الفهرس 
Literature ReviewOnly 14 pages are availabe for public view
 |  | from | 120 |  |  |
| | | from | 120 | | |
Abstract
The present study was concerned with the evaluation and correlation between three important methods National Institute of Health test (NIH), Rapid Focus Fluorescent Inhibition Test (RFFIT), and Passive Hemagglutination test (PHA)-used in potency determination for rabies vaccine preparations. The aim was to know the availability of replacement of the main NIH test for potency evaluation due to its great drawbacks like consumption of large number of animals, non-humane endpoints of the test, and also high variability of the test due to its in vivo nature. The study was also concerned with validation of both in vitro methods – Rapid Focus Fluorescent Inhibition test (RFFIT), and Passive Hemagglutination test (PHA) against the NIH test. In this study, 25 rabies vaccine preparations of a single company were used for potency determination using the three methods mentioned before. The mean of potencies for the three methods were 3.73, 3.51, and 4.50, respectively. All results were within the acceptable limit which not less than 2.5 IU/ml.
Considering validation of the used methods; National Institute of Health (NIH) test was validated using two references and highly qualified rabies vaccine preparation. NIH test is a pharmacopeial described test, so its verification was only needed using reliable references (National Institute for Biological Standards and Controls (NIBSC) reference and National Reference of China), in addition to a highly qualified vaccine (Verorab). The later one was subsequently used as working reference in testing rabies vaccines potency after the verification process. On the other hand, Rapid Focus Fluorescent Inhibition test (RFFIT), and Passive Hemagglutination test (PHA) are WHO described tests, so verification was also applied. In case of Rapid Focus Fluorescent Inhibition test (RFFIT), verification was done statistically using four validation parameters and the results were compared to FDA and ICH validity criteria, while verification of Passive Hemagglutination test (PHA) was done according to WHO guidelines. Results of verification of National Institute of Health (NIH) test were acceptable according to the european pharmacopeia; regarding the two used references as well as the highly qualified rabies vaccine (verorab). As a consequence, verorab vaccine preparation was used as working reference preparation. The potency of verorab “the working reference preparation” was 4.5, while NIBSC and national institute of china were 4, and 3.3, respectively. Rapid Focus Fluorescent Inhibition test (RFFIT) was verified statistically as mentioned before. The results of the four validation parameters were as follows; specificity was 88%, sensitivity was 80%, coefficient of variance was less than 30%, repeatability values were 1%, 2%, and 7%. These results are acceptable according to FDA, ICH guidelines.
Potency determination of the tested vaccine samples using the three methods gave acceptable results. In the main test “National Institute of Health (NIH) test”; the potency of the tested vaccine samples was calculated using Spearman Kärber equation after observation of mice survival. While in case of Rapid Focus Fluorescent Inhibition test (RFFIT), the potency of tested vaccine samples was calculated using Reed Muench equation after microscopic evaluation of neutralized virus in 20 microscopic fields. For potency determination of vaccine samples using Passive hemagglutination (PHA) test, the potency was calculated by determining haemagglutination titre. Results showed strong correlation of potency determination between National Institute of Health (NIH) and either of Rapid Focus Fluorescent Inhibition test (RFFIT) or Passive hemagglutination (PHA). The correlation coefficients produced were equal to 0.93 (National institute of health results versus Rapid focus fluorescent inhibition test results) and 0.98 (National institute of health results versus passive hemagglutination results). The benefits of serological tests were more than those the main test, for example, Rapid Focus Fluorescent Inhibition test (RFFIT) was more accurate, fast, and sensitive than National Institute of Health (NIH). Also Passive hemagglutination test (PHA) was easier, faster, and cheaper than both National Institute of Health (NIH) and Rapid Focus Fluorescent Inhibition test (RFFIT) since although it consumed the same number of animals of RFFIT, but the reagents and materials used in this test were cheaper than those used in RFFIT.
In this study, we experienced decreasing the number of dilutions used for calculation of ED50% in National Institute of Health test (NIH). The results revealed that validity criteria were still met in regimen 2 (using the last three dilutions), regimen 3 (using the first and last two dilutions) and regimen 4 (using the first two and last dilution) with 100% of all samples and were not met in regimen 1 (using the first three dilutions) with 100% for all samples. Anova analysis was done for the study to ensure its validity using NIH as the reference test using two-way ANOVA test, f = 4.1, p = 0.02, which means high significance.
Finally, from this study we can conclude that: (i) both RFFIT and PHA tests could be used for the potency determination of Rabies vaccine products of human use. However, caution had be taken for both tests during assays and validation should be pre-conducted, (ii) for the most tested rabies vaccine preparations, the results of both RFFIT and NIH tests were comparable while PHA still needs further studies in types of antibody production , (iii) both RFFIT and NIH tests have to be carried out to complement and reinforce each other as they provide a comprehensive picture of the product potency, (iv) RFFIT test was more accurate, fast, and sensitive than NIH while PHA was not as accurate as RFFIT. However, it gave reliable results and was easier, faster, and cheaper than RFFIT.