Transitioning from Triton X-100 to Tergitol 15-S-9: impacts on diagnostic assays using viral PCR sample solution

Abstract

In 2019, the European Union banned Triton X-100, a detergent widely used in laboratory diagnostics, including the Viral PCR Sample Solution (VPSS), and urged manufacturers to find environmentally sustainable alternatives. Tergitol 15-S-9 (VPSS2) has been proposed as an alternative surfactant. This multicenter study evaluated the effectiveness of VPSS2, a Tergitol-based viral solution, as a replacement for VPSS. Our results show the equivalent performance of VPSS2 to VPSS for nucleic acid extraction and viral stability over time at different temperatures. The new VPSS formulation was also tested against external quality assurance panels and clinical samples. The results of this work support adopting this modified viral PCR sample solution to replace Triton X-100-containing viral transport solutions.

TWEETABLE ABSTRACT

The European Union has banned Triton X-100. All reagents containing it should be replaced. Could a new Viral PCR Sample Solution (VPSS) containing Tergitol 15-S-9 be a suitable replacement?

METHOD SUMMARY

In this article, we describe the evaluation of a new Tergitol-based Viral PCR Sample Solution (VPSS), here named VPSS2. The effectiveness of VPSS2 in deactivating SARS-COV-2, and the impact on the thermal stability and nucleic acid extraction of various viruses were studied using 17 diagnostic platforms. These platforms, distributed across 13 diagnostic centres in Scotland, included Abbott M2000, Abbott Alinity, ABI 7500, Roche Liat and Cepheid GeneXpert.

Keywords: :

• cycle threshold (Ct)
• SARS-CoV-2
• Tergitol 15-S-9
• Triton X-100
• viral load

The Viral PCR Sample Solution (VPSS) is a liquid formulation created to preserve and transport viral specimens, such as nasopharyngeal swabs in SARS-CoV-2 testing, while also inactivating potential pathogens that might be present in the specimen. It contains Triton X-100 (4-(1,1,3,3-tetramethylbutyl) phenol, ethoxylated), a detergent extensively used in medical diagnostic assays for its ability to release intracellular materials, including nucleic acids [1,2]. However, Triton X-100 has been shown to have environmental and health toxicity, which led to its outlawing by the European Union (EU) in 2019 [3].

Since the EU ban, the pharmaceutical, biomedical and biotechnological industries have been actively searching for novel surfactants with less environmental impact than Triton X-100 [4,5], but work has yet to be conducted on the appropriateness of commercially available alternatives for molecular diagnostics. This study aimed to evaluate the technical suitability of a new viral transport media – here named VPSS2 – with a formulation that replaces Triton X-100 for Tergitol 15-S-9 (polyethylene glycol ether, CAS 68131-40-8).

Tergitol 15-S-9 is a biodegradable detergent with low toxicity, and it has been shown to inactivate SARS-CoV-2 in specific immunoassays [6]. So far, no comprehensive studies have addressed the performance of this surfactant in diagnostic assays. Viral PCR Sample Solution (VPSS, cat. no. BM1675) and its new formulation (here named VPSS2, cat. no. BM1677) were provided by E&O laboratories. Their formulation contains 50% minimum essential media and 50% lysis buffer with either Triton-X-100 (VPSS) or Terigitol 15s9 (VPSS2). A list of the platforms and diagnostic assays can be found in Supplemental Table S1.

VPSS has been manufactured to inactivate viral particles, a crucial step of laboratory safety. We assessed viral inactivation by incubating SARS-CoV-2 in either VPSS, VPSS2 or PBS spiked at a 1:10 ratio for 30–60 min at 6.5–8.0°C. Viral load was determined by real-time PCR, and following buffer exchange, the 50% tissue culture infectious dose (TCID₅₀ endpoint dilution assay) was assessed in Vero E6 (ATCC CRL-1586), as described by Craig et al. [7]. VPSS2 and VPSS demonstrated comparable effectiveness in inactivation of SARS-CoV-2.

Changes in nucleic acid extraction in real-time PCR can impact test result accuracy, sensitivity and reliability. We evaluated the effect of the new formulation (VPSS2) by measuring the amplification of internal controls (IC; murine cytomegalovirus, equine arteritis virus or phocine herpesvirus). Nine real-time PCR platforms from different manufacturers (ROCHE Magna Pure96, Abbott M2000, Easymag (2), Roche COBAS, SEEGENE Starlet, Thermo Fisher Scientific Amplitude, Abbott Alinity M, Cepheid [n = 4] and Roche LIAT [n = 3]) were used. No significant differences were observed between VPSS and VPSS2 (mean Ct values = 27.20 and 27.09, respectively), suggesting VPSS2 is as efficient as VPSS for viral extraction and does not cause extraction inhibition.

VPSS formulations must guarantee the preservation and the integrity of genetic material that will be transported at room temperature (RT). The stability of viral particles in VPSS2 and VPSS was evaluated using DNA and RNA viruses responsible for respiratory or genital infections with high prevalence in the community: SARS-CoV-2, rhinovirus, adenovirus and HSV-2. The conditions tested included different temperatures (RT, 4°C, -20°C and -80°) and incubation times (0, 1, 3, 7, 14, 28 and 60 days), and the Ct values were monitored using RT real-time PCR. All viruses were stable in VPSS2 over time, except that at RT, SARS-CoV-2 lost stability (detected by an increase in Ct) and was undetectable after 2 months (Figure 1).

Figure 1. Stability of viral nucleic acids. Viruses remain stable in ultra-low freezers (left) and at room temperature (right), except for SARS-CoV-2, which rapidly declines over time at room temperature, becoming undetectable after 2 months.

Introducing new reagents can impact the performance of a diagnostic test. To ensure it complied with quality standards, we evaluated VPSS and VPSS2 against external quality controls. For this, quality control for molecular diagnostics produced and delivered 27 external quality assurance panels to 12 NHS Scotland Virology Diagnostic Laboratories. The panels comprised 25 frozen vials of positive and negative SARS-CoV-2 samples (lineages alpha and B.1.153) in VPSS and VPSS2 (the full sample report can be found in Supplemental Table S1 and Summary Statistics, in Supplemental Table S2). A contingency table showing the agreement between VPSS and VPSS2 is shown in Table 1.

Table 1. Contingency table for external quality assurance panels detected by each formulation and summary statistics.

		VPSS2
		Positive	Negative	Total
VPSS	Positive	266	2	268
	Negative	2	54	56
	Total	268	56	324
Summary statistics		Percent	Low limit	High limit
Positive agreement (PPA)		99.3%	97.3%	99.8%
Negative agreement (PNA)		96.4%	87.9%	99.0%
Overall agreement (POA)		98.8%	96.9%	99.5%

PPA = [a/(a+c)]*100; PNA = [d/b+d))]*100; POA = [(a+d)/n]*100.

VPSS2 false negatives: lab code GB240, sample VSERSCV2_21-20 and 21.

VPSS false negative: lab code GB003, sample VSERSCV2_21-09 and lab code GB225, sample VSERSCV2_21-05.

Viral transport media is a key preanalytical factor affecting the performance of nucleic acid amplification tests [8]. The UK Standards for Microbiology Investigations require revalidation of laboratory tests to ensure their fitness for the purpose [9]. This study evaluated for the first time the technical suitability of Tergitol 15-S-9 as a replacement for Triton X-100-containing VPSS in automated diagnostic platforms through viral inactivation, nucleic acid extraction and viral stability studies.

Our results suggest that VPSS containing Tergitol 15-S-9 (VPSS2) is a suitable alternative to Triton X-100-based solutions for molecular diagnostic assays, as demonstrated through various assays and testing platforms. However, some of the limitations in the study, mainly arising from the constraints imposed by the COVID-19 pandemic, should be acknowledged. The overall evaluation was conducted in multiple centers using different protocols and analyzers, with a limited number of viruses and bacterial samples, potentially restricting the generalizability of the findings to a broader range of pathogens. Second, and also due to the considerable strains on the NHS services at the time, we performed studies with an insufficient number of replicates. This is the case of the thermal and temporal stability assessments which, despite showing acceptable results, could have benefitted from a larger sample size and more extended time frames to enhance the reliability of conclusions.

Here, we demonstrate that Tergitol-based VPSS2 performs as well as Triton X-100-containing VPSS. The new formulation meets regulatory and environmental standards, and is an efficient nucleic acid extraction and viral inactivation solution. Viral thermostability of SARS-CoV-2, rhinovirus, HSV-2 and adenovirus was acceptable for laboratory time frames, despite the viral load reduction seen for SARS-CoV-2 at RT over time. The results from assessing the external quality assurance panels in VPSS2 in all laboratories across Scotland reinforce the use of VPSS2. Our data support previous findings on the role of transport media and preanalytical conditions [8,10,11] and provide valuable information for laboratories and researchers regarding viral storage and stability.

Author contributions

A Zorzoli: data curation, data analysis and writing of the original draft. A MacLeanb, S Nicholsonb, A Danielsc, S Hughesb, S Bennet-Slaterb, C Tait-Burkardc, NE Sakkad, R Gunsonb: methodology, investigation, data analysis and editing of the original draft. K Templeton: conceptualization and the overall supervision of the study.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.