Archives of Microbiology & Immunology

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ISSN: 2572-9365

Abstracting and Indexing

Editor In Chief

Masashi Emoto

Professor of Laboratory of Immunology
Department of Laboratory Sciences
Gunma University Graduate School of Health Sciences
Gunma, Japan

Research ArticleOpen Access

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;

 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 drugresistant Mtb


Keywords:

Mycobacterium tuberculosis; Gene mutation; Drug resistant

Untitled Document

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).

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 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.

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

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

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 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

 

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)

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).

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 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.

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.

 

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