Correlation of Sputum mir-144 Copy Levels with Treatment Response among Pulmonary Tuberculosis Patients

Received: 17-Nov-2020 Accepted Date: Dec 21, 2020 ; Published: 28-Dec-2020

Introduction

Globally, Tuberculosis (TB) is the leading cause of mortality from a single infectious agent [1]. The incidence was found to be 10 million affected people in 2018, as per the recent Global TB report 2019 [1]. India is one of the 14 countries mentioned in the three high burden countries given by the World Health Organisation (WHO) for the period 2016-2020 namely TB, Multi-Drug Resistant TB (MDR-TB), and Human Immunodeficiency Virus/Tuberculosis (HIV/TB) co-infection [1]. With the emergence of molecular biology in the field of TB diagnostics, newer technologies are explored to provide a faster and accurate diagnosis of TB.

Up to 60% of the human genome is estimated to be under the regulation of miRNAs [2]. Discovered more than 25 years ago, these small non-coding miRNAs are critical in the embryogenesis of the lung [3]. miRNAs have been linked to the pathogenesis of various respiratory diseases like lung cancer, asthma, Chronic Obstructive Pulmonary Disease (COPD), pneumonia, pulmonary tuberculosis, and Pulmonary Hypertension (PH) [3-6]. If the triggers and the mechanisms behind their dysregulation are known, targeted therapies are possible. Few studies have probed the role of miRNAs in the pathogenesis of tuberculosis [7-8]. To date, multiple serum miRNAs have been studied for their role in molecular pathogenesis, diagnosis of tuberculosis, and their potential to discriminate latent TB from active tuberculosis, using different sequencing mechanisms [9-14].

While many studies have evaluated the role of circulating serum miRNAs in TB, their significance in the sputum sample has been analyzed only in a few studies [15-16]. Sputum culture evaluation is considered the gold standard in the diagnosis of Pulmonary Tuberculosis (PTB). But owing to the slow doubling time of Mycobacterium tuberculosis, results take approximately 6 weeks. Sputum smear microscopy is used for day-to-day practice as it is easy to perform and cheap but lacks sensitivity [17]. The emergence of molecular tests has drastically reduced the diagnosis time, but they too have some drawbacks-minimum volume of sputum needed to run the test and expensive kits/cartridges. Hence our quest for better diagnostic tools for TB remains significant to date.

Mycobacterium tuberculosis (MTB) induces the expression of miR-144*/miR-144-5p in the monocytes and macrophages of human beings, which in turn causes inhibition of DNA Damage Regulated Autophagy Modulator 2 (DRAM2). Because of DRAM2 inhibition, there is decreased autophagy induction leading to increased miR-144* levels in Peripheral Blood Mononuclear Cells (PBMC) of TB patients [18]. The validation of circulating serum miRNAs with sputum miRNAs gains importance since it is a non-invasive test and all our current diagnostic tools are sputum-based assays. Recently, Yan Lv et al., have observed a significant correlation of sputum and serum miRNA 144 levels with initial treatment response i.e., after 1 month of antitubercular therapy [16]. As the treatment duration of PTB varies between 6-8 months, correlation of miR-144 copy levels with sputum smear microscopy throughout the course is needed to ascertain that the initial down-regulation after ATT noted by previous studies was not by chance. Hence, we conceptualized a study to correlate the sputum miR-144 copies with treatment response during the full course of anti-TB chemotherapy. The initial results of our study have been previously reported in the form of an abstract [19].

Materials and Methods

Study Design

This is an analytical (observational) study to explore the correlation between sputum miR-144 levels with response to PTB treatment.

Subjects

Adult (>18 years) patients attending Pulmonary Medicine Outpatient Department or admitted under Pulmonary Medicine Department with cough for 2 weeks or more, along with fever, loss of appetite and weight, malaise, and night sweats, and diagnosed to have sputum smear-positive/smear-negative Pulmonary tuberculosis, as per Revised National Tuberculosis Control Program (RNTCP)-Technical and Operational Guidelines (TOG) for TB control in India 2016, were enrolled in the study [20]. Both new cases and previously treated patients were enrolled, after written informed consent. Patients with the following conditions were excluded from the study: Pregnant women, patients with Drug Resistant-TB (DR-TB), Extrapulmonary Tuberculosis (EPTB), HIV/TB co-infection, or any immunosuppressive conditions, taking immunosuppressive therapy, malignancy (of any organ), psychiatric illness, hemolytic anemia, thalassemia, ischaemic heart disease, type 2 diabetes mellitus, grave’s disease and those who were not willing to participate. Patients, newly diagnosed with any of the above-mentioned conditions during the study period, were also excluded. The study was approved by the Institutional Human Ethics Committee (Faculty/2016/03/27) and was conducted in compliance with the Declaration of Helsinki [21].

Initially, 12 controls (Stable Asthmatics or COPD) were enrolled in the study for analyzing the difference in baseline miR-144 copies among PTB patients and controls. The enrollment of the patients was continued after determining an initial difference between the two patient cohorts. Thus, a total of 46 patients and 12 controls were enrolled for this study.

Data Collection and Follow up

Sputum AFB samples were collected as per RNTCP protocol (2 samples-one ‘spot’ and one early morning sample) along with one sputum sample for miR-144. All sputum samples were positive for AFB at the baseline, except for one. Computed-Tomography (CT) of Thorax followed by Bronchoalveolar Lavage (BAL) was done for the smear-negative patient and BAL was found to be positive for AFB. Patients were started on Category I (6 months duration) or II treatment (8 months duration) as per RNTCP. One patient did not follow up after the first month. Two patients remained sputum AFB positive at the end of the Intensive Phase (IP) but their sputum Cartridge Based-Nucleic Acid Amplification Test (CB-NAAT) sample did not detect resistance to Rifampicin. All patients showed clinical improvement at the end of treatment and were sputum AFB negative. The end of treatment sputum sample for miR-144 analysis (third sample) was collected for all these patients.

RNA Isolation

miRNA was isolated using the HELINI™ PureFast Total RNA (miRNA) Mini spin Prep Kit (Helini, Biomolecules, Pvt India Ltd, India; Cat. No. 2008-25/50/100 preparations) according to the manufacturer’s protocol.

Quantitative Real-Time PCR

Helini microRNA Real-Time PCR kit (Helini, Biomolecules, Pvt India Ltd, India), which includes cDNA mix, miR- 144-3p cDNA primer mix, internal control-cDNA primer mix, probe PCR Master mix, and miR-144-30 probe primer mix was used. Sputum miRNAs were quantified using CFX96TM Real-Time Systems (BIORAD, California, USA). All the steps were followed according to the manufacturer’s instructions. Each reaction contained 2.5 microliters (μl) of sputum miRNA-cDNA in a total of 25 μl reaction. The qRT-PCR reaction was performed using the same kit with the following conditions: Taq enzyme activation at 95°C for 15 min., denaturation at 95°C for 20 sec., annealing/data collection at 56°C for 20 sec., and extension at 72°C for 20 sec.. Standard curves for absolute quantification was generated using commercially available synthetic miRNA oligonucleotides provided in the kit.

Results are reported as miRNA copy number per nanoliter of sputum, calculated based on the known copy number of the standards provided in the kit.

Analysis

Data were analyzed using IBM SPSS ver. 16.0 (IBM Co., Armonk, NY, USA). Quantitative data were recorded as mean ± standard deviation. Comparisons at baseline, end of IP, and end of anti-TB treatment were conducted by paired t-test. The other group comparisons were conducted by the use of the independent-samples t-test in one-way repeated measures of ANOVA (Post-hoc test: Bonferroni and Hochberg). Results were considered to be statistically significant when the p-value was <0.05.

Discussion

Baseline Characteristics

We found no significant difference between the age and sex of the patients and their respective baseline sputum miR- 144 copy levels. A similar finding was observed by Yanaihara et al., in which, they observed no age or sex difference between the serum miRNA levels and survival of adenocarcinoma patients postoperatively [22]. In a study correlating active TB and miR-29a expression profile using PBMCs, male patients were shown to have higher expression as compared to females, at baseline, and after 2 months of IP [23]. Circulating miR-144 was found to be elevated in a few other conditions due to its diverse roles in the pathogenesis of human diseases, from hemolytic anemia to malignancy [24-33].

Sputum miR-144 in PTB Diagnosis and Prognosis

Several studies have reported the ability to circulate miRNAs to differentiate between controls, Latent Tuberculosis Infection (LTBI), and TB disease status [34-37]. In our study, a statistically significant difference in sputum miR-144 copy levels was found between controls and active PTB patients (p<0.001).

A recent study using PBMCs and pleural fluid showed that there are changes in the systemic effects of miRNAs upon therapy [38]. They assessed miR-424 and miR-164a at baseline and observed that the values returned to normal after two months of successful therapy. In a similar study assessing four serum miR profiles, miR-16 showed down-regulation while miR-155 showed up-regulation upon treatment completion for active TB [37]. Though circulating miR levels were found to be stable, correlation with simultaneously collected sputum AFB samples was not done. Since Indian National guidelines stipulate sputum assessment for PTB patients during treatment and at the end of treatment, simultaneous sputum miR sample collection throughout antimycobacterial therapy is a viable option given the difficulties in processing serum samples like expertise, specialized equipment, and training needed to isolate specific cell types from the blood.

A report by Lv et al., correlated the serum and sputum expression levels of miR-144 levels and compared it with the control group [16]. This study has shown that sputum and serum miR-144 can be interchangeably used in the diagnosis and follow up of PTB. To assess treatment response, they followed up for one month, collected serum and sputum samples, and observed that both showed significant down-regulation of miR-144 expression profiles among responders. While the ability of sputum miR-144 to differentiate between controls and disease population is well understood in this study, the potential of miR-144 as a prognostic marker needed further evaluation.

The importance of follow-up sputum examination as and when there is clinical and/or microbiological deterioration can never be overemphasized in this era of drug resistance in TB. We followed up with the patients for their entire treatment period (6 or 8 months, based on whether they were a new case or previously treated). The down-regulation of sputum miR-144 among patients who had sputum for AFB smear conversion (done as a part of RNTCP guidelines) at the end of IP was found to be statistically highly significant (p<0.001).

As a Surrogate Marker for Non-Responders

The potential of miR-144 as a surrogate biomarker for clinical and microbiological deterioration during treatment needs to be noted. The ends of CP/end of treatment miR-144 copy levels were comparable to that of controls. The miR-144 copy levels of two patients who had smear positivity at the end of IP were found to be down-regulated at the end of treatment. Their values were comparable to that of the control group (69.24 ± 7.99 vs 61.72 ± 3.46, mean ± SD). Also, no statistical difference was observed between their sputum miR-144 copy levels to that of the rest 43 patients who had achieved smear conversion within two months of therapy (p=0.196).

To summarize, sputum miR-144 copy levels were able to differentiate between controls and PTB patients. It exhibited a significant down-regulation, in line with the sputum AFB conversion-at the end of IP and the end of CP/end of treatment. Furthermore, the downregulation of miR-144 copy levels was less among the two patients who did not have smear conversion at the end of IP as compared to those who had smear conversion. For these non-responders, as the clinical and microbiological improvement was noted at the end of treatment, miR-144 copy levels also showed downregulation and became comparable to that of the rest.

Limitations

Our study mostly included Tamilian South Indian population and thus cannot be extrapolated for other ethnic populations. Further, we limited our scope to study only drug-sensitive and PTB population as it was an exploratory approach towards treatment response for the entire therapy duration. Hence the impact of Extrapulmonary TB (EPTB), Human Immunodeficiency Virus (HIV)-TB, and Drug-Resistant TB (DR-TB) subpopulations on sputum miR-144 copy levels were not studied. Though two patients were sputum AFB positive at the end of IP, none developed drug resistance, and hence the analysis of sputum miR-144 copy levels among the DR-TB subpopulation could not be performed.

Directions for Future Research

Similar longitudinal studies among multi-ethnic cohorts including the challenging DR-TB, HIV-TB subpopulations will provide more insight into the usage of miR-144 copy levels. Nevertheless, in our quest for better diagnostic and prognostic biomarkers to achieve global targets envisioned in the WHO’s END TB strategy, molecular tests like this requires due consideration for their simple and non-invasive nature.

Declarations

Acknowledgment

We acknowledge Professor and ex-Head of the Department of Pulmonary Medicine Dr.K. Surendra Menon’s administrative support throughout the study period. We also acknowledge Mr. John’s support in the sample collection process.

Declaration of Conflict Of Interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Grant and Funding Information

Faculty intramural research grant by Sri Balaji Vidyapeeth.

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