MyTB database.

The MyTB database will be used to obtain information such as patient’s registration ID, identification number, date of TB notification, date of TB diagnosis, sociodemographic characteristics, home address and patients’ clinical background. The factors obtained include age at diagnosis, gender, ethnicity, nationality, type of TB, type of case, method of tracing, status of comorbidities (diabetes, smoking), BCG scar, HIV status, DST results and treatment outcome.

Patients’ medical record.

The patients’ identification number obtained through the MyTB database will be used to retrieve their medical record from the clinic, including telephone number and other contact details.

Sputum sample collection and culture.

Each of the participants will be required to provide their early morning sputum samples on the day of TB diagnosis at TB Clinic. This step is routinely done in all TB patients. Sputum samples from the patients throughout Kuala Lumpur are routinely sent out to the Institute of Respiratory Medicine (IPR) in Jalan Pahang, Kuala Lumpur, a culture centre which equipped with a Level 2 risk laboratory. Sputum obtained will proceed to culture by using conventional method or automated method Bactec MGIT 960 by medical laboratory technologist in IPR. The Laboratory Turn Around Time (LTAT) for this step is six to eight weeks. Cultures displaying mycobacterium morphology suggestive of TB will be carefully transported to the National Public Health Laboratory (MKAK) for DNA extraction and genomic analysis.

The bacterial clinical isolates examined in this study were obtained from patients as part of routine diagnostic requests. The patient receives proper instructions for collecting sputum specimens, which include using a sterile, leak-proof container and properly identifying the specimen. The lab then receives the specimens. The laboratory places the specimen in a biological safety cabinet and processes it using the standard method of digestion and decontamination with 4% NaOH. The conventional method involves inoculating the suspension onto OGAWA media. When using automated culture, we top up the suspension with phosphate buffer (pH 7) to a volume of 50 ml, then centrifuge it at 3000 x g for 15 minutes. A volume of 500 and 100 μl of the suspension is inoculated in BACTEC MGIT 960 (Becton Dickinson, Franklin Lakes, NJ, USA) and on solid culture medium Löwenstein-Jensen (LJ). The Laboratory Turn Around Time (LTAT) for this step is six to eight weeks. Cultures with suggestive mycobacterium morphology are transported to the National Public Health Laboratory (NPHL) with proper cautions for identification tests. NPHL conducts identification tests for Mycobacterium tuberculosis complex (MTBC) using immunochromatography (ICA) assays and then follows up with a line probe assay (LPA) for MTBC-negative cases. All isolates identified as MTBC are proceed to antibiotic susceptibility testing for first-line anti-TB drugs, including streptomycin, isoniazid, rifampicin, and ethambutol. The results are reported using the SIMKA 3 web-based online system. Then, the MTBC isolates are kept in skim milk media, catalogued, and stored at −80 °C. Any isolates in this study will be found and re-subcultured on LJ medium for further use.

DNA extraction.

Mycobacterium tuberculosis complex (MTB) cultures will undergo molecular extraction. DNA will be extracted using Promega Maxwell RSC Viral Total Nucleic Acid Multi-Pack Kit. Colonies will be emulsified in extraction buffer and lysed at 56°C for 10 minutes. This procedure will be conducted in a biological safety level 3 facilities. The lysate will be transferred to Maxwell Cartridge and the remaining process is fully automated (follow manufacturer protocol). Extracts will be stored at -20°C.

Genomic investigation.

Mycobacterium culture will be analysed using Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeats (MIRU-VNTR) typing as the first-line genotype method followed by WGS for any cluster or selected TB cases. The isolates will be characterized by a standardised set of 24 VNTR loci. PCR fragments for VNTR loci will be loaded onto the QiaXcel Analyser for the fragment separation and allele calling.

For isolates undergoing whole genome sequencing, quantification and quality assessment of each extracted DNA will be performed using Qubit fluorometer and agarose gel electrophoresis. MTBC genomic DNA samples with the purity of 1.8–2.0 (OD260/280) and 2.0–2.2 (OD260/230) at concentration >100 ng/μL will be used for the subsequent whole genome sequencing experiments.

The MTB genome libraries will be prepared using the Nextera DNA Flex library preparation kit according to the man-ufacturer’s instructions (Illumina, Inc., San Diego, CA). The library concentration will be measured using the Qubit Fluorometer. The library pool will be sequenced using the Illumina MiSeq platform.

Bio-informatics analysis.

A phylogenetic tree will be constructed using the MIRU-VNTRplus to identify closely related MIRU-VNTR patterns [19]. Sequence data by WGS will be analysed using open-source bioinformatics software by illumina.

Social network analysis questionnaire.

Eligible patients, as according to the inclusion criteria, will be required to complete a social network analysis (SNA) questionnaire once. This SNA questionnaire will assess patients’ demographic background, medical history, drug and alcohol history, residence, travelling history, places of social aggregation and social networks using open-ended questions. This questionnaire is adopted from Gardy et al. (2011) and modified according to local suitability, especially on the type of common social activities related to the Malaysian culture [14]. Two versions of this questionnaire (English and Malays) will be available to facilitate the interview process.

All the information in the SNA questionnaire will be extracted and entered using the Excel 2016 spreadsheet to create an attribute table. Using the attribute table, a sociomatrix will be created to define and quantify the two-mode relationship between all patients. Relevant models will be created to identify the most effective combination of attribute data necessary to calculate the relevant SNA metrics. The information on clinical history and exposure to TB patient will be cross-validated by referring to patient medical records, contact investigation reports (TBIS 10C-2).

Each of the eligible patients will be contacted via telephone by the trained research assistant or the first author to make aware of the study and the needs of interview. Patients will be excluded if they do not agree to participate. For those willing to participate, the research assistant will set an appointment for the interview. The eligible patients will be interviewed at their own preferential time and method to ensure good response rate. Interviews will be conducted either through telephone call, face to face interviews in the clinic or online survey form. Each participant will be given explicit information about the study background, the purpose of the study and the data collection process. To increase trustworthiness of the study to the patients, the research assistance will provide a study poster that include all necessary information and contact details of all the researcher involves via WhatsApp. Informed consent will be taken before they enrolled in the study. Each participant will be given full autonomy to participate or withdraw from the study at any time without jeopardising their ongoing anti-TB treatment.

Visualization of TB cases distribution in Kuala Lumpur.

This study will visualize areas with high incidence TB cases using home addresses obtained from the MyTB database. The existing physical address will be cleaned to remove unnecessary noise to improve TB case locations accuracy. Then the cleaned address will utilised for geocoding to get the longitude and latitude for each address. The cleaned and geocoded will be aggregated and applied on Tableau to highlight the TB hotspots based on the density number of TB cases. The data on vagabonds and unknown addresses will be excluded from the analysis. Fig 1 depicted the flow of the data collection activities planned for this study.

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Fig 1. Data collection framework.

https://doi.org/10.1371/journal.pone.0307921.g001

Dissemination and data sharing.

The Malaysian National Medical Research Registry has registered this study, which has received ethical approval for the observational study from two human ethics committees: the Medical Research and Ethics Committee, Ministry of Health Malaysia (NMRR-19-2818-50518-IIR), and the Human Research Ethics Committee of Universiti Sains Malaysia (USM/JEPeM/19090558). The study will adhere to the Declaration of Helsinki principles and the Malaysian Good Clinical Practice Guidelines. It will be reported in accordancewith the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: Guidelines for Reporting Observational Studies to enhance reporting transparency [27]. Data and resources will be shared with other eligible investigators through academically established means. The datasets used and analysed during the study will be available from the corresponding author upon reasonable request. All aspects of the study, including results, will be strictly confidential, and only the study team will have access to information on participants, except as required by law. In publication and presentation of the results, no reference will be made to individual subjects in a form that will expose their identity. A report of the study may be submitted for publication. However, individual participants will not be identifiable in such a report.

Data storage and management.

Principal investigators (PI) or research assistants will enter all data, with the study PI verifying data accuracy. Data quality control measures will address missing data, outliers, and discrepancies, ensuring protected health information access is limited to research assistants and the PI. Unique identifiers will safeguard participant anonymity, with data stored securely and accessible only to the PI. Following the study, data and molecular samples will be securely disposed of, with a commitment to maintaining participant privacy and adhering to publication policies that protect subject information. Data quality control measures will include queries to identify missing data, outliers, and discrepancies. Only research assistants and PI will have access to protected health information. After enrolment, a unique identifier will be assigned to each study subject. The data from all sites will be uploaded and stored in a password-encrypted external hard disk which will only be accessible to the PI. All computers use in this study will be password protected and encrypted per university policy to increase security. The PI will ensure that the anonymity is maintained. Patients will not be identified by name in any reports on this study. The study PI will have access to the final study dataset. All data collection forms and questionnaires will be maintained in lockable filing cabinets within the corresponding author’s office. All data will be kept for two years after the completion of the study. To re-identify data at a later stage for feedback to participants, the data will be stored in the identifiable or potentially identifiable (coded) form. The molecular samples will be kept at National Public Health Laboratory (MKAK) and will be disposed of after the study period ended. After the prescribed storage time, all hard paper copies will be shredded before placement in a secure paper recycling bin. All soft copy data will be deleted. With regards to the publication policy, no personal information of subjects will be published to protect the privacy and confidentiality of the subject’s personal information. Permission from the Director-General of Health, Malaysia, or relevant authorities will be obtained prior to publication. The respondents can request for published findings.