Socio-Demographic Characteristics and Risk Factors Contributing Pulmonary Tuberculosis Infection and Recent Transmission

Host factors, environmental factors, genetic diversity and distinct phylogeo-graphic distribution of Mycobacterium tuberculosis (MTB) contribute to re-gional differences in drug resistance. Bangladesh remains among the top 20 high Multi drug resistant tuberculosis (MDR-TB) burden countries of the world. This cross sectional study was conducted to identify the socio demographic characteristics and the risk factors contributing Pulmonary tuberculosis (PTB) infection. These characteristics and risk factors were further investigated among the clustered isolates. Total 60 culture isolates consist of 40 RR and 20 rifampicin sensitive (RS) isolates were enrolled in this study. Laboratory works were done in National Tuberculosis Reference Laboratory (NTRL) and Department of Microbiology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh. All 60 samples were con-firmed as MTB by MPT 64 antigen detection. Two samples were excluded for possible cross contamination and two for failing to give PCR product for most of the locus. So, finally 56 samples were further analyzed for results.


Introduction
Drug-resistant Tuberculosis (TB) threatens global TB care and prevention, and remains a major public health concern in many countries. Globally in 2016, an estimated 4.1% of new cases and 19% of previously treated cases had MDR/Rifampicin resistant TB (RR-TB) [1]. MDR-TB is TB that is resistant to both rifampicin and isoniazid. Both MDR & RR-TB require treatment with second-line drugs. In 2015, the incidence of MDR/RR-TB was 6/100,000 population in Bangladesh. Distribution of MDR/RR-TB was 1.6% in new cases of TB and 29% in previously treated cases of TB in 2015. In the same year, total estimated cases of MDR/RR-TB were 5100 [2] [3]. MDR-TB can occur via two mechanisms: through the selection of resistant M. tuberculosis bacilli during poorly managed anti-TB treatment or via direct transmission from an infectious patient to another. The risk of drug resistance is higher among TB patients who have previously received anti TB chemotherapy. Early studies suggested that effective treatment of drug-susceptible TB would prevent the emergence of resistance [4].
Some studies suggested that failure to diagnose and treat MDR-TB might result in "hot spots" of disease [5].
By incorporating molecular biology methods, epidemiological studies have reached the molecular level. Molecular epidemiology has emerged from a combination of genotyping techniques and conventional epidemiological approaches. This new discipline has established as a resource for understanding the issues in the epidemiology of TB. It comprises sources of infection, to quantify recent transmission, linking transmission and factors for transmission, to evaluate reinfection from relapse. It also tracks the geographic distribution, clonal expansion of strains [6] and helps to identify major clones in association with multidrug resistance [7].
The Mycobacterium tuberculosis Complex (MTBC) consists of a group of highly related mycobacterial lineages (99.9% nucleotide identity). This Complex comprises obligate human pathogens Mycobacterium tuberculosis sensustricto and Mycobacterium africanum. The geographical spread of these lineages differs markedly. Some lineages exhibit a global distribution and others a strong geographical restriction [8] [9].
Delays in the diagnosis and control measures can result in further spread of TB, even among individuals who are at low risk [10]. TB transmission can be extended to social and casual contacts, which are more difficult to trace by classical epidemiological investigation [11] [12]. If asymptomatic individuals remain undetected for a prolong time, they can remain contagious and be the source of unsuspected TB outbreaks [13].

Ethical Considerations
Before starting this study, the research protocol was approved by the Institution- study for further analysis. So, according to this inclusion criterion, all MTB culture positive samples during this study period were included. These patients were enrolled in this study and were further interviewed for socio-demographic data and risk factors according to a predesigned data sheet which included particulars of the patients, socio-demographic characteristics, risk factors and laboratory findings. Informed written consent or assent was taken before collecting these data. The results of these socio-demographic data and risk factors are discussed in result section (see Table 1 & Table 2). At NTRL, isolates were cultured on both Lowenstein-Jensen (L-J) media and Mycobacteria Growth Indicator Tube (MGIT) and were identified as AFB by Z-N staining and as Mycobacterium tuberculosis by MPT 64 antigen test (Standard Diagnostic Inc., Korea) following standard operating procedure. Genotyping of the MTB isolates were done at BSMMU by 24 locus MIRU-VNTR analysis as described by Supply et al. [14].

Data Analysis
Data were entered and analysed with IBM SPSS ver.23 software. Among clustered 4 patients 3 (75%) were male. All of them were between 20 -29 years of age. All of them were BCG vaccinated and resided within Dhaka city.

Results
All isolates of the patients were rifampicin resistant and members of Beijing lineage. Only important risk factor was found to be previous history of anti-TB treatment among 3 out of 4 patients ( Table 2). A study in a rural area of Bangladesh found similar findings except prevalent age group which was 46 -60 years for most of the cases [17].

Discussions
Overall, most of the previous Bangladeshi studies found more or less similar findings for socio demographic data and risk factors.
Out of 38 RR isolates, 2 clusters were found; consist of 2 isolates each. All 2 clusters were Beijing genotypes. A cluster was defined as two or more isolates from different patients with identical MIRU patterns, whereas nonclustered patterns are referred to as unique. The underlying assumption is that genotypes evolve on the same timescale as the process of disease transmission so that each cluster of isolates represents a set of cases that arose recently through transmission, but isolates that are not connected via recent transmission are different through accumulated mutations [13]. Based on this finding, recent transmission index (RTI) was found to be low as 0.105% or 10.5% by "n methods" (Appendix II) [18]. Low transmission rate (6.5%) was also found in study held in rural area of Matlab, Bangladesh [17].
A study was held by incorporating data from 6 countries of different geographic location with different MDR-TB burden ratio. The findings were that 96% of TB cases were due to direct transmission. The highest transmission (99%) was estimated in Uzbekistan and lowest (48%) was in Bangladesh though it is a country of high MDR-TB burden. The other countries were included in this study were Ethiopia, Malawi, Peru and Philippines showed the transmission rate 92%, 82% , 95% and 76% respectively [19]. In Iraq, a study found 33.6% transmission rate [20] Low index found in the present and previous studies of Bangladesh indicates that the drug resistance TB is occurring in our area is by reactivation or acquisition rather than transmission.
The population characteristics and risk factors of clustered cases of the present study were further investigated for epidemiological linkage. Direct transmission link could not be identified through investigation. However, all of these patients resided in the different parts of Dhaka city. And all of these patient's ages were between 20 -29 years. These factors are in favors of transmission. Dhaka city is one of the most overpopulated cities of the world. There might be chance to come in contacts of the patients unknowingly in any occasion. Others characteristics of these patients included the male predominance, all were BCG vaccinated and no apparent risk factors were found excluding anti-TB treatment. Three of them were previously treated whereas 1 was a new case. All the clustered isolates were Beijing strains.
Other previous studies in Bangladesh also could not draw any direct epidemiological linkage regarding clusters. However one of the study found clustered cases among the 19 -40 years of age group and both of the study identified Beijing as the most predominant lineage among clustered isolates [16] [17].
In this study, all the clustered persons were found to be BCG vaccinated. It is hypothesized that the Beijing strains originated in China and disseminate to the region [21]. It is also hypothesized that mass BCG vaccination may create a se-