Development of dried tube specimens for Xpert MTB/RIF proficiency testing

DeGruy, Kyle; Klein, Katherine A.; Rey, Zilma; Hall, Patricia; Kim, Andrea; Alexander, Heather

doi:10.4102/ajlm.v9i1.1166

Cited by 3 publications

(7 citation statements)

References 13 publications

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“…Eight Stored isolates with known genomic and phenotypic results obtained from the WHO-institute of tropical medicine (ITM)-Antwerp (https://www.itg.be/E/ administrator-of-the-worlds-largest-collection-of-tbcstrains) for routine quality control of laboratory processes were used for this study. The production of the DTS followed some of the steps that have been described by Kyle et al 2020 [12]. Briefly, MTB isolates were grown on MGIT with confirmation of MTB-complex (MTBC) done Using SD bioline test (catalog number and description: 08FK50 TB Ag MPT64 Rapid) and Zeihl Neelsen as described in SRL standard operating procedures (SOPs) "S1 File" and WHO guidelines [13,14].…”

Section: Laboratory Methodsmentioning

confidence: 99%

“…Using proof of inactivation and pretest results, five stocks with the lowest cycle threshold (Ct) value means and standard deviation of � 3 were selected to comprise the PT panel as shown in Table 1. A standard deviation of � 3 was selected due to consistency in homogeneity obtained with it during previous PT panel preparations [12]. Samples selected included: two MTB-detected rifampicinsensitive, two MTB-detected rifampicin-resistant and one NTM as the negative sample.…”

Section: Laboratory Methodsmentioning

confidence: 99%

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Advancing proficiency testing for ultra in resource-limited settings using dried tube specimen: A study by SRL-Uganda

Kabugo

Namutebi²,

Adam³

et al. 2023

PLoS ONE

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Background Proficiency testing (PT) has been hard to set up due to cost limitations and technical capacity. Conventional Xpert MTB/RIF PT programs use liquid and culture spots which require stringent storage and transportation conditions with cross-contamination chances prevalent. These setbacks prompted the use of dried tube specimens (DTS) for Ultra assay PT. For continuity of PT provision, stability of DTS and compatibility with testing protocols when kept for a long period needs to be established. Methods DTS were prepared from known isolates inactivated using a hot air oven at 85°C. 100μl of bacterial suspensions were aliquoted and dried inside a Biosafety cabinet. Panel validation was done to establish the baseline Deoxyribonucleic acid (DNA) concentration in terms of cycle threshold (Ct) value. DTS aliquots were shipped to participants to test and report within six weeks. The remaining DTS were kept at 2–8°C and room temperature for one year with testing at six months. Twenty (20) DTS samples per set remaining at one year were heated at 55°C for two weeks before testing. The means of the different samples were compared to validation data using paired t-tests. Boxplots were designed to visualize the differences in the medians of the DTS. Results Overall mean Ct value increased by 4.4 from the validation to testing after one year at the different storage conditions. Samples heated at 55°C showed a 6.4 Ct difference from validation data. Testing done at six months on 2–8°C stored items showed no statistical difference. At all the remaining testing times and conditions, P-values were less than 0.008 although the absolute mean Ct when compared showed slight increments and accommodated differences for the detection of MTB and rifampicin resistance. Median values for samples stored at 2–8°C were lower compared to those at room temperature. Conclusion DTS stored at 2–8°C remain more stable for one year compared to higher temperatures and can be consistently used as PT materials in more than one PT round for biannual PT providers.

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Section: Laboratory Methodsmentioning

confidence: 99%

Section: Laboratory Methodsmentioning

confidence: 99%

Advancing proficiency testing for ultra in resource-limited settings using dried tube specimen: A study by SRL-Uganda

Kabugo

Namutebi²,

Adam³

et al. 2023

PLoS ONE

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“…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 Mycobacteria were grown in BACTEC® MGIT 7 mL tubes (Becton, Dickinson and Company, Sparks, Maryland, United States) and inactivated using a 2:1 ratio of Xpert MTB/RIF sample reagent (Cepheid, Sunnyvale, California, United States) to MGIT culture incubated for 2 hours with intermittent vortexing.…”

Section: Methodsmentioning

confidence: 99%

“…Dried tube specimens were validated prior to distribution by testing 10% of DTS produced with the Xpert MTB/RIF assay (Cepheid, Sunnyvale, California, United States), as described previously. 10 To test DTS, the DTS were rehydrated with 2.5 mL of sample reagent, shaken vigorously 20 times and incubated at room temperature for 15 minutes with additional shaking repeated after 10 min. The Xpert MTB/RIF cartridges were inoculated with approximately 2.0 mL of rehydrated samples using the transfer pipette provided with the kit, and tested immediately on a GeneXpert IV or GeneXpert VIII using GeneXpert DX software version 4.0 (Cepheid, Sunnyvale, California, United States) according to manufacturer instructions.…”

Section: Methodsmentioning

confidence: 99%

“…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 CDC-prepared DTS panels in an Xpert MTB/RIF PT programme.…”

Section: Introductionmentioning

confidence: 99%

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

Klein¹,

DeGruy²,

Rey³

et al. 2020

Afr. J. Lab. Med.

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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.

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The development and implementation of a proficiency testing program for SARS-CoV-2 using dried tube specimens in resource-limited countries

Lutaaya,

Guido,

Ssentamu

et al. 2024

BMC Infect Dis

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Introduction When COVID-19 hit the world in 2019, an enhanced focus on diagnostic testing for SARS-CoV-2 was essential for a successful pandemic response. Testing laboratories stretched their capabilities for the new coronavirus by adopting different test methods. The necessity of having external quality assurance (EQA) mechanisms was even more critical due to this rapid expansion. However, there was a lack of experience in providing the necessary SARS-CoV-2 EQA materials, especially in locations with constrained resources. Objective We aimed to create a PT (Proficiency testing) programme based on the Dried Tube Specimens (DTS) method that would be a practical option for molecular based SARS-CoV-2 EQA in Low- and Middle-Income Countries. Methods Based on previous ISO/IEC 17043:2010 accreditation experiences and with assistance from the US Centers for Disease Control and Prevention, The Supranational Reference Laboratory of Uganda (adapted the DTS sample preparation method and completed a pilot EQA program between 2020 and 2021. Stability and panel validation testing was conducted on the designed materials before shipping to pilot participants in six African countries. Participants received a panel containing five SARS-CoV-2 DTS samples, transported at ambient conditions. Results submitted by participants were compared to validation results. Participants were graded as satisfactory (≥ 80%) or unsatisfactory (< 80%) and performance reports disseminated. Results Our SARS-CoV-2 stability experiments showed that SARS-CoV-2 RNA was stable (-15 to -25 °C, 4 to 8 °C, (18 to 28 °C) room temperature and 35 to 38 °C) as well as DTS panels (4 to 8 °C, 18 to 28 °C, 35 to 38 °C and 45 °C) for a period of 4 weeks. The SARS-CoV-2 DTS panels were successfully piloted in 35 test sites from Zambia, Malawi, Mozambique, Nigeria, and Seychelles. The pilot results of the participants showed good accuracy, with an average of 86% (30/35) concordance with the original SARS CoV-2 expectations. Conclusion The SARS-CoV-2 DTS PT panel is reliable, stable at ambient temperature, simple to prepare and requires minimal resources.

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Development of dried tube specimens for Xpert MTB/RIF proficiency testing

Cited by 3 publications

References 13 publications

Advancing proficiency testing for ultra in resource-limited settings using dried tube specimen: A study by SRL-Uganda

Advancing proficiency testing for ultra in resource-limited settings using dried tube specimen: A study by SRL-Uganda

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

The development and implementation of a proficiency testing program for SARS-CoV-2 using dried tube specimens in resource-limited countries

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