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      False Rifampicin Resistance in Xpert Ultra Applied to Lymph Node Aspirate: A Case Report

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          Abstract

          A 36-year-old male patient was diagnosed with tuberculosis in Antwerp, Belgium, in May 2018. His lymph node aspirate initially tested rifampicin resistant in Xpert MTB/RIF Ultra, but tested susceptible in all other tests including targeted deep sequencing due to a rare matrix effect in the Xpert MTB/RIF Ultra reaction tube.

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          Evaluation of the Xpert MTB/RIF Ultra Assay for Direct Detection of Mycobacterium tuberculosis Complex in Smear-Negative Extrapulmonary Samples

          Daniel Perez-Risco, David Rodriguez-Temporal, Ivan Valledor-Sanchez … (2018)
          The rapid detection of Mycobacterium tuberculosis complex (MTUBC) in clinical samples is essential for successful treatment. New techniques such as real-time PCR have been developed in order to facilitate rapid diagnosis, but their sensitivity is low in extrapulmonary specimens, due to the low bacillary load in such samples. A next-generation assay has recently been developed to try to overcome this limitation. The aim of this study was to analyze the effectiveness of the Xpert MTB/RIF Ultra (GX-Ultra) for the detection of MTUBC DNA in 108 smear-negative extrapulmonary specimens that were MTUBC culture positive. In addition, 40 extrapulmonary culture-negative samples and 20 samples with nontuberculous mycobacteria were tested to evaluate the specificity of the assay. All samples were collected between May 1999 and May 2017. The GX-Ultra detected DNA of MTUBC in 82 extrapulmonary specimens that were MTUBC culture positive (75.9% sensitivity; 95% confidence interval [CI], 66.6 to 83.4%). The assay was negative for all clinical specimens that were MTUBC culture negative and the samples with nontuberculous mycobacteria (100% specificity). Furthermore, two (1.8%) samples presented mutations related to rifampin resistance. The highest sensitivity was obtained in samples of lymph nodes (94.1%) and nonsterile fluids (93.7%), followed by tissue specimens (86.6%), stool material (80%), abscess aspirates (64.7%), and sterile fluids (60.5%). Pleural fluids, one of the least optimal samples for detecting DNA of MTUBC, were GX-Ultra positive in 10/21 (47.6%) of cases. In summary, GX-Ultra showed excellent specificity and high sensitivity in paubacillary specimens, making it a useful tool for rapid diagnosis of extrapulmonary tuberculosis.
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            Assessment of the Xpert MTB/RIF Ultra assay on rapid diagnosis of extrapulmonary tuberculosis

            Xiaocui Wu, Guangkun Tan, Rongliang Gao … (2019)
            To evaluate the diagnostic performance of Xpert MTB/RIF Ultra for EPTB (Extrapulmonary Tuberculosis) patients on different types of extrapulmonary specimens from different anatomic sites.
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              How Well Do Routine Molecular Diagnostics Detect Rifampin Heteroresistance in Mycobacterium tuberculosis?

              Kamela C. S. Ng, Philip Supply, Frank G. J. Cobelens … (2019)
              Rifampin heteroresistance—where rifampin-resistant and -susceptible tuberculosis (TB) bacilli coexist—may result in failed standard TB treatment and potential spread of rifampin-resistant strains. The detection of rifampin heteroresistance in routine rapid diagnostic tests (RDTs) allows for patients to receive prompt and effective multidrug-resistant-TB treatment and may improve rifampin-resistant TB control. The limit of detection (LOD) of rifampin heteroresistance for phenotypic drug susceptibility testing by the proportion method is 1% and, yet, is insufficiently documented for RDTs. We, therefore, aimed to determine, for the four RDTs (XpertMTB/RIF, XpertMTB/RIF Ultra, GenoTypeMTBDR plus v2.0, and GenoscholarNTM+MDRTBII), the LOD per probe and mutation, validated by CFU counting and targeted deep sequencing (Deeplex-MycTB). We selected one rifampin-susceptible and four rifampin-resistant strains, with mutations D435V, H445D, H445Y, and S450L, respectively, mixed them in various proportions in triplicate, tested them with each RDT, and determined the LODs per mutation type. Deeplex-MycTB revealed concordant proportions of the minority resistant variants in the mixtures. The Deeplex-MycTB-validated LODs ranged from 20% to 80% for XpertMTB/RIF, 20% to 70% for Xpert Ultra, 5% to 10% for GenoTypeMTBDRplusv2.0, and 1% to 10% for GenoscholarNTM+MDRTBII for the different mutations. Deeplex-MycTB, GenoTypeMTBDR plus v2.0, and GenoscholarNTM+MDRTBII provide explicit information on rifampin heteroresistance for the most frequently detected mutations. Classic Xpert and Ultra report rifampin heteroresistance as rifampin resistance, while Ultra may denote rifampin heteroresistance through “mixed patterns” of wild-type and mutant melt probe, melt peak temperatures. Overall, our findings inform end users that the threshold for reporting resistance in the case of rifampin heteroresistance is the highest for Classic Xpert and Ultra to resolve phenotypic and genotypic discordant rifampin-resistant TB results.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Open Forum Infect Dis
                Journal ID (iso-abbrev): Open Forum Infect Dis
                Journal ID (publisher-id): ofid
                Title: Open Forum Infectious Diseases
                Publisher: Oxford University Press (US )
                ISSN (Electronic): 2328-8957
                Publication date Collection: June 2020
                Publication date (Electronic): 02 June 2020
                Publication date PMC-release: 02 June 2020
                Volume: 7
                Issue: 6
                Electronic Location Identifier: ofaa204
                Affiliations
                [1 ] Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine , Antwerp, Belgium
                [2 ] Department of Global Health and Social Medicine, Harvard Medical School , Boston, Massachusetts, United States
                [3 ] HIV/TB Unit, Department of Clinical Sciences, Institute of Tropical Medicine , Antwerp, Belgium
                Author notes
                Correspondence: Kamela C. S. Ng, PhD, MSc, Department of Global Health and Social Medicine, Harvard Medical School, 641 Huntington Avenue, Boston, MA 02115 ( kamela_ng@ 123456hms.harvard.edu ).
                Author information
                http://orcid.org/0000-0003-2049-3116
                Article
                Publisher ID: ofaa204
                DOI: 10.1093/ofid/ofaa204
                PMC ID: 7307434
                SO-VID: c4a11c1c-dff8-41df-802e-1e6140ae88d9
                Copyright © © The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence ( http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                History
                Date received : 14 February 2020
                Date: 26 May 2020
                Date accepted : 29 May 2020
                Date: 22 June 2020
                Page count
                Pages: 3
                Funding
                Funded by: Erasmus Mundus Joint Doctorate Fellowship;
                Award ID: 2016-1346
                Funded by: European Research Council, DOI 10.13039/501100000781;
                Categories
                Subject: Brief Report
                Subject: AcademicSubjects/MED00290

                Keywords: extrapulmonary tuberculosis,false mutant melting temperature,false rifampicin resistance,lymph node,xpert ultra
                Data availability:
                Keywords: extrapulmonary tuberculosis, false mutant melting temperature, false rifampicin resistance, lymph node, xpert ultra

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