Abstract
Introduction
Early detection of drug resistance in patients with tuberculosis (TB) is crucial for prompt and effective treatment. This study evaluated the performance of Xpert MTB/XDR assay (Xpert XDR) for detecting resistance to isoniazid (INH), fluoroquinolones (FLQ), aminoglycosides (AMG), and ethionamide (ETH) in patients with pulmonary TB (PTB) in Bangladesh.
Methods
Xpert XDR was performed on sputum samples from 793 Xpert MTB/RIF positive patients with PTB enrolled between April 2021 and March 2023. Results were compared with phenotypic drug susceptibility test (pDST) performed on Lowenstein–Jensen (L–J) media for the detection of resistance to INH, FLQ, AMG, and ETH. The performance of the assay was also compared between newly diagnosed or rifampicin (RIF)-sensitive versus re-treated or RIF-resistant patients with PTB.
Results
Of 793 samples tested by Xpert XDR, indeterminate results for INH, FLQ, AMG, and ETH were observed for 3 (0.4%), 5 (0.6%), 33 (4.2%), and 0 (0%) isolates, respectively. The assay’s sensitivity and specificity compared to pDST was 94.0% (95% CI 90.5–96.4; 264/281) and 97.3% (95% CI 95.4–98.5; 495/509), respectively for INH; 86.0% (95% CI 78.2–91.8; 98/114) and 99.3% (95% CI 98.3–99.3; 669/674), respectively for FLQ; 85.7% (95% CI 42.1–99.6; 6/7) and 99.9% (95% CI 99.3–100.0; 752/753), respectively for AMG; and 25.0% (95% CI 19.0–31.7; 48/192) and 96.7% (95% CI 94.9–98.0; 581/601), respectively for ETH. Agreement of Xpert XDR with pDST was almost perfect for detecting resistance to INH, FLQ, and AMG (kappa: 0.91, 0.89, and 0.86, respectively), but fair for ETH (kappa: 0.28). Xpert XDR performed significantly better among re-treated or RIF-resistant patients with TB compared to newly diagnosed or RIF-sensitive cases.
Conclusions
Given the high performance, Xpert XDR assay can be programmatically implemented nationwide for rapid and accurate detection of resistance to INH, FLQ, and AMG in patients with PTB, aiding clinicians in selecting appropriate regimens for the treatment of drug-resistant TB.
Supplementary Information
The online version contains supplementary material available at 10.1007/s40121-025-01127-0.
Keywords: Xpert MTB/XDR, Diagnostic performance, Drug resistance, Tuberculosis, Bangladesh
Key Summary Points
| Why carry out this study? |
| Early detection of tuberculosis and rapid, accurate identification of drug resistance are vital for effective treatment and management of the patients. |
| Xpert MTB/XDR (Xpert XDR), a World Health Organization-recommended molecular assay, detects resistance to isoniazid (INH), fluoroquinolones (FLQ), aminoglycosides (AMG), and ethionamide (ETH). However, its performance in Bangladesh remains unassessed. |
| What was learned from the study? |
| This study aimed to evaluate the performance of Xpert XDR assay in Bangladesh by comparing its results with the gold-standard phenotypic drug susceptibility test for INH, FLQ, AMG, and ETH resistance. |
| The Xpert XDR assay showed high accuracy in detecting resistance to INH, FLQ, and AMG but performed poorly for ETH. |
| The assay can be widely adopted across the country for rapid and reliable detection of resistance to INH, FLQ, and AMG, which will help clinicians in selecting effective treatment regimens for drug-resistant TB. |
Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be one of the deadliest infectious diseases globally. Prior to the COVID-19 pandemic, TB caused more deaths than any other infectious diseases, including HIV/AIDS. Multidrug-resistant (MDR) or rifampicin-resistant (RR)-TB presents a major global public health challenge due to low treatment success rates and limited access to appropriate care. According to the World Health Organization (WHO), in 2023, an estimated 400,000 people developed MDR/RR-TB globally, yet only about 40% were diagnosed and treated [1]. Bangladesh is among 30 countries facing a high burden TB and MDR/RR-TB. In Bangladesh, an estimated 5000 cases of MDR/RR-TB occurred in 2023, but only about one-third were diagnosed [2]. This significant gap is primarily caused by the lack of early diagnostic tools for drug-resistant TB (DR-TB), which contributes to treatment failure and the ongoing transmission of DR-TB.
Early detection of TB, along with rapid and accurate drug resistance data, is crucial for effective patient treatment and management. Culture and phenotypic drug susceptibility testing (pDST) on solid or liquid media are considered as ‘gold standard’, however, these methods are often impractical due to the lengthy time required to obtain results (2–8 weeks for culture and an additional 4–6 weeks for pDST) as well as the need for specialized infrastructure and trained personnel [3, 4]. The Xpert MTB/RIF (Xpert) and Xpert MTB/RIF Ultra (Xpert Ultra) assays (Cepheid, CA, USA), endorsed by the WHO, are molecular tests that can rapidly detect TB and provide drug resistance information for only rifampicin (RIF) within 2 h [5, 6]. These tests utilize automated semi-nested real-time quantitative PCR and require minimal operator training. However, the Xpert/Xpert Ultra assays cannot detect resistance to isoniazid (INH) and other second-line drugs, creating a significant gap in the drug resistance data needed to effectively treat DR-TB patients. The Xpert MTB/XDR (Xpert XDR) assay (Cepheid, CA, USA) introduced as a solution to these limitations, is a 10-color reflex test that can detect TB and assess resistance to INH, ethionamide (ETH), fluoroquinolones (FLQ), and aminoglycoside (AMG), such as amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) in less than 90 min [7]. According to recent WHO guidelines, Xpert XDR cartridge can be utilized as a reflex test to complement existing technologies like Xpert and Xpert Ultra, enabling rapid identification of resistance to INH and other second-line drugs [8].
The Xpert XDR assay detects INH resistance by targeting four regions: the inhA promoter, KatG gene, fabG1 gene, and the oxyR-ahpC intergenic region. It also detects ETH resistance through mutations in the inhA promoter [9]. FLQ resistance is identified by detecting mutations in the gyrA and/or gyrB genes, while AMG resistance is detected by rrs and eis gene promoter regions [7]. The performance of the Xpert XDR assay and the distribution of resistance mutations can vary across geographical regions. Current data indicate that the assay’s sensitivity for detecting resistance to INH, FLQ, AMG, and ETH ranges between 86.7–98.3%, 80–95%, 70–95%, and 50–65%, respectively, while its specificity ranges from 88–100% [10–16]. However, no data are yet available regarding the assay’s performance for detecting resistance to these drugs in Bangladesh. This study aimed to evaluate the performance of the Xpert XDR assay in Bangladesh by comparing it with the gold-standard pDST for detecting resistance to INH, FLQ, AMG, and ETH. Furthermore, the study aims to identify the mutational profiles linked to resistance against these drugs as determined by the assay.
Methods
Study Population and Specimen Collection
Sputum was collected from Xpert-positive pulmonary TB patients as part of a study for determination of isoniazid resistance pattern in Bangladesh conducted between April 2021 and December 2022, and from an ongoing study investigating the transmission dynamics of RIF-resistant TB in the country. Patients were consecutively enrolled in the study if not yet started anti-TB treatment and were able to provide at least 3 ml of sputum. The present study was approved by the Institutional Review Board of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) (Protocol number: PR-21008 and PR-22047), and was performed in accordance with the Helsinki Declaration of 1964 and its later amendments. Informed written consents/assents were obtained from participants prior to enroll in the study. Collected sputum samples were transported to the Mycobacteriology Laboratory of icddr,b for Xpert XDR testing along with culture and pDST. A total of 793 Xpert-positive pulmonary TB patients, with available results for both Xpert XDR and pDST, were included in this study analysis. Detailed clinical and demographic information was obtained through questionnaires from all the participants.
Xpert XDR Assay
The Xpert XDR assay was conducted on unprocessed raw sputum by following the instructions of the manufacturer [17]. Sample reagent and sputum specimen were combined at a 2:1 ratio in a 15-ml centrifuge tube. The mixture was then vigorously vortexed initially and again after a 10-min incubation at room temperature. After an additional 5-min incubation, 2.0 ml of the sample was transferred into the Xpert XDR cartridge, which was then loaded onto the GeneXpert platform equipped with ten-color modules. Within approximately 90 min, results were generated, indicating the detection of M. tuberculosis and resistance to INH, FLQ, AMG, and ETH.
Sputum Processing and Culture
Sputum samples were decontaminated and processed as previously described [18]. To each sputum sample, an equal volume of N-acetyl-l-cysteine (NALC)-NaOH-Sodium citrate solution (1% NALC, 4% NaOH, and 2.94% Na-citrate) was added in a 50-ml centrifuge tube. The mixture was vortexed thoroughly and incubated at room temperature for 15 min. After incubation, the tube was centrifuged at 3000g for 15 min at 4 °C. The supernatant was then discarded, and the pellet was inoculated onto Lowenstein-Jensen (L-J) media slants. These slants were incubated for up to 8 weeks at 37 °C, with weekly observations to monitor for visible growth of M. tuberculosis.
Phenotypic Drug Susceptibility Testing
pDST was conducted on culture-positive isolates using the L–J proportion method, as previously described [19], following WHO-recommended critical concentrations for isoniazid (INH) (0.2 μg/ml), levofloxacin (LEV) (2 μg/ml), moxifloxacin (MOX) (1 μg/ml), kanamycin (KAN) (30 μg/ml), and ethionamide (ETH) (40 μg/ml) [20]. Isolates were classified as drug-resistant if 1% or more colonies grew on a medium containing the drug, compared to a drug-free medium. Under FLQ, pDST was performed for both LEV and MOX. An isolate resistant to either LEV or MOX was categorized as FLQ-resistant, while sensitive to both was classified as FLQ-sensitive. We tested pDST against KAN under the AMG. Any isolate resistant to KAN was considered AMG-resistant, whereas sensitive to KAN was classified as AMG-sensitive.
Quality Control
To ensure quality control, we tested a susceptible strain (H37Rv, from the American Type Culture Collection, ATCC) and a laboratory-characterized resistant strain (SB256) using both the L–J proportion method and the Xpert XDR assay. H37Rv is a known susceptible strain, while SB256 is resistant to INH, LEV, MOX, KAN, and ETH, respectively. Both the L–J proportion method and the Xpert XDR assay confirmed sensitive results in H37Rv and resistant results in SB256 for these drugs [4]. In addition, the Xpert XDR cartridge has built-in quality controls which contain Sample Processing Control (SPC) and Probe Check Control (PCC). The SPC verifies that the sample processing is adequate and ensures the appropriate amplification of the PCR reaction. Whereas, PCC checks the probe integrity, dye stability, reagent rehydration, and PCR tube filling in the cartridge, demonstrating the result is valid [17].
Statistical Analysis
Demographic, clinical, and laboratory data were entered and analyzed using SPSS 20.0 (Statistical Package for the Social Sciences Inc., Chicago, IL, USA). The results of Xpert XDR assay were compared with the gold-standard L–J proportion method to determine the assay’s sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for detection of resistance to INH, FLQ, AMG, and ETH. The assay’s performance was analyzed across different patient groups, including new versus re-treated TB cases and RIF-sensitive versus RIF-resistant TB patients. Proportions were compared using the Chi-square test or Fisher’s exact test, with a p value of less than 0.05 considered statistically significant. Cohen’s kappa statistic was used to evaluate the two methods’ agreement. The kappa values were interpreted as follows: ≥ 0.81 (almost perfect), 0.61–0.8 (substantial), 0.41–0.6 (moderate), 0.21–0.4 (fair), and < 0.2 (slight) [21].
Results
Demographic and Clinical Characteristics of the Pulmonary TB Patients
The study included 793 Xpert-positive pulmonary TB patients, with 73.6% (584/793) being male. The mean age of the patients was 36.6 years, and the majority (45.5%, 361/793) were between 21 and 40 years of age. Approximately 61.4% (487/793) of the patients resided in urban areas. Smoking was reported in 48.3% (383/793) of patients, while drug abuse and alcohol consumption were observed in 5.5% (44/793) and 7.2% (57/793) of cases, respectively. Around 23% (182/793) patients had a previous history of TB. Diabetes was present in 24.2% (192/793) of the patients. Geographically, the majority of patients were from the Dhaka division (69.1%, 548/793), while the Barishal division had the fewest cases (1.5%, 12/793) (Table 1).
Table 1.
Demographic and clinical characteristics of the enrolled Xpert MTB/RIF-positive pulmonary tuberculosis patients (n = 793)
| Variable | Label | Number of patients (n = 793) | Frequency (%) |
|---|---|---|---|
| Sex | Male | 584 | 73.6 |
| Female | 209 | 26.4 | |
| Age | ≤ 20 | 123 | 15.5 |
| 21–40 | 361 | 45.5 | |
| 41–60 | 228 | 28.8 | |
| > 60 | 81 | 10.2 | |
| Dwelling | Urban | 487 | 61.4 |
| Rural | 306 | 38.6 | |
| Previous history of TB | Yes | 182 | 23.0 |
| No | 611 | 77.0 | |
| Smoking | Yes | 383 | 48.3 |
| No | 410 | 51.7 | |
| Diabetes | Yes | 192 | 24.2 |
| No | 527 | 66.5 | |
| Not known | 74 | 9.3 | |
| Previous history of TB | Yes | 182 | 23.0 |
| No | 611 | 77.0 | |
| Drug abuse | Yes | 44 | 5.5 |
| No | 749 | 94.5 | |
| Alcohol consumption | Yes | 57 | 7.2 |
| No | 736 | 92.8 | |
| Geographic distribution | Dhaka | 548 | 69.1 |
| Chattogram | 92 | 11.6 | |
| Sylhet | 55 | 6.9 | |
| Barishal | 12 | 1.5 | |
| Mymensingh | 22 | 2.8 | |
| Rajshahi | 27 | 3.4 | |
| Rangpur | 13 | 1.6 | |
| Khulna | 24 | 3.0 |
TB tuberculosis
Drug Susceptibility Results of the Cultured Isolates by L–J Proportion Method
pDST against INH, FLQ, AMG and ETH by the L–J proportion method revealed that 46.4% (368/793) of the isolates were sensitive, and 0.5% (4/793) were resistant to all anti-TB drugs (Table 2). Overall resistance rate to any INH, FLQ, AMG, and ETH was 35.8% (284/793), 14.4% (114/793), 0.9% (7/793), and 24.2% (192/793), respectively. The proportion of isolates sensitive to all drugs were significantly higher among new than the re-treated TB patients (p < 0.001), whereas the resistance rate of all drugs, except ETH, was significantly higher among re-treated TB patients compared to newly diagnosed TB patients (p < 0.001). Mono drug resistance to INH, FLQ and ETH was observed in 17.7% (140/793), 4.4% (35/793, and 12.6% (100/793) isolates, respectively. Mono resistance to AMG was not detected in any of the isolates. About 18.2% (144/793) isolates showed poly-drug resistance in different variations (Table 2).
Table 2.
Resistance pattern of isoniazid, fluoroquinolone, aminoglycoside, and ethionamide among patients with pulmonary tuberculosis as determined by the gold standard Lowenstein–Jensen proportion method
| Resistance pattern | Total cases (n = 793) n (%) |
New cases (n = 611) n (%) |
Re-treated cases (n = 182) n (%) |
P value |
|---|---|---|---|---|
| All sensitive | 368 (46.4) | 423 (69.2) | 59 (32.4) | < 0.001 |
| All resistant | 4 (0.5) | 0 (0) | 4 (2.2) | < 0.001 |
| Any resistance | ||||
| INH | 284 (35.8) | 164 (26.8) | 120 (65.9) | < 0.001 |
| FLQ | 114 (14.4) | 67 (11.0) | 47 (25.8) | < 0.001 |
| AMG | 7 (0.9) | 0 (0) | 7 (3.8) | < 0.001 |
| ETH | 192 (24.2) | 149 (24.4) | 43 (23.6) | 0.825 |
| Mono-drug resistant | ||||
| INH | 140 (17.7) | 88 (14.4) | 52 (28.6) | < 0.001 |
| FLQ | 35 (4.4) | 29 (4.7) | 6 (3.3) | 0.418 |
| ETH | 100 (12.6) | 94 (15.4) | 6 (3.3) | < 0.001 |
| AMG | 0 (0) | 0 (0) | 0 (0) | – |
| Poly-drug resistant | ||||
| INH + FLQ | 55 (6.9) | 26 (4.3) | 29 (15.9) | < 0.001 |
| INH + FLQ + AMG | 2 (0.3) | 0 (0) | 2 (1.1) | 0.009 |
| INH + FLQ + ETH | 12 (1.5) | 7 (1.1) | 5 (2.7) | 0.115 |
| INH + ETH | 70 (8.8) | 43 (7.0) | 27 (14.8) | < 0.001 |
| FLQ + ETH | 6 (0.8) | 5 (0.8) | 1 (0.5) | 0.676 |
| INH + AMG | 1 (0.1) | 0 (0) | 1 (0.5) | 0.080 |
INH isoniazid, FLQ fluoroquinolones, AMG aminoglycoside, ETH ethionamide
Performance of the Xpert XDR Assay
Of 793 TB patients, 269 (33.9%) were resistant to RIF, while the remainder were RIF-sensitive. Samples yielding an “Indeterminate” result during Xpert XDR testing were excluded from the analysis. The indeterminate rates for detecting resistance to INH, FLQ, AMG, and ETH were 0.4% (3/793), 0.6% (5/793), 4.2% (33/793), and 0% (0/793), respectively. All indeterminate results from the Xpert XDR assay yielded definitive resistant or sensitive outcomes when tested by pDST (Supplementary Table S1). Consequently, final results for Xpert XDR in detecting resistance to INH, FLQ, AMG, and ETH were available for 790, 788, 760, and 793 patients, respectively.
Table 3 presents the overall performance of the Xpert XDR assay, as well as its performance in new and re-treated pulmonary TB patients for detecting resistance to INH, FLQ, AMG, and ETH. Compared to the L–J proportion method, the overall sensitivities and specificities of the Xpert XDR assay were 94.0% (95% Confidence Interval, CI 90.5–96.4) and 97.3% (95% CI 95.4–98.5), respectively for detecting INH resistance; 86.0% (95% CI 78.2–91.8) and 99.3% (95% CI 98.3–99.8), respectively for detecting FLQ resistance; 85.7% (95% CI 42.1–99.6) and 99.9% (95% CI 99.3–100.0), respectively for detecting AMG resistance; and 25.0% (95% CI 19.0–31.7) and 96.7% (95% CI 94.9–98.0), respectively for detecting ETH resistance. Overall, the Xpert XDR assay had ‘almost perfect’ agreement with L–J proportion method for the determination of resistance to INH (k value = 0.91), FLQ (k value = 0.89), and AMG (k value = 0.86). However, the assay exhibited ‘fair’ agreement for detecting ETH (0.28) resistance.
Table 3.
Performance of the Xpert MTB/XDR assay compared with the gold standard Lowenstein–Jensen proportion method for detection of resistance to isoniazid, fluoroquinolone, aminoglycoside, and ethionamide
| Method | Patient group | Drugs | L-J proportion method | Sensitivity % (95% CI) |
Specificity % (95% CI) |
PPV (95% CI) |
NPV (95% CI) |
Level of agreement (k-value) | ||
|---|---|---|---|---|---|---|---|---|---|---|
| R | S | |||||||||
| Xpert MTB/XDR assay | Overall (n = 793) | INH (n = 790) | R | 264 | 14 |
94.0 (90.5–96.4) |
97.3 (95.4–98.5) |
95.0 (91.8–96.9) |
96.7 (94.8–97.9) |
0.91 |
| S | 17 | 495 | ||||||||
| FLQ (n = 788) | R | 98 | 5 |
86.0 (78.2–91.8) |
99.3 (98.3–99.8) |
95.2 (89.1–97.9) |
97.7 (96.4–98.5) |
0.89 | ||
| S | 16 | 669 | ||||||||
| AMG (n = 760) | R | 6 | 1 |
85.7 (42.1–99.6) |
99.9 (99.3–100) |
85.7 (45.3–97.8) |
99.9 (99.2–100) |
0.86 | ||
| S | 1 | 752 | ||||||||
| ETH (n = 793) | R | 48 | 20 |
25 (19.0–31.7) |
96.7 (94.9–98.0) |
70.6 (59.4–79.8) |
80.1 (78.8–81.4) |
0.28 | ||
| S | 144 | 581 | ||||||||
| New (n = 611) | INH (n = 610) | R | 149 | 13 |
91.4 (86.0–95.2) |
97.1 (95.1–98.4) |
92.0 (87.0–95.2) |
96.9 (94.9–98.1) |
0.89 | |
| S | 14 | 434 | ||||||||
| FLQ (n = 607) | R | 53 | 5 |
79.1 (67.4–88.1) |
99.1 (97.9–99.7) |
91.4 (81.5–96.2) |
97.5 (96.0–98.4) |
0.83 | ||
| S | 14 | 535 | ||||||||
| AMG (n = 582) | R | 0 | 1 | – |
99.8 (99.1–100) |
– |
100 (99.4–100) |
– | ||
| S | 0 | 581 | ||||||||
| ETH (n = 611) | R | 29 | 12 |
19.5 (13.4–26.7) |
97.4 (95.5–98.7) |
70.7 (55.9–82.2) |
79.0 (77.6–80.3) |
0.22 | ||
| S | 120 | 450 | ||||||||
| Re-treated (n = 182) | INH (n = 180) | R | 115 | 1 |
97.5 (92.8–99.5) * |
98.4 (91.3–100) |
99.1 (94.3–99.9) |
95.3 (86.9–98.4) |
0.95 | |
| S | 3 | 61 | ||||||||
| FLQ (n = 181) | R | 45 | 0 |
95.7 (85.5–99.5) * |
100 (97.3–100) |
100 (92.1–100) |
98.5 (94.5–99.6) |
0.97 | ||
| S | 2 | 134 | ||||||||
| AMG (n = 178) | R | 6 | 0 |
85.7 (42.1–99.5) |
100 (97.9–100) |
100 (54.1–100) |
99.4 (96.5–99.9) |
0.92 | ||
| S | 1 | 171 | ||||||||
| ETH (n = 184) | R | 19 | 8 |
44.2 (29.1–60.1) * |
94.2 (89.0–97.5) |
70.4 (52.8–83.4) |
84.5 (80.7–87.7) |
0.44 | ||
| S | 24 | 131 | ||||||||
*Significant differences of proportion between the new and re-treated group of TB patients
RIF rifampicin, INH isoniazid, FLQ fluoroquinolones, AMG aminoglycoside, ETH ethionamide, R resistant, S sensitive, L-J Lowenstein–Jensen, PPV positive predictive value, NPV negative predictive value, CI confidence interval
When comparing sensitivity between new and re-treated groups of TB patients, the Xpert XDR assay demonstrated significantly higher sensitivities in re-treated cases for detecting resistance to INH, FLQ, and ETH. Sensitivity for AMG resistance among newly diagnosed TB patients could not be assessed due to the absence of resistant cases in this group. The sensitivities for detection of INH resistance in the new versus re-treated groups of TB patients were 91.4% (95% CI 86.0–95.2) vs. 97.5% (95% CI 92.8–99.5) (p < 0.05); for FLQ sensitivities were 79.1% (95% CI 67.4–88.1) vs. 95.7% (95% CI 85.5–99.5) (p < 0.05); and for ETH they were 19.5% (95% CI 13.4–26.7) vs. 44.2% (95% CI 29.1–60.1) (p < 0.001), respectively (Table 3). The sensitivities for detecting resistance to INH, FLQ, and ETH were even higher in the re-treated group compared to the assay’s overall sensitivities, whereas there were no significant differences on the specificities for detection of the resistance of all four drugs between new and re-treated TB patients (Table 3).
The performance of Xpert XDR assay was also compared between RIF-resistant and sensitive pulmonary TB patients (Table 4). Sensitivities were significantly higher among the RIF-resistant than the RIF-sensitive TB patients for detecting resistance to INH, FLQ, and ETH. Sensitivity for AMG could not be determined as well since there was no any resistant cases in the newly diagnosed TB patients. The sensitivities for detection of INH resistance in the RIF-sensitive versus RIF-resistant cases were 85.2% (95% CI 72.9–93.4) vs. 96.0% (95% CI 92.6–98.2) (p < 0.001); for FLQ sensitivities were 63.6% (95% CI 45.1–79.6) vs. 95.0% (95% CI 87.7–98.6) (p < 0.001); and for ETH they were 13.0% (95% CI 7.6–20.3) vs. 46.4% (95% CI 34.3–58.8) (p < 0.001), respectively (Table 4), whereas specificities did not differ significantly between the groups for detecting the resistance of all four drugs (Table 4).
Table 4.
Performance of the Xpert MTB/XDR assay compared with the gold standard Lowenstein–Jensen proportion method for detection of resistance to isoniazid, fluoroquinolone, aminoglycoside, and ethionamide among rifampicin-resistant and rifampicin-sensitive patients with pulmonary tuberculosis
| Method | Patient group | Drugs | L–J proportion method | Sensitivity % (95% CI) |
Specificity % (95% CI) |
PPV (95% CI) |
NPV (95% CI) |
Level of agreement (k-value) | ||
|---|---|---|---|---|---|---|---|---|---|---|
| R | S | |||||||||
| Xpert MTB/XDR assay | RIF-sensitive (n = 521) | INH (n = 521) | R | 46 | 8 | 85.2 (72.9–93.4) | 98.3 (96.7–99.3) | 85.2 (74.1–92.0) | 98.3 (96.8–99.1) | 0.83 |
| S | 8 | 459 | ||||||||
| FLQ (n = 517) | R | 21 | 5 | 63.6 (45.1–79.6) | 99.0 (97.6–99.7) | 80.8 (62.9–91.3) | 97.6 (96.2–98.4) | 0.69 | ||
| S | 12 | 479 | ||||||||
| AMG (n = 494) | R | 0 | 0 | – |
100 (99.3–100) |
– |
100 (99.3–100) |
– | ||
| S | 0 | 494 | ||||||||
| ETH (n = 521) | R | 16 | 2 |
13.0 (7.6–20.3) |
99.5 (98.2–99.9) |
88.9 (65.1–97.2) |
78.7 (77.6–79.9) |
0.18 | ||
| S | 107 | 396 | ||||||||
| RIF-resistant (n = 269) | INH (n = 266) | R | 218 | 6 |
96.0 (92.6–98.2) * |
84.6 (695–94.1) |
97.3 (94.6–98.7) |
78.6 (65.6–87.6) |
0.78 | |
| S | 9 | 33 | ||||||||
| FLQ (n = 268) | R | 76 | 0 |
95.0 (87.7–98.6) * |
100 (98.1–100) |
100 (95.3–100) |
97.9 (94.7–99.2) |
0.96 | ||
| S | 4 | 188 | ||||||||
| AMG (n = 263) | R | 6 | 1 |
85.7 (42.1–99.6) |
99.4 (96.5–100) |
85.7 (45.4–97.7) |
99.4 (96.2–100) |
0.85 | ||
| S | 1 | 255 | ||||||||
| ETH (n = 269) | R | 32 | 18 |
46.4 (34.3–58.8) * |
91.0 (86.2–94.6) |
64.0 (51.7–74.7) |
83.1 (79.7–86.0) |
0.41 | ||
| S | 37 | 182 | ||||||||
*Significant differences of proportion between the RIF-sensitive and RIF-resistant group of TB patients; RIF rifampicin, INH isoniazid, FLQ fluoroquinolones, AMG aminoglycoside, ETH ethionamide, R resistant, S sensitive, L–J Lowenstein–Jensen, PPV positive predictive value, NPV negative predictive value, CI confidence interval
Mutational Profiling of Xpert XDR Assay
The Xpert XDR assay detected INH resistance in 278 isolates, with 81.7% (227/278) showing mutations in either the katG gene alone or in combination with other mutations (Table 5). Among the mutations associated with INH resistance, the second most frequent was the inhA promoter region, observed in 25.2% (70/278) of the isolates. Double mutations were also identified, with the most prevalent combination being mutations in both the inhA promoter and the katG region, detected in 7.2% (20/278) isolates. Additionally, a few mutations were identified in the oxyR-ahpC intergenic regions and the fabG1 region. A total of 103 FLQ-resistant isolates were identified, with the majority (97.1%, 100/103) exhibiting mutations in the gyrA gene. One isolate (1.0%) had a mutation in the gyrB gene, and two isolates (1.9%) showed double mutations in both the gyrA and gyrB genes. Regarding AMG resistance, 85.7% (6/7) of the isolates had mutations in the rrs region, while 14.3% (1/7) had mutations in the eis promoter region. For ETH resistance, all 68 resistant isolates (100%) exhibited mutations in the inhA promoter region (Table 5).
Table 5.
Distribution of mutation in genes detected by Xpert MTB/XDR assay among INH, FLQ, AMG, and ETH-resistant isolates
| Drugs | Mutation detected in gene target | No. of isolates | % |
|---|---|---|---|
| INH (n = 278) | Only inhA promoter | 36 | 12.9 |
| Only katG | 169 | 60.8 | |
| Only fabG1 | 1 | 0.4 | |
| Only oxyR-ahpC intergenic region | 2 | 0.7 | |
| inhA promoter and katG | 20 | 7.2 | |
| inhA promoter and fabG1 | 3 | 1.1 | |
| inhA promoter and oxyR-ahpC intergenic region | 8 | 2.9 | |
| katG and oxyR-ahpC intergenic region | 35 | 12.6 | |
| katG and fabG1 | 1 | 0.4 | |
| inhA promoter, fabG1 and oxyR-ahpC intergenic region | 1 | 0.4 | |
| inhA promoter, katG and oxyR-ahpC intergenic region | 2 | 0.7 | |
| FLQ (n = 103) | Only gyrA | 100 | 97.1 |
| Only gyrB | 1 | 1.0 | |
| Both gyrA and gyrB | 2 | 1.9 | |
| AMG (n = 7) | rrs | 6 | 85.7 |
| eis promoter | 1 | 14.3 | |
| ETH (n = 68) | inhA promoter | 68 | 100.0 |
INH isoniazid, FLQ fluoroquinolones, AMG aminoglycoside, ETH ethionamide
Discussion
Rapid transmission and increasing levels of drug resistance render TB one of the biggest challenges in global public health today. In high TB and MDR-TB burden countries such as Bangladesh, prompt and reliable detection of resistance to second-line anti-TB drugs as well as INH is essential, ensuring timely and appropriate treatment for patients. In this study, we evaluated the performance of the Xpert XDR assay in detecting resistance to INH, FLQ, AMG, and ETH among pulmonary TB patients in Bangladesh.
In our study, the Xpert MTB/XDR assay demonstrated good sensitivity for detecting resistance to INH (94.0%), FLQ (86.0%), and AMG (85.7%), but poor sensitivity for ETH (25.0%) when compared to the L–J proportion method. Specificity was nearly perfect across all drugs, ranging from 96.7 to 99.9%. Our findings on INH resistance align with previous studies, which reported sensitivities of 86.7 to 98.3% and specificities of 88 to 100% across regions such as India, China, USA, and several African countries including Uganda, Moldova, and South Africa [10–13]. For FLQ, the assay showed relatively lower sensitivity but maintained high specificity, consistent with earlier studies reporting sensitivities of 80 to 95% and near-perfect specificities [10–13]. The performance for AMG resistance was also in line with studies from China, South Africa, and India, which reported sensitivities of 85 to 90% and similar specificities [13, 14]. While some studies have found higher (> 90%) [12] or lower (70%) [15] sensitivities for AMG, others, such as one from Uganda, could not assess AMG sensitivity due to the absence of resistant isolates [11]. For ETH resistance, the assay performed poorly in our study, with sensitivity of 25.0%. Previous studies also reported low sensitivity (50–65%), though still higher than ours. However, the specificity for ETH in our study (around 97%) was consistent with findings from India, China, and African countries [10, 12, 13, 15, 16].
The mutational profiles detected by the Xpert XDR assay for each drug were analyzed. For INH resistance, the assay targets the inhA promoter, katG, fabG1, and the oxyR-ahpC intergenic region [8, 9]. Among INH-resistant isolates, around 82% had katG mutations and 25% had inhA promoter mutations, consistent with data from Bangladesh and global studies reported previously [22]. However, rare mutations in genes such as ndh, nat, mshA, kasA, and others, not targeted by the assay, may also contribute to INH resistance [23–25]. This explains why 17 isolates were classified as INH-sensitive by Xpert XDR assay but resistant by pDST. Additionally, about 30% of INH-resistant cases globally lack identifiable genetic mutations, suggesting a potential role of non-genetic factors in the resistance mechanism [26].
FLQ resistance is primarily associated with mutations in gyrA and gyrB, both targeted by the Xpert assay [8]. In our study, 97.1% of FLQ-resistant isolates had gyrA mutations, with fewer involving gyrB. This aligns with previous findings linking gyrA mutations to high resistance levels, while gyrB mutations are less frequent and associated with low resistance [27, 28]. However, FLQ resistance without gyrA or gyrB mutations has been attributed to mechanisms like efflux pumps, with Rv1634 and PstB potentially implicated [29, 30]. This may explain why 16 isolates classified as FLQ-sensitive by XDR assay but resistant by pDST in our study. Additionally, heteroresistance with < 10% resistant subpopulations can evade detection by molecular tests, leading to false sensitive results [3].
For AMG, the Xpert XDR assay targets the rrs gene and eis promoter [8]. Among AMG-resistant isolates, around 86% had rrs mutations, consistent with its role as a key resistance site [31]. The low prevalence of eis mutations reflects trends previously found in Bangladesh, where rrs mutations predominated in KAN resistance [32].
The Xpert XDR assay targets only the inhA promoter to determine ETH resistance. Consequently, all ETH-resistant isolates in our study had mutations in this region. While inhA promoter mutations are common, other genes like ethA, ethR, ndh, and mshA also contribute to ETH resistance [33]. This narrow focus likely explains the assay’s low sensitivity, as it may miss other resistance mechanisms. Furthermore, the assay uses the same inhA region to assess both INH and ETH resistance, leading to detection of ETH-resistant isolates only in INH-resistant cases [17]. None of the ETH-resistant isolates in our study were INH-sensitive, consistent with findings from Truden et al., where all 14 ETH-resistant isolates were also INH-resistant [15]. This highlights a significant limitation, as the assay may fail to detect ETH resistance in INH-sensitive TB cases. The lower sensitivity of the Xpert XDR assay for ETH observed in our study raises concerns about its reliability for guiding physicians’ treatment decisions. Previous studies have shown that pDST is unreliable for detecting ETH resistance, making it an unsuitable benchmark for evaluating the Xpert XDR assay’s performance [34]. In the absence of other rapid molecular tools, sequencing remains the most accurate method for detecting ETH resistance and can help physicians make more informed treatment decisions [12].
We also assessed the performance of the Xpert XDR assay among various patient groups, including new and re-treated TB patients, as well as RIF-sensitive and RIF-resistant TB patients. The assay demonstrated significantly higher sensitivity for detection of resistance to INH, FLQ, and ETH in re-treated or RIF-resistant TB patients compared to newly diagnosed or RIF-sensitive TB patients. Sensitivity for AMG resistance could not be assessed in newly diagnosed or RIF-sensitive patients due to the absence of resistant cases in these groups. The reasons for the assay’s higher sensitivity in re-treated or RIF-resistant patients remain unclear and warrant further investigation. Globally, and in Bangladesh, drug-resistant TB and multidrug-resistant TB (MDR-TB) are more commonly observed among re-treated patients [1, 35]. Given these findings, the Xpert XDR assay offers significant value for the rapid detection of drug resistance, particularly in patients with treatment failure, relapse, or RIF-resistant TB. Its use would enable clinicians to promptly initiate appropriate anti-TB regimens, improving outcomes and curbing the spread of drug-resistant TB.
In Bangladesh, routine testing of drug susceptibility is often not recommended in programmatic settings due to resource limitations and the nationwide scarcity of pDST facilities. This poses significant challenges in the timely detection of DR-TB. In 2023, the estimated number of patients with MDR/RR-TB in the country was approximately 5000. However, only 54% of these patients underwent additional testing for FLQ resistance [2]. As a result, nearly half of the RIF-resistant cases remained unaware of their FLQ resistance status, hindering proper classification as pre-XDR-TB and preventing these patients from receiving the appropriate treatment regimen. Moreover, a recent study in Bangladesh revealed that the prevalence of RIF-sensitive but INH-resistant TB (Hr-TB) was approximately 4.5%, more than double the prevalence of MDR-TB [36]. Unfortunately, Hr-TB frequently goes undetected in routine diagnostic settings, leaving many patients without access to the WHO-recommended treatment regimen for this form of TB.
To address these diagnostic gaps, WHO has recently recommended using the Xpert XDR cartridge as a reflex test to complement existing technologies, such as the Xpert or Xpert Ultra assays [37]. Recently, National Tuberculosis Control Programme (NTP) has initiated the programmatic use of the Xpert XDR assay to test for resistance to second-line drugs, but limited only to patients with RIF-resistant TB. Based on our study’s superior performance of the Xpert XDR assay among re-treated TB patients, we recommend that the NTP expand its use beyond RIF-resistant cases to include all previously treated TB patients. This approach would facilitate the accurate identification of resistance to second-line drugs and Hr-TB, ensuring timely and appropriate treatment for all affected individuals.
The Xpert MTB/XDR assay is a more cost-effective alternative to other drug susceptibility testing methods. Currently, the cost per XDR cartridge is approximately $14.80, a reduction from its previous price of $19.80 [38]. In contrast, other methods such as Line Probe Assay (LPA, Hain Lifescience GmbH, Germany), liquid culture-based pDST (BACTEC MGIT 960 system, BD Bioscience, USA), and targeted next-generation sequencing are considerably more expensive [39]. Additionally, these tests require specialized laboratory infrastructure, trained personnel, and longer turnaround times, making them less feasible than Xpert XDR assay for routine use in low-resource settings.
Our study had a few limitations. First, there were a limited number of AMG-resistant isolates, which may not fully represent the Xpert XDR assay’s sensitivity for detecting AMG resistance. Second, we did not conduct additional sequencing of isolates with discordant results between the Xpert XDR assay and pDST. Additionally, indeterminate results from the Xpert XDR assay that were classified as either sensitive or resistant by pDST were not further investigated. This limitation leaves gaps in our understanding of the genetic mechanisms underlying resistance in these cases. A more detailed investigation using whole-genome sequencing or targeted sequencing of these discordant isolates could help identify novel gene targets or mutations not currently detected by the Xpert XDR assay. Such research would provide valuable insights into additional resistance mechanisms and aid manufacturers in potential assay improvements.
Conclusions
In conclusion, the Xpert XDR assay demonstrated high performance in detecting resistance to INH, FLQ, and AMG in this study, though its accuracy for ETH was suboptimal. Notably, the assay exhibited significantly higher accuracy among re-treated or RIF-resistant TB patients compared to newly diagnosed or RIF-sensitive cases. With its rapid turnaround time, user-friendly operation, and reliable detection of resistance to INH, FLQ, and AMG, the Xpert XDR assay can be implemented programmatically nationwide. This would enable the timely identification of resistance to key drugs, empowering clinicians to select appropriate treatment regimens and improving outcomes of TB patients.
Supplementary Information
Below is the link to the electronic supplementary material.
Author Contributions
Conceptualization: S. M. Mazidur Rahman, Sayera Banu; Methodology: S. M. Mazidur Rahman, Noshin Nawer Ruhee, Amiyo Haider, Md Jahid Hasan, Rumana Nasrin; Data analysis and interpretation: S. M. Mazidur Rahman, Noshin Nawer Ruhee, Amiyo Haider; Investigation: S. M. Mazidur Rahman, Sayera Banu, Noshin Nawer Ruhee, Amiyo Haider, Shahriar Ahmed, Aung Kya Jai Maug; Original draft preparation: S. M. Mazidur Rahman; Writing, review and editing: Noshin Nawer Ruhee, Amiyo Haider, Rumana Nasrin, Ahammad Shafiq Sikder Adel, Mohammad Khaja Mafij Uddin, Shahriar Ahmed, Aung Kya Jai Maug, and Sayera Banu; Supervision: S. M. Mazidur Rahman, Sayera Banu; Resources: Sayera Banu. All authors have read and agreed to the published version of the manuscript.
Funding
This research was produced with the support of the United States Agency for International Development (USAID) under the terms of USAID’s Alliance for Combating TB in Bangladesh activity. Views expressed herein do not necessarily reflect the views of the U.S. Government or USAID. icddr,b acknowledges with gratitude the commitment of USAID to its research efforts. icddr,b is also grateful to the Governments of Bangladesh and Canada for providing core/unrestricted support. The Rapid Service Fee was funded by the authors.
Data Availability
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Declarations
Conflict of interest
S. M. Mazidur Rahman, Noshin Nawer Ruhee, Amiyo Haider, Md Jahid Hasan, Rumana Nasrin, Ahammad Shafiq Sikder Adel, Mohammad Khaja Mafij Uddin, Shahriar Ahmed, Aung Kya Jai Maug, and Sayera Banu declare that they have no competing interests.
Ethical Approval
The present study was approved by the Institutional Review Board of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) (Protocol number: PR-21008 and PR-22047), and was performed in accordance with the Helsinki Declaration of 1964 and its later amendments. Informed written consents/assents were obtained from participants prior to enrolling in the study.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.