Abstract
Dried culture spots (DCS) of inactivated Mycobacteria strains designed as part of an external quality assessment (EQA) program for the GeneXpert system has applications to other molecular tuberculosis (TB) diagnostic platforms. DCS tested on the GenoType MTBDRplus and Mycobacterium CM assays performed well with MTBDRplus version 2 but require increased bacterial concentration for use with version 1.
TEXT
Quality assurance (QA) refers to an umbrella of quality checks within a medical laboratory encompassing the entire testing process from specimen collection to result reporting (i.e., preanalytical, analytical, and postanalytical steps) to ensure high-quality testing (1). External quality assessment (EQA), sometimes referred to as proficiency testing, is just one component of a QA program and is defined as a system for objectively checking the performance of a laboratory using an external agency or facility (2). This is often challenging and costly for tuberculosis (TB) laboratories but is particularly exacerbated in low-resource settings (3), which may not always be equipped with an adequate level of biosafety to receive live Mycobacterium tuberculosis (MTB) cultures, and stringent requirements for transportation of such materials exist (4). Several TB EQA molecular schemes exist (http://www.cap.org; 5, 6), but some provide the EQA material in a format containing preextracted DNA, making them inappropriate for the monitoring of the entire testing process from extraction to detection.
To address the complexities of ongoing molecular diagnostic EQA systems in TB laboratories, a verification (fit-for-purpose) (7) and EQA program (pre-/postanalytics) (8) consisting of dried culture spot (DCS) material was developed for the molecular GeneXpert instrument performing the Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) (9). The major advantage of this approach is that the DCS contain whole, inactivated, and quantitated mycobacterial strains that can be safely transported as one would transport documents by mail, without the requirement of cold-chain transport. The spots are easy to use and robust, with a minimum shelf life of 9 months confirmed to date (10).
The DCS EQA program has been successfully implemented in 207 National Health Laboratory Service (NHLS) GeneXpert testing laboratories in South Africa and 82 non-NHLS sites in 21 countries. The Global Laboratory Initiative (GLI) (advisors to the World Health Organization) has also endorsed the DCS for verification of the GeneXpert instrument. As the DCS material is not platform specific, we determined the potential application of the GeneXpert DCS program for the molecular line probe assay (LPA), Genotype MTBDRplus versions 1 and 2 (Hain Lifescience, GmbH, Nehren, Germany).
DCS panels (each comprising 4 spots) were initially tested in a research laboratory in Johannesburg and then piloted in four routine service TB laboratories in South Africa, namely, the Mycobacteriology Referral Laboratory, Johannesburg; Ampath, Pretoria; Lancet, Johannesburg; and Centre for Clinical Tuberculosis Research (CCTR) Laboratory, Task Applied Science, Cape Town.
The Xpert MTB/RIF DCS panels (7), comprising M. tuberculosis rifampin (RIF)-resistant (Xpert probe D mutant)/isoniazid (INH)-susceptible, M. tuberculosis RIF-susceptible/INH-susceptible, and nontuberculous mycobacteria (NTM) (namely M. kansasii, M. intracellulare, and M. fortuitum), were tested on the MTBDRplus versions 1 and 2 . This involved resuspending the DCS in a 50-ml Nunc tube containing 2 ml of phosphate-buffered saline (PBS) for 15 min, with intermittent vortexing. This was followed by manual extraction (for MTBDRplus version 1) or GenoLyse extraction (for MTBDRplus version 2) of the entire lysate, as per the manufacturer's instructions. The extracted material was then amplified and hybridized as per the standard manufacturer's protocol for versions 1 and 2. The species identifications of the negative results for the MTBDRplus version 2 (TUB band absent) were determined using the GenoType Mycobacterium CM assay (Hain Lifescience, Germany).
Table 1 lists the observed findings for the initial research laboratory testing and Table 2 for the pilot site testing.
TABLE 1.
Initial results on use of the DCS material on the GenoType MTBDRplus versions 1 and 2 tested in a research laboratory
| No. of DCS tested by GenoType MTBDRplus versiona | DCS strain | Observed result on GenoType MTBDRplus assay (no. observed/total no. [%]) |
|---|---|---|
| Version 1 | ||
| 8 | M. kansasii | 16/16 (100) negative |
| 8 | M. intracellulare | 16/16 (100) negative |
| 16 | M. tuberculosis RIFr, INHs | 15/16 (94) positive, RIFr INHs; 1/16 (6) negative |
| 16 | M. tuberculosis RIFs, INHs | 8/16 (50) positive, RIFs INHs; 2/16 (12.5) positive, RIF and INH inconclusive; 6/16 (37.5) negative |
| Version 2 | ||
| 8 | M. kansasii | 24/24 (100) negative |
| 8 | M. intracellulare | 24/24 (100) negative |
| 8 | M. fortuitum | 24/24 (100) negative |
| 40 | M. tuberculosis RIFr, INHs | 40/40 (100) positive, RIFr INHs |
| 16 | M. tuberculosis RIFs, INHs | 16/16 (100) positive, RIFs INHs |
Number of DCS tested per strain dependent on available stock.
TABLE 2.
Pilot EQA GenoType MTBDRplus results for the DCS prepared and tested in four routine service laboratories
| No. of sites by GenoType MTBDRplus version | No. of DCS tested | DCS strain | Observed result (no. observed/total no. [%]) |
|---|---|---|---|
| Version 1 | |||
| 2 | 6 | M. intracellulare | No reportable result (blank strips) |
| 6 | M. tuberculosis RIFr, INHs | No reportable result (blank strips) | |
| 12 | M. tuberculosis RIFs, INHs | No reportable result (blank strips) | |
| Version 2 | |||
| 2 | 6 | M. intracellulare | 6/6 (100) negative |
| 6 | M. tuberculosis RIFr, INHs | 5/6 (83) positive, RIFr INHs; 1/6 (17) positive, RIFr INHs (WT7 present, MUT2A present) | |
| 12 | M. tuberculosis RIFs, INHs | 12/12 (100) positive, RIFs INHs | |
The species of all DCS that tested negative on version 2 of the LPA in the research laboratory (Table 1) were further identified with the GenoType Mycobacterium CM assay. Of the eight M. kansasii, M. intracellulare, and M. fortuitum spots tested, the species of 6 from each were successfully identified (18/24 [75%]).
The M. intracellulare DCS tested on LPA version 2 at two of the pilot sites (Table 2; n = 3 per site) were also tested on the Mycobacterium CM assay, and the species of 5/6 (83%) were correctly identified. One DCS (17%) was incorrectly reported as M. avium.
Although partially successful in the initial laboratory testing phase, the two pilot sites performing version 1 failed to produce interpretable results. This was due to the low bacillary load on the DCS designed for detection at a minimum of 150 CFU/ml, which is the lower limit of detection of the Xpert MTB/RIF test (9). Since the LPA version 1 is validated only for use with smear-positive specimens, which has a limit of detection of 10,000 bacteria/ml3 (11), it is feasible to accept that version 1 will not detect M. tuberculosis on the DCS below this lower limit.
The reverse is true for the use of DCS with the MTBDRplus version 2 assay, as all (80/80) DCS results from the research laboratory and 96% (23/24) of the pilot site results were correctly reported. Rifampin resistance was identified by an rpoB wild-type 7 (WT7) missing band and a mutant 2A (MUT2A) band present by the LPA, which corresponds to the Xpert MTB/RIF probe D mutant. One of the pilot sites detected an RIF-resistant DCS as having a WT7 band and MUT2A band present. This may have been due to incorrect interpretation or contamination, but the finding was not considered clinically relevant. Of the three sites (one research laboratory and 2 routine service laboratories) that performed Mycobacterium CM assay testing on the negative results by MTBDRplus version 2, only one incorrect species identification was reported, possibly due to a mislabeled laboratory specimen.
Overall, the DCS program appears to be suitable not only for the Xpert MTB/RIF assay but also for the MTBDRplus assay version 2, due to their similar sensitivities, and it provides measurements for the entire testing process (DNA extraction through result reporting), as opposed to just the amplification and hybridization steps on DNA-prepared EQA materials. The added advantage of the DCS technology is the ability to transport inactivated tuberculosis-positive specimens at room temperature, thereby reducing the overall program costs. Even though the GenoType MTBDRplus version 1 assay has been discontinued in several countries (Hain Lifescience, South Africa [Pty], Ltd., personal communication), if laboratories wish to use the DCS with this version, an increased bacterial concentration will need to be spotted onto the paper card.
Currently, the result reporting of the GeneXpert MTB/RIF EQA DCS program is automated and managed remotely through TBGxMonitor for real-time turnaround of the results, but it could be expanded to provide a more automated result reporting for the MTBDRplus assays. Other M. tuberculosis strains could also be included in the DCS panels that are suitable for second-line drug resistance testing, such as those with the MTBDRsl assay (Hain Lifescience, GmbH, Nehren, Germany).
ACKNOWLEDGMENTS
We thank Pedro da Silva (Mycobacteriology [TB] Referral Laboratory, National Health Laboratory Service, Johannesburg), Cornelius Clay (Ampath National Reference Laboratory, Pretoria), Sharmila Naidoo (Lancet Laboratories, Johannesburg), Marinus Barnard (CCTR Laboratory Task Applied Sciences, Cape Town), Saloshini Ramsamy (Clinical Laboratory Service, Johannesburg), and Ruth Mohlabeng for assistance with LPA testing. We also thank Hain Lifescience, South Africa (Pty) Ltd., Johannesburg, for technical support.
REFERENCES
- 1.World Health Organization. 2007. Policy and procedures of the WHO/NICD Microbiology External Quality Assessment Programme in Africa: years 1 to 4 (2002–2006). World Health Organization, Geneva, Switzerland: http://whqlibdoc.who.int/hq/2007/who_cds_epr_lyo_2007.3_eng.pdf. [Google Scholar]
- 2.World Health Organization. 2011. Overview of external quality assessment (EQA): module 10, content sheet 10-1. World Health Organization, Geneva, Switzerland: http://www.who.int/ihr/training/laboratory_quality/10_b_eqa_contents.pdf. [Google Scholar]
- 3.Parsons LM, Somoskovi A, Gutierrez C, Lee E, Paramasivan CN, Abimiku A, Spector S, Roscigno G, Nkengasong J. 2011. Laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities. Clin Microbiol Rev 24:314–350. doi: 10.1128/CMR.00059-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Noble MA. 2013. An overview of the essential elements of a PT/EQA program. U.S. Centers for Disease Control and Prevention, Atlanta, GA. [Google Scholar]
- 5.Quality Control for Molecular Diagnostics. 2015. EQA programmes. Quality Control for Molecular Diagnostics, Glasgow, Scotland: http://www.qcmd.org/index.php?pageId=3&pageVersion=EN. [Google Scholar]
- 6.UK National External Quality Assessment Service for Microbiology. 2013. Schemes. UK NEQAS for Microbiology, London, United Kingdom: http://www.ukneqasmicro.org.uk/index.php/schemes. [Google Scholar]
- 7.Scott LE, Gous N, Cunningham BE, Kana BD, Perovic O, Erasmus L, Coetzee GJ, Koornhof H, Stevens W. 2011. Dried culture spots for Xpert MTB/RIF external quality assessment: results of a phase 1 pilot study in South Africa. J Clin Microbiol 49:4356–4360. doi: 10.1128/JCM.05167-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Scott L, Albert H, Gilpin C, Alexander H, DeGruy K, Stevens W. 2014. Multicenter feasibility study to assess external quality assessment panels for Xpert MTB/RIF assay in South Africa. J Clin Microbiol 52:2493–2499. doi: 10.1128/JCM.03533-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Helb D, Jones M, Story E, Boehme C, Wallace E, Ho K, Kop J, Owens MR, Rodgers R, Banada P, Safi H, Blakemore R, Lan NT, Jones-Lopez EC, Levi M, Burday M, Ayakaka I, Mugerwa RD, McMillan B, Winn-Deen E, Christel L, Dailey P, Perkins MD, Persing DH, Alland D. 2010. Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. J Clin Microbiol 48:229–237. doi: 10.1128/JCM.01463-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Gous N, Cunningham B, Kana B, Stevens W, Scott LE. 2013. Performance monitoring of Mycobacterium tuberculosis dried culture spots for use with the GeneXpert system within a national program in South Africa. J Clin Microbiol 51:4018–4021. doi: 10.1128/JCM.01715-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Lawn SD, Kerkhoff AD, Vogt M, Ghebrekristos Y, Whitelaw A, Wood R. 2012. Characteristics and early outcomes of patients with Xpert MTB/RIF-negative pulmonary tuberculosis diagnosed during screening before antiretroviral therapy. Clin Infect Dis 54:1071–1079. doi: 10.1093/cid/cir1039. [DOI] [PMC free article] [PubMed] [Google Scholar]