Association of the quantitative oral health status of patients with COVID-19 with their organ status and immunity: A single-centre observational study

Objectives To evaluate the prevalence of oral complications in patients with severe COVID-19; investigate the association between their oral health, organ status, and immunity; and determine whether the resazurin disc test is an effective substitute for the Oral Assessment Guide. Research methodology/design A single-centre observational study. Setting Intensive care unit with restricted access specialising in extracorporeal membrane oxygenation for COVID-19 treatment. Main outcome measures We investigated the oral health of 13 patients with COVID-19 receiving extracorporeal membrane oxygenation therapy between April and December 2021 using the Oral Assessment Guide and colour reactive resazurin disc test. The Sequential Organ Failure Assessment and Prognostic Nutritional Index were used to assess organ status and immunity, respectively. The correlation of oral health status with organ status and immunity was investigated. Results High bacterial levels, revealed by the resazurin disc test, were associated with elevated Oral Assessment Guide scores, indicating oral health deterioration, particularly in terms of teeth and dentures. Increased Sequential Organ Failure Assessment scores and decreased Prognostic Nutritional Index were correlated with poor oral health, revealed by the Oral Assessment Guide and resazurin disc test. Conclusion Poor oral health is an important risk factor for severe COVID-19 complications in patients admitted to an intensive care unit. The Oral Assessment Guide and resazurin disc test can evaluate oral conditions; however, the resazurin disc test is quantitative and does not require salivary specimens to be transferred outside the patient ward for evaluation. The resazurin disc test can be a useful substitute for the Oral Assessment Guide in intensive care units with restricted access. Implications for clinical practice The resazurin disc test can be used for quantitative assessment of patients’ oral condition in isolation wards. Multidisciplinary management of patients with COVID-19 should be promoted and involve oral healthcare providers such as dentists and dental hygienists.


Introduction
For unknown infectious diseases, qualitative and quantitative evaluations of patients' specimens are essential for elucidating disease pathogenesis and thereby obtaining adequate information for effective treatment. However, irrespective of whether the causative bacteria or virus has been identified, the access of medical staff to wards and the transfer of specimens out of wards for quantitative testing may be restricted based on infectious disease control laws. These events occurred during the COVID-19 outbreak. Donning of personal protective equipment (e.g. gloves, gowns, face shields, and surgical masks) was required for entry into isolation wards (D'Amico et al., 2020), thereby resulting in high medical costs and waste generation.
Oral health deterioration is associated with mortality among patients with COVID-19 who are receiving extracorporeal membrane oxygenation (ECMO) in an intensive care unit (ICU), regardless of the duration of ECMO (Yoshino et al., 2021). Based on the findings of another study (Yoshino et al., 2022) that investigated the immunomodulatory relationship between the oral and gut microbial compartments, the oral-gut axis may be associated with mortality in patients with COVID-19. In both studies, an oral assessment was performed only by permitted dedicated nurses wearing personal protective equipment and not by oral care specialists. Thus, despite using the standardised Oral Assessment Guide, the subjective opinion of individual nurses may have influenced the results. The use of a more objective, easy, and inexpensive method of assessing oral conditions is therefore recommended.
The resazurin disc test involves the use of a sheet of sterile filter paper (8-mm diameter) with sucrose as the carbon source and resazurin sodium salt as the indicator (RD test 'Showa'; GC Showayakuhin Corporation, Tokyo, Japan) to quantify cariogenic bacteria in saliva (e.g. Streptococcus mutans and Lactobacillus) based on the degree of sheet discolouration (Kanazawa and Kuramata, 1966). This product is widely used in Japan to monitor oral hygiene status, and a resazurin chromogenic agar assay has recently been reported as effective for the early screening of carbapenemase among Enterobacteriaceae bacteria (Teethaisong et al., 2016). This test utilises the property of cariogenic bacteria to quickly activate and discolour resazurin when incubated in a sucrose culture medium at 37 • C with no aeration. A drop of the saliva specimen is placed on the sheet, which is then sealed with a film and incubated. Instead of an incubator, the sheet can also be applied to the inside of a patient's upper arm. Several methods of oral assessment using saliva exist, such as polymerase chain reaction and other new molecular techniques; however, most necessitate special equipment, whereas some require incubators (Kaczor-Urbanowicz et al., 2017). The resazurin disc test does not require special equipment or the transport of the specimens from the bedside and can potentially be used to quantitatively evaluate oral deterioration rapidly and easily in isolation wards.
Therefore, this study aimed to evaluate prevalence of oral complications in patients with severe COVID-19 receiving ECMO in the ICU and investigated the association of their oral condition with their organ status and immunity. The resazurin disc test was employed to quantitatively assess oral health deterioration. The organ status was investigated using the Sequential Organ Failure Assessment (SOFA) score, whereas the immunity was investigated using Prognostic Nutritional Index (PNI).

Objectives
The aim of this study was to quantitatively evaluate the oral health of patients with COVID-19 and investigate the association of their oral health with their organ status and immunity.

Setting
This single-centre observational study was conducted in the Fukuoka University Hospital ECMO centre, which is an ICU with restricted access that specialises in ECMO treatment.

Ethical approval
This study was approved by the Clinical Research and Ethics Centre of Fukuoka University (No. U21-03-006). The patients' surrogates provided informed consent.

Participants
Adult patients with COVID-19 treated at the ECMO centre between April and December 2021 who were able to undergo an oral assessment and the resazurin disc test were included in this study.

Data collection
The following patient data were collected: age, sex, in-hospital mortality, Acute Physiology and Chronic Health Evaluation II score, length of stay at the centre, details of mechanical ventilation, and details of the ECMO treatment and duration. We investigated the correlations among the findings of the oral assessment, the resazurin disc test, SOFA, and PNI assessment, which were performed on the same day for each participant.
Objective oral assessments were conducted, as described previously (Yoshino et al., 2021). The oral health status of all eligible patients was evaluated by nurses on the first day of admission using the Oral Assessment Guide from Fukuoka University (OAG-F) scoring system, which is always used to assess oral health at this centre; thereafter, all patients underwent an oral assessment daily. The OAG-F scoring system has the following eight categories: voice, swallowing, lips, tongue, saliva, mucous membranes, gums, and teeth and dentures; each category is scored on a three-point scale. The higher the score, the worse the condition.
The resazurin disc test was performed before initiating oral health care. The patients' saliva was collected. A drop of the saliva was placed on the test sheet, which was then sealed with film and applied to the patient's inner upper arm for 15 min during oral health care. The number of cariogenic bacteria on the test sheet ( Fig. 1) was scored as 1, 2, or 3 (i.e. the resazurin disc score), indicating low, middle, and high bacterial levels, respectively. All patients were tested twice a week.
The SOFA score is widely employed in the daily monitoring of acute morbidity in critical care units. It is based on six different scores for the respiratory, cardiovascular, hepatic, haemostatic, renal, and neurological systems, respectively. Each system is scored from 0 to 4 with each increase in score reflecting worsening organ dysfunction (Vincent et al., 1998). Investigators have recently reported that SOFA scores can be used to assess the severity of COVID-19 (Yang et al., 2021). Therefore, in the present study of patients with severe COVID-19, the total SOFA scores were investigated.
PNI is used to indicate a patient's immune and nutritional status based on albumin and lymphocyte counts in the blood (Kosovali et al., 2021). A low PNI indicates immune dysregulation and is a prognostic factor for malignancy and infection (Hua et al., 2019;Li et al., 2018;Zhou et al., 2017). The PNI can also be used to predict mortality in patients with COVID-19 in the ICU (Kosovali et al., 2021). In this study, the PNI was employed as an indicator of immunity. The PNI was calculated as 10 × serum albumin (g/dL) + 0.005 × total lymphocytes count (per mm 3 ) (Onodera et al., 1984).
Oral health care was administered based on a previously described protocol (Yoshino et al., 2021). In brief, all patients received continuous oral care comprising cleaning the oral mucosa and tongue using a sponge brush 3-8 times a day. The frequency and methods of oral care were adjusted based on the ventilator status and oral assessment results of the patients.

Outcome measures
The OAG-F score is based on a nurse's assessment of a patient's oral health status, and the total score and categorical score were determined. The resazurin disc score was assessed as a quantitative indicator of oral hygiene status. The total SOFA score was examined as an indicator of organ status. PNI was surveyed as an indicator of immunity.

Statistical analyses
The correlations among the resazurin disc, total OAG-F, total SOFA, and PNI scores were determined using Spearman's rank correlation coefficient. Absolute rs values were classified as follows: 0-0.19, a very weak correlation; 0.2-0.39, a weak correlation; 0.40-0.59, a moderate correlation; 0.6-0.79, a strong correlation; and 0.8-1.00, a very strong correlation. Multiple linear regression analysis was conducted to examine the relationship between the resazurin disc score and OAG-F categorical scores. P values of < 0.05 were considered to indicate a statistically significant difference. Data were analysed using JMP version 14.0 (JMP Institute, Tokyo, Japan).

Characteristics of the patients
Thirteen patients were enrolled in this study (Table 1). The median age of the patients was 53 (interquartile range: 46-60) years, and 92% of the patients were men. Two patients died during hospitalisation in the centre. The median length of the hospital stay of all patients was 27 (interquartile range: 13-79) days.
In 105 instances, both oral evaluation and resazurin testing were performed on the same day; therefore, the OAG-F scores and the resazurin disc scores on these days were subjected to correlation analysis. The SOFA scores were concurrently obtained on 100 days, and these scores were included in the correlation analysis with the OAG-F scores and the resazurin disc scores. The PNI was evaluated on 103 of the 105 days, and the results were correlated with the OAG-F scores and the resazurin disc scores.

Correlations between the resazurin disc and OAG-F scores
A significant positive weak correlation was found between the resazurin disc and total OAG-F scores (Fig. 2). Multiple regression analysis revealed that the 'teeth and denture' score among the OAG-F scores affected the resazurin disc score (Table 2).

Correlations between the total SOFA, resazurin disc, and total OAG-F scores
High resazurin disc scores were weakly correlated with high SOFA scores (Fig. 3a). High SOFA scores were weakly associated with high total OAG-F scores (Fig. 3b).

Fig. 1.
The resazurin disc test. The number of cariogenic bacteria was determined at three levels (low, medium, and high), based on the discolouration level, and was scored as 1, 2, or 3 (i.e. resazurin disc score), respectively.

Correlations between the PNI, resazurin disc, and total OAG-F scores
A decreased PNI score indicated immune dysregulation. A significant negative, weak correlation was found between the PNI and resazurin disc scores (Fig. 4a). The PNI and total OAG-F scores had a significant negative moderate correlation (Fig. 4b).

Discussion
Accurate diagnosis and treatment of unknown infectious diseases require thorough and frequent examination of patient specimens. However, during the COVID-19 outbreak, the entry of medical staff into isolation wards and the transfer of specimens for evaluation were restricted.
The usefulness of the OAG-F scoring system, an objective measure to assess the oral health of patients with infectious diseases receiving ICU management, has been previously reported (Yoshino et al., 2021). It involves a quantitative assessment, although it can be time-consuming. Furthermore, the evaluation is subjective and based on the judgement of the examiner. Additional objective and quantitative evaluations are therefore desirable for greater accuracy. In this study, the resazurin disc test was employed to quantitatively evaluate the oral health status of patients without requiring their specimens to be transferred outside a unit for testing. The unique aspect of this method is that the oral health status can be quantitatively measured by means of a colour reaction using the patient's body as an incubator for the disc. Furthermore, compared with the OAG-F scores, it has a better discriminatory value for early and accurate detection of oral deterioration, regardless of the professional administering the test.
In the present study, a high total OAG-F score, which indicates oral health deterioration, was statistically associated with an elevated resazurin disc score, thereby suggesting that the resazurin disc test is an effective substitute for the OAG. Furthermore, the film can be applied to the patient's arm before starting oral care. This allows the clinician to administer oral care while waiting for the colour reaction, thereby making the process efficient.
The resazurin disc test also showed a correlation between elevated resazurin disc scores and impaired dental conditions, as indicated by the OAG-F scores. This finding suggested that the morphologic complexity of the oral cavity may contribute to worsening of patients' oral hygiene and an increase in the oral microbial load. Furthermore, for patients with severe COVID-19 receiving ECMO in the ICU, the presence of many remaining teeth or extensive prostheses may impair the dental condition and prevent effective oral hygiene intervention by nurses. The dental condition of each patient is different; therefore, professional oral care requires relevant expertise and skills. Referring professional oral care to nurses can lead to a greater personal burden on them. Regular assessment and professional oral care by oral healthcare providers such as dentists and dental hygienists is therefore necessary. Collaboration between the medical teams may enable the delivery of safer and higher quality medical care to patients with severe COVID-19.
Poor oral health according to high OAG-F scores is an important risk factor for severe complications in patients with COVID-19 receiving ECMO management in the ICU (Yoshino et al., 2021). The existence of a poor oral-gut axis, accompanied by immunity disturbances in patients with severe COVID-19, was confirmed through a blood-based   inflammatory parameter, neutrophil-to-lymphocyte ratio, and impairment criteria, based on the gut microbial community (i.e. the Bristol Stool Form Scale) (Yoshino et al., 2022). In this study, we identified weak but significant correlations between the SOFA score, which facilitates reliable and accurate quantification of sepsis and organ dysfunction, and higher resazurin disc and OAG-F scores. This finding suggested that poor oral health had reproducible relevance with organ failures and immune dysregulation. Yang et al. (2021) reported that the SOFA score can be applied to predict patient severity and multiple organ failure. They also suggest that organ failure in COVID-19 patients primarily occurs with a cytokine storm and immunosuppression.
In this study, we hypothesised that oral deterioration may be associated with a deterioration in the systemic status due to immune dysregulation. Kosovali et al. (2021) reported that a PNI score ≤ 42 is an independent risk factor for mortality in patients with COVID-19 admitted to the ICU. Thus, PNI can serve as a predictor of mortality (Kosovali et al., 2021). The PNI score was below 42 at all points measured in the present study, although the mortality rate was low at 15% (Table 1). This finding is inconsistent with that of Kosovali et al. (2021). This difference may be attributable to the small sample size (n = 13), the lifesaving effects of ECMO treatment, despite the severe conditions, and malnutrition due to severe COVID-19. However, the correlation between immune dysregulation and oral deterioration observed in the present study is consistent with previous findings (Yoshino et al., 2022).
The described quantitative evaluation methods for evaluating patients' general status and local conditions, especially oral health conditions, helped detect the coexistence of oral dysbiosis, organ failure, and impaired immunity, and thereby predict the outcomes of patients with severe COVID-19. Furthermore, combining routine oral hygiene intervention and systemic therapy could lead to the development of more effective treatments. Therefore, a multidisciplinary collaboration involving oral care professionals such as dentists and dental hygienists, as well as physicians, nurses, pharmacists, nutritionists, and professionals from other related disciplines, could help improve the management of patients with severe infectious diseases.

Limitations
This study has a few limitations, including its small sample size. Furthermore, the genetic characteristics of the host and viral variants were not considered. Other factors, such as the type of drug used, could have been considered to increase the value of the study. However, the various conditions would not provide a sufficient amount of data for evaluation. In this pilot study, we aimed to evaluate the changes in the oral and systemic status on a broad scale. In addition, 6 of the 13 patients used mouthwash for oral care owing to family preference. The possibility of differences between the antimicrobial mouthwash and water influencing the RD scores cannot be ruled out; however, this was outside the scope of our study and, therefore, not examined. We did not include a control group. No standard evaluations of the periodontal status were conducted because of the limited access of oral health care specialists in the centre.

Conclusion
Considering the restricted access to the ICU, the resazurin disc test was particularly useful as it could be completed within the ward. Among patients with severe COVID-19 in an infectious disease isolation ICU, oral deterioration was correlated with organ failure and impaired immunity. The resazurin disc test could therefore be an effective substitute for the OAG scoring system. A multidisciplinary collaboration involving oral care professionals such as dentists, dental hygienists, and nutritionists could improve the management of critical patients with infectious diseases.

Ethical statement
This study was approved by the Clinical Research and Ethics Centre of Fukuoka University (No. U21-03-006). The patients' surrogates provided informed consent.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fukuoka University Hospital) for his support in data collection.

Funding
This work was supported by the Grant of the Clinical Research Promotion Foundation (2021) [grant number  and JSPS KAKENHI [Grant Number 22K10303]. The sponsor had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Clinical Trial registration number
Not applicable.