Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT00284700 |
| Other study ID # |
97-004 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
December 1997 |
| Est. completion date |
May 2001 |
Study information
| Verified date |
January 2006 |
| Source |
International Centre for Diarrhoeal Disease Research, Bangladesh |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
Helicobacter pylori is recognized as a major gastrointestinal pathogen in developing
countries. This microorganism infects up to 60% of children less than five years in those
countries and is strongly associated with chronic gastritis and peptic ulcer disease in
children and adults. The progression of gastritis to atrophy often leads to decreased gastric
acid output, which is a well-known risk factor for anemia. Gastric acid is essential for
increasing the bioavailability and absorption of non-heme dietary iron, the most important
source of iron in developing countries. Numerous reports suggest that iron malabsorption
secondary to low gastric acid output is a problem in developing world countries. It has been
further observed that iron deficiency anemia is resistant to iron therapy particularly in
these countries. In a recently completed study we observed an association of anaemia with H.
pylori infection. We hypothesize that the poor bioavailability of iron in these countries
could be related to H. pylori -induced low gastric acid output and we propose to investigate
the role of H. pylori infection as a cause of anemia and treatment failure of iron
supplementation in Bangladesh. A prospective, randomized, double-blind, placebo-controlled
field trial is proposed among four groups ( 65 each) of H. Pylori infected children of 2-5
years of age with iron deficiency anemia. The children will be assigned to one of the four
therapies: antibiotics alone (for H. Pylori eradication), antibiotic plus iron therapy, iron
therapy alone, or placebo. Hemoglobin concentration, serum ferritin concentration, and
transferrin receptor will be measured before and at 1 and 3 month after the intervention. We
also propose a complementary study in an additional 20 children with H. Pylori infection and
iron deficiency anemia to assess iron absorption with application of double stable isotopes.
The change in hematological parameters will also be compared among the groups before and
after the therapy. The results of this study are expected to have implications in the
prevention and treatment of iron deficiency anemia in developing countries.
Description:
We propose to complementary study:
1. A prospective, double-blind, placebo-controlled field trial in two hundred sixty
children of a peri-urban community near Dhaka City is proposed.
2. The assessment of iron absorption before and after anti-infective therapy in 20 children
with H. Pylori infection and iron deficiency anemia
3. Field trial
1.1 Description of the study population Children will be recruited from Nandipara, a
peri-urban community situated 7 miles northeast of Dhaka City. This community has a
population of 3,500 in an area of approximately 2.5 square miles. Among the residents,
70% of males are classified as day laborers, 20% are rickshaw pullers, and 5% are
carpenters or service holders. Fifteen percent of women are day laborers and 85% are
housewives. The average family size is 4.5 members. The slum has a municipal water
supply for drinking and cooking and most families live in poorly constructed houses. The
great majority of houses are mud walled with thatched dry leaves and bamboo or tin
roofs. The population less than five years of age is about 700. The prevelance of H.
pylori infection in less than five children in this community is 50%.44 A weekly clinic
has been run by the International Centre for Diarrhoeal Diseases Research, Bangladesh
(ICDDR,B) for minor case of illness in the population since 1986.
All children in the community will initially be screened for iron deficiency anemia and
H. pylori infection. After explanation of the study and the tests, informed consent will
be obtained from the parents or legal guardians. Demographic information including name,
date of birth, sex, weight, height, economic status of the parents, nature of feeding,
and previous medication in the past 1 month will be recorded.
Screening for iron deficiency anemia 50 µl capillary blood samples obtained by
fingerstick will be assessed for hemoglobin (Hgb) concentration. Measurement of
hemoglobin concentration is the most useful test in screening iron deficiency anemia
since it directly reflects the quantity of most abundant essential iron compound in the
body. Children with hemoglobin concentration below 110g/l will be considered as anemic.
In children with low Hgb identified through the initial screening, the diagnosis of iron
deficiency anemia will be confirmed by venepuncture Hgb, serum ferritin concentration,
and serum transferrin receptor concentration. Haemoglobin concentration can be
transiently depressed due to inflammation or infection, and in such a situation does not
indicate pathological anemia. Of all the biochemical tests for assessing iron status, a
low SF is the most specific test for iron deficiency. The major disadvantage of SF is
that it is readily elevated in response to any infector inflammatory condition. One
major advantage of sTfR is that unlike SF, sTfR is not significantly effected by
infection or the inflammatory process. However, sTfR levels are increased in situations
of increased RBC turnover such as hemolytic anemia, i.e. thalassemia, etc. A uniform
cutoff value of >8.5 mg/l indicates an elevated sTfR and therefore iron deficiency or
hemolytic anaemia.
Measurement of serum transferrin receptors also becomes a promising new test for the
evaluation of iron status particularly in epidemiological assessment of anemia.
Performed in conjunction with serum ferritin measurements, the serum transferrin
receptor serves to distinguish true iron deficiency from the anemia from the anemia of
chronic diseases or inflammatory diseases and, therefore, offers a major advantage in
establishing the true prevalence of iron deficiency anemia in population studies. The
combination of low hemoglobin, a low serum ferritin, and an elevated serum transferrin
receptor are diagnostic of iron deficiency anemia. The diagnosis of iron deficiency will
be made when the following criteria are met: transferrin receptor > 8.3 mg/l, serum
ferritin concentration <12ng/ml, and hemoglobin concentration(<11 gm/dl).
Screening of H. Pylori infected children The anemic children will be screened for H.
Pylori infection using a urea breath test (UBT) which is considered by many of the
reference diagnostic test for H. pylori infection. We have recently established the test
at ICDDR,B.44 In brief the procedures are as follows.
Urea Breath Test A breath sample will be collected for base line 13 CO2 in a vacutainer
following a fasting period of 2 hr. A volume of 100 ml of whole milk will then be
allowed to be consumed by the children. Ten min later 40 mg of 13 C urea (99% 13C,
Tracer Technologies, Boston, Massachusetts) will be given after dissolving in 25 ml of
water. After administration of the substrate, breath samples will again be collected in
duplicates after 30 min. Samples will be collected through two-way pediatric mask with
an attached non-return valve into a vacutainer and will be stored for shipment to the
University of Alabama, to measure the 13C concentration of a respiratory CO2 mass
spectrometer. The excess over the baseline will be expressed as parts per thousand(delta
0/00). A breath test in which the excess over a baseline is 5.00/00 at 30 min will be
regarded as positive for H. pylori infection.
1.2 Selection of Study children Iron deficiency anemic children with weight for age >60%
of National Center for Health Statistics (NCHS), with no evidence of deficiency diseases
or systemic infection and whose parents give informed consent will be enrolled in this
study.
Exclusion criteria The children with following problems will be excluded.
- Acute infection or apparent inflammatory process
- Signs of vitamins deficiency
- Severe anemia (Hemoglobin <70 G/l)
- Severe malnutrition (marasmus, marasmic kwasiorkar or kwasiorkar)
- Presence of hook worms and /or Giardia lamblia (cyst or vegetative form) in a stool
microscopic examination
- Presence of fat in a stool microscopic examination
- Presence of occult blood in a stool as demonstrated by Guaiac test
- History of taking antibiotics or any drugs for any cause in the preceding month
1.3 Procedure A detailed medical history will be recorded. Body weight and height will
be measured upon enrolment into the study. Naked weights will be obtained to the nearest
10 g using a balance scale (Gebrüder Soehnle, Murrhardt, Germany) and the mean of three
consecutive measurements recorded. Recumbent length will be measured in children less
than two years of age using a slideboard infantometer (Harpenden, St. Albans, England).
In older children, standing height is to be determined using a locally constructed
instrument in which a metal tape measure is extended between a footplate and head bar
that has an attached fluid level to ensure a horizontal position. The mean of two
consecutive height measurements to the nearest 0.1 cm is recorded as the observed value.
Measurements are compared to the standards according to the National Center for Health
Statistic data and the nutritional status assessed by Z-score. , Percent of median
equivalents of standard deviation units will be determined and categorized according to
the scheme of Waterlow.
All children will be subjected to physical examination by one of the investigators. The
children will be assigned to one of the four groups according to randomization:
- Anti Hp group
- Fe group
- Anti -Hp group + Fe group
- Placebo group
In order to compare the overall prevalence of resistance to iron therapy among iron
deficient children, there will be a fifth group comprising children with iron deficiency
anemia but without H. pylori infection. This group will be treated with iron therapy.
Anti Hp therapy: Clarithromycin 15 mg/kg/day in two divided doses for 10 days plus
omeprazole 20 mg single dose for 14 days. ,
Iron therapy: An iron dose of 3 mg/kg/day (or about 30 mg/day) as ferrous sulphate as
advocated by Dallman41 will be fed in a single dose in the morning in one half hour
before food. The iron therapy will be given for 90 days.
Placebo Glucose syrup
1.3.1 Supplementation The intervention syrup(s) will be fed daily in a single dose in
morning in an empty stomach (Half an hour before food). The children in the placebo
group will receive same amounts of glucose syrup. All therapies will be kept in
identical dark brown containers and will be fed by a health worker who will be kept
unaware about the nature of the study and of the contents inside the bottle. Twenty
four-hour food intakes will be recorded on a precoded form by dietary recall.
The children will be followed for three months after the end of supplementation. Urea
breath test will be repeated at the end and at 1 and 3 months after the supplementation.
Venous blood (500 µl) will be taken for determination of serum ferritin, transferrin
receptor and hemoglobin concentration at the end and after 1 and 3 months of
supplementation. Children in the placebo group will be given a three month course of
supervised iron supplementation at the end of study period.
1.4 Sample size calculation The sample size is calculated on the basis of (i) prevalence
of H. Pylori infection, (ii) prevalence of non response to iron therapy.
Sample size calculation based on the prevalence on H. Pylori infection
Considering the prevalence of H. Pylori infection 50% and iron deficiency anemia 70%,44
we anticipate H. Pylori related anemia in 50% of infants and children in this community.
We expect a reduction in prevalence of H. Pylori related iron deficiency anemia after
therapy with antibiotic plus iron therapies 25%. To detect a difference of this
magnitude at 5% level and at 80% power the sample size is calculated as follows:
N =[P1(100-P1) +P2(100-P2)]*8/(P1-P2)2
Where P1= prevalence of H. Pylori related anemia P2= prevalence of H. Pylori related
anemia after therapy
=[50(50) +25(75)] * 8 / (25)2
=56 in each group
Based on a previous study in this community we anticipate a drop out rate of
approximately 15%. With a (Akramuzzaman SM, personal communication), the necessary
sample size will be 65 children in each group and the total sample of children will be
260.
Calculation based on prevalence of non-responder to iron therapy
Observation from an ongoing study with an oral iron supplementation programme in anaemic
patients being conducted in Dinajpur (a northern district of Bangladesh) suggests that
70% of the iron supplemented group do not respond ( S. Rahman, personal communication).
The prevalence of H. Pylori infection in the proposed study population is approximately
50%,35 and we expect that after oral iron supplementation the non responders will be 40%
less among the H. pylori negative group, compared to the H. pylori positive group. To
detect a difference of this magnitude at 5% level and at 80% power the sample size is
calculated as follows:
N = [P1(100-P1) +P2(100-P2)]*8/(P1-P2)2
Where P1= prevalence of anemia after iron supplementation in H. pylori infected children
P2= prevalence of anemia after iron supplementation in H. pylori non-infected children
- [(70)(30) +(42)(58)] * 8 / (35)2 =46 in each group
With 15% drop out rate, the number of children will be 53 in each group i.e. total
sample of 212 children.
We will take the larger sample size, i.e. 260 children for this study.
1.5 Randomization A master randomization chart will be prepared by an appropriately
trained person who is not connected with the study using random permuted block numbers
taking a size of three. Bottles containing anti-Hp therapy, iron therapy or placebo will
be identical in appearance and will be arranged in sequence that corresponds to the
randomization chart and will be serially numbered. The serial number of the bottles will
correspond to the serial number of the patients enrolled in the study. As one group of
children would receive anti-Hp therapy and iron therapy simultaneously, there will be
two bottles (bottles A or B) for each group of children to make the study double blind.
For children belonging to anti-Hp plus Iron therapy, there will be one bottle containing
anti-Hp therapy or and the other containing iron therapy. Children belonging to iron
therapy or anti-Hp therapy alone, will be getting one bottle containing the medication,
while the other will contain glucose syrup. As the duration of anti-Hp therapy is 14
days, the children belonging to this group will be provided glucose syrup for the rest
of the month in order to make the duration of therapy equal among the four groups.
1.6 Laboratory test Hb concentration will be determined by the cyanmethemoglobin method,
serum ferritin will be determined by a radio immunometric assay. Serum transferrin
receptor will be measured by enzyme-linked immunosorbent assay (ELISA). Stool will be
tested for the presence of occult blood by the guaiac reaction (Occultotest, Ames
Co.,Elkhart, Inc.,. USA). Serum pepsinogen and gastrin will be measured by
radioimmunoassay.
1.7 Withdrawal from the study - The noncompliance of subject, either because the leaves
the study area for other place before the end of the study or
- because the child requires unscheduled treatment for serious interim infection.
Treatment of withdrawal during analysis Results of all randomized children will be
included in the analysis of the study. Data from the patients withdrawn will be included
up to the time of withdrawal. A supplemental analysis in which such patients are
excluded will also be made
1.8 Outcome variables
Major
- The incidence of H. Pylori infection following the therapy.
- Rise of serum hemoglobin, serum ferritin concentration or reduction of level of serum
transferrin receptors.
- The number of children in each group with IDA as primarily defined before and after
the intervention
Minor
- Change of anthropometric indices
2. Iron absorption study Twenty additional children with a positive Urea breath test and iron
deficiency anemia will be selected for the Iron absorption study. The purpose of the study
and all procedures related to iron absorption study will be explained to the parents and
consents will be obtained. Before commencing the iron absorption study, all children will be
given a commercial whey adjusted cows' milk formula with 8 mg Fe/l (Nidina 1; Nestle, Paris,
France) for a period of at least three weeks. After 21 days of formula feedings they will be
admitted to the Metabolic Ward of Clinical Research and Service Center.
Erythrocyte incorporation of labeled iron (radio stable) has been used successfully to
examine absorption from the gut. , The erythrocyte incorporation method involves oral
administration of an isotope of iron followed by measurement of the isotope ratio of iron in
erythrocytes at a selected time after administration. We will follow the technique for
measuring iron absorption in children by a double stable isotope technique, developed
recently by Kastenmayer et al.
2.1 Method for Iron absorption study
Preparation of labeled iron sulfate solutions Enriched 57Fe and 58Fe-ferric oxide (Fe2O3)
will be purchased from Oak ridge National Laboratory (Oak Ridge, TN, USA). The isotope
compositions of iron in the 58Fe preparation (as provided by Oak ridge National Laboratories)
are (mg): (weight%) 54 Fe (0.65), 56 Fe (35.6)57 Fe (3.47), and 58 Fe(60.28). The isotope
compositions of iron in the 57Fe (weight%) are; 54 Fe(0.65), 56Fe(35.60), 57Fe (3.47), and
58Fe (60.28).
Ferric oxide will be dissolved in concentrated H2SO4 by bubbling carbon monoxide through the
solution over heat for approximately four hours. The solution will then be derated by passing
purified nitrogen through it for several minutes. The volume will then be adjusted using
derated water. Two ml aliquot of the resultants 57 FeSO4 and 58 FeSO4 solutions will be
transferred into opaque ampules. Each ampule will be purged with nitrogen and sealed. Ampules
will contain either 200 µg/ml of total iron (as ferrous sulphate) and 150 µg/ml of 57Fe or
700 µg/ml of total iron (as ferrous sulphate) and 420 µg/ml of 58Fe. Aliquot from an
individual ampule will be used to prepare 57Fe and 58Fe-spiked working solutions.
Stability and safety of parenteral 57 Fe solutions Prior to i.v. administration to the
infants the 57FeSO4 solution will be tested for pyrogenicity, sterility, and stability.
Limulus amoebocyte lysate (LAL) testing and repeated culture for bacterial and fungal growth
to confirm that the 57FeSO4 solutions are pyrogen-free and sterile at the concentrations to
be used in the study. The nutrient content of the parenteral solutions has been previously
described. The total iron content of the 57FeSO4 -nutrient solution will be determined by
atomic absorption spectrometry before and after filtration (0.22 µ filters), and over time at
room temperature.
2.2 Study design of iron absorption study Infants enrolled in the study will receive a
combination of parenteral and enteral feeds. The 57FeSO4 solution will be provided
parenterally while the 58FeSO4 will be given via the enteral route. Erythrocyte incorporation
of 57Fe and 58Fe will then be measured from blood samples taken two weeks after initial
infusions. The percentage of the I.V administered dose presents in the circulating
erythrocytes will be used as a reference for the percentage of absorbed iron (from the orally
administered dose) present in the circulating erythrocytes. The "corrected" percent
erythrocyte 58Fe and 57Fe incorporation (58 Fe inc-cor/ 57Fe inc-cor) will be calculated
mathematically as described below.
Prior to iv administration to the children, 0.7-0.8 ml of the 57FeSO4 solution(0.2mg/ml) will
be added to a burytrol containing 30 ml of the nutrient solution used for parenteral feeding,
using sterile techniques. The iron-nutrient mixture, which has been used safely previously,
will be infused slowly into a peripheral vein of the children over a 12-24 hour period with
the aid of an infusion pump.
Soon after the start of the parenteral infusion of the 57FeSO4 solution, the enteral 58FeSO4
will be given to the children. During this time, parenteral infusion of 57FeSO4 will be
continued. After checking to ensure that the nasogastric feeding tube is properly located in
the stomach, the tube will be aspirated to check stomach contents. If the stomach is empty,
1.5 ml of the 58 FeSO4 will be delivered directly via a syringe into the feeding tube. The
tube will then be flushed with 0.5 ml of sterile water. The dosage of parenteral 57FeSO4 will
be 0.15mg/kg (57Fe-enriched ferrous sulfate solution), which would provide 0.12 mg/kg of
57Fe. The dosage of enteral will be 1.5mg/kg (58Fe-enriched ferrous sulfate solution), which
will provide 0.8mg/kg of 58Fe. The children will be discharged with therapy for Helicobacter
pylori infection (Klarithromycin plus omeprazole) for 10 days.
After one month of therapy the mothers will be requested to bring their children at the CRSC.
A repeat UBT and iron absorption study will be performed.
2.3 Blood sampling Blood samples will be taken before administration of the stable isotopes
(baseline) and 14 days later in both occasions. Blood sample size will be 200-300 µl up per
sample.
2.4 Calculation of Erythrocyte Iron Incorporation The quantity administered 58Fe and 57Fe
(mg) incorporated into erythrocytes at specified time 't' after administration of the dose of
labeled iron will be calculated in a manner similar to that used by Zlotkin et al 1995.
58 Fe inc. =Rt58/54 -Ro58/54 xFe circ x 0.00322 Ro58/54
57Feinc =R157/54-Ro57/54 x Fe circ x 0.0218 Ro57/54
In this equation, 58 Feinc and 57Feinc will be expressed in mg. R158/54, R157/54 and R057/ 54
are the isotope ratios expressed on weight basis for 58Fe/54Fe and 57Fe/54Fe, which will be
determined on digested blood samples at time "t" after dosing and at a baseline (pre 58Fe and
pre 57Fe doses) respectively; Fe circ is the quantity of the circulating iron. Fe circ at the
time "t"; 0.0032 and 0.0218 are the natural abundances (weight fractions) of 58 Fe and 57 Fe.
The quantity of total circulating iron, Fe circ, expressed in mg, will be estimated as
Fe circ. =BV x Hb X 3.47
Where BV is the blood volume in liters, assumed to be 0.085l/kg of the body weight; Hb is the
hemoglobin concentration in g/l; 3.47 is the concentration of iron in Hb (mg/g).
The quantity of administered 58Fe or 57Fe incorporated into erythrocytes, % Fe inc, will be
expressed as a percentage of the administered doses of 58Fe or 57Fe. Feinc.
- 58 Fe inc = 58Fe inc x 100 or Fe inc
- 57 Fe inc = 57 Fe inc x 100 57 Fe inc
The corrected percent erythrocyte for analysis of the stable isotope ratios in erythrocytes
will be calculated using the percentage of erythrocyte incorporation of i.v infused 57 Fe.
- 58Fe inc-cor = % 58Fe inc X 100.
- 57 Feinc
Mass spectrometric analysis Preparation of blood samples for mass spectrometric analysis and
preparation of enriched standards will be followed as per the procedures described by
Kastenmayer et al. Samples of heparinized whole blood will wet ashed with concentrated HNO3
at a ratio of 1:3 (blood: acid)(vol/vol) on a hot plate over low heat for 20-30 min. Digested
solutions will be adjusted with deionized water to yield an iron concentration of 10 µg/ml.
Isotope-spiked standards for an instrument calibration will be prepared in the same manner as
with the unknown samples. Isotope ratios for 58Fe/54Fe and 57Fe/54Fe in blood before and
after concurrent enteral (58Fe) and parenteral (57Fe) administration of isotopes will be
determined by a computer-controlled thermal ionization mass spectrometer using an Elan Model
250 ICP/MS system, (SCIEX, Inc., Thornhill, Ontario, Canada), operated in the isotope ratio
mode, as followed by Zlotkin et al. in the Department of Pediatric and Nutritional Science,
University of Tronoto, Canada.
Intubation test A standard intubation test as described earlier14 will be performed in this
subset of children after completion of iron absorption study. In brief, as soft gastric tube
(18G) will be placed in most dependent part of stomach. Gastric acid samples will be
collected from children lying in the left recumbent position using manual suction. The
position of the tube will be confirmed by auscultation, aspiration of gastric juice, and
nearly complete recovery of water injected in the tube. Samples of gastric juice will be
discarded during the first 10 min after intubation. Thereafter, samples will be collected and
saved in 15- min aliquot for 90 min. After 90 min of collection (basal sample), pentagastrin
(peptavlon, ICI) will be injected subcutaneously at a dose of 0.6 ug/kg. The sample following
pentagastrin stimulation for 60 min will be termed as "stimulated sample". The volime of
gastric secretion in each of the 15 min collections will be rcorded and expressed as
mililitre per hour. The concentrations of acid in each will be measured by titration of 1 mL
gastric juice with 0.01N sodium hydroxide to pH 7.4, using an automated titrator (Metrohm,
Herisau, Switzerland). Total acid output was calculated for each time pint by multiplying
volume of gastric juice and acid concentration. Basal (BAO) and stimulated acid output (SAO)
were calculated based on 2 samples collected for baseline measurement and the 4 samples
collected after administration of pentagastrin respectively, and were expressed as mMol/h.
