ABSTRACT
Background:
The Mycobacterium tuberculosis (MTB) is a threat to human health. However, the drug-resistant Mycobacterium tuberculosis (DR-MTB) is more threat than MTB. In the patients with pulmonary tuberculosis (PTB), the reasons of recurrence are key points for prevention and treatment.
Objective:
This study aimed to compare the characteristics of DR-MTB from patients with PTB between primary treatment (PT) and retreatment (RT).
Materials and Methods:
Patients with DR-PTB, including primary treatment (DR-PTB-PT) and retreatment (DR-PTB-RT), were retrospectively studied from January 2020 to December 2023. Demographic data were collected. All the isolates were cultured using Roche culture method, drug susceptibility test (DST) using the proportion method, and detect the gene mutation using the real-time fluorescent polymerase chain reaction melting curve analysis method (MCA).
Results:
There were 123 patients with DR-PTB-PT and 133 patients with DR-PTB-RT included in this study. There were no significant differences on mean age, gender, living regions, and residence between two groups. There was a significant difference on phenotypic (P < 0.05) between two groups. There were positive correlations between the extrapulmonary sites accompanied by MTB, comorbidity, and DR-PTB types. The phenotypic DR types and comorbidity were significant risk factors for DR-PTB-RT.
Conclusion:
The codons of RpoB529–533, KatG315, Emb306, and GyrA88–94 were main gene mutation sites in the DR-PTB-PT and DR-PTB-RT in the local southwest regions of China. The poly-DR and comorbidity were the risk factors of DR-PTB-RT.
Keywords: Drug resistance, mycobacterium tuberculosis, primary treatment, pulmonary, reoccurrence
Introduction
MTB can affect the lung as well as extrapulmonary regions, such the skin, bones, soft tissues, lymph nodes, and urine. In 2015–2025, the World Health Organization (WHO) achieved a significant milestone: a 50% decrease in the incidence rate and a 75% decrease in the number of deaths, the incidence rate and death rate decreased by 8.7% and 19%, respectively, between 2015 and 2022.[1] Drug-resistant TB is around 50% of previously treated cases in the countries that made up the former Soviet Union, while the incidence of DR-TB is steady (3–4% for newly diagnosed cases and 18–21% for previously treated cases).[2] According to a meta-analysis, the combined percentage of pre-XDR-TB in the 22 nations was 26%, while the proportion of XDR-TB in MDR-TB cases was 9% in patients receiving treatment for MDR-TB. Additionally, the combined percentage of resistance to fluoroquinolones was 27%, and the proportion of second-line injectable drug resistance was 11%.[3,4] The rates of resistance to ethambutol (EMB), streptomycin (SM), and pyrazinamide (PZA) for MDR-TB were 47.2%, 42.4%, and 28.9%, respectively, while the rates of resistance to fluoroquinolones (FQs) and second-line injectable drugs were 4.1–7.1% and 9.0–14.1%, respectively, and the rate of extensively drug-resistant TB (XDR-TB) was 1.9%, among other drugs.[5] An estimate of 121,600 MDR-TB cases were calculated for China in 2012, as well as in new and previously treated cases, which was 5.4%, 12.8%, and 28.5%, respectively.[6]
China’s southwest area has a greater incidence than the other regions. Research on spinal tuberculosis,[7] genetic diversity of DR-MTB,[8] molecular characteristics of drug-resistant extrapulmonary Mycobacterium tuberculosis (DR-EPTB),[9] and whole genome sequencing (WGS) of DR-EPTB has been conducted. However, over the long decades,[10] there has been little information available about the characteristics of DR-PTB, such as primary treatment (DR-PTB-PT) and retreatment (DR-PTB-RT), in the southwest of China. This information was particularly lacking during the COVID-19 pandemic. The purpose of this study was to examine the features of DR-PTB-PT and DR-PTB-RT in China’s southwest.
The study is benefit for doctor to promote diagnosis and treatment of DR-PTB. DR-PTB needs early and precise diagnosis, and timely and standard treatment. The study of mutant gene sites can help doctor to recognize the reason of drug resistance to make right decision. The study reminds patient to pay attention to comprehensive treatment for diseases except for DR-PTB. The standard, full-course, and comprehensive treatment is necessary for the patient with DR-PTB and comorbidity, which may reduce the reoccurrence rate of DR-PTB.
Materials and Methods
Study population
From January 2020 to December 2023, the medical records of consecutive patients admitted to Chongqing Public Health Medical Center for DR-PTB were gathered. According to the WHO definition,[11] DR-PTB was diagnosed when a clinician decided to treat the patient with a full course of antituberculosis treatment after receiving a culture-positive specimen, histological evidence, or compelling clinical evidence of active MTB illnesses. The ethics committee had approved the research and the date was 2023-05-07. This study was approved by the Medical Ethics Committee of Chongqing Public Health Medical Center (2023-057-01-KY).
Clinical specimen collection
Sputum can be obtained using fiberoptic bronchoscopy or nebulization for tuberculosis patients who do not produce it. Patients who do not produce qualifying sputum should be given further instructions before having the specimen collected for analysis.
Specimen separation and culture
The specimen underwent preprocessing in accordance with the alkali treatment-neutralization centrifugation method’s specifications. Following centrifugation, the sediment was resuspended and subsequently infected with neutral Roche medium and MGIT960 Mycobacterium Growth Indicator Tubes (Bacton Dickinson and Company, USA) in accordance with the MGIT960 system’s operating instructions. The inoculated Roche media was incubated at 37°C in a biochemical incubator, and the inoculated MGIT960 Mycobacterium growth indicator tubes were incubated in a BACTEC MGIT960 apparatus (Bacton Dickinson and Company 960, USA). Any presence of any of the two culture methods was viewed as positive, and the results were documented as needed.
Phenotypic DST (p-DST)
The isolates were determined by the proportion method for DST.[12] If the resistance rate was >1% (resistance rate = number of colonies grown on drug-containing medium/number of colonies on control medium*100%), the test strain was considered to be resistant to the drug. The p-DST detection drugs included the isoniazid (INH), rifampicin (RIF), ethambutol (EMB), streptomycin (SM), rifapentine (RFT), para-aminosalicylic acid (PAS), capreomycin (CPM), levofloxacin (LVFX), ofloxacin (OFX), protionamide (1321Th), and dipasic (Dip).
HR-TB was described as rifampicin-susceptible, mono-isoniazid-resistant TB. Mono-rifampicin-resistant, isoniazid-susceptible TB was referred to as RR-TB. MDR-TB was classified as having both rifampicin and isoniazid resistances. Pre-XDR-TB was defined as resistance to rifampicin and isoniazid along with resistance to either one of the fluoroquinolones (ofloxacin, levofloxacin, or moxifloxacin) or second-line injectables (amikacin, capreomycin, or kanamycin). XDR-TB was defined as MDR-TB, plus any fluoroquinolone, plus at least one of the drugs, such as bedaquiline and linezolid. The poly-DR-TB was defined as resistance to multiple drugs but not meet to definition of HR-TB, RR-TB, MDR-TB, pre-XDR-TB, and XDR-TB.
Molecular detection
The MCA (Xiamen Zhishan Biotechnology Co., Ltd.) determines whether a sample has mutations by directly detecting and comparing the difference in melting point values between the test sample and the positive control. The specific loci were as follows: RR screening for mutations in the 27-amino acid codon region 507–533 of the MTB rpoB gene (the 81-bp core region of RRDR), and HR screening for mutations in the MTB ahpC promoter region (-44~ -30 and -15~ -3 sites), the inhA94 codon, the inhA promoter region (-17~ -8 sites) and the katA promoter region (-17~ -8 sites), and the katA promoter region (-17~ -8 sites). The screening of ethambutol resistance was performed by detecting mutations in codons 306, 406, and 497 of the MTB embB gene, and the screening of fluoroquinolone resistance was performed by detecting mutations in codons 88–94 of the MTB gyrA gene.
The specific operations were as follows: the precipitate after centrifugation was retained; 1 ml of sputum treatment solution was added and placed at 100°C for 10 min; nucleic acid extraction was carried out for the heated and sterilized specimen according to the instruction manual of extraction kit. The purified DNA was retained in centrifugation radius. The purified DNA was centrifuged in a low-temperature and high-speed centrifuge with a radius of 8.8 cm and a speed of 13000r for 2 min; the supernatant was taken and finally subjected to PCR according to the instruction manual of the gene detection kit. The results were automatically read by the software and categorized into “no MTB detected,” “unclear,” “sensitive,” “resistant,” and “no MTB detected.” If the result of drug resistance was “unclear,” it was not included in this study. The drugs of MCA detection included INH, RIF, EMB, and LVFX.
Statistical analysis
All data were presented as mean ± standard deviation (SD) or frequency. Statistical analysis for possible significant associations between the different characteristics and different TB types was performed using t test and Pearson’s Chi-squared test. The univariate analysis was used to compare the different characteristics between the DR-PTB-PT and DR-PTB-RT. All characteristics underwent multivariate logistic regression analysis. A P value of < 0.05 was considered statistically significant.
Results
Demography
There were 256 samples, including 123 patients with DR-PTB-PT and 133 patients with DR-PTB-RT in this study. The mean age of 256 patients, including 30 female and 226 male, was 51.69 ± 14.15 years old (21–81 years old). Table 1 displays distribution of patients in different regions. Most patients lived in Chongqing regions and accounted for 80.86%. The pleura was the most common extrapulmonary sites affecting MTB in all the patients, followed by the intestine and peritoneum. The diabetes was the most common comorbidity in all the patients, followed by the high blood pressure (HBP) and pneumoconiosis. There were no significant differences of gender distribution in the different characteristics between two groups.
Table 1.
Demographic characteristic of patients with DR-PTB (n=256)
| Characteristics | DR-PTB-PT (n=123) | DR-PTB-RT (n=133) | P |
|---|---|---|---|
| Mean age (years) | 51.31±14.34 | 52.05±14.01 | >0.05 |
| Sex, M/F | 109/14 | 117/16 | >0.05 |
| Extrapulmonary sites infecting MTB | >0.05 | ||
| Throat (M/F) | 2/0 | 2/0 | |
| Pleura (M/F) | 7/1 | 16/2 | |
| Intestine (M/F) | 5/0 | 10/2 | |
| Pericardium (M/F) | 1/0 | 3/1 | |
| Peritoneum (M/F) | 2/0 | 4/0 | |
| Cerebrospinal fluid (M/F) | 1/0 | 3/1 | |
| Urinary (M/F) | 1/0 | 1/1 | |
| Comorbidity | >0.05 | ||
| COPD (M/F) | 2/0 | 9/1 | |
| Pneumoconiosis (M/F) | 5/0 | 12/0 | |
| CRI (M/F) | 1/0 | 2/1 | |
| Lung cancer (M/F) | 1/0 | 2/0 | |
| Diabetes (M/F) | 16/0 | 43/4 | |
| HBP (M/F) | 5/0 | 13/3 | |
| Viral hepatitis (M/F) | 5/0 | 8/0 | |
| CHD (M/F) | 1/0 | 2/2 | |
| Others | 2/2 | 12/4 | |
| Regions | |||
| Chongqing City (M/F) | 89/11 | 94/13 | >0.05 |
| Sichuan Province (M/F) | 19/2 | 22/2 | >0.05 |
| Guizhou Province (M/F) | 1/1 | 1/1 | - |
| Residence | |||
| Urban (M/F) | 40/5 | 42/5 | >0.05 |
| Rural (M/F) | 69/9 | 75/11 | >0.05 |
Characteristics of DR between DR-PTB-PT and DR-PTB-RT
The poly-DR and pre-XDR were mainly DR types. The patients with pre-XDR accounted for 58% in the DR-PTB-PT and 51% in the DR-PTB-RT [Figures 1 and 2]. The fewest DR type was HR in two groups. The molecular DR types were mainly the INH, followed by RPF and EMB in two groups. RpoB529–533 condon was the most common gene mutation sites for RIF in two groups [Figure 3]. KatG315 condon was the most common gene mutation sites for INH in two groups, followed by the InhA promoter region [Figure 4]. Emb306 condon was the most common gene mutation sites for EMB in two groups [Figure 5]. GyrA88–94 condon was the most common gene mutation sites for LVFX in two groups [Figure 6]. In the poly-DR, the INH + RIF + SM + EMB + RFT + Dip drug resistance was the most common type, followed by INH + RIF + SM + RFT + Dip and INH + SM + Dip in the patients with DR-PTB-PT [Figure 7], while INH + SM + RIF + EMB + RFT + Dip was the most common, followed by INH + SM + RIF + RFT + Dip and INH + SM + Dip in the patients with DR-PTB-RT [Figure 8]. There were significant differences on the phenotypic DR (P < 0.05) between two groups [Table 2]. The mono-resistance was 8.94% in the DR-PTB-PT and 8.27% in the DR-PTB-RT; the poly-resistance was 27.64% and 39.85%, respectively; the MDR was 5.69% and 0.00%, respectively, and the pre-XDR was 57.72% and 51.88%, respectively.
Figure 1.
The percentages of DR types in the patients with DR-PTB-PT
Figure 2.
The percentages of DR types in the patients with DR-PTB-RT
Figure 3.
Numbers of patients with different gene mutation sites of RIF in two groups
Figure 4.
Numbers of patients with different gene mutation sites of INH in two groups
Figure 5.
Numbers of patients with different gene mutation sites of EMB in two groups
Figure 6.
Numbers of patients with different gene mutation sites of LVFX in two groups
Figure 7.
Numbers of patients with different types of poly-DR in the DR-PTB-PT
Figure 8.
Numbers of patients with different types of poly-DR in the DR-PTB-RT
Table 2.
Comparison of characteristics of DR types between PTB-PT and PTB-RT (n=256)
| Characteristics | DR-PTB-PT (n=123) | DR-PTB-RT (n=133) | P |
|---|---|---|---|
| Phenotypic DR | <0.05 | ||
| RR | 8 | 7 | |
| HR | 3 | 4 | |
| MDR | 7 | 0 | |
| Poly-DR | 34 | 53 | |
| Pre-XDR | 71 | 69 | |
| Molecular DR | >0.05 | ||
| RIF | 119 | 127 | |
| INH | 134 | 144 | |
| EMB | 82 | 89 | |
| LVFX | 65 | 68 | |
| Gene mutation sites of molecular DR | |||
| RIF | >0.05 | ||
| RpoB529–533 | 81 | 90 | |
| RpoB513–520 | 17 | 11 | |
| RpoB521–528 | 8 | 10 | |
| RpoB507–512 | 12 | 11 | |
| INH | >0.05 | ||
| KatG315 | 98 | 104 | |
| InhA promoter region (-17～-8) | 32 | 35 | |
| InhA94 | 1 | 1 | |
| EMB | >0.05 | ||
| Emb306 | 62 | 69 | |
| Emb497 | 10 | 12 | |
| Emb406 | 7 | 5 | |
| LVFX | |||
| GyrA88–94 | 65 | 68 | - |
Correlation between characteristics and DR-TB types
The correlations between the different characteristics and DR-PTB types were analyzed. The extrapulmonary sites affecting MTB and comorbidity were positive correlations with DR-PTB types [Table 3]. Using multivariable logistic regression for analyzing the details, the poly-DR (P < 0.05) and comorbidity (P < 0.01) were the correlations with the DR-PTB-RT [Table 4].
Table 3.
Correlation analysis of the risk factor for PTB types in patients (n=256)
| DR-PTB types | χ 2 | P | ||
|---|---|---|---|---|
|
| ||||
| DR-PTB-PT (n=123) | DR-PTB-RT (n=133) | |||
| Gender | 0.010 | >0.05 | ||
| Male | 109 | 117 | ||
| Female | 14 | 16 | ||
| Age (years) | ||||
| ≤ 30 | 15 | 15 | 0.027 | >0.05 |
| 31–60 | 73 | 78 | ||
| ≥ 61 | 35 | 40 | ||
| Residence | 0.004 | >0.05 | ||
| Rural | 78 | 86 | ||
| Urban | 45 | 47 | ||
| Regions | -0.010 | >0.05 | ||
| Chongqing City | 100 | 107 | ||
| Sichuan Province | 21 | 24 | ||
| Guizhou Province | 2 | 2 | ||
| Phenotypic DR types | 0.000 | >0.05 | ||
| RR | 8 | 7 | ||
| HR | 3 | 4 | ||
| MDR | 7 | 0 | ||
| Poly-DR | 34 | 53 | ||
| Pre-XDR | 71 | 69 | ||
| No. of molecular DR kinds | -0.009 | >0.05 | ||
| 1 | 19 | 22 | ||
| 2 | 18 | 21 | ||
| 3 | 34 | 34 | ||
| 4 | 52 | 56 | ||
| Extrapulmonary sites infecting MTB | 0.320 | <0.01 | ||
| Throat | 2 | 2 | ||
| Pleura | 8 | 18 | ||
| Intestine | 5 | 12 | ||
| Pericardium | 1 | 4 | ||
| Peritoneum | 2 | 4 | ||
| Cerebrospinal fluid | 1 | 4 | ||
| Urinary | 1 | 2 | ||
| Comorbidity | 0.632 | <0.01 | ||
| COPD | 2 | 10 | ||
| Pneumoconiosis | 5 | 12 | ||
| CRI | 1 | 3 | ||
| Lung cancer | 1 | 2 | ||
| Diabetes | 16 | 47 | ||
| HBP | 5 | 16 | ||
| Viral hepatitis | 5 | 8 | ||
| CHD | 1 | 4 | ||
| Others | 4 | 16 | ||
DR=Drug resistance, RR=Rifampicin resistance, HR=Heart rate, MDR=Multidrug resistance, COPD=Chronic obstructive pulmonary disease, CRI=Chronic renal insufficiency, HBP=High blood pressure, CHD=Coronary heart disease
Table 4.
Multivariable logistic regression for risk factor of PTB types in patients (n=256)
| OR (95% CI) | P | |
|---|---|---|
| Gender | ||
| Male | 1.205 (0.502–2.889) | >0.05 |
| Female | Ref. | |
| Age (years) | ||
| ≤ 30 | 1.242 (0.467–3.307) | >0.05 |
| 31–60 | 1.087 (0.599–1.972) | >0.05 |
| ≥ 61 | Ref. | |
| Residence | ||
| Rural | 1.200 (0.502–2.889) | >0.05 |
| Urban | Ref. | |
| Regions | ||
| Chongqing City | Ref. | |
| Sichuan Province | 0.744 (0.369–1.500) | >0.05 |
| Guizhou Province | 0.448 (0.022–9.195) | >0.05 |
| Phenotypic DR types | ||
| RR | 0.450 (0.076–2.648) | >0.05 |
| HR | 0.358 (0.048–2.693) | >0.05 |
| MDR | - | >0.05 |
| Poly-DR | 0.370 (0.138–0.990) | <0.05 |
| Pre-XDR | Ref. | |
| No. of molecular DR kinds | ||
| 1 | 2.227 (0.535–9.260) | >0.05 |
| 2 | 1.598 (0.469–5.445) | >0.05 |
| 3 | 1.810 (0.770–4.256) | >0.05 |
| 4 | Ref. | |
| Extrapulmonary sites infecting MTB | ||
| Yes | Ref. | |
| No | 1.402 (0.561–3.504) | >0.05 |
| Comorbidity | ||
| Yes | 17.706 (8.783–35.697) | <0.01 |
| No | Ref. |
DR=Drug resistance, RR=Rifampicin resistance, HR=Heart rate, MDR=Multidrug resistance, CI=Confidence interval, PTB=Pulmonary tuberculosis, MTB=Mycobacterium tuberculosis
The DR types and gene mutations of patients with primary treatment or retreatment twice
In the DR-PTB-PT, there were six patients who received no retreatments after the primary treatment; the rest patients were retreated for PTB and there were 16 patients who accepted retreatment twice. The DR-types and gene mutations of six patients with primary treatment and 16 patients with twice retreatments are displayed in Table 5.
Table 5.
The DR types and gene mutations of patients who accepted primary treatment (n=6) and patients who accepted retreatment twice (n=16)
| Patients’ types | Primary treatment | The first retreatment | The second retreatment | |||
|---|---|---|---|---|---|---|
|
| ||||||
| DR types | Gene mutations | DR types | Gene mutations | DR types | Gene mutations | |
| Primary treatment | ||||||
| 1 | Pre-XDR | rpoB513–520 katG315 embB306 | ||||
| 2 | Pre-XDR | rpoB529–533 inhA promoter region (-17~-8) gyrA88–94 | ||||
| 3 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 | ||||
| 4 | Pre-XDR | rpoB507–512 katG315 embB406 gyrA88–94 | ||||
| 5 | Pre-XDR | rpoB513–520 gyrA88–94 | ||||
| 6 | Pre-XDR | rpoB529–533 katG315 gyrA88–94 | ||||
| Retreatment twice | ||||||
| 1 | Poly-DR | rpoB521–528 katG315 | Poly-DR | rpoB521–528 katG315 | Poly-DR | rpoB521–528 katG315 |
| 2 | HR | katG315 | HR | katG315 | HR | katG315 |
| 3 | Poly-DR | rpoB529–533 inhA promoter region (-17~-8) | Poly-DR | rpoB529–533 inhA promoter region (-17~-8) | Poly-DR | rpoB529–533 inhA promoter region (-17~-8) |
| 4 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 |
| 5 | Poly-DR | rpoB529–533 embB306 gyrA88–94 | Poly-DR | rpoB529–533 embB306 gyrA88–94 | Poly-DR | rpoB529–533 embB306 gyrA88–94 |
| 6 | Pre-XDR | rpoB529–533 ahpC promoter region (-44~-30 and -15~3) gyrA88–94 | Pre-XDR | rpoB529–533 ahpC promoter region (-44~-30 and -15~3) gyrA88–94 | Pre-XDR | rpoB529–533 ahpC promoter region (-44~-30 and -15~3) embB497 gyrA88–94 |
| 7 | Poly-DR | rpoB529–533 katG315 embB497 | Poly-DR | rpoB529–533 katG315 embB497 | Poly-DR | rpoB529–533 katG315 embB497 |
| 8 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 |
| 9 | Poly-DR | rpoB529–533 inhA promoter region (-17~-8) katG315 embB306 | Poly-DR | rpoB529–533 inhA promoter region (-17~-8) katG315 embB306 | Poly-DR | rpoB529–533 inhA promoter region (-17~-8) katG315 embB306 |
| 10 | Pre-XDR | rpoB529–533 inhA promoter region (-17~-8) embB306 gyrA88–94 | Pre-XDR | rpoB529–533 inhA promoter region (-17~-8) embB306 gyrA88–94 | Pre-XDR | rpoB529–533 inhA promoter region (-17~-8) embB306 gyrA88–94 |
| 11 | Pre-XDR | rpoB529–533 katG315 embB306 | Poly-DR | rpoB529–533 katG315 embB306 | Poly-DR | rpoB529–533 katG315 embB306 |
| 12 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 | Pre-XDR | rpoB529–533 katG315 embB306 gyrA88–94 |
| 13 | Poly-DR | inhA promoter region (-17~-8) | Poly-DR | inhA promoter region (-17~-8) | Poly-DR | inhA promoter region (-17~-8) |
| 14 | RR | rpoB529–533 | RR | rpoB529–533 | RR | rpoB529–533 |
| 15 | Pre-XDR | rpoB521–528 rpoB529–533 katG315 embB306 gyrA88–94 | Pre-XDR | rpoB521–528 rpoB529–533 katG315 embB306 gyrA88–94 | Pre-XDR | rpoB521–528 rpoB529–533 katG315 embB306 gyrA88–94 |
| 16 | Pre-XDR | rpoB529–533 katG315 gyrA88–94 | Pre-XDR | rpoB529–533 katG315 gyrA88–94 | Pre-XDR | rpoB529–533 katG315 gyrA88–94 |
DR=Drug resistance, RR=Rifampicin resistance, HR=Heart rate, MDR=Multidrug resistance, XDR=Extended detection and response
Discussion
According to the WHO report in 2022, 7.5 million people were newly diagnosed with TB and officially notified as a TB case and the total number of deaths caused by TB was 1.30 million.[1] In the TB cases, the DR-TB is more threat to the public health. In 2022, the estimated proportion of people with TB who had MDR/RR-TB was 3.3% among new cases and 17% among those previously treated.[1] In this study, there were 123 patients who were diagnosed DR-PTB cases at first and never accepted any antituberculosis drug treatment, which means all the 123 patients were infected by DR-MTB at the first infection. All the patients relapsed and 10 patients relapsed twice. This search was to determinate the characteristics of DR types and gene mutations between DR-PTB-PT and DR-PTB-RT.
There were some studies on the DR types of DR-PTB between the patients with primary treatment and retreatment. In the primary treatment, the most common were SM and EMB, followed by RIF 3.89% in the northwest Ethiopia, while in the retreatment INH was the most common[13]; INH and SM were the highest resistance rates in the primary treatment in Gabon, which was the same in the retreatment[14]; in Iraq the SM was the most common, followed by INH, RIF, and EMB, while the INH was the most, followed by RIF and SM.[15] In China, the DR types were various according to the different provinces; for example, in Heilongjiang Province the SM was the most common, followed by INH and RIF.[16] In this study, the INH was the most common, followed by RIF and EMB, and the DR types in the retreatment were the same as the primary treatment. In this study, there were some details that need to be concerned: firstly, the pre-XDR cases accounted for 57.7% in the DR-PTB-PT and 51.1% in the DR-PTB-RT; secondly, the MDR case was 0 in the DR-PTB-RT, while in the DR-PTB-PT was 6.5%; thirdly, the poly-DR cases raised from 34 in the DR-PTB-PT to 54 DR-PTB-RT. These data showed that the DR was cumulative and evolutional process.
In this study, the codons 529–533 of rpoB gene was the main mutation for RIF, codon 315 of katG gene for INH, codon 306 of emb gene for EMB, and codons 88–94 of gyrA were for LVFX, in the DR-PTB-PT and DR-PTB-RT. The gene mutation sites were various in the different regions and countries. In the southern coastal region of India, the codon 531 of rpoB gene and codon 315 of katG gene were found to have the highest mutation frequency for RIF resistance and INH resistance, and inhA promoter was for INH resistant[17]; in Lisbon, Portugal inhA was main gene mutation site for INH, rpoB was for RIP, rpsL for SM and pncA was for PZA[18]; the codon 531 of rpoB gene was the main mutation for RIF, codon 315 of katG gene for INH, and codon 306 of emb gene for EMB in Jayapura, Papua, Indonesia, which was the same as this study.[19] There was a research mainly on EMB resistance in China, which showed that 82.6% of EMB-resistant isolates harbored at least one mutation within embCAB and most of the mutations were located in the embB and embA upstream region.[20] There was another article that compared the gene mutations between the new cases and retreatment, which showed that the codon 531 of rpoB was slightly higher in new treatment cases; the codon 315 of katG was mainly in new cases, while in the retreatment was katGS315T1 mutation.[21] All the above researches showed that the gene mutation sites between the primary treatment and retreatment were almost the same and rare change.
There are some detection methods on DR-MTB, for example, detection of the deletion of region of difference 105 (RD105), mycobacterial interspersed repetitive-unit variable-number tandem-repeat (MIRU-VNTR), X-pert and restriction fragment length poly-morphism (RFLP) analysis. The X-pert is a good technique to detect the DR-MTB, but there were some researches that show this method for detection of STB was false result,[22] and when detecting the STB the samples should be pus rather than tissue samples.[23] The WGS is a new method and has been used for detection of DR-MTB, but it is very expensive and is not be used widely. This MCA method was reported by E. Lyon[24] and was used by Chinese researcher for detection of DR-MTB; the dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.[25] This study used the MCA method to detect the DR-PTB, which showed the effectiveness and application.
The recurrence of PTB is a continuous threat to human health. According to researches, the recurrence of PTB was correlated with many factors. For example, the MDR was related to retreatment in northeast China[26]; the lost to follow-up was correlated with retreatment in Serbia[27]; retreatment was related to the resistance to any TB drug or MDR in South Africa[28]; in Iran DR was correlated with retreatment[29]; and in Shandong Province of China the smoking, cavity, and comorbidity lead to an increased risk of drug resistance among retreated PTB.[30] In this study, the poly-DR and comorbidity were the risk factors of DR-PTB-RT, which were different with the previous studies. The reasons maybe include: firstly, the patients were infected DR-MTB at first, which means that the recurrence was a high probability; secondly, most patients had more than one kind of comorbidity.
There were some researches about the reasons of PTB reoccurrence. In Hunan province of China, the main mechanism leading to TB recurrences was endogenous relapse, and in the reoccurrence, the FQs resistance was high frequency using the whole gene sequencing method.[31] In South Africa, the injectable drugs were discontinued frequently due to adverse effects and both AMK and PAS were frequently discontinued due to patient refusal,[32] which may be one of reasons of PTB reoccurrence. In Chongqing city of China, the DR-TB was higher in the patients of 15–64 years old.[33] A retrospective study from Ethiopia displayed that the factors of recurrence of MDR-TB included age, weight, social drug usage, clinical complications, MDR-TB category, education level, previous TB history, and place of residence, and the weight increment, smokers and alcohol users, clinical complications of hemoptysis and pneumonia, patients with pulmonary disease who had a history of previous anti-TB treatment, and being rural residents are prognostic factors.[34]
Conclusions
The codons of RpoB529–533, KatG315, Emb306, and GyrA88–94 were the main gene mutation sites in the DR-PTB-PT and DR-PTB-RT in the local southwest regions of China. The poly-DR and comorbidity were the risk factors of DR-PTB-RT.
Limitations and future aspects
The limitations of this study included three points: 1) the samples were small, especially the twice re-treatment cases; 2) the lacks of deep research of reoccurrence reasons and recurrent mechanism; 3) the lacks of research how the comorbidity affects the reoccurrence. The research of future aspects will focus on the following points: 1) collecting the more reoccurrence samples; 2) using the whole gene sequencing method to study the mechanism of reoccurrence; 3) determining the mechanism of comorbidity affecting the reoccurrence.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Conflicts of interest
There are no conflicts of interest.
Acknowledgements
We thank all the patients who agreed to participate in this study.
Funding Statement
This study was supported by Chongqing Medical Scientific Research Project (Joint Project of Chongqing Health Commission and Science and Technology Bureau) (No. 2024MSXM061) and The Project for Cultivating High-Level Medical Talents of Middle-aged and Young People of Chongqing (No. YXGD202426).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.