DETECTION OF POLIO VIRUS ANTIBODIES LEVEL AMONG CHILDREN IN KANO STATE, NIGERIA

* Aminu AI 1 , Aliyu FA 1 , Mukhtar MD 1 , Gajida AU 2 and Aminu M 3 . 1. Department of Microbiology, Bayero University, Kano, Nigeria. 2. Aminu Kano Teaching Hospital, Kano, Nigeria. 3. Department of Microbiology, Faculty of Science, Ahmadu Bello University, Zaria, Nigeria. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The study was conducted to detect poliovirus antibody and its level in children aged < 5 years in three local government areas (LGAs) of Kano state, Nigeria. The study was a hospital-based study and purposive sampling was used in selecting one health institution each from the three LGAs. Random sampling was employed in selecting 423 children whose blood samples were collected and analyzed for the presence of poliovirus antibody and its level using polyclonal Elisa detection kits. The study revealed that 94.1% of the children were seropositive for poliovirus with mean titer value of 1.6938, and age was significantly associated with seroprevalence rates with 33.4% and 37.4% of the seropositive children in the age groups 0-12 and 13-24 months respectively (p<0.05). Also, no significant difference existed in the seropositivity rates of the children regarding their sex as well as their mother"s educational level (p>0.05), although the father"s educational level significantly affected it (p<0.05). The Poliovirus antibody mean titer level significantly increases in routine and campaign oral poliovirus (OPV) immunization as the number of the vaccine doses increases (p<0.05). The study detected a high seroprevalence rates to poliovirus among children in Kano state and recommends that fathers should be considered as a target group in the course of enlightenment campaign as well as the need for more efforts to improve literacy level of women and to target the unimmunized children whose lack of immunity poses a risk of re-emergence of the poliovirus infection in Nigeria.

…………………………………………………………………………………………………….... Introduction:-
Poliomyelitis is a disease caused by poliovirus which according to Mark (2006) and Tapani (2006) have been known to cause paralysis especially in unimmunized or sub-optimally immunized children. The World Health Organization (WHO) recorded that there were an estimated 350,000 case of polio worldwide in 1988 and this number reduced to 291 in 2012, representing a 99% reduction, although in 2013 and 2014 there was a bounce back in some countries towards more cases (WHO, 2014). In 2015 only 2 countries remained where the disease is endemic-Afghanistan and Pakistan, whereas Nigeria was declared wild polio free in 2015 and was reported as the first time that Nigeria has interrupted transmission of wild poliovirus, bringing the country and the African region closer than ever to being certified polio free (WHO, 2015).

1989
Poliomyelitis is a highly contagious disease caused by three serotypes (types 1, 2 & 3) of poliovirus which are spread by faecal-to-oral transmission (Shibuya and Murray, 2004;WHO, 2014). Children are thought to play a dominant role in the transmission of Polioviruses within populations (Fine and Ritchie, 2006), and age of children, number of doses the children had taken and educational level of the children"s fathers have been identified as significant risk factors on the prevalence of poliovirus antibodies (Mawashi et al., 2015).
In 1988 the World Health Assembly launched a campaign to eradicate polio by 2000 and CDC (2014) revealed that the Polio eradication programs most important step is the interruption of endemic transmission of polio virus which according to Maigari et al. (2015) was mainly based on successful administration of polio vaccine through the massive immunization programs targeted towards preventing poliomyelitis.
Earlier studies revealed that reduction in poliomyelitis cases were due to extensive use of two vaccines, the live attenuated oral Poliovirus vaccine (OPV) or the Sabin vaccine, and the inactivated Poliovirus vaccine (IPV), or the Salk vaccine (Friedrich, 2000). Thus, the Global Polio Eradication Initiative aimed at immunization of children with multiple doses of OPV, via both routine immunization (RI) and supplementary immunization activities (SIAs) (CDC, 2010).
Pasca et al. (1994) earlier revealed that it is customary in any country where a comprehensive program to vaccinate children against Polioviruses was undertaken to investigate the immune status of the eligible children or even the entire population to Polioviruses from time to time. The WHO (2015) further expounded that Polio sero-prevalence surveys (SPS) revealed a positive correlation between number of OPV doses received and immunity levels. Specifically children receiving >7 OPV doses have higher antibody titres of 90% and above, and this also addresses the unfounded concerns on "too many OPV doses" often expressed by sceptical health professionals and outright non-compliant parents.

Materials and Methods:-Study Design:-
The study was a cross-sectional health facility based study conducted at three local government areas (LGAs) which were selected from three senetorial zones of Kano state. Purposive sampling method was used in selecting the health facility while random sampling was used in selecting the children that participated in the study. Thus, at each LGA the most popular Community hospitals which provide free medical services, have a large turnover in pediatric OPD and which is patronized by patients from all the area were selected. The hospitals selected were; Kura General Hospital, Dambatta General Hospital and Wudil General Hospital.

Study Area:-
The study was conducted in Kano State Nigeria located at latitude 9 0 55"N and 8 0 53"E at a latitude of 1300 meters, 4100ft above sea level. The climate condition is characterized by breeze harmattan between October and March and a rainy period between April and September with anannual rainfall of about 1300mm. According to Bureau of statistics (2008) the state has an estimated land mass of about 130,913 square kilometers, and a population of 9,383,682 people with population density of 403 per square kilometers, based on the 2006 national population census. The main occupation is farming with most of the urban populations being civil servants and traders. The adult literacy rate was estimated at 29.1% and 16.5% for male and female respectively (Bureau of statistics, 2008). Dambatta LGA is located at the latitude 12.

1990
Ethical Clearance and Consent:-Ethical approval and permission to conduct the study was received from the Ethics Committee of Kano State Ministry of Health, Hospital management Board, and the selected LGAs hospitals of the study (appendices I). Provision of Helsinki declaration concerning research on human subjects were also strictly adhered to and the parent of the children consented prior to sample collection

Study Population:-
The subjects for the study were children < 5 years of age, who were residents of the selected LGAs, and attending the health facilities from June to October 2015.
Inclusion and Exclusion Criteria:-Any child in the specified age group of < 5 years with a history of polio immunization brought to the General Outpatient Department (OPD) of the selected hospital and residing in the LGA for at least six months preceding the study and whose Parent/Guardian gave consent for participation was included in the study. All infants or children outside the specified age group or who reside outside the specified LGAs, and those whose Parent/Guardian do not provide consent for their participation in the study were excluded. In addition, children with contraindication for venipuncture, very sick requiring hospitalization and those diagnosed or suspected of having congenital immunodeficiency disorder were also excluded.

Sample Size Estimation:-
The sample for the study was calculated based on 51% sero-prevalence rates of polio type 3 among children aged 6-9 months in Kano reported by Iliyasu et al.

Sample Collection and Processing:-
A total of 423 blood samples were collected from the children upon the consent of their parents from June to October 2015. Standard antiseptic procedures were strictly adhered to before and after the study. Exactly 2mls of blood was aseptically collected by venipuncture in a plain vacutainer tube as described by Jawetz et al. (2010). Immediately after collection, the blood samples were allowed to clot at room temperature for 30 minutes and kept in the refrigerator at 4-8°C for up to 6 hours at the LGA hospital. Thereafter they were transported to Aminu Kano Teaching Hospital (AKTH) Laboratory in cold cryoboxes in which ice-packs were earlier placed so as to preserve the samples as described by WHO (2000). The tubes containing the samples were centrifuged at 3000 revolutions per minute for five minutes. Exactly 2ml of serum from each sample was pipetted into a cryovial, labeled with respective ID number of the study subject. Cryovials were put in cryoboxes and stored at -60 0 C at AKTH until all samples are collected for processing (Iliyasu et al., 2014).
Prior to sample collection, an interviewer administered questionnaire adapted from Iliyasu et al. (2014) was used in generating data for the study. Some of the aspects reflected in the questionnaire include physical examination of the children including weight and length, their mothers and fathers educational level and immunization history.

Detection of Antibodies to Polio Virus:-
The poliovirus antibody and its level was detected from the processed blood samples using polyclonal Enzyme linked immunosorbent assay (ELISA) detection test kits (Demeditec Diagnostics GmbH, Germany) which detects antibodies against the three types of polio simultaneously, due to a past illness or to immunity by vaccination (http://www.demeditec.com). The Polio IgG antibody test kit is based on the principle of the enzyme immunoassay (EIA) in which Polio antigen bounded on the surface of the microtiter strips reacts and bind with IgG antibodies present in diluted patient serum or ready-to-use standards upon addition into the wells. A ready-to-use anti-human-IgG peroxidase conjugate is added followed by the addition of substrate (TMB) solution which induces the development of a blue dye in the wells. The color development is terminated by the addition of a stop solution, which changes the color from blue to yellow and the resulting absorption of the dye is measured at the wavelength 1991 of 450 nm and the concentration of the IgG antibodies is directly proportional to the intensity of the color (http://www.demeditec.com).
The assay was carried out according to manufacturer"s instructions (http://www.demeditec.com). Ninety six (96) wells that contained an immobilized polio antigen were labeled 1-12 vertically and A-G horizontally. The wells A1-A5 were labeled as substrate blank, negative control, cut-off standard, weak positive control, and positive control respectively. Both the samples the standards and controls were diluted 1:101. Then for each diluted sample 100μl of it was added to the respective labeled well, similarly the ready-to-use standards and controls were pipetted respectively into the wells. One well was left empty for the substrate blank. The plate was covered with the re-usable plate cover and incubated at room temperature for 60 minutes and the wells were then emptied by dumping. Then 300μl of diluted washing solution was added and procedure was repeated totally three times, and rests of the washing buffer were afterwards removed by gentle tapping of the microtiter plate on a tissue cloth. Then 100μl each of ready-to-use conjugate was pipette into all of the wells excluding the substrate blank well and then the plate was covered with the re-usable plate cover and incubated at room temperature for 30 minutes. The wells of the plates were then emptied by dumping, and 300μl of diluted washing solution was added. This procedure was repeated three times and rests of the washing buffer were afterwards removed by gentle tapping of the microtiter plate on a tissue cloth. Finally 100μl each of the ready-to-use substrate was pipetted into the wells including the substrate blank well. The plate was covered with the re-usable plate cover and incubated at room temperature for 20 minutes in the dark (drawer). Then 100μl each of the ready-to-use stop solution was pipetted into all the wells to terminate the substrate reaction. This was thoroughly mixed and the bottom of the plate was wiped. The reading was performed and measured spectrophotometrically at the wavelength of 450 nm. The concentration of the IgG antibodies is directly proportional to the intensity of the color and the results were read and interpreted according to the manufacturer"s instructions by comparing the optical density (OD) of the cut-off standard values to that of the sample OD values as indicated below: 1. Cut-off standard (OD) value = 0.391. 2. Sample OD value greater than (>) cut-off standard OD value (0.391) = Positive result. 3. Sample OD value less than (<) cut-off standard OD value (0.391) = Negative result.
Data Analysis:-Data generated from the study was analysed using SPSS version 17 Software. Results generated were presented as percentages, while chi-squire and student T-test were used in determining any significant association with regards to demographic and other associated risk factors among the studied children. A p-value of 0.05 or less was considered significant at 95% confidence interval.

Results:-
The results of the study revealed that, of the 423 studied children, 398 (94.1%) had antibodies to polio virus with a mean titer value of 1.6938. The mean weight of the studied children was 10.93kg and their mean height was 11.143cm. Table 1 revealed that of the 398 children seropositive to poliovirus, 205 (51.5%) were males and 193 (48.5%) were females (P=0.734). Results shown on Table 2 further revealed that children in the age groups 0-12 and 13-24 months had higher significant seroprevalence rates of 33.8% (133/398) and 37.4% (149/398) respectively compared with children in the age groups 37-48 and 49 -60 years who both had prevalence rates of 1(4.0%) (P=0.044). The results also revealed that polio virus antibody was uniformly distributed among the children in the three LGAs (p>0.05), with 34.2%, 33.4% and 32.5% in Dambatta, Kura and Wudil respectively (Table 3).

Results on
There was no significant difference in the prevalence of polio virus antibody among the studied children regarding their mothers" educational status, although 317 (79.6%) of the seropositive children had mothers whose educational level was identified as primary, and 81(20.4%) having mothers with educational status identified as secondary or tertiary level (P=0.310) ( Table 4). However, there was a significant relationships between the prevalence of polio virus antibody among the studied children with regards to their fathers" educational status (P=0.001) with 160 (40.2%) of them having fathers whose educational was identified as secondary or tertiary level ( Table 5).
The results further revealed that children that received at least four doses of routine OPV had a significantly higher mean antibody titer value of 2.0913 compared to those with 1-3 doses who had mean titer values of at least 0.889 (P 1992 = 0.001) (Table 6). Similarly, with regards to the number of campaign OPV, children that received > 7 doses of the OPV had significantly higher titer values of 1.8929 compared to those with 1-3 doses who had at least a titer value of 1.4929 (P = 0.001) ( Table 7).

Discussion:-
This study revealed that 94.1% of the blood samples collected from children <5 years of age from the three senatorial zones of Kano state were sero-positive to poliovirus antibody (IgG). These finding were similar to earlier studies which reported that greater than 90% of school age children, adolescent and young adults had detectable antibodies to Poliovirus (Kelley et al., 1991;Orenstein et al., 1988) The study further revealed that 5.9% of the studied children were seronegative to poliovirus serotypes indicating that these children constituted the unimmunized and unprotected children who could serve as a source of re-emergence of poliovirus infection which could obstruct the success of polio immunization programme. The study further revealed that although the seronegative children have been documented with a history of poliovirus immunization, their seronegative status indicated that they did not either respond properly to the vaccine administered to them or the vaccine might have lost its immunogenicity at the time of administration or it may even be that their immunization history was incorrectly documented. Earlier, a study in Delta state revealed only 4% (8) of the children had no detectable antibody (Danboraye et al., 2011). In another study, 7.9% seronegativity to poliovirus antibodies was reported by Adeniji et al. (2015) in a study conducted in North-central and South-western Nigeria among 229 Children between 10 months and 13years. Also, in a study conducted in Zaria, North Western Nigeria between 2008 and 2009, 1.9% of 264 studied children aged 1-10 years had no antibodies to all the three poliovirus serotypes (Giwa et al., 2012).
The findings of this study revealed that prevalence of poliovirus antibodies was significantly associated with younger children compared to the older ones. This indicates that parents and care givers are more likely to expose their children especially those in the earlier months of life to receive both routine and campaign OPV, hence the need to put more effort to reach children in this category as they are the most vulnerable to receive the immunization. In a similar study Mawashi et al. (2015) earlier explained that the high seroprevalence of poliovirus antibodies among children in lower age groups such as 12-23 months might be as a result of antibody boosting through routine and campaign OPV and probable exposure to wild poliovirus (WPV) through unsanitary habits. of antibody boosting resulting from contagious to the virus in an endemic area as such seropositivity will not decrease with increasing age of the study participants. In similar studies in 2013, Iliyasu et al. (2014) also reported that the seroprevalence was unexpectedly low among infants aged 6-9 months, and remained high among children aged 36-47 months. They further explained that the marked drop in seroprevalence for all 3 serotypes in the group aged 6-9 months between 2011 and 2013 brought into focus the quality of IPD operations and the need to devise new strategies to reach the younger children, especially newborn babies and infants, most of whom would be with their mothers and therefore be in the category of "child absent" during IPDs. Thus, the evidence provided by the study yielded an increase in resources for demand-creation activities targeting mothers and caregivers of children in this age group. As a result an improvement was recorded in 2014, compared with 2013 (Iliyasu et al., 2014). In this regard, the baseline seroprevalence among infants aged 6-9 months in 2014 was reported to be better than that in 2013 by Craig et al. (2016).
The findings of this study confirms earlier observations by other studies that gender was insignificantly associated with seroprevalence of poliovirus antibodies among children (P=0.734). Although studies have shown that more females were immune than males, yet no significant association exist in relation to polio specific IgG. In contrast Iliyasu et al. reported that male gender tended to have higher seroprevalence for the three serotypes, although not statistically significant (Iliyasu et al., 2014). Baba et al. (2012) uphold the same view that no correlation existed between gender and antibody to the poliovirus serotypes.
In this study the difference in seropositivity among the three LGAs was not statistically significant (P=0.086). This study is in line with the study by Yusuf et al. (2015) who reported that there was no association among seroprevalence of poliovirus antibody and location of child's place of residence. Although, a study by Mawashi et al. (2015) stated that Children in the urban areas had more poliovirus antibody prevalence than those from their rural counterparts.
The level of mother"s education does not significantly affect sero-prevalence to polio virus among studied children in this study (P=0.310), with only 81 (20.4%) of the children having mothers with secondary level of education and above. However, the study revealed that the father"s educational level significantly affects seroprevalence rates. Children whose fathers received education up to secondary level and above had the highest poliovirus antibody prevalence of 40.2% ( P=0.001). Yusuf et al. (2015) reported that children whose fathers were educated up to tertiary level were all seropositive in their study. This study had reaffirmed what was already known, that Father"s educational level positively affects child"s antibody prevalence. Thus, results from the study emphasize Father"s educational level as an important factor in the course of a successful immunization programmes. This observation may not be unconnected to the fact that in the area where this study was carried out men predominate in the affairs of the family. Earlier, Vincelik et al. (2007) stated that educational and cultural differences in communities were found to play a significant role in transforming attitudes and behaviors towards vaccination. Mohammed et al.
(2014) further reported that noncompliant (to immunization) heads of households had low level of education compared to compliant heads of households in Sokoto State, Nigeria. Similar report revealed that low level of parents' education was found to be associated with non-vaccination during mass campaign in India (Singh et al., 1997). However, according to Omer et al. (2009) only in few instances in developed countries, had it been seen that advanced educational status was a major factor in vaccine refusals.
The number of routine OPV doses received by a child was shown in this study to be a very significant factor to the development of polio virus antibody as revealed by the antibody titre mean value. Children who did not receive even a single dose of OPV had a significantly lower mean antibody titer value of 0.8890 compared to those who had received four (4) doses of routine OPV recording a mean titre value of 2.0913 (P=0.001). Likewise, the mean titer value increases significantly with increase in the number of campaign OPV doses (P=0.001). This study therefore revealed that the mean antibody titer increases with increase in the number of both routine and campaign OPV doses received by the study participants with the highest mean titer value reported in children that received complete doses of OPV. Similar reports by Ilyasu et al.