Molecular characterization and drug susceptibility profile of Mycobacterium tuberculosis isolates from Northeast Bangladesh

Accepted Manuscript Molecular characterization and drug susceptibility profile of Mycobacterium tuberculosis isolates from Northeast Bangladesh

Mohammad Khaja Mafij Uddin, Moshtaq Ahmed, Mohammad Riazul Islam, Arfatur Rahman, Razia Khatun, M.D. Anwar Hossain, Aung Kya Jai Maug, Sayera Banu PII: DOI: Reference:

S1567-1348(18)30536-7 doi:10.1016/j.meegid.2018.07.027 MEEGID 3599

To appear in:

Infection, Genetics and Evolution

Received date: Revised date: Accepted date:

15 February 2018 24 May 2018 22 July 2018

Please cite this article as: Mohammad Khaja Mafij Uddin, Moshtaq Ahmed, Mohammad Riazul Islam, Arfatur Rahman, Razia Khatun, M.D. Anwar Hossain, Aung Kya Jai Maug, Sayera Banu , Molecular characterization and drug susceptibility profile of Mycobacterium tuberculosis isolates from Northeast Bangladesh. Meegid (2018), doi:10.1016/j.meegid.2018.07.027

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ACCEPTED MANUSCRIPT Molecular characterization and drug susceptibility profile of Mycobacterium tuberculosis isolates from Northeast Bangladesh Mohammad Khaja Mafij Uddin a, Moshtaq Ahmed a, Mohammad Riazul Islam b, Arfatur

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Rahman a, Razia Khatun a, Md. Anwar Hossain c, Aung Kya Jai Maug c, Sayera Banu

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*Correspondence to: Sayera Banu, Infectious Diseases Division, icddr,b

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68, Shaheed Tajuddin Ahmed Sarani, Dhaka-1212, Bangladesh

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e-mail: [email protected]

Mohammad Khaja Mafij Uddin

Infectious Diseases Division, icddr,b

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Authors Affiliations:

68, Shaheed Tajuddin Ahmed Sarani, Dhaka-1212, Bangladesh

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Moshtaq Ahmed

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e-mail: [email protected]

Infectious Diseases Division, icddr,b

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68, Shaheed Tajuddin Ahmed Sarani, Dhaka-1212, Bangladesh e-mail: [email protected]

Mohammad Riazul Islam b

Dept. of Biochemistry and Molecular Biology, University of Dhaka

Dhaka 1000, Bangladesh e-mail: [email protected]

a, *

ACCEPTED MANUSCRIPT Arfatur Rahman a

Infectious Diseases Division, icddr,b

68, Shaheed Tajuddin Ahmed Sarani, Dhaka-1212, Bangladesh e-mail: [email protected]

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Infectious Diseases Division, icddr,b

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68, Shaheed Tajuddin Ahmed Sarani, Dhaka-1212, Bangladesh

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e-mail: [email protected]

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Md. Anwar Hossain c

Damien Foundation Bangladesh

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H# 106, R # 25, Block - A, Banani, Dhaka - 1213

Damien Foundation Bangladesh

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c

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e-mail: [email protected]

Aung Kya Jai Maug

H# 106, R # 25, Block - A, Banani, Dhaka - 1213

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a

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e-mail: [email protected]

Sayera Banu

Infectious Diseases Division, icddr,b

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68, Shaheed Tajuddin Ahmed Sarani, Dhaka-1212, Bangladesh e-mail: [email protected]

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Razia Khatun

ACCEPTED MANUSCRIPT Abstract

Tuberculosis (TB) remains a major public health problem worldwide including in Bangladesh. Molecular epidemiological tools provide genotyping profiles of Mycobacterium tuberculosis (M. tuberculosis) strains that can give insight into the transmission of TB in a specific region. The

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objective of the study was to identify the genetic diversity and drug susceptibility profile of M.

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tuberculosis strains circulating in the northeast Bangladesh. A total of 244 smear-positive sputum specimens were collected from two referral hospitals in Mymensingh and Netrakona

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districts. The isolated strains were genotyped by deletion analysis, spoligotyping, and MIRUVNTR typing. We also analyzed the distributions of drug susceptibility pattern and demographic

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data among different genotypes. All isolates were identified as M. tuberculosis and among them

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167 strains (68.44%) were ‘ancestral’ and the remaining 77 (31.56%) were ‘modern’ type.

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Spoligotyping analysis yielded 119 distinct patterns, among them, 86 isolates had unique patterns and the remaining 158 were grouped into 33 distinct clusters containing 2-18 isolates. The

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predominant spoligotypes belong to the EAI lineage strains, comprising 66 (27.04%) isolates followed by Beijing (7.38%), T1 (6.15%), CAS1-Delhi (5.33), LAM9 (3.28%), MANU-2 and

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X2. MIRU-VNTR analysis revealed 167 isolates (68%) had unique patterns, whereas 77 (32%)

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were grouped into 26 clusters and the rate of recent transmission was 20.9%, suggesting that the majority of TB cases in this region are caused by the reactivation of previous TB infections rather than recent transmission. About 136 (55.7%) isolates were sensitive to four anti-TB drugs, 69 (28.3%) were resistant to one or more (except rifampicin and isoniazid combination) drugs and 39 (15.9%) were MDR. In conclusion, our study provides a first insight into molecular characterization and drug resistance profile of M. tuberculosis strains in northeast Bangladesh which will ultimately contribute to the national TB control program.

ACCEPTED MANUSCRIPT Keywords: Mycobacterium tuberculosis, Epidemiology, Deletion analysis, Spoligotyping,

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MIRU-VNTR, Drug resistance

ACCEPTED MANUSCRIPT 1. Introduction Tuberculosis (TB) is one of the major causes of death in developing countries and remains one of the major public health problems in Bangladesh, ranking seventh among 22 high TB burden countries with 225/100,000 TB incident cases and 45/100,000 mortality rate. The emergence of

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drug-resistant TB is becoming one of the major threats to TB control in Bangladesh. According

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to the World Health Organization (WHO) global TB report 2016, 3.9% of the new cases and

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21% of the previously treated cases were multi-drug resistant TB (MDR-TB)/ rifampicin resistant (RR-TB) (Kamal et al., 2015; World Health). Recent drug resistance surveillance

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reported that 2.3% of new and 13.8% of previously treated cases were MDR-TB in Bangladesh

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(Banu et al., 2017). In the last couple of decades, understanding TB epidemiology and the route of TB transmission has significantly increased due to the introduction of molecular methods for

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genotyping of M. tuberculosis (Arnold, 2007; Banu et al., 2012b; Jagielski et al., 2016; Liu et al.,

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2016; Supply et al., 2006). Most of the previously published M. tuberculosis epidemiological investigations were done using IS6110 based restriction fragment length polymorphism (RFLP),

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but the use of this method is declining due to the requirement of high-quality DNA, laborious

al., 2001).

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laboratory procedure and low copy number of IS6110 in many South Asian countries (Siddiqi et

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Spacer oligonucleotide typing (Spoligotyping) and Mycobacterial Interspersed Repetitive UnitsVariable Number of Tandem Repeats (MIRU-VNTR) typing methods are frequently being used in recent epidemiological studies. Spoligotyping is a polymerase chain reaction (PCR) and hybridization based technique that allows simultaneous detection and typing of M. tuberculosis complex into various lineages and sub-lineages (Brudey et al., 2006; Kamerbeek J, 1997).This method is useful for TB transmission surveillance and outbreak investigation. Spoligotyping is

ACCEPTED MANUSCRIPT quick, easy to perform and requires very small amount of DNA. On the other hand, discriminatory power of this method is lower than other available genotyping methods (Banu et al., 2012b; Jagielski et al., 2016). MIRU-VNTR is also a PCR based technique using primers specific for the flanking regions of the VNTRs. The size of the amplicon reflects the number of

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VNTR copies present in the specimens. MIRU-VNTR typing is also easy to perform, highly

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reproducible, and more discriminative than spoligotyping, but it is laborious and time consuming

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because of screening of large number of loci for each specimen. Recently 15-loci and 24-loci MIRU-VNTR typing have shown more discriminatory power than that of 12-loci (Mazars et al.,

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2001; Tarashi et al., 2017). In many settings, MIRU-VNTR along with spoligotyping has been

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used for epidemiological analyses (Banu et al., 2012a; Banu et al., 2013; Banu et al., 2015; Banu et al., 2012b; Oelemann et al., 2007). PCR based deletion analysis includes the region of

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difference (RD9), which differentiates members of the M. tuberculosis complex, whereas

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tuberculosis specific deletion 1 (TbD1) analysis discriminates between ancestral (BOV, Afri, EAI lineages) and modern (Beijing, CAS, T, H, X, LAM, MANU lineages) M. tuberculosis

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strains (Brosch et al., 2002; Shabbeer et al., 2012; Smith et al., 2009).

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Although Bangladesh is a TB endemic country, the data related to molecular epidemiology is limited. Very few studies from limited areas of Bangladesh have reported epidemiological and

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transmission information of TB. A study conducted in rural Matlab, Bangladesh, showed that EAI lineage was more prevalent (25%) among the investigated strains. A similar finding was reported in a pilot study conducted in an urban slum of Dhaka, Bangladesh (Banu et al., 2013). On the other hand, the Beijing genotype was documented as a more prevalent strain (19%) in a study conducted at a tertiary referral hospital in Dhaka (Banu et al., 2012a). Another study conducted in Dhaka Central Jail reported Beijing as the most predominant lineage, accounting

ACCEPTED MANUSCRIPT for 31% of all strains (Banu et al., 2015). The genetic information in association with drug resistance patterns of clinical M. tuberculosis isolates in Bangladesh is rare. The aim of the present study was to investigate the genetic diversity among the M. tuberculosis isolates circulating in the northeast part of Bangladesh. The study also aimed to explore the distribution

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of drug resistance patterns among the different lineages and sub-lineages defined by

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spoligotyping.

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2. Materials and Methods 2.1. Study population

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A total of 244 confirmed acid-fast bacilli (AFB) smear-positive sputum specimens were

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collected throughout the year 2004 from two TB and Leprosy referral hospitals situated in Mymensingh and Netrakona with a capacity of 100 and 52 beds respectively. Most of the

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patients admitted to these hospitals were from the northeast part of Bangladesh that includes

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Mymensingh, Netrokona, Kishoreganj, Jamalpur and Tangail districts. All the collected specimens were transported to the Mycobacteriology Laboratory of International Centre for

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Diarrhoeal Disease Research, Bangladesh (icddr,b), in Dhaka. In these studied areas, TB and

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leprosy control programs are headed by the Damien Foundation, Bangladesh. Sociodemographic and clinical data were collected from all the patients based on the structured

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questionnaire. The study protocol was reviewed and approved by the Research Review Committee and the Ethical Review Committee of icddr,b.

2.2. Culture and drug susceptibility testing All sputum specimens were processed with N-Acetyl-L-Cysteine (NALC)-Sodium Hydroxide (NaOH) method which is widely used and the recommended procedure for M. tuberculosis

ACCEPTED MANUSCRIPT identification (Uddin et al., 2013). Briefly, sputum specimens were decontaminated and digested with an equal volume of 4% NaOH and 2.9% Sodium-Citrate along with 1% NALC. The specimens were then neutralized with phosphate buffer saline (PBS; PH 6.8) and centrifuged at 3000g for 15 minutes at 4°C. The pellet was re-suspended in 1.5 mL of PBS and inoculated on

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Lowenstein-Jensen (L-J) slants. The L-J slants were incubated at 37°C up to eight weeks and

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checked weekly for visible colony. The standard proportion method was followed for drug

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susceptibility testing against four anti-TB drugs (Banu et al., 2017). The concentrations of the tested drugs were as follows: Isoniazid (I) (0.2 mg/l), Rifampicin (R) (40 mg/l), Ethambutol (E)

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(2 mg/l) and Streptomycin (S) (4 mg/l). An isolate was considered as resistant to a particular

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drug when the growth rate was ≥1% compared to the control. Similarly, when the growth rate

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was <1%, the isolate was considered as sensitive.

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2.3. Genomic DNA extraction

DNA was extracted from fresh culture cells by re-suspending mycobacterial colonies in 100-

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200 µL of distilled water and heating with heat block at 95°C for 30 minutes. After

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centrifugation at 10000 rpm for 5 minutes, the supernatant containing the genomic DNA was

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stored at -20°C for further use.

2.4. Deletion analysis

Deletion analysis with RD9 and TbD1 was done using the PCR described previously (Brosch et al., 2002). Internal and flanking primer sequences for RD9 and TbD1 were obtained from the following websites: http://www.genolist.pasteur.fr/tuberculist/ and http://www.sanger.ac.uk/projects/m_bovis/.

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2.5. Spoligotyping Spoligotyping was performed using commercially available kit (Isogen Biosciences, BV, Bilthoven, Netherlands) following the standard protocol described previously (Kamerbeek et al.,

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1997). In short, M. tuberculosis DNA was amplified by biotinylated Dra and Drb primers using

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the PTC-200 (Bio-Rad Laboratories, Inc., PA., USA) PCR system. The amplified PCR products

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were hybridized with nitrocellulose membrane that is covalently linked with 43 synthetic oligonucleotides corresponding to 43 spacers. The hybridized products were identified by the

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enhanced chemiluminescence system (Amersham, UK). Different spoligo patterns were

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converted into octal code and major spoligotypes were determined based on phylogenetic clades using freely accessible SITVITWEB database (http://www.pasteur-

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guadeloupe.fr:8081/SITVIT_ONLINE/) (Demay et al., 2012).

2.6. MIRU-VNTR typing

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MIRU-VNTR typing was performed for 12 loci (2,4,10,16,20,23,24,26,27,31,39 and 40) using

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the standard protocol described previously (Banu et al., 2004; Mazars et al., 2001; Supply et al., 2000). Data regarding MIRU 20 locus was excluded from the analysis as the primers failed to

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produce specific PCR products. MIRU-VNTR copy number was determined as described previously by Supply et al. (Supply et al., 2000). MIRU-VNTRplus web application (http://www.miru- vntrplus.org/MIRU/index.faces) was used to define the clusters and to build an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree. The rate of recent transmission was calculated using the formula:[T(c) - N(c)]/ T(a), where T(c) is the total number of

ACCEPTED MANUSCRIPT clustered strains, N (c) is the number of clusters and T(a) is the total number of strains (Brudey et al., 2004).

2.7. Statistical analysis

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All the data were entered and analyzed using the Statistical Package for Social Sciences

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(SPSS) version 20.0 (IBM Corp, Armonk, NY, USA). Chi-square tests or Fisher’s exact tests

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were used to identify any statistical significance of the difference between clustered and non-

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clustered strains. P-value < 0.05was considered to be statistically significant.

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3. Results 3.1. Demographic characteristics of patients

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Of the 244 patients, 213 (87.3%) were from the northeast part of Bangladesh and the

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remaining 31 (12.7%) were from other regions of the country. Among these patients, 154 (63.1%) were male and 90 (36.9%) were female. The age of the patients ranged from 11 to 90

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years with the mean age of 41.26 years. More than half of the patients (54.9%) were from 20-45

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years, 38.1% were >45 years and only 7.0% were below 20 years of age. According to the treatment history, 45.9% were new cases and 54.1% of the patients were previously treated as

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TB patients (Table 1).

3.2. Deletion analysis All 244 isolates were confirmed as M. tuberculosis on the basis of the presence of RD9 region which is usually conserved in M. tuberculosis. Among the strains, 167 (68.44%) were TbD1

ACCEPTED MANUSCRIPT intact, indicating that these strains were ‘ancestral’ type whereas in 77 strains (31.56%), this region was deleted indicating that these were ‘modern’ type (Table 2).

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3.3. M. tuberculosis lineages determined by Spoligotyping

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Spoligotyping analysis revealed 119 distinct spoligo patterns of which 86 had unique patterns

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and the remaining isolates were grouped into 33 distinct clusters containing 2 to 18 isolates (Fig.

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1 and Table 2). Based on the SITVITWEB database, a total of 134 (54.92%) isolates were subdivided into 17 specific lineages comprising 40 different Spoligotype International Type

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(SITs). Specific lineages could not be described for the remaining 110 (45.08%) isolates which were considered as “orphan” (Table 2). There were six different specific sub-lineages among the

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EAI lineage strains, including EAI5, EAI6-BGD1, EAI1-SOM, EAI7-BGD2, EAI3-IND and

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EAI2-Manila types. Among EAI genotypes, the largest sub-lineage belonged to the EAI5 family, consisting of 24 (9.84%) isolates and the second largest sub-lineage was the EAI6-BGD1 or

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‘Matlab’ type consisting of 22 (9.08%) isolates. A total of 18 (7.38%) isolates belonged to the

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Beijing genotypes. The other common lineages were T1 (6.15%), CAS1-Delhi (5.33), EAI1SOM (4.92%), LAM9 (3.28%), EAI7-BGD2 (1.63%), X3 (1.63%), EAI3-IND (1.23%), H3

(Table 2).

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(1.23%), T4 (1.23%), CAS2 (0.41%), EAI2-Manila (0.41%), MANU-2 (0.41%), and X2 (0.41%)

3.4. Regional distribution of M. tuberculosis lineages Geographical distribution of M. tuberculosis lineages is shown in Fig. 2. The ancient EAI strains were predominantly found in Tangail (39.3%), Netrakona (32.3%) and the fewest in the

ACCEPTED MANUSCRIPT Jamalpur district (18.7%). On the other hand, the Beijing genotype was predominantly found in Mymensingh (10.8%) with the fewest in Tangail district (3.6%). The T strains were found in all regions but predominantly in Tangail (10.7%). Other lineages including CAS, MANU, LAM 9,

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X contributed to less than 10% of all isolates.

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3.5. Cluster analysis by MIRU-VNTR typing

The MIRU-VNTR analysis revealed 193 distinct patterns including 77 (31.5%) strains in 26

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clusters and 167 (68.5%) were unique strains. The largest cluster contained 9 strains and the

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other clusters were composed of 2 to 5 strains (Fig. 1 and Table 3). The 18 Beijing strains were further grouped into 17 distinct patterns including one cluster of 2 strains by MIRU-VNTR

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typing. The rate of recent transmission was 20.9%, where T (c) = 77, N(c) = 26 and T(a) = 244.

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Based on the geographical locations, we could not document any relationship among the patients who had similar MIRU-VNTR patterns. All these patients were from the different villages (Fig.

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3).

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3.6. Phylogenetic analysis

Genetic relatedness among the studied strains was determined based on the UPGMA tree formed by MIRU-VNTRplus database using 11-loci MIRU-VNTR (MIRU 2,4,10,16,23,24,26,27,31,39 and 40) and spoligotyping data. When spoligotyping and MIRUVNTR were used independently, the clustering rate was 64.7% and 31.5% respectively. On the other hand, with the combined use of MIRU-VNTR and spoligotyping, the clustering rate reduced to 16.8%. In the combined analysis, 41 strains were grouped into 17 clusters consisting

ACCEPTED MANUSCRIPT of 2-5 strains (Fig. 1 and Table 3). We also analyzed the geographical location, age, sex, treatment history and drug resistance profile of the clustered and non-clustered strains determined by the combined use of spoligotyping and MIRU-VNTR. We could not identify any

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epidemiological linkages among the clustered strains (Table 5).

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3.7. Drug resistance patterns among the spoligo lineages

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Drug susceptibility test results revealed that 136 (55.8%) isolates were sensitive to all four first-line drugs, 69 (28.3%) were resistant to any one or more (except I+R combination) drugs

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and 39 (15.9%) were MDR (Table 4). Of the 69 resistant cases, 31 (44.9%) were mono-resistant,

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25 (36.3%) were resistant to double drugs and 13 (18.8%) were resistant to triple drugs. Out of 39 MDR cases, 6 (15.4%) belonged to EAI, 8 (20.6%) belonged to Beijing, 6 (15.4%) belonged

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to T, 2 (5%) belonged to CAS, 7 (17.95%) were orphan and 10 (25.65%) belonged to other

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lineages (LAM, X, H, MANU). Among the Beijing genotype, 8 (42.1%) were MDR cases, 3 (15.8%) were sensitive to all drugs, 4 (21.07%) were mono-resistant, 2 (10.54%) were resistant

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to two drugs and 2 (10.54%) were resistant to three drugs. Among 136 sensitive strains, 74

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(54.41%) belonged to orphan group, 45 (33.08%) belonged to EAI family and remaining 17 (12.5%) belonged to other lineages.

4. Discussion The present study gives an insight into the molecular characterization of M. tuberculosis strains circulating in the northeast part of Bangladesh. This study was performed in two referral

ACCEPTED MANUSCRIPT hospitals in Mymensingh and Netrakona districts where most of the patients were admitted from the northeast part of Bangladesh. In this study we found that 167 (68.44%) strains were ‘ancestral’ and 77 (31.56%) strains belonged to the ‘modern’ type. The percentage of the ancestral strains is similar to our previous

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study conducted in a rural area of Bangladesh where the ancestral strain was 65% (Banu et al.,

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2012b). However, compared to present study, higher percentage of modern strains (60-70%) was

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found in other studies conducted in a tertiary care hospital and the central jail in Dhaka , Bangladesh, (Banu et al., 2012a; Banu et al., 2015).

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Spoligotyping analysis revealed that the most predominant lineage was the EAI genotype

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comprising 66 strains (27.04%) of all isolates. All the EAI genotypes correspond to the ‘ancestral’ M. tuberculosis type. We also found that EAI5 and EAI6-BGD1 were the most

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predominant sub-lineages of EAI, consisting of 24 (9.84%) and 22 (9.02%) isolates respectively.

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The EAI lineage strain was also predominantly found in previous studies in Bangladesh as well as in neighboring countries (Banu et al., 2012a; Banu et al., 2013; Banu et al., 2012b; Devi et al.,

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2015; Parwati et al., 2008). This data clearly suggests that the high prevalence of TB is due to the

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‘ancestral’ or ‘EAI’ strains of M. tuberculosis in the northeast part of Bangladesh and may have a long history in this geographical region (Banu et al., 2013; Banu et al., 2012b; Rahim et al.,

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2007). The Beijing family strains accounted for 7.38% of all isolates which was the second most prevalent strain found in the studied region. This finding was comparable with the previous studies conducted in Bangladesh (Banu et al., 2013; Banu et al., 2012b; Rahim et al., 2007). Previously conducted studies in an urban hospital and also in the largest prison in Bangladesh, the Beijing strains were 19% and 31% respectively (Banu et al., 2004; Banu et al., 2012a). Geographical location as well as duration of the study may be the reasons for the lower rate of

ACCEPTED MANUSCRIPT Beijing strains in the current study. The ill-defined T lineage, specifically T1, was also predominant in our study. Among the 18 strains belonging to the T family, 15 (83%) were from T1 sub-lineage. The T lineage exists predominantly in Central and South American, African and European countries (Weniger et al., 2010). Although there is no geographical linkage between

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also consistent with the previous findings (Banu et al., 2012a).

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Bangladesh and these countries, T lineage was also circulating in the studied region, which is

The MIRU-VNTR analysis revealed 193 distinct patterns and the majority of the strains had

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unique profiles, indicating a lack of transmission in this region. In the present study, the rate of

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MIRU-VNTR clustering was higher compared to other studies conducted in Bangladesh (Banu et al., 2004; Banu et al., 2015; Banu et al., 2012b). Usually clustering indicates the rate of recent

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transmission of TB. The rate of clustering depends on numerous factors including incidence rate

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of a particular area, heterogeneity of the population, sampling procedures, duration of the study and inclusion of inpatient and outpatient isolates (Jagielski et al., 2016). In developed countries

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the rate of recently transmitted cases is reported to vary from 10% to 58% (Murray and Nardell,

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2002; Yang and Gao, 2018) and from 20% to 36% in developing countries (Godfrey-Faussett et al., 2000; Narayanan et al., 2002). In the present study, the rate of recent transmission was 20.9%

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which is higher than previous studies in Bangladesh. The rate of recent transmission was different such as 6.5% in a rural Matlab area and 9.6% in Dhaka Central Jail (Banu et al., 2015; Banu et al., 2012b). There could be two possible reasons for the higher clustering in this studied population. Firstly, the previous transmission may have occurred before our sampling procedure and secondly, the examined isolates were collected from the inpatient hospitals but other studies were conducted in specific areas of the country. On the other hand, the rate of recent

ACCEPTED MANUSCRIPT transmission was considerably lower than other studies conducted in different countries; like 3841 % in New York (Alland et al., 1994) and 36% in France (Gutierrez et al., 1998). In a study conducted at two outpatient hospitals in Sao Paulo, Brazil, the authors reported 32% of recent transmission rate (Ferrazoli et al., 2000). Another study performed at a tertiary care hospital in

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Brazil over one year of period found 19% recent transmission, which is very similar to our

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finding (Fandinho et al., 2000). We found a considerably higher (55.84%) clustering rate among

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the younger age group (15-45 years), which is also consistent with several other studies (Goldblatt et al., 2014). Frequent migration for their employment is likely to be a significant

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cause of higher clustering rate among the younger age group.

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In the present study, 90% of MDR-TB patients were previously treated which indicate acquired MDR-TB is higher among these populations. We found an overall drug resistance (resistant to

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any drug) level as 28.28%, and MDR-TB as 15.98%. The rate of MDR-TB is higher than the

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nationwide survey but lower than the previous study conducted in the tertiary hospital in Dhaka, Bangladesh (Banu et al., 2012a; Kamal et al., 2015). There might be numerous factors behind the

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higher drug resistance and MDR-TB, such as the type of infections, treatment history, admission

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of severely ill patients, DOTS strategies, and also the demographic profile of these regions which may be different from the nationwide survey as well as from other studies. Among the MDR

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cases, Beijing genotype constitutes highest number of MDR (20.6%) cases followed by both EAI and T1 (15.4%). Our data also showed that among the Beijing genotype, 42.1% of the cases were MDR. This data demonstrates that the emergence of MDR strains belonging to the Beijing, EAI and T families remains a threat to the local TB control program. Our study has few limitations. Firstly, the use of agarose gel electrophoresis based 12-loci MIRU-VNTR typing method instead of 15-loci or 24 -loci MIRU–VNTR typing with automated system. In many studies the use of an

ACCEPTED MANUSCRIPT automated method with 15 or 24- loci MIRU-VNTR showed more discriminatory power than the use of 12-loci (Vadwai et al., 2012). Secondly, the study specimens were only collected from admitted patients of the two referral hospitals. This may not reflect the original clustering rate.

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5. Conclusions

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The present study demonstrated that the ‘ancestral’ strain was more prevalent than the ‘modern’

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strain, suggesting that drug susceptible ‘ancestral’ types are circulating in this area. The current study also revealed that the EAI family is the most predominant lineage spread over the northeast

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part of Bangladesh. Along with EAI, the Beijing strains were also common in this region and

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have a high degree of transmission potential as well as spreading MDR-TB. In conclusion, our study findings suggest that the majority of TB transmission in this region was due to the

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reactivation of previous TB infection. In addition, a lower rate of recent transmission was found

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in this studied region. The genetic profile and strain diversity information obtained from the present study could be useful for larger population based epidemiological studies on tuberculosis

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in Bangladesh, ultimately contributing to the national TB control program.

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Acknowledgements

This study was supported by WHO and Gates Foundation. icddr,b acknowledges with gratitude the commitment of WHO and Gates Foundation to their research efforts. icddr,b is also grateful to the Governments of Bangladesh, Canada, Sweden and the UK for providing core/unrestricted support. The authors are thankful to all the staff of TB and Leprosy hospitals in Mymensingh and Netrakona leaded by Damien Foundation, Bangladesh for their support during the study period. We are also grateful to all the subjects who were participated in this study to make it success. In

ACCEPTED MANUSCRIPT addition, we would like to express our sincere thanks to all laboratory staff for their excellent

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work on this study.

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Arnold, C., 2007. Molecular evolution of Mycobacterium tuberculosis. Clinical Microbiology

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Table and figures

Variable

Label Male Female Youth (<20) Adult (20-45) Old (>45) New case Previously treated Mymensingh Netrokona Kishoreganj Tangail Jamalpur Others

Age

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Treatment history

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Sex

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Table 1. Socio-demographic and clinical characteristics of 244 tuberculosis patients from greater Mymensingh districts of Bangladesh

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Geographic

Number of patient (n= 244)

Frequency %

154 90 17 134 93 112 132 65 65 39 28 16 31

63.1 36.9 7.0 54.9 38.1 45.9 54.1 26.6 26.6 16.0 11.5 6.6 12.7

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24 22 18 15 13 12 8 4 4 3 3 3 1 1 1 1 1 110

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TbD1 Type Ancestral (TbD1+) Modern (TbD1-)

167 77

Percentage (%)

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No. of isolates (n=244)

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Categories Spoligo Lineages EAI5 EAI6-BGD1 Beijing T1 CAS1-Delhi EAI1-SOM LAM9 EAI7-BGD2 X3 EAI3-IND H3 T4 Beijing- like CAS2 EAI2-Manila Manu2 X2 Orphan

9.84 9.02 7.38 6.15 5.33 4.92 3.28 1.63 1.63 1.23 1.23 1.23 0.41 0.41 0.41 0.41 0.41 45.08

68.44 31.56

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No of

Clustered

Unique

Cluster size

Clustering

pattern (n)

cluster (n)

Isolate (n)

Isolates (n)

(n)

rate (%)

MIRU

193

26

77

167

2-9

31.5

Spoligo

119

33

158

86

2-18

64.7

MIRU-Spoligo

220

17

41

203

2-5

16.8

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Methods

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MIRU, Mycobacterial Interspersed Repetitive Units

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No of

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of both method among the studied strains.

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Others(LAM ,X,H,M ANU)

Orphan

Overall

2 (10. 5) 1 (5.5)

0

19

0

18

2 (14. 3) 0

0

14

0

17

5 (4.5)

1 (0.9 ) 1 (0.4 )

11 0

E

SI

SR

SE

RE

IE

S IE

5 (7.6)

3 (4.5 ) 0

1 (1.5 ) 0

0

1 (1.5)

0

0

0

2 (3.1)

1 (5.2)

0

1 (5.2 ) 0

0

1 (5.5 ) 0

1 (5.2 ) 0

3 (4.5 ) 0

0

0

0

7 (6.3)

2 (1.8 ) 6 (2.5 )

0

1 (7.1)

16 (6.5)

0

1 (7.1 ) 0

1 (0.4 )

4 (22. 3) 3 (21. 4) 3 (17. 6) 4 (3.6)

6 (5.4 ) 8 (3.3 )

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CAS (1-Delhi,2)

66

R

3 (15. 8) 0

0

0

0

0

1 (5.5 ) 0

0

0

0

0

0

2 (1.8 ) 5 (2.1 )

1 (0.9 ) 2 (0.8 )

1 (0.9 ) 1 (0.4 )

0

16 (6.5)

1 (0.4 )

0

12 (4.9)

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S, Streptomycin; I, Isoniazid; R, Rifampicin; E, Ethambutol; Res, Resistance; Sen, Sensitive; MDR, Multi-drug resistant

To tal

SR E 0

I

IP

T (1,4)

Triple Res

S

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Beijing (Beijing, Beijing-like)

45 (68 .2) 3 (15 .8) 5 (27 .8) 5 (35 .7) 4 (23 .6) 74 (67 .2) 13 6 (55 .7)

M DR 6 (9. 1) 8 (42 .1) 6 (33 .4) 2 (14 .3) 10 (58 .8) 7 (6. 3) 39 (16 .0)

Double Res

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EAI (2-M anila,3IND,5,SOM , 6-BGD1,7BGD2)

Mono Res

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Lineage

Resistance pattern, n (%)

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All Se n, n (% )

24 4 (10 0)

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Geographic

TbD1

T

IP

CR

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Treatment history Drug resistance

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Sex

<20 20-45 >45 Male Female Previously treated New cases Any S resistance Any I resistance Any R resistance Any E resistance MDR Mymensingh Netrokona Kishoreganj Tangail Jamalpur Others Ancestral Modern

Clustered Yes (n=41) (%) No (n=203) (%) 4 (23.5) 13 (76.5) 17 (12.6) 117 (87.4) 20 (21.5) 73 (78.5) 22 (14.8) 132 (85.2) 19 (21.1) 71(78.9) 20 (15.1) 112 (84.9) 21 (18.7) 91 (81.3) 13 (15.1) 73 (84.9) 14 (18.9) 60 (81.1) 7 (15.9) 37 (84.1) 8 (12.5) 56 (87.5) 7 (17.9) 32 (82.1) 12 (18.5) 53 ( 81.5) 15 (23.0) 50 (77) 2 (5.1) 37 (94.9) 4 (14.2) 24 (85.8) 4 (25.0) 12 (75.0) 4 (12.9) 27 (87.1) 30 (17.9) 137 (82.1) 11 (14.2) 66 (85.8)

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Age (years)

Label

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Characteristics

p-Value 0.16

0.23 0.56 0.73 0.69 1.00 0.38 1.00 0.82 0.17 0.06 0.91 0.58 0.71 0.596

S, Streptomycin; I, Isoniazid; R, Rifampicin; E, Ethambutol; Res, Resistance; Sen, Sensitive;

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MDR, Multi-drug resistant

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CE

PT

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M

AN

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Fig. 1. UPGMA tree showing the genetic relationship among 244 isolates based on 12-loci MIRU-VNTR and their corresponding spoligotypes. The bars denote the cluster isolates, from right to left: strain name, SIT based on SITVITWEB, MIRU-VNTR and Spoligo pattern.

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Fig. 2. Geographical location of various lineages among the studied strains

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Fig. 3. Map of the greater Mymensingh districts (northeast Bangladesh) showing the location of the clustered strains. Individual color and shape denoted as different clusters.

ACCEPTED MANUSCRIPT Highlights 

Investigation of genetic diversity and drug resistance profile among the Mycobacterium tuberculosis isolates of Northeast Bangladesh.

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‘Ancestral’ strain is more prevalent than the ‘modern’ strain, suggesting that drug

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susceptible ‘ancestral’ types are circulating in this area. EAI lineages have been identified as predominant followed by Beijing and T family.

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Drug resistances TB are common in Beijing genotype.

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Reactivation of previous TB infection plays an important role in TB transmission in

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northeast part of Bangladesh.