Mycobacterial genomic DNA from used Xpert MTB/RIF cartridges can be utilised for accurate second-line genotypic drug susceptibility testing and spoligotyping

Abstract

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 cartridges was, without downstream DNA extraction or purification, suitable for both genotypic DST (MTBDRplus, MTBDRsl), which may permit patients to rapidly receive a XDR-TB diagnosis from a single specimen, and spoligotyping, which could facilitate routine genotyping. To determine the limit-of-detection and diagnostic accuracy, CEs from dilution series of drug-susceptible and -resistant bacilli were tested (MTBDRplus, MTBDRsl). Xpert TB-positive patient sputa CEs (n = 85) were tested (56 Xpert-rifampicin-susceptible, MTBDRplus and MTBDRsl; 29 Xpert-rifampicin-resistant, MTBDRsl). Spoligotyping was done on CEs from dilution series and patient sputa (n = 10). MTBDRplus had high non-valid result rates. MTBDRsl on CEs from dilutions ≥103CFU/ml (CT ≤ 24, >“low” Xpert semiquantitation category) was accurate, had low indeterminate rates and, on CE from sputa, highly concordant with MTBDRsl isolate results. CE spoligotyping results from dilutions ≥103CFU/ml and sputa were correct. MTBDRsl and spoligotyping on CE are thus highly feasible. These findings reduce the need for additional specimen collection and culture, for which capacity is limited in high-burden countries, and have implications for diagnostic laboratories and TB molecular epidemiology.

Rapid Tuberculosis Diagnostics Including Molecular First- and Second-Line Resistance Testing Based on a Novel Microfluidic DNA Extraction Cartridge

“…However, the cartridges had to be punctured with a syringe to extract DNA. 22,23 A strength of this TB-Diskebased workflow is that, besides providing DNA for TB testing, a separate external reaction tube containing DNA for additional tests provides maximal flexibility; and no puncturing of the cartridge is required. After diagnosis of TB, Inh, Rif, and SLD resistance markers can be immediately investigated by PCR, LPA, or potentially targeted next-generation sequencing in a stepwise algorithm.…”

Review and Updates on the Diagnosis of Tuberculosis

“…It is based on a hemi-nested real-time polymerase-chain-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 ].…”

Extract from used Xpert MTB/RIF Ultra cartridges is useful for accurate second-line drug-resistant tuberculosis diagnosis with minimal rpoB-amplicon cross-contamination risk

“…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).…”

“…Previous work showed that mycobacterial genomic DNA can be recovered from the rear diamond-shaped chamber of used Xpert MTB/RIF (Xpert) cartridges after the test is complete. This diamond cartridge extract (dCE) is useful for downstream testing with the MTBDRsl line probe assay (LPA) (Hain Lifescience, Germany), the only World Health Organization (WHO)-endorsed molecular test for second-line drug resistance, and 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.…”