Short-Term Storage Does Not Affect the Quantitative Yield of Mycobacterium tuberculosis in Sputum in Early-Bactericidal-Activity Studies

Abstract

dEarly-bactericidal-activity (EBA) studies measure the change in mycobacterial load in sputum over time to evaluate antituberculosis drugs. We investigated whether a delay in sputum processing influences the quantitative results of sputum mycobacterial culture. We identified pretreatment smear-positive sputum samples collected overnight and processed at a single laboratory. Sputum volume, time from sputum collection to processing, CFU counts/ml of sputum, and time to culture positivity (TTP) data were retrieved. We obtained 817 TTP and 794 CFU results from a total of 844 sputum samples. Contamination did not occur more frequently with prolonged storage (TTP, 2.0%; CFU, 2.4%). Sample volumes were <5 ml in 5%, 5 to 10 ml in 46%, and >10 ml in 49%. Delays to processing were 0, 1, 2, and 3 days in 696 (43.2%), 722 (44.8%), 128 (7.9%), and 65 (4.0%) samples, respectively. TTP and CFU did not significantly differ between days of delay to processing (P ‫؍‬ 0.098 and P ‫؍‬ 0.908, respectively), but there was a nonsignificant trend toward a prolonged TTP over time (P ‫؍‬ 0.052, Jonckheere-Terpstra trend test). Sputa of <5 ml in volume showed a significantly prolonged TTP compared to sputum of >5 ml (113 h versus 99 h; P < 0.01) but no significant decrease in CFU. Sputum can be stored under refrigerated conditions for deferred processing for at least 3 days. This means that central laboratories can be used for quantitative mycobacterial study endpoints when delays to processing are not expected to exceed a few days. Care should be taken to collect sputum of sufficient volume. Sputum culture for Mycobacterium tuberculosis remains a cornerstone of clinical mycobacteriology. The detection of viable M. tuberculosis in sputum establishes the diagnosis of pulmonary tuberculosis (TB), and sustained sputum culture conversion is a marker of clinical improvement and cure. Storage of sputum for deferred culture may be considered where decentralized health care centers rely on a central laboratory service, but this may result in loss of viability and false-negative cultures (1-4) or overgrowth of contaminants, particularly if sensitive liquid media are used (5-7). Beyond its clinical purpose, sputum culture can quantify the viable M. tuberculosis sputum load expressed as CFU per ml of sputum counted on agar plates or as times to culture positivity (TTP) of a volume of decontaminated sputum inoculated in liquid mycobacterial culture (8). High sputum mycobacterial loads at diagnosis have been associated with treatment failure and recurrence (9, 10). Early-bactericidal-activity (EBA) studies are considered a key step in the clinical evaluation of individual antituberculosis agents and new combination regimens and describe the change in log CFU and TTP on consecutive sputum samples collected over up to 14 treatment days (2, 11).EBA studies commonly include small groups of sputum smear-positive, treatment-naïve patients who are given investigational treatments under controlled conditions in hospital. The change of the bacterial sputum loa...

The early bactericidal activity of antituberculosis drugs

“…This might lead to an interval of several days before sputum specimens are delivered to the laboratory with a likely loss of viability of a proportion of bacilli and an increased risk of contamination. This was recently evaluated during series of EBA studies during which 817 TTP and 794 CFU results were processed [68]. Time elapsed before processing varied from 0 to 3 days and specimens were maintained on ice at 2-8˚C when any delay occurred in processing of the specimen.…”

Laboratory Diagnosis of Mycobacterium tuberculosis Infection and Disease in Children

“…If transport to the laboratory is delayed by Ͼ1 h, specimens should be refrigerated at 4°C as well as upon arrival in the laboratory until they are processed. One study in adults showed that mycobacterial load and culture time to positivity were not significantly affected by refrigerated storage for Յ3 days (12). If prolonged storage or transport is unavoidable, preservatives can be added to the specimens to inhibit growth of contaminant bacteria and thus improve the yield from culture.…”

Folding of 16S rRNA in a Signal‐Producing Structure for the Detection of Bacteria

“…For example, a typical sputum sample of a Mycobacterium tuberculosis -infected individual may contain 10 5 –10 6 cells/ml. [7] Using RNAse inhibitors that prevent RNA degradation in the whole-cell assay may further improve the detection limit. The assay is selective, since little or no fluorescence increase was observed in the presence of RNA from a non-targeted bacterium.…”