Analysis of molecular mechanisms of drug resistance of Mycobacterium tuberculosis in patients with pulmonary tuberculosis and its pharmacoeconomics

Purpose: To investigate the molecular mechanisms of drug resistance of Mycobacterium tuberculosis in patients with pulmonary tuberculosis and its pharmacoeconomics. Methods: Data pertaining to patients with primary tuberculosis treated in the First Affiliated Hospital of Zhaoqing Medical College, Zhaoqing, China from January 2020 to June 2021 were retrospectively analyzed. Sputum specimens were collected from all eligible patients, and 151 uncontaminated specimens with good bacteriophage activity were screened. Results: A total of 107 Mycobacterium tuberculosis strains were isolated from the 151 specimens, 31 of which strains were resistant to varying degrees to rifampicin, isoniazid, streptomycin, and ethambutol with an overall resistance of 28.97 %. There were 16 strains with rpoB mutation, 22 strains with katG mutation, and 8 strains with inhA mutation. The difference in the sputum negative rate, lesion absorption rate, and tuberculosis cavity closure rate, and total medical cost between the two group were not statistically significant (p > 0.05). The incidence of adverse reactions in the FDC group was significantly lower than that in the blister pack group (p < 0.05). Conclusion: The total resistance of Mycobacterium tuberculosis in primary tuberculosis patients remains at a high level, and the development of resistance is associated with mutations in rpoB, katG, and inhA genes. FDC regimen provides more pharmacoeconomic and therapeutic benefits than blister pack regimen.


INTRODUCTION
China has a heavy clinical burden of tuberculosis, with the third-highest prevalence worldwide [1]. Pulmonary tuberculosis is managed using first-line anti-tuberculosis drugs, such as rifampicin, isoniazid, streptomycin, and ethambutol. However, clinical practice has revealed an increasing trend in drug resistance in pulmonary tuberculosis, due to factors such as age and improper anti-tuberculosis treatment [2]. Prior research has shown that drug resistance gene mutations are a significant molecular mechanism of drug resistance in pulmonary tuberculosis [3].
It has been reported that, compared with ordinary tuberculosis, the long course and treatment difficulties for multidrug-resistant tuberculosis impose a serious economic burden on patients and their families, which consequently impairs patient compliance and complicates the treatment [4]. Blister pack and fixed-dose combination (FDC) medications are the current treatment regimens for pulmonary tuberculosis with promising efficacy [5]. Accordingly, this study was designed to compare FDC and blister pack regimen as well as analyze the molecular mechanism of drug resistance of Mycobacterium tuberculosis and some pharmacoeconomics outcomes.

Clinical data
The medical data of patients with primary tuberculosis treated in the First Affiliated Hospital of Zhaoqing Medical College, from January 2020 to June 2021 were retrospectively analyzed. Sputum specimens were collected from all patients, and a total of 151 specimens with no contamination and good bacteriophage activity were screened.

Inclusion criteria
Patients aged 18 -60 years, with a confirmed diagnosis of pulmonary tuberculosis [6], with positive acid-fast bacilli smear or a positive mycobacterial culture, with complete clinical data, and who received FDC or blister pack medications (2HRZE/4HR protocol) were included.

Exclusion criteria
Patients with disseminated tuberculosis, tuberculous pleurisy, or extrapulmonary tuberculosis, with contaminated strains or a low survival of strains after culture, who were pregnant or lactating, with allergies to antituberculosis drugs, or who failed to complete the prescribed course of treatment were excluded.
There were 92 males and 59 females, aged 20 to 58 years, with a mean age of 51.29 ± 5.73 years, and the duration of disease ranged from 1 to 24 months, with a mean duration of 7.79 ± 4.96 months. The study complied with the relevant regulations of hospital clinical research ethics and it was approved by the ethics committee of the First Affiliated Hospital of Zhaoqing Medical College (approval no. 2019-12-26). The study protocol was conducted in strict accordance with the guidelines of Helsinki Declaration [7].

Species identification
Non-tuberculous mycobacteria and Mycobacterium bovis were excluded using pnitro benzoic acid (PNB) selective medium and 2-thiophenecarboxylic acid hydrazide (TCH) selective medium. The colonies of Mycobacterium tuberculosis cultured for 2 -3 weeks were ground in a sterile glass grinder, diluted with 0.9 % saline containing 0.05 % Tween 80, and made into 1 mg/mL bacterial suspension using the McFarland standards to adjust the turbidity of the suspension. After diluting the concentration to 10 -2 mg/mL using a 22SWG standard inoculation loop, 0.01 mL of the solution was inoculated into PNB and TCH medium and incubated at 37 o C with 5 % CO2 for 4 weeks. In the case of Mycobacterium bovis, no colony growth was observed on PNB and TCH media. Colonies growing only on TCH medium were considered Mycobacterium tuberculosis, and those growing on both PNB and TCH media were considered non-tuberculous mycobacteria.

Drug susceptibility test
In accordance with the National Bacteriological Procedures for Tuberculosis (NBPT), drugcontaining media were prepared by dissolving rifampicin (40 μg/mL), isoniazid (0.2 μg/mL), streptomycin (4 μg/mL), and ethambutol (2 μg/mL) in Roche media (Zhuhai Yinke Medical Engineering Co., Ltd.), solidified at 85 o C for 50 min and kept at 4 o C away from light after inspection to ensure no contamination. The bacterial solution was diluted to a concentration of 10 -2 mg/mL and 10 -4 mg/mL using a 22SWG standard inoculation loop, and inoculated evenly into the slant of drugcontaining media and blank medium respectively by streaking, and incubated at 37 o C and 5 % CO2 for 4 weeks. Drug resistance (DR) was calculated using Eq 1. DR (%) = (CD -CM) / CB x 100 ……… (1) where CD represent the colonies grown in drug, CM is the containing medium and CB are the colonies grown in blank medium The percentage of drug resistance greater than 1 % was considered resistant to the drug. Monoresistance cases refer to resistance to a single first-line anti-tuberculosis drug, poly-resistance cases refer to resistance to two or more first-line drugs but not to both isoniazid and rifampicin, while multidrug resistance cases refer to resistance to first-line anti-tuberculosis drugs including at least isoniazid or rifampicin.

Drug-resistant gene determination
The DNA extraction fresh colonies were collected using the inoculation loop, transferred in a centrifuge tube containing 1.5 mL of sterile saline, and inactivated at 100 °C for 30 min. The solution was then centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. A 400 μL volume of TE buffer (Beijing Solabao Biotechnology Co., Ltd., item No. T1120) was added to the solution and the cell pellet was suspended evenly, followed by the addition of lysozyme (Beijing Ita Biotechnology Co., Ltd., Item No. SY3854), and the solution was then incubated overnight at 37 o C. The DNA of Mycobacterium tuberculosis was extracted according to the instructions of cetyltrimethylammonium bromide (CTAB) kit (Shanghai JingAn Biological Engineering Co. Ltd., Item No. JK-EA02057), and stored at -20 o C, prior to the determination of DNA concentration using the ultra-micro UV-Vis spectrophotometer (METTLER TOLEDO, Switzerland, model UV5Nano) and 10-fold dilution. The primers for polymerase chain reaction (PCR) amplification were synthesized by Shanghai Bioengineering Co ( Table 1) cycles, 68 o C for 10 min. Then 2 μL of amplification products were subjected to agarose gel electrophoresis, and the gel imaging system was used for photography. The PCR products were subjected to DNA sequencing by Shanghai Bioengineering Co. Ltd and compared with the standard sequences of rpoB, katG, and inhA genes in the NCBI database.

Treatments
The blister pack group was treated with 0.3 g of bulk isoniazid tablets (NMPA approval no. H51020436) daily, 0.45 g of rifampin capsules (NMPA approval no. H51022410) daily, 0.5 g of pyrazinamide tablets (NMPA approval no. H51022733) daily, and 0.75 g of ethambutol hydrochloride tablets (NMPA approval no. H51020720) daily. After 2 months of treatment with the drugs, isoniazid tablets and rifampin capsules were administered for another 2 months. The FDC group was given an antituberculosis fixed-dose combination manufactured by Shenyang Hongqi Pharmaceutical Co. Ethambutol hydrochloride + pyrazinamide + rifampin + isoniazid tablets (NMPA approval no. H20090219) were administered for 2 months, 4 tablets daily, and isoniazid tablets (NMPA approval no. H20103325) were administered for 4 months, 2 tablets daily.

Sputum negative rate
Negative sputum culture for 2 consecutive months and no re-positive cases observed were considered a successful conversion from positive to negative.

Lesion absorption
Chest CT showing lesion reduction > 70 % indicates significant absorption, a reduction of 50 to 70 % indicates partial absorption, a reduction of < 50 % indicates no absorption, and a significant enlargement of more than 50 % indicates deterioration. The total effectiveness (T) was calculated with the formula in equation 2.
Where S is significant absorption, and P is partial absorption.

Tuberculosis cavity closure
Closure of the tuberculosis cavity or disappearance of the cavity shown by chest CT is considered a tuberculosis cavity closure. The diameter of the cavity, if equal to 1/2 or less of the original diameter is considered a reduced tuberculosis cavity. Reduction or enlargement of the diameter of the cavity less than 1/2 of the original diameter is considered an unchanged cavity, while an enlargement of the diameter of the cavity by 1.5 times of the original diameter and above is considered increased cavity. The total effectiveness (T1) was calculated as in Eq 3.
where C is the cavity closure, and R is reduced cavity.

Adverse reactions
The occurrence of gastrointestinal reactions, liver damage, allergic reactions, hyperuricemia, and leukopenia in patients during treatment were recorded.

Medical cost
The total cost includes direct medical cost, direct non-medical cost, indirect cost, and hidden cost. The direct medical cost includes medication fee, examination fee, bed fee, nursing fee, hospitalization fee, and fee for adverse reaction management.

Statistical analysis
The Statistical Packages for the Social sciences (SPSS) 20.0 software was used for statistical analyses. Normally distributed measurement data are expressed as mean ± standard deviation (SD), and independent samples t-test was used for comparison between the two groups. Count data are expressed as frequencies or composition ratios and analyzed using chi-square test. A difference was considered statistically significant at p < 0.05.

Species identification
After preliminary identification using PNB and TCH selective media, a total of 107 strains of Mycobacterium tuberculosis, 39 strains of Mycobacterium bovis, and 5 strains of nontuberculous mycobacteria were isolated from the 151 sputum specimens, of which 16 strains with rpoB mutation ( Table 2).    Table 5).

Efficacy of treatment regimens
The difference in the sputum negative rate, lesion absorption rate, and tuberculosis cavity closure rate between the two groups did not come up to the statistical standard (P > 0.05) ( Table 6).

Incidence of adverse reactions
The incidence of adverse reactions in the FDC group (7.89 %) was significantly lower than that in the blister pack group (18.67 %) (p < 0.05) ( Table 7).

Costs treatment protocol
The total cost per capita in the blister pack and FDC groups was 9813.14 ± 249.26 Yuan and (9693.28 ± 236.37) yuan, respectively. The FDC group had a lower total cost per capita than the blister pack group, but the difference was not statistically significant (t = 135.27, p = 0.216).

Cost-effectiveness and sensitivity of the two treatment regimens
A cost-effectiveness analysis was performed on the two treatment regimens. The cost (C) / effective (E) ratio was 105.15 for the blister pack group and 100.92 for the FDC group, and the ∆C/∆E for the blister pack group was 125.29 with the lowest cost FDC group as a parameter.
A univariate sensitivity analysis assuming a 15 % decrease in drug price and no change in treatment effect found that the C/E ratios for the blister pack and FDC groups were 89.33 and 85.70, respectively, and the ΔC/ΔE for the blister pack group was 113.87 using the lowest cost FDC group as a parameter (Table 8).

DISCUSSION
The global tuberculosis report released in October 2020 stated that TB remains the leading cause of negative health status of people and one of the top 10 causes of death worldwide [9]. Mycobacterium tuberculosis drug resistance or multidrug resistance is a major contributor to mortality in patients with tuberculosis [10]. In this study, an analysis of sputum specimens from patients with primary tuberculosis admitted to the hospital from January 2020 -June 2021 revealed that the overall drug resistance prevalence of pulmonary tuberculosis was 28.97 %, which was higher than that reported by Yuan Wei et al [11] and by Li et al [12]. The development of drug resistance in Mycobacterium tuberculosis is considered to be attributed to mutations in drug resistance genes, and drug resistance-related genes include rpoB, katG, and inhA [13]. Drug resistance gene testing of Mycobacterium tuberculosis is of great clinical value to provide guidance for proper clinical regulation of drug use [14]. In the present study, gene sequencing after PCR amplification of drug-resistant Mycobacterium tuberculosis DNA from 31 extracted strains revealed that the degree of mutation rates of rpoB, katG, and inhA genes were 51.61, 70.97, and 25.81 %, respectively, which were basically consistent with the results of the previous study [15]. The results suggest an association between the development of drug resistance in Mycobacterium tuberculosis patients with primary tuberculosis and mutations in the rpoB, katG, and inhA genes, with higher mutation rates in the rpoB and katG genes in mono-and multidrug-resistant strains and higher mutation % in the rpoB gene in polyresistant strains, in accordance with previous findings [16].
Research has demonstrated that blister pack medication significantly improves compliance and provides high overall clinical efficacy when compared to traditional dosage forms [17]. Fixeddose combination (FDC) is a synthetic formulation with a fixed-dose based on compounded formulations. An application of FDC to the treatment of patients with pulmonary tuberculosis was found to be moderately effective and to reduce the economic burden of the disease by improving patient compliance [18]. The results of sputum negativity rate, lesion absorption rate, and cavity closure rate in the present study were consistent with the results of previous studies. The above indexes were slightly higher in the FDC group than in the blister pack group, suggesting that FDC may be a preferred treatment option for the patients. Furthermore, the FDC group showed a significantly lower incidence of adverse reactions than the blister pack group, which was attributed to the reduction of anti-tuberculosis drug doses and the improvement of drug safety by FDC. For tuberculosis patients, especially those with drug resistance, the economic burden is a major factor impacting their treatment efficacy and relapse [19][20]. In the present study, the total cost per capita in the FDC group was lower than that in the blister pack group, but no significant difference was detected, and cost-effectiveness analysis revealed a C/E ratio of 105.15 and 100.92 in the blister pack and FDC groups, respectively, indicating a better performance of FDC regimen versus the blister pack method.

Limitations of this study
The limitations of this study include the absence of pharmacoeconomic analysis of other treatment regimens for patients with primary tuberculosis and treatment regimens for patients with relapsed tuberculosis, which will be further analyzed in future studies.

CONCLUSION
The development of resistance is associated with mutations in the rpoB, katG, and inhA genes. The FDC regimen provides more pharmacoeconomic and therapeutic benefits when compared to the blister pack medication regimen. There will be need for further studies for a more detailed pharmacoeconomic analysis.

Ethical approval
This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhaoqing Medical College, China (approval no. 2019-12-26).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.