Background
Tanzania is among the 30 countries with the highest tuberculosis (TB) burden worldwide according to the World Health Organization (WHO), with 63,151 TB cases in 2015 [
1]. Of these, 60,563 were new cases, and 1580 were previously treated [
1]. Retreatment cases are of particular concern to public health due to the failure of treatment with first-line drugs that makes it crucial to closely monitor TB cases, which includes drug susceptibility testing (DST) performed by a TB laboratory. In Tanzania, the central tuberculosis referral laboratory (CTRL) is responsible to perform DST nationwide. DST relies in turn on sputum samples, but these often do not reach the CTRL or, when they do, samples show overgrowth with other bacteria such as nontuberculous mycobacteria from the environment or commensal bacteria from the airways [
2,
3]. To correctly identify a positive sputum sample, viability must be preserved and contamination with environmental and commensal bacteria suppressed during transportation [
4,
5]. Cetylpyridinium chloride (CPC) with sodium chloride is a simple reagent with low toxicity to
Mycobacterium tuberculosis that has a mild decontamination effect on bacteria and fungi [
6]. The use of Difco neutralization buffer with CPC treated samples before inoculation has been observed to further increase the culture yield [
7].
Though CPC can preserve sample viability and reduce contamination, which makes it an effective reagent for storage of sputum samples at ambient temperature for several days [
6,
8], rigorous evaluation of the effect of CPC treatment on culture and molecular test performance has been limited in sub-Saharan Africa. We aimed to systematically study the effect of CPC treatment and decontamination time on culture results compared to non-CPC samples, tested if the use of Difco neutralization buffer with CPC treated samples before inoculation can further increase the culture yield, and investigated the performance of Xpert MTB/RIF in CPC treated samples.
Discussion
Our study conducted in the high TB incidence country Tanzania using 200 samples, equally split into a CPC and non-CPC study arm, showed that preservation of sputum samples in CPC increases the TB culture yield and decreases contamination, and that CPC pretreatment has no negative effect on the performance of Xpert MTB/RIF.
Sputum samples can lose viability during long-duration transportation over large distances, which increases contamination by environmental and commensal bacteria present in the sputum and suppresses growth of TB [
6,
10]. CPC is known to be an effective sputum transportation reagent, which decreases contamination by microflora during transportation. However, this has been reported only in studies outside of sub-Saharan Africa [
5,
10‐
12]. Sub-Saharan African countries like Tanzania have a distinct hot and humid climate with a high chance of environmental bacteria to contaminate solid TB cultures [
5]. In contrast to previous studies [
4,
5,
7,
10,
11,
13], we prospectively collected and split the sputum samples into two equal aliquots which allowed a direct comparison between CPC treated and non-CPC samples. Our study also extended sputum sample storage with CPC beyond the usually reported 8 days [
4,
10,
11,
13] to systematically investigate the CPC storage time of 14 and 21 days. Finally, we also tested the effect of CPC treatment on the performance of the molecular assay Xpert MTB/RIF.
The CPC treated samples showed higher culture recovery and lower contamination compared to non-CPC samples, with no negative effect on sample viability even when stored at ambient temperature (25 °C) for up to 21 days. Although TB culturing is resource-intensive and require expensive laboratory facilities, TB cultures remain essential for drug susceptibility testing, drug resistance surveillance and molecular epidemiological studies [
2,
3]. In our study, 15 min of sample decontamination with 1% NaOH yielded optimal culture recovery, contamination, and culture negativity rates. CPC treated samples should be preferably decontaminated using a lower NaOH concentration than the NaOH concentration routinely used for decontamination (1% versus 1.5%) due to the weak decontaminating properties of CPC [
4,
11]. We observed that sputum samples stored with CPC as a preservation method can be stored up to 21 days at room temperature in CPC without reducing the culture recovery rate, consistent with the finding from a previous study conducted in a region of the former Soviet Union [
4]. However, the latter study did not systematically test the effect of storage time on culture yield, but used a range of 7 to 36 days between sample collection and processing.
Surprisingly, we did not observe a superior culture recovery using the neutralizing Difco buffer compared to the standard PBS buffer procedure. The Difco buffer possibly neutralizes the weak anti-bacterial activity of CPC, which might have a negative effect on the culture yield. Our finding is in contrast to a previous study which found that neutralization of CPC treated sputum samples showed a higher culture yield compared to the standard PBS procedure [
7]. This difference might be partially explained by the different sampling method (random assignment to CPC or non-CPC treatment versus splitting of sputum samples in our study), and the different decontamination time used (10 min versus 15 min in our study) [
7].
We did not observe a negative effect of CPC treatment on the performance of the molecular test assay Xpert MTB/RIF. Xpert is a semi-automated molecular assay which detects both
M. tuberculosis complex species and rifampicin drug resistance [
14]. It has been endorsed by WHO and is currently being scaled-up in high TB incidence countries [
15]. Our results support the current WHO guidelines for surveillance of drug resistance in TB stating that sputum samples can also produce reliable results when tested using Xpert MTB/RIF even after a month of CPC sputum sample storage [
16]. Four samples had a positive Xpert MTB/RIF results, but the sputum cultures were either contaminated or negative. However, as all samples were smear microscopy positive they were likely to be Xpert MTB/RIF true positive. A limitation of this analysis is that we performed Xpert MTB/RIF on CPC treated sputum samples only.
Conclusion
In conclusion, in sub-Saharan African settings in which sample collection sites can be distant from laboratories CPC, can be used to preserve sample viability during storage and transportation at ambient temperature for at least 7 to as many as 21 days, with a high culture recovery at an optimal decontamination time of 15 min. Furthermore, CPC treated samples can also be used for molecular analysis such as Xpert MTB/RIF. CPC can be used at minimal costs (~0.01 USD per sample) as preservative in sputum samples during transportation in hot humid areas for laboratories and public health surveillance, sample storage, transport using postal services, and for drug resistance surveys. Unfortunately, CPC treated sputum samples can only be cultured on solid media, but not in liquid culture systems such as BACTEC MGIT due to incompatibility with CPC [
16,
17].
Future research should focus on the use of CPC or other preservation methods [
18] for the use in liquid culture systems such as MIGT as a more sensitive culture detection and more standardized drug susceptibility testing technique than solid culture media. Moreover, addition of neutralizing buffer solutions after pretreatment with CPC to increase culture recovery warrants further exploration.
Acknowledgements
We thank all tuberculosis patients for participating in this study and providing sputum samples. We are grateful to the District and Regional TB coordinators of the Temeke District, as well as the Central Tuberculosis Reference Laboratory and the National TB Programme in Tanzania for their support. This work was supported by funding from the Rudolf Geigy Foundation (Basel, Switzerland).
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