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.).

Keywords: AFB scanty; OMNIgene.SPUTUM; Xpert; cetylpyridinium chloride; ethanol; isolate; molecular tests; short/long-fragment PCR; sputum; storage.

Copyright © 2019 Sanoussi et al.

Figures

FIG 1

Flow diagram for specimens and methods.

FIG 2

Algorithm for the choice of the suitable reagent for the storage of sputum for TB diagnostics. 1, Molecular tests on the stored sputum (direct molecular tests); 2, from up to 28 days (present study) to 2 to 7 years (20); 3, previously showed by Affolabi et al. (21); 4, extra washing required before inoculation on culture medium (2, 21, 25); 5, reduced/delayed growth after OMNI storage (2, 22, 23); 6, CPC is not compatible with direct MGIT inoculation (24), with reduced/delayed positivity (25), but washing off the CPC prior to inoculation in MGITs (2, 21, 25) increases the culture positivity rate (25); 7, cost OMNI > CPC > ETOH, shipping restriction for specimens stored with CPC and OMNI (IATA category B); 8, when Xpert CT values are <10, a RIF-resistant result should be confirmed on a dilution (1/100) of the remaining sample or a new sample if no leftover remains; 9, not optimal with higher temperature (≥37°C tested in this study), ETOH has shipping restrictions: dangerous good if total volume is >100 ml; 10, short decontamination required before inoculation on culture medium (21); LJ, Löwenstein-Jensen medium; MGIT, mycobacterial growth indicator tube (manual or automated); LJ/MGIT, for LJ or MGIT; CPC/OMNI/ETOH, use either CPC, OMNI, or ETOH; CPC/OMNI, use either CPC or OMNI.