Each subject was evaluated by the researcher on contact. The purpose of the study was explained to each participant. Demographic data such as name, sex, age marital status and occupation were obtained. A detailed history was obtained from the participants with emphasis on the putative risk factors for HBV infection. Physical examination was also conducted with particular attention to features of liver disease. (See appendix 1)
None of the subjects had received prior hepatitis B vaccination. Ten mililitres (mls) of venous blood was drawn from the antecubital fossa under strict aseptic conditions and observing universal precautionary measures. Two mls of blood were put in an ethylenediaminetetraacetic acid (EDTA) bottle for haematological studies. Another 4 mls of blood were put in a plain bottle and sent for liver function tests. The rest was put in another plain bottle and spun at a rate of 4000rpm for 5 minutes, then the serum was separated and tested for the serological markers. The samples that were not tested immediately were frozen and stored at -4o C and thawed when ready for testing. All samples were retested for HBsAg. Only the samples found positive for HBsAg were tested for the other serological markers. The tests were done by the researcher with the aid of the laboratory scientist at the Department of Medicine Research Laboratory.
Details of the assay procedure are as contained in appendix 2.
Assay for serological markers
Immunochromatographic method was employed in all the assays for serological markers. This immunoassay is approved by WHO and has a sensitivity of 99.0%, specificity of 97.5% and accuracy of 98.5% when compared with enzyme linked immunosorbent assay (ELISA).120 Test kits were made by Linear Chemicals SL, Montgat, Barcelona, Spain.
HBsAg
A rapid qualitative immunoassay for the detection of HBsAg was used with test kits for HBsAg, Batch no. 4210220. It is based on the immunochromatographic sandwich principle which employs a unique combination of monoclonal dye conjugates and polyclonal solid phase antibodies to HBsAg to selectively identify HBsAg.
Principle
This is a qualitative lateral flow immunoassay in which a membrane is pre-coated with anti HBsAg and colloidal gold conjugates. During testing, the serum or plasma specimen migrates upwards on the membrane chromatographically by capillary action and reacts with the particle coated with anti HBsAg antibody to generate a colour line. The presence of a coloured line in the test region indicates a positive result while its absence signifies a negative result. To serve as a procedural control, a coloured line will always appear in the control line region indicating that the proper volume of specimen has been added and capillary action has occurred.
HBeAg
This employs the same principle as above and test kits used were made by the same company (HBe Ag test kit, Batch no 4250220). The membrane here is pre-coated with anti HBeAg antibodies which reacts with serum or plasma.
Anti HBc IgG
The membrane is pre-coated with anti HBc to the test line region. During the test, the serum or plasma specimens react with the gold colloid conjugated HBc antigen in the microwell. In the presence of anti HBc IgG, a complex of antigen, antibody and indicator is formed and the mixture migrates upwards chromatographically towards the test region. At the test region, there is no available antigen to react with the antibody and thus no colour change at the test region. This indicates a positive result. In the absence of anti HBc IgG, the antigen and indicator complex migrate to the test region where the antigen reacts with the pre-embedded antibody giving a colour change signifying a negative result. Test kits used were Anti HBc kites, Batch no 4220220.
Anti HBc IgM
For this assay, the test region is pre-coated with a combination of synthetic and recombinant hepatitis B ‘core’ antigen. The microwell contains colloidal gold as indicator. Serum specimen containing anti HBc IgM antibody migrates towards the test region and forms an antigen-antibody complex and with the aid of the indicator produces a colour at the test region. This shows a positive result. The
reverse is the case for a negative result. Test kits used were Anti HBc IgM kits, Batch no 4320220.
Anti HBe antibody
This is a colloidal gold enhanced immunoassay for the detection of antibodies to the hepatitis B ‘e’ antigen and the principle is similar to anti HBc IgG antibody as described above. The nitrocellulose membrane is immobilised with mouse anti HBeAg (anti HBeAg McAb) on the test region. The presence of the colour indicator at the test region denotes a negative result while its absence signifies a positive result. Test kits used were anti HBe kit, Batch no 4350220
The liver function tests and haemoglobin estimation were done at the Department of Medicine, UNTH Research Laboratory. The tests were done by an autoanalyzer (Humalyzer 2000 by Human, Westbaden, Germany).
Statistical analysis.
The statistical analysis was done with Statistical package for social sciences (SPSS) version 11.5. Normally distributed variables were expressed as mean +/- standard deviation (SD) The t test and chi square were used to test for significance. The Pearson correlation was used to test for correlation for parametric variables. P-values < 0.05 were accepted as significant.
CHARPTER FOUR RESULTS Subjects
Two hundred and four (204) subjects were recruited into the study but 53 subjects did not meet the study criteria. Sixteen were found to be HBsAg negative on retesting, 5 were symptomatic with fever and jaundice at the time of presentation, 3 subjects though asymptomatic, were found to have shrunken liver on examination and subsequent ultrasound examination revealed features suggestive of liver cirrhosis. Twenty nine subjects were excluded because they had hepatomegaly and/or splenomegaly. One hundred and fifty one subjects comprising 103 (68.2%) males and 48 (31.8%) females fulfilled the study criteria and completed the study. The sex ratio of male to female was 2.1:1. The mean age of the subjects was 33.66±12.43 years. There was statistical difference between the mean age of the males (35.82±14.00 years) and the females (29.04±5.99 years), p = 0.002. Seventy three (48.3%) of the study population were single, while 70 (46.4%) were married. Table 1 shows the details of the demographic data of the study population.
The age group 15-29 years with 66 subjects accounted for 43.7% of the study population. This was followed by the 30-44 years age group constituting 57 (37.7%). The age ranges 45-59 years and 60-74 years had 21 (13.9%) and 7 (4.6%) respectively. (Table 2)
Of the 151 subjects, 28 (18.5%) were artisans, 27 (17.9%) were traders, and 25 (16.6%) were professionals/business people. The three groups constituted 53%
of the study population. Twenty four (15.9%) were students, 23 (15.2%) were unemployed, while 11 (7.3%) were unskilled labourers. Eleven (7.3%) belonged to the armed forces including the police. Only 2 (1.3%) belonged to the medical profession. (Table 3)
Risk factors
For the risk factors for acquisition of hepatitis B virus infection evaluated in this study (see Table 4), 32 (21.2%) gave a past history of jaundice. Only 8 subjects (5.3%) had received blood transfusion in the past. Fifty seven (37.7%) of the study population, had multiple sexual partners. Seventy two subjects (47.7%) admitted to having received injections from quacks and medicine stores. There was a positive history of scarification marks and tattoos in 13 (8.6%), and communal use of toothbrush in 27 (17.9%).
Biochemical parameters
With respect to the liver function tests, 126 subjects had normal ALT with a mean of 9.86±3.06 U/L, while 25 had elevated values; mean 30.40±10.57, (p<0.001). The results for AST follows closely the pattern of ALT. Normal serum albumin was found in 136 subjects with a mean of 40.70±5.02 g/L.
Fifteen subjects had reduced albumin level with a mean of 32.23±1.64 g/L, p<0.001. The rest of the biochemical parameters are shown in Table 5.
Serological markers
The pattern of serological markers of hepatitis B virus infection in the subjects is illustrated in Table 6. All the subjects were positive for HBsAg. AntiHBc IgG was found in 147 (97.4%) of the subjects, while none of the subjects was positive for antiHBc IgM antibody. Significantly, only 13 (8.6%) of the subjects were positive for HBeAg. A high prevalence of antiHBe antibody was found with a positive result in 114 (75.5%) of the group studied. The pattern and combination of serological markers as seen in this study is shown in Table 7.
One hundred and fourteen (114) subjects had HBsAg , anti HBc IgG, and anti HBe but without HBeAg. Twenty one (21) subjects had HBsAg and anti HBc IgG as the only positive markers. Three (3) subjects, though positive for surface antigen were negative for all other markers of HBV infection. HBeAg and anti HBe were not found together in any of the subjects. None of the subjects had either only HBsAg or anti HBeAb as the only marker. Thirteen (13) subjects were positive for HBsAg, anti HBc IgG and HBeAg only
Out of the 13 HBeAg positive subjects, 9 were males and 4 were females. The prevalence of HBeAg in the male subjects was 8.7%, while that of the females
was 8.3%. The prevalence in all patients studied was 8.6%. There was no association between gender and HBeAg positivity, p = 0.936. (Table 8)
A correlation analysis for the association between age range and HBeAg positivity was performed. There was no correlation, p = 0.279. (Table 9).
Of the liver function tests, the subjects who were positive for HBeAg had abnormal values for total serum bilirubin, (mean 24.72±4.89 μmol/L), conjugated bilirubin, (mean 15.52±4.73 μmol/L), ALT, (mean 31.54±8.79 U/L), AST, (39.46±12.67 U/L). The corresponding mean values for these parameters for HBeAg negative subjects were 13.62±5.08 μmol/L, 6.43±3.80 μmol/L, 11.54±6.7.14 U/L and 14.76±7.79 U/L respectively. The difference between these means were statistically significant, p<0.001. Although the values for alkaline phosphatase and total serum proteins were within normal limits for both groups, the differences in their absolute values were significant, p<0.004. The values for serum albumin for both groups were within normal range and the difference did not reach statistical significance, p = 0.241. (Table 10)
Table 1: Demographic data of the study population.
SUBJECTS
Z
SCORE
p-VALUE ALL
N=151
MALE N=103
FEMALE N=48 Age (years)±SD
Range Gender (%) Marital status:
Single (%) Married (%) Separated (%) Widowed (%)
33.66±12.43 18-73
73 (48.3) 70 (46.4) 3 (2.0) 5 (3.3)
35.82±14.00 18-73
103 (68.2%)
55(75.3) 43 (61.4) 0 (0) 5 (100)
29.04±5.99 18-40 48 (31.8%)
18 (24.7) 27 (38.6) 3 (100) 0 (0)
3.213 0.002
<0.001
Table 2: Age and sex distribution of the study population
AGE RANGE (YEARS)
SEX
TOTAL (%)
MALE FEMALE
15-29 30-44 45-59 60-74 TOTAL
41 34 21 7 103
25 23 0 0 48
66 (43.7) 57 (37.7) 21(13.9) 7 (4.6) 151 (100)
Table 3: Occupational distribution of the study population
OCCUPATION NUMBER PERCENTAGE (%)
Unemployed Students
Unskilled labourers Artisans
Traders
Armed forces personnel Professionals/business people Medical professionals
Total
23 24 11 28 27 11 25 2 151
15.2 15.9 7.3 18.5 17.9 7.3 16.6 1.3 100
Table 4: Risk factors for HBV infection.
RISK FACTORS
NUMBER POSITIVE (%)
NUMBER
NEGATIVE (%)
CHI
SQUARE p-VALUE
Past history of jaundice Past history of blood transfusion
Sexual partners None One Two Multiple Scarification marks Injections from quacks Communal use of tooth brush
32 (21.2) 8 (4.3)
37 (24.5) 35 (23.2) 22 (14.6) 57(37.7) 13 (8.6) 72 (47.7)
27 (17.9)
119 (78.8) 143 (94.7)
138 (91.4) 79 (52.3)
124 (82.1)
50.126 120.695
16.603
103.477 .325
62.311
<0.001
<0.001
0.001
<0.001 0.569
<0.001
Table 5: Results of the laboratory tests of the study population.
TEST
NORMAL ABNORMAL
p-VALUE N
MEAN
VALUE±SD N
MEAN VALUE±SD Total S Bil (µmol/L)
Conj S Bil (µmol/L) Alk Phos (U/L) ALT (U/L) AST (U/L)
Total S Protein (g/L) Serum albumin (g/L) Haemoglobin (g/L) Males (normal 14-16) Females ( normal 12-14)
115 102 126 126 123 143 136
45 25
11.91±3.31 4.82±2.52 61.20±16.37 9.86±3.06 12.81±3.79 70.55±6.03 40.70±5.02
14.85±0.82 12.69±0.71
36 49 25 25 28 8 15
58 23
23.10±4.17 ↑ 12.20±4.04 ↑ 102.94±6.55 ↑ 30.40±10.57 ↑ 34.63±13.21 ↑ 57.38±1.60 ↓ 32.13±1.64 ↓
12.47±1.04 10.98±0.72
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
<0.001
<0.001
Abbreviations are:S Bil, Serum bilirubin; Conj S Bil, conjugated serum bilirubin; Alk Phos, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Total S Protein, total serum protein. ↑ elevated; ↓ reduced; N, number of subjects. See appendix 3 for range of normal values.
Table 6: Serological markers of hepatitis B virus infection in the study population
SEROLOGICAL MARKER
NUMBER POSITIVE (%) NUMBER NEGATIVE (%) HBsAg
Anti HBc IgM Anti HBc IgG HBeAg
HBeAb
151 (100) 0 (0) 147 (97.4) 13 (8.6) 114 (75.5)
0 (0) 151 (100) 4 (2.6) 138 (91.4) 37 (24.5)
Table 7: Pattern of serological markers in the study group HBV MARKER
HBsAg AntiHBc IgM
AntiHBc IgG
HBeAg HBeAb NUMBER
+ + + + +
– – – – –
+ + + – –
+ – – – –
– + – – +
13 114 21 3 0
Table 8: Association between gender and HBeAg positivity SEX
TOTAL (%)
MALE (%) FEMALE (%)
HBeAg positive Negative Total
9 (8.7) 94 (91.3) 103 (100)
4 (8.3) 44 (91.7) 48 (100)
13 (8.6) 138 (91.4) 151 (100) Pearson chi square .007, df 1, p = 0.938
Table 9: Association between HBeAg positivity and age range
AGE RANGE (YEARS)
HBeAg
TOTAL
POSITIVE NEGATIVE
15-29 30-44 45-59 60-74 Total
5 4 4 0 13
61 53 17 7 138
66 57 21 7 151 Chi square 3.841. df 3, p=0.279
Table 10: Relationship between HBeAg positivity and liver function tests.
Test
HBeAg Positive N = 13
HBeAg Negative N = 138
p-Value Mean value
±SD
Mean value
±SD Total S Bil (μmol/L)
Conj S Bil (μmol/L) S Alk Phos (U/L) S ALT (U/L) S AST (U/L) S Prot (g/L) S Albumin (g/L)
24.72±4.89 15.52±4.73 86.31±15.01 31.54±8.79 39.46.±12.67 64.77±5.86 38.15±4.96
13.62±5.08 6.43±3.80 66.40±21.53 11.54±7.14 14.76±7.79 70.33±6.49 40.01±5.27
<0.001
<0.001 0.001
<0.001
<0.001 0.003 0.241 Abbreviations are: S Bil, serum bilirubin; conj S Bil, conjugated serum bilirubin; S
Alk phos, serum alkaline phosphatase, S ALT, serum alanine aminotransferase; S AST, serum aspartate aminotransferase; S prot, serum protein; S albumin, serum albumin. See appendix 3 for range of normal values.
Figure 1. Hepatitis B Virus genome.
Robbin’s Pathological Basis of Diseases. 6th ed. 1999. 858.
Figure 2: Geographical distribution of HBV infection
WHO. Introduction of hepatitis B vaccine into childhood immunization services. WHO/V&B, Geneva.
Figure 3. Serological markers in chronic HBV infection
Centre for disease Control and Prevention. Hepatitis B In: Epidemiology and Prevention of Vaccine Preventable Diseases. 7th ed. 2003
68%
32%
male female
Figure 4: sex distribution of the study population