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Comparison of contact sensitivity in patch test with the aqueous and acetone extracts of Parthenium hysterophorus.

Byline: Saulat Zahra, Lamees Mahmood Malik, Abduraheem Khan and Muhammad Jahangir

Abstract Objective To compare the frequency of patch test positivity to Parthenium hysterophorus by using aqueous and acetone extracts of fresh plant in patients of allergic contact dermatitis (ACD).

Patients and methods 115 clinically diagnosed patients of allergic contact dermatitis involving [greater than or equal to]40% of body area were enrolled from dermatology outdoor department. After taking informed consent patch test was performed with aqueous and acetone extracts of fresh plant. Aqueous allergen was prepared in three serial dilutions as 0.1%,1%, and 10% strength. 1% acetone extract allergen was taken as gold standard for comparison as well as positive control while pure aqua was taken as negative control. Patch test results were interpreted according to ICDRG criteria

Results 115 patients of ACD consisted of 88 males and 27 females with the age range of 20 to 75 years (mean 42+-10.20 years). Patch test showed positive contact sensitivity in 56 (49%) patients with 1% acetone extract, in 52 (45%) patients with 10% aqueous extract, in 44 (38%) patients with 1% aqueous extract and in 13 (11.3%) patients with 0.1% aqueous extract. Irritant reaction was seen in 20 (17%) patients with 1% acetone extract, in 12 (10%) patients with 10% aqueous extract and in 5 (4%) patients with 1% aqueous extract.

Conclusion Contact sensitivity to P. hysterophorus can be diagnosed with patch test by applying either aqueous or acetone extracts of fresh plant. Aqueous extract is safe (as it produced less number of irritant reactions) and almost equally sensitive allergen. Thus it can be a readily available alternate allergen for patch test in diagnosing parthenium sensitivity.

Key words

Parthenium hysterophorus, patch test, aqueous extract, contact sensitivity.

Introduction

Parthenium hysterophorus (congress grass, congress weed, carrot weed, wild feverfew) a member of compositae family is an exotic weed that was accidentally introduced in Asia through imported food grains.1 In Pakistan it is found in rural as well as urban areas as a wild weed, also used in bouquets as filler.2 It is the leading cause of plant induced airborne contact dermatitis and has achieved major weed status in India as well as in Pakistan and Australia within the past few decades.3,4,5

The weed can affect human health, animal husbandry, crop production and biodiversity.2 After 1-10 years of exposure, 10-20% of the population will develop severe allergenic reactions.1 There may be hay fever, asthma or dermatitis. When testing for sensitivity to parthenium weed, a prevalence rate of 37% was found.2,3

The severity of dermatitis in Pakistan and India is greater in comparison to America because the plant grows more vigorously and contains large amounts of the sesquiterpene lactone, parthenin, which is absent in the plants in South America.1,2

Another contact sensitizer responsible for dermatitis, parthenolide, is an oil soluble oleoresin present throughout the plant and pollens. Up to one-third of the oil soluble fraction can be removed with water alone.6,7,8

Parthenium sensitivity can be diagnosed with patch test. The allergens, responsible for dermatitis are extractable in various solvents such as acetone, alcohol, ether, and water.8,9,10

Among these aqueous extract of fresh plant in serial dilutions will be used in this study. It is easily prepared, cheap and cost-effective. Thus it may prove to be an alternative, equally sensitive and readily available allergen for the diagnosis of parthenium sensitivity with patch test.

This study was undertaken to compare the frequency of patch test positivity to P. hysterophorus by using aqueous and acetone extracts of fresh P. hysterophorus in patients of allergic contact dermatitis.

Patients and methods

It was a cross-sectional comparative study conducted at the Department of Dermatology, Jinnah Hospital Lahore for duration of six months from 20th February to 19th August, 2010. Calculated sample size was 115 cases, with 6% margin of error and 95% confidence level. It was a nonprobability purposive sampling technique. Included patients were of either sex and any age presenting in dermatology outdoor department and clinically diagnosed as cases of allergic contact dermatitis involving [greater than or equal to]40% body area (according to rule of 9).

All the patients on oral steroid above 15 mg prednisolone/day or on immunmodulator drugs during previous six weeks and patients with active eczematous skin eruption on the back or denuded areas on the back were excluded from the study. Also previously diagnosed patients suffering from chronic morbid conditions like diabetes mellitus, chronic renal failure, chronic liver failure, sarcoidosis, SLE; and pregnant ladies were not patch tested.

Fresh plant was taken, washed and all parts crushed into small pieces. It was blended in electric blender for 1 minute into paste like material. This material was weighed and diluted with pure water as 1:10 parts and kept overnight to achieve maximum solubility of the allergen. After 12 hrs it was filtered with common filter paper to prepare the final allergen to be applied in patch test. Further allergens were prepared in 2 rising dilutions with water by w/v as 1:100, 1:1000.

Standard IQ chambers were used for patch testing. Filter discs (Whatman 3 MM size) were placed into the IQ chambers and saturated with the prepared aqueous extract allergens. 1% acetone extract of P. hysterophorus was taken as gold standard allergen for the diagnosis of parthenium dermatitis. After taking informed consent from the patient, allergens for the patch test were applied. Patch test record sheets were used for the proper record of the allergens tested their position and the test results.

Patches were applied to patients' back for 48 hours and then removed. Baseline readings were recorded half an hour after removal of patches to let erythema of patches and tape (if any) to settle down. Two other readings were recorded at 72 and 120 hours after application of patches.

These readings were interpreted according to International Contact Dermatitis Research Group (ICDRG) criteria (Table 1). To reduce the chance of false positive reactions, type 3, 4 and 5 were considered as positive reactions.

Reaction of type 1 and 2 were considered negative.

Data were collected and entered by using SPSS version 10. Qualitative analysis of each allergen was accessed in terms of ICDRG criteria as positive, negative or irritant. Tables of percentages and frequency of patch test results with each dilution with reference to time were generated. Positivity of patch test with acetone and aqueous extracts of P. hystetrophorus was compared by using chi square analysis.

Results

115 cases of allergic contact dermatitis involving [greater than or equal to]40% body area were patch tested. The demographic profile of study population is shown in Table 2. Patients of allergic contact dermatitis (who had positive patch test reactions) were categorized according to their clinical pattern of disease as airborne contact dermatitis (ABCD), photocontact dermatitis (PCD), adult-onset atopic dermatitis (AOAD), and mixed pattern contact dermatitis (MCD).

40% patients were of ABCD who presented with erythematous papules and plaques on exposed areas i.e. face, upper eyelid, sides of neck, V of chest, flexures of forearm and cubital fossae.

25% patients were diagnosed as cases of PCD that presented clinically with erythematous papules and plaques (few showing vesicles) on the sun-exposed regions of the body. 21% patients had clinical features of both ABCD and PCD in mixed pattern and were categorized as MCD. Severely itchy and lichenified papules and plaques involving cubital fossae, popliteal

Table 1 Recording of Patch test reactions according to International Contact Dermatitis Research Group (ICDRGI Criteria.

Skin changes###Interpretation

1 .No skin lesions###Negative (-)

###Doubtful reaction

2. Faint erythema only###(+1-)

3. Palpable erythema,###Weak positive

infiltration, possibly papules###reaction (+)

4. Erythema, infiltration,###Strong positive

papules, vesicles###(++)

5. Intense erythema, infiltration Extreme positive

and coalescing vesicles###(+++)

###Irritant reaction

6.###(IR)

7. Not tested###NT

Table 2 Demographic profile of study population

- (n=1 15).

Gender distribution

Males###88 (76.5%)

Females###27 (23.5%)

Male female ratio###3:1

Age distribution

Range (years)###20-75

Mean (years)###40 + 16

Completed study###102 (87%)

Failure to complete study###13 (11%)

Pattern of eczema

Airborne contact dermatitis###40%

Photocontact dermatitis###25%

Adult-onset atopic dermatitis###14%

Mixed pattern###21%

fossae and hands i.e. AOAD pattern was present in 14% of patients of allergic contact dermatitis.

Detailed five days readings of patch test in terms of positive, negative and irritant reactions according to ICDRG criteria i.e. at 48 hrs, at 72 hours and at 120 hours; with all the allergens applied are shown in Table 3.

Patch test reactions at 120 hours with all the allergens applied on patients were taken as final results of study in terms of positive, negative and irritant reactions according to ICDRG criteria. On day 5 i.e. at 120 hrs, with 1% acetone extract allergen, positive reaction was seen in 56 (49%) patients, with 10% aqueous

Table 3 Detailed three days reactions of all allergens applied according to ICDRG criteria (n=1 15).

###Types ofpatch test reactions

Type of allergen applied###At 48 hrs###At 72 hrs###At 120 hrs

###+ve -ye Irritant +ve###-ye Irritant###+ye###-ye###Irritant

1%Acetoneextract###41###54###20###49###46 20###56###39###20

10%Aqueousextract###40###63###12###48###55 12###52###51###12

1% Aqueous extract###32###78###5###37###73###5###44###66###5

O.1%Aqueousextract###0###115###0###5###110###0###13###102###0

Pureaqua###0###0###0###0###0###0###0###0###0

allergen in 52 (45%) patients, with 1% aqueous extract allergen in 44 (38%) patients and with 0.1% aqueous extract allergen in 13 (11.3%) patients (Table 3). 1% acetone extract allergen showed irritant reaction in 20 (17%) patients whereas with 10% aqueous extract allergen. Irritant reaction was seen in 12 (10%) patients. With 1% aqueous extract allergen it was seen in only 5 (4%) patients and with 0.1% aqueous extract allergen and pure aqua no irritant reaction was seen.

Table 4 compares the positive results of different concentrations of aqueous extracts of allergen with the acetone extract, the gold standard.

Discussion

Parthenium hysterophorus is an annual herb belonging to the compositae family. The allergenicity of P. hysterophorus is due to presence of sesquiterpenelactones (SQLs) and parthenolide. Parthenium sensitivity is diagnosed with patch test.

In standard series of allergens, parthenolide and SQL mixture are used for the diagnosis of compositae sensitivity. However, this is not a reliable screen for the diagnosis of parthenium sensitivity since SQLs show cross reactivity. Also, these SQLs are present in non-compositae plants. Thus for a reliable diagnosis of parthenium sensitivity actual plant should be used in patch test.11

For the diagnosis of parthenium sensitivity with patch test, filtered acetone or ethanol extract of dried plant and a short ether extract of fresh plant has been in use for a while. These allergens require proper laboratory set up and are also costly to prepare. On the contrary aqueous extract of plant can be easily prepared and is comparatively inexpensive.1,7

In this study, aqueous extract of fresh P. hysterophorus in three serial dilutions were used in the patch test to diagnose contact sensitivity. For comparison, gold standard 1% acetone extract of fresh P. hysterophorus was used and also was considered as positive control.

Results of patch test were interpreted according to ICDRG criteria. Positive reaction with our gold standard 1% acetone extract was seen in 56 patients on day 5 i.e. at 120 hours, so these patients were considered as confirmed cases of parthenium dermatitis. Out of those 56 cases of parthenium dermatitis (positive with gold standard) 10% aqueous extract allergen produced positive reaction in 52 (92.8%) patients, 1% aqueous extract allergen produced positive reaction in 40 (78.6%) patients while 0.1% aqueous extract allergen produced positive reaction in only 13 (23.2%) patients.

Comparing frequency of irritant reactions, with 1% acetone extract allergen 20 (17%) patients showed irritant reaction, whereas with 10% aqueous extract allergen 12 (10%) patients had irritant reaction while with 1% aqueous extract allergen only 5 (4%) patients had an irritant reaction. With 0.1% aqueous extract allergen no irritant reaction was seen.

Therefore, in our study 10% aqueous allergen proved to be nearly equivalent with 1% acetone extract allergen in diagnosing parthenium sensitivity with patch test. Also it produced less number of irritant reactions comparative to our gold standard i.e. 1% acetone extract allergen.

Comparison of patch test results between serial dilutions of aqueous extract allergens showed that although 1% aqueous extract allergen produced less positive reactions than 10% aqueous extract allergen yet it had lesser number of irritant reactions. However, 0.1% aqueous extract allergen proved to be very poor allergen for the patch test in diagnosing parthenium sensitivity.

Our results were comparable to a number of national and international studies. Verma et al. used fresh plant extracts of P. hysterophorus in various dilutions for the patch test to determine titre of contact hypersensitivity (TCH) in patients of parthenium dermatitis.12 In this study patch test reactions were positive with undiluted allergen (UD) in 2 (4.8%) patients, with 10% dilution in 15 (35.7%) patients, with 1% dilution in 20 (47.6%) patients and with 0.1% dilution in 5 (11.9%) patients, respectively.

Similarly in another study done by Sharma et al, patch test was done with 1% and 0.5% dilutions of an acetone extract of P. hysterophorus along with the Indian standard series (which includes aqueous extracts of parthenium, xanthium and chrysanthemum) in 72 patients of airborne contact dermatitis.13

All their patients showed positive contact sensitivity to the 1% dilution of acetone extract of plant while with 0.1% dilution of acetone extract 67 patients had positive contact sensitivity reactions; whereas only 45 patients showed positive contact sensitivity with the Indian standard series of parthenium allergen.

A multiphase study done by Nadeem et al. in Pakistan, patch test was done using actual leaf and flower of P. hysterophorus, with 1% parthenium in petrolatum base for the diagnosis of contact sensitivity to P. hysterophorus. In their study overall 77% patients showed positive patch test reactions to P. hysterophorus.5

Various studies exist on patch test diagnosis of contact sensitivity to P. hysterophorus on national as well as international levels but comparison of serial dilutions of aqueous extract of fresh P. hystertophorus was only done in our study for the first time in Pakistan.

Today P. hysterophorus has become a growing concern in our community due to its serious impact on humans, animals and even on natural ecosystem. We should create public awareness regarding health hazards of P. hysterophorus. There is no curative treatment of parthenium dermatitis yet and only preventive measures are helpful. Thus it is very important to detect cases of parthenium dermatitis at earlier stages. Our study has helped us to devise an inexpensive and readily available method for diagnosing the cases of parthenium sensitivity with patch test.

Conclusion

Aqueous extract of fresh P. hysterophorus is easy to produce and cheaper as compared to the previous allergens in use. It is nearly as sensitive as 1% acetone extract of plant in diagnosing parthenium sensitivity with patch test. Aqueous extract allergen also produces less number of irritant reactions compared to the gold standard 1% acetone extract. So it is a safe allergen, as well.

Thus aqueous extract of fresh plant is a good alternative, almost equally sensitive and less irritant, readily available allergen to be used in patch test for the diagnosis of P. hysterophorus sensitivity.

References

1. Lakshmi C, Srinivas CR. Parthenium: A wide angle view. Indian J Dermatol Venereol Leprol 2007;73:296-306.

2. Steve WA, Navie SC. Parthenium weed: a potential major weed for agro ecosystem in Pakistan. Pak J. Weed Sci Res 2006;12:19- 36.

3. Javaid A, Anjum T. Parthenium hysterophorus L.- a noxious alien weed. Pak J Weed Sci Res 2005;11:81-7.

4. Suraj VD, Shenoi SD, Prabhu S et al. Clinical evaluation of patients patch tested with plant series a prospective study. Indian J Dermatol 2011;56:383-8.

5. Nadeem M, Rani Z, Kazmi HA et al. Parthenium weed a growing concern in Pakistan. J Pak Assoc Dermaol 2005;15:4-8.

6. Agarwal KK, D'Souza M. Airborne contact dermatitis induced by parthenium: a study of 50 cases in South India. Clin Exp Dermatol 2009;34:4-6.

7. Sharma VK, Sethuraman G. Parthenium dermatitis. Dermatitis 2007;18:183-90.

8. COMPOSITAE-18 Parthenium (Daisy or Sunflower family) URL: www .http://BoDD. cf. ac.uk/Bot DermFolder/BotDermC/COMP-18.

9. Khan MS, Ahmad S. Pharmacognostical, phytochemical, biological and tissue culture studies on Parthenium hystertophorus Linn: A review. Internet J Alternative Medicine2009;6(2).

10. Sharon EJ, Tamar Z. ACDG Allergens: Compositae mix and sesquiterpene lactones [Electronic] Publication Date: Jun 15, 2007. Vol: 15 Issue: 6.URL: http://www.skinandaging.com/article/7316.

11. Nandakishore T, Pasricha JS. Pattern of cross-sensitivity between 4 Compositae plants, Parthenium hysterophorus, Xanthium strumarium, Helianthus annuus and Chrysanthemum, in Indian patients. Contact Dermatitis 1994;30:162-7.

12. Verma KK, Manchanda Y, Dwivedi SN.Failure of titre of contact hypersensitivity to correlate with clinical severity and therapeutic response in contact dermatitis caused by parthenium. Indian J Dermatol Venereol Leprol 2004;70:210-3.

13. Sharma VK, Sethuraman G, Tejasvi T.Comparison of patch test contact sensitivity to acetone and aqueous extracts of Parthenium hysterophorus in patients with airborne contact dermatitis. Contact Dermatitis 2004;50:230-2.
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Author:Zahra, Saulat; Malik, Lamees Mahmood; Khan, Abduraheem; Jahangir, Muhammad
Publication:Journal of Pakistan Association of Dermatologists
Article Type:Report
Geographic Code:9PAKI
Date:Sep 30, 2012
Words:2907
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