Molecular Detection and Drug Resistance in Multidrug-Resistant Tuberculosis (MDR-TB) Patients in Bangladesh: A Case Report

Abstract

Bangladesh is facing an emerging issue of Multidrug resistant tuberculosis (MDR-TB) as M. tuberculosis strains resistant to at least isoniazid and rifampicin, the two most potent first line anti TB drugs. Available evidence from Bangladesh indicates that the high prevalence of MDR-TB is due to lack of adherence to treatment, misdiagnosis and lack of surveillance systems that lead to high morbidity and mortality of patients with MDR-TB. This is because the traditional culture-based approach that is the most used method is slow and fails to detect drug resistant strains quickly. Beginning with molecular diagnostics, namely GeneXpert, polymerase chain reaction (PCR), and whole genome sequencing (WGS), M. tuberculosis drug resistance mutations have been detected. These technologies enable rapid identification of resistant strains and thus provide a timely and appropriate treatment intervention. For example, GeneXpert not only can deliver its results within hours, but do so much more quickly than conventional methods. Escape from constraints impose deep constraints on bacterial genomics inferring comprehensive insights into the genetic composition of the bacteria and enables specific mutation determinations of drug resistance allowing for discrimination of treatment regimens based upon specific mutations of patients. This is particularly significant with regards to genetic mutations that can lead to failure of treatment. Several mutations in genes such as rpoB, katG, and inhA are strongly linked to resistance to first line drugs and poor outcome. Molecular diagnostics of these mutations in Bangladesh not only expose us to the MDR TB epidemiology but also highlight the fact that personalized treatment strategies are essential to fight the rising health crisis. Improving MDR-TB management and reducing its burden in Bangladesh and other high burden areas requires integrating of advanced molecular techniques to routine clinical practice.

Keywords

Introduction

This is however one of the enduring and big global health challenges as the World Health Organization (WHO) says that 1.6 million people died of TB in 2021 and about 10.6 million new cases were recorded in that year alone [1]. In Bangladesh, the incidence rate of TB is among the highest in the world. The number of new TB cases in 2021 was estimated at 300,000 in Bangladesh, where it is estimated that 300,000 new TB cases occurred in 2021 [1]. But since TB is already a complicated disease to treat, it was made more so by the emergence of Multidrug Resistant TB (MDR-TB), a term used to describe TB that is resistant to at least isoniazid and rifampicin. This resistance is usually due to the wrong use or misuse of drug and treatment noncompliance resulting in treatment failure and a further increase of transmission rate [2,3,4]. Mycobacterium tuberculosis has developed the molecular basis of resistance to anti tuberculosis drugs by specific genetic mutations that leads to drug resistant tuberculosis. One of the important properties of mycobacterium tuberculosis is that there are mutations in the rpoB gene, which are associated with rifampicin resistance; and mutations in the katG and inhA genes, which correspond to isoniazid resistance [5,6]. In addition to being used by MDR strains [7] to survive in the presence of antibiotics, these genetic alterations also aid in the transmissibility and pathogenicity of such strains. This has resulted in increased selection pressure occasioned by poor treatment regimens leading to proliferation of these strains and complicating global efforts to control TB [5,2]. In Bangladesh, there exists significant gaps in diagnosis, treatment and monitoring of TB, especially in the rural areas which have poor access to molecular diagnostic tools [8,9]. However, it is based on the traditional methods of diagnosis that often leads to delayed initiation of treatment, even MDR TB [8,9]. In addition, there are no comprehensive surveillance systems to monitor drug resistance patterns, and thus the implementation of targeted interventions [8,9] is made difficult. These gaps must be addressed to effectively control TB and reduce MDR-TB burden in Bangladesh and other such high burden settings.

Case Presentation

When presenting a case of a patient with TB, it is important to discuss several demographic, clinical and diagnostic factors related to the disease complexity.

Patient Demographics

The case patient is a 45-year-old male of low socio-economic background, who had poor access to healthcare services. Past medical history is a history of previous diagnosis of pulmonary tuberculosis for which he received a standard drug regime. Unfortunately, he was one of the unlucky patients who suffered treatment failure, a not uncommon outcome in TB prevalent areas and areas with limited healthcare resources [10]. In the light of the above, this socio demographic profile has great importance as often socio-economic factors correlate with treatment adherence [10] as well as access to healthcare, which are the main determinants of TB outcomes.

Clinical Symptoms & Diagnostic Findings

The patient had typical symptoms of TB such as a persistent cough (>3 weeks), significant weight loss, intermittent fever and night sweats. The clinical presentation of most TB patients is highly suggestive of active TB infection hence these are the manifestation of TB-infection [10]. Sputum smear microscopy for AFB confirming the presence of Mycobacterium tuberculosis was one of the diagnostic findings. Bilateral infiltrates with cavitary lesions are characteristic of advanced pulmonary TB [11], which was found on chest X rays. However, sputum smear microscopy is still heavily relied upon in many settings and is counterproductive in terms of sensitivity in situations where the disease is extrapulmonary, or in patients with HIV coinfection [12]. In addition, the patient’s history of treatment failure also puts him in the group of patients with multidrug resistant TB (MDR-TB), therefore, its treatment is complicated. Various factors like noncompliance with the prescribed regime, poor drug supply and emergence of drug resistance strain because of improper treatment protocol [10], are indicated to be responsible for treatment failure. All in all, this case illustrates the multiple challenges to management of TB, especially in high TB burden settings. However, socio-economic factors, clinical presentation and diagnostic challenges synergistically impose a need for better diagnostic tools and treatment strategies to fight TB.

Molecular and Genetic Analysis

According to the molecular and genetic analysis for the diagnosis of multidrug resistant tuberculosis (MDR-TB), sputum sample collection and testing of suspect patients is necessary to identify drug resistance and get insight into a course of treatment.

Sample Collection

Patients suspected of being infected with MDR TB, especially failure of treatment, provide sputum samples. This collection is essential because it provides the biological material requisite for molecular testing that is essential for proper diagnosis and management of TB [13,14]. Inadequate samples can result in missed or indeterminate answers [13], therefore, the collection process needs to be such that samples are representative and of sufficient quality to have reliable results.

Molecular Testing Methods

1. Mycobacterium tuberculosis and rifampicin resistance:

This geneXpert MTB/RIF test is a widely used automated molecular test for the rapid detection of Mycobacterium tuberculosis and its resistance to rifampicin as a first line drug. The GeneXpert assay is designed to detect mutations in rpoB gene, a gene where mutations occur that result in rifampicin resistance. High sensitivity and specificity of the GeneXpert test for rifampicin resistance is shown to have been reported of between 98 and 100?curacy for the identification of rpoB gene mutations [14,15]. Yet, it is worth highlighting that false positives can also occur when low amounts of bacteria or certain mutations existed and were not detected by the assay [16].

2. Line Probe Assay (LPA):

This is used test for the katG and inhA genes mutations related to isoniazid resistance. To complement the results from the GeneXpert test [15,17], LPA offers a rapid and reliable method for detection of resistance mutations. Both molecular techniques combining provide an overall enhanced diagnostic accuracy for detecting MDR-TB.

3. It is a more advanced technique which provides whole genome sequence information on resistant M. tuberculosis strains. It is WGS that allows for a more comprehensive identification of the mutations than can be achieved by GeneXpert and LPA, and consequently a richer understanding of the underlying resistance mechanisms it play [13,15]. This is a good method, but especially in epidemiological studies and for studying the dynamics of MDR TB strains transmission.

Findings

• The molecular test results usually disclose the existence of certain mutations in the rpoB gene that indicate rifampicin resistance. In addition, such isoniazid resistance is often due to mutations in the katG gene or the inhA promoter region [15,18]. The findings are crucial given that treatment decisions must account for the fact that MDR-TB placement implores the use of second line drugs, which are also more expensive as well as significantly more toxic than first line therapies [13,15]. Finally, the incorporation of the molecular testing methods, i.e. GeneXpert, LPA, and WGS into the TB diagnostic workflow has the potential to bolster the diagnostic capacity and sensibility to detect drug resistant strains. In regions with high burden of TB such as Bangladesh, timely and accurate diagnosis of the disease will result in improved outcome for patients and better control of TB transmission.
• For the management of multidrug resistant tuberculosis (MDR-TB), it is important to identify genetic mutations linked to drug resistance to enable the development of an appropriate treatment regimen. This section describes the results regarding detection of mutations in the rpoB and katG genes and pharmacological management and treatment outcomes of a patient infected with MDR-TB.
• Specific mutations in the rpoB gene that resulted in direct rifampicin resistance were detected on molecular testing. These mutations, at codons [5,16] and, to a lesser extent, [5,31], are frequently linked to the development of rifampicin resistance as a principal TB treatment drug [19,20]. In addition, mutations in the katG gene were found that are responsible for isoniazid resistance. In particular, the katG mutation is important, as it is responsible for the catalytic activation of isoniazid and mutation of the enzyme leads to an ineffective response to drug action [21, 22]. Through these mutations, it is evident how complicated MDR-TB is and how clinical diagnostics must be precisely understood before treatment can begin.

Pharmacological Management & Treatment Outcome: A new treatment plan is started following the identification of these mutations, according to those recommendations from the World Health Organization (WHO) for the treatment of MDR-TB. She was started on a regimen with second line anti TB drugs like bedaquiline, linezolid and delamanid specifically indicated for treating resistant strain of M. tuberculosis [23, 24]. This approach corresponds to the National TB Program of Bangladesh guidelines which recommend using second line drug for MDR TB [19,25]. It was monitored treatment outcome closely and improvements were significant. Following a preset treatment duration, the patient experienced marked reduction of bacterial load indicated by subsequent sputum cultures which became negative for M. tuberculosis. This is consistent with other studies that show high treatment success rates, when appropriate second line therapies are used [25,24]. Conversion to negative sputum culture is a critical milestone in the management of MDR-TB and a measure of successful treatment as well as lower probability of transmission to others [23, 25]. The patient’s treatment outcome is good because of the identification of some particular genetic mutations involved in drug resistance which later combined with using a WHO recommended treatment regimen. In the literature, this case provides an example of the utility of molecular diagnostics in enabling proper implementation of TB treatment strategies in the face of increasing drug resistance.

DISCUSSION

This discussion about molecular TB diagnosis, including Bangladesh, raises important challenges and opportunities with regards to improving TB management, specifically MDR-TB management.

Molecular TB Diagnosis in Bangladesh:

However, costs and infrastructure hurdles resulting from the limited implementation of molecular diagnostic tools like GeneXpert reduces their use in Bangladesh. The rapid, accurate detection of Mycobacterium tuberculosis and rifampicin resistance by the GeneXpert MTB/RIF assay has been recognized and access is limited particularly in rural areas where the supply of healthcare resources is scarce [26,27]. The adoption of these technologies is hindered by the high cost of these technologies combined with inadequate training and support for the healthcare providers [28]. As a result, MDR TB is left undetected early and morbidity and transmission in the community increases 29.

Comparison with Global Studies:

Studies comparing the prevalence of rpoB and katG mutation in South Asia with the rest of the regions indicate a significantly higher number of these mutations in this region. For instance, MDR-TB challenges are not unique to India and Nepal but cases report for India and Nepal have shared similar resistance pattern [30,31] which underscores the regional dimension of such a problem. Indeed, South Asian countries have been noted to have high alarmingly high prevalence of rifampicin resistance often linked to mutations in rpoB gene [32]. This local phenomenon is congruent with global results of a link between a high TB load and drug resistance [33].

Clinical Implications:

Molecular study findings point to the requirement of routine molecular screening in such high-risk TB patients particularly those with histories of treatment failure or exposure to known MDR TB cases. Timely identification of drug-resistant strains can be achieved by using routine molecular diagnostics, which can facilitate prompt initiation of appropriate treatment regimens [34]. At the same time, the TB control policies are also strengthened to introduce more advanced molecular diagnostics to improve patient outcomes and reduce the overall burden of TB in Bangladesh [35]. Such policies could include melding molecular testing with the existing National TB Program (NTP) format so that all suspected cases receive swift and proper diagnostic testing [36]. Finally, molecular TB diagnostics offer great potential but will include large challenges to implement them in Bangladesh. Integrating molecular TB testing into routine practice will help Bangladesh tackle the obstacles to access and strengthen its TB control efforts and improve patient outcomes for those with MDR TB.

CONCLUSION

The findings from this study underscore the critical association between multidrug-resistant tuberculosis (MDR-TB) and specific genetic mutations in Bangladesh. The presence of mutations in the rpoB and katG genes highlights the urgent need for enhanced molecular diagnostics to effectively manage and control the spread of MDR-TB. The study has demonstrated that MDR-TB in Bangladesh is strongly associated with specific genetic mutations, particularly in the rpoB gene, which is linked to rifampicin resistance, and the katG gene, which is associated with isoniazid resistance. These findings are consistent with global trends indicating that drug resistance in Mycobacterium tuberculosis is a significant public health challenge, particularly in regions with high TB burdens.

Future Directions

To combat the rising incidence of MDR-TB, it is essential to expand access to molecular testing methods such as GeneXpert and Line Probe Assays (LPA) to district-level hospitals across Bangladesh. This expansion will facilitate early detection of drug-resistant strains and enable timely initiation of appropriate treatment regimens. Additionally, further research into new drug combinations is necessary to enhance treatment efficacy against MDR-TB, particularly considering emerging resistance patterns.

Call for Policy Changes

There is a pressing need for government funding to support the integration of molecular diagnostics into national TB programs. Such funding would ensure that advanced diagnostic tools are available and accessible to all patients, particularly in rural and underserved areas. Furthermore, strengthening patient compliance monitoring is crucial to reduce the spread of MDR-TB. Implementing robust adherence strategies, including patient education and support systems, will be vital in ensuring that patients complete their treatment regimens effectively. In summary, addressing the challenges of MDR-TB in Bangladesh requires a multifaceted approach that includes expanding molecular diagnostics, researching new treatment options, and implementing policy changes to support effective TB management. By taking these steps, Bangladesh can significantly improve its TB control efforts and reduce the burden of drug-resistant tuberculosis.

REFERENCES

1. Yang Z., Wang L., Dong H., & Xin Z.. Design, synthesis, and molecular docking studies of chroman-4-one linked thiosemicarbazide derivatives as inhibitors of katg and anti-mycobacterium tuberculosis agents. Heterocycles 2023;106(6):1047. https://doi.org/10.3987/com-23-1485
2. Kozobkova N.. Photoinactivation of mycobacterium tuberculosis and mycobacterium smegmatis by near-infrared radiation using a trehalose-conjugated heptamethine cyanine. International Journal of Molecular Sciences 2024;25(15):8505. https://doi.org/10.3390/ijms25158505
3. Gatwiri W. and Kagia R.. Identification of potential compounds for the management of multidrug-resistant tuberculosis using computational methods. F1000research 2023;12:298. https://doi.org/10.12688/f1000research.130024.1
4. Kurnianingsih W., Tamtomo D., & Murti B.. The effect of non-compliance with medication on multidrug resistant of tuberculosis. Journal of Epidemiology and Public Health 2020;5(4):442-450. https://doi.org/10.26911/jepublichealth.2020.05.04.06
5. Viana J. and Torres R.. Resistance mechanisms of mycobacterium tuberculosis in the tuberculosis pathogenicity in humans: an overview. Revista Interfaces Saúde Humanas E Tecnologia 2022;10(2):1431-1440. https://doi.org/10.16891/2317-434x.v10.e2.a2022.pp1431-1440
6. Ito H.. Identification of novel antimicrobial compounds targeting mycobacterium tuberculosis s-adenosyl-l-homocysteine hydrolase using dual hierarchical in silico structure-based drug screening. Molecules 2024;29(6):1303. https://doi.org/10.3390/molecules29061303
7. Becerra M., Huang C., Lecca L., Bayona J., Contreras C., Calderón R.et al.. Transmissibility and potential for disease progression of drug resistantmycobacterium tuberculosis: prospective cohort study. BMJ 2019:l5894. https://doi.org/10.1136/bmj.l5894
8. Assemie M., Alene M., Petrucka P., Leshargie C., & Ketema D.. Time to sputum culture conversion and its associated factors among multidrug-resistant tuberculosis patients in eastern africa: a systematic review and meta-analysis. International Journal of Infectious Diseases 2020;98:230-236. https://doi.org/10.1016/j.ijid.2020.06.029
9. Brandão A., Pinhata J., Oliveira R., Galesi V., Caiaffa-Filho H., & Ferrazoli L.. Speeding up the diagnosis of multidrug-resistant tuberculosis in a high-burden region with the use of a commercial line probe assay. Jornal Brasileiro De Pneumologia 2019;45(2). https://doi.org/10.1590/1806-3713/e20180128
10. Andom A.. Understanding barriers to tuberculosis diagnosis and treatment completion in a low-resource setting: a mixed-methods study in the kingdom of lesotho. Plos One 2023;18(5):e0285774. https://doi.org/10.1371/journal.pone.0285774
11. Gómez-Valverde J.. Chest x-ray–based telemedicine platform for pediatric tuberculosis diagnosis in low-resource settings: development and validation study. Jmir Pediatrics and Parenting 2024;7:e51743. https://doi.org/10.2196/51743
12. Kyu H., Maddison E., Henry N., Ledesma J., Wiens K., Reiner R.et al.. Global, regional, and national burden of tuberculosis, 1990–2016: results from the global burden of diseases, injuries, and risk factors 2016 study. The Lancet Infectious Diseases 2018;18(12):1329-1349. https://doi.org/10.1016/s1473-3099(18)30625-x
13. Khan M., Islam M., Hassan J., Paul S., Islam M., Pateras K.et al.. Hierarchical true prevalence, risk factors and clinical symptoms of tuberculosis among suspects in bangladesh. Plos One 2022;17(7):e0262978. https://doi.org/10.1371/journal.pone.0262978
14. Nur T., Hosna A., Rayhan N., & Nazneen N.. Diagnosis of lymph node tuberculosis using the genexpert mtb/rif in bangladesh. Mediscope 2018;6(1):19-23. https://doi.org/10.3329/mediscope.v6i1.38939
15. Saeed M., Hussain S., Riaz S., Rasheed F., Ahmad M., Iram S.et al.. Genexpert technology for the diagnosis of hiv-associated tuberculosis: is scale-up worth it?. Open Life Sciences 2020;15(1):458-465. https://doi.org/10.1515/biol-2020-0052
16. Mechal Y., Benaissa E., Mrimar N., Benlahlou Y., Bssaibis F., Zegmout A.et al.. Evaluation of genexpert mtb/rif system performances in the diagnosis of extrapulmonary tuberculosis. BMC Infectious Diseases 2019;19(1). https://doi.org/10.1186/s12879-019-4687-7
17. Aricha S., Kingwara L., Mwirigi N., Chaba L., Kiptai T., Wahogo J.et al.. Comparison of genexpert and line probe assay for detection of mycobacterium tuberculosis and rifampicin-mono resistance at the national tuberculosis reference laboratory, kenya. BMC Infectious Diseases 2019;19(1). https://doi.org/10.1186/s12879-019-4470-9
18. Nurpermatasari A., Harahap U., & Siagian P.. Identification gene mutations rpob cause of multidrug resistance tuberculosis in haji adam malik hospital. Asian Journal of Pharmaceutical and Clinical Research 2018;11(13):155. https://doi.org/10.22159/ajpcr.2018.v11s1.26595
19. Shibabaw A., Gelaw B., Gebreyes W., Robinson R., Wang S., & Tessema B.. The burden of pre-extensively and extensively drug-resistant tuberculosis among mdr-tb patients in the amhara region, ethiopia. Plos One 2020;15(2):e0229040. https://doi.org/10.1371/journal.pone.0229040
20. Farra A., Koula K., Jolly B., Gando H., Ouarandji L., Mossoro-Kpinde C.et al.. Effectiveness of xpert mtb/rif and the line probe assay tests for the rapid detection of drug-resistant tuberculosis in the central african republic. Plos Global Public Health 2023;3(5):e0001847. https://doi.org/10.1371/journal.pgph.0001847
21. Valencia-Trujillo D.. Phenotypic and genotypic drug resistance of mycobacterium tuberculosis strains isolated from hiv-infected patients from a third-level public hospital in mexico. Pathogens 2024;13(2):98. https://doi.org/10.3390/pathogens13020098
22. Abdul J., Adégbitè B., Ndanga M., Edoa J., Mevyann R., Mfoumbi G.et al.. Resistance patterns among drug-resistant tuberculosis patients and trends-over-time analysis of national surveillance data in gabon, central africa. Infection 2022;51(3):697-704. https://doi.org/10.1007/s15010-022-01941-5
23. Evans D.. Patient and provider costs of the new bpal regimen for drug-resistant tuberculosis treatment in south africa: a cost-effectiveness analysis. Plos One 2024;19(10):e0309034. https://doi.org/10.1371/journal.pone.0309034
24. Zainabadi K.. A bedaquiline, pyrazinamide, levofloxacin, linezolid, and clofazimine second-line regimen for tuberculosis displays similar early bactericidal activity as the standard rifampin-based first-line regimen. The Journal of Infectious Diseases 2023;230(2):e447-e456. https://doi.org/10.1093/infdis/jiad564
25. Javaid A., Ullah I., Masud H., Basit A., Ahmad W., Butt Z.et al.. Predictors of poor treatment outcomes in multidrug-resistant tuberculosis patients: a retrospective cohort study. Clinical Microbiology and Infection 2018;24(6):612-617. https://doi.org/10.1016/j.cmi.2017.09.012
26. Ejeta E., Beyene G., Bonsa Z., & Abebe G.. Xpert mtb/rif assay for the diagnosis of mycobacterium tuberculosis and rifampicin resistance in high human immunodeficiency virus setting in gambella regional state, southwest ethiopia. Journal of Clinical Tuberculosis and Other Mycobacterial Diseases 2018;12:14-20. https://doi.org/10.1016/j.jctube.2018.06.002
27. Farra A., Manirakiza A., Yambiyo B., Zandanga G., Lokoti B., Berlioz-Arthaud A.et al.. Surveillance of rifampicin resistance with genexpert mtb/rif in the national reference laboratory for tuberculosis at the institut pasteur in bangui, 2015–2017. Open Forum Infectious Diseases 2019;6(3). https://doi.org/10.1093/ofid/ofz075
28. Mechal Y., Benaissa E., Mrimar N., Benlahlou Y., Bssaibis F., Zegmout A.et al.. Evaluation of genexpert mtb/rif system performances in the diagnosis of extrapulmonary tuberculosis. BMC Infectious Diseases 2019;19(1). https://doi.org/10.1186/s12879-019-4687-7
29. Asare K.. Comparison of microscopic and xpert mtb diagnoses of presumptive mycobacteria tuberculosis infection: retrospective analysis of routine diagnosis at cape coast teaching hospital. BMC Infectious Diseases 2024;24(1). https://doi.org/10.1186/s12879-024-09566-9
30. Rasool G., Khan A., Mohy-Ud-Din R., & Riaz M.. Detection of mycobacterium tuberculosis in afb smear-negative sputum specimens through mtb culture and genexpert® mtb/rif assay. International Journal of Immunopathology and Pharmacology 2019;33. https://doi.org/10.1177/2058738419827174
31. Cui J.. Using ultrasound guided needle biopsy in conjunction with genexpert mtb/rif to diagnose epididymal tuberculosis. Medicine 2023;102(52):e36344. https://doi.org/10.1097/md.0000000000036344
32. Cheng X.. Comparison of 3 diagnostic methods for pulmonary tuberculosis in suspected patients with negative sputum smear or no sputum. Medicine 2024;103(6):e37039. https://doi.org/10.1097/md.0000000000037039
33. Gupta J.. Comparative evaluation of genexpert with ziehl-neelsen (zn) stain in samples of suspected tuberculosis cases at a tertiary care teaching hospital in central india. Cureus 2024. https://doi.org/10.7759/cureus.71402
34. Hemeg H.. Evaluating the sensitivity of different molecular techniques for detecting mycobacterium tuberculosis complex in patients with pulmonary infection. Polish Journal of Microbiology 2023;72(4):421-431. https://doi.org/10.33073/pjm-2023-040
35. Pongpeeradech N., Kasetchareo Y., Chuchottaworn C., Lawpoolsri S., Silachamroon U., & Kaewkungwal J.. Evaluation of the use of genexpert mtb/rif in a zone with high burden of tuberculosis in thailand. Plos One 2022;17(7):e0271130. https://doi.org/10.1371/journal.pone.0271130
36. Aguilar?Jiménez J.. How has the municipal availability of the genexpert®mtb/rif system affected the detection of drug?resistant tuberculosis in brazil?. Tropical Medicine & International Health 2023;29(1):57-62. https://doi.org/10.1111/tmi.13945.