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 Table of Contents  
Year : 2021  |  Volume : 10  |  Issue : 4  |  Page : 421-427

The espD full gene as a potential biomarker in active pulmonary tuberculosis

1 Department of Medical Microbiology, Faculty of Medicine, Universitas Airlangga; Department of Laboratory of Tuberculosis, Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia
2 Department of Microbiology, Faculty of Medicine, Universitas Ciputra, Surabaya, Indonesia; Department of Bacteriology School of Medicine, Niigata University, Niigata, Japan
3 Department of Pulmonology and Respiratory Medicine, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia
4 Department of Health, Faculty of Vocational Studies, Universitas Airlangga; Department of Proteomic Laboratory, University CoE Research Center for Bio Molecule Engineering, Universitas Airlangga, Surabaya, Indonesia
5 Department of Proteomic Laboratory, University CoE Research Center for Bio Molecule Engineering, Universitas Airlangga; Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia
6 Laboratory of Tuberculosis, Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia
7 Department of Medical Microbiology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia; Department of Bacteriology School of Medicine, Niigata University, Niigata, Japan

Date of Submission28-Sep-2021
Date of Decision11-Oct-2021
Date of Acceptance05-Nov-2021
Date of Web Publication14-Dec-2021

Correspondence Address:
Ni Made Mertaniasih
Jl. Mayjen Prof. Dr. Moestopo No. 47 Surabaya 60131
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_198_21

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Background: Pulmonary tuberculosis (PTB) is still a major health problem worldwide. The espD has a potential to be a new biomarker because it is important for the espA, espC, and ESX-1 protein secretion system that are actively expressed in active multiplication of Mycobacterium tuberculosis complex. Methods: A total of 55 sputum samples and 41 culture isolates from newly diagnosed PTB patients at Dr. Soetomo Academic Hospital were collected from September 2016 to April 2019. The tested samples using polymerase chain reaction targeted 555 bp of espD gene and sequencing. Clone Manager Version 6 and NCBI BLAST were used to align the gene sequence against wild-type M. tuberculosis. The prediction of T-cell epitope in espD gene was detected by GENETYX. The three-dimensional (3D) structure of espD was modeled by SWISS-MODEL and I-TASSER and was visualized with PyMOL. Results: From 55 sputum samples, 43 (78.18%) showed positive results, and all culture isolates showed positive results. In addition, all sequenced samples showed 100% homolog with M. tuberculosis H37Rv gene without detected variant or mutation. There were four T-cell epitopes that could be obtained. The 3D model had a I-TASSER confidence score of 3.91 with estimated RMSD of approximately 14.5 Å. The structure consists of a main fold of a three-stranded antiparallel β-sheet and a long α-helix surrounded by several minor secondary structures. Conclusions: This study provides a brief information about the sequence, epitope prediction, and 3D structure of EspD protein from M. tuberculosis strains in Indonesia.

Keywords: espD full gene, Mycobacterium tuberculosis, polymerase chain reaction, T-cell epitopes, three-dimensional protein structure

How to cite this article:
Mertaniasih NM, Surya Suameitria Dewi DN, Soedarsono S, Kurniati A, Rohman A, Nuha Z, Matsumoto S. The espD full gene as a potential biomarker in active pulmonary tuberculosis. Int J Mycobacteriol 2021;10:421-7

How to cite this URL:
Mertaniasih NM, Surya Suameitria Dewi DN, Soedarsono S, Kurniati A, Rohman A, Nuha Z, Matsumoto S. The espD full gene as a potential biomarker in active pulmonary tuberculosis. Int J Mycobacteriol [serial online] 2021 [cited 2022 Jan 21];10:421-7. Available from: https://www.ijmyco.org/text.asp?2021/10/4/421/332358

  Introduction Top

Pulmonary tuberculosis (PTB) is an infectious disease caused by Mycobacterium tuberculosis. It is still a major health problem causing high mortality worldwide. M. tuberculosis is an intracellular pathogen that coevolved with humans for a long time.[1],[2],[3],[4] The bacilli have evolving mechanisms that interfere with the host-pathogen interplay in the phagosome, which largely depends on the secretion of virulence effector protein translocated through the bacterial cell wall by multiprotein complexes forming a secretory apparatus.[1]

M. tuberculosis complex (MTBC) has a variety of mechanisms to survive within the phagosomal compartment. The ESX-1 system, which is a type VII secretion system, allows specialized protein secretion of 6-kDa early secreted antigenic target (ESAT-6) and 10-kDa culture filtrate protein (CFP-10) through mycobacterial cell envelope.[1]

MTBC genes contain five ESX paralogs, of which two, ESX-1 and ESX-5 are implicated in virulence, ESX-1-encoding core region spanning from espE (rv 3864) to mycP1 (rv 3883c), which also includes trans-acting elements outside the core region, namely extended ESX-1 such as espR (rv 3849) and esp A, C, D (rv 3616c to rv 3614c). Moreover, ESAT-6 (esxA [Rv3875]) and CFP-10 (esxB [Rv3874]) are also included in the ESX-1, both genes form 1:1 heterodimer and are translocated.[1]

Indonesia ranked as the third-highest PTB burden country worldwide for consecutive 2 years, in 2017 and 2018. In 2018, the total incidence rate of PTB cases in Indonesia was about 316 cases per 100,000 population,[5],[6] 312 cases per 100,000 population in 2019.[7] The Republic of Indonesia Ministry of Health (2018) also reported that the number of multidrug-and rifampicin-resistant (MDR/RR)- TB cases in Indonesia was always increasing from 2009 to 2015 until now. Moreover, the case of notification rate per 100,000 people was 161 in 2017, whereas it was 139 in 2016.[8]

Because of the crucial health problems caused by PTB, rapid and accurate detection of M. tuberculosis is needed. Until now, the culture method that is considered the gold standard of PTB diagnosis requires 3–8 weeks to obtain the results; moreover, the smear microscopy has low specificity and sensitivity. Thus, another rapid and more accurate diagnostic method, such as the polymerase chain reaction (PCR) test, is required together with the culture method.[2],[3]

One of the important steps to develop a diagnostic kit of PCR is to decide on a specific target gene. The espD gene is a part of the ESAT-6 system 1, which is usually abbreviated as ESX-1 gene cluster. ESX-1 is part of the T7SS system secretion that transports the protein to the mycobacterial cell envelope, and ESAT-6 and CFP-10, which are virulence proteins of M. tuberculosis that are increased in active PTB, are part of ESX-1.[1],[9],[10],[11]

ESX-1 consists of 20 genes located in the core region, whereas extended ESX-1 consists of four genes as trans-acting elements located outside the core region, which are espD (Rv3614), espC (Rv3615), espA (Rv3616), and espR (Rv3849). The evidence suggests that espD gene could stabilize the cellular level of espA and espC. espA and espC gene secretion requires espD secretion, which as the factors play regulatory role for the virulence secretory such as ESAT-6, CFP-10, and other virulence proteins associated with tissue injury to lung parenchyme tissue. The unique characteristic of espD makes it a potential specific target for the development of PTB diagnostic, which have the role as biomarker potential in active PTB disease process.[9],[10],[12]

Detection and identification of espD full gene as a target of active PTB diagnostic in sputum specimen from suspected active PTB patients could offer important information that will be used to identify M. tuberculosis. Developing a sensitive, specific, and rapid diagnostic to accompany diagnostic-based culture method is critically needed to control incidence and to prevent mortality cases of active PTB. This study aimed to evaluate the value of PCR method using espD full gene as the target for specifying M. tuberculosis. In addition, the study also identify the crucial T-cell epitopes which have the role in inflammatory in active PTB disease process, and the prediction of protein structure of EspD that both related to regulate the secretory protein that involved in the pathogenesis of active PTB.

  Methods Top

A total of 55 sputum samples and 41 MTBC culture isolates from the newly diagnosed active PTB patients in Dr Soetomo Academic Hospital, Surabaya, Indonesia, were collected from September 2016 to April 2019. The active disease process was identified by chest X-rays (CXR), and the confirmed PTB cases were rifampicin-sensitive (RS)-TB, or MDR/RR-TB using GeneXpert (Cepheid, Sunnyvale, CA) and BD BACTEC MGIT 960 system assays. Samples were tested using the PCR method performed at the Laboratory of Tuberculosis, Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia. The study was approved by the ethics committee in health research of Dr Soetomo Academic Hospital, ethical clearance no. 541/Panke. KKE/IX/2016, 618/Panke. KKE/X/2017, and 0410/KEPK/VII/2018. The data from TB patients in this study were obtained from medical record in hospital. All respondent's information is kept confidential and only used for research purposes.

The sputum sample was decontaminated and concentrated using the alkali Petroff method as processed sputum sample.[13] DNA extraction was performed from 3 to 5 μL processed sputum samples using QIAGEN DNA Kit (DNeasy Blood and Easy Kit, Cat No. 69504), followed by the PCR process. The other examination, one loop of MTBC culture isolates conducted DNA extraction with the same method. The primer for the PCR process was designed using Clone Manager software 6 (version 6.00) targeted at 555 bp espD gene of M. tuberculosis H37Rv wild-type strain.

KAPA2G Fast ReadyMix PCR Kit was used as the PCR master mix. The solution mixture consisted of 25 μl of KAPA2G Fast ReadyMix PCR Kit, 1 μl of 10 μM pair of primer, 20 μl of nuclease-free water, and 3 μl of DNA template. The amplification reaction in the thermal cycle started with pre denaturation at 95°C for 3 min and continued by denaturation at 95°C for 10 s, annealing at 58.1°C for 10 s, and extended at 72°C for 15 s. The denaturation, annealing, and extension process was done for 35 cycles and finished with a final extension at 72°C for 10 min. Controls were used in PCR reaction consist of M. tuberculosis H37Rv as a positive control, Mycobacterium fortuitum, Staphylococcus aureus, and PCR mix without DNA template as the negative control.

The sequencing process was performed by sending the PCR products to 1st Base, Singapore, using ABI PRISM 3730xl Genetic Analyzer instrument (Applied Biosystems). The sample sequence was then analyzed using BioEdit and NCBI BLAST tools to determine homology percentage between the sample and the wild-type M. tuberculosis H37Rv (NC_000962.3) sequence referenced from GenBank to detect the mutation or variant of gene.

GENETYX was used to analyze the T-cell epitope of espD gene expression. The three-dimensional (3D) structure of espD was modeled by employing a structural homology modeling with the SWISS-MODEL server[14] and a hierarchical approach to predict protein structure and function on I-TASSER online server[15] and was visualized with the program PyMOL (PyMOL Molecular Graphics System, version 0.99, Schrödinger).

  Results Top

A total of 55 sputum samples and 41 MTBC culture isolates from PTB patients were tested for 555 bp espD gene detected. From 55 sputum samples, there were 43 (78.18%) [Table 1] that showed positive results for 555 bp [Figure 1]; on the other hand, all 41 MTBC culture isolates showed positive results (100%). In addition, all of the sequenced samples had 100% identity to the espD gene of wild-type M. tuberculosis H37Rv (NC_000962.3) sequence [Figure 2].
Table 1: The results of Mycobacterium tuberculosis identification from sputum samples using polymerase chain reaction espD 555 bp full gene and GeneXpert

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Figure 1: Visualization of electrophoresis result of polymerase chain reaction 555 bp full gene targeted. (a) DNA band at 1, 2, 3, 4 showed positive result. (b) DNA band at Px15, PS90, PS91, PS92, PS93, and PS94 showed positive result. M = ladder DNA marker SizerTM 100 bp; K(+) = M. tuberculosis H37Rv ATCC 27294; K− = All reagent without DNA; 1–4 = MTBC culture isolate; PS = MTBC culture isolate; Px = sputum; SAU = Staphylocccus aureus; Frt = Mycobacterium fortuitum

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Figure 2: Multiple alignment using the BioEdit program showed 100% identity between samples with wild-type M. tuberculosis H37Rv (NC_000962.3). PMSG01 MDR, 9, 13, 18, and PP03 are MTBC culture isolates. PMSG01MDR is an MDR-TB sample, whereas others are drug-sensitive samples

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We found that there were concordance positive results between PCR espD detection and GeneXpert on sputum samples, there were 65.45% (36 of 55).

The T-cell epitope prediction of all samples were VGSAAT, VSVSTL, LSARVA, and ALLRKT based on IAD pattern position and DLPG, DAVD, DPIIG, RVAW, DLAR, GLPS, and EVFAT based on Rothbard/Taylor pattern position [Table 2].
Table 2: Prediction of T cell epitope espD gene expression in Mycobacterium tuberculosis of sputum samples and Mycobacterium tuberculosis complex culture isolates

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Prediction of the 3D protein structure of espD that provides new information about the protein structure of espD of M. tuberculosis [Figure 3]. The model had an I-TASSER confidence score (C-score) of-3.91, which is the highest C-score among several model alternatives of EspD protein structure suggested by the program, with an estimated RMSD of approximately 14.5 Å. The structure consists of a main fold of a three-stranded antiparallel β-sheet and a long α-helix (colored in green) surrounded by several minor secondary structures. The figure was prepared using the program PyMOL (PyMOL Molecular Graphics System, version 0.99, Schrödinger).
Figure 3: Three-dimensional protein structure of EspD as modeled by the program I-TASSER

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  Discussion Top

Using PCR, all of the 41 MTBC culture isolates (100%) were positively detected for espD full gene 555 bp, and 43 of 55 sputum samples (78.18%) were positively detected for espD full gene. This difference could be related to difficulties in obtaining the accurate sputum samples from PTB patients, especially from MDR-TB patients; and the other hand, it is revealed espD gene conserved and specific in all MTBC isolates. However, there were 65.45% (36 of 55) positivity concordance between espD-targeted PCR and GeneXpert MTB/RIF in detecting sputum samples. Further study is needed because there is still a shortcoming in the gold standard for TB diagnostic. Therefore, complementary tests for confirming the gold standard are needed.

Among samples with MDR/RR-TB and RS-TB, no different gene sequences were detected in the espD full gene and 100% homolog with wild-type M. tuberculosis H37Rv; hence, it could be assumed that there was no variant or mutant in the espD gene, and it could be a stable gene expression in active PTB. Finding the right targets as potential biomarkers for developing active PTB diagnostic is an important step. Many M. tuberculosis proteins had been examined based on their function and tried to be used as a biomarker for developing diagnostic tools and vaccines.[9],[11]

One of M. tuberculosis cluster genes that have been studied a lot is ESX-1 Type VII secretion system (T7SS). The ESX-1 gene cluster has ESAT-6 and CFP-10 encoded by esxA and esxB, respectively, as a compartment of the potent virulence factors for M. tuberculosis. However, aside from the protein, there is another gene that could affect their function as virulence genes. The espD gene is one of the important genes that helped in ESAT-6 and CFP-10 expression.[9],[11]

The EspD protein within the espA-espC-espD gene cluster has a role in ESX-1-mediated protein transport and virulence in M. tuberculosis. Thus, it has a critical and complex role for modulating the ESX-1 secretion system in M. tuberculosis. The EspD expression is critical for esxA secretion; however, espD secretion does not exclusively require ESX-1.[12],[16]

A previous study proved that espD could not be secreted by the ESX-5 secretory apparatus. However, it is possible that espD may require one or more of the other ESX systems, such as ESX-2, ESX-3, and ESX-4, in MTBC. For maintaining cellular levels of espA and espC, it requires espD. The espD, espA, and espC are essential for ESX-1 function. EspA maintenance requires both espC and espD. espA is more abundant when co-expressed with espC and espD in MTBC. For that purpose, espD gene plays a major role in M. tuberculosis growth.[12],[17] The pathogenic potential of M. tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families.[18]

In this study, there was no variant or mutant of the espD full gene. It is suggested that espD is a stable gene expression in active PTB, which have role alike regulon for expression virulence proteins such as ESAT-6, CFP-10, and others, as well it is found in all MTBC culture isolates that could be as the conserved gene sequenced based on the samples of PTB patients which residence were spread across different areas in Indonesia, that is possible because Dr. Soetomo academic hospital is a tertiary hospital which is the one of top TB referral system in Indonesia. Moreover, in other secretory systems such as esxA, no variant was also found.[4],[19],[20] However, a previous study concluded that the phenotypic characterization of MTBC strains expressing espD variants revealed the existence of a highly complex and multifunctional aspect of espD in MTBC. Thus, further study about those variants is needed.

The variants in regulators, such as espD, espL, espH, espR, and WhiB6, could affect infection outcome. The presence of point mutations, that is, SNP insertion within the binding sites results in transcriptional deregulation. The regulatory proteins involved in the expression of genes encoding the ESX-1 apparatus, espA-espC-espD locus, are crucial for ESX-1 function.[1],[11]

Transcriptional activator espA-espC-espD in ESX-1 secretion functions as the indirect control of PhoP/PhoR 2 component regulatory system, which is also the upregulator for the espA-espC-espD locus. In addition, espR is another transcriptional activator that upregulates ESX-1 actively, by binding espA to increase transcription of espA-espC-espD locus. The espA-espC-espD locus enhances transcriptional repressors and represses the ESX-1 locus.[11]

Disease progression and host-pathogen interaction are influenced by various virulence factors encoded by the bacterial genome; the most important are ESX-1 and other gene clusters from the type VII secretion system. The ESX-1 cluster compresses around 20 genes that encode a specific secretion apparatus, which release effectors into the extracellular milieu and take part in cytosol recognition and stimulation of innate immunity, bacterial escape, phagosome rupture, intracellular spread, and systemic disease. The ESX-1 function leads to transcriptional regulation that is utilized by EspR, Lsr2, CRP, MprA, and mIHF on the espA-espC-espD locus.[21],[22]

Interactions between pathogens and hosts occur at the cell wall and secretion proteins involved in virulence. These proteins often interrupt normal host defenses and allow bacteria to multiply and survive. Many of the virulence factors are secreted by nonclassical secretory systems that facilitate the translocation of proteins across host cell membranes.[23]

The espD gene targets alternative types of machinery, that is, ESX-2, ESX-3, or ESX-4. The espD gene forms a chaperone complex with EspL and stabilizes the espA-espC, espE-espH dimers, espL and espF. A previous study on Mycobacterium reported the conservation of the genes for espD, espH, and espL, which confirmed that future investigation is really needed.[21],[24]

CXR findings in all of the new cases revealed that there are chronic characteristic of active infection lesions in these patients. The other crucial finding was 100% similarity of the espD full gene that was detected in RS-TB and MDR/RR-TB, which CXR shows as the active disease process. It could be assumed that the espD gene and the other genes of the ESX-1 secretion system are essential and can be potentially used as a biomarker for the active PTB disease process that is meaningful for diagnostic and vaccine development and others.

This study was the first report on the T-cell epitope of espD. T-cell recognition on macrophages infected by MTBC is crucial to the confinement of TB infection. In general, T cells are in response to some major antigens in TB such as Ag85a, Ag85b, ESAT-6, and CFP-10, and those responses becoming the basis for TB inflammation response. Based on that, the researcher hopes that those immunodominant antigens could be developed into vaccine.[25],[26],[27] In contrast with esxA T-cell epitope, the espD gene has a higher number of T cell epitope predictions.[28] In addition, this study also reported on the 3D structure of EspD protein by I-TASSER program. A Pfam search[29] using the amino acid sequence of EspD as a query revealed that the sequence could not be assigned to any protein family. Likewise, a 3D structural similarity search using the primary structure of EspD on the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the Protein Data Bank (PDB) database showed that espD has no similarity to other proteins with a known 3D structure.

The espD gene is a part of the espAC operon because it functions as a specific chaperone. However, at this point, the precise function of the EspD protein is unknown.[16],[30] Chen et al. (2011) concluded that EspD is critical for both stabilization and secretion of EspA and EspC. It has a role in stabilizing the intracellular levels of the secreted substrate dimer EspC or EspA.[12]

However, this protein itself is not a substrate of the ESX-1 system. The last eight amino acid residues at the carboxyl terminus (C-terminus) of EspD are unessential for its function and secretion, unlike EsxB and EspC.[12] Multiple chaperones, including EspD and other genes, such as EspG1 and EspH, are responsible for stabilizing their cognate substrates PE35/PPE68, EspC/EspA, and EspF/EspE, respectively.[17]

However, a structural homology search using the fold and function assignment system server[31] with the espD sequence resulted in the Ybab protein from  Escherichia More Details coli (PDB entry 1PUG) as the top hit, albeit with only approximately 10% sequence identity to EspD. Using the SWISS-MODEL server (Biasini et al., 2014) based on the structure of Ybab, a structural homology modeling of EspD was able to predict a model for EspD, but only for 65 amino acid residues (residue numbers 60–124). However, other studies reported that the modeling of EspD protein structure produced a Phyre2 coverage of 38% by residue 65-134.[32],[33],[34]

The predicted model consists of a three-stranded antiparallel β-sheet (residue numbers 64–89) and a long α-helix (residue numbers 98–124) (data not indicated). Moreover, an iterative structural modeling approach with the I-TASSER program[15] predicted several full 3D structures of EspD protein, with the best model showing a confidence score of −3.91 and RMSD of approximately 14.5 Å [Figure 3]. The main fold of this model coincides quite well with the structure predicted by the SWISS-MODEL.

The main structure is surrounded by two short α-helices that come from the N-terminal region of EspD and an α-helix from the C-terminus. Overall, the predicted structure of EspD is similar to Ybab protein from Escherichia coli. Unfortunately, the physiological function of Ybab is currently unclear. These predictions are important for the development of diagnostic based on the espD gene. Thus, the role and function of EspD in M. tuberculosis need to be further characterized. Insight in the crucial T-cell epitopes which have involved in regulated inflammatory in active PTB disease process, and the other hand the prediction of the protein structure of EspD that related to the regulation of the secretory proteins, which both compartments have involved in the pathogenesis of active PTB disease process, more fundamental research is needed in molecular interactions of active disease process. Protein structure interaction modeling is important to understand the effects of mutation and understanding the molecular mechanism of mycobacterial resistance.[35] Besides that, future research is essential to determine the mechanism of co-expression in the proteins of the ESX-1 secretion system of MTBC, that related to active disease PTB process and active multiplication of MTBC in the study of whole-genome sequencing, which could be the basis for the future research of diagnostic, anti-TB drug, and therapeutic vaccine development.

  Conclusions Top

This research showed that the espD full gene in M. tuberculosis is a conserved, specific, and stable gene that could be used as a potential biomarker to determine the active PTB disease process. This study could provide a brief information about the sequence of the espD full gene, epitope prediction, and 3D structure of the EspD protein from M. tuberculosis strains in Indonesia. The crucial T-cell epitopes which have the role in inflammatory progress in active PTB disease process and the prediction of protein structure of EspD that both compartments have related to the regulation of the secretory proteins that involved in the pathogenesis of active PTB. These fundamental findings are needed as a reference for further research in the development of diagnostics and vaccines for preventive and therapeutic. For future study, we need to explore the protein structure prediction and interaction with anti-TB drug for new TB drug development.


The authors would like to thank to Directorate General of Higher Education, Ministry of Research, Technology, and Higher Education of Republic of Indonesia, and Rector of Universitas Airlangga for supporting this research. In addition, the author would like to thank to Director of Dr Soetomo Academic Hospital, Surabaya, Indonesia; Chairman of Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia; and Dean of Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia. We would also like to thank Agnes Dwi Sis Perwitasari as staff of Laboratory of Tuberculosis, Institute of Tropical Disease, Universitas Airlangga. Finally, we also thank a staff of Department of Clinical Microbiology, Dr Soetomo Academic Hospital, Sugeng Harijono, A. Md. A. K.

Ethical clearance

Ethical clearance no. 541/Panke. KKE/IX/2016, 618/Panke. KKE/X/2017, and 0410/KEPK/VII/2018.

Financial support and sponsorship

Directorate General of Higher Education, Ministry of Research, Technology, and Higher Education of Republic of Indonesia and Universitas Airlangga.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]


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