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Cartridge extract extraction procedure. (a) The arrow indicates the diamond-shaped reaction chamber where the PCR amplification takes place and contains cartridge extract with mycobacterial genomic DNA. The needle is placed at the top of the diamond and the film is slowly and carefully pierced. (b) The needle is then slowly inserted deeper into the pocket and cartridge extract mix drawn out without piercing the other side.
Source publication
Xpert MTB/RIF (Xpert) is a widely-used test for tuberculosis (TB) and rifampicin-resistance. Second-line drug susceptibility testing (DST), which is recommended by policymakers, typically requires additional specimen collection that delays effective treatment initiation. We examined whether cartridge extract (CE) from used Xpert TB-positive cartrid...
Context in source publication
Context 1
... of mycobacterial genomic DNA from used Xpert MTB/RIF cartridges. The transparent diamond-shaped reaction chamber on the back of the cartridge was punctured with a sterile fixed-needle insulin syringe (1 ml; 29 G) ( Fig. 1) in a biosafety level 2 cabinet. The full CE volume, typically ~15 µl, was withdrawn and stored in sterile, safe-lock micro-centrifuge tubes at −20 °C prior to analysis. Each cartridge and the surrounding sur- face was wiped down thoroughly with 1% sodium hypochlorite and 70% EtOH before and after extraction and UV sterilization was ...
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Citations
... It is a cartridge based nuleic acid amplification test which is automated and can isolate the genomic material of MTB via sonication, amplify via PCR and identify using fluorescent probes called molecular beacons (Boehme, 2010). Xpert MTB/RIF (cephad), a computerized molecular test for the detection of MTB and RIF resistance directly from clinical specimens is one of the most widely used molecular tests for the diagnosis of TB globally (Huang et al., 2022) In this test specific sequence of the rpoB gene is amplified with no cross contamination issues (Venter et al., 2017). In 2017 WHO approved XPERT MTB/ultra as the initial TB diagnostic test for adults and children, regardless of HIV status (WHO, 2017). ...
Rifampicin resistance in Mycobacterium tuberculosis is a public health concern in many countries including Nigeria. There is an increase of multidrug resistant tuberculosis which results to morbidity and mortality among presumptive tuberculosis patients. Data regarding Rifampicin resistant tuberculosis in the study area are lacking. A one year prospective, cross-sectional, laboratory based study was carried out among patients attending Direct Observed Treatment Short course in Damaturu, Gashua and potiskum specialist hospitals, Yobe State, Nigeria; from January, 2020 to December, 2020. Sputum samples were collected from consented / assented participants and analyzed using MTB/RIF assay (Cepheid, GeneXpert USA). The data were analyzed using Person Chi-Square and p≤0.05 considered the presence of significant relationship and results were presented in tables and charts. Out of 400 studied participants, males had a prevalence of 2.5% (10/400) while females had 0.8% (3/400). Age group 30-39 years had the highest prevalence of 1.5%. The results show significant relationship between age, gender, and marital status, in the study area. This study confirmed the presence of Rifampicin resistant tuberculosis in the study area. Therefore, there’s need to have a public health awareness and strengthen the laboratory capacity for diagnosis and make the services available and accessible to the patients who need them.
... It is based on a hemi-nested real-time polymerasechain-reaction (PCR) assay to amplify an Mtb-specific sequence of the rpoB gene. The turnaround time of this assay is short (2-3 h), and the problem of cross-contamination is eliminated because of self-contained cartridges [30]. It uses three specific primers and five unique molecular probes to ensure a high degree of specificity, and NTM does not confound testing [31]. ...
Diagnosis of tuberculosis, and especially the diagnosis of extrapulmonary tuberculosis, still faces challenges in clinical practice. There are several reasons for this. Methods based on the detection of Mycobacterium tuberculosis (Mtb) are insufficiently sensitive, methods based on the detection of Mtb-specific immune responses cannot always differentiate active disease from latent infection, and some of the serological markers of infection with Mtb are insufficiently specific to differentiate tuberculosis from other inflammatory diseases. New tools based on technologies such as flow cytometry, mass spectrometry, high-throughput sequencing, and artificial intelligence have the potential to solve this dilemma. The aim of this review was to provide an updated overview of current efforts to optimize classical diagnostic methods, as well as new molecular and other methodologies, for accurate diagnosis of patients with Mtb infection.
... 19 In that study, the sensitivity and LPA specificity in smear-negative specimens were respectively 83.3% and 97.9% for detecting INH resistance and 100%, 98%, respectively, for detecting RIF resistance only if the TB PCR results were positive. For the purpose of exploring the feasibility of using direct LPA beyond the current guidelines, we used DNA specimens rather than smear-negative specimens and found that 23 MTBDRplus testing was not feasible with Xpert cartridge extracts due to a large number of rpoB amplicons which could bind to the MTBDRplus probes. However, as rpoB amplicons did not bind to the MTBDRsl probe, MTBDRsl testing was feasible in specimens with an adequate bacillary load (Ct 24). ...
... If the result of PCR test is positive with an adequate Ct value (,25 in our study) the stored sample can be used for LPA, which would enable rapid detection of drug resistance without the additional submission of sputum samples. However, unlike the method using Xpert cartridge remnants, 23 this method requires an additional step of extracting and purifying DNA, which might be impractical in many high-burden settings. ...
BACKGROUND: We investigated the feasibility of early line-probe assay (LPA) using remnant DNA of Mycobacterium tuberculosis from polymerase chain reaction (PCR) test.METHODS: M. tuberculosis DNA specimens with cycle threshold (Ct) values reported and collected from patients with known results for both LPA with culture isolates and phenotype drug susceptibility testing (pDST) were selected. The diagnostic performance of DNA-based LPA according to the Ct value was investigated.RESULTS: A total of 143 respiratory specimens were included. For isoniazid resistance, the accuracy in subgroups with Ct value <25, 25-29 and ≥29 was respectively 96.8%, 65.7% and 13.3%. For rifampicin resistance, accuracy in subgroups with Ct values <29 and ≥29 was respectively 87.8% and 13.3%. When compared to the pDST results, sensitivity, specificity, positive predictive value and negative predictive value in specimens with Ct values <25 was respectively 1.00 (95% CI 0.69-1.00), 0.95 (95% CI 0.76-1.00), 0.91 (95% CI 0.59-1.00) and 1.00 (95% CI 0.83-1.00) for isoniazid resistance. For rifampicin resistance, corresponding values in subgroups with Ct values <29 were respectively 0.89 (95% CI 0.52-1.00), 0.98 (95% CI 0.91-1.00), 0.80 (95% CI 0.50-0.94) and 0.99 (95% CI 0.92-1.00).CONCLUSIONS: DNA-based early LPA with remnant DNA from respiratory samples was feasible and accurate when the Ct values were low.
... The standard N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method is relatively harsh and may reduce the viability of Mtb bacilli by up to 80% [10]. The sputum processing reagent used for the GeneXpert® assays (Xpert MTB/RIF, Xpert MTB/RIF Ultra, and Xpert MTB/XDR) liquefies and inactivates Mtb in raw sputum specimens but does not produce a DNA extract that can be used for WGS [11]. Other sputum processing solutions utilized by automated NAATs do not deplete contaminants and the resulting extract is therefore not suitable for efficient Mtb WGS [2]. ...
Whole genome sequencing (WGS) can investigate the entire Mycobacterium tuberculosis (Mtb) genome but currently requires large amounts of mycobacterial DNA, necessitating culture. Culture-free Mtb WGS could revolutionize the clinical use of WGS but is hampered by the high viscosity, low mycobacterial load, and high contamination with bacterial and human DNA in sputum samples. To improve the sputum liquefaction and decontamination step prior to DNA extraction, we assessed the efficiency of Myco-TB, MycoPrep, and Sputolysin with/without TiKa-Kic in liquefying and decontaminating sputum and aimed to evaluate the effect of these approaches on mycobacterial viability, and Mtb DNA quality and quantity. Experiments using spiked sputum samples showed that Myco-TB and BD MycoPrep with standard (15 min) or increased (30 min) incubation time, but not reduced (7,5 min) incubation time performed well in liquefying and decontaminating sputum. No difference in DNA quality or quantity, contamination, or the amount of human DNA present was observed. In comparison, Sputolysin with/without TiKa-Kic was less effective for liquefaction and decontamination of sputum. PCR amplification of the human GAPDH gene after sputum treatment, showed the presence of human DNA in all samples, regardless of sputum treatment. Focused efforts are needed to deplete contaminating DNA for culture-free Mtb WGS.
... 8,28,29 Systematically collecting two specimens at first visit (one for Xpert and one for smear/LPA/culture/DST when RR-TB is detected), 30 or use of the Xpert cartridge remnant for GenoType MTBDRsl (Hain Lifescience) are both structural approaches to reduce the need for collection of an additional specimen. 31 Strengths of the study include the pragmatic setting, the representative sample of RR-TB patients from three provinces of South Africa, and the prospective assessment of the RR-TB care cascade from diagnosis throughout the entire treatment episode. Furthermore, we determined the WHO minimum indicators for monitoring DR-TB programmes, an exercise for which we could not find a precedent. ...
BACKGROUND: Xpert® MTB/RIF was expected to revolutionise the management of rifampicin‐resistant TB (RR‐TB) by enabling rapid and decentralised diagnosis of rifampicin (RIF) resistance.
METHODS: We performed a care cascade analysis for a cohort of RR‐TB patients managed under programmatic conditions. Cumulative incidences of time to completion of the RR‐TB care cascade steps were estimated, reasons for delay or attrition from the cascade investigated and WHO programme indicators for monitoring of RR‐TB programmes calculated.
RESULTS: Of 502 patients diagnosed with RR‐TB using Xpert, 64% initiated multidrug‐resistant TB (MDR‐TB) treatment immediately, 20% after some first‐line treatment, 16% never initiated MDR‐TB treatment, mainly because of death (44%) or loss to follow‐up (26%) soon after diagnosis. A supplementary sputum sample was collected within 14 days of treatment in 58.8% of cases. Only 63% of RR‐TB cases were assessed for isoniazid resistance, and only 65% of MDR‐TB cases were evaluated for pre‐XDR‐TB (extensively drug‐resistant TB). Treatment was individualised in 57% of pre‐XDR and 68% of XDR‐TB patients. Only 8% completed the entire RR‐TB care cascade as intended.
CONCLUSION: Fidelity to the RR‐TB algorithm was poor, with substantial losses at each step of the cascade, highlighting the fact that implementation of novel technologies needs to be accompanied by health system strengthening to maximise impact.
... While we used the remnants of sputa processed for Xpert, others studies have extracted the TB DNA from used Xpert MTB/RIF [20] and Xpert Ultra cartridges [21] for accurate second-line genotypic drug susceptibility testing and genotyping, with minimal rpoB-amplicon cross-contamination [21]. This approach could likely isolate cleaner DNA than is possible to obtain directly from the inactivated sputum, and thus could improve the sensitivity of subsequent tests and perhaps reduce the number of ambiguous results. ...
Background
The incidence of tuberculosis (TB) remains high in many countries, including some middle- and high-income countries without financial constraints for diagnosis and treatment. The implementation of an improved algorithm for diagnosis using 2 rapid molecular tests should help reduce the TB burden.
Material/Methods
Between April 2018 and March 2019, sputum samples from 711 patients suspected of TB in Nanshan, Shenzhen, China, were included in this prospective study. All sputum samples were examined by smear microscopy, Mycobacterium Growth Indicator Tube (MGIT) 960 culture, and Xpert MTB/RIF. The sputum remnants of Xpert MTB/RIF were used for MTBDRplus to confirm the Xpert results both for the presence of TB bacilli and for resistance to rifampicin (RIF), and also to diagnose multidrug-resistant tuberculosis (MDR-TB).
Results
In total, 200 (28.1%) of the 711 sputa were positive for TB by Xpert MTB/RIF, and the sputum remnants were used for MTBDRplus. The simultaneous use of Xpert MTB/RIF and MTBDRplus directly on sputum samples permitted accurate bacteriologic confirmation of TB in 64% (119/187) of cases and detection of 70% (7/10) of strains that were MDR.
Conclusions
The implementation of 2 rapid nucleic acid-based tests on sputum samples could facilitate the prompt and appropriate treatment of most TB cases.
... spoligotyping 6 , a method useful for monitoring the molecular epidemiology of TB outbreaks. This additional testing does not require extra specimen collection nor additional downstream DNA extraction, both of which can exacerbate patient loss within the diagnostic care cascade. ...
... Crude DNA (heat inactivated for Ultra on sputum from TB patients. Forty used positive Ultra cartridges done on NALC-NaOH decontaminated sputa from pre-treatment TB patients with known drug resistance [5 rifampicin-mono-resistant, 15 MDR, 10 pre-XDR (resistance to rifampicin, isoniazid and either a fluoroquinolones or a second-line injectable), 10 XDR] were collected from November 2015 to September 2017 and dCEs were extracted as described previously 6 (Fig. 1B). To confirm MTBDRsl results from dCEs, MTBDRsl was done per the manufacturer's instructions directly on corresponding decontaminated sputa 18,19 . ...
... Diamond chamber extract. dCEs were extracted from all positive cartridges by puncturing the rear chamber with a sterile 29 G × 1/2′′ 1 ml insulin syringe (Avacare, South Africa) ( Fig. 2A,B) as described previously 6 . The full volume was extracted (~15 µl for Xpert; ~35 µl for Ultra). ...
Xpert MTB/RIF Ultra (Ultra) detects Mycobacterium tuberculosis and rifampicin resistance. Follow-on drug susceptibility testing (DST) requires additional sputum. Extract from the diamond-shaped chamber of the cartridge (dCE) of Ultra’s predecessor, Xpert MTB/RIF (Xpert), is useful for MTBDRsl-based DST but this is unexplored with Ultra. Furthermore, whether CE from non-diamond compartments is useful, the performance of FluoroType MTBDR (FT) on CE, and rpoB cross-contamination risk associated with the extraction procedure are unknown. We tested MTBDRsl, MTBDRplus, and FT on CEs from chambers from cartridges (Ultra, Xpert) tested on bacilli dilution series. MTBDRsl on Ultra dCE on TB-positive sputa (n = 40) was also evaluated and, separately, rpoB amplicon cross-contamination risk . MTBDRsl on Ultra dCE from dilutions ≥103 CFU/ml (CTmin <25, >“low semi-quantitation”) detected fluoroquinolone (FQ) and second-line injectable (SLID) susceptibility and resistance correctly (some SLIDs-indeterminate). At the same threshold (at which ~85% of Ultra-positives in our setting would be eligible), 35/35 (100%) FQ and 34/35 (97%) SLID results from Ultra dCE were concordant with sputa results. Tests on other chambers were unfeasible. No tubes open during 20 batched extractions had FT-detected rpoB cross-contamination. False-positive Ultra rpoB results was observed when dCE dilutions ≤10−3 were re-tested. MTBDRsl on Ultra dCE is concordant with isolate results. rpoB amplicon cross-contamination is unlikely. These data mitigate additional specimen collection for second-line DST and cross-contamination concerns.
... [79] Furthermore, we described innovative approaches for how material typically discarded by laboratories can be used for detailed drug susceptibility testing (thus alleviating the need for additional specimen collection). [80] We recently identified widespread suboptimal performance of the only commercial molecular test for multidrug resistance (MTBDRplus) across dozens of laboratories worldwide and, importantly, demonstrated how this can be corrected by changing the conditions used for DNA amplification. [81] This is now in the process of being incorporated into WHO external quality assessment processes for TB laboratories. ...
The South African Medical Research Council Centre for Tuberculosis Research has a rich history of high-impact research that has
influenced our understating of this hyper-epidemic which is further exacerbated by the emergence and spread of drug-resistant forms of the
disease. This review aims to summarise the past 30 years of research conducted in the Centre which has influenced the way that tuberculosis
(TB) is diagnosed and treated. The review includes the development of new technologies for rapid screening of people with probable TB
and the repurposing of human diagnostics for wildlife conservation.
Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml-1, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.
Objectives
Isoniazid (INH) and second-line drug resistance (DR) detection through line probe assay (LPA) takes long in extrapulmonary (EP) specimens because culture growth needs to be obtained to perform deoxyribonucleic acid (DNA) extraction due to the paucibacillary nature of these specimens. Knowing the DR pattern at the earliest is key to success of the treatment. Delay in appropriate tuberculosis (TB) treatment in EP TB patients runs the risk of DR amplification, significant disease damage, and patient loss to follow-up. Here, LPA was attempted on truenat-derived DNA elute from EP specimens, which, in routine, is discarded after the truenat test, to determine drug sensitivity test (DST) for INH and, where necessary, for second-line drugs (Fluoroquinolones, Kanamycin, amikacin, and capreomycin).
Material and Methods
Truenat, acid-fast bacilli culture, and fluorescent microscopy were performed on all EP samples that were received at the laboratory during June–September 2022. DNA elute that was left over from 59 truenat Mycobacterium tuberculosis (MTB) positive EP samples were subjected to Genotype MTBDR plus Ver 2.0 assay.
Results
MTBDR plus assay (DNA elute) detected MTB and rifampicin (RIF) and INH DST in 47 samples (79.6%) having truenat MTB count of 7.8 × 10 ² colony-forming unit/milliliter and above. It also detected RIF DST in 65.2% truenat RIF indeterminate samples and DST for both RIF and INH in 60% of culture negative EP specimens. DST results by LPA (DNA elute) completely concorded with standard indirect LPA (on 21 culture isolates from smear-negative specimens). The MTBDRsl yield was however relatively low (11.1%), although second line LPA (SLLPA) was performed only on 9 first-line DR samples.
Conclusions
Left-over truenat-derived DNA elute is a significant sample by-product that can significantly speed up and increase the yield of determination of MTB DST in EP samples for RIF and INH, the most critical drugs for TB treatment.
