Exploring Three Methods for Sampling Oral Microbiota in Older People: A Comparative Study

Abstract

A variety of methods are described in the literature for sampling oral microbiota, including their advantages and disadvantages. The aim of this study was to assess which method (swab, paper point, and oral fluid collection) for sampling oral microbiota is most favourable for use on a geriatric population. The oral microbiota of geriatric patients who visited the Geriatric Department of the University Medical Center Groningen (The Netherlands), because of memory complaints, were assessed between April 2022 and April 2023. The samples were collected using three methods: swab, paper point, and oral fluid collection. The participants had to have at least one natural tooth. The samples were checked for 11 oral bacteria commonly associated with oral and general diseases. Of the 30 potentially eligible participants, 28 could be enrolled. A 100% bacteria match was achieved by all 3 sampling techniques between 64% of the cases (18/28) and a 91% match between another 25% (7/28). The swab had the highest positivity rate in this population, as it was easy to perform and usable on edentulous individuals, even on those with (severe) disabilities. Overall, the sampling methods showed comparable microbiota in most cases but differed in the complexity of use. The swab seemed to be the most favourable method among the geriatric participants.

1. Introduction

The human oral cavity, which includes various habitats such as the teeth, the gingival sulci, and the tongue, harbours a myriad of microorganisms collectively known as the oral microbiota [1]. This microbial array is present not only in the oral cavity, but also in adjacent structures such as the tonsils, pharynx, lungs, gut, and oesophagus [1,2]. Moreover, the oral microbiota differs between individuals [3].

In the literature, several studies suggest that the composition of oral microbiota can impact general health [4,5]. It has been shown that periodontitis is linked to several systemic conditions such as cardiovascular diseases, diabetes, and, recently, neurodegenerative diseases [6,7]. To be able to better understand the potential role or the impact of the oral microorganisms on general health, it is needed to further unravel the complex host–microbiome interactions that might influence health and disease [6,7]. In order to understand the potential role or the impact of the oral microorganisms on general health better, the complex host–microbiome interactions that might influence health and disease need to be unravelled further [8]. Sampling methods play a crucial role here, but all the published studies have utilised different sampling and analysis methods [6,9,10,11]. This variety in sampling methods leads to challenges when comparing the results, as well as in determining the optimal and cost-effective sampling approach [12]. To be able to compare studies, it is of utmost importance to know whether the results of different sampling methods can be compared with each other as well as which one is best for certain circumstances or for different kinds of patients [13]. In the near future, the number of older adults with neurodegenerative diseases will increase rapidly because the geriatric population is growing [14]. Regarding the interplay between the oral microbiota and neurogenerative diseases, it is hypothesised that specific oral pathogens are responsible, such as for the onset of Alzheimer’s disease and Parkinson’s disease [6,15]. Thus, a simple and effective microbial sampling method should be used, especially on the geriatric population. However, it is still unclear which is the most appropriate method for sampling the oral microbiota in a geriatric population suffering from disabilities, including those with neurogenerative diseases. It is imperative to use a technique that is not only easy to perform but also provides reliable and reproducible results.

The most commonly mentioned methods to sample the oral microbiota are swabbing (swabbing the oral mucosa with a sponge), paper point analysis (placement of a paper point in the gingival sulcus), and oral fluid collection (spitting oral fluid into a container, e.g., after rinsing the oral cavity) [9,10,11]. A few studies have compared the “collecting oral fluids method” and the “paper point method” [9,10,11], but, to the best of our knowledge, no studies have directly compared the three most commonly used methods on a geriatric population. Therefore, the aim of this study was to assess and compare the results of the three most commonly used methods (swab, paper point, and oral fluid collection) after sampling a geriatric population’s oral microbiota. The most common eleven bacteria thought to be involved in oral and general health were assessed [16].

2. Materials and Methods

2.1. Study Design

This study was conducted among older people who were seen for a first consultation regarding memory complaints at the geriatric department of the University Medical Center Groningen (UMCG, The Netherlands) between April 2022 and April 2023. All the participants’ oral microbiota was analysed using three different sampling methods (swab, paper point, and oral fluid collection).

2.2. Participants

All geriatric patients with memory complaints who were scheduled for an appointment at the geriatric clinic received a letter from the principal investigator containing written information regarding the study. In the letter, the patients were asked if they were willing to participate. Ethical approval for this study was obtained from the Medical Ethics Committee of the UMCG (METC-2020.606).

Inclusion criteria were:

-

Presence of cognitive complaints;

-

Presence of at least 1 natural tooth (to be able to use the paper point method on all patients).

Exclusion criteria:

-

Incompetent to follow or to perform the given sampling instructions correctly (e.g., due to severe cognitive problems and/or physical problems);

-

Incapacitated by law.

Variables such as the participants’ sex, age, smoking habits, medication use, antibiotic use, pocket depth, number of teeth, and presence of dentures were collected.

2.3. Sampling Methods

After consenting to participate in the study in written form, the participant was asked to abstain from eating, drinking (except water), smoking, or performing oral hygiene for least one hour prior to sample collection:

1. Swab: using a swab (Flowflex™ mini sponge, Acon Biotech, Hangzhou, China) attached to a small stick (Figure 1a)), the oral mucosa (cheek mucosa, both sides) was swiped five times whereafter the swab was preserved in a sterile test tube without fluid.

2. Paper point: the deepest pocket (measured with a pocket probe) among all the teeth was sampled. Before sampling, most of the supragingival plaque was carefully removed with a dental probe to gain access to the deepest periodontal pocket. Then, a sterile R&S ISO 40 paper point was inserted into the pocket for 10 s (Figure 1b). The paper point was transferred to a dry test vial.

3. Oral fluid collection: the participants were first asked to rinse their mouth with sterile water and then to spit that water out. They were then instructed to let the oral fluids accumulate in their mouth for 30 s without swallowing. After this time, they had to expectorate the accumulated oral fluid into a sterile 50 mL container (Figure 1c).

All samples were collected by the same operator. The order of sample collection was always the same. This standardised approach was applied to all participants to ensure consistency throughout the study.

All the samples were stored at −80 °C within half an hour after sampling whereupon they were transported on dry ice to ADD Laboral B.V. (Malden, The Netherlands).

2.4. Oral Microbiota

All the samples were screened for 11 of the most commonly tested oral bacteria (see microbiological Q-PCR test) [15]. The selected microorganisms are associated with common health problems in older people, such as neurodegenerative diseases and diabetes [17]. The 11 bacteria belong to the red, orange, and green complexes, as described by Socranski et al. in 1998 [18]. The presence and quantity of each bacterium were assessed and expressed as colony-forming units (CFUs).

2.5. Microbiological Q-PCR Test

The ADD Laboral Paro 11 test panel was performed. The microbiological Q-PCR test aided in identifying the bacteria in the samples. This panel contains the following 11 species: Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn), Parvimonas micra (Pm), Prevotalla nigrescens (Pn), Campylobacter gra-cilis (Cg), Campylobacter rectus (Cr), and Eubacterium nodatum (En).

2.6. Bacterial DNA Extraction

Sample preparation: after thawing (swab, paper point, and oral fluids), the material was prepared as follows:

Swab (dry): An amount of 100 µL of DNAse-free water was added to the test tube with the swab. The tubes were vortexed at maximum speed for 20 s. A total of 50 µL was transferred to a new test tube and used for DNA extraction;

Paper point (dry): no preparation necessary (InstaGene^TM matrix will be added during the extraction phase);

Collected oral fluids: the oral fluid samples were mixed well by vortexing, after which 50 µL of material was transferred to a new test tube and used for DNA extraction.

After the preparation phase, the bacterial DNA in all the material was isolated by adding 150 µL InstaGene™ Matrix (Bio-Rad, Hercules, CA, USA) extraction buffer to the test tubes. DNA extraction was done by following the InstaGene™ Matrix protocol. The Matrix was added directly to the paper points, the 50 µL of oral fluid, and the swabs’ eluate, followed immediately afterwards by vortexing, centrifuging, and incubating at 56 °C for 20 min. The solutions underwent further vortexing, centrifuging, and incubation at 100 °C for eight minutes, followed by final vortexing and centrifuging. The isolated DNA was then used in the PCR reactions.

Bacterial counts were scored digitally and expressed as CFU.

The number of CFUs were noted as follows:

0 CFU	−	Negative
1–10 CFU	+	Minimal amount
10–100 CFU	++	Low amount
100–1000 CFU	+++	Moderate amount
1000–10,000 CFU	++++	Moderately high amount
10,000–100,000 CFU	+++++	High amount
100,000–>1,000,000 CFU	++++++	Very high amount

2.7. The Ease of Use and Cost of the Three Different Sampling Methods

During sampling, the following parameters were noted and scored:

-

Cost of sample material (expensive (>€5) −, fair deal (€2–€5) +/−, cheap (<€2) +);

-

Cost of laboratory analysis (price in euros);

-

Participant comfort (good +, average +/−, poor −);

-

Sampling time length (short + (<30 s), average +/− (30 s–2 min), long − (>2 min));

-

Ease of sampling (for the practitioner) (easy +, average +/−, difficult −);

-

Accessibility of the sample location (good +, average +/−, poor −);

2.8. Sample Size Calculation

The required sample size was determined using a G*Power (version 3.1.9.7). A power analysis was performed with an alpha level of 0.017, 80% power (β = 0.20), and an expected effect size of 0.666. Based on these parameters, the minimum required sample size was 27 participants.

2.9. Statistical Analysis

The raw data were collected and stored in Microsoft Excel. All statistical analyses were performed using IBM SPSS Statistics for Windows (version 23.0; IBM Corp., Armonk, NY, USA). The distribution of continuous variables was assessed using the Shapiro–Wilk test. Descriptive statistics were used to summarise demographic data, oral health characteristics, and the presence of periodontal pathogens. Continuous variables were presented as the mean and standard deviation (SD). Categorical variables were presented as counts and percentages.

Chi-square tests were conducted to assess differences in categorical variables, such as the distribution of sex, between groups. To evaluate potential differences in bacterial detection between the three sampling methods (swab, paper point, and collected oral fluid), a Cochran’s Q test was performed for each bacterial species.

3. Results

3.1. Characteristics of the Participants

A total of 30 eligible consecutive participants were asked to participate (

Table 1. Characteristics of the included participants.

	Participants, n = 28 (%)	p-Value
Gender; female	13 (46%)
Gender; male	15 (54%)	0.705
Age, year, mean (SD)	77.5 (5.3)
Smoking	17 (60.7)
No medication	0 (0.0)
1–4 medications	15 (53.6)
Polypharmacy	13 (46.4)
Antibiotic use (<6 months)	14 (50.0)
Oral status of the participants
Pocket depth, mean (SD)	6.3 (1.0)
Number of teeth, mean (SD)	22.0 (5.4)
Number of carious teeth per participant, mean (SD)	4 (2.1)
Mucosal pathology	7 (8.5)
A maxillary partial denture	4 (14.3)
A mandibular partial denture	3 (10.7)
A complete maxillary denture	2 (7.1)
A complete mandibular denture	0 (0.0)

). All were willing to join the study, but two participants had to be excluded while preparing the samples in the lab due to the fact that sputum seemed to have been collected instead of oral fluid. The remaining 28 participants’ samples were eligible for analysis. The mean age of the participants was 77.5 years (SD 5.3). Of the 28 participants, there were 13 (46%) females and 15 males (54%). No significant gender differences were observed (p = 0.705). Seventeen participants (60.7%) were smokers, 53.6% were taking between one and four medications, and 46.4% used ≥5 medications (polypharmacy). Furthermore, 50% of the participants had used antibiotics within the six-month period before the oral microbiota collection.

In terms of oral health, the mean periodontal pocket depth was 6 mm (SD 1.0) and participants had an average of 22 teeth (SD 5.4). On average, participants had 4.0 (SD 2.1) carious teeth, and mucosal pathology was observed in 8.5% of cases. A partial denture was present in the maxilla of 14.3% of participants and in the mandible of 10.7%. Moreover, 7.1% of participants had a complete maxillary denture. No participants had a complete mandibular denture.

3.2. Comparison of the Different Sampling Methods

Table 2. Bacteria species prevalence found with the three different sampling techniques.

Participant	Number of Teeth	Pocket Depth	Aa	Pg	Tf	Td	Pi	Fn	Pm	Pn	Cg	Cr	En	Match (%)
1 OF			−	−	+++	+++	−	+++	+	+++	+	+	−
1 PP	13	7 mm	−	−	+++++	+++++	−	+++++	++++	++++++	++++	+	+++	91
1 Swab			−	−	+++	+++	−	+++	++	+++	+++	+	+
2 OF			−	+++	+++	+++	−	+++	+	++++	++	++	+
2 PP	28	6 mm	−	+++	+++	+++	−	++++	++	++++	++++	++	++	100
2 Swab			−	+++	+++	+++	−	+++	+	+++	+	++	+
3 OF			−	−	+++	+++	−	++	+	−	+	++	−
3 PP	26	7 mm	−	−	++++	++++	−	++++	+++	+++++	++++	+++	++	82
3 Swab			−	−	++	++	−	++	−	−	+	+	+
4 OF			−	+++	+++	++++	−	+++	+++	+++	++	++	++
4 PP	22	6 mm	−	++	+++	+++	−	++++	+++	++++	+++	++	++	100
4 Swab			−	+++	+++	+++	−	+++	+++	++++	++	++	++
5 OF			−	−	++++	+++	−	+++	++	++++	++	+++	−
5 PP	26	7 mm	−	−	+++++	++++	−	+++++	++++	+++++	++++	+++++	−	100
5 Swab			−	−	+++	++	−	++	++	+++	+	++	−
6 OF			−	−	++++	++++	−	+++	+++	−	++	+++	+++
6 PP	26	7 mm	−	−	++++	+++++	−	+++++	+++	−	+++	++++	+++	100
6 Swab			−	−	+++	+++	−	+++	++	−	++	+++	++
7 OF			−	+++	+++	+++	−	+++	++	+++	+/−	++	++
7 PP	27	8 mm	−	+++++	+++++	++++++	−	+++++	++++	+++++	++++	++++	++++	100
7 Swab			−	++	++	+++	−	++	++	++	+/−	+	++
8OF			−	+++	+++	+++	+++	+++	++	++++	+	++	+
8 PP	25	7 mm	−	++++	++++	++++	++++	++++	+++	+++++	++	+++	+++	100
8 Swab			−	++	++	+++	++	++	++	++++	+/−	++	++
9 OF			−	+++	+++	−	−	+++	+++	+++	+	++	+++
9 PP	17	6 mm	−	+++	+++	−	−	++++	+++	+++++	+++	+++	+++	100
9 Swab			−	++	++	−	−	+	++	++	+	+	++
10 OF			−	−	+++	+++	−	+++	++	−	+	+++	−
10 PP	27	7 mm	−	−	+++	+++	−	+++++	+++	+++++	+++	++	−	91
10 Swab			−	−	++	++	−	++	+	+++	+/−	+	−
11 OF			−	+++	+++	++++	+++	+++	+	++++	+	+++	++
11 PP	17	7 mm	−	+++	++++	+++++	++++	++++	++	+++++	+++	+++++	+++	100
11 Swab			−	++	++	+++	++	+++	−	+++	+/−	++	++
12 OF			−	−	+++	+++	+++	+++	++	++++	+	+++	+
12 PP	25	5 mm	−	−	+++	+++	+++	++++	++	++++	+++	+++++	+	100
12 Swab			−	−	++	+++	++	++	+	++	+	++	+
13 OF			−	−	++++	++++	−	++++	+++	−	−	+++	++
13 PP	27	7 mm	−	−	+++++	++++	+++	+++++	++++	+++	++	++++++	+++	82
13 Swab			−	−	++	++	−	++	++	−	−	+	+/−
14 OF			−	−	++	+++	−	+++	+++	++++	+	++	−
14 PP	26	5 mm	−	−	++	+++	−	++++	++	+++	++	+++	−	100
14 Swab			−	−	++	++	−	++++	+++	++++	+++	+++	−
15 OF			−	−	−	+++	+++	++++	+++	+++++	++	++++	+++
15 PP	25	7 mm	−	−	−	++++	++++	+++++	+++	++++	+++	++++	++	91
15 Swab			−	−	−	+++	+++	++	++	−	+/−	++	++
16 OF			−	+++	+++	++++	−	+++	++	−	−	+++	−
16 PP	18	5 mm	−	+++	++++	++++	−	+++++	++++	++++	++	+++	−	91
16 Swab			−	+++	++	+++	−	++	+/-	+++	−	++	−
17 OF			−	++	+++	+++	−	+++	+++	+++	−	++	+/−
17 PP	21	7 mm	−	+	++++	+++++	−	+++++	++++	+++++	+++	++++	++	73
17 Swab			−	+	−	++	−	++	−	++	+	+	+
18 OF			−	+++	++++	++++	++++	+++	+++	++	++	+++	+
18 PP	25	8 mm	−	+++++	+++++	++++++	++++	+++++	+++	+++	+++	+++++	+++	100
18 Swab			−	++	++	+++	++	++	+	++	+/−	++	+
19 OF			−	−	−	−	−	++	+	−	+	−	−
19 PP	11	5 mm	−	−	−	−	−	++++	++	−	+++	+/−	−	100
19 Swab			−	−	−	−	−	+/−	−	−	+/−	−	−
20 OF			−	++	+++	−	−	+++	++	+++	+	+++	−
20 PP	15	5 mm	−	++	+++	−	−	+++++	+++	++++	+++	++++	−	100
20 Swab			−	++	++	−	−	++	+	++	+/−	+	−
21 OF			++	+++	−	++++	++++	+++	+	−	++	++	+
21 PP	22	7 mm	+++++	+++++	−	++++++	+++++	++++++	+++++	−	++++	+++++	++	100
21 Swab			+	+++	−	++++	+++	++	++	−	+	++	+
22 OF			−	−	++	−	−	+	+/−	++	++	+	−
22 PP	24	5 mm	−	−	+++	−	−	+++	+/−	+++	+++	++	−	100
22 Swab			−	−	++	−	−	+	+/−	++	+	+	−
23 OF			−	+++	++++	+++	+++	++++	++++	++++	+++	+++	+
23 PP	16	7 mm	−	++++	++++	+++++	++++	+++++	++++	++++++	++++	++++	++	100
23 Swab			−	+++	+++	+++	+++	++	++	+++	+	++	+/-
24 OF			−	−	++	+++	−	+++	+	−	+	++	−
24 PP	22	5 mm	−	++	++	++	−	++++	++++	+++	++	++	−	91
24 Swab			−	−	++	++	−	++	+	−	+	+	−
25 OF			−	+++	+++	++++	+++	++	+	−	+	++	+/-
25 PP	28	5 mm	−	+++	+++	++++	+++	++++	++	++++	++	++	+	91
25 Swab			−	++	++	+++	++	++	+	+++	+/−	+	+/-
26 OF			−	+++	++++	−	−	++++	+++	++++	++	++	−
26 PP	24	5 mm	−	++	+++	−	−	+++	+++	+++	++	+++	−	100
26 Swab			−	+++	+++	−	−	++	++	+++	++	+	−
27 OF			−	+++	++++	++++	++++	+++	++	++++	+	++	+++
27 PP	16	7 mm	−	++++	+++++	+++++	++++	+++++	++++	++++++	+++	++++	+++	91
27 Swab			−	++++	++++	+++++	++++	+++	+++	−	+/−	+++	++++
28 OF			−	−	+++	+++	−	++	++	+++	+	++	−
28 PP	10	7 mm	−	−	+++++	+++++	−	+++++	++++	+++++	++++	+++++	−	100
28 Swab			−	−	+++	++	−	++	++	+++	+	+	−

1–28; each participant’s number; OF: collected oral fluids; PP: paper points; Swab: oral swab; Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn), Parvimonas micra (Pm), Prevotalla nigrescens (Pn), Campylobacter gracilis (Cg), Campylobacter rectus (Cr), and Eubacterium nodatum (En). A cell marked with a ‘−’ indicates that no signal was detected. A white cell indicates that no signal was detected using one of the sampling techniques. A grey cell indicates that a signal was only detected using one of the sampling techniques. A red cell indicates a difference in the level of bacterial presence compared with that detected from the other sampling technique.

provides an overview of the results per participant, per sampling method, and per bacterial species. The number of bacteria differed per method, but the composition of the oral flora was comparable in 95% of the cases. Considering the 308 bacteria tests (28 participants × 11 bacteria = 308), 14 did not have all the bacteria in all the samples retrieved by the 3 methods. A 100% match was found in 18 out of the 28 participants’ (64.3%) swabs, paper points, and oral fluid collection. The match between 7 participants (25%) was 91%, 2 participants (7.1%) had an 82% match, and 1 participant’s (3.6%) sampling method match was 73%.

Both the swab and oral fluids had an accuracy of 97% (9 mismatches per sample method over 308 measures). Paper point had an accuracy of 100% in this study.

3.3. Comparing Sampling Methods for Bacterial Detection

Table 3. Comparison of swab, paper point, and oral fluid samples with respect to bacterial presence.

Bacteria Species	p-Value
Aggregatibacter actinomycetemcomitans	1.000
Porphyromonas gingivalis	0.368
Tannerella forsythia	0.368
Treponema denticola	1.000
Fusobacterium nucleatum	0.368
Prevotella intermedia	1.000
Parvimonas micra	0.018
Prevotalla nigrescens	0.021
Eubacterium nodatum	0.097
Eikenella corrodens	0.368
Campylobacter concisus	0.135

shows a comparison of swab, paper point, and oral fluid samples in terms of bacterial presence. Significant differences in detection were observed for Parvimonas micra (p = 0.018) and Prevotella nigrescens (p = 0.021), indicating that the sampling method could affect the ability to identify these particular periodontal pathogens. These results emphasise the importance of considering sample type when conducting microbiological assessments of the oral cavity.

3.4. The Association Between Bacterial Match and Participant Characteristics

Table 4. Bacteria match between sampling technique and smoking, polypharmacy, and antibiotic use.

Bacteria Match (%)	Number of Participants	Number Smoking (%)	Polypharmacy (%)	Antibiotic Use (%)
100	18	10 (59)	9 (69)	9 (64)
91	7	5 (29)	2 (15)	4 (29)
82	2	2 (12)	1 (8)	0 (0)
73	1	0 (0)	1 (8)	1 (7)

shows the specifically studied participant parameter (smokers, polypharmacy, and antibiotic use <6 months before sampling) comparisons with the bacteria matches. Surprisingly, the group with 100% bacteria matches had a high number of smokers, polypharmacy, and the antibiotic use.

3.5. Characteristics of the Sampling Methods

Table 5. An overview of the characteristics per sample method (costs, comfort during sampling, ease of sampling, and accessibility).

	Cost of Sample Material Anno 2024 in The Netherlands (€)	Cost of Laboratory Analysis Anno 2024	Participant Comfort	Time of Sampling	Ease of Sampling (for the Practitioner)	Accessibility of the Sample Location
Swab	(0.03) +	75 euro	+	++	++	++
Paper point	(0.12) +	75 euro	−	−	−	−
Oral fluid collection	(0.15) +	75 euro	++	−	−	+

shows the characteristics of each sampling method. Notably, the costs of the sampling material for paper point (€0.12), the collection of oral fluids (€0.03), and the swab (€0.03) are very minor. Also, the costs of the lab analyses are the same for all three methods (€75). The participants’ comfort and the comfort during sample collection are best with the swab (mean score ++) and oral fluid collection (+) compared with the paper point (−). Most of the participants reflected that they considered the measurement of pockets and inserting paper points as uncomfortable. However, the insertion of a paper point in a pocket itself was usually not perceived as uncomfortable. On the other hand, the swabbing (++) was easier and needed less time compared to the paper point (−) and collection of fluids (−). The accessibility of the sampling site was best with the swab method (++), moderate for the collection of oral fluids (+), and lowest for the paper point method (−). A drawback of sampling oral fluids was that the practitioner had to give clear instructions to the participant and be sure these instructions were understood, which was not always easy for the participants with cognitive impairment or hearing disabilities. Overall, based on ease of use, costs, and comfort for the participant, the swab had the highest positive rating.

4. Discussion

In this study, the three most commonly used sampling techniques for oral microbiota (swab, paper point, and oral fluid) were compared in a geriatric population with memory complaints.

It seemed that, in most cases, comparable results were found for these three bacteria level testing techniques. When looking at the participant level, a 100% match was found in 18 out of the 28 participants. A 95% match was found at the bacteria level.

Our study shows that each sampling method has strengths and degrees of accuracy. Therefore, each specific study’s method choice should be guided by the bacteria to be detected. At the population level, especially for research purposes in comprehensive geriatric populations, swabbing appears to be the most practical and broadly applicable technique. Selecting an appropriate sampling strategy is essential for accurate and comprehensive microbial analysis. Factors to consider include the collection method, the participant population, sample clarity, hydration status, ability to cooperate, and sampling method.

The oral fluid and swab sampling techniques were rated as the most convenient by the participants and practitioner. When placing the paper point in a periodontal pocket, the deepest pocket needed to be found and measured before sampling, which was usually rated as unpleasant. The oral fluid collection method was not always easy as the clinician had to give strict instructions to the patient and the patient did not always understand the instructions. This can be a hassle if the patient is suffering from cognitive impairment or is deaf. In our group, two participants even started gurgling their throats when they had to collect their oral fluids. This resulted in a poor test as the sample was supposed to be oral. Moreover, oral fluids can be dangerous for geriatric patients suffering from dysphagia. The swab was therefore considered the best and easiest method for this geriatric population as it was highly accurate, comfortable, cheap, and easy to use, even in patients with disabilities such as tremor, bent neck, dysphagia, deafness, oral dryness, cognitive impairment, etc. However, when the primary focus is on periodontal microbiota, the paper’s point is still the most appropriate, despite the logistical challenges involved.

Previous studies have investigated subsets of the sampling techniques compared in our study. For example, Boutaga et al. evaluated mouthwash and paper point sampling methods, concluding that mouthwash sampling is a less time-consuming and non-invasive technique that is easy to perform, inexpensive, and cost-effective, which is consistent with our findings [9]. Importantly, they reported similar detection rates for the key bacteria, including Aa, Pg, Tf, Pi, and Pm [9]. Furthermore, Smola et al. compared the paper point and oral swab techniques and found strong agreement in the detection of several relevant bacteria, including Pg, Td, Tf, Fn, and Pi [11]. These results overlap substantially with our own [11]. They concluded that the swab method could be applied easily in clinical practice due to its simplicity and non-invasiveness and could even be self-administered by patients [11]. These findings align with our own, reinforcing the practicality and broader applicability of swab-based sampling, particularly in geriatric elderly or less cooperative populations, such as mentally handicapped, where more invasive techniques may be less feasible [11].

Even though the denture was removed during sampling, its presence may still influence the outcome of the oral microbiota. Some microorganisms that are normally present in the oral mucosa can cause opportunistic infections of the oral cavity or systemic diseases in wearers of partial or total removable dentures [19]. Furthermore, the presence of a complete denture in the mouth decreases salivary flow and pH, thereby influencing the composition of the oral microbiota [20]. Furthermore, the mucous area covered by complete denture does not undergo the cleansing action of saliva or the mechanical cleansing action of the tongue, thus favouring the development of biofilms [21]. It should be noted that older people’s oral tissue loses elasticity and flakes off more easily, becoming more fragile and susceptible to lesions in the oral cavity and favouring the colonisation of opportunistic microbiota [22].

Limitations and Strengths of This Study

This study compared three different oral sampling methods, providing a comprehensive overview of their respective advantages and disadvantages, particularly within the geriatric population. This is a key strength, as it reflects the real-world challenges of sampling older people in clinical and research settings.

A limitation of this study is the relatively small population. The sample size is large enough to detect small differences between the sampling methods, particularly for bacteria with low positivity rates. However, comparable numbers of participants were analysed by other studies of oral microbiota [23,24]. The sampling order was not randomised and no adjustments were made for potential confounders such as smoking, antibiotic use, and polypharmacy, which may have influenced the results. Furthermore, paper points were rather difficult to use and only feasible in those participants who still had their own dentition, or at least one tooth. Next, it is important to note that the scope of our microbiota analysis was rather limited. Due to the high cost of analysing the whole oral microbiome and the limited feasibility of an exploratory study, comprehensive sequencing methods that provide complete microbiome profiles were not undertaken. Instead, we selected a targeted bacterial panel comprising the Socransky complexes, several of which are associated with age-related diseases and periodontitis in the literature [18]. The rationale behind the selection of these bacteria is that the function of viruses and yeasts in periodontitis remains to be fully elucidated, thus leading to their exclusion from this study.

Although this approach does not capture the full microbial diversity of the oral cavity, it aligned well with the study’s objectives. Similar studies have used bacteria panels containing between five and twelve bacteria, primarily in relation to periodontal disease and its systemic associations [25,26]. For example, one study used qPCR to quantify seven bacteria (Aa, Fn, En, Pg, Pi, Tf, and Td) [26].

5. Conclusions

The three studied sampling methods showed comparable microbiota outcomes but differed in feasibility and user experience. At a population level, the swab was considered the most favourable for geriatric patients, based on costs, comfort, time, ease of sampling, and accessibility. Oral fluid collection was generally well-tolerated but required clear instructions and was less feasible for participants with cognitive impairment or dysphagia. The paper point method was least favourable due to lower comfort, longer sampling time, and limited applicability to dentate participants, but remains most suitable when the primary focus is on periodontal microbiota.