Genotyping Multidrug-Resistant Mycobacterium tuberculosis from Primary Sputum and Decontaminated Sediment with an Integrated Microfluidic Amplification Microarray Test

Linger, Yvonne; Knickerbocker, Christopher; Sipes, David; Golova, Julia; Franke, Molly F.; Calderón, Róger; Lecca, Leonid; Thakore, Nitu; Holmberg, Rebecca C.; Qu, Peter; Kukhtin, Alexander; Murray, Megan; Cooney, Christopher G.; Chandler, Darrell P.

doi:10.1128/jcm.01652-17

Cited by 16 publications

(26 citation statements)

References 49 publications

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“…In summary, the assays from Akonni Biosystems (C) were the most accurate but required a significantly longer TAT in comparison to the other developers. This was due to the Akonni test platform requiring a 3-hour DNA microarray hybridization step after amplification by asymmetric PCR [34,35]. The assays from Two Pore Guys (F) were next best in terms of sensitivity when using either PCR (F1) or LAMP (F2) and provided a very short TAT (20 and 35 minutes for LAMP and PCR, respectively).…”

Section: Resultsmentioning

confidence: 99%

“…Akonni have worked to overcome the inherent complexity of microarray-based diagnostics for routine use by combining asymmetric PCR amplification, microarray hybridization and waste fluid retention to within a single microfluidic chamber. The detection of array bound Cy3 labelled PCR products is enabled via a low-cost, field-portable microarray imager that Akonni is developing [35].…”

Section: Discussionmentioning

confidence: 99%

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Assessment of eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settings

et al. 2019

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Nucleic acid amplification technologies (NAATs) are high-performance tools for rapidly and accurately detecting infectious agents. They are widely used in high-income countries to diagnose disease and improve patient care. The complexities associated with test methods, reagents, equipment, quality control and assurance require dedicated laboratories with trained staff, which can exclude their use in low-resource and decentralized healthcare settings. For certain diseases, fully integrated NAAT devices and assays are available for use in environmentally-controlled clinics or emergency rooms where relatively untrained staff can perform testing. However, decentralized settings in many low- and middle-income countries with large burdens of infectious disease are challenged by extreme environments, poor infrastructure, few trained staff and limited financial resources. Therefore, there is an urgent need for low-cost, integrated NAAT tools specifically designed for use in low-resource settings (LRS). Two essential components of integrated NAAT tools are: 1) efficient nucleic acid extraction technologies for diverse and complex sample types; and 2) robust and sensitive nucleic acid amplification and detection technologies. In prior work we reported the performance and workflow capacity for the nucleic acid extraction component. In the current study we evaluated performance of eight novel nucleic acid amplification and detection technologies from seven developers using blinded panels of RNA and/or DNA from three pathogens to assess both diagnostic accuracy and suitability as an essential component for low-cost NAAT in LRS. In this exercise, we noted significant differences in performance among these technologies and identified those most promising for potential further development.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Assessment of eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settings

et al. 2019

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“…The long-term technology objective of this research is to unite automated sputum sample preparation with a M . tuberculosis drug susceptibility test (e.g., [ 35 ]) in a flexible, sample-to-answer diagnostic device. We opted to pursue an “open” sample preparation fluidic architecture of TruTips and reagent plates because the approach (and workstation) provides several straightforward opportunities to increase input sample size (volume), modify reagents, and/or adjust procedural steps to increase extraction efficacy, if needed.…”

Section: Resultsmentioning

confidence: 99%

Automated TruTip nucleic acid extraction and purification from raw sputum

et al. 2018

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Automated nucleic acid extraction from primary (raw) sputum continues to be a significant technical challenge for molecular diagnostics. In this work, we developed a prototype open-architecture, automated nucleic acid workstation that includes a mechanical homogenization and lysis function integrated with heating and TruTip purification; optimized an extraction protocol for raw sputum; and evaluated system performance on primary clinical specimens. Eight samples could be processed within 70 min. The system efficiently homogenized primary sputa and doubled nucleic acid recovery relative to an automated protocol that did not incorporate sample homogenization. Nucleic acid recovery was at least five times higher from raw sputum as compared to that of matched sediments regardless of smear or culture grade, and the automated workstation reproducibly recovered PCR-detectable DNA to at least 80 CFU mL-1 raw sputum. M. tuberculosis DNA was recovered and detected from 122/123 (99.2%) and 124/124 (100%) primary sputum and sediment extracts, respectively. There was no detectable cross-contamination across 53 automated system runs and amplification or fluorescent inhibitors (if present) were not detectable. The open fluidic architecture of the prototype automated workstation yields purified sputum DNA that can be used for any molecular diagnostic test. The ability to transfer TruTip protocols between personalized, on-demand pipetting tools and the fully automated workstation also affords public health agencies an opportunity to standardize sputum nucleic acid sample preparation procedures, reagents, and quality control across multiple levels of the health care system.

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“…While the reference methods typically gave the best results, the Akonni platform had better performance with certain target pathogens. Akonni is heavily invested in MTB diagnostic NAATs and so their best in class performance for MTB likely reflects their expertise in this area [35][36][37].…”

Section: Discussionmentioning

confidence: 99%

Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings

et al. 2019

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Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS.

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Genotyping Multidrug-Resistant Mycobacterium tuberculosis from Primary Sputum and Decontaminated Sediment with an Integrated Microfluidic Amplification Microarray Test

Cited by 16 publications

References 49 publications

Assessment of eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settings

Assessment of eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settings

Automated TruTip nucleic acid extraction and purification from raw sputum

Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings

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