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

Rouxjeane Venter,Tanya Dolby,Natasha Kitchin,Samantha Pillay,John Simpson,Robin M Warren,Brigitta Derendinger,Paul D Van Helden,Ashley Ruiters,Margaretha De Vos,Grant Theron

doi:10.1038/s41598-017-14385-x

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.

Highlights

Of the 10.4 million individuals with active tuberculosis (TB) in 2015, 580 000 were rifampicin (RIF) resistant or multidrug-resistant (MDR), defined as resistance to isoniazid (INH) and RIF1
Our results show that accurate second-line drug testing using MTBDRsl is possible on cartridge extract (CE) from Xpert cartridges that would otherwise be discarded
We defined a threshold at which this approach is feasible, meaning that MTBDRsl assays do not need to be wasted on CE unlikely to give a valid result

Summary

Introduction

Of the 10.4 million individuals with active tuberculosis (TB) in 2015, 580 000 were rifampicin (RIF) resistant or multidrug-resistant (MDR), defined as resistance to isoniazid (INH) and RIF1. We conducted a proof-of-concept evaluation on whether M. tuberculosis-complex genomic DNA in the PCR-reaction mix from used Xpert cartridges (cartridge extract; CE) - that would otherwise be discarded - was detectable in an accurate manner using MTBDRplus and MTBDRsl. The feasibility of genotyping on CE by spoligotyping was tested as this would potentially be useful for research laboratories and programmes seeking to implement routine strain surveillance. Isolates were not available from Xpert TB-positive, RIF-susceptible specimens as culture is not routinely done in these patients11,28.

Results

Conclusion