The Gene Mutation and Drug Resistant of Mycobacterium tuberculosis in Patients of Chongqing

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Arch Microbiol Immunology 2018; 2 (1): 010-017 DOI: 10.26502/ami.93650018
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Archives of Microbiology & Immunology Vol. 2 No. 1 - Mar 2018. 10
Research Article
The Gene Mutation and Drug Resistant of Mycobacterium
tuberculosis in Patients of Chongqing
Yishu Tang*, Peiyang Song, Huiting Su
Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, No. 1 Youyi
Road, Yuzhong District, Chongqing, 400016, China
*Corresponding Author: Yishu Tang, Department of Laboratory Medicine, The First Affiliated Hospital of
Chongqing Medical University, No. 1 Youyi Road,Yuzhong District,Chongqing, 400016, China, Tel: Tel: +86 23
89012735; E-mail: tangyishu111@163.com
Received: 06 March 2018; Accepted: 09 March 2018; Published: 12 March 2018
Abstract
Objective: To detect the infection and drug resistance of Mycobacterium tuberculosis (MTB) in Chongqing and
provide a scientific basis for the prevention and treatment of tuberculosis.
Methods: DNA was collected from all new suspected patients in Chongqing from January 2014 to September 2017,
Genechip technology was used to identify Mtb strain. Genechip technology detects mutations in the ropB gene
(associated with resistance to rifampicin) at locus 511, locus 513, locus 516 locus 526, locus 531 or locus 533.
Genechip technology was also used to detect mutations in the KatG gene and inhA gene．
Results: Genechip revealed that the Mtb infected male accounted for 73.98% and female accounted for 26.12%.The
total drug resistance rate of rifampicin and isoniazid were 11.2% (122/110771). Genechip revealed that ropB gene of
72 strains were mutation. The highest mutation site was 531 (TCG) locus (37.5%, 27/72). The people with katG and
inhA gene mutation were 50 patients. The most common mutation site was 315 (AGC) locus. The cavity, history of
treatment, and irregular medication were the risk factor of drug-resistant Mtb.
Conclusion: Our report demonstrated the infected ratio and the drug resistant types of Mtb in Chongqing district.
We should strengthen health management and provide psychosocial support, in order to reduce the risk of drug-
resistant Mtb
Keywords: Mycobacterium tuberculosis; Gene mutation; Drug resistant

Arch Microbiol Immunology 2018; 2 (1): 010-017 DOI: 10.26502/ami.93650018
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Archives of Microbiology & Immunology Vol. 2 No. 1 - Mar 2018. 11
1. Introduction
Cancer-associated retinopathy (CAR) is a challenging clinical entity with often delayed diagnosis and difficult
prognosis. The condition occurs in patients with typically known systemic malignancy but can precede cancer
diagnosis. The clinical phenotype is varied, but typically includes optic nerve pallor, retinal vascular attenuation and
visual field loss in the absence of obvious peripheral retinal abnormalities. Diagnosis is typically confirmed by
serologic testing for anti-retinal antibodies. Numerous treatment options including systemic immune suppression
with intravenous corticosteroids, intravenous immunoglobulins (IV Ig), and plasmapheresis have been used with
mixed results. Even with treatment of the systemic malignancy, the prognosis typically involves worsening visual
field loss. Anecdotal reports of intravitreal (intraocular) steroid injections have been able to demonstrate stability of
visual field loss. This case report details the course of a patient with serologically confirmed cancer-associated
retinopathy who showed initial improvement and later stabilization of visual acuity, optic nerve structure and
function after a single intravitreal steroid injection in one eye.
2. Materials and Methods
2.1 Research population
From January 2014 to September 2017, the sputum, urine, pleural effusion, cerebrospinal fluid, and puncture fluid
samples were collected from 6557 suspected TB patients (18-75 years) in The First Affiliated Hospital of
Chongqing Medical University. All the patients were negative for hepatitis B virus, hepatitis C virus, human
immunodeficiency virus (HIV),combined tumor and other symptoms of liver damage. This study was approved by
the Institutional Review Board (IRB) committee of The First Affiliated Hospital of Chongqing Medical University.
Written consent given by the patients was waived by the approving IRB.
2.2 Detection by DNA microarray chip
This study was based on the designing of oligonucleotide probes which can specifically detect the specific gene site
of Mtb, and the mutations on the promoter of rpoB, KatG and inhA. Briefly, the DNA microarray chip
technique (20) was used to test mutations in the rpoB gene at the 511, 513, 526, 531 and 533 codons. Common
mutation sites to give an indication of RFP resistance. For INH resistance, the katG315 and inhA-15 mutation sites
were assessed. The Mtb population detection kit and GeeDom Mtb drug detection kits (CapitalBio Corporation,
Beijing, China) were operated according to the instruction of manufactory. The nucleic acid was extracted and PCR
amplification. Once combined with a hybridization buffer (CapitalBio Corporation), the products were placed in a
BioMixer chip hybridization instrument (CapitalBio Corporation) for hybridization. Then products were put in a
slidewasher 8 chip dry cleaning instrument (CapitalBio Corporation) for washing and drying.Finally, the chip was
placed in chip identification system (CapitalBio Corporation) for scanning and interpretation (LuxScanTM 10K/B
software, CapitalBio Corporation).
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2.3 Statistical analysis
Mann-Whitney U tests of SSPS12.0 (SPSS, Inc., Chicago, IL, USA) were used to assess the difference between
different groups. A two-tailed P<0.05 was considered statistically significant. The distribution of the study variables

Arch Microbiol Immunology 2018; 2 (1): 010-017 DOI: 10.26502/ami.93650018
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Archives of Microbiology & Immunology Vol. 2 No. 1 - Mar 2018. 12
was calculated using means with standard deviations for normal continuous variables or using median with quartile
range for skewness variables, and frequencies and percent for categorical variables. For continuous variable
comparisons, Student’s t tests were used when equality of variances was satisfied, otherwise Satterthwaite-tests were
conducted. We assessed effect of each “risk” factor for TB using binary logistic regression. Stepwise logistic
regression was performed in this study.
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3. Results
3.1 Mtb positive patients and positive types distribution
We detected 6557 samples from 2014 to 2017 using Mtb strain identification gene chip and the Mtb positive ratio
was 16.89%. The positive ratio in 2014-2017 was 27.14%, 21.13%, 14.46% and 12.67%, respectively (Table 1). The
sputum, urine, pleural effusion, cerebrospinal fluid and puncture fluid were include in the sample types, which
accounted for 22.9%, 6.5%, 17.5%, 15.8% and 37.3% , respectively (Table 2).
Year Total samples Positive samples Positive (%)
2014 943 256 27.14
2015 1203 254 21.13
2016 2123 307 14.46
2017 2288 290 12.67
2014-2017 6557 1107 16.89
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Table 1: The positive sample in the 6557 patient of Chongqing
Sample type Sputum Urine Pleural Effusion Cerebrospinal Fluid Puncture Fluid
Sample No. 254 72 194 175 412
Positive (%) 22.9 6.5 17.5 15.8 37.3
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Table 2: The sample type distribution in 1107 Mtb positive patient
3.2 The distribution of Mtb infected and drug-resistant patient
We then detected the sensitive situation of REF and INH in the 1107 Mtb positive samples, using the drug sensitive
identification gene chip. The Table 3 demonstrated that infected male accounted for 73.98% (819/1107), and female
accounted for 26.12% (P<0.05). Less than 20 years group, 20-39 years group, 40-60 years group and >60 years

Arch Microbiol Immunology 2018; 2 (1): 010-017 DOI: 10.26502/ami.93650018
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group accounted for 3.52% (39/1107, 18.33% (203/1107), 32.61% (361/1107), 45.52% (504/1107), respectively.
Therefore, the Mtb infected ratio increased by age. The general ratio of INH-resistant and RFP-resistant was 11.02
% ( 122/1107). The INH-resistant and RFP-resistant ratio were 5.32% (59/1107) and 3.41% (38/1107), respectively.
Both INH and RFP resistant ratio was 2.25 % (25/1107). According to the age group, the drug-resistant ratio of the
>60 years group was the highest, accounting for 4.24% (47/1107).
Drug sensitive type Gender Age groups (years)
Male Female <20 20-39 40-60 >60
Both sensitive 744 241 36 176 316 457
Resistant-REF 27 11 1 7 16 14
Resistant-INH 33 26 1 13 21 24
Both resistant 15 10 1 7 8 9
Total 819 288 39 203 361 504
Table 3: The drug sensitive types in 1107 Mtb positive patient
3.3 The mutation site of ropB, katG and inhA gene
The Table 4 showed the people with ropB gene mutation were 72 patients. Of these, the patients with single
mutation were 68, double mutations were 3 patients, and triple mutations were 2 patients. The highest mutation site
was 531 (TCG) locus (37.5%, 27/72). The people with katG and inhA gene mutation were 50 patients. Of these, the
katG and inhA gene mutation ratio were 44.0% (22/50) and 42% (21/50), respectively. Meanwhile, both catch and
inhale gene mutation ratio was 14% (7/50). The most common mutation site was 315 (AGC) locus.
Gene Mutation site Mutation type Positive
samples Mutation
rate (%)
rpoB 511 (CTG) T→C 11 9.02
513 (CAA) C→A, A→C 3 2.45
516 (GAC) A→T, A→G 7 5.74
526 (CAG)
C→T, C→G, A→T, A→G 13 10.66
531 (TCG) C→T, C→G 27 22.13
533 (CTG) T→C 7 5.73
526, 533 1 0.82
526, 531 1 0.82
511, 516, 531 1 0.82

Arch Microbiol Immunology 2018; 2 (1): 010-017 DOI: 10.26502/ami.93650018
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511, 516 1 0.82
katG 315 (AGC) G→C, G→A 22 18.03
inhA -15 (ACG) C→T 21 17.21
katG, inhA 315, -15 7 5.73
Table 4: The distribution of ropB, inhale, katG gene mutation
3.4 The risk factor analysis of drug- resistant Mtb
The Table 5 demonstrated the age, marital status, cavity, history of treatment, and regular medication were
statistically different between drug-resistant and control group (drug-sensitivity) (P<0.05). Otherwise, educational
level and occupation have no difference between drug-resistant and control group (drug-sensitivity) (P>0.05).
Social demographic characteristics Case group (%)
n=122 Control group (%)
n=985 P OR (95% CI)
Age(year)
<40 30 212 0.121
1.011 (0.720-
1.430)
≥40 92 773 0.023
0.452 (0.312-
0.897)
Marital status
Unmarried/other 45 321 0.223
0.776 (0.579-
1.324)
Married 77 664 0.012
0.440 (0.305-
0.636)
Educational level
Higher education 21 143 0.582 1.132 (0.728-
1.760)
Secondary education or below 101 842 0.457 1.101 (0.643-
1.574)
Occupation
Staff 10 124 0.267
1.818 (0.857-
3.859)
Housekeeping, domestic chores and
unemployed
76 578 0.056
0.962 (0.460-
2.011)
Worker 22 209 0.302
1.261 (0.574-
2.772)

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other 14 74 0.295
1.661 (0.783-
3.292)
History of treatment
Yes 51 671 0.023
1.394 (0.759-
2.583)
No 71 314 0.507
1.952 (0.843-
2.351)
Cavity
Yes 42 269 0.031
1.467 (1.018-
2.113)
No 80 716 0.418
1.699 (0.986-
2.459)
Regular medication
Yes 73 809 0.673
1.363 (0.824-
2.087)
No 49 176 0.016
2.115 (1.225-
3.195)
Table 5: Results of risk factors for MDR-TB by logistic regression analysis
4. Discussion
The spread of Mtb seriously affected the people in the world. Moreover, the drug-resistant Mtb and its co-infection
with HIV have seriously affected TB prevention and treatment [11]. Inherently, this has meant deterioration in the
control of epidemics.
RFP and INH are primarily first-line anti-TB drugs. However, the effectiveness of the drugs has been greatly
affected by the increase in drug resistance. A previous study has also suggested that in TB clinical strains
demonstrate high levels of RFP (13.3%), INH (24.6%), and multi-drug (10.5%) resistance [5].
The most of RFP resistance related gene mutations located in the rpoB gene. The mutations on the 531 Ser, 526 His
and 516 Asp codons accounted for 85% of the strains resistant to drugs. INH is another first-line anti-TB drug that is
used together with RFP. Most INH resistance related to gene mutation were identified in the katG315 and inhA-15
mutations [11]. The primary mutation mechanism of INH-resistance in the MTB katG gene investigated was 315
AGC→ACC, Ser→Thr (S315T).
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We detected the suspected Mtb patients in Chongqing using gene chip method and the positive ration was 16.89 %.
The general ratio of INH-resistant and RFP-resistant was 11.02 %,which was less than 13.49% in the report of Pang
Y et al. [13]. The reason might be that we recruited new patients not retreatment patients. The Mtb infected male

Arch Microbiol Immunology 2018; 2 (1): 010-017 DOI: 10.26502/ami.93650018
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Archives of Microbiology & Immunology Vol. 2 No. 1 - Mar 2018. 16
accounted for 73.98% and female accounted for 26.12%.The most Mtb infected people were men and the ratio of
infected men/women was 2.5. The data showed no difference with the report of Pang Y et al. [13]. Our report
demonstrated the history of treatment was the risk factor of the drug-resistant , which consisted with the report of the
report of Lomtadze N et al. [14]. Moreover, irregular medication was another risk factor, which consisted with the
report of the report of DIANDÉ S et al. [15]. The irregular medication caused disease relapse and drug-resistant Mtb
became the dominant bacteria. The cavity was also another risk factor in our report, which In accordance with the
study of Ahmad AM et al. [16]. There were lots of drug-resistant Mtb in the cavity, so the Mtb might spread easily.
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Our report demonstrated the infected ratio and the drug resistant types of Mtb in Chongqing district. The risk factors
associated with drug resistant are complex; we should reinforce early detection, rapid diagnosis, and standardize
therapy, in order to make sure that the patients take the full course of the treatment to reduce the risk of drug
resistant types of Mtb.
Acknowledgements
This work was supported by a grant from National Natural Science Foundation of China (No 81501818 of Yishu
Tang) and the National Key Clinical Specialties Construction Program of China.
Conflict of interest
The authors declare no conflicts of interest.
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Citation: Yishu Tang, Peiyang Song, Huiting Su.The Gene Mutation and Drug Resistant of
Mycobacterium tuberculosis in Patients of Chongqing. Archives of Microbiology & Immunology 2 (2018):
010-017.