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

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 (CT) 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 CT 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.)

Acquired rifampicin resistance during first TB treatment: magnitude, relative importance, risk factors and keys to control in low-income settings

“…preservation of 0.5 mL diagnostic (new or recurrence) sputum added to 1 mL denatured ethanol in hermetically closing cryovials. 13 To identify specimens unequivocally, transparent Scotch tape covered cryovial labels with laser-preprinted specimen-unique identification codes routinely used already on request forms, registers and treatment cards. Cryovials were kept at the clinics at room temperature in special collection boxes, arranged sequentially (code-defined) for easy retrieval in case of recurrence/LTFU.…”

Apport Du Genexpert MTB/Rif Dans La Detection De L’infection a Mycobacterium Tuberculosis Et La Resistance a La Rifampicine a L’hopital General De Reference De Kirungu-Moba, Sud-Est De La Republique Democratique Du Congo

“…La durée de transport et de stockage des crachats est de ce fait fonction de la technique utilisée pour la conservation. D'où la pratique de l'utilisation de cétylpyridinium (CPC), PrimeStone ® MTN, l'éthanol et l'Omnigene ® Sputum dans les situations exigeant la prolongation de la durée de conservation et de transport des échantillons [73], [75], [76]. Les résultats des analyses de culture microbienne et de Xpert MTB/RIF sont similaires à ceux du transport et conservation des échantillons dans les conditions standards (transport au frais à la température ambiante ou de 2 à 8°C) [74], [75]…”

High Throughput Sequencing for Clinical Tuberculosis: An Overview

“…When intended for culture, multiple samples are ideal to increase the chance of enough viable organism for growth. If not used immediately, samples are stored at −80 • C. Sputum, specifically, is usually stored with cetylpyridinium chloride (CPC), ethanol, or commercially available products such as intended to help preserve M. tuberculosis DNA, decrease contamination, and improve culture yield [32,33]. DNA extraction on sputum or culture isolates has used a variety of methods and kits including ethanol precipitation with pre-treatment steps [7,27,34], the Maxwell 16 FFPE Tissue LEV DNA Purification Kit (Promega, Madison, WI, USA) [18][19][20], QIAamp DNA-Mini-Kit (Qiagen) [35], and the Roche Cobas Amplicor extraction kit [13].…”