The Effectiveness of Brushing and Flossing Sequence on Control of Plaque and Gingival Inflammation: Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for The Effectiveness of Brushing and Flossing Sequence on Control of Plaque and Gingival Inflammation (NCT03989427), a randomized na study. The primary outcome is Change From Baseline in Mean BPI Index Scores at 2 Weeks.

2. Clinical Question

This is a comparison of sequence order, rather than a comparison between flossing and no flossing: both sequences include brushing and flossing. The trial therefore addresses whether the order in which those activities are performed is associated with different changes in the recorded indices under the study conditions. The registry also lists change in the mean RMNPI index score at 2 weeks as a secondary outcome. The posted results do not establish how the findings would apply beyond this study population or setting.

3. Trial Design

The registry describes a randomized, single-masked crossover clinical trial sponsored by Melaka Manipal Medical College. Enrollment was 30, and the record lists the study as completed. The listed start date is 2019-05-20 and the primary completion date is 2019-12-15. The phase is listed as NA.

The two sequences were brushing first and flossing later (BF), and flossing first and brushing later (FB). Both descriptions include brushing with the modified Bass method and flossing; the BF description specifies the spool method for flossing, and the FB description also specifies the spool method. The intervention descriptions refer to Colgate toothpaste, toothbrushes and dental floss. Each description gives a 2-week period. The primary outcome description says that measurements were taken at baseline and 2 weeks, followed by a washout period of one week, and then measurements were taken again at baseline and 2 weeks after crossover.

A crossover design compares conditions across periods, allowing participants to receive the sequences in turn. Randomization concerns assignment to the sequence; it does not by itself explain how period, sequence or carryover effects were handled in the analysis. Those analysis details are not reported in the registry record. The masking field is recorded as single, but the identity of the masked party is not reported in the registry record.

The primary effectiveness population is described as participants who received at least one intervention and completed all study visits. The posted outcome summaries list 30 in each sequence. The registry record does not report the detailed participant flow or explain how incomplete visits or missing measurements were handled. The record identifies two outcome measures and two reported analyses, with one secondary outcome posted.

4. Participant Flow and Accounting

The registry reports how participants moved through each stage of the study. All counts below are exactly as posted; missing counts are not inferred from other stages.

Before assignment: 30 of 221 participants were randomized. Of those not randomized, 120 did not meet inclusion criteria and 71 declined to participate.

First Intervention (2 Weeks)

Washout (1 Week)

Second Intervention (2 Weeks)

Analysis population for the primary outcome, as defined in the registry: All participants who received at least one intervention each and completed all study visits were included in the effectiveness analysis.

5. Baseline Characteristics

The table reproduces the baseline characteristics posted in the registry, which describe the participants analysed at baseline and help show whether the randomized groups were comparable.

6. Primary Endpoint

The registry lists the primary outcome as Change From Baseline in Mean BPI Index Scores at 2 Weeks, measured over Baseline and 2 weeks.

The primary endpoint is change from baseline in mean BPI score at 2 weeks. BPI, or Bleeding Point Index, measures bleeding on probing. The registry describes assessment of buccal, lingual and proximal tooth surfaces. Bleeding on probing is used as an objective indicator of inflammation, so the index provides a measured outcome relevant to the trial's focus on gingival inflammation.

The registry describes BPI scores as ranging from 0% to 100% of bleeding surfaces in an individual with teeth. It also states that higher mean scores represent a poorer outcome, while a greater reduction from baseline represents a better outcome. The endpoint is a change score, not simply the level of BPI at follow-up. Its interpretation depends on the direction used to calculate change; the registry does not further state that sign convention in the posted summary.

Measurements were scheduled at baseline and 2 weeks for each intervention period, with a washout period of one week between periods. Comparing changes can account for the starting score in each period, but it does not by itself show whether the analysis accounted for the paired nature of a crossover study or for period and sequence effects. The registry record does not report those model details.

The outcome is an index-based measure, not a direct report of symptoms or a clinical diagnosis. Its value in this trial is as a standardized way to compare observed changes in bleeding across the two brushing-and-flossing sequences.

7. Results Posted in the Registry

• Flossing First and Brushing Later (FB): mean -1.03 versus 0.40 on control; difference -1.42 (95% CI -2.54 to -0.31), standardized effect d = -0.65; z-test p = 0.012.

8. Secondary Efficacy Endpoints

The registry also posts results for secondary outcomes. For each outcome below, the endpoint type, the test applied to the posted summary statistics, the result and its interpretation are given in turn. Secondary endpoints are usually not adjusted for multiple testing, so they support rather than confirm the primary finding.

Change From Baseline in Mean RMNPI Index Score at 2 Weeks

• Endpoint type: Continuous: a mean value per participant, compared as a difference in means.
• Test: Unadjusted two-sample z-test for the difference in means, using the posted standard errors.
• Result:
  • Flossing First and Brushing Later (FB): mean 0.10 versus 0.04 on control; difference 0.06 (95% CI -3.41 to 3.52), standardized effect d = 0.01; z-test p = 0.973.
• Interpretation:
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between Flossing First and Brushing Later (FB) and the control. The interval gives the range of effects that remain compatible with the data.

The registry posts one secondary endpoint, change from baseline in mean RMNPI score at 2 weeks. Its unadjusted comparison is close to no estimated difference, but the confidence interval is broad and includes differences in either direction. The sponsor-reported analysis also has a broad interval spanning values on both sides of no difference. These results are less precise than a narrow interval would be; they do not establish that the sequences produce identical plaque-index changes.

The secondary outcome does not reproduce the pattern of the primary BPI comparison, whose unadjusted interval excludes no difference. That contrast in patterns should be interpreted cautiously. The endpoints measure different indices, and the registry record does not report a multiplicity adjustment or a hierarchy for interpreting primary and secondary findings. A secondary result should not be used to confirm or overturn the primary finding on its own.

Unadjusted secondary results carry less weight when several outcomes are examined because repeated testing can increase the chance of a result appearing notable by chance. The record lists two outcome measures and two analyses, but it does not state whether these were the complete prespecified analysis family or whether any correction was planned. The secondary findings are therefore best read as additional descriptive evidence, with their broad uncertainty and analysis context in mind, rather than as definitive confirmation of a sequence effect.

Secondary outcomes registered for this trial, for reference (only those above were recalculated):

• Change From Baseline in Mean RMNPI Index Score at 2 Weeks

9. Statistical Interpretation

10. Statistical Methods Explained

The posted summaries report means and standard errors for continuous outcomes. A mean describes the average recorded change within a sequence. A standard error describes the estimated precision of that mean, rather than the spread of individual participants' measurements. It is therefore not interchangeable with a standard deviation. The primary summaries list 30 observations for each sequence, but the registry does not report the participant-level data or the method used to account for repeated measurements in the crossover design.

What does the mean difference represent?

The recalculated primary contrast is defined as the FB mean minus the BF mean. A negative estimate therefore means the posted FB mean change is lower than the posted BF mean change on that stated contrast. The estimate is -1.424. Reversing the order of subtraction reverses the sign while leaving the underlying comparison conceptually the same. The registry-reported ANOVA gives a positive net difference of 1.424, which is consistent with the opposite contrast orientation. The outcome description says that greater reduction from baseline is better, but the record does not specify the arithmetic sign convention for the change score. Interpretation should therefore retain the stated contrast and outcome coding rather than infer a clinical direction from the sign alone.

What does a confidence interval add?

A confidence interval gives a range of values compatible with the observed estimate under the calculation's assumptions. For the recalculated primary comparison, the 95% interval is -2.536 to -0.312. Because the interval does not include no difference for that contrast, the unadjusted calculation is inconsistent with no difference at the corresponding conventional test threshold. This statement is about the calculation and its assumptions; it does not prove that the design, measurement or analysis is free from bias. The interval is not a measure of the proportion of participants who improved, and it does not show the spread of individual responses.

Why distinguish the recalculation from the registry analysis?

The recalculation is an unadjusted comparison from the posted summary statistics. It does not reproduce the sponsor's ANOVA or any model that might use participant-level data. In a crossover trial, measurements from the same participant across periods are related. A summary-level comparison that treats the two sequence summaries as independent cannot make use of that within-participant structure unless the required paired information is available. The registry does not report the covariance, model specification, period adjustment, sequence adjustment or carryover assessment needed to evaluate those features. The sponsor-reported result should consequently be kept distinct from the recalculated result, rather than treated as a direct reproduction of the sponsor's analysis.

What does the p-value say?

A p-value describes how incompatible the observed data are with a specified null model, assuming that model and the analysis assumptions are correct. It is not the probability that the null hypothesis is true, nor a measure of the size or importance of an effect. The unadjusted recalculated primary p-value is 0.012; the sponsor-reported p-value is 0.022. These are results from different reported calculations and should not be substituted for one another. The record labels the sponsor analysis as equivalence, but the equivalence margin and the test details are not reported in the registry record.

How should the secondary measure be viewed?

RMNPI is the posted secondary outcome, also expressed as change from baseline in a mean index score at 2 weeks. Its unadjusted recalculated contrast is 0.059, with a 95% confidence interval from -3.405 to 3.523 and p = 0.973. This wide interval includes values on either side of no difference and does not provide a precise estimate of the sequence contrast. Its sponsor-reported ANOVA gives a net difference of -0.058, a 95% confidence interval from -3.335 to 3.219 and p = 0.971. These results are not evidence that the sequences are identical; they show that the reported data and calculations do not distinguish a difference precisely.

Multiplicity arises when several outcomes or comparisons are examined. The registry lists two outcomes and two analyses, with one secondary outcome posted. It does not report an adjustment for multiple testing or identify a hierarchy for interpreting outcomes. Unadjusted p-values do not account for the chance of false positive findings across a family of tests, so they warrant more caution when read alongside other outcomes. The registry record also does not report a sample-size justification, interim analyses, a prespecified statistical analysis plan, missing-data methods or the details of the equivalence margin. These gaps limit how fully the posted tests can be assessed.

11. Confidence Intervals and Precision

The primary unadjusted estimate is -1.424 for the FB-minus-BF contrast, and its 95% confidence interval runs from -2.536 to -0.312. The interval excludes no difference for this contrast, but it spans effects of differing magnitudes. It conveys uncertainty in the estimated mean difference under the recalculation's assumptions, not uncertainty about every aspect of the study or the probability that a particular value is true.

The sponsor-reported primary confidence interval is 0.221 to 2.628 around a net mean difference of 1.424. The reversed sign is consistent with the contrast being stated in the opposite group order. The endpoints and p-value differ from those of the unadjusted recalculation. The registry record does not include enough information about the sponsor's model to determine why these interval estimates differ. They should be read as separate analyses, not combined into a single interval.

For the secondary RMNPI comparison, the recalculated estimate is 0.059, with a 95% confidence interval from -3.405 to 3.523. That range includes no difference and values in either direction, indicating limited precision for the contrast. The sponsor-reported interval is -3.335 to 3.219 around -0.058. Neither interval provides a narrow estimate of the secondary difference.

Confidence intervals describe sampling uncertainty conditional on the analysis assumptions. They do not incorporate unreported sources of uncertainty such as missing-data handling, carryover, measurement limitations or the choice of analysis model. The record does not report a clinically important difference threshold, so the clinical importance of estimates within these ranges cannot be judged from the intervals alone.

12. Planning a Similar Trial: Sample Size

This is a planning illustration, not a re-evaluation of the original trial. It asks: if a new study wanted to detect a difference like the one observed, or a smaller one, how large would it need to be? It is deliberately not “observed power” of the finished trial, which is not a meaningful quantity.

The planning figures are participants per arm, not total events. This is a continuous-outcome planning illustration, and the figures are based on the stated fraction of the observed effect. The listed assumptions are a two-sided significance level of 0.05, equal allocation between the two arms and a normal approximation. The trial's original design assumptions and sample-size justification are not reported in the registry record.

The 80% power column gives the illustrative number of participants per arm for a design with an 80% chance of detecting the specified effect under the planning assumptions, if that effect and the assumptions are correct. The 90% power column gives the corresponding planning figure for a 90% chance. These are design calculations, not a guarantee that a future study will detect an effect. They are not observed power for this completed trial.

The rows consider planning for 100%, 75% and 50% of the observed effect. A smaller target effect requires more participants to detect it at the same stated power, because a smaller signal is harder to distinguish from sampling variation. The displayed figures can therefore rise sharply when planning for a smaller fraction. The observed effect is itself uncertain, particularly when it comes from a small study, so planning directly from it may be unstable. These illustrations are not evidence that the trial had adequate or inadequate power, and they should not be read as a reconstruction of the investigators' original design.

The primary and secondary outcomes have different observed estimates and uncertainty. Their planning illustrations consequently differ greatly. Such differences reflect the effect and variability estimates used in the calculation, not a ranking of clinical importance. The registry record does not report whether the trial's recruitment target was based on either outcome.

13. How the Sample Size Is Derived

Power is the probability that a study of a given size rejects the null hypothesis when a specific true effect exists. It depends on four things: the effect to detect, the variability of the outcome, the significance level (here a two-sided α of 0.05, so z_1−α/2 = 1.960) and the desired power (z_1−β = 0.842 for 80% and 1.282 for 90%).

Formula

n is the number of participants in each arm, Δ is the difference in means to detect and σ is the common standard deviation.

Worked calculation for this trial

1. Observed difference Δ = -1.424; pooled standard deviation σ = 2.197.
2. n = 2 × (1.96 + 0.842)² × 2.197² / -1.424² = 37.4, rounded up to 38 participants per arm for 80% power.

The smaller-effect rows in the table above repeat the same calculation with the effect multiplied by 0.75 and 0.5 (for ratios, applied to the logarithm of the ratio).

R code

alpha <- 0.05
z_a   <- qnorm(1 - alpha / 2)          # two-sided
delta <- -1.424                      # observed difference in means
sigma <- 2.19692057207356                     # pooled standard deviation
n_per_arm <- function(power, frac = 1) {
  ceiling(round(2 * (z_a + qnorm(power))^2 * sigma^2 / (delta * frac)^2, 6))
}
for (f in c(1, 0.75, 0.5))
  cat(sprintf("%3.0f%% of observed effect: n per arm = %s (80%% power), %s (90%% power)\n", 100 * f,
              format(n_per_arm(0.80, f), big.mark = ","), format(n_per_arm(0.90, f), big.mark = ",")))

# Check against R's built-in function (t-based, so very slightly larger)
cat("power.t.test:", round(power.t.test(delta = delta, sd = sigma, power = 0.80)$n, 1), "\n")

# Simulation: exact two-sample t-test using simulated means and variances
set.seed(3989427)
n    <- n_per_arm(0.80)
reps <- 20000
m_diff <- rnorm(reps, delta, sigma * sqrt(2 / n))             # difference in sample means
s2     <- sigma^2 * rchisq(reps, 2 * (n - 1)) / (2 * (n - 1))   # pooled sample variance
tstat  <- m_diff / sqrt(s2 * 2 / n)
cat(sprintf("Simulated power at n = %s per arm: %.3f (%d replicates)\n", format(n, big.mark = ","), mean(abs(tstat) > qt(0.975, 2 * (n - 1))), reps))

Output from running the code

100% of observed effect: n per arm = 38 (80% power), 51 (90% power)
 75% of observed effect: n per arm = 67 (80% power), 89 (90% power)
 50% of observed effect: n per arm = 150 (80% power), 201 (90% power)
power.t.test: 38.3 
Simulated power at n = 38 per arm: 0.795 (20000 replicates)

The simulation draws many sets of group means and variances at the planned sample size and counts how often the two-sample t-test rejects the null. It should land close to the target power; at very small required sample sizes it can fall below it, because the normal approximation is rough there.

14. Analyses Reported by the Sponsor

The sponsor-reported primary analysis is an ANOVA labelled as an equivalence analysis. It reports a net mean difference of 1.424, a 95% confidence interval from 0.221 to 2.628 and p = 0.022. The unadjusted recalculation reports the contrast as FB minus BF, giving -1.424, a 95% confidence interval from -2.536 to -0.312 and p = 0.012. The opposite signs are consistent with reversing the order of groups in the contrast. The confidence limits and p-values are not identical.

The recalculation uses posted means and standard errors and is not a reproduction of the sponsor's ANOVA. The registry does not report the model details needed to explain the differences in interval width and p-value. The sponsor's analysis is the trial's reported analysis, while the recalculation is a supplementary unadjusted comparison. Both should be described with their contrast orientation and method clear.

The equivalence label requires particular care. A conclusion of equivalence depends on a prespecified margin and an analysis designed to test that margin. Neither the margin nor its justification is reported in the registry record. A confidence interval and p-value alone, without the margin and the applicable test details, do not establish equivalence. The RMNPI sponsor analysis is likewise labelled equivalence, but the same limitation applies.

15. Multiplicity, Missing Data and Analysis Populations

The registry lists two outcome measures and two analyses, including the primary BPI change and the secondary RMNPI change. The secondary result is posted, but the registry record does not report a multiplicity adjustment, a testing hierarchy or a prespecified rule for interpreting multiple outcomes. When several hypotheses are tested, unadjusted p-values do not account for the increased opportunity to obtain a notable result by chance. This matters especially when interpreting secondary findings alongside the primary outcome.

The primary analysis population is described as participants who received at least one intervention and completed all study visits. The posted arm summaries each list 30. The registry record does not report how many participants were screened, randomized to each sequence, excluded from the effectiveness analysis, or missing particular measurements. It also does not report an imputation method, a sensitivity analysis for missing data or whether the posted summaries use all measurements available at each visit. The stated analysis-population description is not enough to assess the effect of incomplete data.

The record identifies the design as crossover and describes a washout period of one week. However, it does not report whether the analysis accounted for within-participant correlation, period effects, sequence effects or possible carryover. It does not report the statistical model, the analysis plan, interim analyses, or a sample-size justification. The sponsor analysis is labelled equivalence, but the equivalence margin and its rationale are not reported.

These omissions do not show that such procedures were absent; they mean that the registry record does not provide enough detail to assess them. They limit reproducibility and make it difficult to determine how well the posted estimates address the crossover structure and the several outcomes.

16. Safety Results

The registry reports serious adverse events as affected participants divided by participants at risk.

Deaths

Other (non-serious) adverse events

Participants with at least one non-serious event at or above the registry's 0% reporting threshold.

The registry's adverse-event record lists 30 participants at risk for serious adverse events in the combined brushing-and-flossing group, with 0 affected and 0 deaths. It also lists 30 at risk for other adverse events, with 0 affected. No serious adverse-event terms are listed.

These are the adverse-event counts recorded for the study, not proof that no event of any kind occurred beyond what was captured and reported. The record does not provide further detail about the method or period of adverse-event collection, or about how events were attributed to either sequence. It also does not present separate safety counts for BF and FB. The figures should therefore be read narrowly as the posted registry counts, rather than as a comparative safety analysis or a complete characterization of safety.

17. What the Result Does, and Does Not, Mean

The posted data show a difference between the two sequence summaries for the primary BPI change under the unadjusted recalculation. The recalculated estimate, its confidence interval and the sponsor-reported ANOVA are statistical descriptions of the observed data. They do not, by themselves, show that sequence order caused the difference or establish how important that difference is. The registry does not report a threshold for a clinically important change.

The primary outcome is an index of bleeding on probing, and the secondary outcome is a plaque index. These are measured indices, not direct evidence about every aspect of oral health or about long-term outcomes. The RMNPI comparison is much less precise, with an interval spanning values on either side of no difference. It should not be interpreted as proof of no secondary difference.

The crossover design and the reported washout period are relevant to interpretation, but the registry does not describe the analysis of repeated measurements or possible period and carryover effects. The statistical summaries cannot resolve those questions. The posted analysis also does not establish equivalence: the required margin is not reported, despite the sponsor analysis label.

The results apply directly to the recorded study population, procedures and measurement periods. The record does not provide enough information to determine how representative the participants were of other populations or settings, or whether the findings persist beyond the reported measurement periods. The statistics do not support a recommendation about which sequence an individual should use.

18. Why This Trial Matters Statistically

This trial raises an important statistical distinction: a randomized crossover comparison can generate repeated measurements within participants, while the posted summary-level recalculation compares means and standard errors. Those summaries are useful for describing the observed contrast, but they cannot show whether the analysis fully used the paired structure. Reporting the model, period and sequence terms, and any carryover assessment would make the result easier to evaluate.

The different sign conventions in the sponsor analysis and the recalculation illustrate why the group order for a contrast should always be stated. A positive and negative estimate can describe the same comparison when the order is reversed, but the interval and test statistics should still be interpreted according to the method that produced them. Clear labeling helps prevent readers from mistaking an orientation difference for a disagreement in direction.

The equivalence label also highlights a broader point: equivalence is not established merely because an estimate appears small or because an analysis has a nonsignificant result. Interpretation depends on a prespecified margin and an appropriate analysis. The registry record does not report the margin, making the label difficult to assess.

Finally, the primary and secondary outcomes have different precision and patterns. A careful reading keeps the outcome hierarchy, uncertainty and unadjusted testing in view rather than treating every p-value as equally conclusive. The registry's missing details about multiplicity and missing data further emphasize the value of transparent statistical reporting.

19. Limitations

• The posted summary statistics are the only data these results rest on; patient-level data, full protocols and statistical analysis plans are not available.
• Recalculated results are simple, unadjusted calculations from posted summaries. They do not replace the sponsor's prespecified analysis.

Several limitations specific to this record affect interpretation. The trial enrolled 30, and the effectiveness population is defined by receipt of at least one intervention and completion of all study visits. Although the arm summaries list 30 for each sequence, detailed participant flow and missing-measurement information are not reported in the registry record.

The study is identified as a crossover trial and describes a washout period of one week. The registry does not report the statistical model or whether it accounts for within-participant correlation, period effects, sequence effects or carryover. The unadjusted recalculation from summary statistics therefore cannot be assumed to reproduce an analysis that uses the crossover structure.

The sponsor's primary analysis is labelled as equivalence, but no equivalence margin or rationale is reported. The registry also does not report the original sample-size justification, the planned multiplicity approach, interim analyses, or a detailed statistical analysis plan. These omissions limit assessment of whether the reported tests match the intended design and analysis.

The masking field is single, but who was masked is not identified. The record does not report a clinically important BPI difference threshold. The posted outcome periods are short, and the registry does not report follow-up beyond the measurement schedule described. Finally, the participant characteristics and details needed to assess generalizability are not reported in the registry record.

20. Related Tutorials

21. Related Statistical Calculators

22. Sources

• ClinicalTrials.gov: The Effectiveness of Brushing and Flossing Sequence on Control of Plaque and Gingival Inflammation — NCT03989427.
• Registry record last updated 2020-06-02; status last verified 2020-05.
• Results first posted 2020-06-02.
• Data retrieved from ClinicalTrials.gov on 2026-11-09.
• Group sizes, means, event counts and sponsor-reported p-values are reproduced as posted. Effect estimates, confidence intervals and p-values not attributed to the sponsor are independently calculated from the posted summary statistics.

23. Record Summary

NCT03989427 is a completed randomized, single-masked crossover trial sponsored by Melaka Manipal Medical College. It compared brushing followed by flossing with flossing followed by brushing. Enrollment was 30. The primary outcome was change from baseline in mean BPI score at 2 weeks; change in mean RMNPI score was the posted secondary outcome.

The unadjusted primary recalculation estimates an FB-minus-BF difference of -1.424, with a 95% confidence interval from -2.536 to -0.312 and p = 0.012. The sponsor-reported ANOVA gives a net difference of 1.424, a 95% confidence interval from 0.221 to 2.628 and p = 0.022. The secondary comparison is imprecise. Important details about the analysis model, missing data, multiplicity and the equivalence margin are not reported in the registry record.