Exploring the methods of salivary analysis in the diagnosis of Alzheimer's disease: A scoping review

Dania Awais; Farah Ayman Sarhan; Laila Sami Al-Majali; Mariam Almuzaien; Marwan Amjad Awad; Mostafa Nagi Amer; Marwan Mansoor Ali Mohammed

doi:10.4103/abhs.abhs_62_22

REVIEW ARTICLE

Year : 2023 | Volume : 2 | Issue : 2 | Page : 51-57

Exploring the methods of salivary analysis in the diagnosis of Alzheimer's disease: A scoping review

Dania Awais, Farah Ayman Sarhan, Laila Sami Al-Majali, Mariam Almuzaien, Marwan Amjad Awad, Mostafa Nagi Amer, Marwan Mansoor Ali Mohammed
Department of Oral and Craniofacial Health Sciences, College of Dental Medicine, University of Sharjah, Sharjah, United Arab Emirates

Date of Submission	25-Nov-2022
Date of Decision	20-Jan-2023
Date of Acceptance	26-Jan-2023
Date of Web Publication	01-Mar-2023

Correspondence Address:
Dr. Marwan Mansoor Ali Mohammed
Department of Oral and Craniofacial Health Sciences, College of Dental Medicine, University of Sharjah, Sharjah
United Arab Emirates

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/abhs.abhs_62_22

Abstract

Alzheimer's disease is a common type of dementia. It is a progressive neurodegenerative disease that starts with mild memory loss and may progress to loss of ability to convey a conversation and respond to the surrounding. Early detection can help in the management of this disease. In this review, we attempted to find the most accurate method for salivary biomarkers detection, which can aid in the early diagnosis of Alzheimer's disease. For this scoping review; PubMed, Scopus, and Google Scholar online databases were searched to find studies and articles that test the salivary biomarkers for the diagnosis of Alzheimer's disease. Eleven studies were eligible for our research. Ten were case–control, and one was a cohort study. Seven of the studies used the enzyme-linked immunosorbent assay (ELISA) method, five of which showed diagnostic potential. Two studies used Luminex assay and photomultiplier tube, showing similar results to ELISA. AbsoluteIDQ®p150 Kit and faster ultra-performance liquid chromatography-mass spectrometry analysis and pattern recognition techniques tested different metabolites with potential diagnostic significance. Furthermore, another study using an extended gate ion sensitive field effect transistor sensor related salivary trehalose sugar to Alzheimer's disease. Different reliable biomarkers were detected using ELISA kit and appear to be effective in disease detection. Further studies and clinical trials on a large scale are required to determine the most accurate method of detecting Alzheimer's disease.

Keywords: Alzheimer's, biomarker, dementia, enzyme-linked immunosorbent assay, saliva

How to cite this article:
Awais D, Sarhan FA, Al-Majali LS, Almuzaien M, Awad MA, Amer MN, Ali Mohammed MM. Exploring the methods of salivary analysis in the diagnosis of Alzheimer's disease: A scoping review. Adv Biomed Health Sci 2023;2:51-7

How to cite this URL:
Awais D, Sarhan FA, Al-Majali LS, Almuzaien M, Awad MA, Amer MN, Ali Mohammed MM. Exploring the methods of salivary analysis in the diagnosis of Alzheimer's disease: A scoping review. Adv Biomed Health Sci [serial online] 2023 [cited 2023 Jun 9];2:51-7. Available from: http://www.abhsjournal.net/text.asp?2023/2/2/51/370883

Background

Alzheimer's disease is an incurable disease that imprisoned around 5.6 million people in the US in 2016 ^[1]. Alzheimer's disease is a progressive neurodegenerative disorder characterized by disturbances in memory and cognitive function. It is a form of dementia, commonly affecting individuals in the elderly population. There is currently no known cure for Alzheimer's disease, leading to concern among individuals regarding the potential onset of the disorder in loved ones ^[2]. Alzheimer's disease is usually caused by a combination of genetic, environmental, and lifestyle factors. However, the early onset of Alzheimer's disease is usually caused by genetic mutations ^[3]. The symptoms of Alzheimer's disease vary from one person to another but may include memory loss, confusion, shortened attention span, language difficulties, anxiety, restlessness, and repetitive statements or movements ^[4]. All of these can negatively affect the ability of a person to carry out everyday tasks. In addition to affecting the people around them when their loved one becomes unable to recognize them. This indicates the need to develop reliable means of early detection that will allow us to intervene quickly enough to slow the disease's progression and development. Biomarker molecules in the saliva samples could be used for early detection of Alzheimer's disease. As a collection of saliva samples is convenient and noninvasive, therefore preferred by patients ^[5]. Numerous different methods are being used to identify salivary biomarkers for the detection of Alzheimer's disease however each one of them has a different level of accuracy and sensitivity.

This review aims to find the most accurate method for using salivary biomarkers in the early diagnosis of Alzheimer's disease.

Materials and Methods

Focused question

Which method of salivary biomarkers identification yields the most accurate results for early detection of Alzheimer's disease?

Search strategy

The online searching of the databases PubMed, Scopus, and Google Scholar was used to extract articles for this review. The search was performed on the literature published from the year 2000 up to and including the year 2021 to determine the best method used to detect Alzheimer's disease using salivary biomarkers.

The keywords used were as follows: “Alzheimer's disease,” “salivary biomarkers,” “tau,” “ELISA,” “Simoa,” “Sandwich,” “Luminex,” “Ellman colorimetric method,” “Nanobead,” “Aβ42.” The same keywords were used for all three databases. However, the most commonly used ones were “Alzheimer's disease” and/or “salivary biomarkers.”

Eligibility criteria

The selected studies were restricted to salivary biomarkers rather than blood biomarkers and cerebrospinal fluid (CSF). Sufficient data was mentioned in the statistical analysis of each study. The aim of the selected studies was focused on detecting Alzheimer's disease from salivary biomarkers. Studies only published in the English language were eligible. Literature reviews, systematic reviews, articles with weak evidence, and nonpeer-reviewed articles were excluded. Studies including least feasible methods, biomarkers detected in nonsalivary tests, studies testing young populations (kids and teenagers), editorials, and testing made to prove other disease outcomes were excluded as well as studies where patients' results are under previous intervention for Alzheimer's disease.

Data extraction

Six investigators, working individually, reviewed the articles that were collected from the databases. This was done in three steps. First, the duplicated articles were removed manually. Then, the titles and abstracts were screened according to our inclusion criteria, which are: patients diagnosed with Alzheimer's disease, studies using control groups, elderly patients (60 years and above), male or/and female patients, articles with good outcomes, and clear significant results, and studies where the testing method has a clear outcome and consistent results. Finally, the full texts of the articles were screened according to the same inclusion criteria mentioned above.

The investigators (DA, FS, LM, MA, MA, and MN) evaluated the eligibility of the articles.

Studies quality assessment

We critically appraised eleven included studies (ten were case–control studies and one was a cohort study) using 11 questions based on the Critical Appraisal Skills Program (CASP) Checklist for the case–control studies and 12 questions based on the CASP Checklist for the cohort study. We calculated the overall evidence of each one to determine which ones have strong, moderate, or weak evidence as mentioned in [Table 1].

Table 1: Evidence strength and results of critical appraisal of the included studies.

Click here to view

This study was preregistered in OSF registries osf.io/byx95.

Results

Selection

A total of 202 articles were initially found through electronic databases (n = 202). After removing 58 duplicates, we ended up with 144 articles for screening. The records were screened, and 73 were excluded due to age <60 (n = 4), studies not focusing on salivary biomarkers (n = 44), studies about non-Alzheimer's patients (n = 24), and studies not in the English language (n = 1) leaving us with 71 articles (n = 71) [Figure 1].

Figure 1: Preferred reporting items for systematic reviews and meta-analyses flow chart of the selection process of the included studies.

Click here to view

After full-text reading, another 60 articles were considered ineligible and excluded, due to the following reasons studies not about salivary biomarkers (n = 34), including patients with age >60 (n = 7), non-Alzheimer's patients (n = 18), small sample size (n = 1). Eleven articles are included in our scoping review. Our eligibility criteria are included in our Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) diagram, and all the evidence from the studies we used was critically appraised and tabulated [Figure 1].

Critical appraisal

We critically appraised the studies using 11 questions based on the CASP checklist for the case-control studies and 12 questions based on the CASP checklist for the cohort study ^[6]. Eight articles scored 67–100 and were considered to have strong evidence. The remaining three articles scored 34–66 and were given a moderate evidence rating [Table 1].

Study characteristics

Ten of the included studies were case-control studies and the remaining one was a cohort study. Three of the studies were performed in Spain ^[7],[8],[9]. Two of the studies were done in the Republic of Korea ^[10],[11], two were done in the USA ^[12],[13] and one was done in Canada ^[14], Austria ^[15], China ^[18], and Brazil^[17] [Table 1].

Study outcomes

Two studies ^[8],[9] using enzyme-linked immunosorbent assay (ELISA) showed that lactoferrin levels in Alzheimer's disease groups were significantly lower compared with healthy control.

Two studies; one using ELISA^[17] and another one using Luminex assay^[13] found that the p-tau/t-tau ratio is increased in Alzheimer's disease compared to healthy control.

Three studies ^{[12],[14],[17]} using ELISA found that there is an increase in the concentration of Aβ42 biomarker in Alzheimer's disease patients compared to control groups. Moreover, the study was done by Shi et al. ^[13] using Luminex assay failed to detect Aβ42. However, another study^[7] using specific Aβ ELISA only showed a significant difference between Alzheimer's disease mild-stage patients and control groups.

A study using photomultiplier tube^[10] found a higher level of Aβ42 in severe stages than moderate stages as opposed to a study using specific Aβ ELISA^[7] showing an increase for only mild-stage Alzheimer's disease patients.

In a study using ELISA ^[11], Aβ42 was not detected and p-tau/t-tau ratio levels were not significantly different between Alzheimer's disease patients and healthy controls. However, using an extended gate ion sensitive field effect transistor (EG-ISFET) sensor showed higher current in normal controls than Alzheimer's disease patients showing that Alzheimer's disease patients have higher salivary trehalose than normal controls.

In another study done by Marksteiner et al. ^[15]., they test three salivary metabolites; acyl-alkyl-phosphatidyl-cholines (PCae), diacyl-Phosphatidylcholines (PCaa), and Lyso-acyl-Phosphatidylcholines (lyso-PC-a). The analysis of saliva (PCae) is the only one that showed a significant decrease of PCae metabolites in Alzheimer's disease patients compared to controls.

Another study^[18] which combines faster ultra-performance liquid chromatography-mass spectrometry (FUPLC-MS) and pattern recognition techniques were able to find 3 metabolites; sphinganine-1-phosphate, ornithine, and phenyllactic acid as potential biomarkers for early diagnosis of Alzheimer's disease.

The study design, methods, findings, and biomarkers in all included studies are summarized in [Table 2].

Table 2: Evidence table showing tabulated results of the included studies.

Click here to view

Discussion

This review aims to determine the most effective method for the detection of Alzheimer's disease using salivary biomarkers by analyzing published literature. This review prioritizes the methods that are less invasive than collecting CSF ^[18]. All studies agreed that using saliva samples is a noninvasive, reliable, and low-cost approach ^{[14],[19],[20]} for diagnosing Alzheimer's disease. Although it should be kept in mind that samples need to be processed rapidly due to the instability of the biomarkers ^[21]. However, since the number of studies on this topic is limited, this area needs additional research and more clinical investigations.

The studies included in this review test and evaluate different methods for the detection of a variety of biomarkers which are lactoferrin, p-Tau to t-Tau ratio, Aβ42, salivary metabolites; acyl-alkyl-phosphatidyl-cholines (PCae), diacyl-Phosphatidylcholines (PCaa), Lyso-acyl-Phosphatidylcholines (lyso-PC-a), sphinganine-1-phosphate, ornithine, and phenyllactic acid. In a recent systematic review, they concluded that salivary biomarker Aβ42 would be the most promising for the diagnosis of Morbus Alzheimer ^[22].

In one study saliva samples from Alzheimer's disease and amnestic mild cognitive impairment (aMCI) patients were compared with control subjects. Lactoferrin expression was examined for the first time after SDS-PAGE fractionation and MS detection, showing reduced levels of lactoferrin in aMCI and Alzheimer's disease relative to the control group. Further ELISA confirmation testing in individual donor samples showed that in aMCI and Alzheimer's disease patients, the salivary lactoferrin levels in comparison with the stable control group were significantly reduced ^[7].

Another study found that using FUPLC-MS combined with a pattern recognition approach could be an advanced tool to help us find metabolites and classify the sample groups ^[18].

An EG-ISFET biosensor that detects a sugar-like component such as trehalose may be used as an alternative diagnostic method for Alzheimer's disease ^[11]. On the other hand, salivary p-tau was found to be expressed higher in Alzheimer's disease group than in the PD and control groups. Cell-based EG-ISFET biosensors were only used in this study ^[11].

Two studies that used salivary metabolites require further research as no other literature has been published to support the results of these studies. ELISA is the method that appears to be repeatedly used in the majority of the published literature to identify the different biomarkers. Although the method used was ELISA the biomarkers identified were different and this shows that there is no universal biomarker that can be used as a definitive marker of Alzheimer's disease even though the preferred method appears to be ELISA.

The results of our scoping review matter since it sheds light upon the fact that salivary biomarkers have a lot of potential in being a noninvasive method for the detection of Alzheimer's disease in the future once a definitive method and specific biomarkers have been identified. For this to happen a lot more research needs to be carried out by repeated experiments using the existing methods as well as the possible development of more advanced methods and techniques for the identification of salivary biomarkers. The use of saliva in the detection of Alzheimer's disease opens the window for many potential patients to seek investigation and allow early intervention and treatment to help with the mitigation of further progression of the disease.

Since there are a limited number of studies performed it is difficult to conclude as to which method is most accurate. Not much clinical evidence is provided when it comes to method accuracy therefore our search spectrum was limited. ELISA kits for diagnostic use have yet to be standardized and validated. It will, however, be important to establish standardized salivary tests and perform studies with a larger population to determine the exact cut-off values of salivary biomarkers ^[4],[16].

Most research concentrated on analyzing biomarkers rather than determining the precision of the techniques used to detect the biomarkers. Therefore, it is recommended that further experiments and studies are needed to determine the most accurate technique that is specific or nonspecific to a biomarker.

A comparison of the techniques can help and is needed to determine the most accurate technique for detecting biomarkers of Alzheimer's disease since during our research for articles we did not come across any studies comparing different methods of detection of salivary biomarkers. It is recommended that studies should compare the already existing methods as well as experiment with new methods of detection to determine a definitive method of detection that has high accuracy and could be used as a universal method.

Conclusion

Using salivary biomarkers is an easier, less expensive, and noninvasive method to detect Alzheimer's disease. Although a specific most accurate technique of detecting Alzheimer's disease has not been determined. The majority of the studies have used an ELISA kit which is currently considered the gold standard. However, there has not been a study that compared the different techniques used for these biomarkers.

We have concluded that further studies and clinical trials are required to determine the most accurate method of detecting Alzheimer's disease in patients using salivary biomarkers to bring awareness to the disease and help with early detection. This could provide an easier, more affordable way to detect this disease.

Study limitations

The low number of studies that are dealing with the topic.

Authors' contribution

DA, FAS, LSM, MA, MAA, MNA and MMAM conceived the research concept, DA, FAS, LSM, MA, MAA, MNA developed the research design, data collection and data analysis. DA, FAS, LSM, MA, MAA, MNA prepared the first draft of the manuscript, MMAM reviewed and approved the final draft of the manuscript. All authors are responsible for the manuscript's contents.

Ethical statement

This scoping review study was reported according to PRISMA guidelines.

Data availability statement

All relevant data are available within the manuscript.

Financial support and sponsorship

Not applicable.

Conflict of interests

No conflict of interests declared.

References

1.	François M, Bull CF, Fenech MF, Leifert WR. Current state of saliva biomarkers for aging and alzheimer's disease. Curr Alzheimer Res 2019;16:56-66.
2.	Reale M, Gonzales-Portillo I, Borlongan CV. Saliva, an easily accessible fluid as diagnostic tool and potent stem cell source for alzheimer's Disease: Present and future applications. Brain Res 2020;1727:146535.
3.	Landrigan PJ, Sonawane B, Butler RN, Trasande L, Callan R, Droller D. Early environmental origins of neurodegenerative disease in later life. Environ Health Perspect 2005;113:1230-3.
4.	Schepici G, Silvestro S, Trubiani O, Bramanti P, Mazzon E. Salivary biomarkers: Future approaches for early diagnosis of neurodegenerative diseases. Brain Sci 2020;10:245.
5.	Gug IT, Tertis M, Hosu O, Cristea C. Salivary biomarkers detection: Analytical and immunological methods overview. TRAC Trends Analyt Chem 2019;113:301-16.
6.	Critical Appraisal Skills Programme. CASP; 2022. Available from: https://casp-uk.net/casp-tools-checklists/. [Last accessed on 2022 Oct 03].
7.	Bermejo-Pareja F, Antequera D, Vargas T, Molina JA, Carro E. Saliva levels of Abeta1-42 as potential biomarker of alzheimer's disease: A pilot study. BMC Neurol 2010;10:108.
8.	Carro E, Bartolomé F, Bermejo-Pareja F, Villarejo-Galende A, Molina JA, Ortiz P, et al. Early diagnosis of mild cognitive impairment and alzheimer's disease based on salivary lactoferrin. Alzheimers Dement (Amst) 2017;8:131-8.
9.	González-Sánchez M, Bartolome F, Antequera D, Puertas-Martín V, González P, Gómez-Grande A, et al. Decreased salivary lactoferrin levels are specific to alzheimer's disease. EBioMedicine 2020;57:102834.
10.	Kim CB, Choi YY, Song WK, Song KB. Antibody-based magnetic nanoparticle immunoassay for quantification of alzheimer's disease pathogenic factor. J Biomed Opt 2014;19:051205.
11.	Lau HC, Lee IK, Ko PW, Lee HW, Huh JS, Cho WJ, et al. Non-invasive screening for Alzheimer's disease by sensing salivary sugar using Drosophila cells expressing gustatory receptor (Gr5a) immobilized on an extended gate ion-sensitive field-effect transistor (EG-ISFET) biosensor. PLoS One 2015;10:e0117810.
12.	Sabbagh MN, Shi J, Lee M, Arnold L, Al-Hasan Y, Heim J, et al. Salivary beta amyloid protein levels are detectable and differentiate patients with alzheimer's disease dementia from normal controls: Preliminary findings. BMC Neurol 2018;18:155.
13.	Shi M, Sui YT, Peskind ER, Li G, Hwang H, Devic I, et al. Salivary tau species are potential biomarkers of alzheimer's disease. J Alzheimers Dis 2011;27:299-305.
14.	Lee M, Guo JP, Kennedy K, McGeer EG, McGeer PL. A method for diagnosing alzheimer's disease based on salivary amyloid-β protein 42 levels. J Alzheimers Dis 2017;55:1175-82.
15.	Marksteiner J, Oberacher H, Humpel C. Acyl-alkyl-phosphatidlycholines are decreased in saliva of patients with alzheimer's disease as identified by targeted metabolomics. J Alzheimers Dis 2019;68:583-9.
16.	Liang D, Lu H. Salivary biological biomarkers for alzheimer's disease. Arch Oral Biol 2019;105:5-12.
17.	Santos GA, Olave E, Pardi PC. Salivary biomarkers in alzheimer's disease. Int J Morphol 2020;38:230-4.
18.	Liang Q, Liu H, Zhang T, Jiang Y, Xing H, Zhang AH. Metabolomics-based screening of salivary biomarkers for early diagnosis of alzheimer's disease. Rsc Adv 2015;5:96074-9.
19.	Gleerup HS, Hasselbalch SG, Simonsen AH. Biomarkers for alzheimer's disease in saliva: A systematic review. Dis Markers 2019;2019:4761054.
20.	Serafín V, Gamella M, Pedrero M, Montero-Calle A, Razzino CA, Yáñez-Sedeño P, et al. Enlightening the advancements in electrochemical bioanalysis for the diagnosis of alzheimer's disease and other neurodegenerative disorders. J Pharm Biomed Anal 2020;189:113437.
21.	Hrubešová K, Fousková M, Habartová L, Fišar Z, Jirák R, Raboch J, et al. Search for biomarkers of alzheimer's disease: Recent insights, current challenges and future prospects. Clin Biochem 2019;72:39-51.
22.	Wolgin M, Zobernig M, Dvornyk V, Braun RJ, Kielbassa AM. Systematic review on saliva biomarkers in patients diagnosed with morbus alzheimer and morbus parkinson. Biomedicines 2022;10:1702.