Registration

5.10:1: i Clinical management

On recruitment into the study, an interviewer administered structured questionnaire was administered to subjects. Data collected includes; duration of marriage, number of sexual partners, history of abnormal vaginal discharge, history suggestive of pelvic inflammatory disease (PID), chronic pelvic pains, termination of pregnancy, contraceptive use and age of first sexual intercourse. Thereafter, the weight and height of subjects were measured and body mass index calculated as described below.

5.10: 1 ii Measurements of anthropometric parameters

1. The weight was measured using RZY 160 combined weight and height measuring scale, manufactured in 2014 by Seca^R, England which was standardized and subjects’

weights were measured with minimum clothing on. Measurements were taken to the nearest 0.1kg.

2. Height was measured to the nearest 0.1m also using the RZY 160 combined weight and height measuring scale. Patients were without shoes, head gear or cap before measurements were taken.

3. Body mass index was calculated as weight in kg divided by the square of height in meters.

4. Patients were classified as underweight when BMI was <18.5kg/m², subjects with BMI between 18.5 – 24.9kg/m² were termed normal, 25.0 – 29.9kg/m² as overweight and subjects with BMI ≥ 30kg/m² as obese.

34 5.10:1 iii Sample collection

After the weight and height measurement, the Patient was taken to a designated Consulting room and made comfortable by sitting down and the interviewer administered questionnaire was administered. After resting for about five minutes, the left ante-cubital fossa was exposed and a tourniquet was applied about 2-4cm above the area in order to make the vein prominent. With the least discomfort to the patient, five milliliters of venous blood samples was collected from each subject using a sterile 5ml hypodermic needle after swabbing the area with methylated spirit. The sample obtained was placed in a labeled plain sterile vercutainer for analysis of antibodies to chlamydia trachomatis. This was kept upright in a specimen tray and transported to the Department of Chemical Pathology of Lagos State University Teaching Hospital for further processing.

5.10:2 Laboratory estimation of chlamydia trachomatis antibodies

On arrival at the laboratory, the blood specimen was allowed to clot and then centrifuged at 10,000 revolutions per minute for 5 minutes using a Hettich^R universal 320 centrifuge. After centrifugation, the serum was withdrawn with a pipette and placed in a labeled micro-vial and stored in the freezer at -4 °C. The samples were collected and processed in this manner for the first four months of the study and analyzed for Chlamydia antibodies in batches.

Serological assay was done using ELISA Ig G kit, produced by Perfemed Group, Inc. South San Francisco, CA 94080, USA. This was both a qualitative and quantitative ELISA test kit with a sensitivity of 91.1% and a specificity of 98.5%. The Ig G kit contained microwell strips,

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sample diluents, calibrator, negative control, positive control, washing concentrate, enzyme conjugate, tetramethylbenzidine (TMB) chromogenic substrate and stop solution.

The principle of the test is based on the purified Chlamydia trachomatis antigen coated on the surface of the microwells. The diluted patient’s serum is added to the wells and the Chlamydia trachomatis Ig G specific antibody, if present, binds to the antigen. All unbound materials are washed away. After adding enzyme conjugate, it binds to the antibody-antigen complex. Excess enzyme conjugate is washed off and TMB chromogenic substrate is added.

The enzyme conjugate’s catalytic reaction is stopped at a specific time. The intensity of the colour generated is proportional to the amount of Ig G specific antibody in the sample. The results are read by a microwell reader compared in a parallel manner with calibrator and controls. The results are then presented using Ig G index.

5.10:3 Laboratory procedure

This was commenced by bringing the frozen samples to room temperature and allowed to thaw in the storage sample trays; 200μl of sample diluents (blue solution) was placed into appropriately labeled microvials already arranged in trays. The 1 in 40 dilution was prepared by adding 5μl of each Patient’s samples, the negative control, the positive control and calibrator to 200μl of the sample diluents then mixed well then 100 μl of the 1 in 40 diluted sera, calibratror and control was dispensed on to the 96 ELISA microwell plates using an automated pipette. This was then left to incubate for 30 minutes at room temperature to allow for the antigen-antibody reaction. The plated 96 ELISA microwell was placed in a biotec automatic ELISA washer with the washing buffer and allowed to run three times.

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After washing, the solution became clear i.e. the microwells contained a clear solution and 100 μl of the enzyme conjugate was dispensed to the each of the wells and allowed to incubate at room temperature for another 30 minutes and then turned pink. The enzyme conjugate was washed off from all the wells by placing in the automatic ELISA washer and allowed to run thrice, thereafter, 100 μl tetramethylbenzidine (TMB) chromogenic substrate was dispensed to the mixture and allowed to stand for 15 minutes at room temperature.

Finally, 100 μl of the stop solution was added in order to stop the reaction. A colour change was observed in the positive microwell while the negative ones remained clear. This was then read immediately using the ELISA microwell reader at 450nm and printed out.

5.10: 4 Calculations of results

The results were represented using the Chlamydia Ig G index as stated by the manufacturer.

The mean optical density of all the calibrators was first calculated and this was multiplied by Factor (f) printed on the label of calibrator in order to have the cut off optical density.

Mean calibrator OD obtained was 2.00

Factor (F) on the label = 0.45

2.00 x 0.45= 0.9

Cut off optical density (OD) = 0.9

In order to calculate the Chlamydia Ig G index, the OD of the each patient’s sample which was obtained from the ELISA reader’s print out was divided by the cut off OD value.

37 Chlamydia Ig G index = Patient’s OD (print out value)

Cut off OD

E.g: Patient’s optical density (OD) (print out value) = 1.624 Cut off OD = 0.9

= 1.624

0.9

= 1.80

Patient’s Chlamydia Ig G index is 1.80 (POSITIVE)

Results summary

Negative = Chlamydia Ig G index of 0.99 or less

Positive = Chlamydia Ig G index of 1.00 or greater

5.11: Data processing and statistical analysis

The data obtained was processed and analyzed using the Statistical Package for Social Sciences (SPSS), version 23.0 (Chicago, Illinois Inc) 2015. Percentages, mean and standard deviation of numerical variables were determined. The histogram plot of outcome variable (Chlamydia index) was drawn to determine if it followed a normal distribution. Mann Whitney U test was used to compare the median of chlamydia index of independent groups.

Chi squared test and Fischers’ exact test (for 2 x 2 tables with expected frequencies less than 5) were used to compare categorical variables as necessary. Kappa was used to assess the

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agreement between classification of tubal blockage using chlamydia index and tubal blockage using hysterosalpingogram. Kappa values can be interpreted thus; ≤ 0 indicates no agreement, 0.01-0.20 shows none to slight agreement, 0.21- 0.40 shows fair agreement, 0.41-0.60 shows moderate agreement while 0.61 – 0.80 shows substantial agreement and 0.81 – 1.0 shows an almost perfect agreement.⁷⁵ All (independent) variables that were significant (p < .05) on bivariate analysis were entered at once at the beginning to assess their predictive ability. The quality of the model was adjudged good when the Omnibus tests of model coefficients were significant (i.e., p < .05) and the Hosmer–Lemeshow goodness-of-fit test value was >0.05. Confidence interval was set at 95% for all statistical tests. P value of < 0.05 was considered statistically significant.

5.12 Ethical Consideration

Approval for the study was obtained from the Health Research and Ethics Committee of the Lagos State University Teaching Hospital, Ikeja, Lagos State. The research participants for the study were assured of confidentiality and data collected were deidentified so that the identity of the subjects was not revealed. This can be seen as appendices I and ii, found on pages 62 and 64 respectively.