About the Author(s)


Danielle Martin symbol
Department of Clinical Microbiology and Infectious Diseases, National Health Laboratory Service and Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

Rispah Chomba symbol
Department of Clinical Microbiology and Infectious Diseases, National Health Laboratory Service and Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

Tshegofatso Pelego symbol
National Institute of Communicable Diseases, Centre for Tuberculosis, Johannesburg, South Africa

Sanelisiwe T. Duze Email symbol
Department of Clinical Microbiology and Infectious Diseases, National Health Laboratory Service and Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

Citation


Martin D, Chomba R, Pelego T, Duze ST. In silico and in vitro validation of a duplex polymerase chain reaction assay for detecting Cronobacter sakazakii in powdered infant milk. Afr J Lab Med. 2025;14(1), a2902. https://doi.org/10.4102/ajlm.v14i1.2902

Original Research

In silico and in vitro validation of a duplex polymerase chain reaction assay for detecting Cronobacter sakazakii in powdered infant milk

Danielle Martin, Rispah Chomba, Tshegofatso Pelego, Sanelisiwe T. Duze

Received: 13 June 2025; Accepted: 16 Sept. 2025; Published: 30 Nov. 2025

Copyright: © 2025. The Authors. Licensee: AOSIS.
This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/).

Abstract

Background: Cronobacter sakazakii causes life-threatening infections in neonates, primarily transmitted through contaminated powdered infant formula (PIF). In low- and middle-income countries, limited surveillance and diagnostic capacity hinder accurate detection of C. sakazakii, highlighting the need for rapid and affordable testing methods.

Objective: To validate a duplex polymerase chain reaction (PCR) assay for rapidly detecting Cronobacter spp. and C. sakazakii in PIF using both in silico and in vitro approaches.

Methods: This study was conducted in South Africa between March and August 2022. Seven gene targets and their primers were selected from published literature. To assess sensitivity and specificity, in silico PCR was performed using genome sequences of Cronobacter and non-Cronobacter species from the National Center for Biotechnology Information database. The best-performing primers were selected for an in vitro analysis using bacterial isolates and PIF samples from the Infection Control Service Laboratory. The specificity of the assay was assessed using eight foodborne pathogens, and further evaluated using PIF samples artificially contaminated with Cronobacter spp., Bacillus cereus, and Salmonella Typhi.

Results: The best-performing primers, lpfA_1, fimG, and fimp1, showed 100% sensitivity and specificity. Duplex PCR assay successfully detected both Cronobacter spp. and C. sakazakii with no cross-reactivity with non-Cronobacter pathogens, and remained effective in the presence of contaminants such as B. cereus and Salmonella Typhi.

Conclusion: The validated duplex PCR assay offers a rapid, specific, and affordable assay for detecting Cronobacter spp. and C. sakazakii in PIF.

What this study adds: This assay combined in silico and in vitro validation of a rapid and affordable PCR assay for PIF screening in resource-limited settings.

Keywords: Cronobacter spp.; Cronobacter sakazakii; powdered infant formula; polymerase chain reaction; specificity; sensitivity.

Introduction

Cronobacter sakazakii is a Gram-negative, opportunistic, motile, food-borne bacterium within the Enterobacterales family.1 Originally described as ‘yellow-pigmented Enterobacter cloacae’, it was renamed Enterobacter sakazakii in 1980, and later reclassified under the genus Cronobacter.2,3 The genus currently comprises seven species: Cronobacter condimenti, C. dublinensis, C. malonaticus, C. muytjensii, C. sakazakii, C. turicensis, and C. universalis.3 Among these, only C. sakazakii, C. turicensis, and C. malonaticus are associated with human diseases, particularly C. sakazakii, the most clinically significant species, often associated with neonatal meningitis.4,5,6,7 The majority of C. malonaticus infections occur in adults and are less severe.6

Cronobacter sakazakii is a clinically significant pathogen, particularly in neonates and infants, where infections can lead to meningitis, necrotising enterocolitis, septicaemia, and even death.8,9 Premature infants, or those with low birthweight or a compromised immune system, are at an even greater risk of infection, and outbreaks of disease have been reported in hospital units for newborns.10,11 Cronobacter infections are often attributed to contaminated powdered infant formula (PIF), which is a nonsterile product.1,11,12 Cronobacter sakazakii has also been isolated from breast milk expressed with contaminated pump equipment,13,14 as well as other environmental sources, including pacifiers, bottles, feeding utensils, and kitchen sink surfaces, all of which serve as potential points of contamination for PIF.14

Despite its severity, the public health impact of C. sakazakii is often underestimated, particularly in low- and middle-income countries because of the absence of active surveillance systems, underreporting of cases, and inadequate diagnostic infrastructure.15 Although C. sakazakii has been detected in various food products and environments across Africa, including South Africa,16,17,18 Egypt,19,20,21 Côte d’Ivoire,22 and Nigeria,23 epidemiological data remain scarce. This diagnostic gap underscores the need for rapid, accurate, and affordable detection methods to ensure the safety of PIF and enable timely public health interventions, as C. sakazakii infections are associated with high morbidity and mortality.7 Moreover, survivors often suffer irreversible neurological sequelae, such as brain abscesses, quadriplegia, developmental delays, hydrocephalus, and motor impairment.5,24,25,26

Traditional culture-based methods, such as those recommended by the US Food and Drug Administration, the International Organization for Standardization, and the International Dairy Federation (ISO/TS 22964:2006), are widely used for detecting C. sakazakii in PIF and are considered reliable.27 However, these methods are labour-intensive, require a pre-enrichment step, and can take up to six days to yield results. Additionally, traditional biochemical tests and chromogenic media used for confirmation are limited by phenotypic overlap between Cronobacter spp. and other Enterobacteriaceae, which can lead to misidentification.27,28 Furthermore, competition from faster-growing organisms and interference from background microflora can reduce recovery rates and hinder accurate detection.29

Molecular methods, such as polymerase chain reaction (PCR), offer higher sensitivity and specificity, along with faster turnaround times. Existing PCR assays often lack species-level specificity and may require multiple reactions to differentiate closely related Cronobacter spp.4,30,31 Moreover, the limited availability of rapid and accurate detection methods is compounded by the high cost and technical complexity of the existing assays, making routine testing especially challenging in resource-limited settings. This study, therefore, aimed to optimise and validate an accurate and affordable duplex PCR assay for the rapid and simultaneous detection of Cronobacter spp. and C. sakazakii in PIF, ideal for use in resource-limited settings.

Methods

Ethical considerations

An ethics waiver (reference number: W-CBP-220422-03) was obtained from the Witwatersrand Human Research Ethics Committee (Medical), University of the Witwatersrand, Johannesburg, South Africa. The study was conducted between March 2022 and August 2022, and involved no human participants or animal research. Bacterial isolates and PIF samples were sourced from the Infection Control Service Laboratory of the National Health Laboratory Service. The PIF samples were tested for contaminants during routine food testing at the Infection Control Service Laboratory before use in artificial contamination experiments. All of the experiments complied with institutional and laboratory biosafety and ethical guidelines. The data generated contained no personal identifiers and were managed following institutional data protection policies.

Study design

This study was a laboratory-based validation combining in silico and in vitro approaches to evaluate the performance of primers for a duplex PCR assay that can rapidly detect Cronobacter spp. and C. sakazakii in PIF. The study was conducted at the Infection Control Service Laboratory of the National Health Laboratory Service at the University of the Witwatersrand in Johannesburg, South Africa, between March 2022 and August 2022.

Sample collection

This study used a collection of 27 complete Cronobacter genome sequences (20 C. sakazakii, 2 C. malonaticus, 1 C. turicensis, 1 C. dublinensis, 1 C. universalis, 1 C. muytjensii, and 1 C. condiment), and 20 complete genome sequences of non-Cronobacter foodborne pathogens, including three Salmonella Typhi, two Shigella sonnei, two Listeria monocytogenes, three E. cloacae, two Escherichia coli, three Bacillus cereus, two Klebsiella pneumoniae, and three Staphylococcus aureus. The genome sequences were downloaded as FASTA files from the National Center for Biotechnology Information database (NCBI: https://www.ncbi.nlm.nih.gov) and were used in the in silico PCR analysis. In addition, 11 previously characterised foodborne pathogens and four PIF samples (PIF7069, PIF6748, PIF7070, and PIF6749) from two brands were obtained from the Infection Control Service Laboratory through routine diagnostics; these were used for the in vitro PCR analysis. The foodborne pathogens included three Cronobacter isolates (C. turicensis, C. sakazakii M0020, and C. sakazakii M0022) and eight non-Cronobacter isolates (Salmonella Typhi, S. sonnei, L. monocytogenes, E. cloacae, E. coli, B. cereus, K. pneumoniae, and S. aureus). Routine food testing confirmed that PIF7069, PIF6748, and PIF7070 were free of bacterial contamination, whereas PIF6749 was positive for B. cereus.

Gene targets and primer selection

A literature search through PubMed, Google Scholar, and Scopus was performed to identify gene targets for the molecular detection of Cronobacter spp. and C. sakazakii. The search was performed using the following terms: ‘Cronobacter sakazakii’, ‘Cronobacter spp.’, ‘PCR’, ‘gene target’, ‘species-specific primers’, ‘genus-specific primers’. Seven gene targets were selected based on published studies that reported their potential diagnostic value in differentiating Cronobacter spp., specificity, sensitivity, and availability of the primer sequences. These seven gene targets included six species-specific genes (cgcA, gyrB, rpoB, fimp1, fimG, lpfA_1) and one genus-specific gene (grxB). The corresponding primer sequences for each gene target were downloaded and used in the in silico PCR analysis (Table 1).

TABLE 1: Gene targets and primer sequences for detecting Cronobacter spp. and Cronobacter sakazakii.
In silico PCR analysis

Genome sequences of the 27 Cronobacter species and 20 non-Cronobacter foodborne pathogens were used in the in silico PCR analysis to determine the sensitivity and specificity of the primer sequences for each gene target. The in silico PCR was performed using an online platform available at http://insilico.ehu.es/PCR/, which simulates theoretical PCR amplification based on primer sequences and genome sequences.35 Each primer pair was tested against each of the 47 genome sequences, using default settings to determine the presence or absence of an amplification product matching the expected amplicon size. The in silico PCR analysis provides the predicted start position and size of the amplicon. Results were recorded as positive (+) if the expected product size was detected, and negative (-) if no product was observed. Thereafter, the sensitivity and specificity of each primer set were calculated using Equations 1 and 2. The species-specific primers with the highest sensitivity and specificity for C. sakazakii detection were selected for further validation in vitro.

PCR optimisation and specificity

Cronobacter turicensis, C. sakazakii M0020, and C. sakazakii M0022, and eight non-Cronobacter foodborne pathogens were cultured on nutrient agar (Thermo Fischer Scientific, Waltham, Massachusetts, United States) and incubated overnight at 36 °C. Crude genomic DNA was extracted by suspending two loopfuls of bacterial colonies in 200 µL of tris-ethylenediaminetetraacetic acid (TE) buffer, followed by incubation at 95 °C for 25 min, vortexing, and centrifugation at 12 000 rpm for 3 min. The supernatant was collected and stored at 8 °C until further use. Two singleplex PCR assays targeting the lpfA_1 and grxB genes, were optimised in a 25 µL reaction volume containing 12.5 µL of 2X PCR Master Mix (Thermo Fischer Scientific, Waltham, Massachusetts, United States), 2 µL of DNA, 0.2 µM primers, and nuclease-free water. The PCR cycling conditions were 95 °C for 3 min, 35 cycles of 95 °C for 30 s, 51 °C for 30 s, 72 °C for 60 s, followed by a final extension at 72 °C for 10 min. Amplicons were resolved by gel electrophoresis on a 2% agarose gel stained with GelRed™ (Anatech, Johannesburg, South Africa), using 1X Tris-Borate-EDTA buffer at 110 V, and visualised using the GelDoc system (Bio-Rad Laboratories, Hercules, California, United States).

The duplex PCR assay targeting both lpfA_1 and grxB was optimised under the same conditions, with each primer added at a final concentration of 0.2 µM. In all PCR reactions, C. turicensis and C. sakazakii M0020 were used as positive controls for the grxB and lpfA_1, and nuclease-free water was used for the negative control. The specificity of the duplex PCR assay was evaluated using genomic DNA from C. sakazakii M0022, C. turicensis, and eight non-Cronobacter foodborne pathogens listed above. The specificity of the assay was calculated using Equation 2.

Artificial contamination

To artificially contaminate the PIF samples with C. sakazakii, 5 µL of a suspension (108 colony-forming units/mL) was added to 10 g of each sample in a sterile glass bottle with 100 mL buffered peptone water, followed by incubation for 6 h at 36 °C. This resulted in an estimated final concentration of approximately 5.0 × 106 colony-forming units/mL in the pre-enrichment. The PIF7070 sample was also inoculated with 5 µL of Salmonella Typhi (108 colony-forming units/mL). After incubation, 40 mL of the pre-enrichment broth was centrifuged at 3000×g for 10 min. The pellet was resuspended in 200 µL of supernatant, and genomic DNA was extracted using the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany), followed by PCR amplification using the duplex PCR assay. Powdered infant formula samples PIF7070 and PIF6749 were specifically used to evaluate assay interference. The experiment was performed in duplicate.

Data analysis

All data were collected and recorded using Microsoft Excel® 2010. For both in silico and in vitro PCR assays, amplification results were recorded based on the presence or absence of the expected amplicon size. Similarly, results from the artificially contaminated PIF samples were classified as positive or negative according to band visualisation on a 2% gel electrophoresis. A true positive was defined as a C. sakazakii or Cronobacter spp. yielding a positive amplification with the expected amplicon size. A true negative was defined as a non-Cronobacter foodborne pathogen (or genome sequence) with no amplification, confirming assay specificity. False positives were recorded when amplification occurred in non-Cronobacter spp., and false negatives when the assay failed to amplify the target genes for Cronobacter spp. The results were tabulated, and the sensitivity and specificity were calculated using Equation 1 and Equation 2:

Results

In silico PCR performance

In total, seven primer pairs corresponding to the seven gene targets for Cronobacter spp. and C. sakazakii detection were evaluated against 47 genome sequences using in silico PCR. Species-specific primers targeting the lpfA_1, fimG, and fimp1 genes demonstrated 100% sensitivity and specificity for C. sakazakii detection during in silico PCR. The cgcA and gyrB primers had 100% specificity, with sensitivities of 94.7% and 89.5%, each missing one or two C. sakazakii sequences during the in silico PCR (Table 2). The rpoB primers exhibited the lowest performance, with sensitivity and specificity of 78.9% and 92.6%, whereas the genus-specific grxB primers detected all Cronobacter spp. except C. dublinensis and C. condiment, resulting in 92.3% sensitivity and 100% specificity (Table 2). Based on these results, lpfA_1 and grxB primers were selected for in vitro testing.

TABLE 2: Summary of the sensitivity and specificity of each primer pair based on in silico polymerase chain reaction analysis.
In vitro PCR performance

Two singleplex PCR assays targeting the lpfA_1 and grxB genes were successfully optimised and effectively detected the respective genes in C. sakazakii M0022, C. sakazakii M0020, and C. turicensis (Figure 1a). Additionally, a duplex PCR assay targeting both genes was optimised, demonstrating simultaneous detection of both the lpfA_1 and grxB genes in C. sakazakii M0022, C. sakazakii M0020, while only detecting the grxB gene in C. turicensis (Figure 1b).

FIGURE 1: (a) Gel electrophoresis of the singleplex polymerase chain reaction (PCR) assays targeting the lpfA_1 (881 bp; Lanes 2–5) and grxB (378 bp; Lanes 6–9) genes: Lane 1, DNA ladder (100 bp, Thermo Fischer Scientific, Waltham, Massachusetts, United States); Lane 2, Cronobacter sakazakii M0022; Lane 3, C. turicensis; Lane 4, C. sakazakii M0020; Lane 5, negative control; Lane 6, C. sakazakii M0022; Lane 7, C. sakazakii M0020; Lane 8, C. turicensis; and Lane 9, negative control. (b) Duplex PCR assay targeting C. sakazakii and Cronobacter spp. in a single reaction: Lane 1, DNA ladder (Fastruler, Thermo Fischer Scientific, Waltham, Massachusetts, United States); Lane 2, C. sakazakii M0022; Lane 3, C. sakazakii M0020; Lane 4, C. turicensis; and Lane 5, negative control.

The duplex PCR assay was 100% specific for Cronobacter spp. and C. sakazakii, with successful amplification of the grxB gene in C. sakazakii M0022, C. sakazakii M0020, and C. turicensis, as well as the amplification of the lpfA_1 gene in C. sakazakii M0022 and C. sakazakii M0020, and no amplification in any of the eight non-Cronobacter foodborne pathogens (Figure 2a). Moreover, the lpfA_1 and grxB genes were successfully detected in the PIF samples artificially contaminated with C. sakazakii M0022 (Figure 2b, Table 3), with no interference from the non-Cronobacter foodborne pathogens in PIF samples PIF6749 (containing B. cereus) and PIF7070 (containing Salmonella Typhi). These results demonstrate the assay’s reliability and suitability for detecting Cronobacter spp. and C. sakazakii in PIF, even in the presence of other foodborne pathogens.

FIGURE 2: (a) Gel electrophoresis showing the specificity of the polymerase chain reaction (PCR) assay: Lane 1, DNA ladder (100 bp, Promega, Madison, Wisconsin, United States); Lanes 2–9, non-Cronobacter species (Escherichia coli, Listeria monocytogenes, Bacillus cereus, Salmonella Typhi, Klebsiella pneumoniae, Staphylococcus aureus, Enterobacter cloacae, Shigella sonnei); Lane 10, Cronobacter turicensis; Lane 11, C. sakazakii M0022; Lane 12, C. sakazakii M0020; and Lane 13, negative control. (b) Polymerase chain reaction results from the artificially contaminated powdered infant formula (PIF) samples: Lane 1, DNA ladder (Fastruler, Thermo Fischer Scientific, Waltham, Massachusetts, United States); Lane 2 and Lane 5, PIF contaminated with C. sakazakii M0022; Lane 3, PIF with B. cereus and C. sakazakii M0022; Lane 4, PIF with Salmonella Typhi and C. sakazakii M0022; Lane 6, C. sakazakii M0020; Lane 7, C. turicensis; and Lane 8, negative control.

TABLE 3: Cronobacter sakazakii detection in artificially contaminated powdered infant formula samples.

Discussion

In silico PCR is a valuable tool used to predict amplification efficiency, specificity, and amplicon size by aligning primers against target genomes, facilitating the selection of optimal primer pairs for PCR assays.36 In our study, primers targeting the rpoB, cgcA, and gyrB genes exhibited suboptimal sensitivity and specificity for C. sakazakii detection. The rpoB gene requires two separate PCR reactions to differentiate between C. sakazakii and C. malonaticus, limiting its diagnostic utility.31 Our findings are consistent with previous studies reporting limited species-level resolution of the cgcA and gyrB primers.31,33

In contrast, primers targeting the fimp1, fimG, and lpfA_1 showed 100% specificity and sensitivity in silico. However, the short amplicon size of fimp1 (103 bp) reduces its suitability for conventional PCR. Prior studies investigating the suitability of fimG and lpfA_1 genes for C. sakazakii detection using bioinformatics and in vitro approaches demonstrated superior sensitivity for lpfA_1 over fimG, supporting the selection of the lpfA_1 gene for further validation.4 The genus-specific grxB gene, while slightly less sensitive, showed broad detection across Cronobacter genomes and 100% specificity within our study sample, reinforcing its value for genus-level identification. Our findings are consistent with previous studies that have successfully employed the grxB gene for the detection of Cronobacter spp.34,36,37,38

In vitro validation confirmed the efficacy of the selected primers in detecting Cronobacter spp. and C. sakazakii. Singleplex and duplex PCR assays targeting lpfA_1 and grxB successfully detected C. sakazakii and C. turicensis with high specificity. The duplex assay enabled simultaneous detection of both targets in artificially contaminated PIF, even in the presence of other foodborne pathogens such as B. cereus and Salmonella Typhi. These results corroborate the in silico findings and demonstrate the assay’s robustness and reliability.

Importantly, the duplex PCR assay reduced turnaround time to approximately 12 h, compared to the 3–6-day protocol required by the conventional culture-based method recommended by the United States Food and Drug Administration.27,39 This time efficiency, combined with high specificity and affordability, makes the assay particularly suitable for resource-limited settings where rapid screening is essential for public health interventions and outbreak response.

Although culture-based methods remain the cornerstone of microbiological diagnostics, particularly for strain isolation, antimicrobial susceptibility testing, and whole-genome sequencing, which are essential for surveillance and outbreak investigations. The duplex PCR assay reported in this study will complement culture-based methods by serving as a rapid screening tool for C. sakazakii in PIF, enabling early detection of contaminated PIF products. The assay will facilitate timely interventions and support targeted use of culture-based confirmation, especially in resource-limited settings where laboratory capacity and turnaround time are constrained. By identifying positives quickly, this PCR assay will enable resource-limited laboratories to prioritise samples for culture, thereby optimising resource use and improving overall diagnostic turnaround time.

Limitations

This study had a few limitations. The in vitro sensitivity of the duplex PCR assay could not be fully assessed because of the limited availability of C. sakazakii and other Cronobacter spp. isolates, despite efforts to source additional strains from public and private laboratories. However, incorporating in silico validation of the primers using publicly available genome sequences from multiple Cronobacter species allowed us to evaluate primer specificity across diverse species. In silico analysis thus provided a valuable alternative for assay validation. This approach also supports the growing utility of bioinformatics tools in enhancing assay design. Another limitation of this study is that the sensitivity of the assay was determined using high bacterial concentrations, which may not fully represent the variable contamination levels of C. sakazakii typically found in PIF, as contamination may occur at low levels, and accurate detection under such conditions is critical for food safety. Future work should therefore include the determination of the assay’s limit of detection using serial dilutions of C. sakazakii spiked into PIF. While this study focused on conventional PCR to ensure affordability and accessibility in basic laboratory settings, it may not fully meet the throughput and automation needs of modern food safety laboratories. Adapting the assay into a real-time multiplex PCR assay will allow for quantitative detection, improved sensitivity, and reduced turnaround time. Additionally, integrating automated DNA extraction workflows could streamline sample processing and minimise human error, making the assay more suitable for high-throughput environments and outbreak response scenarios. These enhancements would support broader implementation in both resource-limited and well-equipped laboratories, contributing to more efficient monitoring of Cronobacter contamination in infant formula.

Conclusion

This assay combined in silico and in vitro validation of a rapid and affordable duplex PCR assay for the simultaneous detection of Cronobacter spp. and C. sakazakii in PIF. The performance of the assay was not affected by the presence of non-Cronobacter species such as B. cereus and Salmonella Typhi. This duplex PCR assay can facilitate the diagnosis of C. sakazakii in PIF, particularly in resource-limited settings, contributing to improved epidemiological surveillance and screening of Cronobacter spp. in PIF. Its adaptability for routine diagnostics and outbreak investigations further underscores its potential for integration into national food safety monitoring programmes.

Acknowledgements

The authors would like to acknowledge the staff of the Infection Control Service Laboratory of the National Health Laboratory Service for providing us with the bacterial isolates and powdered infant formula samples used in this study. This article is partially based on Danielle Martin’s thesis entitled, ‘Optimizing a PCR assay for rapid detection of Cronobacter sakazakii in powdered infant formula’, towards the degree of BHSc Honours in the Department of Clinical Microbiology and Infectious Diseases, University of the Witwatersrand, South Africa, submitted in December 2022, with supervisors Rispah Chomba, Tshegofatso Pelego and Sanelisiwe T. Duze. The thesis is not publicly available.

Competing interests

The author reported that they received funding from National Research Foundation which may be affected by the research reported in the enclosed publication. The author has disclosed those interests fully and has implemented an approved plan for managing any potential conflicts arising from their involvement. The terms of these funding arrangements have been reviewed and approved by the affiliated university in accordance with its policy on objectivity in research.

Authors’ contributions

All of the authors contributed to this manuscript. S.T.D. and R.C. conceptualised the original idea for the study and supervised this study. D.M. and T.P. carried out the laboratory work, data collection, and analysis; D.M. and S.T.D. wrote the manuscript; and R.C., D.M., T.P., and S.T.D. reviewed the final draft of the article.

Funding information

This work was supported by the National Research Foundation (NRF) under Sanelisiwe T. Duze (Grant Number 138279).

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

Disclaimer

The views and opinions expressed in this article are those of the authors and are the product of professional research. The article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. The authors are responsible for this article’s results, findings, and content.

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