharma et al., reported a low prevalence (varied from 0.14-5.3%) and high prevalence (varied from 13-24%) of MDR-TB among new cases from different parts of India.¹² Our findings are in line with the study by Jain et al., who reported a high prevalence of MDR-TB among new patients in their study area.¹³

However, our observations may not be generalizable to the entire country or region due to the limited data reported and cannot conclude anything about the transmission of MDR-TB. More studies and rigorous testing of these isolates using molecular epidemiological tools, like Line probe assay and whole-genome sequencing should be done to identify any additional drug resistance patterns.

Conclusion

The growing number of MDR-TB in treatment-naive patients helps in circulating drug-resistant TB in the community and hinder the global and national target of the End TB Strategy adopted by WHO and the Government of India, respectively. Hence, continuous surveillance of MDR-TB in every newly notified TB case should be carried out by the joined teamwork of public health centres (PHCs), health care workers, primary care physicians, private practitioners and national TB services to manage TB and provide appropriate and exact treatment to interrupt the chain of transmission of DR-TB in the community to attain the nation’s target of ending TB by 2035.

Conflicts of interest

None.