Abstract
Background
Over 20% of the world population suffers from immunoglobulin E (IgE)-mediated allergic diseases such as asthma, rhino-conjunctivitis, eczema, and anaphylaxis. In India, approximately 20% of the population suffers from allergic rhinitis and 15% from bronchial asthma. 1 Even though there has been enormous development in the field of in vitro diagnostic testing, the skin prick test is the most important diagnostic tool to detect IgE-mediated disease, reliability of the results of which heavily depends on the technique and the materials used. 2 Knowledge of the potency of allergen extracts used for diagnostic and therapeutic products is mandatory to gain a better evaluation and treatment of allergic individuals. 3 Very few studies have been published on allergy skin testing in Indian subjects.
The quality of some allergen extracts is strictly monitored within the United States by the U.S. Food and Drug Administration (USFDA) through its Center for Biologics Evaluation and Research which dictates reference tests and substances to be used for extract quality control before batch release. 2 In India, since the first registration for commercial manufacture and supply of allergen extracts in 1972, the allergen preparations do not have any special status and they need to comply with general requirements for medicinal products. Over the past 10 years, there have been attempts to standardize the extracts using standard immune-biochemical methods. 4 However, the manufacturers are not constrained to adhere to any standardized protocol for preparation of allergen extracts. In view of the above, there could be a great deal of variability in the quality of the marketed allergen extracts in India.
Despite increasing widespread use of allergen extracts in clinical practice, there has been no study done so far to compare the potency of the allergenic extracts in the Indian market with internationally approved extracts. With this background, we decided to undertake a double-blinded randomized control trial to compare the biological activity (by prick skin testing) of nonstandardized allergen extracts of
Methods
Aim
To compare the skin test response to Indian manufactured allergen extracts of DP and DF against USFDA-approved comparator by skin prick testing.
Design of the Study
Double-blind randomized control trial.
Setting and Characteristics of Studies Included in the Trial
Adults of age 17 years and above who were evaluated in the outpatient department for various allergic disorders and referred for skin prick testing to the allergy testing lab of the Department of Pulmonary medicine, CMC, Vellore were approached to participate in the study.
Patients referred for allergy testing were tested with an allergy panel which included an allergen extract of USFDA-approved dust mite and the tests were carried out as per internationally accepted protocol. 5 A positive test is a wheal diameter of 3 mm or greater relative to the negative saline control. Individuals who tested positive for the dust mite mixture, namely, DP and DF, and were willing to give informed consent, were then included in the trial.
Study participants received prick skin testing with the USFDA-approved allergens and test allergen extracts from Indian market at full concentration as well as in various dilutions on the same day.
The Skin Prick Allergen Extracts
DP and DF allergen extracts were obtained from 2 Indian pharmaceutical sources. Details of standardization of these products were not specified. The allergens were coded as follows:
C-DP and C-DF (nonglycerinated allergen extracts of DP and DF from Creative Diagnostic Medicare Private Limited D-296, Vashi Plaza, Sector 17, Navi Mumbai). B-DF (nonglycerinated allergen extracts of DF from Trivandrum Bioproducts and Diagnostics Private Ltd., Plot No 22 and 23, Industrial Development Plot, Manvila, Kulathoor (p.o), Thiruvananthapuram) S-DP and S-DF (USFDA-approved allergen extracts of DP and DF from Jubilant Hollister Stier, Spokane, WA, USA)
The following dilutions were made of the allergen extracts: 1:1, 1:10, 1:100, and 1:1000.
Masking
The indigenous allergen extracts and their dilutions and the USFDA-approved extracts and their dilutions were stored in 5 mL identical coded bottles and capped with a dropper, for blinding purposes. The codes were kept at the Biostatistics department of CMC. Investigators and patients were blinded to the allergens and their strength. Unblinding was done only after the statistical analysis was carried out.
Ethical Issues
The study protocol was approved by the institutional review board of CMC, Vellore.
Technique
The prick tests were performed on the skin surface over the upper back of the patient. After cleaning the test site with alcohol swab, the skin prick tests were performed by 1 of the 2 investigators. Circles were drawn with a ball point pen, 2 cm apart to ensure uniform spacing between the applications. The allergen extract was applied within the circle using the dropper on to the skin as per standard protocols.6,7 The time of the first prick was noted. Individual lancets (Manufacturer—MEDIPOINT Lancet, Mineola, NY) were used for each prick. A single technique skin prick of piercing the skin at 45° to 60° angle to the surface using the lancet was used as described in the accepted international protocol. 5 Allergen response was measured at roughly 18 minutes from the initial prick. After careful blotting of the excess allergens, the wheal diameter (Figure 1) was measured in both the longitudinal and the transverse axis in millimeters, and the readings were recorded in the clinical reporting form. The positive reaction was considered as 3 mm above the negative control measured for the subject according to international protocol. 5 After the test, the subjects were duly instructed and discharged in a satisfactory condition. The mean wheal surface area was calculated for each of the measurements. The 2 investigators who performed the test did a pilot testing on 10 subjects to ensure that their technique was similar and the wheal size comparable.
The photograph shows the wheal and erythema due to the prick skin testing.
Statistical Analysis
Relative skin test reactivity was compared using the mean wheal surface areas produced by reactivity of the test allergens and the standard allergens at full strength and at individual dilutions. To determine the diagnostic test accuracy, the Friedman rank sum test with a Tukey adjustment for multiple comparisons was used to study the difference in the distribution of wheal surface areas. Determination of statistical significance was kept at a test level alpha of 0.05. The null hypothesis is that there is no difference between the means of the wheal surface areas produced by skin reactivity to the USFDA comparator and Indian allergens. All statistical calculation was performed with R Version 3.3.2.
Results
Demographic Data of Study Patients
The 197 patients enrolled in the study were between 17 and 77 years of age with an average age of 37.2 years. Around 62% of study participants were males. Among the study population, 66% were diagnosed with asthma, 24% with food allergies, 7% with drug allergies, and 25% with skin allergy or eczema. Around 9% of patients were current or exsmokers. Among the study population, 54% lived in urban settings, 31% in rural, and 15% partially lived in urban and rural settings (Table 1).
Demographic Data of the Study Population.
Abbreviation: SD, standard deviation.
Adverse Events
Only 1 patient had a local reaction of intense erythema and itching on the testing site which faded with a single dose of oral antihistamine.
Potency Comparison Between Test Allergens and Comparator
Prick testing results of mean wheal sizes were compared between indigenous extracts and standard extracts. They showed significant differences across all dilution with significant
Comparison of DF Extracts
The S-DF extract had a mean of 109.07 (standard deviation [SD] 162.73) at full strength. However, B-DF extract had a mean wheal size of only 7.69 (SD 9.91) and C-DF had a mean wheal size of 31.01 (SD 51.04). This difference was significant between both the S-DF and C-DF (
Comparison of mean wheal surface area of
Mean Wheal Sizes of the Allergen Extracts Across All Dilutions Along With the
Abbreviations: B-DF,
Comparison of DP Extracts
The S-DP extract had a mean wheal size of 167.66 (SD 270.80) at full strength. In comparison, the indigenous C-DP had a mean wheal size of 39.37 (SD 51.74) in full strength. The mean wheal size of S-DP was significantly higher than C-DP across all dilutions, which was highly significant with
Comparison of mean wheal surface area of
The full strength indigenous extracts produced wheal area considerably smaller than even the 1:1000 dilutions of the Food and Drug Administration (FDA) standardized extracts, indicating that their potency in terms of allergen content was in each case well below one of thousands of the FDA standardized extract.
Even between the 2 indigenous extracts, there were statistically significant differences between the allergen extracts with C-DF showing larger mean wheal sizes at all dilutions than B-DF (Figure 2).
Discussion
This is the first double-blind randomized control study from India to report the differences in biopotency between indigenous allergens and an USFDA-approved allergen. Our study was done as a single center study to prevent the bias of the variations in the techniques.
Biological methods of comparison have been performed in both United States and Europe with 3-fold dilution series 8 and hence, we decided to follow a similar procedure.
The study was also done with a single batch of all the products obtained from the 3 manufacturers. Batch-to-batch variability could lead to significant variations as has been found in a study done by Pagani et al. 3 from Italy. Researchers in this study analyzed different batches and noticed significant differences between batches. The reasons for the differences were found to be dependent on both the type of allergens and the manufacturers.
Our study showed that the biological activity of the full strength of the indigenous allergen was statistically significantly lower than the USFDA-approved antigen extracts even at 1:1000 dilutions. This lack of potency may have been in part due to the indigenous extracts, unlike the FDA standardize extract, being in a nonglycerinated solution.9
A similar variation in potency of
The results of our study has significant implications in the diagnosis and treatment of allergic disorders in India. House dust mites (DP and DF) are major allergens responsible for nearly 80% of the sensitization among allergic individuals. 11 The results of the study should discourage the clinician from using indigenous allergens.
Based on above observations, we recommend that Indian manufacturers prioritize the standardization of their allergen extracts with international standards. Protocols could involve either in vivo standardization as we have done or in vitro (Radio Allergen Sorbent Test, ImmunoCap, or ELISA inhibition assays). 12
Results of the study also urgently call for coordinated efforts like development of “certified reference materials for allergenic products and validation of methods for their quantification (CREATE)” project in Europe which implements a synergistic in vivo and in vitro validated methods, for standardization of the indigenous extracts by various stakeholders. 13 There is also a need to do similar studies with other allergen extracts in India such as House dust mite, Bermuda grass and cat allergen, all of which showed varying potency when European and Mexican extracts were compared to USFDA standard allergens.2,10
The study has limitations in that it has been assumed that the USFDA-approved allergen extract is the gold standard for skin prick testing. This is, however, debatable. But the study was constructed pragmatically to obtain comparative results using the best standard extracts available. Another limitation of our study was that we were not able to include all the marketed DP and DF allergens in India.
Conclusion
Our study shows that the Indian manufactured allergen extracts of DP and DF were less potent than USFDA-approved allergen extracts. Therefore, using the tested Indian allergen extracts for testing would give unreliable results, and so there is an urgent need for standardization of allergens by the concerned statutory bodies.
Availability of Data and Material
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.