Measles, a disease caused by Measles virus of genus Morbillivirus is a highly contagious disease, which is transmitted through respiratory droplets of infected person (Rasool
Recently, several disastrous epidemics have been exploded in different areas of the world including Europe and China. About 1.5 million deaths were reported in 2010 only. According to a report of WHO in 2012, about 1.2 million individuals were died of measles and majority of them were young children up to 5 years of age. According to an estimate, the case fatality ratios (CFRs) of measles are about 0.1% in developed countries and up to 30% in immigrant people (Perry and Halsey, 2004). During the time period of 1999–2005, measles mortality and morbidity rate has been reduced up to 60%; however, yet causalities due to measles are still far above the ground in various parts of the world (Cohen
Measles can lead to many severe complications including pneumonia, encephalitis and even death. After infection, contagious encephalitis may also develop about 1/1,000 registered measles patients and mortality rate is about 2–3 deaths/1,000 measles cases (Gindler
Several studies recommend that the routine vaccination program is the only way to achieve high level of immunity in the community (Shakurnia
The significance of an early and accurate diagnosis cannot be overlooked to adopt the effective control measures against a disease (Moss and Strebel, 2011). Clinicians can perform these EIAs very easily with only single sample of minute quantity and carried out after 4 weeks of manifestation of rash (Ratnam
Antibodies against Measles can also be detected using indirect haemagglutination assay (IHA). IHA is found to be simple, specific and cost effective therefore, it was chosen for the assessment of serum samples in present study. After optimization of test conditions, IHA was used for the evaluation of humoral immune response against measles and for assessment of certain risk factors associated with the disease in District Sargodha and Khushab of Punjab Pakistan.
Children of 1 to 10 years of age from different areas of district Sargodha and Khushab, Punjab Pakistan were selected as target population. They were further divided and assessed on the basis of different parameters
Indirect haemagglutination antibodies (IHA) titers of all the serum samples were measured against measles virus antigens by applying the technique explained by Sakata and Sugiura (1988). The test was performed using micro-titration plates each comprising of 96 U-shaped wells. The highest dilution of each serum sample showing a clear haemagglutination pattern was considered as end point and taken as positive whereas button formation as negative. The IHA antibodies titre was exhibited as the reciprocal of its end point dilution. The IHA titers of all the serum samples thus obtained were recorded and Geometric mean titers (GMTs) were calculated and analyzed on the basis of gender, locale, age groups and vaccination status. The data was analyzed statistically by independent t-test and a one way Analysis of Variance (ANOVA). A value of p ≤ 0.05 was considered as significant (Ruzauskas, 2005).
Indirect Haemagglutination Assay was optimized using gluteraldehyde (0.1%) and tannic acid (10 mg/dl) with RBCs obtained from different species including sheep, chicken and rabbit. Following optimization of test conditions, it was used for the sero-diagnosis and evaluation of humoral immune response against measles in Sargodha and Khushab districts of Punjab, Pakistan. 2% sheep erythrocyte suspension fixed with 0.1% gluteraldehyde and tanned with tannic acid (10 mg/dl), and adsorbed with ultra-sonicated measles antigen showed the clear and reproducible results in terms of agglutination and button formation as compared to 1% sheep RBCs.
IHA antibodies titre of 16 or more was considered as positive and < 8 as negative. Similarly, GMT value of 8 was also taken as negative. A total of 231 serum samples from district Sargodha (n = 134) and Khushab (n = 97) were collected from urban, peri-urban and rural areas and processed through IHA. The GMT of all 231 samples of district Sargodha and Khushab showed non-significant (p > 0.05) difference among males and females. The mean GMT of male was 77.51; while that of female was 66.15.
Comparison of GMTs of vaccinated and non-vaccinated children of both districts revealed a highly significant (p < 0.01) difference. The GMT of vaccinated individuals was higher than that of non-vaccinated individuals. Similarly, a highly significant (p < 0.01) difference was found in antibody titre among individuals of urban, peri-urban and rural areas. The mean antibody titre of individuals of urban area was highest when compared to individuals from peri-urban and rural areas. There was also a significant difference in the antibody titre of individuals of district Sargodha and Khushab. The mean antibody titre of individuals of district Khushab was high than that of district Sargodha. The samples obtained from both districts were divided into 3 age groups
Overall comparison of GMTs of both districts on the basis of different parameters.
Geometric Mean Titre (GMT) and P-values | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Gender | Vaccination status | Age groups (Years) | Locale | District | |||||||
Male | Female | Yes | No | 1–3 | 4–6 | 7–10 | Urban | Peri-urban | Rural | Sargodha | Khushab |
77.51 | 66.15 | 103.65 | 26.52 | 21.90 | 61.96 | 105.03 | 109.42 | 66.56 | 43.56 | 54.64 | 98.21 |
0.660NS | 0.704NS | 4.675 | 5.673 | 0.001** | 0.007** | –2.580 | –2.399 |
The GMT of 231 samples of district Sargodha was compared and there was non-significant difference in the results of GMT of both males and females of district Sargodha. The GMT of male was 56.54; whereas that of female was 52.00. GMT of individuals of district Sargodha was compared on the basis of different area like urban, peri-urban and rural. The results revealed that there was a highly significant difference in the antibody titre of individuals in urban, peri-urban and rural areas. The GMT of individuals of urban area was higher than that of peri-urban and rural areas (Table II).
Comparison of GMTs on the basis of different parameters in district Sargodha.
Geometric Mean Titre (GMT) and P-values | |||||||||
---|---|---|---|---|---|---|---|---|---|
Gender | Vaccination status | Age groups (Years) | Locale | ||||||
Male | Female | Yes | No | 1–3 | 4–6 | 7–10 | Urban | Peri-urban | Rural |
56.54 | 52.00 | 71.72 | 24.04 | 15.86 | 54.27 | 72.72 | 100.00 | 49.09 | 21.95 |
0.265NS | 0.276NS | 2.783 | 3.628 | 0.037* | 0.001** |
The GMTs of 97 samples of district Khushab was compared and results revealed that there was non-significant difference among males and females of district Khushab. The mean GMT of male was 104.77; while that of female was 87.57. GMTs of vaccinated and non-vaccinated individuals of district Khushab revealed a highly significant difference. The GMT of vaccinated individuals was higher than that of non-vaccinated individuals. In district Khushab, GMT of individuals of urban area was higher than that of peri-urban and rural areas. Similarly, GMT in individuals of age 7–10 years was higher than that of other two age groups (Table III).
Comparison of GMTs on the basis of different parameters in district Khushab.
Geometric Mean Titre (GMT) and P-values | |||||||||
---|---|---|---|---|---|---|---|---|---|
Gender | Vaccination status | Age groups (Years) | Locale | ||||||
Male | Female | Yes | No | 1–3 | 4–6 | 7–10 | Urban | Peri-urban | Rural |
104.77 | 87.57 | 155.47 | 29.23 | 34.92 | 69.17 | 159.78 | 146.00 | 96.52 | 42.60 |
0.517NS | 0.562NS | 4.239 | 4.634 | 0.009** | 0.029* |
In present study, standardization and optimization of IHA was carried out to assess humoral immune response to measles among children because measles is a highly infectious disease and one of the major reasons of mortality and morbidity among children in the whole world, mainly in developing countries. In urbanized countries, especially Europe and US, measles infection has been controlled through immunization. However, still developing countries are being affected by measles due to inadequate vaccine exposure and inappropriate management of vaccines (Merajuddin
Measles epidemics were and are still incident in various regions of the world with large ratio of reported cases and mortalities in a short duration of time. This huge gap is due to different reasons including duplicity in healthcare system, poor healthcare facilities, lower immunization coverage, undermined usual immunization, negligence among parents, and lack in quantity of vaccinators (Niazi and Sadaf, 2014). The efficiency of measles vaccine and improvement of immunity against measles among individuals of early age was not most favourable according to WHO strategies and un-vaccinated individuals were at high risk of measles infection (Zahoor
Considering the significance of this infection particularly in the circumstances of present epidemics, this study was carried out with the aim of optimization of Indirect Hemagglutination Assay for the evaluation of humoral immune response against measles in district Sargodha and Khushab. Erythrocytes of various species including sheep, chicken and rabbit were used to perform IHA (Rasool
In this study, after optimization and standardization of IHA with 2% sheep RBC’s fixed with 0.1% gluteraldehyde and adsorbed with measles antigen, IHA titres were calculated from the sera samples (n = 231) collected from children of district Sargodha and Khushab to assess the immunity against measles. The results of this study suggested that there was greater variation in the GMTs of vaccinated and non-vaccinated children of both districts
In conclusion, it was accomplished that IHA with 2% sensitized sheep erythrocytes provided the most clear, consistent and reproducible results as compared to 1% sensitized sheep RBCs. Furthermore, it was found to be an inexpensive and valuable sero-diagnostic tool and hence can be used for the evaluation of humoral immune response against measles among different populations. Furthermore low antibody titers in non-vaccinated children indicate their susceptibility to measles infection with wild type of virus. So there is an urgent need for mass scale vaccination keeping in view the international heath standards to save our future generations against this devastating disease.