Validation of a noninvasive aMMP‐8 point‐of‐care diagnostic methodology in COVID‐19 patients with periodontal disease

Abstract Objectives The aim of this study was to validate an active matrix metalloproteinase (MMP‐8) point‐of‐care diagnostic tool in COVID‐19 patients with periodontal disease. Subjects, Materials, and Methods Seventy‐two COVID‐19‐positive and 30 COVID‐19‐negative subjects were enrolled in the study. Demographic data were recorded, periodontal examination carried out, and chairside tests run for evaluating the expression of active MMP‐8 (aMMP‐8) in the site with maximum periodontal breakdown via gingival crevicular fluid sampling as well as via a mouth rinse‐based kit for general disease activity. In COVID‐19‐positive patients, the kits were run again once the patients turned COVID‐19 negative. Results The overall (n = 102) sensitivity/specificity of the mouthrinse‐based kits to detect periodontal disease was 79.41%/36.76% and that of site‐specific kits was 64.71%/55.88% while adjusting for age, gender, and smoking status increased the sensitivity and specificity (82.35%/76.47% and 73.53%/88.24, respectively). Receiver operating characteristic (ROC) analysis for the adjusted model revealed very good area under the ROC curve 0.746–0.869 (p < .001) and 0.740–0.872 (p < .001) (the aMMP‐8 mouth rinse and site‐specific kits, respectively). No statistically significant difference was observed in the distribution of results of aMMP‐8 mouth rinse test (p = .302) and aMMP‐8 site‐specific test (p = .189) once the subjects recovered from COVID‐19. Conclusions The findings of the present study support the aMMP‐8 point‐of‐care testing (PoCT) kits as screening tools for periodontitis in COVID‐19 patients. The overall screening accuracy can be further increased by utilizing adjunctively risk factors of periodontitis. The reported noninvasive, user‐friendly, and objective PoCT diagnostic methodology may provide a way of stratifying risk groups, deciding upon referrals, and in the institution of diligent oral hygiene regimens.


| INTRODUCTION
Respiratory failure due to acute respiratory distress syndrome (ARDS) has been implicated to be the most prominent cause for COVID-19-related mortality (Malek et al., 2020). Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) reportedly has the potential to induce pulmonary tissue alterations via numerous pathways, of which one involves matrix metalloproteinases (MMPs; Malek et al., 2020). This pathophysiology is an uncanny reflection of that involving SARS CoV-2.
MMPs lead to the degradation of extracellular matrix and modify the immune response. Besides mediating pulmonary tissue remodeling, these factors consequentially relate to increased vascular permeability along with endothelium damage (Malek et al., 2020). Mechanical ventilation for ARDS management can further cause lung injury as a result of ventilationinduced MMP-8 elaboration (Kong et al., 2020;Malek et al., 2020). In fact, the eventual mortality of patients has been associated with the elevated levels of MMP-2, MMP-9, MMP-8, and TIMP-1, as observed in the early stages of sepsis (Lauhio et al., 2011). MMP-8 expression has also been evidenced to be indicative of systemic compromise and multiorgan pathoses (Lauhio et al., 2011). There is ample evidence to support the association between compromised clinical outcomes and elevated levels of certain systemic inflammatory biomarkers. Reportedly, MMPs also play a role in enhancing early viral entry into cells (Yates et al., 2020).
Periodontitis is one of the most prevalent chronic inflammatory oral diseases. Proteinases, pertinent of which, collagenase, responsible for bringing about matrix degradation in the presence of periodontitis is primarily derived from polymorphonuclear leukocytes (PMNs) of the diseased periodontium. Periodontopathogens of potency can elaborate proteases responsible for activating latent MMP-1 and -8 by direct proteolysis while instigating their secretion from gingiva-derived fibroblasts and oral keratinocytes, as well as via infiltrating inflammatory cells (Ding et al., 1996;Sorsa et al., 1995Sorsa et al., , 2006. Subsequent to PMN release, these latent MMPs transform into their activated counterparts as a result of proteolytic cleavage or reactive oxygen species interaction (Ding et al., 1996). Elevated levels of PMN-derived collagenolytic  have been evidenced in the saliva, gingival crevicular fluid (GCF), and gingival tissue of periodontitis patients Gangbar et al., 1990;Kiili et al., 2002;Lee et al., 1995;Mancini et al., 1999;Romanelli et al., 1999;Sorsa et al., 2015Sorsa et al., , 2006. The rapidity of PMN degranulation accompanied by the enzyme release upon microbial phagocytosis provides further strength to the validity of this pathway in the pathophysiology of periodontitis ).
An active MMP-8 (aMMP-8) point-of-care (PoC) test, has been validated across countries in both the adolescent and adult populations as a tool to define active and inactive sites of periodontal disease, evaluate prognosis, and assess patients during the treatment and maintenance phases (Alassiri et al., 2018;Heikkinen et al., 2016;Izadi Borujeni et al., 2015;Johnson et al., 2016;Lähteenmäki et al., 2020;Leppilahti et al., 2018;Lorenz et al., 2017;Nwhator et al., 2014;Räisänen et al., 2018Räisänen et al., , 2019Sorsa et al., 2020. This PoC testing methodology, in particular, exhibits a sensitivity of 76%-83% and specificity of 96% with results being reported in a time frame of 5-7 min (Sorsa et al., , 2017. Of late, numerous hypotheses and some studies have suggested the possibility of a link between periodontal disease and COVID-19 adverse outcomes Gupta, Saarikko et al., 2022;. In fact, SARS-CoV-2 has also been recovered from GCF, further strengthening its rationale to be utilized as a diagnostic fluid (Gupta, Mohindra et al., 2021). Identification of this potential corisk factor by means of a PoC tool would be invaluable to healthcare personnel and medical professionals in deciding referrals, identifying patient risk groups, and reinforcing oral hygiene measures .
It could also mitigate and predict the risk of adverse systemic effects of periodontitis and potentially also the risk of severe COVID-19 infections, especially among older patients and those with underlying health conditions. Moreover, it could also increase awareness of the periodontal disease among individuals and encourage and warn them to seek oral care before periodontal disease has progressed to a severe stage. aMMP-8 could then be GUPTA ET AL. | 989 used as a potential biomarker for COVID-19 infection and help in understanding the inflammatory response against the virus.
The current study thus aimed to assess whether such a PoC diagnostic methodology could be utilized as a screening tool to assess active periodontal disease in patients suffering from COVID-19.

| MATERIALS AND METHODS
The cross-sectional analytical study was carried out by the Unit of patients were then either quarantined at home or admitted to the hospital. The patient information sheet was given to all the patients and written informed consent was obtained from all the subjects. Pregnant ladies, patients less than 18 years old, and those unwilling or not in a position to give written informed consent were excluded from the study. The sample size was based on convenient sampling as close proximity to a potentially infectious person is required to conduct intraoral examination and aMMP-8 analysis. Demographic data were recorded, and chairside tests were run for evaluating the expression of aMMP-8 in the site with the maximum periodontal breakdown as well as via a mouth rinse-based kit for general disease activity. In the COVID-19 positive patients, the kits were run again once the patients recovered and tested COVID-19 negative. Hence the subjects were recalled for the second examination at 17 days. The clinical examination was carried out by a single examiner (S. G.), while the kits were run by another examiner (M. S.).

| aMMP-8 PoC mouth rinse sample collection and qualitative analysis
Mouthrinse sample collection was conducted step by step according to the manufacturer's instructions for PerioSafe lateral-flow immunoassay kits (PerioSafe; Dentognostics GmbH, Solingen, Germany) utilizing two anti-aMMP-8 monoclonal antibodies as described previously (Hanemaaijer et al., 1997;Sorsa et al., 1999). Patients were made to rinse with tap water for 30 s and then spit it out. After 1 minute, they were asked to rinse their mouth for 30 s with the rinsing solution provided in the test kit and spit it out in the measuring cup provided in the kit. About 2-3 ml of sample solution was collected in the syringe provided in the kit and the filter attached.
The test cassette was kept on a horizontal surface and 3-4 drops of the sample solution were poured into the round opening of the test cassette. The results were read after exactly 5 min.

| aMMP-8 PoC site-specific sample collection and qualitative analysis
Site-specific sample collection was conducted step by step according to the manufacturer's instructions for ImplantSafe lateral-flow immunoassay kits (ImplantSafe; Dentognostics GmbH, Jena, Germany) utilizing, similar to the PerioSafe kit, two anti-aMMP-8 monoclonal antibodies as described previously (Hanemaaijer et al., 1997;Sorsa et al., 1999). The sampling site was prepared by the removal of excess saliva with a short, gentle blast of air/cotton swab.
A sterile collection strip provided in the kit was placed apically as deeply as possible into the sulcus at the sampling site using tweezers, with its blue end pointing away from the tooth. The strip was left there for 30 s and then pulled out. It was placed in the vial containing elution fluid provided in the kit. The vial was gently turned upside down five times to make sure the strip was totally immersed in the fluid. Once the strip starts floating, it was tipped upside down five times again. The samples were analyzed by a dipstick, which was dipped with the yellow absorption zone facing down, into the elution fluid until the liquid was visible in the readout window. After that, the dipstick was removed from the elution fluid and placed on a level surface. The results were read after exactly 5 min.
The results in both cases were read as a single blue line indicating aMMP-8 levels of less than 20 ng/ml (negative); and two blue lines as aMMP-8 levels of more than 20 ng/ml (positive), indicating active periodontal disease (Figures 1 and 2).

| Statistical analysis
Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 27.0; IBM Corp., Armonk, NY. Results on continuous measurements were presented on mean ± SD (min-max) and categorical as frequency (percentage). The normality of the data was assessed using the Shapiro-Wilk test/Kolmogorov-Smirnov test.
Bivariate associations were examined using Fisher's exact test/χ 2 test. Kruskal-Wallis test was used to compare the variables at a different level of periodontal disease. McNemar's test was used to compare the distribution of patients diagnosed as positive and negative based on kit results among the same patients who were tested for COVID-19 initially and turned out negative later.
Comparison was done with an intention to treat basis, that is it was done on those selective patients on whom before and after the kit was used. Criterion validity in terms of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) with confidence intervals (CI 95%) were computed using a two-way table with clinical diagnosis as a gold standard test. These diagnostics characteristics were calculated for aMMP-8 PoC mouth rinse and site-specific test kits with and without adjusting for the patient's age, gender, and smoking status. Adjusting for the three variables was calculated by logistic regression and the cut-offs for predicted probabilities based on the Youden's index were further utilized in ROC analysis to assess the model's ability to classify patients' disease and health. The sensitivity of the test is its ability to identify correctly those patients who have the disease, whereas the specificity of the test is its ability to identify correctly those patients who do not have the disease. These two parameters are the most accepted ways to quantify the intrinsic diagnostic accuracy and validity of a medical test. However, in clinical practice, even if the sensitivity and specificity of a test are known, the clinician really wants to know how good the test is at predicting abnormality (i.e., what proportion of patients with an abnormal test result is truly abnormal). The PPV is the proportion of patients with a positive test result who truly have the disease and the NPV is the proportion of patients with negative test results who truly do not have the disease. However, PPVs and NPVs vary with the prevalence of a condition within a population while sensitivity and specificity are prevalence-independent test characteristics. The significance level adopted was 5%.         compared with a site-specific test (p = .003). The sensitivity of the kits in the COVID negative cohort is similar to that reported in the literature (Sorsa et al., , 2017 Keskin et al., 2020;Lee et al., 1995;Leppilahti et al., 2014Leppilahti et al., , 2015Mancini et al., 1999;Räisänen et al., 2021;Romanelli et al., 1999;Sorsa et al., 2020). Abbreviations: aMMP-8, active matrix metalloproteinase 8; CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value. a Age, gender, and smoking status adjusted logistic regression model, the optimal cut-off for probabilities by Youden's index.

Figure 4 and
GUPTA ET AL.
| 995 mediated via the MMP pathway. Not only this, but MMP-8 levels have also been evidenced to be increased alongside MMP-9, MMP-2, and TIMP-1 during early sepsis, a condition which has eventually been related to patient mortality (Malek et al., 2020).
The MMP pathway of inflammation is not only at play during acute presentations but also emerges and assumes importance in a more chronic and low-grade variety such as that involving obesity.  (Griffith et al., 2020). Moreover, the proportion of smokers was relatively low in this sample. Nevertheless, taking into account these three risk factors of periodontitis age, gender, and smoking status together with the aMMP-8 POCT was able to increase the accuracy of identifying periodontal disease and health. This is in agreement with and further extends previous studies demonstrating the benefits of assessing aMMP-8 POCT measurements together with the other risk factors of periodontitis when screening for periodontitis and related history of occurred attachment loss .
COVID-19-related adverse outcomes seemed to have an association with the severity of periodontitis, this is in agreement with a few previous studies suggesting a similar relationship (Gupta, Saarikko et al., 2022;Marouf et al., 2021). Chest findings on CT scans are fast emerging as a standardized methodology to diagnose COVID-19 infections in patients who have tested negative on RT-PCR but otherwise remain symptomatic in favor of COVID-19 (Pakdemirli et al., 2020). In our study, the majority of patients were symptomatic with a significant number presenting with ground-glass opacities as CT-chest findings. Demographic and comorbidity-related factors, however, remained unassociated with the severity of periodontitis in patients who were deemed to be COVID-19 negative. This finding also differs from a number of established reports in the literature but can be attributed to the small sample size.
Seeing as the presence of periodontitis seems to correlate with a worsening of COVID-19 related adverse outcomes, the utilization of the aMMP-8 PoC diagnostic kits, assumes importance as a screening tool to diagnose active periodontal disease states at both the fullmouth and site-specific levels. As Sampson et al. (2020) discuss, a raised level of association between severe COVID-19 infection and the presence of chronic periodontitis might be accounted for by the phenomenon first identified by Limeback, (1988aLimeback, ( , 1998b)-there is greater potential for aspiration of periodontitis-associated organisms directly into the lungs if subjects have chronic periodontitis (Scannapieco & Genco, 1999).
There was a significant association between mouth rinse and site- eventually decreased the power to find significant differences as well as decreased the diagnostic accuracy. Regardless, as our results show, such a noninvasive screening methodology can prove to be invaluable in times of a pandemic wherein the simplicity of its conduction and output may enable any medical or paramedical professional as well as laymen to diagnose the presence of active disease and take appropriate action.

| CONCLUSIONS
The findings of the present study recommend the utilization of an

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on reasonable request from the corresponding author. The data are not publicly available due to privacy and ethical restrictions.

ETHICS STATEMENT
The study was conducted according to the guidelines of the