Rong-Bo Zhang, Chao-Pin Li, Yu-Bao Cui, Department of Etiology and Immunology, School of Medicine, Anhui University of Science and Technology, Huainan 232001, Anhui Province, China
Yong Huang, Shandong Institute of Parasitic Diseases, Jining 272033, Shandong Province, China
Correspondence to: Dr. Chao-Pin Li, Department of Etiology and Immunology, School of Medicine, Anhui University of Science and Technology, Huainan 232001, Anhui Province, China.  cpli @aust.edu.cn
Telephone: +86-554-6658770    Fax: +86-554-6662469
Received: 2003-08-02    Accepted: 2003-09-18

Abstract
AIM: To explore the value of avidin-biotin system enzyme-linked immunosorbent assay (ABC-ELISA) in diagnosis of intestinal acariasis.

METHODS: Mite-specific IgG levels in serum of 48 patients with intestinal acariasis were measured with ABC-ELISA. The sensitivity of this method was compared with that of staphylococcal protein A enzyme-linked immunosorbent assay (SPA-ELISA).

RESULTS: The positive rate of mite-specific IgG detected with ABC-ELISA and SPA-ELISA was 89.58% (43/48) and 56.25% (27/48), respectively. The positive rate with ABC-ELISA was statistically higher than that with SPA-ELISA (x²=13.50, P<0.01).

CONCLUSION: ABC-ELISA is an effective method for the diagnosis of intestinal acariasis.

Zhang RB, Huang Y, Li CP, Cui YB. Diagnosis of intestinal acariasis with avidin-biotin system enzyme-linked immunosorbent assay. World J Gastroenterol  2004; 10(9): 1369-1371
http://www.wjgnet.com/1007-9327/10/1369.asp

INTRODUCTION
Mites could live in intestinal tract and cause intestinal acariasis with most frequent symptoms of abdominal pain, diarrhea and pyohemofecia^[1-3]. Intestinal acariasis is traditionally diagnosed by identification of adult or larval mites, eggs or hypopus in either single or multiple fecal specimens under microscopy. The detection of the parasites is increased on examination of multiple fecal samples obtained on three different days. However, it is not easy to detect mites technically. Thus, the present study intended to develop and evaluate an enzyme-linked immunosorbent assay for mite-specific antibody detection in serum samples using Dermatophagoides farinae extract.

MATERIALS AND METHODS
Serum
A total of 78 serum specimens were collected from 48 patients with intestinal acariasis and 30 health blood donors. All of the subjects investigated were asked to provide stools for detection of mites by saturated saline floatation method. The 48 patients with mites in stools were grouped as experimental group, while the health blood donors without mites as control group.

Reagents 
Reagents for ABC-ELISA and SPA-ELISA were provided by Shanghai Institute of Biological Products (Batch No. 990004 and 990012). Dermatophagoides farinae extract was made according to NIBSC82/518 approved by World Health Organization (WHO) in 1984. The mites were cultured in the initial medium for several months. A 48-h maceration in a borate buffer (pH 8.5) was centrifuged. The supernatant was neutralized and precipitated with a series of acetone. The precipitated fraction at 800 mL/L acetone was isolated, washed and dried. This purified extract was lyophilized or stored as a solution in the presence of 500 mL/L glycerol and 50 mL/L phenol^[4-7].

Methods
ABC-ELISA procedure  Initially 0.1 mL Dermatophagoides farinae extract with protein concentrations of 62.5 mg/mL was added in each well on a 40-well plate coated with enzyme, and the plate was placed at 4 °C overnight. Then the plate was washed with PBS (pH=7.4), sera of the patients with intestinal acariasis were diluted at 1:40 and added in duplicated wells, and incubated in water bath at 37 °C for 60 min. Following washing of the plate, bio-SAH IgG at the concentration of 1:40 was added and incubated at 37 °C for 60 min, avidin-HRP at the concentration of 1:20 was added for reaction at 37 °C for 30 min, and the substrate OPD-H₂O₂ was added and incubated at 37 °C for 30 min. Lastly, 2 mol/L H₂SO₄ was added to terminate the reaction. Sera of the control group were detected with the same procedure^[8,9].
SPA-ELISA procedure  Concentrations of antigen and sera dilution in SPA-ELISA method were similar to those in ABC-ELISA. SPA-ELISA was performed as routine. Values of optic densities (OD) in each well were determined, the value of 2.1 times or more of  the negative control was considered as positive. 

RESULTS
General data  
A total of 48 patients (male 32 and female 16) with intestinal acariasis were selected as mites were found in their stools. The  48 patients consisted of 13 workers in traditional Chinese medical storehouses, 22 workers in rice storehouse or mill, 8 miners, 2 workers in machine factory and 3 with other occupations. The mites in stool samples included Acarus siro, TyroPhagus putrescentiae, Dermatophagoides farinae, D. pteronyssinus, Glycyphagus domesticus, G.ornatus, Carpoglyphus lactis and Tarsonemus granaries.

ABC-ELISA data
In ABC-ELISA, the mean OD value of mite-specific IgG in sera of the 48 patients was 0.358±0.124 (0.176-0.615), while that in 30 controls was 0.112±0.065 (0.085-0.253). The Absorbent values in 43 of 48 patients were at least 2.1 times of 0.112, the positive rate of the patients detected by ABC-ELISA was 89.58%. Interestingly, the A value of one case in normal control group was 0.253, and the false positive rate was 3.33%.

SPA-ELISA data 
In SPA-ELISA, the mean OD value of mite-specific IgG in sera of the 48 patients was 0.225±0.147 (0.133-0.574) and that in the control group was 0.078±0.047 (0.043-0.172). The A values in 37 patients were at least 2.1 times of 0.078, the positive rate of the patients detected by SPA-ELISA was 56.25%. However, the A values of 2 cases in control group were 0.168 and 0.172, respectively, which were higher than 0.1638. The false positive rate in SPA-ELISA was 6.67%.

Comparison between ABC-ELISA data and SPA-ELISA data 
The positive rate of the patients detected by ABC-ELISA and SPA-ELISA was 89.58% (43/48) and 56.25% (27/48) with a significant difference (x²=13.50, P<0.01). Although only 25 cases were positive in both ABC-ELISA and SPA-ELISA, the positive number of patients was 45 (93.75%) by ABC-ELISA or SPA-ELISA (Table 1).

Table 1  Intestinal acariasis detected with ABC-ELISA and SPA-ELISA

^b: x²=13.50, P<0.01.

DISCUSSION
ABC-ELISA and SPA-ELISA were used to detect mite-specific antibody IgG in serum samples from 48 patients with intestinal acariasis, and the positive rate in ABC-ELISA and SPA-ELISA was 89.58% and 56.25%, respectively. ABC-ELISA had high specificity in diagnosis of intestinal acariasis. Moreover, ABC-ELISA in detection of mite-specific antibody was easy to perform, inexpensive, and numerous samples could be performed   simultaneously. The test could be carried out quickly for the  diagnosis, particularly for individuals who suffered from recurrent diarrhea, chronic abdominal pain, malabsorption and stunting due to infection.
      The occurrence of false positive in two methods in diagnosis of intestinal acariasis might be associated with mites’ invasive locus, stool examination techniques and dilution of sera. In addition to gastrointestinal tract, acaroid mites could infest   other organs of the human body such as respiratory tract and urinary tract, no matter where they were parasized, mite-specific antibody would occur in peripheral blood^[10-18]. Although saturated saline floatation method is useful in examination of feces, some large and heavy mites may be missed because of difficult drift. Sera dilution at 1:40 in the present study was lower than that in routine ELISA with dilution at 1:100 to 1:200. It was suggested that much more times repeating stool examination should be carried out in the normal control group to avoid false positive detection^[19-24].
      Our study showed 8 species of mites in human stools. We used Dermatophagoides farinae extract as coating antigen only, which might decrease the detectable rates of mite-specific antibody, because common antigen might exist in Dermatophagoides farinae and other seven species of mites^[25-28]. In addition, the number of mites in intestinal tract might affect the levels of mite-specific antibodies and detectable rates. In conclusion, ABC-ELISA method is effective in diagnosis of intestinal acariasis.  

REFERENCES
1    Li CP, Wang J. Intestinal acariasis in Anhui Province. World J Gastroenterol 2000; 6: 597-600
2    Li CP, Cui YB, Wang J, Yang QG, Tian Y. Acaroid mite, intestinal and urinary acariasis. World J Gastroenterol 
      2003; 9: 874-877         
3    Li CP, Wang KX. A study on treatment of intestinal acariasis. Shijie Huaren Xiaohua Zazhi 2000; 8: 919-920
4    Nuttall TJ, Lamb JR, Hill PB. Characterization of major and minor Dermatophagoides allergens in canine atopic dermatitis.
      Res Vet Sci 2001; 71: 51-57
5    Basomba A, Tabar AI, de Rojas DH, Garcia BE, Alamar R, Olaguibel JM, del Prado JM, Martin S, Rico P. Allergen
      vaccination with a liposome-encapsulated extract of Dermatophagoides pteronyssinus: a randomized, double-blind,
      placebo -controlled trial in asthmatic patients. J Allergy Clin Immunol 2002; 109:943-948
6    Akcakaya N, Hassanzadeh A, Camcioglu Y, Cokugras H. Local and systemic reactions during immunotherapy with 
      adsorbed extracts of house dust mite in children.  Ann Allergy Asthma Immunol 2000; 85: 317-321
7    Hillier A, Kwochka KW, Pinchbeck LR. Reactivity to intradermal injection of extracts of Dermatophagoides farinae,
      Dermatophagoides pteronyssinus, house dust mite mix, and house dust in dogs suspected to have atopic dermatitis: 
      115 cases (1996-1998). J Am Vet Med Assoc 2000; 217: 536-540  
8    Guo H, Zhao ZF, Shi DZ. A study on the culture medium antigens of Cystcercus cellulosae for detecting antibodies of
      cysticercosis by means of ABC-ELISA. Southeast Asian J Trop Med Public Health 1997; 28(Suppl 1): 125-127 
9    Koldas M, Uras F. Avidin-biotin ELISA for measurement of prothrombin in human plasma. Thromb Res 
      2001; 102: 221-227         
10  Beco L, Petite A, Olivry T. Comparison of subcutaneous ivermectin and oral moxidectin for the treatment of notoedric
      acariasis in hamsters. Vet Rec  2001; 149: 324-327
11  Hiraoka E, Sato T, Shirai W, Kimura J, Nogami S, Itou M, Shimizu K. A case of pulmonary acariasis in lung of Japanese
      macaque. J Vet Med Sci 2001; 63: 87-89
12  van der Geest LP, Elliot SL, Breeuwer JA, Beerling EA. Diseases of mites. Exp Appl Acarol 2000; 24: 497-560
13  Hammerberg B, Bevier D, DeBoer DJ, Olivry T, Orton SM, Gebhard D, Vaden SL. Auto IgG anti-IgE and IgG x IgE immune
      complex presence and effects on ELISA-based quantitation of IgE in canine atopic dermatitis, demodectic acariasis and
      helminthiasis. Vet Immunol Immunopathol 1997; 60: 33-46
14  Morris DO, Dunstan RW. A histomorphological study of sarcoptic acariasis in the dog: 19 cases. J Am Anim Hosp Assoc
      1996; 32: 119-124
15  Jungmann P, Guenet JL, Cazenave PA, Coutinho A, Huerre M. Murine acariasis: I. Pathological and clinical evidence
      suggesting cutaneous allergy and wasting syndrome in BALB/c mouse. Res Immunol 1996; 147: 27-38
16  Jungmann P, Freitas A, Bandeira A, Nobrega A, Coutinho A, Marcos MA, Minoprio P. Murine acariasis. II. Immunological
      dysfunction and evidence for chronic activation of Th-2 lymphocytes. Scand J Immunol 1996; 43: 604-612 
17  Ponsonby AL, Kemp A, Dwyer T, Carmichael A, Couper D, Cochrane J. Feather bedding and house dust mite sensitization
      and airway disease in childhood. J Clin Epidemiol 2002; 55: 556-562
18  Paufler P, Gebel T, Dunkelberg H. Quantification of house dust mite allergens in ambient air. Rev Environ Health 
      2001; 16: 65-80
19  Gonin P, Trudel L. Detection and differentiation of Entamoeba histolytica and Entamoeba dispar isolates in clinical samples
      by PCR and enzyme-linked immunosorbent assay. J Clin Microbiol 2003; 41: 237-241
20  Molinari JL, Garcia-Mendoza E, de la Garza Y, Ramirez JA, Sotelo J, Tato P. Discrimination between active and inactive
      neurocysticercosis by metacestode excretory/secretory antigens of Taenia solium in an enzyme-linked immunosorbent
      assay. Am J Trop Med Hyg 2002; 66: 777-781
21  Levett PN, Branch SL. Evaluation of two enzyme-linked immunosorbent assay methods for detection of immunoglobulin 
      M antibodies in acute leptospirosis. Am J Trop Med Hyg 2002; 66: 745-748
22  Van Gool T, Vetter H, Vervoort T, Doenhoff MJ, Wetsteyn J, Overbosch D. Serodiagnosis of imported schistosomiasis by 
      a combination of a commercial indirect hemagglutination test with Schistosoma mansoni adult worm antigens and an
      enzyme-linked immunosorbent assay with S. mansoni egg antigens. J Clin Microbiol 2002; 40: 3432-3437 
23  Odashima NS, Takayanagui OM, Figueiredo JF. Enzyme linked immunosorbent assay (ELISA) for the detection of IgG, 
      IgM, IgE and IgA against Cysticercus cellulosae in cerebrospinal fluid of patients with neurocysticercosis. Arq
      Neuropsiquiatr 2002; 60: 400-405
24  Starke-Buzetti WA, Machado RZ, Zocoller-Seno MC. An enzyme-linked immunosorbent assay (ELISA) for detection of
      antibodies against Toxocara vitulorum in water buffaloes. Vet Parasitol 2001; 97: 55-64
25  Varela J, Ventas P, Carreira J, Barbas JA, Gimenez-Gallego G, Polo F. Primary structure of Lep d I, the main 
      Lepidoglyphus destructor allergen. Eur J Biochem 1994; 225: 93-98
26  Kemp SF, Lockey RF, Fernandez-Caldas E, Arlian LG. Skin test and crossreactivity studies with Euroglyphus maynei and
      Dermatophagoides pteronyssinus. Clin Exp Allergy 1997; 27:893-897
27  Garcia-Robaina JC, Eraso E, De la Torre F, Guisantes J, Martinez A, Palacios R, Martinez J. Extracts from various mite
      species contain cross-reactive and noncross-reactive IgE epitopes. A RAST inhibition study. J Investig Allergol Clin
      Immunol 1998; 8: 285-289
28  Smith W, Mills K, Hazell L, Hart B, Thomas W. Molecular analysis of the group 1 and 2 allergens from the house dust 
      mite, Euroglyphus maynei. Int Arch Allergy Immunol 1999; 118: 15-22

   Edited by Ren SY and Wang XL  Proofread by Xu FM  

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