Dried Culture Spots for Xpert MTB/RIF External Quality Assessment: Results of a Phase 1 Pilot Study in South Africa

Abstract

Implementation of Xpert MTB/RIF requires quality assessment. A pilot program using dried culture spots (DCSs) of inactivated Mycobacterium tuberculosis is described. Of 274 DCS results received, 2.19% generated errors; the remainder yielded 100% correct Mycobacterium tuberculosis detection. The probe A cycle threshold (C T ) variability of three DCS batches was <3.47. The study of longer-term DCS stability is ongoing.The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) (1, 3-5, 9, 12, 13, 15, 19, 25) for the diagnosis of Mycobacterium tuberculosis has recently been endorsed by the WHO (28), and recommendations for data collection to quantify the impact of this GeneXpert (GX) technology are provided (26). Guidance, however, with respect to appropriate external quality assessment (EQA) programs is lacking (17). Current international tuberculosis (TB) EQA programs focus on microscopy, culture, and susceptibility testing laboratories (24) and highlight the difficulties in expansion due to labor-intensive preparatory work and the high cost and regulations associated with shipping drug-resistant isolates (27).Criteria for a verification ("fit for purpose") and EQA program suited to the characteristics of the Xpert MTB/RIF assay (3, 8) will require the following elements. (i) The testing material must contain whole M. tuberculosis (8).(ii) Transportation of EQA material needs to be safe. (iii) The testing procedure needs to be safe and compatible with the Xpert MTB/ RIF current testing protocol. (iv) Health care workers who do not have laboratory skills must be able to perform the testing in nonlaboratory settings. (v) Finally, the programs will need to be cost-effective and sustainable. Such a program using whole inactivated M. tuberculosis spotted onto filter paper was developed and piloted in South Africa as part of the National Health Laboratory Service (NHLS) GX rollout. H37Ra]) and well-characterized local clinical strain MYCTU 15, and (iii) the ATCC 25618 (H37Rv) laboratory strain grown for single-cell-organism suspensions (11). The MGIT cultures S-MYCTU-02-P2 and MYCTU 15 and clinical isolates were pooled in their respective batches (with strains kept separate and not mixed), centrifuged (3,000 ϫ g for 15 min at 4°C) to pellet cells, and resuspended in 40 ml phosphate-buffered saline (PBS) followed by addition of 80 ml (2:1 ratio of buffer to culture) of the Xpert sample reagent (SR) buffer. For the H37Rv strain, 200 ml of culture was harvested (by centrifugation at 3,500 ϫ g) at room temperature for 10 min, and cells were resuspended in PBS to 40 ml followed by addition of 80 ml SR buffer (2:1 ratio of buffer to cells). Both MGIT-grown and H37Rv strain cultures were inactivated in SR buffer for 2 h at room temperature, with intermittent mixing. The inactivated material was washed twice with sterile PBS and resuspended in final volumes of 10 ml (S-MYCTU-02-P2 and MYCTU 15) and 40 ml (H37Rv) PBS. For confirmation of inactivation, washed cultures (0.5 ml) were reinoculated into new MGIT tubes in Bactec cabinets fo...

Performance Monitoring of Mycobacterium tuberculosis Dried Culture Spots for Use with the GeneXpert System within a National Program in South Africa

“…Overall, most of the DCS results were in the low semiquantitative category (mean C T range, 22.6 to 24.7). (3). The stability of intact mycobacterial cells on filter paper for up to 6 months at all temperatures (4°, RT, and 37°C) was well within the expected time limits for shipping to testing in a national program.…”

“…By 31 March 2013, approximately 2,315,380 Xpert MTB/RIF cartridges had been sold globally, over half of which had been procured for use in South Africa alone (2). In parallel to this implementation, a GeneXpert instrument verification program consisting of inactivated Mycobacterium tuberculosis organisms spotted onto filter cards, termed dried culture spots (DCSs), was developed and successfully used to verify and ensure that newly placed instruments during phase I of the implementation (n ϭ 26 sites) were "fit for purpose" before clinical specimen testing (3). A website (www.tbgxmonitor.com) was developed to automatically perform statistical analyses and to generate verification reports in real time.…”

Advances in tuberculosis diagnostics: the Xpert MTB/RIF assay and future prospects for a point-of-care test

“…Rigorous quality assessment programmes will also be needed, following, for example, a model developed in South Africa that used dried culture spots of inactivated M tuberculosis on filter paper. 76 This is essential to ensure that results are accurate.…”

Tuberculosis control in South Africa: Successes, challenges and recommendations

“…Between March 2011 and April 2013 >1.3 million Xpert MTB/RIF tests were done in SA, which accounted for more than half of the global usage of Xpert MTB/RIF. SA also pioneered the use of dried culture spots for verification and external quality assurance of Xpert MTB/RIF [4] and implemented a central data monitoring system to remotely monitor the performance of Xpert MTB/RIF, the proportion of positive tests and the proportion with rifampicin resistance.…”