Simulation Scenarios

THE STUDY AREA

The study was conducted at the University of Uyo Teaching Hospital, Uyo, in Akwa Ibom State. Uyo is the capital city of Akwa Ibom State, and it is bounded in the north by Itu Local Government Area, in the south by Ibesikpo Asutan Local Government Area, in the east and west by Uruan and Abak Local Government Areas, respectively. It is located on an elevation of about 60.96 metres (200 feet) above sea level and lies between latitude 4⁰20’ and 5⁰12’ North; and longitude 7⁰31’ and 8⁰11’ East. It is predominantly a civil service town. There are two major seasons; a rainy season between May and October and a dry season which spans November to April. Borehole water is the main source of domestic water supply constituting about 81.40% of drinking water source in the town. Public pipe-borne water and piped water within households constitute 3.72% each.¹¹² Rain water is used in many homes during the rainy season.

The Teaching Hospital is the only tertiary health institution in the state. It is located in the outskirts of the town, along Uyo - Abak road, about 5 km from the centre of Uyo, in a semi-urban setting. The hospital has a total bed capacity of 250 with various departments and areas of specialties. The Children Emergency Unit (CHEU) is located on the ground floor of the major hospital block. It has a waiting area, a consulting area, the nurses’ station, and the main emergency unit; with a treatment room and a side laboratory. It also has a transition room for fairly stable patients who await transfer to the paediatrics ward. It has a bed capacity of 17, and an average of 75 patients (aged between 1 day and 18 years) is seen per month. It operates a 24-hour service run by senior registrars, registrars and house officers. Consultant ward-rounds are done at

least three times each week. There are also trained nurses of all cadres in attendance.

ETHICAL APPROVAL

Approval was given by the hospital's ethical committee for this prospective study to be conducted in the Children Emergency Unit (CHEU) of the University of Uyo Teaching Hospital. Appendix 11.

SAMPLE SIZE

Calculated using the formula

n = Z²P(1-P) d²

Where n = Minimum sample size

Z = Standardized normal deviation (1.96)

P = Best estimate of population prevalence rate (82%) ¹⁰ d = Tolerable error margin (5%)

The best estimate of population prevalence rate for H. pylori in Nigerian children = 82%.¹⁰

Therefore, minimum sample size

n = (1.96)² × 0.82 x 0.18 = 227 (0.05)²

INCLUSION CRITERIA

The following categories of eligible subjects were to be recruited for the study:

(i) All consecutive infants and children between 6 months and 18 years of age who presented at the Children Emergency Unit and required venipuncture for intravenous access and/or blood-letting for any other investigations.

(ii) All children whose parents gave informed consent for their inclusion in the study and all eligible children from 12 years of age who gave their informed consent for inclusion in the study.

(iii) All eligible children who did not have clinically detectable icterus.

EXCLUSION CRITERIA

(i) All neonates and infants less than six months old who presented at the unit. This was to avoid the possibility of assessing transplacentally transferred maternal IgG antibodies to H. pylori.

(ii) All children for whom informed consent for their inclusion in the study was refused.

(iii) All clinically icteric children. This was the caution of the manufacturer of the ELISA’s test kit in order to avoid false positive results.

METHODOLOGY

The study was carried out between 26^th August 2008 and 23^rd January 2009. The study population consisted of children between the ages of 6 months and 15 years who required venipuncture, and for whom an informed consent was obtained. Although children older than 15 years (up to 18 years) are also seen in the emergency unit, they did not form part of the study population. Only five of such older children were admitted during the study period but they were not eligible. Three had sickle cell anaemic crisis with clinically detectable icterus and were excluded from the study, while the remaining two refused their consent for inclusion in the study. Thus, children aged

6 months to 15 years formed the study population. Informed consent was obtained from the parent(s)/guardian(s) and also from the children who were up to 12 years old and above.

A clinical history was obtained from the care giver and/or the patient including the educational level and occupation of the parent(s)/guardian(s), the type of accommodation and household density (including bed-sharing if applicable), the source of household water supply, type of waste disposal system used; and any history of diarrhoea and/or dysentery in the previous 6 months. Information was also obtained about the type of school attended and the school environment, in the cases of children who had started school. The provisional diagnoses of the subjects were also documented. These were recorded on the study proforma. Appendix V.

The child’s social class was determined using the parents/guardians social class according to the social classification scheme proposed by Oyedeji ¹¹³ (Appendix IV). The scheme classified the occupational as well as the educational level of parents on a score of 1 to 5, with the highest educational attainments (university degree or above) being class 1. The score for the parents’ (or surrogates’) occupational status plus those of their educational levels will be added together and the mean of the total of these four scores to the nearest whole number was regarded as the child’s social status score. For a child whose father had occupational status score of 3 with educational level score of 2 and the mother having occupational score of 4 and educational level of 3; the child’s

social status score would be (3+2+4+3/4) = 3 when taken to the nearest whole number.

Axillary temperature of each subject was measured using a digital clinical thermometer and the temperature was recorded to the nearest 0.1⁰C. Anthropometric measurements of the children were also taken. For infants, the infant scale (RGZ-20) was used. The pointer was adjusted to the zero point before commencement of weighing. The naked infant was placed in the basin scale and allowed to stay steady, without swinging, and the weight was read off to the nearest 0.1 kilogram. For older children who could stand unsupported, the bathroom scale Hanson Emperors Marketing Co Ltd, Model: H89 DK Blue was used and weight measured with the children wearing only under-wears and without shoes. Dressing gowns were provided for older girls. Weights were read with precautions against errors due to parallax by reading at right angle to the pointer. The scale’s pointer was adjusted to the zero point before commencement of each measurement. For children who were too ill to stand for weight measurements and whose weight plus that of the mother/caregiver would not reach the 120kg maximum limits of the bathroom scale, the weight was estimated by weighing the naked child being carried by the mother, and then weighing the mother alone. The difference was taken as an estimate of the child’s weight. For example, weight of child and mother = a, mother’s weight = b, child’s weight = a-b.

Length was measured for infants and children less than 2 years¹¹⁴ with the use of an infantometre. The child was put in supine position with the head placed against a fixed edge in such a way that the occiput aligns with the pupils and the child was held steady in that position while an assistant held the child’s feet with legs extended and ankles at right angle to a vertical movable platform. The length of the subject was then marked off and measured with a tape-rule from the fixed occipital end to the point of the marks of the feet. This was recorded to the nearest centimetre. Similar procedures were carried out for older children who were too ill to stand for their heights to be measured.

A few tall subjects who could not stand for height measurement had their supine length estimated while on their beds. Children who were aged 2 years¹¹⁴ and above stood against a graduated vertical height-measure placed against the wall and positioned such that the heels, back and occiput touched the wall and the occiput held against a movable firm wood with pupils aligned to occiput. The height was then read off to the nearest centimetre. The body mass index figures were read off from a standard normogram. Anthropometric measurements so obtained were also recorded on the study proforma. Appendix V.

About 2.0mls of blood was collected from each subject into a plain specimen bottle and taken to the laboratory within 30 minutes for centrifuging to separate the serum. Serum samples were then transported to the Akwa Ibom State cold store and stored frozen at -20⁰C till sufficient samples were pooled for analysis. The Vic Torch H. pylori IgG was used for the analysis.

The H. pylori IgG reagents were stored unopened at 4⁰C (this was the manufacturer's instruction). The vacuum sealed micro plate strips were kept sealed till the time of analysis. The controls and additive for Sample Diluents were also stored unopened at 4⁰C.

TEST PRINCIPLE

H. pylori causes an immunologic response during infection and specific antibodies of the different classes of IgG, IgA and IgM are produced by the patient. Enzyme-Linked Immunosorbent Assay (ELISA) is currently used to screen patients affected by gastritis or peptic ulcers for acute active infection due to some H. pylori virulent strains.

In particular, the presence of IgA and IgM antibodies seem to be correlated to the acute phase of illness. On the other hand, IgG antibodies are present at different titres shortly after the primary infection and remain in the patient's blood for many years after the infection.

The micro plate wells are coated with immobilized H. pylori antigens. Specific antibodies against H. pylori, if present in the specimen will bind to the corresponding antigens.

After removing the specimen, the presence of specific antibodies against H. pylori would be revealed by the specific antihuman IgA, IgG or IgM conjugated to peroxidase.

Excess conjugate was removed and the substrate A/B added. A blue colour then developed, the intensity of which was proportional to the antibody content of the specimen. The enzyme reaction was then blocked by the addition of the stop solution.

The blue colour turned to yellow and the absorbance was read at 450nm. Evaluation was carried out using the intensity of the cut-off control according to the evaluating criteria of the test. The cut-off value for positive antibody activity (AA) was taken at greater than 20.

Repeated freezing and thawing of the samples as well as microbial contamination were avoided. Also icteric or turbid samples were not used, in order to avoid wrong results (manufacturer’s precautionary advice). Antibody determinations were run with 1:21 diluted serum specimens. The controls (supplied in a ready to use dilution) and Additive for Sample Diluents were stored at 4⁰C and brought to room temperature prior to being pipetted into the wells. Any remaining stock was stored back immediately. The substrate A/B was used within 10 minutes (as directed) after preparation and then stored away from direct light.

REAGENT PREPARATION Sample Diluents with Additive

The Sample Diluents was prepared by adding the Additive for Sample Diluents (2mls) directly to the Sample Diluents (20mls).

The wash buffer was prepared by diluting the concentrated Wash Buffer (e.g. 20mls concentrated solution plus 480mls distilled water).

The substrate A/B was prepared during the last washing step after conjugate incubation.

Number of wells

processed Volume

needed(mls) Substrate

solution B(mls) Substrate

solution A(mls)

8 (1 strip) 1 1 0.050

16 (2 strips) 2 2 0.100

32 (4 strips) 4 4 0.200

48 (6 strips) 6 6 0.300

SAMPLE PREPARATION

The assay was run with a 1:21 sample dilution carried out directly in the wells. In the absence of an ELISA micro plate washer, manual washing was done in 4 cycles, by dispensing and aspirating 0.3mls per well per cycle. The liquid washed out from the plates was considered potentially infected and inactivated with a sodium hypochlorite solution at a final concentration of 2.5% before being discarded.

ASSAY PROCEDURE

All reagents, controls and samples were brought to room temperature (18-30⁰C) about two hours before use, and then mixed carefully. The controls and samples were duplicated. Distribution and incubation time were the same for all wells in the same analysis. The excess of wash buffer was eliminated from the micro plate after washing by blotting it gently on an absorbent paper pad.

ASSAY SCHEME

Reagents A1 Blank Controls Sample

Sample Diluents with Additive

- - 100l

Controls - 100l -

Sample - - 5l

The strips were then covered with adhesive film and incubated in a humid chamber at 37⁰C ± 1⁰C at 30 min. ± 2 min.

The Adhesive was thereafter peeled out and the reaction solution aspirated from all the wells.

Washing was done in 4 cycles using 300l of the diluted wash buffer, allowed to stand for 1 minute each time, before aspirating.

Enzyme Conjugate - 100l 100l

The strips were again covered with adhesive film and incubated at 37±1⁰C for 30±2min.

The adhesive film was then pulled out and the reaction solution aspirated from all wells

Washing was again done in 4 cycles with 300l of diluted wash buffer, and allowed to stand for 1 minute each time and then the reaction solution was aspirated.

Substrate A/B 100l 100l 100l

Incubated 15 min. at room temperature, protected from light.

With the plates left uncovered

Stop solution 100l 100l 100l

The absorbance of each well was read against A1 blank well at 450nm within 5-10

min. after stopping.

EVALUATION OF RESULTS

Every absorbance was read against the reagent blank (substrate A/B and stop solution) as reference value (index).

The presence or absence of 1gG antibodies against H. pylori was established by comparison of the absorbance obtained for the cut-off control with that of each single specimen. The test reference values allowed for direct comparison of test results and thereby minimised variations from run to run, allowing for consistent reporting of results. The test reference values were obtained by dividing the absorbance obtained for each specimen by the absorbance value obtained for cut-off control. An absorbance value lower than 10% or less of the cut-off control value was considered non-reactive, and values higher than the cut-off control were considered as reactive. All specimens giving absorbance values located within the value of the cut-off control and 10% below this value were considered as possibly reactive. This is because at the early stage of infection antibody concentrations may still be very low, and may thus fall between the mean absorbance of negative specimens and the cut-off control.

Negative control: Index has to be ≤20

Positive control: Index has to be >20 Manufacturer’s values Cut-off control range: 20 -21

EXAMPLE OF EVALUATION

Specimen OD 450nm Test Reference Value (Index)

Absorbance positive control

1.847 21

Absorbance cut-off control

1.767 >20

Absorbance negative control

1.760 ≤20

A non-reactive result indicates the absence of the IgG antibodies against H. pylori infection within the limits of the test. A reactive result indicates the definite presence of IgG antibodies against the pathogen and this is chosen for the test evaluation since it is generally associated with the presence of antibodies due to a previous infection.¹¹⁵ Highly and very highly reactive results (for this test, these were values above 20).

STATISTICAL ANALYSES

Statistical analyses were performed using the SPSS software package (Statistical Package for Social Sciences) 14.0 Software. Data have been summarised into frequency tables and chart as appropriate.

The seropositivity for H. pylori was computed according to age. Correlates of H. pylori were evaluated by a comparison of proportions of children with and without infection using Chi-square(χ²) test and Fisher’s exact test as appropriate. Multivariate adjusted Odds Ratio (OR) with their 95% confidence intervals were estimated by multiple logistic regression analysis. Controlling variables for evaluation with multiple logistic models were age, gender and social class. The statistical significance of the adjusted seropositive rates among comparison groups was also tested. p-value of ≤0.05 was the significant level.