Diagnostic accuracy of LiquidArray MTB-XDR VER1.0 for the detection of Mycobacterium tuberculosis complex, fluoroquinolone, amikacin, ethambutol, and linezolid susceptibility

Drug susceptibility testing (DST) is essential for effectively starting people on effective tuberculosis (TB) regimens. No accuracy data exists for the new high-throughput LiquidArray MTB-XDR (LA-XDR) test, which detects Mycobacterium tuberculosis complex (MTBC) and susceptibility to the fluoroquinolones, amikacin, ethambutol, and linezolid (the latter two drugs have no rapid molecular DSTs available). We enrolled (n=720) people with presumptive TB who provided two sputa for Xpert MTB/RIF Ultra and culture (MTBC reference standard). Phenotypic DST and Sanger sequencing served as a composite reference standard. Manual FluoroLyse and automated GenoXtract-fleXT (fleXT) DNA extraction methods were compared. For MTBC, LA-XDR using fleXT-extracted or FluoroLyse-extracted DNA had similar sensitivities (85–87%; which improved upon eluate retesting) and specificities (99%). Drug susceptibility sensitivities varied: 94% (86, 98) for fluoroquinolones, 64% (45, 80) for amikacin, and 88% (79, 93) for ethambutol (specificities 97–100%). LA-XDR detected 86% (6/7) phenotypically resistant linezolid isolates. LA-XDR with fleXT had indeterminate proportions of 8% (21/251) for fluoroquinolones, 1% (2/251) for ethambutol, 25% (63/251) for amikacin, and 37% (93/251) for linezolid. In a hypothetical population of 100 smear-negative fluoroquinolones-resistant cases, 24% (24/100) could be missed due to an unsuccessful result (1 fleXT error and, for LA-XDR, 2 invalid results, 15 MTBC-negative, 6 fluoroquinolone-indeterminate, 1 false-susceptible). LA-XDR met the minimum WHO target product profile for a next-generation sputum-based moderate complexity DST with high sensitivity for fluoroquinolones and ethambutol resistance, moderate sensitivity for amikacin resistance, and promise for linezolid resistance, for which more data are needed. Improved MTBC detection would reduce missed resistance.

In a hypothetical population of 100 smear-negative fluoroquinolonesresistant cases, 24% (24/100) could be missed due to an unsuccessful result (1 fleXT error and, for LA-XDR, 2 invalid results, 15 MTBC-negative, 6 fluoroquinolone-indeterminate, 1 false-susceptible).LA-XDR met the minimum WHO target product profile for a next-generation sputum-based moderate complexity DST with high sensitivity for fluoroquinolones and

Introduction
Tuberculosis (TB) remains a devastating global health emergency, with an estimated 7.5 million new cases and 1.3 million deaths in 2022, and an estimated 410,000 multidrug-resistant (MDR) TB cases were reported 1 .The COVID-19 pandemic exacerbated TB management leading to underdiagnosis 1 .Between 2019 and 2022, there was a 15% decrease in drug-resistant (DR)-TB cases potentially obscured by COVID-19 2,3 .In 2022, the World Health Organization (WHO) endorsed a 6-month treatment regimen that incorporates fluoroquinolone (FQ) and linezolid (LZD) for MDR/RR-TB, replacing the longer 9-month or 18-month options 4 .
Current rapid molecular WHO-approved assays face limitations in detecting ethambutol (EMB) resistance, newer and repurposed drugs such as LZD resistance as the two can only be targeted by sequencing or phenotypic drug susceptibility testing (pDST) 5,6 .LiquidArray MTB-XDR VER1.0 (LA-XDR) assay (Bruker/Hain Lifescience GmbH, Nehren, Germany) is a highthroughput (24-94 tests within 3-5 hours) centralised DST.It is a successor to the MTBDRsl line probe assay 7 , integrates within laboratory information systems facilitating rapid results dissemination and, by minimising handson manipulation 8 (only required during initial instrument setup and PCR plate transfer), reduces contamination risk 9 .
LA-XDR utilises the same DNA extraction platform as the WHOrecommended moderate complexity FluoroType MTBDR Ver2.0 10,11 , detects Mycobacterium tuberculosis complex (MTBC), and mutations conferring resistance to FQ in gyrA and gyrB, amikacin (AMK) in rrs, EMB in embB, and LZD in rplC, and rrl genes in both sputum and isolates 5,[12][13][14] .It's rapid DST, including for drugs for which there are no rapid tests like LZD and EMB offering a crucial component for effective TB management.
Undiagnosed resistance enhances the risk of selection of untreatable strains 13 , in other words, especially as rapid tests for new drugs do not yet exist, one of the best ways to protect them is to maximise the number of effective drugs (for example, fluoroquinolone resistance often precedes bedaquiline resistance 15 ).This is the first evaluation of LA-XDR diagnostic performance in MTBC and resistance detection using two distinct DNA extraction methods, the manual FluoroLyse and automated GenoXtract fleXT (fleXT).

Study Samples group.
Group A samples consisted of 87% (572/659) of NHLS samples from individuals with presumptive TB following Xpert MTB/RIF Ultra (Ultra) testing irrespective of smear status (Supplementary Figure 1).13% (87/659) of samples from the NHLS were delegated to Group B were Ultra rifampicin-resistant with either FQ-resistance or AMK-resistance confirmed by either Genotype MTBDRsl (MTBDRsl) or pDST at Stellenbosch University (Supplementary Methods).Group C samples (Supplementary Figure 2) all from FIND consisted of 8% (61/720) of sputa and corresponding isolates with specific resistance confirmed by pDST or whole genome sequencing.Seven LZD phenotypic-resistant isolates from the Stellenbosch University biobank were included.

FluoroLyse and GenoXtract fleXT DNA extraction.
DNA was extracted from sputa sediments and isolates using both the manual FluoroLyse and automated fleXT platform with GenoXtract X2 Extraction VER1.0 (Bruker/Hain Lifescience GmbH, Nehren, Germany) according to the manufacturer's instructions 17 .A sample consisting of saline was included as a negative control, as well as the provided positive control.At the end of extraction, fleXT reports if the processing was successful or not (reported as an error with code).

LiquidArray MTB-XDR testing.
MTBC and drug susceptibility detection was done using the FluoroCycler XT-96 (Bruker/Hain Lifescience GmbH, Nehren, Germany) instrument.PCRs were set up by the fleXT instrument using DNA extracted using fleXT.A manual PCR reaction was set up with 20 µl of FluoroLyse-extracted DNA added to a pre-mix of 14 µl AM-B and 6 µl AM-A solution per sample.
Automated results interpretation was done by the FluoroSoftware XT-IVD (Supplementary Figure 3).The LA-XDR test results can be categorised into successful (MTBC detected with DST-determinate; MTBC not detected), MTBC detected with DST-indeterminate, invalid and error (applicable to samples extracted with fleXT only).

MGIT960 and phenotypic DST
pDST was done on Group A isolates if the index assay identified resistance.
Group B underwent pDST for all drugs for LA-XDR detects resistance, while (Supplementary Methods) Group C only had LZD pDST done (samples were already characterised for FQ, AMK and EMB resistance by either WGS or pDST).pDST used the following WHO critical concentrations of kanamycin 5µg/ml, EMB 5µg/ml, ofloxacin 2µg/ml, moxifloxacin 2µg/ml, AMK 5µg/ml, and LZD 1µg/ml 18 .Negative control was done using the H37Rv (NC_000962) pan-susceptible MTBC strain.

Discrepant result analysis.
Repeat testing was done with remnant DNA to resolve result discrepancies, including false-negatives and false-positives detected by LA-XDR using FluoroLyse-or fleXT-extracted DNA.The repeat results were not included in the main analyses but are shown in Supplementary Tables 2-3.

Data Analysis.
Statistical analysis, conducted with Stata 18.0 (StataCorp LP, Texas, USA), involved a two-sample test of proportions.Primary analyses assessed sensitivity and specificity for MTBC using a culture reference standard in Group A and B samples.To assess differences in sensitivity and specificity for the two DNA extraction methods, a McNemar test for paired data was done.P-values ≤0.05 were considered significant.

Ethics statement.
This study was done according to relevant guidelines and regulations, approved by the Stellenbosch University Health Research Ethics Committee (N16/04/045 and N09/11/296) and Western Cape Health Research Committee (2016RP18637).Permission was granted to access anonymised to-be-discarded residual samples collected in routine diagnostic practice with waived informed consent.

Error, invalid, and indeterminate results from fleXT and LA-XDR
DNA extraction: Four people did not have sputum extracted using FluoroLyse and 16 people did not have sputa extracted using fleXT.fleXT had an error proportion of 4.5% (30/668) for sputum compared to 2.2% (7/322; p=0.045) for isolates (Supplementary Tables 4).
Linezolid: The assay exhibited a specificity of 100% (95% CI: 97, 100) for both methods.However, sensitivity could not be precisely assessed due to few resistant cases.LA-XDR detected 86% (6/7) of phenotypic-resistant isolates, with one being susceptible.All isolates with resistance detected by LA-XDR had the C-154-R rplC resistance-associated variant 22 .

Discussion
Our data shows that using sputum, LA-XDR has: 1) high sensitivity for MTBC detection using either DNA method, 2) comparable FQ and AMK sensitivity and specificity to MTBDRsl, 3) high sensitivity and specificity for EMB resistance and 4) potential as a rule-in test for LZD resistance detection, particularly in where the rplC C-154-R variant is most frequent.Additionally, LA-XDR has relatively low indeterminate rates, however, due to suboptimal sensitivity for MTBC (~80% in smear-negatives), at least 20% of people will not have a susceptibility result generated.

LA-XDR sensitivity for MTBC (87%) is comparable to that of other WHOapproved high throughput, moderate complexity NAAT platforms (Abbott RealTime MTB-RIF/INH, Roche COBAS MTB-RIF/INH, BD MAX MDR-TB and
FluoroType MTBDR) 9,23 .As expected, sensitivity in smear-negative specimens was diminished (80% vs. 96% for smear-positives), meaning that 20% (28/137) of smear-negative TB cases will be missed (and therefore, due to non-detection of MTBC DNA by LA-XDR, not have resistance or susceptible results generated, potentially resulting in missed resistance).Our results indicate that retesting smear-negative samples almost half the time leads to successful MTBC detection, which is valuable information for refining diagnostic algorithms.Additionally, if LA-XDR is employed as a reflex test following an initial positive TB screening, we recommend repeating the test using the remaining DNA extract.
Sputum FQ and AMK susceptibility sensitivity and specificity were comparable to those for MTBDRsl and like those for Xpert MTB/XDR 6,24,25 .
FQ sensitivity (94%, independent of DNA extraction method) met the WHO target product profile (TPP) of >90% sensitivity 26 .LA-XDR is promising in detecting EMB resistance, with sensitivity and specificity estimates of 88% and 98%, respectively.This is important because EMB is included in the management of MDR-TB in most clinical settings.The LA-XDR is the only option for programmes to do EMB DST and will fill the existing diagnostic gap as there is a high EMB resistance prevalence among MDR-TB 27 .
LZD is a key WHO group A drug regimen, LA-XDR has 100% specificity which makes it a valuable tool for ruling out LZD resistance.The test accurately detected the rplC mutation 5,28 in phenotypic-resistant isolates and detected the most resistant specimens, demonstrating its potential for LZD susceptibility testing, especially in the context of no other commercially available molecular tools for susceptibility testing for this drug.On sputa samples, more than half (141/227) had indeterminate LZD results, possibly due to a high detection call threshold or unclear variant significance.
We demonstrated low AMK sensitivity estimates (64%), likely due to selection bias in FIND Biobank samples from Eastern Europe with the prevalent C-14-T eis promoter variant 22 undetected by LA-XDR.WHO classifies the C-14-T eis promoter mutation as provisionally associated with resistance, it can only confer resistance if linked to a functional eis 22 A substantial proportion (77/106) of indeterminate AMK results (MTBCpositive) were from smear-negative sputa samples, likely due to low bacillary load or difference detection thresholds.
In summary, our data shows that, while LA-XDR is adept at detecting and ruling in resistance, its potential impact is, like many molecular DSTs, undermined by its ability to detect TB (required to generate a DST result) (Supplementary Table 6).For example, in a hypothetical population of 100 smear-negative cases with FQ resistance, 24% (24/100) will be missed.
Our study had limitations, even though we had a large sample size, our cohort had no clinical specimens with LZD resistance, but we included phenotypic-resistant isolates detected by LA-XDR.Using Ultra test results for selection of Group A and B samples introduces variability, and likely could overestimate MTBC sensitivity, but reflects real-world scenarios where LA-XDR would likely be used in many settings.Lastly, we did not evaluate the utility of repeating LA-XDR on sputum, except for falsenegative results.
In conclusion, the LA-XDR meets the criteria WHO TPP for a sputum-based next-generation WHO moderate complexity NAAT technology.The LA-XDR can be a valuable tool for MTBC detection and drug susceptibility profiling, especially for LZD and EMB resistance, for which no other commercial molecular DSTs are presently available.Future work should focus on improving the detection of smear-negative TB cases, ensuring that this technology can be effectively utilised in a broader range of clinical scenarios.ɫ Within-row comparisons for Sputum vs Isolate.
# No LZD resistance samples were evaluated hence sensitivity is incalculable.

Figure 2 .
Figure 2. Forest plots showing diagnostic accuracy estimates

Figure 2 Forest
Figure 2

Tables 452 Table 1 :
Participant demographic and clinical characteristics.Most participants 453were men, HIV-negative and culture-negative.Data are % (n/N) or median (IQR).

Table 2 .
Diagnostic accuracy of LiquidArray MTB-XDR for detecting MTBC from smear-positive and smear-negative sputum samples by DNA extraction method.The sensitivity and specificity are higher in smear-positive than that of smearnegative regardless of DNA extraction methods employed.Data are % (95% CI) and n/N.

Table 3 .
Diagnostic accuracy of LiquidArray MTB-XDR for the detection of drug resistance to FQ, AMK, EMB, and LZD compared to composite reference standard by sample type and DNA extraction method.The sensitivity and specificity are higher in isolates than that of sputum regardless of DNA extraction methods employed.The FluoroLyse DNA extraction method is comparable to the GenoXtract fleXT except for the amikacin and ethambutol.