A global proficiency testing programme for Xpert® MTB/RIF using dried tube specimens, 2013–2015

Background Proficiency testing (PT) is an important quality assurance measure toward ensuring accurate and reliable diagnostic test results from clinical and public health laboratories. Despite the rapid expansion of the Xpert® MTB/RIF assay for the detection of tuberculosis in resource-limited settings (RLS), low-cost PT materials for Xpert MTB/RIF external quality assessment (EQA) are not widely available. Objective We sought to determine whether a dried tube specimen (DTS)-based PT programme would be a feasible option to support Xpert MTB/RIF EQA in RLS. Methods Between 2013 and 2015, the United States Centers for Disease Control and Prevention developed and conducted a voluntary EQA programme using DTS-based PT material. Eight rounds of PT, each comprising five DTS samples, were provided to enrolled testing sites. After each round, participant results were compared to expected results, scored as satisfactory or unsatisfactory, and sites were provided with performance reports. Results Programme enrolment increased from 102 testing sites in seven countries to 441 testing sites in 14 countries over the course of three years. In each PT round, approximately 90% of participating sites demonstrated satisfactory performance. In seven of the 14 enrolled countries, the proportion of sites with a satisfactory score increased between the first round of participation and the most recent round of participation. Conclusion This programme demonstrated that it is possible to implement an Xpert MTB/RIF PT programme for RLS using DTS, that substantial demand for Xpert MTB/RIF PT material exists in RLS, and that country performance can improve in a DTS-based PT programme.


Introduction
The World Health Organization recommends the Xpert ® MTB/RIF (Mycobacterium tuberculosis/ rifampicin) assay (Cepheid, Sunnyvale, California, United States) as one of the initial diagnostic tests for all individuals evaluated for tuberculosis. 1,2 The Xpert MTB/RIF assay is an integrated sputum processing and real-time polymerase chain reaction system that rapidly and simultaneously detects M. tuberculosis and the presence of mutations conferring resistance to rifampicin. 3 The roll-out of the Xpert MTB/RIF assay to resource-limited settings (RLS) with a high tuberculosis burden has been remarkable. By the end of 2016, 6659 GeneXpert instruments had been purchased at concessional prices for use at multiple levels of the tiered laboratory network. 4 External quality assessment is a critical component of a laboratory quality management system. Proficiency testing (PT) is one means of conducting external quality assessment for tests in clinical laboratories. Proficiency testing provides objective evidence of accurate testing and may identify areas in the diagnostic process, including pre-analytic, analytic, and post-analytic phases, where quality improvement is needed. 5,6 The rapid scale-up of Xpert MTB/RIF testing in RLS has not coincided with the same investment in quality assurance activities for the assay. Few PT providers for the Xpert MTB/RIF assay offer materials to sites in RLS, leaving a substantial gap in external quality assessment implementation that could otherwise help to confirm site competency in assay administration and accuracy of test results for limited-resource countries. 7,8,9 http://www.ajlmonline.org Open Access The United States Centers for Disease Control and Prevention (CDC) developed a PT panel for Xpert MTB/RIF, using a dried tube specimen (DTS)-based method. 10 The DTS technique has successfully been used to develop PT panels for HIV, malaria and syphilis diagnostic assays in RLS. 11,12,13 Our goal in this evaluation was to assess the performance and utility of CDCprepared DTS panels in an Xpert MTB/RIF PT programme.

Ethical considerations
No patient specimen collection was required and no patient identifiers were recorded on quality assurance tools. Participation in the Xpert MTB/RIF quality assurance and PT programme was voluntary and free of charge. No incentives were provided. This study was approved by the CDC Center for Global Health Division of Human Research Protection (CGH HSR tracking number 2014-082).

Dried tube specimen production
The method for producing DTS was described previously by DeGruy et al. 10 Briefly, DTS were produced by chemically inactivating liquid cultures of well-characterised mycobacterial strains using equipment and supplies commonly found in laboratories conducting tuberculosis liquid culture. 14  Frequencies for results in each category were calculated by PT sample and in aggregate for all samples. The mycobacterial strain used to produce the PT sample and the mean cycle threshold (Ct) of probe A during validation of the PT sample were included in the analysis to look for trends between these variables and the amount and type of discordance observed. In line with previous studies, probe A was selected for most analyses as it was the first probe to reach the detection threshold. 7 We conducted additional statistical analyses to test for an association between the mean Ct of probe A during validation and several types of discordance including false-negative M. tuberculosis complex detection, false-positive rifampicin resistance, and indeterminate rifampicin resistance. Logistic regression of the type of discordance on mean Ct of probe A was conducted using R glm (R Foundation for Statistical Computing, Vienna, Austria). 16 The proportion of participating sites with a satisfactory score ranged from a minimum of 88.1% in round 2015-B to a maximum of 93.1% in round 2014-A (Table 1). Individual countries had as few as 50% and as many as 100% of their participating sites achieve satisfactory scores. In seven of the 14 enrolled countries, the proportion of sites with a satisfactory score increased between the first round of participation and the most recent round of participation. Of the remaining seven countries, the proportion of sites with a satisfactory score remained constant in three countries but decreased in four countries (

Discussion
We have shown that it is feasible to implement an Xpert MTB/RIF PT programme for RLS using DTS. Over the eight rounds of PT panels provided across a period of three years, the number of countries participating in the programme doubled and the number of participating sites increased fivefold. In each PT round, at least two-thirds of enrolled sites participated in PT panel testing and reported test results. Lower participation rates were seen in rounds during which certain countries or their enrolled sites were subsequently unable to participate due to logistical challenges, such as a lack of funding for in-country panel distribution, lack of staff, PT kits being lost in the mail between the country coordinator and the enrolled testing site, non-functioning Xpert instruments or computers, and stockouts (inability to procure test cartridges). Further work is needed to investigate and address the reasons for non-participation in proficiency testing as part of a comprehensive continuous quality improvement package.

Limitations
Several challenges arose during operation of the DTS-based Xpert MTB/RIF PT programme. The participant consensus for sample 2015-B-1 was below 80% concordance with expected results, mainly due to false-negative M. tuberculosis complex results (failure to detect M. tuberculosis complex when the expected result was 'MTB detected'). Although this sample was produced using the same method as other DTS samples, the probe A Cts during validation were on average higher than those of other M. tuberculosis complex samples produced. This indicates 2015-B-1 contained fewer inactivated organisms and thus less DNA than other samples, potentially contributing to the higher proportion of falsenegative M. tuberculosis complex results received. 20 To investigate the low concordance of 2015-B-1, three remaining aliquots were tested and confirmed the lower than average semi-quantitative result. Due to the low participant consensus, the decision was made to award all sites that submitted results for 2015-B-1 full credit (20 points). Based on this experience, we have implemented additional DTS validation criteria to ensure DTS contain enough inactivated organism to perform reliably. Beginning in 2016, only DTS samples with an average Ct value of less than 23 for the first probe detected are included in the distributed PT panels. The mean Ct value of 23 was selected based on the findings of previous investigators, who observed good concordance with mean Ct values up to 23. 7 In addition, recording and reporting of results was also a recurring challenge. At times, no explanation was provided for missing or incomplete results, and it was not possible to determine whether a site attempted to test the sample or if the test was unsuccessful and the result not reported. More clearly defined reporting language and procedures were added to report forms in future PT rounds to reduce the reliance of free text data entry and improve our ability to provide assistance on determining the root causes of discordant results and unsuccessful tests.

Recommendations and next steps
The steady increase in enrolment and number of sites participating in this DTS-based Xpert MTB/RIF PT programme demonstrates that there is a substantial demand for routine PT material for the Xpert MTB/RIF assay. The ease of largescale batch preparation of DTS using readily available supplies and equipment, and the successful establishment of country coordinator roles for in-country management of PT programme operations, indicate that a DTS-based Xpert MTB/RIF PT programme could be a feasible option for countries wanting to manage their own Xpert MTB/RIF PT programme. Future work will focus on piloting the transfer of the DTS-based PT package to national and regional programmes as part of a comprehensive continuous quality improvement package, as well as developing a web-based PT data entry and reporting system.

Conclusion
A continuous quality improvement package for Xpert MTB/ RIF in RLS that includes routine PT material is an important contributor to ensuring provision of accurate results. A DTSbased PT programme for Xpert MTB/RIF is a useful tool for monitoring and improving Xpert MTB/RIF performance. The simplicity of producing DTS and use of common mycobacteriology laboratory supplies make DTS a feasible way for countries to provide Xpert MTB/RIF PT material to their testing sites.