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Desensitising Celiac Disease Patients With the Human Hookworm (NaCeD)

This study has been completed.
Sponsor:
ClinicalTrials.gov Identifier:
NCT01661933
First Posted: August 10, 2012
Last Update Posted: October 20, 2014
The safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Listing a study does not mean it has been evaluated by the U.S. Federal Government. Read our disclaimer for details.
Collaborator:
National Health and Medical Research Council, Australia
Information provided by (Responsible Party):
Dr John Croese, The Prince Charles Hospital
Results First Submitted: September 21, 2014  
Study Type: Interventional
Study Design: Intervention Model: Single Group Assignment;   Masking: Single (Outcomes Assessor);   Primary Purpose: Prevention
Condition: Celiac Disease
Intervention: Biological: Necator americanus

  Participant Flow
  Hide Participant Flow

Recruitment Details
Key information relevant to the recruitment process for the overall study, such as dates of the recruitment period and locations
Of 20 potential candidates, 2 could not be contacted (address changed), 1 was not interested, and 5 were unavailable (travel-2, pregnancy-2 or study-1 commitments). Twelve enrolled in September 2012. 8 committed to 3 endoscopic procedures and 2 committed to two endoscopies.

Pre-Assignment Details
Significant events and approaches for the overall study following participant enrollment, but prior to group assignment
No text entered.

Reporting Groups
  Description
Necator Americanus, Gluten Challenge Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, a detailed assessment including histology was performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).

Participant Flow for 3 periods

Period 1:   Gluten Micro-challenge
    Necator Americanus, Gluten Challenge
STARTED   12 [1] 
COMPLETED   12 [2] 
NOT COMPLETED   0 
[1] All 12 enrolled completed hookworm inoculation and gluten micro-challenge.
[2] Two gluten intolerant participants did not proceed to the low-dose challenge.

Period 2:   Low-dose Gluten Challenge
    Necator Americanus, Gluten Challenge
STARTED   12 [1] 
COMPLETED   10 [2] 
NOT COMPLETED   2 
Physician Decision                2 
[1] One was symptomatically intolerant to gluten >25 mg. One had active CeD pre- & post-micro-challenge
[2] Two had challenge interrupted for 1-2 weeks. All completed challenge.

Period 3:   High-dose Gluten Challenge
    Necator Americanus, Gluten Challenge
STARTED   10 [1] 
COMPLETED   8 
NOT COMPLETED   2 
Withdrawal by Subject                2 
[1] 2 of 10 available after post-GC-1g did not enroll for reasons unrelated to gluten.



  Baseline Characteristics
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Population Description
Explanation of how the number of participants for analysis was determined. Includes whether analysis was per protocol, intention to treat, or another method. Also provides relevant details such as imputation technique, as appropriate.
Mean age 53 years, range 39-67; females 9.

Reporting Groups
  Description
Gluten Micro-challenge Single arm, vertical. All twelve healthy adults enrolled subjects were successfully inoculated with hookworm and there was no serious adverse response. Individual hemoglobin levels were all normal and the group mean had significantly increased unexpectedly at completion of the study. Histology was not graded until after low-dose challenge but retrospectively confirmed enrollment Marsh scores of M0-8, M1-1, M2-2 and M3a-1. All participants were complying with a gluten-free diet, were symptomatically well and had a normal anti-tTG.

Baseline Measures
   Gluten Micro-challenge 
Overall Participants Analyzed 
[Units: Participants]
 12 
Age 
[Units: Participants]
 
<=18 years   0 
Between 18 and 65 years   11 
>=65 years   1 
Age 
[Units: Years]
Mean (Full Range)
 53 
 (39 to 67) 
Gender 
[Units: Participants]
 
Female   9 
Male   3 


  Outcome Measures
  Hide All Outcome Measures

1.  Primary:   Duodenal Villus Height:Crypt Depth   [ Time Frame: Week -24 to -36 ]

Measure Type Primary
Measure Title Duodenal Villus Height:Crypt Depth
Measure Description Biopsies were fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 µm) were stained with H&E. Slides from both time-points were de-identified, shuffled and graded by Dr John Croese after which results from poorly orientated slides were verified by Dr Andrew Clouston. The Vh:Cd ratios were measured on 5 randomly selected well-orientated sites. The null hypothesis is that hookworm infection will not protect against mucosal damage following 12-week exposure to gluten in celiac disease.
Time Frame Week -24 to -36  

Population Description
Explanation of how the number of participants for analysis was determined. Includes whether analysis was per protocol, intention to treat, or another method. Also provides relevant details such as imputation technique, as appropriate.
All 10 of 12 participants who completed micro-challenge successfully progressed and completed low-dose challenge.

Reporting Groups
  Description
Necator Americanus, Gluten Challenge Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, assessments including histology were performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).

Measured Values
   Necator Americanus, Gluten Challenge 
Participants Analyzed 
[Units: Participants]
 10 
Duodenal Villus Height:Crypt Depth 
[Units: Ratio]
Mean (95% Confidence Interval)
 
Baseline   2.73 
 (2.31 to 3.15) 
Low-dose gluten   2.73 
 (2.31 to 3.15) 


Statistical Analysis 1 for Duodenal Villus Height:Crypt Depth
Groups [1] Necator Americanus, Gluten Challenge
Statistical Test Type [2] Superiority or Other
Statistical Method [3] t-test, 2 sided
P Value [4] 0.693
[1] Additional details about the analysis, such as null hypothesis and power calculation:
  No text entered.
[2] Details of power calculation, definition of non-inferiority margin, and other key parameters:
  No text entered.
[3] Other relevant method information, such as adjustments or degrees of freedom:
  No text entered.
[4] Additional information, such as whether or not the p-value is adjusted for multiple comparisons and the a priori threshold for statistical significance:
  No text entered.



2.  Secondary:   Intraepithelial Lymphocyte Count   [ Time Frame: Week-24 and -36 ]

Measure Type Secondary
Measure Title Intraepithelial Lymphocyte Count
Measure Description Biopsies were fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 µm) were stained with anti-CD3. All slides were de-identified and graded by Dr John Croese. The IEL percentages were measured on 2 or more randomly selected well-orientated villi. The null hypothesis is that hookworm infection will not protect against mucosal IEL influx following 12-week exposure to gluten in celiac disease.
Time Frame Week-24 and -36  

Population Description
Explanation of how the number of participants for analysis was determined. Includes whether analysis was per protocol, intention to treat, or another method. Also provides relevant details such as imputation technique, as appropriate.
No text entered.

Reporting Groups
  Description
Necator Americanus, Gluten Challenge Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, a detailed assessment including histology was performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).

Measured Values
   Necator Americanus, Gluten Challenge 
Participants Analyzed 
[Units: Participants]
 10 
Intraepithelial Lymphocyte Count 
[Units: Percentage of epithelial cells]
Mean (95% Confidence Interval)
 
Baseline   33.40 
 (27.12 to 39.68) 
Low-dose gluten   35.00 
 (28.72 to 41.28) 


Statistical Analysis 1 for Intraepithelial Lymphocyte Count
Groups [1] Necator Americanus, Gluten Challenge
Statistical Test Type [2] Superiority or Other
Statistical Method [3] t-test, 2 sided
P Value [4] =0.837
[1] Additional details about the analysis, such as null hypothesis and power calculation:
  No text entered.
[2] Details of power calculation, definition of non-inferiority margin, and other key parameters:
  No text entered.
[3] Other relevant method information, such as adjustments or degrees of freedom:
  No text entered.
[4] Additional information, such as whether or not the p-value is adjusted for multiple comparisons and the a priori threshold for statistical significance:
  No text entered.



3.  Secondary:   Number of Participants With 2 Points Increase in Marsh Score Post GC-1g   [ Time Frame: Longitudinal change between week-24 and week-36 ]

Measure Type Secondary
Measure Title Number of Participants With 2 Points Increase in Marsh Score Post GC-1g
Measure Description The Marsh score is a defined but qualitative assessment assigned a value to allow for comparison. The scores were evaluated by consensus between the primary (chief) investigator and the study pathologist. The Marsh score was graded 0, 1, 2, 3A (assigned-4), 3B (-5) and 3C (-6); rage 1-6 with normal=0 and severe inflammation=6. Because the scoring is vulnerable to artefact, only a 2-point shift was regarded as a significant intra-individual change. The scores were graded after week-36 on biopsies de-identified shuffled. An upward shift was interpreted to reflect a significant worsening of gluten-associated inflammation. The comparison reported evaluated changes from baseline (week-24) to post-low-dose gluten challenge (week-24; GC-1g). The objective for using the Marsh score was to identify individuals who might have experienced a severe worsening in pathology due to GC-1g that might not be reflected in the Vh:Cd group analysis.
Time Frame Longitudinal change between week-24 and week-36  

Population Description
Explanation of how the number of participants for analysis was determined. Includes whether analysis was per protocol, intention to treat, or another method. Also provides relevant details such as imputation technique, as appropriate.
The outcome score was the number with a 2-point increase in Marsh 3 score post GC-1g.

Reporting Groups
  Description
Necator Americanus, Pre-gluten Challenge Single arm, vertical. Ten of 12 participants enrolled based on symptomatic tolerance of gluten and satisfactory histology during and after micro-challenge. 2 were withdrawn pre-GC-1g, one who was symptomatically intolerant of gluten and one who had M3a after micro-challenge. Pre-GC1g scores.

Measured Values
   Necator Americanus, Pre-gluten Challenge 
Participants Analyzed 
[Units: Participants]
 10 
Number of Participants With 2 Points Increase in Marsh Score Post GC-1g 
[Units: Participants]
 1 


Statistical Analysis 1 for Number of Participants With 2 Points Increase in Marsh Score Post GC-1g
Groups [1] Necator Americanus, Pre-gluten Challenge
Statistical Test Type [2] Superiority or Other
Statistical Method [3] Binomial distribution
P Value [4] =0.99
[1] Additional details about the analysis, such as null hypothesis and power calculation:
  The null hypothesis was that irrespective of hookworm infection, GC-1g would result in a 2-point or more deterioration in the Marsh score. A binomial (yes=deterioration or no=no deterioration) distribution was applied to pre- and post-GC-1g paired biopsies.
[2] Details of power calculation, definition of non-inferiority margin, and other key parameters:
  No text entered.
[3] Other relevant method information, such as adjustments or degrees of freedom:
  No text entered.
[4] Additional information, such as whether or not the p-value is adjusted for multiple comparisons and the a priori threshold for statistical significance:
  No text entered.



4.  Secondary:   Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL)   [ Time Frame: Anti-tTG IU/mL levels pre-trial, mid-trial and after 3 gram/day gluten challenge ]

Measure Type Secondary
Measure Title Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL)
Measure Description The trial was extended with pre-trial and mid-trial anti-tTG antibody levels used to compare with the post-trial levels. Anti-tTG is a serological measure of tissue transglutaminase-2 antibodies. In active celiac disease, levels are increased. In treated disease, levels are low (normal cut-off was <15 IU/mL). A significant increase compared to baseline in tTG can be expected 2 weeks after consuming 3g of gluten daily for 2 weeks in people with celiac disease who have been maintaining a gluten-free diet, but who are not taking other treatment.
Time Frame Anti-tTG IU/mL levels pre-trial, mid-trial and after 3 gram/day gluten challenge  

Population Description
Explanation of how the number of participants for analysis was determined. Includes whether analysis was per protocol, intention to treat, or another method. Also provides relevant details such as imputation technique, as appropriate.
2 of 12 participants did not progress past baseline micro-challenge, 2 of 10 participants did not progress past low-dose challenge.

Reporting Groups
  Description
Necator Americanus, Gluten Challenge Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, a detailed assessment including histology was performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).

Measured Values
   Necator Americanus, Gluten Challenge 
Participants Analyzed 
[Units: Participants]
 8 
Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL) 
[Units: International Units/mL]
Mean (95% Confidence Interval)
 
Baseline   4.12 
 (3.12 to 5.10) 
Low-dose gluten   2.99 
 (2.00 to 3.94) 
After 3 G gluten daily   2.11 
 (1.16 to 3.07) 


Statistical Analysis 1 for Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL)
Groups [1] Necator Americanus, Gluten Challenge
Statistical Test Type [2] Superiority or Other
Statistical Method [3] Mixed effects model.
P Value [4] =0.005
[1] Additional details about the analysis, such as null hypothesis and power calculation:
  No text entered.
[2] Details of power calculation, definition of non-inferiority margin, and other key parameters:
  No text entered.
[3] Other relevant method information, such as adjustments or degrees of freedom:
  No text entered.
[4] Additional information, such as whether or not the p-value is adjusted for multiple comparisons and the a priori threshold for statistical significance:
  No text entered.




  Serious Adverse Events


  Other Adverse Events


  Limitations and Caveats
  Hide Limitations and Caveats

Limitations of the study, such as early termination leading to small numbers of participants analyzed and technical problems with measurement leading to unreliable or uninterpretable data
Open study. Small number. Historical controls.


  More Information
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Certain Agreements:  
Principal Investigators are NOT employed by the organization sponsoring the study.
There is NOT an agreement between Principal Investigators and the Sponsor (or its agents) that restricts the PI's rights to discuss or publish trial results after the trial is completed.


Results Point of Contact:  
Name/Title: Dr John Croese. The Chief Investigator
Organization: The Prince Charles Hospital
phone: +61733716164
e-mail: jcroese@bigpond.com


Publications:

Responsible Party: Dr John Croese, The Prince Charles Hospital
ClinicalTrials.gov Identifier: NCT01661933     History of Changes
Other Study ID Numbers: AU/3/BOBD012
First Submitted: August 6, 2012
First Posted: August 10, 2012
Results First Submitted: September 21, 2014
Results First Posted: October 13, 2014
Last Update Posted: October 20, 2014