Relevance of Monitoring Blood and Salivar Levels of Drugs Used in Rheumatic Autoimmune Diseases: Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for Relevance of Monitoring Blood and Salivar Levels of Drugs Used in Rheumatic Autoimmune Diseases (NCT03122431), a randomized phase 4 study. The primary outcome is Serum Levels of Hydroxycloroquine.

2. Clinical Question

3. Trial Design

The registry describes a completed Phase 4 study with randomized allocation, no masking, and a parallel model. Enrollment is listed as 93. The record gives a start date of 2017-06-05 and a primary completion date of 2020-12-30. The sponsor is University of Sao Paulo General Hospital.

The project description covers monitoring drug concentrations in blood and saliva in rheumatic autoimmune diseases. It names hydroxychloroquine, thalidomide, biologic agents, and glucocorticoids among the intended areas of study. It describes high-performance liquid chromatography coupled to mass spectrometry for evaluating some drugs and ELISA capture methodology in relation to biologic agents. Those broader aims should be distinguished from the primary result posted for serum hydroxychloroquine levels.

The registry describes separate subprojects rather than a single comparison covering all the listed conditions and drugs. One arm concerns people with active, refractory cutaneous lupus eligible for thalidomide 100mg/day for 12 months. The two inactive-SLE arms concern standard-dose hydroxychloroquine at 400mg/day and a reduced regimen of 400mg 3 times a week. The primary comparison with posted serum-level summaries is between those two hydroxychloroquine arms. The record does not describe a thalidomide comparator arm.

Although the study is labeled randomized, details of how assignment was conducted, which subprojects involved random allocation, and the allocation procedure are not reported in the registry record. The original design assumptions and the planned sample size for each subproject are also not reported in the registry record. The posted primary arm summaries list 41 participants for standard dose and 32 for reduced dose; the registry separately lists overall enrollment as 93. These figures describe different registry entries and should not be treated as a complete account of participant flow.

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.

Overall Study

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 Serum Levels of Hydroxycloroquine, measured over 12 months.

The primary endpoint is the mean serum level of hydroxychloroquine at 12 months, measured in ng/mL using LCMS, as described in the registry. A serum concentration is a continuous measure: it can take a range of values rather than being only a category such as present or absent. The registry reports a mean and standard deviation for each of the two hydroxychloroquine arms.

In general, measuring drug concentration can help characterize exposure. The project description frames blood and saliva monitoring as a way to investigate adherence and possible mechanisms related to effectiveness and adverse effects. That rationale does not make a measured serum level a direct measurement of adherence, nor does it establish a link between a particular level and a clinical outcome. Those conclusions require relevant data and analyses, which are not reported in the registry record.

The posted endpoint is specifically serum hydroxychloroquine, not salivary concentration and not a clinical disease outcome. The registry does not report a separate endpoint result for salivary levels in this comparison. The endpoint therefore addresses a difference in measured serum levels between the two inactive-SLE hydroxychloroquine arms, rather than whether one regimen changes disease activity, prevents adverse effects, or improves adherence.

7. Results Posted in the Registry

• Inactive SLE With Reduced Dose of HCQ: mean 569.00 versus 991.60 on control; difference -422.60 (95% CI -678.09 to -167.11), standardized effect d = -0.76; z-test p = 0.001.

8. Statistical Interpretation

9. Statistical Methods Explained

The primary outcome is continuous, so its arm-level summaries are expressed as means and standard deviations. A mean describes the average measured level in an arm. A standard deviation describes how spread out measurements are around that mean. In general, a mean difference compares the two arm means on the measurement’s original scale, so the reported contrast remains in ng/mL rather than being a percentage or a standardized clinical effect.

How should the mean difference be read?

The comparison is defined as reduced dose relative to standard dose. The reported estimate is -422.6 ng/mL. Its negative direction indicates that the reduced-dose mean is below the standard-dose mean. This is a between-arm difference in the posted summaries; it is not an estimate of the change experienced by each participant over time. The registry record does not report paired measurements or a within-person change analysis.

The two arm means are 991.6 ng/mL and 569.0 ng/mL, and the corresponding standard deviations are 576.3 ng/mL and 533.4 ng/mL. In general, a mean difference is sensitive to the values of observations and may not describe the typical participant when measurements are widely spread or skewed. The registry record does not report the distribution of individual levels, a median, or a distributional diagnostic, so those features cannot be assessed from the posted summaries.

What does the confidence interval add?

A confidence interval gives a range of values compatible with the estimate under the statistical procedure and assumptions used. The reported 95% confidence interval for the mean difference extends from -678.1 to -167.1 ng/mL. In general, a confidence interval is not a guarantee that the true value lies in the interval, and it is not a range containing a specified proportion of individual measurements. It describes uncertainty in the estimated contrast, not the spread of participant-level values.

The interval lies below zero, which is consistent with a lower mean in the reduced-dose arm. Its endpoints also show that the point estimate should not be treated as exact. The width reflects uncertainty in estimating the difference. It does not include every possible source of uncertainty: for example, the registry does not report enough detail to assess missing-data assumptions or the full analysis plan.

How should the p-value be interpreted?

The unadjusted p-value is 0.001. In general, a p-value describes how unusual data at least as discrepant as the observed data would be under a specified null model and its assumptions. It is not a probability that the observed conclusion is wrong, nor is it a measure of effect size. The estimate and confidence interval are needed to understand the direction and scale of the difference. The value is labeled unadjusted, and no multiplicity adjustment is reported.

The registry reports a pooled standard deviation of 558.0 for the comparison and a standardized effect value, but the latter is not needed to interpret the difference on the original ng/mL scale. Standardized effects express a difference relative to variability and can aid comparisons across measures with different units. They should not replace the original-scale result when the clinical meaning of the measurement is the focus.

What assumptions cannot be checked?

General methods for comparing means rely on assumptions about the observations and the statistical model. Independence of observations, the distribution of measurement errors, and the handling of missing measurements can affect inference. The registry record does not report diagnostic checks, transformations, covariate adjustment, or a detailed model specification. The posted summaries do not permit those assumptions to be evaluated at the participant level.

Over-dispersion is a general concern when observed variability exceeds what a chosen model allows. If a model understates variability, standard errors and confidence intervals may be too optimistic. The posted standard deviations convey arm-level variability, but the record does not report an over-dispersion assessment or a model-based variance diagnostic. It is therefore not possible to determine from the registry record whether this issue was relevant to the study’s own analysis.

The study is identified as randomized, but the record does not report the allocation procedure, stratification, concealment, or whether randomization applied to this specific comparison. Nor does it report the original analysis plan. Those details matter when interpreting causal contrasts and assessing whether the posted comparison follows the intended design. The registry record reports no posted analyses, so the displayed comparison should be read as a recalculation from the posted arm summaries, not as a complete account of the sponsor’s analysis.

In general, random allocation can support comparisons between groups when carried out and analyzed appropriately, but the registry label alone does not document those procedures. The difference in serum levels is a direct description of the two posted arm summaries. Claims about causal effects, clinical outcomes, or mechanisms require more information than this comparison provides.

10. Confidence Intervals and Precision

The estimated mean difference for reduced dose relative to standard dose is -422.6 ng/mL. Its 95% confidence interval is -678.1 to -167.1 ng/mL. The interval gives a range of plausible values for the underlying mean contrast under the statistical method and its assumptions. Because the range lies below zero, its direction is consistent with lower mean serum levels in the reduced-dose arm.

The interval is not a range of individual participants’ levels. The standard deviations, 576.3 ng/mL in the standard-dose arm and 533.4 ng/mL in the reduced-dose arm, describe variation among measurements within arms. Confidence intervals and standard deviations answer different questions: one expresses uncertainty in the estimated group contrast, while the other describes spread in the observed measurements.

Precision is limited by the width of the interval: the posted estimate is not known with exactness. The interval also depends on the analysis assumptions. The registry record does not report missing-data procedures, model diagnostics, or a detailed analysis plan, so the interval cannot address uncertainties associated with those unreported choices. It also does not show whether the difference is clinically meaningful, since the registry record does not give a threshold for clinical importance.

The unadjusted p-value of 0.001 provides another summary of the comparison under a null model, but it does not replace the confidence interval. The interval communicates both direction and a range for the estimated difference; the p-value alone does not communicate the size or precision of that difference.

11. 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 illustrations express sample size as participants per arm, not total events. They use the observed mean difference as a reference and consider planning for 100% of that observed effect, 75% of the observed effect, and 50% of the observed effect. For 100% of the observed effect, the illustration gives 28 participants per arm for 80% power and 37 participants per arm for 90% power. For 75% of the observed effect, it gives 49 participants per arm for 80% power and 66 participants per arm for 90% power. For 50% of the observed effect, it gives 110 participants per arm for 80% power and 147 participants per arm for 90% power.

The planning assumptions are a two-sided significance level of 0.05, equal allocation between the two arms, and a normal approximation. The effect to detect is the stated fraction of the observed effect. In plain terms, the 80% power column gives the illustrative participants per arm for a design with an 80% chance of detecting an effect of the specified size under these assumptions, if that effect is truly present. The 90% power column uses a higher target chance of detection under the same stated framework.

Smaller effects need more participants to be detected with the same target power under the same assumptions, as reflected in the increasing planning figures for smaller fractions. These are planning illustrations based on the observed effect, not observed power. The trial’s original sample-size justification and design assumptions are not reported in the registry record. The curve represents the relationship between the effect size considered and the corresponding planning requirement under the stated assumptions; it is not evidence about the power achieved by the completed study.

12. 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 = -422.600; pooled standard deviation σ = 557.975.
2. n = 2 × (1.96 + 0.842)² × 557.975² / -422.600² = 27.4, rounded up to 28 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 <- -422.6                      # observed difference in means
sigma <- 557.974833598772                     # 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(3122431)
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 = 28 (80% power), 37 (90% power)
 75% of observed effect: n per arm = 49 (80% power), 66 (90% power)
 50% of observed effect: n per arm = 110 (80% power), 147 (90% power)
power.t.test: 28.4 
Simulated power at n = 28 per arm: 0.793 (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.

13. Multiplicity, Missing Data and Analysis Populations

The registry lists 2 outcome measures but no secondary outcomes with posted results. It lists no posted analyses. The primary serum hydroxychloroquine result is accompanied by an unadjusted p-value, and the registry record does not report whether the study team conducted other analyses or adjusted for multiple comparisons. A multiplicity strategy, including any prespecified hierarchy or correction, is not reported in the registry record.

Multiplicity matters in general when several outcomes, groups, time points, or comparisons are tested. Repeated testing can increase the chance of obtaining a statistically notable result by chance. The project description covers several drugs and monitoring aims, but the record does not specify a complete planned testing family for those aims. It is therefore not possible to assess from the registry record whether the posted p-value was intended as the sole primary test or how it relates to other planned analyses.

Missing-data handling is not reported in the registry record. The record does not state how many endpoint measurements were unavailable, why any measurements might be missing, whether imputation was used, or how missingness was addressed in the analysis. The primary arm summaries list 41 participants for standard dose and 32 for reduced dose, but the registry does not describe whether these are all randomized participants, all participants with an observed endpoint, or another analysis population.

Analysis-population definitions are also not reported in the registry record. There is no stated intention-to-treat, per-protocol, or safety population definition for the primary comparison. Although the study is labeled randomized, the record does not explain how randomized participants were handled after allocation or whether participants were analyzed according to assigned group. These omissions limit assessment of how the reported comparison relates to the trial’s original design and the completeness of the endpoint data.

14. 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 5% reporting threshold.

Most frequent non-serious adverse event terms

The registry reports serious adverse events and deaths separately from other adverse events. For the thalidomide group, 20 participants are listed as at risk for serious adverse events, with 0 affected; 20 are listed as at risk for other adverse events, with 12 affected; and deaths are listed as 0. For the standard-dose hydroxychloroquine group, 41 are listed as at risk for serious adverse events and 0 affected, 41 are listed as at risk for other adverse events and 0 affected, and deaths are listed as 0. For the reduced-dose hydroxychloroquine group, the corresponding figures are 32 at risk and 0 affected for serious events, 32 at risk and 0 affected for other events, and 0 deaths.

These are registry counts, not estimates of comparative risk. The record does not provide event descriptions for the other adverse events in the thalidomide group, and no top serious-event terms are listed. Zero serious events recorded should be read as the posted count, not proof that no risk exists. The record does not report additional detail on event ascertainment or follow-up for these safety summaries.

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

The primary posted result is a difference in mean serum hydroxychloroquine levels at 12 months between the standard-dose and reduced-dose inactive-SLE arms. The reduced-dose arm has a lower posted mean, and the estimated mean difference is -422.6 ng/mL, with a 95% confidence interval from -678.1 to -167.1. The unadjusted p-value is 0.001. Together, these statistics describe a difference in measured concentration in the posted summaries, with uncertainty represented by the interval.

This concentration result does not establish that one regimen is more effective or safer, and it does not show that a participant took or did not take medication as prescribed. A serum level may be relevant to drug exposure, but the registry does not report a validated threshold linking the observed levels to adherence or clinical outcomes. It also does not report a salivary-level comparison that would show how blood and saliva measurements relate in these arms.

The project description includes broader interests in adherence, pharmacokinetic and pharmacodynamic modeling, mechanisms of efficacy and adverse effects, and several drug classes. The posted primary result does not resolve those questions. The registry record does not report detailed clinical outcome results, secondary results, or analyses of possible mechanisms. The safety counts are descriptive and do not provide a comparative analysis of adverse-event risk.

Interpretation is also bounded by unreported information about missing data, analysis populations, allocation procedures, and the original statistical plan. The posted comparison should be understood as a statistical description of the listed arm summaries, not a complete account of every intended analysis or a basis for individual treatment decisions.

16. Why This Trial Matters Statistically

This study is statistically interesting because it places a laboratory concentration measure at the center of a monitoring question. Serum drug levels are continuous measurements, so differences in group means can be expressed on the original measurement scale and accompanied by a confidence interval. That makes it possible to discuss both the direction of the contrast and the uncertainty around its estimated size.

The record also illustrates the distinction between a broad research rationale and the result that can be interpreted directly. The project description proposes blood and saliva monitoring and discusses adherence, mechanisms, effectiveness, adverse effects, and multiple drug classes. The primary result available for interpretation is a serum hydroxychloroquine comparison between two inactive-SLE dose groups. Keeping those scopes separate helps prevent a concentration difference from being overread as evidence about clinical benefit or harm.

The marked spread in the arm-level measurements is another important statistical feature. Means summarize group levels, while standard deviations describe variability around those means. The confidence interval for the mean difference adds information about precision, but it cannot replace distributional information or address unreported analysis decisions. The sample-size illustrations also show how planning requirements depend on the effect size targeted; smaller target effects require more participants under the stated assumptions.

Finally, this record highlights why registry detail matters for interpreting trial results. Randomized allocation is recorded, yet the allocation procedure, analysis populations, missing-data handling, and multiplicity plan are not reported. Those details affect how confidently readers can connect a statistical comparison to the trial design. The result is informative about the posted serum-level contrast, while its broader interpretation remains limited by what the registry does not report.

17. 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.

The primary result addresses serum hydroxychloroquine levels in the two inactive-SLE arms. It does not report a direct comparison of salivary and serum measurements, although the project description emphasizes both. The registry also describes thalidomide, biologic agents, and glucocorticoids as areas of interest, but the posted primary comparison does not provide results for those drug classes. Further outcomes and analyses are not reported in the registry record.

The arm descriptions identify different hydroxychloroquine regimens, but the registry does not report the rationale for the reduced regimen, the criteria for maintaining or changing doses, or additional treatment details. It also does not report the method used to measure adherence or a prespecified serum concentration target. Consequently, the observed mean difference cannot be translated into a conclusion about adherence or an intended therapeutic range.

The record’s randomized label is not accompanied by details of sequence generation, allocation concealment, or the application of randomization to this specific comparison. Masking is listed as none. The record does not explain how these design features were handled in analysis, nor does it state whether covariates were adjusted for. Missingness, participant flow, and analysis-population definitions are not reported in the registry record.

The result is based on posted group summaries rather than participant-level data. The registry does not report the distribution of individual levels, potential influential observations, or diagnostic checks. No adjustment for multiplicity is reported, and no secondary results are posted. Finally, the safety counts are not accompanied by detailed event descriptions for other adverse events in the thalidomide group. These limitations restrict interpretation to the posted summaries and counts.

18. Related Tutorials

19. Related Statistical Calculators

20. Sources

• ClinicalTrials.gov: Relevance of Monitoring Blood and Salivar Levels of Drugs Used in Rheumatic Autoimmune Diseases — NCT03122431.
• Registry record last updated 2021-12-16; status last verified 2021-05.
• Results first posted 2021-12-16.
• 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.

21. Record Summary

The registry describes a completed Phase 4, randomized, unmasked, parallel study sponsored by University of Sao Paulo General Hospital. Overall enrollment is listed as 93. The project concerns drug-level monitoring in rheumatic autoimmune diseases, including SLE, juvenile SLE, and cutaneous lupus.

The primary posted result concerns serum hydroxychloroquine at 12 months in inactive-SLE standard-dose and reduced-dose arms. The reported means are 991.6 ng/mL and 569.0 ng/mL, respectively. The mean difference for reduced dose relative to standard dose is -422.6 ng/mL, with a 95% confidence interval from -678.1 to -167.1 and an unadjusted p-value of 0.001.

The registry also lists safety counts for the hydroxychloroquine and thalidomide groups. The original sample-size justification, detailed analysis plan, missing-data handling, analysis populations, and allocation procedures are not reported in the registry record. No secondary results or posted analyses are listed.