Antibodies prevalence against Haemophilus influenzae type b in Jeddah population,Saudi Arabia. I. Total antibodies

2.1 Participants and samples

One thousand three (1003) venous blood samples (2–5 mL) were obtained by venepuncture with informed consent from consecutive healthy individuals attending the local medical facility in Jeddah, Saudi Arabia, for routine medical check-up, from the period of February 2014 to January 2016. Volunteers with immune disorders or who had receiving immunomodulators were excluded based on medical history data and participants’ answers. The participants include 490 males and 513 females with ages ranged from one year to 91 years old for the both. The average age for all participants is 42.4 years, includes the average 42.8 years for male participants and the average 42 years for female participants. Most of participants are Saudi nationals 964 with 486 males and 478 females. Other nationalities (39 samples) include 1 American female (Sample# 398), 2 Egyptian males (Sample# 6 and 208), 9 Egyptian females (Sample# 15, 49, 115, 186, 323, 427, 462, 498, and 1599), 1 French female (Sample# 555), 1 Indian male (Sample# 130), 2 Indian females (Sample# 472 and 608), 1 Indonesian female (Sample# 85), 1 Jordanian female (Sample# 143), 2 Malaysian females (Sample# 22 and 598), 1 Macedonian female (Sample# 1091), 1 Moroccan male (Sample# 197), 7 Moroccan females (Sample# 75, 131, 151, 210, 292, 549, and 1244), 6 Pakistani females (Sample# 8, 596, 601, 640, 1121, and 1642), 1 Palestinian female (Sample# 511), and 3 Filipino females (Sample# 86, 602, and 611). Samples have been age and gender-stratified as shown in results section.

All sera were separated from clotted whole-blood samples by centrifugation at room temperature, and kept frozen to - 20 ∘ C in 1.5 mL Safe-Lock Eppendorf tubes. All samples were diluted to 1:100 in phosphate buffer saline (PBS).

2.2 Standard curve

Human anti-Hib polyribosyl phosphate (PRP) IgG ELISA Kit, Human anti-Hib polyribosyl phosphate (PRP) IgM ELISA Kit, and Human anti-Hib polyribosyl phosphate (PRP) IgA ELISA Kit were used to plot standards curves as described in kits pamphlet.

2.3 ELISA assay to evaluate total anti-PRP IgG

ELISA assay to evaluate total anti-PRP IgG antibody concentrations were designed as described by [16, 17, 18, 19, 20, 21, 22, 23, 24]. CPS at concentration of 17 μ g/mL were dissolved in 1% ( N -(3-dimethylaminopropyl))- N - ethyl-carbodiimide hydrochloride (EDC) then dispensed (50 μ l/well) in CovaLink NH microwell plates and incubated overnight at 37 ∘ C. PBS was prepared by reconstitute of 1 tablet in 200 mL of distilled water, the resulting solution is 1X PBS. Then, 2 g (1%) of gelatin and 20 μ l (0.05%) of Tween 20 were added to PBS to make washing solution. After overnight incubation, plates contents were discarded and plates washed three times by washer machine with 50 μ l of washing solution. Blocking solution was prepared by adding 4 g (2%) of gelatin to 200 mL of washing solution. Plates blocked by addition of 100 μ l/well of blocking solution and incubated for 60 min. at 37 ∘ C. After incubation, plates contents discarded and patted dry on fresh paper towels. Each serum (50 μ l) was added in duplicate to microtiter plates wells and incubated for 60 minutes at 37 ∘ C. After incubation, plates contents were discarded and wells washed 3 times and patted dry on fresh paper towels [18]. Anti-IgG antibody were prepared based on 1:50,000 dilution in washing solution. Anti-IgG antibody was added (50 μ l/well), incubated for 30 minutes at 37 ∘ C. After incubation, plates contents were discarded and wells washed 3 times and patted dry on fresh paper towels [21].

Streptavidin-peroxidase were prepared based on 1:50,000 dilution in washing solution. Streptavidin-peroxidase was added (50 μ l/well), incubated for 30 minutes at 37 ∘ C. After incubation, plates contents were discarded and wells washed 4 times and patted dry on fresh paper towels. TMB substrate was added (50 μ l/well), incubated for 15 minutes in the dark. The reaction started and liquid in the wells turn blue. To stop the reaction, 50 μ l of 1% sulphuric acid was added to each well, tapped gently to mix, the enzyme reaction stopped and liquid in the wells turned yellow. Absorbance of the entire plates was read on reader machine at 450 nm using a single wavelength within 10 minutes after stop solution addition [20, 22].

ELISA assay to evaluate total anti-PRP IgM antibody concentrations were designed as described by [16, 23]. All steps were as above mention then followed by anti-IgM antibody based on 1:1,500 dilution.

ELISA assay to evaluate total anti-PRP IgA antibody concentrations were designed as described by [16, 17]. The steps as above mentioned then followed by anti-IgA antibody based on 1:50,000 dilution, then all steps were done the same.

2.4 Statistical analysis

T -test has been used to compare different data throughout the study, while regression test has been used to test relationship between parameters.

3.1 Evaluation of total anti-PRP IgG

A total of 1,003 sera samples from routine medical check-up of healthy individuals (490 males and 513 females) attending the local medical facility in Jeddah, Saudi Arabia in the period from February 2014 to January 2016, and stratified by age in 5-year intervals showed 2.41 μ g/ml average GMC of IgG with 0.47 average standard deviation (SD) and 0.67 average standard error of the mean (SEM) (Table 4).

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There were 11 participants have less than 0.15 μ g/ml of anti-PRP IgG (in the range from 0.08 to 0.13 μ g/ml); 5 males (IgG in the range from 0.08 to 0.13 μ g/ml) and 6 females (IgG in the range from 0.1 to 0.13 μ g/ml), representing about 1.1% of 1,003 participants enrolled in this study (1.02% of male participants and about 1.17% of female participants) (Table 4 and Fig. 1). The difference between average results of 0.15 μ g/ml IgG in both genders (about 0.11 ± 0.03 μ g/ml for males and around 0.12 ± 0.04 μ g/ml for females) was statistically non-significant. Male ages ranged from 57 to 91 years and female ages ranged from 33 to 64 years, so there was no participant younger than 33 years old with 0.15 μ g/ml IgG concentration in both genders. All participants who have low IgG except two females are over 50 years old. All low IgG participants are Saudi citizen except one Malaysian female aged 33 years. If the Malaysian participant excluded, the ages range of Saudi females will be 35–64 years (Table 4 and Fig. 1).

Results showed that 205 participants (representing about 20.44% of 1,003 participants enrolled in this study have concentration of IgG ⩾ 0.15 and 1 μ g/ml (range 0.16–0.99 μ g/ml). These participants include 107 males; representing 21.84% of male participants (range 0.16–0.96 μ g/ml) and 98 females; representing 19.1% of female participants (range 0.17–0.99 μ g/ml). Ages of male ranged from 1 to 86 years and females from 1 to 88 years. The average concentrations of 1 μ g/ml $>$ anti-PRP IgG ⩾ 0.15 μ g/ml are statistically the same in both male (0.56 ± 0.23) and female (0.58 ± 0.23) (Table 4 and Fig. 1).

Moreover, a total of 787 participants have IgG concentration ⩾ 1 μ g/ml, which represent about 78.46% of 1003 participants, 379 males (range 1.01–10.05 μ g/ml) and 408 females (range 1.01–10.02 μ g/ml). Ages ranged from 1–88 years in male, and from 2–91 years in female. IgG concentration averages are the same in the both genders (3.03 ± 0.54 for males and 2.95 ± 0.54 for females) (Table 4 and Fig. 1).

Average of IgG in males and females for all ages were ranged from 0.08–10.05, 0.1–10.02 μ g/ml, respectively. Statistically, the concentration averages in both genders for all ages were the same (2.4635 ± 0.47 for males and 2.4634 ± 0.48 for females) (Table 4 and Fig. 1). Generally, the GMC of IgG shows that 205 participants (20.44%) maintain seroprotective levels of ⩾ 0.15 μ g/mL, and 787 (78.5%) show seroprotective level of ⩾ 1 μ g/mL, the level of ⩾ 0.15 μ g/mL described as short protective and ⩾ 1 μ g/mL described as long protective, although the both are considered protective [8]. Only 11 participants (1.1%) have IgG level 0.15 μ g/mL, this percentage is less than what was found in a study conducted in UK in about one year following Hib vaccine introduction, which found that 5% of adults had antibody levels 0.15 μ g/mL [27]. The result of total IgG in 1 year old infants in recent study, which 100% of them developed $>$ 0.15 μ g/mL IgG, is the same to what has been found in vaccinees infants in Brazil study [26]; this phenomenon of high IgG is well known in infants who are recently received the full series of combined Hib vaccines. In contrast, the study that was conducted in Egypt before the introduction of Hib vaccine, shows that 34.46% of 1–15 years old participants have results of 0.15 μ g/mL of IgG [17]. However, recent study has no participants with 0.15 μ g/mL of IgG for the same age category, the lowest result for this age category is 0.16 μ g/mL, this results reflect the efficiency of vaccination program in Saudi community, at least in young and adult ages. Comparing to Cuban 4–5 years old children, recent study reflects less percentage of children that have IgG $>$ 1 μ g/mL (87.1%) against 99.7% in Cuba [27]. This variance may be resulted from any factors that may affect the velocity of immunity response to vaccines (e.g. cross-immunogenicity, subclinical diseases, genetic makeup, etc.) and/or immunogenetic background variants. In Paradiso et al. study, the mean IgG concentration of age category 1–2 years is 3.42 μ g/mL, it is seem comparable to what recorded in the US (4.4 μ g/mL) [11], but it is less than what had been found in Saudi Arabia (14.4 μ g/mL) in the year of officially introduction of Hib vaccines [10]. This phenomenon of relatively low antibody level has been noticed in countries using all types of Hib vaccine on a wide scale and in a long term [12]. According to Hazlewood et al. [25], adult-type IgG response is only achieved by age 8–10 years, in recent study the concentration of total IgG reached 1.7 μ g/mL in 1 year old ages and continue to elevate up to adulthood ages and beyond (one participant in age category 86–91 years old reached 4.6 μ g/mL), these data may totally reflect the strong herd immunity in the Saudi community against Hib.

In 5 years’ age interval categories, the IgG mean of age category 86–91 years ( n = 7) was the lowest mean (1.54 μ g/mL), and if we exclude the single relative extreme high value (4.61 μ g/mL) the mean reduces to 1.03 μ g/mL ( n = 6), while the highest mean was 2.94 μ g/mL in age category 51–55 years ( 0.05). Again, in 5 years’ age interval categories, the mean of IgM in 81–85 year age category was the lowest (0.594 μ g/mL) compared to other age categories, while the highest mean of IgM was 1.241 μ g/mL in age category 6–10 year ( 0.001). The relatively low levels of IgG and IgM in elderly participants may explain the relatively high rate of Hib infections in elderly, as described by [28] that more likely cases of pneumonia in older adults, and the unusual case of chronic prostatitis caused by H. influenzae in an elderly Saudi patient in 2013 [29], and a rare infection of the skin and soft tissues (necrotizing fasciitis) caused by Hib [30], and several fluoroquinolone-resistant clones appeared to have invaded the population of elderly patients in a particular area, Sapporo city in Japan [31].

No statistically difference was detected between male and female for GMC of IgG and IgA, despite of an Indian study that showed considerable diversity in nasal carriage of H. influenzae among female and male children [32]. A higher concentration of IgG ( ⩾ 5 μ g/mL) is needed to control the colonization (e.g. nasal carriage), and many factors other than serum antibodies concentration may affect the colonization, such nasal microbiome, locally produced mucosal antibodies, antibody subclass predominate, and community hygiene [33]. Antibody titres against H. influenzae were higher in smoker men than in smoker women [34]. All IgG mean in age categories 1–30, 31–60, and 61–91 years old also statistically the same between males and females, despite the prevalence of males that suffered from Hib meningitis over females in the study that conducted in Saudi Arabia in 1999-2001 [6]. Also, in age category 65–91 years in recent study, the IgG level is statistically the same, which is contrary to what described by [34], that is antibody titres against H. influenzae were higher in men than in women in elderly smoking population, and the levels were higher in smokers compared to non-smokers.

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The results of all participants of 5 years interval show 0.91 μ g/mL GMC of IgM (Table 5). There were 13 participants have less than 0.15 μ g/mL of anti-PRP IgM, 8 males (range 0.049–0.139 μ g/mL) and 5 females (0.091–0.146 μ g/mL), representing 1.3% of total 1003 participants that enrolled in the study (1.63% of male participants, 0.97% of female participants). The average results in both gender was statistically the same. Male ages ranged from 1 to 64 year, while female ages ranged from 37 to 67 year, so there is no younger than 37 years old with 0.15 μ g/mL IgM concentrations in female. All low IgM participants except two males and one female are over 50 years old, and all of them are Saudi (Table 5 and Fig. 2). However, the results show that about 686 participants have concentration of IgM ⩾ 0.15 and 1 μ g/mL. they were include 366 males, represent 74.69% of 490 males (range 0.15–0.95 μ g/mL) and 319 females, represent 62.18% of 513 females (range 0.15–0.99 μ g/mL), and both genders represent 68.2% from all 1003 participants. The female average concentration of IgM is statistically higher than the male average concentration ( 0.05) (Table 5 and Fig. 2). Also, the results show that the total of 305 participants have IgM concentration ⩾ 1 μ g/mL, represent 30.4% of 1003 participants (117 males) represent 23.88% of males (range 1.01–7.77 μ g/mL) and 188 females, represent 36.65% of females (range 1.01–4.93 μ g/mL). Ages ranged from 3–88 year in male, and from 2–84 year in female. No statistical differences between males and females of IgM concentration averages of ⩾ 1 μ g/mL (Table 5 and Fig. 2). The average of IgM concentration in both genders within all ages were statistically ( 0.001) higher in female (1.03 μ g/mL) than male (0.81 μ g/mL) with average of 0.93 μ g/mL in the both (Table 5 and Fig. 2).

The average of IgM concentration in male of all categorized ages show the highest in age category 6–10 year, followed by age category 16–20 year, while the lowest was seen in the age category 81–85 year, followed by age category 51–55 year. There is no significant differences between averages of IgM concentration in age groups 1–5 and 6–10 year. While, age group 6–10 year average is higher than the average of age groups 26–30, 46–50, 51–55, 56–60, and 81–85 year ( 0.05) as seen in (Table 5 and Fig. 2). However, the average of IgM concentration in female of all categorized ages show the highest in age category 1–5 year, followed by age category 11–15 year. While the lowest was seen in age category 86–91 year, followed by age category 66–70 year. There is no significant differences between averages of IgM concentration in age groups 1–5 and 6–10 year, while 1–5 year is higher than 21–25, 36–40, and all higher age categorise ( 0.05), (Table 5 and Fig. 2).

The results of total IgM show that 990 (98.7%) have seroprotective level of ⩾ 0.15 μ g/mL, which is higher than the percentage (64.2%) that described by Matos et al. [26]. Regarding the age category 1–15 year, 210 participants showed IgM level of ⩾ 0.15 μ g/mL out of 211 participants, this is also higher than the percentage described by Redwan and Elsawy for the same age category [17]. The rising of IgM level in current study reflects the efficiency of Hib vaccination program in Saudi Arabia and the related high herd immunity in the community. Three hundred five participants (30.4%) have long immunity level of ⩾ 1 μ g/m, and only 13 participants (1.3%) have IgM level 0.15 μ g/mL. The recent study is comparable with the results of Brazilian study which indicate that 18.88% of vaccinated infants developed IgM concentration of ⩾ 1 μ g/mL [26]. These data prove the prevalence of IgG antibodies over IgM in response to Hib, this prevalence of IgG may be explained by the ability of IgG to opsonized Hib for polymorphonuclear leukocytes, and the lack of this ability in IgM [35]. IgM average is 0.92 μ g/mL, the male average is 0.805 μ g/mL and the female average is 1.031 μ g/mL.

The GMC of total IgM was 0.91 μ g/mL. Female GMC was 1.02 μ g/mL and it is higher than male GMC (0.8 μ g/mL) ( 0.001), which may have resulted from the different nature of immune system in female and increased number of childbearing women with autoimmune diseases [36]. According to age categories 1–30, 31–60 year IgM in female participants are higher than male participant ( 0.001 and 0.01 respectively), while the age category 61–91 year were same for both genders. We could not find any publications that studied the differences in anti-Hib IgM levels between male and female, so our study may consider the first measured these differences.

The results of all participants of age 5 years interval show 0.34 μ g/mL GMC of IgA (Table 6). There were 364 participants have less than 0.15 μ g/mL of anti-PRP IgA, which representing 36.3% of 1003 participants. Out of these, 183 males, represent 37.35% (range 0.003–0.14 μ g/mL), and 181 females, represent 35.28% (range 0.003–0.14 μ g/mL). Male ages ranged from 1 to 91 year, while female ages ranged from 1 to 85 year. The average results of male and female were statistically the same (Table 6 and Fig. 3).

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Also, the results show that 567 participants have concentration of IgA ⩾ 0.15 and 1 μ g/mL, represent 56.5% of 1003 participants. These participants include 283 males, represent 57.76%, and 284 females, represent 55.36%. The average concentrations of IgA in both gender were not significant (Table 6 and Fig. 3). While, 72 participants have IgA concentration ⩾ 1 μ g/mL (range 1–3.86 μ g/mL), which represent 7.2% of 1003 participants, within those 25 were males represent 5.10% (range 1–3.64 μ g/mL), and 47 females represent 9.16% (1–3.86 μ g/mL). Regarding IgA concentration ⩾ 1 μ g/mL, the average concentration is the same in both genders (Table 6 and Fig. 3).

The overall average concentration of IgA was higher in female (0.38 μ g/mL) comparing to male (0.32 μ g/mL), with average of 0.35 μ g/mL in the both ( 0.05). Male IgA concentration was ranged 0.003–3.65 μ g/mL and female ranged from 0.003–3.86 μ g/m (Table 6 and Fig. 3).

The average concentration of IgA in male of all categorized ages show the highest in age category 86–91 year, followed by age category 46–50 year. While the lowest seen in age category 1–5 year, followed by age category 36–40 year. The age group 1–5 year is statistically less than age groups 6–10, 21–25, 46–50, 86–91 year ( 0.05), (Table 6 and Fig. 3). While, the average concentration of IgA in female of all categorized ages show the highest in age category 21–25 year, followed by age category 51–55 year. The lowest concentration was seen in age category 76–80 year, followed by age category 1–5 year. Similar to male, the age group 1–5 year was significantly less than age groups 11–15, 21–25, 36–40, 51–55 year ( 0.05). Also, the average concertation of age group 76–80 year was significantly less than age groups 21–25, 36–40, and 51–55 year ( 0.05) (Table 6 and Fig. 3).

In ages from 1 to 16 year (the expected ages that received Hib vaccines in Saudi Arabia), the average of IgA is 0.3 μ g/mL, which consider seroprotective and it seem agree with the data that Hib vaccination provokes a combination of IgM, IgG and IgA antibodies [37]. To the best of our knowledge, there were no previous studies that focusing on the levels of total prevalence of IgA in different age categories, so our study considers the first in this point.

Using regression analysis, very weak negative relationship between age and IgG concentration distribution ( R 2 = 0.0003), a weak negative relationship was detected between IgM concentration distribution and age ( R 2 = 0.0684), and a very weak positive correlation between age and IgA concentration R 2 = 0.0014 (Fig. 4).