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      Storage of Sputum in Cetylpyridinium Chloride, OMNIgene.SPUTUM, and Ethanol Is Compatible with Molecular Tuberculosis Diagnostic Testing

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          Abstract

          We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples).

          ABSTRACT

          We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples). Each patient’s sputum was stored with either CPC, ETOH, or OMNI for 28 days at ambient temperature, with subsequent rpoB amplification targeting a short fragment (81 bp, GeneXpert MTB/RIF [Xpert]) or a long fragment (1,764 bp, in-house nested PCR). For 36 patients, Xpert was also performed at baseline on all 108 fresh sputum samples. After the 28-day storage (D28), Xpert positivity did not significantly differ between storage methods. In contrast, higher positivity for rpoB nested PCR was obtained with OMNI ( n = 125, 94%) than with ETOH ( n = 114, 85.7%; P = 0.001). Smears with scanty acid-fast bacilli (AFB) had lower rpoB PCR positivity with ETOH storage ( n = 10, 41.7%) than with CPC ( n = 16, 66.7%; difference, 25%; 95% confidence interval [CI], 3.5 to 46.5; P = 0.031) or OMNI ( n = 16, 69.6%; difference, 26.1%; 95% CI, 3.8 to 48.4; P = 0.031), with no difference between CPC and OMNI. Poststorage, the threshold cycle ( C T ) values significantly decreased compared to those prestorage with ETOH (difference, −1.1; 95% CI, −1.6 to −0.6; P = 0.0001) but not with CPC ( P = 0.915) or OMNI ( P = 0.33). For one patient’s ETOH- and CPC-stored specimens with a C T of <10, Xpert gave results of rifampin false resistant at D28, which was resolved by repeating Xpert on a 1/100 diluted specimen. In conclusion, 28-day storage of sputum in OMNI, CPC, or ETOH at ambient temperature does not impact short-fragment PCR (Xpert), including for low smear grades. However, for long-fragment PCR, ETOH yielded a lower PCR positivity for low smear grades, while the performance of OMNI and CPC was excellent for all smear grades. (The study has been registered at ClinicalTrials.gov under registration number NCT02744469.)

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          Most cited references 15

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          Disputed rpoB mutations can frequently cause important rifampicin resistance among new tuberculosis patients.

          Greater Mymensingh area, Bangladesh.
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            Inactivation of Mycobacterium tuberculosis for DNA typing analysis.

            DNA fingerprinting analysis of Mycobacterium tuberculosis is used for epidemiological studies and the control of laboratory cross-contamination. Because standardized procedures are not entirely safe for mycobacteriology laboratory staff, the paper proposes a new technique for the processing of specimens. The technique ensures the inactivation of M. tuberculosis before DNA extraction without the loss of DNA integrity. The control of inactivated cultures should be rigorous and should involve the use of two different culture media incubated for at least 4 months.
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              Newly developed primers for comprehensive amplification of the rpoB gene and detection of rifampin resistance in Mycobacterium tuberculosis.

              New rpoB gene primers for detecting Rif(r) in Mycobacterium tuberculosis complex bacteria achieved 100% specificity and 88% (fresh sputa) and 92% (ethanol-preserved sputa) diagnostic sensitivity and detected up to 4 CFU/sample. Of the 99 Rif(r) isolates examined, 97% had mutations within cluster I, 2% at codon 176, and 1% at codon 497.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                J Clin Microbiol
                J. Clin. Microbiol
                jcm
                jcm
                JCM
                Journal of Clinical Microbiology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0095-1137
                1098-660X
                15 May 2019
                25 June 2019
                July 2019
                25 June 2019
                : 57
                : 7
                Affiliations
                [a ]Laboratoire de Référence des Mycobactéries, Cotonou, Benin
                [b ]Unit of Mycobacteriology, Institute of Tropical Medicine, Antwerp, Belgium
                [c ]Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
                Carter BloodCare & Baylor University Medical Center
                Author notes
                Address correspondence to C. N’Dira Sanoussi, ndirasanoussi@ 123456gmail.com .

                Citation Sanoussi CN, de Jong BC, Affolabi D, Meehan CJ, Odoun M, Rigouts L. 2019. Storage of sputum in cetylpyridinium chloride, OMNIgene.SPUTUM, and ethanol is compatible with molecular tuberculosis diagnostic testing. J Clin Microbiol 57:e00275-19. https://doi.org/10.1128/JCM.00275-19.

                Article
                00275-19
                10.1128/JCM.00275-19
                6595455
                31092592
                Copyright © 2019 Sanoussi et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                Page count
                supplementary-material: 1, Figures: 2, Tables: 4, Equations: 0, References: 25, Pages: 9, Words: 6031
                Product
                Funding
                Funded by: Directorate General for Development (DGD) Belgium;
                Award ID: FA4
                Award Recipient : Award Recipient : Award Recipient : Award Recipient :
                Funded by: European Research Council-INTERRUPTB starting grant;
                Award ID: 311725
                Award Recipient : Award Recipient : Award Recipient :
                Categories
                Mycobacteriology and Aerobic Actinomycetes
                Custom metadata
                July 2019

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