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Evaluation of extracts from used Xpert MTB/RIF cartridges for detection of resistance to second-line anti-tuberculosis drugs in patients with multidrug-resistant tuberculosis in Ethiopia

BMC Microbiology volume 25, Article number: 26 (2025) Cite this article

Abstract

Background

Early and accurate diagnosis of drug resistance, including resistance to second-line anti-tuberculosis (TB) drugs, is crucial for the effective control and management of pre-extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB). The Xpert MTB/XDR assay is the WHO recommended method for detecting resistance to isoniazid and second-line anti-TB drugs when rifampicin resistance is detected. Currently, the Xpert MTB/XDR assay is not yet implemented in Ethiopia, thus the MTBDRsl assay continues to be used. However, the MTBDRsl assay requires additional patient visits and specimen collection, which can lead to delays in diagnosis and treatment initiation.

Objective

This study aimed to evaluate the feasibility of using extracts from used Xpert MTB/RIF cartridges for detecting resistance to second-line anti-TB drugs by MTBDRsl assay in patients with rifampicin resistant-TB (RR-TB) in Eastern and Western Oromia, Ethiopia.

Methods

A cross-sectional diagnostic evaluation study was conducted from June 2020 to May 2021 at two TB Referral Laboratories in Eastern and Western Oromia, Ethiopia. Sputum samples from RR-TB patients were split, with one aliquot being subjected for Xpert testing and the other being cultured on Lowenstein-Jensen media. DNA extracted from the used Xpert cartridges was amplified by PCR and tested by MTBDRsl assay, and the results were compared to those obtained from culture isolates. To establish the detection limits, the MTBDRsl assay was performed on cartridge extracts (CEs) from a series of dilutions of drug-susceptible and multidrug-resistant TB strains.

Results

The MTBDRsl on CEs from dilutions at ≥ 102 CFU/mL (CT ≤ 22) accurately identified susceptibility and resistance patterns for fluoroquinolones (FQL) and second-line injectable drugs (SLIDs). The MTBDRsl on rifampicin-resistant CEs from sputum samples (n = 40) yielded 100% interpretable results for FQL and 90% (4 indeterminate) interpretable results for SLIDs. All interpretable CE results showed complete agreement with the MTBDRsl results from the culture isolates.

Conclusion

This study demonstrated the feasibility of using extracts from used Xpert MTB/RIF cartridges for detecting resistance to second-line anti-TB drugs using the MTBDRsl assay. This approach could mitigate the need for additional specimen collection and allow for earlier treatment initiation, potentially improving patient outcomes and reducing the transmission of drug-resistant TB strains.

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Introduction

Tuberculosis (TB) is one of the world’s leading causes of death from a single infectious agent [1]. Globally, in 2022, there were an estimated 410,000 (95% UI: 370 000–450 000) multidrug-resistant or rifampicin-resistant TB (MDR/RR-TB) cases [2]. Among the 450,000 MDR/RR-TB cases estimated to have occurred in 2021, only 37% were detected, and 36% were treated [3]. In Ethiopia, drug-resistant TB (DR-TB) poses daunting challenges because of the complex requirements for diagnosis and treatment. A systematic review conducted in 2022 reported that 2.64% of new TB cases and 11.54% of previously treated cases had MDR/RR-TB in Ethiopia [4].

Early and accurate diagnosis of drug resistance, including resistance to second-line anti-TB drugs, is crucial for the effective control and management of pre-extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) [5, 6]. Conventionally, the diagnosis of DR-TB has relied heavily on culture-based phenotypic drug susceptibility testing (DST). However, phenotypic DST results are obtained only after weeks to months of incubation, and many developing countries lack the resources to establish the laboratory conditions required for phenotypic DST [7,8,9,10]. For these reasons, the use of rapid molecular tests, including Xpert MTB/RIF, Xpert Ultra, and Xpert MTB/XDR assays (Cepheid, Sunnyvale, CA, USA), as well as Hain GenoType MTBDRplus and MTBDRsl line probe assays (Hain Life-Science GmbH, Nehren, Germany), is increasing worldwide to reduce the diagnostic turnaround time [11,12,13,14,15,16,17,18,19,20,21].

In 2021, the World Health Organization (WHO) recommended the Xpert MTB/XDR assay as a low-complexity molecular sputum-based reflex test for the detection of resistance to isoniazid, ethionamide, fluoroquinolones (FLQ), and second-line injectable drugs (SLIDs), such as kanamycin, amikacin, and capreomycin [22]. However, in Ethiopia, the Xpert MTB/XDR has not yet been widely implemented as part of the DR-TB program, and the MTBDRsl assay continues to be used for detecting resistance to second-line anti-TB drugs.

The challenge with the MTBDRsl assay is it often requires additional patient visits and specimen collection, which could lead to patient loss and delayed treatment initiation. Moreover, MTBDRsl shows suboptimal sensitivity on direct specimens, and culture is often required prior to DNA extraction. Therefore, direct testing of the first specimens is highly warranted to ensure that more patients receive comprehensive DST to reduce delays in the diagnosis and treatment of pre-XDR-TB and XDR-TB.

Previous studies have shown the feasibility of using Xpert cartridge extracts (CEs) for detecting resistance to FQL and SLIDs using the MTBDRsl [23, 24]. The MTBDRsl on CEs does not require additional patient visits or additional specimen collection. Moreover, the detection of resistance to second-line anti-TB drugs from CEs on the first available specimen could reduce the number of patients lost during the diagnostic cascade and allow earlier diagnosis of pre-XDR or XDR-TB, leading to earlier effective treatment initiation, reduced health system costs, and improved clinical outcomes. Therefore, this study aimed to evaluate the feasibility of using extracts from used Xpert MTB/RIF cartridges for detecting resistance to second-line anti-TB drugs using the MTBDRsl assay in patients with RR-TB in Eastern and Western Oromia, Ethiopia.

Materials and methods

Study design and settings

A cross-sectional diagnostic study was conducted at two TB Referral Laboratories in Eastern and Western Oromia, Ethiopia, from June 2020 to May 2021. Jimma University Mycobacteriology Research Center (JUMRC) and Adama Regional Laboratory (ARL) were the study sites.

Study subjects

All Xpert confirmed RR-TB patients referred to JUMRC and ARL during the study period were included. In accordance with the national guidelines, these RR-TB patients were referred to reference TB laboratories for screening for resistance to core second-line anti-TB drugs before the initiation of MDR/RR-TB treatment.

Inclusion and exclusion criteria

All RR-TB patients confirmed by Xpert MTB/RIF and referred to JUMC and ARL from different MDR-TB Treatment Initiation Centers (TICs) in Eastern and Western Oromia, Ethiopia, were included in the study. Xpert RR-TB patients who had already started second-line anti-TB treatment, who had insufficient sputum volume and who had negative or contaminated culture results were excluded from the study.

Study procedures

Sputum samples were collected from eligible participants at TICs and sent to the reference labs. The samples were divided into two aliquots: one for Xpert testing and one for culture on Lowenstein-Jensen (LJ) media. When Xpert detected rifampicin resistance, the cartridges were stored at 4° C until extractions for 24 h.

MTBDRsl assay on cartridge extracts

Mycobacterial DNA was recovered from used Xpert cartridges following a previously described procedure [23]. The cartridge and its surroundings were thoroughly cleaned with 1% sodium hypochlorite and 70% alcohol. Using a sterile fixed-needle insulin syringe, a transparent, diamond-shaped reaction chamber on the back of the cartridge was punctured in a biosafety cabinet. After puncturing, the entire volume of the cartridge (~ 15 µL) was carefully withdrawn and stored in 1.5 mL conical tubes at -20 °C until analysis. Finally, 5 µL of CE was used for PCR amplification. No DNA extraction or additional purification steps were performed on the CEs. After extracting from the cartridges, all subsequent procedures, including master mix preparation, PCR amplification, hybridization, detection, and result interpretation, were performed using the same protocols and reagents/consumables as those for MTBDRsl on sputum or culture isolates [25].

Culture and identification

Sputum samples were decontaminated, neutralized, centrifuged and inoculated on LJ media for culture. Positive cultures were confirmed as Mycobacterium tuberculosis (MTB) complex via Ziehl-Neelsen staining and the SD Bioline Ag MPT64 Rapid test (Standard Diagnostics, Seoul, South Korea).

MTBDRsl assay on culture isolates

DNA was extracted from confirmed MTB complex isolates, and the MTBDRsl assay was performed according to manufacturer’s instructions [25]. The results were interpreted on the basis of the wild type deletion and the presence of mutant bands.

Analytical validation of MTBDRsl on CEs on control strains

A series of dilutions of drug-susceptible H37Rv and MDR-TB strains were tested by Xpert to evaluate the detection limits of the MTBDRsl on CEs.

Data analysis procedures

The data were entered into EpiData version 3.1 and analyzed by SPSS version 23. Descriptive statistics were performed using frequencies and percentages to describe the sociodemographic characteristics of the study population. The yields of the MTBDRsl assay performed on CEs were compared with those of the MTBDRsl assay performed on culture isolates.

Results

Baseline characteristics of study participants

During the study period, a total of 62 sputum samples (RR-TB positive by Xpert) were referred to JUMRC and ARL for second-line DST using the MTBDRsl assay. Among these, 40 samples that were confirmed Xpert RR-TB and culture positive for MTB were included in this study. The remaining 22 samples were excluded from the analysis: 12 were rifampicin-susceptible on repeat Xpert testing, six were culture-negative, and four had contaminated cultures. Among the 40 patients included in the study, 70% (28) were males, 95% (38) were HIV negative, and 75% (30) had a previous history of TB treatment. The median age of the patients was 30 years (Table 1).

Table 1 Sociodemographic and clinical characteristics of the study participants (N = 40)
Full size table

Analytical validation of MTBDRsl on CEs using control strains

The MTBDRsl assay on CEs from both drug-susceptible and MDR-TB strains accurately determined susceptibility and resistance patterns for FQL and SLIDs at dilutions of ≥ 102 CFU/mL, corresponding to a cycle threshold (CT) of ≤ 22 (Table 2).

Table 2 Results of MTBDRsl performed on cartridge extracts from a dilution series of H37Rv and MDR-TB strains in triplicate (n = 3)
Full size table

Feasibility of MTBDRsl on extracts from cartridges

The MTBDRsl performed on CEs from used Xpert cartridges on sputum yielded 100% (40/40) interpretable results for FQL and 90% (36/40) interpretable results for SLIDs. Four CEs had an indeterminate MTBDRsl results for SLIDs (Fig. 1). Three of these CEs were missing the rrs locus control band, whereas one had a weak band for this control. Consistent with the results of MTBDRsl from the culture isolates, no resistance was detected for FQL or SLIDs in any of the CEs, with interpretable MTBDRsl results.

Fig. 1
figure 1

Results of MTBDRsl performed on extracts from used Xpert cartridges (N = 40). FQL, fluoroquinolones; SLIDs, second-line injectable drugs

Full size image

MTBDRsl on cartridge extracts versus the Xpert semiquantitative category

Among the 40 Xpert-positive samples, 37.5% (15) had a high bacillary load (CT < 16), 52.5% (21) had a medium bacillary load (CT, 16–22), and 10% (4) had a low bacillary load (CT, 22–28). All CEs with Xpert semiquantitative grades of at least medium (CT ≤ 22) had interpretable MTBDRsl results. The four CEs with indeterminate MTBDRsl results had low Xpert semiquantitative grades (CT, 22–28) (Fig. 2).

Fig. 2
figure 2

Xpert semiquantitative grades versus MTBDRsl results for extracts from used Xpert cartridges

Full size image

Concordance of MTBDRsl results on cartridge extracts and culture isolates

The MTBDRsl on CEs showed 100% (40/40) concordance with the MTBDRsl results from culture isolates for FQL. After excluding the four indeterminate CE results, the MTBDRsl on CEs showed 100% (36/36) concordance with the MTBDRsl results from culture isolates for SLIDs. Although the data are not shown here, the average time to result was two days for CEs, whereas it was 24 days for the culture isolates.

Discussion

Drug-resistant TB is a major challenge for global TB control efforts. A large proportion of pre-XDR/XDR-TB cases remain undiagnosed and continue to transmit the disease in the community. Early detection of resistance to second-line anti-TB drugs among rifampicin resistant TB (RR-TB) patients is critical for determining the best treatment regimen composition. To facilitate this, the WHO has endorsed the Xpert MTB/XDR assay for the detection of resistance to isoniazid and second-line anti-TB drugs [22]. However, in Ethiopia, the Xpert MTB/XDR has not yet been widely implemented as part of the drug resistant TB program, and the MTBDRsl assay continues to be used for detecting resistance to second-line anti-TB drugs. This entails additional patient visits, specimen collection, and sometimes culture before DNA extraction. This could lead to patient loss and delay treatment initiation. Hence, it is crucial to adopt approaches that ensure comprehensive DSTs by directly testing the first specimens that patients provide during their first visit. In this study, we evaluated the feasibility of using CEs from used Xpert cartridges for detecting resistance to second-line anti-TB drugs using the MTBDRsl assay in patients with RR-TB in Eastern and Western Oromia, Ethiopia.

Our validation experiments revealed that MTBDRsl on CEs correctly identified susceptibility and resistance patterns for FLQ and SLIDs at dilutions ≥ 102 CFU/mL (CT ≤ 22). CEs are readily available after Xpert tests and can be analysed immediately. This could reduce the need for additional specimen collection and culture. It also offers cost reduction by minimizing the expenses associated with laboratory processing and reagents. Moreover, it requires minimal biosafety precautions, making it particularly advantageous in resource-limited settings where access to advanced laboratory infrastructure and resources is limited [23, 26]. Additionally, it enables swift treatment initiation by shortening the turnaround time to obtain DST results. Overall, it optimizes patient management, mitigates the emergence of drug resistance and reduces the transmission of drug-resistant TB.

In this study, MTBDRsl on CEs yielded 100% interpretable results for FQL and 90% for SLIDs. This finding is supported by a previous study conducted in South Africa where MTBDRsl on CEs showed better performance for the detection of resistance to FQL compared to SLIDs [23]. All CEs with interpretable results demonstrated complete agreement with the MTBDRsl from the culture isolates. The concordance rate observed in this study is higher than the values reported from South Africa (90% for FLQs and 84% for SLIDs) [23]. The reason for this variation should be explored in the future with larger studies.

In the present study, four CEs yielded indeterminate results for SLIDs. All four extracts were sensitive to FQL and low-level kanamycin, with a visible TUB band, amplification control, and conjugate control when MTBDRsl was performed on culture isolates. All four CEs had low Xpert semiquantitative grades (CT, 22–28). Three of these extracts showed an absence of rrs locus control hybridization, whereas the remaining one had a faint band for this control. Ideally, the absence of a locus control band is linked to mutations or deletions in the locus control region, as well as the complete or partial deletion of a target gene [27]. However, in our study, this explanation seems unlikely since the MTBDRsl on culture isolates demonstrated the rrs locus control band. Considering the low bacillary load detected by Xpert, the indeterminate results could be attributed to the insufficient amount of mycobacterial DNA present in the CEs. A previous study noted that individuals with a low bacillary load, including those with negative and scanty smear microscopy results, had a significantly greater proportion of indeterminate MTBDRsl results [28]. In such cases, the WHO recommends repeating MTBDRsl on culture isolates [29].

The time to detect resistance to second-line anti-TB drugs was significantly shorter when MTBDRsl was performed on CEs than when MTBDRsl was applied to cultured isolates. This enables rapid detection of pre-XDR or XDR-TB and significantly reduces treatment delays. Despite the launch and recommendation of the MTBDRsl assay, patients diagnosed with RR-TB still await culture to proceed with downstream molecular assays. Performing MTBDRsl on the first available specimen could reduce the number of patients lost during the diagnostic cascade and allow earlier diagnosis of pre-XDR or XDR-TB, leading to earlier effective treatment initiation and better clinical outcomes.

The process of collecting CEs from Xpert cartridges may increase the risk of amplicon cross-contamination. To mitigate this risk, it is essential to use a dedicated biosafety cabinet or room for the CE, decontaminate the workspace with UV light and appropriate disinfectants before and after extraction. Furthermore, CE extraction should be conducted with the greatest care, as it may increase the risk of needlestick injury.

This study should be interpreted with certain limitations. The standard phenotypic DST was not carried out; MTBDRsl results from culture isolates were used for comparison. Moreover, the effect of transporting and storing the whole cartridges at ambient temperatures on the assay’s performance was not evaluated.

Conclusion

This study revealed that MTBDRsl performed on cartridge extracts showed a high level of agreement with MTBDRsl performed on culture isolates for detecting resistance to second-line anti-TB drugs. At CT values ≤ 22, MTBDRsl on CEs is effective for detecting resistance to second-line anti-TB drugs, offering swift treatment initiation, reducing patient visits and losses, and requiring minimal biosafety precautions. However, further study with a larger sample size is needed to assess the effects of transporting and storing cartridges at ambient temperature for an extended period on the performance of MTBDRsl on cartridge extracts. Furthermore, we recommend future studies evaluate the diagnostic performance of the Xpert MTB/XDR assay on cartridge extracts obtained from Xpert Ultra.

Data availability

All the data generated or analyzed during this study are included in this published article.

Abbreviations

CEs:

Cartridge extracts

CT:

Cycle threshold

DR-TB:

Drug resistant tuberculosis

DST:

Drug susceptibility testing

XDR-TB:

Extensively drug-resistant tuberculosis

FQL:

Fluoroquinolones

MDR-TB:

Multidrug resistant tuberculosis

RR-TB:

Rifampicin resistant tuberculosis

SLIDs:

Second-line injectable drugs

TB:

Tuberculosis

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Acknowledgements

The authors would like to thank the staff members of all MDR-TB treatment centers for their kind cooperation during the data collection process. We are also grateful to the data collectors.

Funding

This work was supported by Jimma University-Institute of Health, JUMRC, and ARL. The funders had no role in the study design, data collection and analysis, preparation of the manuscript or decision to publish.

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Author notes

  1. Tilahun Ketema and Mulualem Tadesse contributed equally to this work.

Authors and Affiliations

  1. Mycobacteriology Research Center, Institute of Health, Jimma University, Jimma, Oromia, Ethiopia

    Mulualem Tadesse, Zegeye Bonsa, Getu Balay, Wakjira Kebede & Gemeda Abebe

  2. School of Medical Laboratory Sciences, Faculty of Health Sciences, Jimma University, Jimma, Oromia, Ethiopia

    Mulualem Tadesse, Wakjira Kebede, Mekidim Mekonnen & Gemeda Abebe

  3. Armauer Hansen Research Institute, Addis Ababa, Ethiopia

    Tilahun Ketema

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Contributions

TK, GA, MT, ZB, WK, and MM conceived and designed the study. TK, ZB, and GB, were involved in participant recruitment and laboratory investigations. TK, ZB, and MT analyzed the data. ZB drafted the manuscript. GA, MT, GB, WK, and MM reviewed the manuscript. All the authors have read and approved the final version of the manuscript.

Corresponding author

Correspondence to Zegeye Bonsa.

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Ethical approval and consent to participate

The study was carried out in compliance with the Declaration of Helsinki. The Institutional Review Board (IRB) at the Institute of Health, Jimma University, Ethiopia, approved the study protocol (Reference No. IHRPG/45/2020). Informed consent was obtained from all patients for the use of their data for research purposes.

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Not applicable.

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The authors declare no competing interests.

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Ketema, T., Tadesse, M., Bonsa, Z. et al. Evaluation of extracts from used Xpert MTB/RIF cartridges for detection of resistance to second-line anti-tuberculosis drugs in patients with multidrug-resistant tuberculosis in Ethiopia. BMC Microbiol 25, 26 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03746-6

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