Phase IIa Clinical Study of Recombinant Mycobacterium Tuberculosis Allergen ESAT6-CFP10: Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for Phase IIa Clinical Study of Recombinant Mycobacterium Tuberculosis Allergen ESAT6-CFP10 (NCT02329730), a randomized phase 2 study. The primary outcome is Number of Healthy Participants Negative for Reaction at 24 Hours After Intradermal Injection With ESAT6-CFP10.

2. Clinical Question

3. Trial Design

The registry identifies this as a Phase IIa, randomized, open-label, parallel-group study of recombinant ESAT6-CFP10. The study status is completed. The listed start is 2014-02 and the primary completion is 2014-06. The overall enrollment is 144, and the condition is tuberculosis. The sponsor is Anhui Zhifei Longcom Biologic Pharmacy Co., Ltd.

The study included both healthy participants and participants with tuberculosis. The brief summary describes dose groups ranging from low to high, with administration of ESAT6-CFP10 and comparator injections by the intradermal route. Intervention entries list ESAT6-CFP10 concentrations of 1μg/ml, 5μg/ml, 10μg/ml, and 20μg/ml, paired with either tuberculin purified protein derivative or placebo. The arm descriptions distinguish healthy participants, a first part of tuberculosis participants, and a second part of tuberculosis participants; details of those descriptions are incomplete in the registry record.

For the primary endpoint, the posted results cover the healthy-participant groups at the four concentrations, each with 14.0 participants. The listed control for these comparisons is 1μg/ml ESAT6-CFP10. The registry describes measuring skin induration and redness at 24 hours, and also identifies blisters and lymphangitis as positive reactions. The brief summary additionally describes interferon gamma testing before the skin test and at 72h±2h and 144h±2h. These design features indicate that the record covers dose evaluation and safety as well as the primary skin-reaction outcome; they do not establish that every planned comparison or analysis was completed or reported.

Randomization means assignment is intended to reduce systematic differences between groups, but the registry record does not report the sequence-generation method, allocation concealment, or implementation details. Masking is listed as none. The record does not report the protocol’s sample-size justification or a detailed statistical analysis plan.

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.

Only the first 6 of 12 groups are shown.

Overall Study

Reasons for not completing this period:

Analysis population for the primary outcome, as defined in the registry: The percentage of negative(negative number/total number\*100%) is the specificity of ESAT6-CFP10 .

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.

Population: Each group of baseline analysis population apply full analysis set (FAS).FAS are the participant, following the principle of ITT, who receive at least once drug and have curative effect evaluation of drug treatment .Missing data of the curative effect part in FAS population use Last observation carried forward(LOCF)method to supplement.

6. Primary Endpoint

The registry lists the primary outcome as Number of Healthy Participants Negative for Reaction at 24 Hours After Intradermal Injection With ESAT6-CFP10, measured over 24 hours after intradermal injection.

The primary endpoint is the number of healthy participants negative for reaction 24 hours after intradermal injection with ESAT6-CFP10. The registry describes measuring the longitudinal and transverse diameters of induration and redness with a vernier caliper. It defines induration, redness, blisters, and lymphangitis as positive reactions. A participant without a positive reaction is counted as negative.

The registry states that the percentage of negative results is the specificity of ESAT6-CFP10. This endpoint is therefore used to describe how often healthy participants do not show the specified reaction after injection. It is a count endpoint in the posted results, with the number of negative participants reported alongside the group size. The registry’s definition gives the interpretation of the count, but no full diagnostic-accuracy framework or threshold for interpreting specificity is reported.

In the 1μg/ml reference group, 13.0 of 14.0 participants were negative. At 5μg/ml, 13.0 of 14.0 were negative; at 10μg/ml, 14.0 of 14.0 were negative; and at 20μg/ml, 13.0 of 14.0 were negative. The posted endpoint thus describes similar counts across these small groups, with one group recording all participants as negative. It does not, by itself, answer questions about clinical outcomes or establish that one concentration is preferable.

As a general statistical concept, a binary endpoint reduces each participant’s observed response to a category such as negative or positive. That makes group counts straightforward to describe, but the resulting precision depends on the number of participants and the distribution of responses.

7. Results Posted in the Registry

• 5μg/ml ESAT6-CFP10: 92.9% (13/14) versus 92.9% (13/14) on control; risk difference 0.0 percentage points (95% CI -19.1 to 19.1), risk ratio 1.00 (0.81–1.23); unadjusted two-proportion z-test p = 1.000.
• 10μg/ml ESAT6-CFP10: 100.0% (14/14) versus 92.9% (13/14) on control; risk difference 7.1 percentage points (95% CI -6.3 to 20.6), risk ratio 1.08 (0.93–1.25); unadjusted two-proportion z-test p = 0.309.
• 20μg/ml ESAT6-CFP10: 92.9% (13/14) versus 92.9% (13/14) on control; risk difference 0.0 percentage points (95% CI -19.1 to 19.1), risk ratio 1.00 (0.81–1.23); unadjusted two-proportion z-test p = 1.000.

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.

the Number of Healthy Participants Negative for IFN-γ(Gamma Interferon ) Before Injection

• Endpoint type: Binary: the number of participants with the event, compared as proportions.
• Test: Unadjusted two-proportion z-test, with a Wald 95% confidence interval for the risk difference.
• Result:
  • 5μg/ml ESAT6-CFP10: 92.9% (13/14) versus 85.7% (12/14) on control; risk difference 7.1 percentage points (95% CI -15.6 to 29.9), risk ratio 1.08 (0.84–1.40); unadjusted two-proportion z-test p = 0.541.
  • 10μg/ml ESAT6-CFP10: 92.9% (13/14) versus 85.7% (12/14) on control; risk difference 7.1 percentage points (95% CI -15.6 to 29.9), risk ratio 1.08 (0.84–1.40); unadjusted two-proportion z-test p = 0.541.
• Interpretation:
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between 5μg/ml ESAT6-CFP10 and the control. The interval gives the range of effects that remain compatible with the data.
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between 10μg/ml ESAT6-CFP10 and the control. The interval gives the range of effects that remain compatible with the data.

the Number of Tuberculosis (TB) Participants Positive for IFN-γ(Gamma Interferon ) Before Injection

• Endpoint type: Binary: the number of participants with the event, compared as proportions.
• Test: Unadjusted two-proportion z-test, with a Wald 95% confidence interval for the risk difference.
• Result:
  • 5μg/ml ESAT6-CFP10: 89.5% (17/19) versus 100.0% (18/18) on control; risk difference -10.5 percentage points (95% CI -24.3 to 3.3), risk ratio 0.89 (0.77–1.04); unadjusted two-proportion z-test p = 0.157.
  • 10μg/ml ESAT6-CFP10: 85.7% (18/21) versus 100.0% (18/18) on control; risk difference -14.3 percentage points (95% CI -29.3 to 0.7), risk ratio 0.86 (0.72–1.02); unadjusted two-proportion z-test p = 0.095.
• Interpretation:
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between 5μg/ml ESAT6-CFP10 and the control. The interval gives the range of effects that remain compatible with the data.
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between 10μg/ml ESAT6-CFP10 and the control. The interval gives the range of effects that remain compatible with the data.

the Number of Healthy Participants Negative for IFN-γ(Gamma Interferon ) at 72 Hours After Intradermal Injection With ESAT6-CFP10

• Endpoint type: Binary: the number of participants with the event, compared as proportions.
• Test: Unadjusted two-proportion z-test, with a Wald 95% confidence interval for the risk difference.
• Result:
  • 5μg/ml ESAT6-CFP10: 85.7% (12/14) versus 92.9% (13/14) on control; risk difference -7.1 percentage points (95% CI -29.9 to 15.6), risk ratio 0.92 (0.71–1.20); unadjusted two-proportion z-test p = 0.541.
  • 10μg/ml ESAT6-CFP10: 92.9% (13/14) versus 92.9% (13/14) on control; risk difference 0.0 percentage points (95% CI -19.1 to 19.1), risk ratio 1.00 (0.81–1.23); unadjusted two-proportion z-test p = 1.000.
• Interpretation:
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between 5μg/ml ESAT6-CFP10 and the control. The interval gives the range of effects that remain compatible with the data.
  • The 95% confidence interval includes no difference, so these data do not show a statistically significant difference between 10μg/ml ESAT6-CFP10 and the control. The interval gives the range of effects that remain compatible with the data.

The secondary outcomes span adverse events and IFN-γ measurements before injection and at later time points, in healthy participants and participants with tuberculosis. At a broad level, the posted secondary counts show variation across groups and time points rather than a single consistent pattern that can be interpreted as a dose response. These outcomes provide additional descriptions of response and safety, but they do not replace or validate the primary endpoint.

The healthy-participant baseline and 72-hour outcomes include risk-difference estimates whose intervals encompass zero. The tuberculosis-participant baseline results also show uncertainty around the comparisons. The registry lists a 144-hour secondary outcome, but no results for that outcome appear in the posted secondary results described in the record. The secondary findings thus offer incomplete coverage of the outcome schedule stated in the trial summary.

The primary result concerns negative skin reactions in healthy participants at 24 hours. IFN-γ status is a different measure, assessed at different times and in different participant groups. Any relationship between those findings and the primary skin-reaction count would require a prespecified analysis and appropriate participant-level information; such an analysis is not reported. The secondary outcomes can therefore be read as separate descriptive outcomes, not as a combined explanation for the primary result.

The registry reports seven secondary outcomes, but the number of analyses reported is zero. The posted comparisons and planning illustrations do not amount to a reported sponsor analysis plan. Unadjusted secondary results carry less weight because multiple outcomes, time points, and group contrasts create opportunities for chance findings. Without a reported multiplicity approach, secondary differences should be treated as exploratory descriptions rather than independent confirmation. The registry record does not report a hierarchy of outcomes or a rule for interpreting secondary findings alongside the primary result.

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

• the Number of Participants With Adverse Events
• the Number of Healthy Participants Negative for IFN-γ(Gamma Interferon ) Before Injection
• the Number of Tuberculosis (TB) Participants Positive for IFN-γ(Gamma Interferon ) Before Injection
• the Number of Healthy Participants Negative for IFN-γ(Gamma Interferon ) at 72 Hours After Intradermal Injection With ESAT6-CFP10
• the Number of Tuberculosis (TB) Participants Positive for IFN-γ(Gamma Interferon ) at 72 Hours After Intradermal Injection With ESAT6-CFP10
• the Number of Healthy Participants Negative for IFN-γ(Gamma Interferon ) at 144 Hours After Intradermal Injection With ESAT6-CFP10
• the Number of Tuberculosis (TB) Participants Positive for IFN-γ(Gamma Interferon ) at 144 Hours After Intradermal Injection With ESAT6-CFP10

9. Statistical Interpretation

10. Statistical Methods Explained

The posted primary results are binary counts: each participant is classified as having a negative or positive reaction, and the number of negative participants is reported for each concentration. The comparison measure is a risk difference, which expresses the difference between the group proportions for the negative outcome. A positive risk difference means a larger proportion had a negative reaction in the named concentration group than in the 1μg/ml reference group; a negative value means a smaller proportion. The posted risk differences are 0.0 for 5μg/ml, 0.071 for 10μg/ml, and 0.0 for 20μg/ml.

Each estimate is accompanied by a confidence interval. As a general statistical explanation, a confidence interval describes a range of values compatible with the observed data under the method’s assumptions. It conveys uncertainty around an estimate, rather than listing the range of individual participant responses. In these results, all three primary confidence intervals include zero: -0.191 to 0.191 for 5μg/ml, -0.064 to 0.206 for 10μg/ml, and -0.191 to 0.191 for 20μg/ml. The intervals therefore do not distinguish the observed risk differences from zero at the level represented by the intervals.

The registry posts unadjusted p-values of 1.0, 0.309, and 1.0 for the comparisons with 5μg/ml, 10μg/ml, and 20μg/ml, respectively. Generally, a p-value measures how unusual the observed data, or more extreme data, would be under a specified null model. It is not the probability that the null hypothesis is true, and it does not report the probability that the result will replicate. Because the p-values are unadjusted, they do not account for the number of comparisons or outcomes examined.

The results include risk ratios and, for some comparisons, odds ratios as well as risk differences. These are alternative ways of expressing associations between group and binary outcome. The risk difference is particularly direct for this endpoint because it describes the absolute difference in the frequency of negative reactions. The risk ratio expresses relative frequencies, while the odds ratio compares odds. Each measure answers a different scale-based question; none changes the small group sizes or removes uncertainty. The confidence intervals for the ratio measures are also broad in the posted comparisons.

The comparisons are simple group-level contrasts, not a reported adjusted model. The registry record does not report covariate adjustment, stratification, or a method for handling repeated or correlated observations. No additional analysis details are reported. The study is listed as randomized, but randomization alone does not reveal whether all randomized participants were included in each endpoint analysis, or how exclusions and missing measurements were handled.

For the primary endpoint, each group contains 14.0 participants. With a small denominator, changing the outcome of a single participant can materially change the observed group proportion. This general feature of binary outcomes helps explain why the intervals remain wide even where the counts are similar. The 10μg/ml group has 14.0 negative participants out of 14.0; that observed count does not mean the underlying response probability is known without uncertainty.

The registry also includes planning illustrations for selected secondary comparisons. Their stated assumptions are a two-sided significance level of 0.05, equal allocation between two arms, and a normal approximation. The illustrations use 100%, 75%, and 50% of the observed effect as effect sizes to detect. For the baseline healthy-participant IFN-γ comparison of 5μg/ml versus 1μg/ml, the listed values are 294 participants per arm for 80% power and 392 participants per arm for 90% power when planning for 100% of the observed effect; 561 and 750 participants per arm for 75%; and 1,345 and 1,800 participants per arm for 50%. These are planning illustrations from the observed effect, not the trial’s design justification and not observed power. The protocol’s sample-size justification is not reported in the registry record.

Generally, over-dispersion matters when observed variability exceeds what a statistical model assumes; if ignored, uncertainty can be understated. The posted primary outcome is binary, and the record does not report an over-dispersion assessment or a model requiring one. No such assessment should be inferred from the counts. The analysis details needed to evaluate model assumptions are not reported in the registry record.

11. Confidence Intervals and Precision

The primary comparisons have limited precision. For 5μg/ml versus 1μg/ml, the risk difference is 0.0 and the confidence interval is -0.191 to 0.191. For 20μg/ml versus 1μg/ml, the estimate and interval are the same. For 10μg/ml versus 1μg/ml, the risk difference is 0.071, with an interval from -0.064 to 0.206. Each interval includes zero and allows for differences in either direction.

As a general explanation, an interval’s width reflects statistical uncertainty under the calculation method. Here, each primary group has 14.0 participants, and the estimates are based on counts of negative reactions. Such a small group size gives limited information about the underlying response frequency. The fact that one group recorded 14.0 negative reactions out of 14.0 does not eliminate uncertainty about responses beyond the observed participants.

The intervals should not be treated as ranges containing a fixed proportion of individual responses, nor as proof that a particular value is true. They describe uncertainty around the risk-difference estimates. Their inclusion of zero means the posted data are compatible with no difference under the stated comparison measure, while their endpoints also allow nonzero differences. This is why the observed point estimates alone should not be read as definitive evidence of a dose-related pattern.

The registry does not report a prespecified clinically meaningful difference for this endpoint, so the practical importance of the interval limits cannot be determined from the record. No sample-size justification for the primary endpoint is reported. The precision assessment is therefore based on the posted group counts and comparison intervals, not on evidence that the trial was designed to rule out a particular size of difference.

12. Multiplicity, Missing Data and Analysis Populations

The registry lists 15 outcome measures and seven secondary outcomes, but reports zero analyses. It does not describe a multiplicity adjustment, a testing hierarchy, or a prespecified rule for interpreting results across endpoints, doses, populations, and time points. The posted p-values are unadjusted. When many comparisons are considered, unadjusted tests do not control the chance of at least one apparently notable result across the full set. This is a general concern, not evidence that any particular result is false.

The record also does not report how missing outcome measurements were handled, whether participants withdrew, or whether all randomized participants were included in each analysis. The registry’s overall enrollment is 144, while the primary endpoint groups each contain 14.0 participants. The record does not explain how these endpoint groups relate to all enrolled participants or identify an analysis population such as intention-to-treat or per-protocol. It is therefore not possible to determine from the registry record whether the endpoint counts include all eligible randomized participants.

For an intradermal reaction measured after injection, completeness and timing of measurement can affect which observations contribute to the endpoint. The brief summary specifies assessment times, but the record does not describe missed visits, unusable measurements, or rules for classifying missing skin-reaction data. It also does not report whether any outcome data were imputed.

These omissions make it difficult to assess the planned inferential framework and the effect of missingness or multiplicity on interpretation. The posted group counts and recalculated unadjusted comparisons remain useful descriptions of the available results, but they cannot substitute for a complete analysis plan or participant flow account. The protocol’s statistical plan, sample-size justification, interim analyses, and analysis-population definitions are not reported in the registry record.

13. Safety Results

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

Other (non-serious) adverse events

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

Most frequent non-serious adverse event terms

The safety results list serious adverse events as zero in each of the 12 adverse-event groups. The listed groups include healthy participants and participants with tuberculosis at the ESAT6-CFP10 concentrations, with tuberculin purified protein derivative or placebo as described. The at-risk counts are 14 in each of the listed healthy and tuberculosis groups paired with TB-PPD, and 8 in each tuberculosis group paired with placebo. These figures describe the recorded serious-event counts; they do not establish the absence of risk.

The registry also lists other adverse events in the groups, with counts ranging from 0 to 6. These are not serious adverse events, and the record does not provide serious-event terms in its list of top serious terms. Death counts are not reported in the registry record. The posted safety information should therefore be read as a set of observed counts over the recorded study groups, not as a comprehensive characterization of safety.

Generally, observing no serious adverse events in a study does not prove that serious events cannot occur. The interpretation depends on the number of participants, the length and completeness of follow-up, and the way events were collected. Those details are not fully reported in the registry record. The safety counts do not support medical advice or conclusions about use outside this study.

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

The primary result describes the number of healthy participants without a positive skin reaction 24 hours after receiving one of four ESAT6-CFP10 concentrations. The counts were similar in the 1μg/ml, 5μg/ml, and 20μg/ml groups, while the 10μg/ml group had one more negative response than the reference group. The risk-difference estimates and confidence intervals express the uncertainty around these observed group contrasts.

The data do not establish that the concentrations produce identical responses. The intervals are compatible with zero differences as well as with differences in either direction, and the primary groups are small. Nor do the results establish a meaningful dose-response pattern. The observed values are not consistently increasing or decreasing across the concentrations, and the record does not report a prespecified dose-response analysis.

The primary endpoint is a skin reaction in healthy participants, which the registry connects to specificity. It is not a direct measure of tuberculosis treatment, disease progression, or clinical benefit. The study’s stated purpose includes evaluating safety and informing an appropriate dose range for Phase IIb trials, but the posted endpoint counts alone do not establish an appropriate dose range.

Generally, a randomized comparison can help reduce systematic differences between assigned groups, but its reliability also depends on the conduct of randomization, outcome measurement, and analysis. The registry lists the study as randomized and open-label, but does not report the randomization procedures, assessor masking, missing-data handling, or analysis populations. The results should be understood within those reporting limits.

Nothing in these findings supports a recommendation for individual care. They describe recorded outcomes in this completed Phase IIa study. Further interpretation would require the full protocol and analysis details, complete outcome reporting, and evidence from additional research; such details and findings are not reported in the registry record.

15. Why This Trial Matters Statistically

This trial is statistically informative as an example of the distinction between a point estimate and the precision of that estimate. The primary risk differences are close to zero, but the confidence intervals remain broad enough to include nonzero differences in either direction. The apparent closeness of the group counts should not be confused with precise evidence that the groups are equivalent.

The study also illustrates the limits of small groups for binary outcomes. With 14.0 participants in each primary group, a small change in the number classified as negative can substantially change the group estimate. Confidence intervals help make that uncertainty visible, while p-values alone would not describe the range of plausible differences or their magnitude.

Another statistical lesson is that planned sample size and observed results serve different purposes. The registry’s planning illustrations use the observed effect and stated assumptions to show participant counts for different target power levels. They are not the trial’s sample-size justification and do not describe observed power. The protocol rationale for the enrolled size is not reported in the registry record.

Finally, this record presents multiple populations, outcomes, and assessment times without a reported analysis plan or multiplicity strategy. That makes transparent separation of the primary endpoint from the secondary outcomes especially important. The primary result can be described on its own terms, while the secondary findings remain exploratory in the absence of a documented hierarchy and adjustment approach. These distinctions help prevent descriptive patterns from being overinterpreted as confirmatory evidence.

16. 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 registry record leaves several trial-specific limitations unresolved. Although the study is listed as randomized, the method for generating and concealing allocation is not reported. Masking is listed as none, and the record does not state whether outcome assessors were masked. Because the primary endpoint involves measuring skin induration and redness, the lack of reported assessor-masking details limits assessment of possible measurement influence.

The overall enrollment is 144, but the primary endpoint results are posted for four healthy-participant groups of 14.0 each. The record does not explain how the primary endpoint sample relates to overall enrollment or give participant flow by arm. It also does not identify analysis populations or report losses to follow-up, exclusions, or missing measurements. The arm descriptions are incomplete, and the relationship between all listed interventions and the posted endpoint groups is not fully explained.

The primary outcome description specifies reaction criteria but does not report a decision process for borderline measurements or inter-rater assessment. The record does not report a protocol-defined clinically meaningful difference, sample-size justification, or full statistical analysis plan. The posted analyses are unadjusted, and no multiplicity approach is described despite multiple outcomes and time points.

Safety reporting is also limited: serious-event counts are listed as zero in each group, but death counts are not reported, and follow-up duration and completeness are not fully described. These gaps do not negate the observed counts; they constrain how far those counts can be generalized or interpreted.

17. Related Tutorials

18. Related Statistical Calculators

19. Sources

• ClinicalTrials.gov: Phase IIa Clinical Study of Recombinant Mycobacterium Tuberculosis Allergen ESAT6-CFP10 — NCT02329730.
• Registry record last updated 2015-04-09; status last verified 2015-03.
• Results first posted 2015-04-09.
• 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.

20. Record Summary

NCT02329730 is a completed randomized, open-label, parallel Phase IIa study of intradermal recombinant ESAT6-CFP10 in healthy participants and participants with tuberculosis. The registry lists overall enrollment of 144 and a study period from 2014-02 to 2014-06.

The primary endpoint counts healthy participants with no positive reaction 24 hours after injection. The four concentration groups each include 14.0 participants; negative-reaction counts are 13.0, 13.0, 14.0, and 13.0 across 1μg/ml, 5μg/ml, 10μg/ml, and 20μg/ml, respectively. The posted unadjusted risk-difference comparisons have confidence intervals that include zero.

The record includes secondary IFN-γ and adverse-event outcomes, but reports zero analyses and does not report the statistical plan, sample-size justification, missing-data handling, or analysis populations. The results are therefore best read as descriptive, with substantial uncertainty around the primary group comparisons.