Research Article, J Athl Enhanc Vol: 12 Issue: 5
The Reliability of the Cube Reader LFD to Measure Salivary Cortisol and Salivary Immunoglobulin A in Inter-County Hurling Players
Declan O Rahilly1,2*, Niamh Whelan1,2, Siobhan Moane3
1Department of Sport and Early Childhood, Technological University of Shannon: Midlands Midwest, Limerick, Ireland
2ACTIVE Research Group, Technological University of Shannon: Midlands Midwest, Limerick, Ireland
3LIFE Health & Biosciences Research Institute, Technological University of Shannon: Midlands Midwest, Limerick, Ireland
*Corresponding Author: Declan O Rahilly,
Department of Sport and Early
Childhood, Technological University of Shannon: Midlands Midwest, Limerick,
Ireland; ACTIVE Research Group, Technological University of Shannon:
Midlands Midwest, Limerick, Ireland E-mail: Declan.ORahilly@TUS.ie
Received date: 24 August, 2023, Manuscript No. JAE-23-111207;
Editor assigned date: 28 August, 2023, PreQC No. JAE-23-111207 (PQ);
Reviewed date: 11 September, 2023, QC No. JAE-23-111207;
Revised date: 18 September, 2023, Manuscript No. JAE-23-111207 (R);
Published date: 25 September, 2023, DOI: 10.4172/2324-9080.1000083
Citation: Rahilly DO, Whelan N, Moane S (2023) The Reliability of the Cube Reader LFD to Measure Salivary Cortisol and Salivary Immunoglobulin A in Inter-County Hurling Players. J Athl Enhanc 12:4
Abstract
The aim of this study was to assess the reliability of evening salivary cortisol and salivary Immunoglobulin A (s-IgA) samples measured using the Soma Cube Reader Lateral Flow Device (SCR LFD) in inter-county hurling players. Forty (n=40) male inter- county hurling players provided two saliva samples concurrently to assess for reliability. Good reliability, ICC (r=0.792, P<0.001) was found for salivary cortisol. Similarly, good reliability, Intra class Correlation Coefficient (ICC) (r=0.810, P<0.001) was found for s-IgA. The limit of agreement between test 1 and 2 for salivary cortisol and s-IgA was graphed using a Bland Altman plot. The results showed the data for both tests fell within the 95% Confidence Intervals (CI) range. These findings show the soma cube reader LFD to have good reliability when measuring evening salivary cortisol and s-IgA samples in inter-county hurling players, which is important as training, is scheduled in the evening for these amateur players.
Keywords
Player monitoring; Stress; Immune function; Fatigue; Training.
Introduction
The collection and analysis of saliva in the assessment of physiological biomarkers such as cortisol and Immunoglobulin A (IgA), is a developing trend in sports and exercise [1]. Cortisol is a stress hormone and is released in times of physical and psychological stress with the purpose to mobilise substrates and influence the immune system [2,3]. Previous research by Vining et al., as well as Aardal and Holm have shown that salivary cortisol levels are stronger than serum cortisol [4,5]. Furthermore, according to Vining et al., salivary cortisol is a better measure of the adrenal cortex function. Cortisol is produced in the adrenal cortex and is sensitive to circadian rhythm. During nightly sleep, while the body is in a fasted state, cortisol concentration levels are suppressed. However, it is well-known that cortisol levels increase upon awakening and begin to decrease an hour after wakening [6]. On the other hand, IgA is secreted at mucosal surfaces such as saliva; tears and sweat with small amounts found in blood and has been described as the first line of immune defence against Upper Respiratory Tract Infections (URTI) [7-9]. Moreira et al., reported that those who participate in high-intensity exercise or no exercise were at greater risk of contracting a URTI. However, those who participated in moderate exercise had the lowest risk of a URTI [10].
Although the benefits of analysing saliva in sport and exercise have previously been established by Diaz et al., The popularity of saliva testing is primarily due to it being non-invasive and time efficient [11-13]. Previously, the Enzyme-Linked Immunosorbent Assay (ELISA) has been common practice in research to conduct salivary analysis [14,15]. The ELISA method has long been considered the ‘gold standard’ for salivary analysis. However, given the need for specialised laboratory equipment and personnel as well as the lengthy experimental testing time associated with conducting salivary analysis, make this method un-practical in an applied sporting environment.
Coad et al., reported a bio-sensory device called the Individual Profiling Lateral Flow Device (IPRO LFD) to be reliable (r=0.89) when measuring salivary Immunoglobin A (s-IgA) in physically active men and women. Furthermore, the validity of the device was compared against ELISA with the result showing a strong positive correlation (r=0.93) for s-IgA. Due to technological advancements, a new cube reader has been developed by Soma Bioscience Ltd. A comparative study of the IPRO LFD and cube reader lateral flow device by Dunbar et al., showed the cube reader had good validity for measuring cortisol (r=0.96) and s-IgA (r=0.98) when compared to the IPRO LFD device [16]. The validity of the Cube Reader for analysing salivary cortisol has been examined by Mitsuishi et al., in healthy Japanese university students [17]. It was found that when compared to the ELISA method, the Cube Reader had values (r=0.5-0.75). These results show that the Cube Reader can be used to measure salivary cortisol.
The cube reader LFD is an immunoassay, similar to ELISA. However, the cube reader LFD contains an immune-chromatographic test strip, an immune-chromatographic bio-sensor and reagent buffers. Given that the cube reader is portable, time efficient and does not require specialised laboratory equipment, makes it more suitable to be used in an applied sports setting. To date, salivary testing has been conducted in team sports such as Australian Rules Football (AFL), professional soccer, Rugby union and basketball [18,19]. In Gaelic games, only one study by McGuire et al., has utilised salivary testing with inter-county male Gaelic football players [20]. The purpose of this study is to assess the reliability of evening salivary cortisol and salivary immunoglobin A samples measured using the cube reader LFD (Soma Bioscience, Wallingford, United Kingdom) in highly trained inter-county male hurling players. The studies previously mentioned all conducted testing in the morning as cortisol is highest in the morning. The time of testing of the current study kept in line with the training schedule of the team. As a result of previous research examining morning samples, a dearth of research exists that has examined evening salivary sample and the reliability of the cube reader LFD to measure evening salivary samples.
Materials and Methods
Participants
Forty (n=40) participants (18.59 ± 0.50 years, 179.97 ± 6.12 cm, 76.94 ± 6.41 kg) who were highly trained inter-county male hurling players participated in the study. To be included in the study, participants had to be current members of a male inter-county Hurling team, aged >18 years and train >6 hrs per week. The study was approved by the Technological University of the Shannon: Midlands Midwest Ethics Committee (4.1.4) and all procedures were in accordance with the Declaration of Helsinki.
Procedures
The reliability of salivary cortisol and salivary Immunoglobulin A (s-IgA) using the cube reader (Soma Bioscience, Wallingford, United Kingdom) was analysed in highly trained inter-county male hurling players. Reliability was assessed by determining the variation in salivary cortisol and s-IgA between two separate saliva samples collected concurrently (test 1 and test 2) in the mouth at once and analysed using the Soma Bioscience Oral Fluid Collector (OFC) method.
Salivary collection protocol: Prior to saliva collection, the players were instructed to go about their day as normal eating and hydrating as normal before training. If they encountered any stressful situation prior to training this was recorded as it could affect the cortisol reading. During saliva collection, players placed two separate OFC swabs (Soma Bioscience, Wallingford, United Kingdom) on top of their tongue and closed their mouth until both swabs collected 0.5 ml of fluid and the stem turned blue. The players were instructed to not suck on the swab or move it around their mouth to ensure consistency. Once the stem turned blue, the swab was then placed in the OFC buffer bottle of assays and mixed for 2-minutes in line with the manufacturer’s guidelines. Two drops of the solution were added to the sample window of the Lateral Flow Device (LFD) and left incubate for 15-minutes. The LFD device was then placed in the LFD cube reader with the result given in seconds. All saliva samples were collected in one session which was an evening training session, keeping in line with the regular schedule of the team.
Statistical analysis
The normality of data was assessed using the Shapiro-Wilk test. Reliability of the Soma Bioscience cube reader was assessed using an Inter-class Correlation Coefficient (ICC) analysis, with 95% Confidence Intervals (CI) reported. A Bland Altman plot was used to show the agreement between test 1 and test 2 with 95% CI reported. Alpha intervals were set at p<0.05 for all correlation tests. All of the statistical analysis was conducted using the Statistical Package for the Social Sciences ((SPSS) version 28, Chicago, IL, USA). The strength of the interpretation of the Intra-class Correlation Coefficient (ICC) was evaluated as: <0.5 poor reliability, 0.5-0.75 moderate reliability, 0.75-0.9 good reliability and >0.9 excellent reliability.
Results
Descriptive statistics for test 1 and test 2 of salivary cortisol and salivary immunoglobulin A are shown in Table 1. Good reliability, ICC (r=0.792, P<0.001) was found between test 1 and test 2 for salivary cortisol. Furthermore, good reliability (r=0.810, P<0.001) was found between test 1 and test 2 for s-IgA. A Bland-Altman plot was used to illustrate the agreement between test 1 and test 2 for cortisol and s-IgA. Figure 1 shows the variability of the data for salivary cortisol with most of the data falling within the 95% CI range. The range was set from 2.37 to -2.30 and two outliers are shown on the plot. Figure 2 shows the variability of the data for s-IgA with all the data falling within the 95% CI range. The range was set from 172.5 to -151.4 (Figures 1 and 2) (Tables 1 and 2).
Figure 1: Bland and Altman scatter plot. Test 1 and Test 2 for salivary cortisol. The difference between the 2 tests is presented on the y-axis with the mean value of the 2 tests on the x-axis. The broken lines represent 95% CI.
Figure 2: Bland and Altman scatter plot. Test 1 and Test 2 for s-IgA. The difference between the 2 tests is presented on the y-axis with the mean value of the 2 tests on the x-axis. The broken lines represent 95% CI.
| Cortisol t1 | Cortisol t2 | s-IgA t1 | s-IgA t2 | |
|---|---|---|---|---|
| Mean ± SD | 3.66 ± 1.67 | 3.62 ± 1.97 | 196.59 ± 152.54 | 186.07 ± 111.16 |
| Mean 95% CI Lower | 3.188 | 3.16 | 165.61 | 150.48 |
| Mean 95% CI Upper | 4.2 | 4.37 | 239.74 | 226.69 |
| Maximum | 8 | 8.9 | 954.4 | 583.4 |
| Minimum | 1.3 | 1.4 | 41.3 | 39.2 |
| Note: CI= Confidence Intervals; s-IgA= salivary Immunoglobulin A. | ||||
Table 1: Descriptive statistics of test 1 and test 2 for salivary cortisol and salivary Immunoglobulin A (s-IgA).
| ICC | 95% CI Upper limit | 95% CI Lower limit | |
|---|---|---|---|
| Salivary cortisol | 0.792 | 0.884 | 0.639 |
| s-IgA | 0.81 | 0.894 | 0.669 |
| Note: ICC= Intraclass Correlation Coefficient; 95% CI= 95% Confidence Intervals; s-IgA= salivary Immunoglobulin A. | |||
Table 2: Reliability values for salivary cortisol and s-IgA.
Discussion
The aim of the current study was to compare two evening samples of salivary cortisol and s-IgA collected concurrently to assess the reliability of the cube reader LFD (Soma Bioscience, Wallingford, United Kingdom). The main finding from the current study was the cube reader LFD showed good reliability and no systematic bias when measuring salivary cortisol and s-IgA. These findings would suggest that the cube reader LFD is a reliable instrument to assess salivary markers in highly trained male inter-county Hurling players.
The Intra-class Correlation Coefficient (ICC) provides a measurement on the agreement between values [21]. Within the current study, the ICC for salivary cortisol of 0.792 and s-IgA 0.810, represent good reliability. These results support the findings of previous research by Coad et al., who found s-IgA had an ICC value (r=0.89). Fisher and McLellan also found salivary cortisol had ICC value (0.85) [22]. Furthermore, Fisher and McLellan showed the Lateral Flow Device (LFD) was valid when compared against the ELISA method with no significant difference found between both methods (r=0.45). To further assess the reliability of the cube reader LFD, the Bland-Altman analysis was used to assess systematic bias. According to Bland and Altman, the Bland-Altman plot is used for two testing points [23]. It shows the level of agreement between both time points and can identify any outliers that fall outside the 95% CI. Within the current study, two outliers were identified when testing the difference between test 1 and test 2 of salivary cortisol with no outliers identified in the analysis of s-IgA. The Bland-Altman plot for salivary cortisol Figure 1 and for s-IgA Figure 2 show no systematic error and an agreement between test 1 and test 2 as the points are evenly distributed either side of the zero line. These findings are similar to those of Coad et al., who presented similar findings for s-IgA. Also, Mitsuishi et al., used a Bland-Altman plot to assess the reliability of the cube reader LFD against the ELISA method with no systematic error reported.
The findings of the current study are in agreement with those previously published by Coad et al; Fisher et al; Dunbar et al., and Mitsuishi et al., who all found a LFD to be reliable at measuring salivary cortisol and s-IgA. Due to the high demands that are placed on inter-county players, the results of the current study are significant. During periods of intense activity such as pre-season or in-season competition, the cube reader can provide real time information on the internal status of players. Previous research conducted by Cormack et al., in AFL and Rowell et al., in professional soccer assessed salivary cortisol. Similarly, Morgans et al., in professional soccer and Tiernan et al., professional Rugby union have assess s-IgA [24,19]. The purpose of the aforementioned research was to assess the recovery status of players which is an important component at reducing the risk of injury and/or illness especially during periods of high workload or during competition [25,26]. To date, only one study has investigated the seasonal variation in salivary cortisol and s-IgA with inter-county male players. As the reliability of the cube reader LFD has been establish in this current study with inter-county male players, it is important that salivary testing be conducted in GAA to objectively establish the recovery status of the players.
Practical Applications
The time of testing in the current study differs from those previously published. Given the amateur status of Gaelic Athletic Association (GAA) players, it is important that testing fits into schedule of the players. Therefore, keeping with the training schedule of the players, testing was conducted in the evening, which would be common practice among inter-county teams. As a result, establishing the reliability of the cube reader LFD for use in inter-county players to measure evening salivary samples is significant. The cube reader LFD is a time efficient tool that can be used to monitor salivary cortisol and s-IgA with inter-county male players. The stress and immune function of players can be established rapidly which provides objective feedback to a coach. When used in conjunction with subjective monitoring, will give a full rounded view of the players recovery status. This can have the potential to reduce the risk of injury and/or illness.
Conclusion
As GAA players are amateur by status, train similar to professionals but lack the time for recovery, it is essential that the players are monitored. Previous research by Moreira et al., has highlighted the effect different stressors such as deprived sleep and psychological challenges can have on mucosal immunity. Although the timing of testing in the current study differs from those previously published, establishing the reliability of the cube reader will provide confidence to practitioners when using this method in GAA. The cube reader provides results of the physiological status of the player within 15 minutes of testing. Previously, to establish this level of information, the ELISA method was used. Given that this unpractical in applied setting, the results of this study helps bridge the gap between lab and field based measures. Further testing should be conducted in a similar cohort to provide greater confidence to practitioners when using the cube reader to assess salivary markers.
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