Transmission and drug-resistance of tuberculosis in Luodian revealed by whole genome sequencing based molecular epidemiology study

doi:10.21203/rs.3.rs-26012/v1

BACKGROUND

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), remains a severe public health problem globally. Guizhou has the fourth highest TB report rate of pulmonary TB around China. Uncovering the current status of TB epidemic, and distinguishing disease caused by recent or remote infections are the key issue to formulate effective prevention and control strategy. However, these data are limited in Guizhou. In this study, we aimed to investigate the transmission and drug-resistance profiles of TB in Luodian, a highest TB incidence and resources limited area in Guizhou, China.

METHODS

During 22 May 2018 to 21 April 2019, individuals with positive MTB culture were enrolled, all of them accepted the standardized interview. MTB isolates were performed whole genome sequencing. The prevalence of MTB genotypes, the genomic cluster rate and drug-resistance conferring mutations were analyzed based on the sequencing data.

RESULTS

A total of 107 cases were enrolled, of which 64.5% were male, and the median age of the patients was 51 years old (interquartile range, 40–65 years old). 84% patient were new case while 16% were retreated cases. All cases excepted three came from nine towns, and 55.1% of cases were from Longping and Bianyang. The phylogeny tree showed that 53.3% of strains were Lineage 2 (Beijing genotype), while 46.7% were Lineage 4 (Euro-American genotype). Among Lineage 2 strains, 66.7% were modern Beijing. Seven clusters with genomic distance within 12 SNVs were identified. The clusters included 14 strains, accounting for a cluster rate of 13.1%. The distance of clustered cases was between 2.1 to 71 kilometers (Km), with a media distance of 14 Km (interquartile range, 2.8–38 Km). Cases of two clusters came from the same town. Based on the gene mutations associated to drug-resistance, we predicted that 4.8% was resistant to isoniazid, 3.7% to rifampicin, 3.7% to streptomycin, and only one strain (0.9%) was multidrug resistance (MDR).

CONLUSIONS:

The study found high transmission and low drug-resistance rate in Luodian, and sublineages of modern Beijing branch had recent expansion in Luodian. this work also may serve as a genomic baseline to study the evolution and spread of MTB in Guizhou.

Infectious Diseases

Epidemiology

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains the globally severe public health problem, is the top cause of death induced by a single pathogen. In 2018, there were 10 million people fell ill with TB worldwide, and China has the second largest number of new TB cases following India, which account for 9% of the global burden¹. According to the fifth national tuberculosis epidemiological survey, there was geographical variation of the incidence rate across China, western part was the area with the most serious prevalence of pulmonary TB². Guizhou province located in the southwest of China, with low levels of socioeconomic development. Guizhou has the fourth highest TB report rate of pulmonary TB in China, estimated at 102.5 per 10,000 population (average rate in China was 55.6 per 10,000)³. Uncovering the current status of tuberculosis epidemic in a region and distinguishing tuberculosis caused by recent infection or remote infection are the basis and key to formulate effective prevention and control strategy⁴. However, these data are limited in Guizhou province.

The genotyping of MTB has been served as an essential tool to investigate the key issues in TB epidemiology, for example to assess the recent transmission, identify risk factors associated to recent transmission, and detect outbreak. Recently, Whole genome sequencing (WGS) was considered as an ultimate tool for genotyping MTB strains owing to provide about 90% of genomic information^{5, 6}, and it has advantages including high resolution and be phylogenetic informative as compared to conventional genotyping methods in identifying outbreaks⁷ and tracing transmissions⁶.

To estimate the recent transmission of pulmonary TB (PTB) in Guizhou, we conducted a retrospective study of pulmonary tuberculosis in Luodian county, which has a report rate at 271.1 per 10,0 00, being the region with the most severe tuberculosis epidemic in Guizhou⁸.

Setting and study population

Luodian county located at the south of Guizhou province, with a population of around 353,000. Passive case finding strategy was used to identify suspected pulmonary TB with symptoms according to the guidelines of Chinese National Center for Disease Control and Prevention(CDC)⁹. All suspected TB cases are referred to the only one comprehensive TB designated hospital, were requested to give three sputum samples collected at morning, night, and spot before taking medicine. All samples should be sent for smear, Ziehl-Neelsen staining and microscopy, then samples with smear-positive were cultured on Lowen-stein-Jensen medium. During 22 May 2018 to 21 April 2019, individuals with sputum culture positive were enrolled, all of them accepted the standardized interview.

Whole genome sequencing

Genomic DNAs were extracted with the cetyltrimethylammonium bromide-lysozyme (CTAB) method¹⁰. Then, the genomic DNAs were sent for whole genome sequencing. A 300 base pair (bp) paired-end library was constructed for each DNA sample. Sequencing was carried out on the Illumina Hiseq 2000 with 100 or 115 cycles, with an excepted coverage of 100. To analyze single nucleotide polymorphisms (SNPs), we mapped reads to H37Rv reference genome (GenBank assession: AL123456.3) with Bowtie using non-gapped alignments and identified SNPs as previously described¹¹. All homozygous heterozygous calls and SNPs with coverage higher than three were called by Samtools/Bcftools. Any SNPs in the PE/PPE genes and drug resistant associated genes were discounted. Using an in-house Perl script, SNP lists for individual strains were combined into a single nonredundant list, and corresponding base calls were recovered for each strain. The concatenated sequences of strains were used to generate a Maximum Likelihood (ML) phylogeny by MEGA5¹². To test the reproducibility of WGS and data processing, two strains were sequenced and analyzed twice, Fig. 2. Genomic cluster was defined as strains with genomic difference of 12 SNPs or less, and the unique was strains with genetic difference of more than 12 SNPs¹³.

The drug resistance data were predicted for rifampicin (rpoB RRDR), isoniazid （katG, inhA, and its promoter）, streptomycin (rpsl and gidB), ethambutal (embA, embB, and embC), pyrazinamide (pncA and rpsA), fluoroquinolones (gyrA and gyrB), second-line injection antibiotics (rrs, eis-promoter and tlyA) based on the mutations reported to be highly associated with drug resistance (http://www.tbdreamdb.com)¹⁴.

Data collection and analysis

Patients’ demographic and clinical characteristics were collected from the national TB registration system and the Luodian hospital clinical system. Data were analyzed using R (version 3.2.1). the fisher’s exact test was used to assess possible association factors of genomic clustering and genotype. P <0.05 was considered statistically significance.

Demographic characteristics of PTB cases

During 22 May 2018 to 21 April 2019, 116 MTB clinical strains were isolated and 112 were successfully sequenced. Finally, 107 cases were enrolled after excluding five cases due to low data quality (Figure 1). Of enrolled patients, 64.5% (69/107) were male, and the median age of the patients was 51 years (interquartile range, 40-65 years). Almost all patients were local residents except one that was from neighbor county and two didn’t have detail information. Two of 107 cases were high school students, 97 were farmers, and eight were lacked information of occupation. 84% (89/107) patient were new case while 16% (17/107) were retreated cases. All local cases came from nine areas, and Longping and Bianyang accounted 55.1% of cases. Table 1.

Basic description of phylogeny tree and clustering by WGS

To describe the genetic structure of these strains, we constructed a maximum likelihood Phylogenetic tree based on the minimum SNP difference between each pair of all 107 strains. Phylogenetic tree of all 107 strains was shown in Figure 2. 53.3% (57/107) of them were Lineage 2 (Beijing genotype), while 46.7% (50/107) were Lineage 4 (Euro-American genotype). Among Lineage 2 strains, 66.7% (38/57) were modern Beijing (Figure 2).

In study of tuberculosis molecular epidemiology, the recent transmission rate was indicated by cluster rate, here we calculated the genomic-cluster rate. By the cluster definition as genetic distance was 12 SNPs or less, seven clusters with genomic distance within 12 SNVs were identified. The clusters include 14 strains, accounting for a cluster rate of 13.1% (14/107). The genetic difference between strains in each cluster ranged from 0 to 11 SNPs, four pairs were less than 5 SNPs, and three pairs were 8, 9, and 11 SNPs respectively.

The distance of clustered cases was between 2.1 to 71 kilometers, with media as 14 kilometers (interquartile range, 2.8-38 kilometers). Cases of two clusters came from the same town (Figure 1). To further understand the factor related to genomic clustering, we analyzed the relationship between the cluster and bacteriologic, demographic, clinic, and geographic factors. However, there was no statistically significance. Supplementary file.

For understanding if genotype of strains in Luodian was different with that of strains isolated from other areas, we constructed the phylogenetic trees based WGS of our strains together strains collected from other parts of China¹⁵. The result indicated that almost all Luodian’s strains were mixed with strains from other regions. However, among the modern Beijing branch, there were three sublineages containing 22 strains exclusively from Luodian. In one sublineage, the genetic distance of strains was very small, suggesting potential recent expansion occurred in Luodian area during recent decades. Figure 3.

Drug-resistance related gene mutation

China was one of the countries with high burden of TB drug-resistance. In most county of Guizhou province like Luodian, TB therapy mainly relied on empirical strategy due to clinical resources limited. Understanding the level of regional drug resistance is important for clinical treatment. Hence, in order to realize the situation of TB drug-resistance rate in Luodian, we checked the genetic drug-resistance profile based on WGS data. After checking 16 drug-resistance related genes, a number of 39 strains were identified with 54 mutations in seven resistance conferring genes. supplementary file. However, only 13 mutations in ten strains were highly related to drug-resistance, including Ser315Thr (n=4) and Ser315Asn (n=1) of katG, Ser450Leu (n=4) of rpoB, Lys43Arg (n=1) and Lys88Arg (n=3) of rpsl. By these mutations, drug-resistance profiles of all strains were predicted as follows, 4.8% (5/107) was resistant to isoniazid, 3.7% (4/107) to rifampicin, 3.7% (4/107) to streptomycin. But only 0.9% (1/107) was multidrug resistance (MDR). Figure 2. We further checked the genotype of these ten drug-resistant strains, the results showed that four strains belonged to lineage 4, while four to ancient Beijing and two to modern Beijing.

Our results showed that MTB strains isolated from Luodian were less likely belonging to Beijing family (53.3%) compared to those from other central, southern or estern China ( 66.0% in Hunan and Fujian¹⁶, 84.1% in Shenzhen¹⁷, 79.4% in Shandong¹⁸). The prevalence of Beijing family was similar to that of the neighbor province (53.9% in Sichuan¹⁹, 55.3% in Guangxi²⁰, 59.6% in Yunnan²¹) and to the neighbored Zunyi city ( 48.0%) of Guizhou province ²². Our results also revealed that among Beijing strains, modern Beijing sublineage (66.7%) was more dominated than ancient Beijing genotype (33.3%), which was close to data from Shenzhen ( modern Beijing 61.5%)¹⁷.

We found that 13.1% (14/107) of the pulmonary TB cases had genomic-clustered strains in Luodian county during a period less than one year. The cluster rate was lower than the results of other studies, including one conducted in Shenzhen (25.2% , 105/417) during five years¹⁷, and another one from Spain (33.5%, 109/325) in a 3-year population-based study²³. The cluster rate in current study likely be underestimated by following reasons. Firstly, we could not include all incident PTB cases. Due to resources limited, only samples that smear positive would be sent to the higher level hospital for culturing, this test only detected about 18.8%of active TB cases²⁴. In addition, the rate of culture positive was influenced by the reservation and transferring of samples to other hospital. Secondly, the study was last only one year, while molecular epidemiology study of TB usually choose the sampling time of two years or more^{25, 26}. However, we still found that seven genomic clusters containing 14 strains. Although we did not identify any risk factor related to genomic clustering since short study period, we could conclude that the transmission rate of TB in Luodian is likely much higher, further study is needed to more comprehensively investigate of TB transmissions in this area.

Our results revealed that three local sublineages of modern Beijing branch contained strains from Luodian exclusively by reconstructing phylogeny tree along with strains from other regions of China. This phenomenon should be taken caution because modern Beijing genotype got increasing prevalence globally and had caused outbreaks in different locations worldwide²⁷, and it is being found as significantly associated with recurrent TB²⁸, linked to multidrug resistant (MDR) TB²⁹, and more likely to be highly virulent than ancient strains³⁰.

Based on gene mutation that referring to anti-TB drug resistance, we found that 4.8% of strains resistant to isoniazid, 3.7% to rifampicin, 3.7% to streptomycin, only 0.9% were MDR, and none of strains was resistant to any second line drugs. These data indicated drug-resistance in Luodian was at a very low level, as compared to data of whole Guizhou province which was 16.3% to isoniazid, 13.5% to rifampicin, 15.9% to streptomycin, and 9.4% of strains were MDR³¹. This discrepancy may be due to that TB patient in Luodian prefer conservative treatment to chemical medicine because most of population in this region were minority and had native living habit.

This retrospective genomic epidemiological study in Luodian of Guizhou, which is limited in resources, provides a primary map of the transmission and drug-resistance profile of PTB in Luodian of Guizhou of China. We found that 13.1% of the population with PTB had genomic-clustered MTB strains, which suggested on-going transmissions were occurring in this area, and it needed to take caution for further expansion of modern Beijing branch. This work also may serve as a genomic baseline to study the evolution and spread of MTB in Guizhou.

TB: Tuberculosis; MTB: Mycobacterium tuberculosis; MDR: multidrug resistance; WGS: Whole genome sequencing; PTB: pulmonary TB; CDC: Center for Disease Control and Prevention; CTAB: cetyltrimethylammonium bromide-lysozyme; bp: base pair; SNPs: single nucleotide polymorphisms; ML: Maximum Likelihood; Km: kilometer.

Acknowledgements

The authors would like to thank Tian Tian and ZhengQin Yang in Luodian hospital for data collection from clinic system，thank Xuefeng Fu for transferring samples, and special thanks are extended to the participants who enrolled in this study.

Author’s contribution

LC and PX designed this survey, XL and QL collected data, ML, TL, and PX managed and analyzed data, QG and WC controlled the quality of data analysis, ML drafted the manuscript. PX and LC edited the manuscript. All authors interpreted the results, revised the report and approved the final version.

Funding

This project was funded by the National Natural Science Foundation of China (No. 81760003; No.31700130), Science and Technology program in Guizhou (No. [2019]1465), the Project from Health and Family Planning Commission of Guizhou province (No. gzwjkj2018-1-024), and Guizhou Province Governor for special study of Clinical Application (No. Qian co he chengguo [2020]4Y012). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

Data sharing is not applicable to this article as no datasets were generated or analyzed during the study.

Ethics approval and consent to participate

The project proposal was approved by the Institutional Review Board of Zunyi Medical University, Guizhou, China. Written informed consent was obtained from all participants once they agree to take part in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Public Health Clinical Center, Fudan University, Shanghai, 200032, China. ²Key Laboratory of Infectious Disease & Biosafety, Institute of life Sciences, Zunyi Medical University, No.6 West Xuefu Road, Xinpu District, Zunyi, Guizhou Province, 563000, China.³ Department of Infectious Diseases, Hospital of Luodian County, No.96 Jiefang East Road, Luodian 550100, Guizhou, China. ⁴ Department of Respiratory Medicine, Affiliated Hospital of Zunyi Medical University, No.149 Dalian Road, Zunyi 563000, Guizhou, China.⁵ Department of TB Control, Center of Disease Control and Prevention, Guiyang 550004, Guizhou, China.⁶Department of Pathogenic Biology, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, No.17 People's South Road, Chengdu 610041, China.

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Table 1. Characteristics of genomic-clustered and unique cases of pulmonary Tuberculosis and genotype of MTB in Luodian, China

	No.(%)
	L2 (n=57)	L4 (n=50)	Clustered (n=14)	Unique (n=93)
Bacteriologic factor
Genotype (N=107)
Modern Beijing	38 (66.7)	/	8	30 (32.3)	38 (35.5)
Ancient Beijing	19 (33.3)	/	0	19 (20.4)	19 (17.8)
Non-Beijing	/	50 (100.0)	6 (42.9)	44 (47.3)	50 (46.7)
Demographic factor
Gender (N=107)
Male	34 (59.6)	35 (70.0)	6 (42.9)	63 (67.7)	69 (64.5)
Female	23 (40.4)	15 (30.0)	8 (57.1)	30 (32.3)	38 (35.5)
Age, years (N=107)
15-24	3 (5.3)	5 (10.0)	1 (7.1)	7 (7.5)	8 (7.5)
25-34	8 (14.0)	5 (10.0)	2 (14.3)	11 (11.8)	13 (12.1)
35-44	8 (14.0)	4 (8.0)	2 (14.3)	10 (10.8)	12 (11.2)
45-54	12(21.1)	15 (30.0)	2 (14.3)	25 (26.9)	27 (25.2)
≥55	26 (45.6)	21 (42.0)	7 (50.0)	40 (43.0)	47 (43.9)
Occupation (N=105)
Student	1 (1.8)	2 (4.0)	1 (7.1)	2 (2.2)	3 (2.9)
Farmer	54 (98.2)	48 (96.0)	13 (92.9)	89 (97.8)	102 (97.1)
Clinic factor
TB history (N=106)
New case	49 (87.5)	40 (80.0)	11 (78.6)	78 (84.7)	89 (84.0)
Retreated case	7 (12.5)	10 (20.0)	3 (21.4)	14 (15.2)	17 (16.0)
Geographic factor
Living town (N=105)
Bianyang	14 (25.9)	15 (30.0)	2 (14.3)	27 (29.0)	29 (27.1)
Fengting Zhen	5 (9.2)	2 (4.0)	1 (7.1)	6 (6.5)	7 (6.5)
Fengting Xiang	1 (1.9)	1 (2.0)	1 (7.1)	1 (1.1)	2 (1.9)
Hongshuihe	1 (1.9)	6 (12.0)	1 (7.1)	6 (6.5)	7 (6.5)
Longping	20 (37.0)	10 (20.0)	5 (35.7)	25 (26.9)	30 (28.0)
Luokun	7 (13.0)	2 (4.0)	2 (14.3)	7 (7.5)	9 (8.4)
Maojing	1 (1.9)	5 (10.0)	1 (7.1)	5 (5.4)	6 (5.8)
Moyang	5 (9.2)	8 (16.0)	1 (7.1)	12 (12.9)	13 (12.1)
Muyin	0	1 (2.0)	0	1 (1.1)	1 (0.9)

Supplementary.docx

Transmission and drug-resistance of tuberculosis in Luodian revealed by whole genome sequencing based molecular epidemiology study

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Abstract

BACKGROUND

METHODS

RESULTS

CONLUSIONS:

Figures

Introduction

Material and methods

Results

Discussion

Conclusion

Abbreviation

Declarations

References

Tables

Supplementary Files

Status:

Version 1