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Staphylococci adherence test for serodiagnosis of amoebiasis
Journal of Microbiological Methods 10 (1989) 5 3 - 5 7
53
Elsevier MIMET 00322
Staphylococci adherence test for serodiagnosis of amoebiasis S.C. Parija l, S. K a s i n a t h a n 2 a n d R.S. R a o 1 1Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India," 2Department of Biology, Pondicherry University, Pondicherry, India (Received 21 December 1988; revision received 8 April 1989; accepted l0 April 1989)
Summary The staphylococci adherence test (SAT) using axenic amoebic antigen slides and Staphylococcus aureus (Cowan's strain I) bearing Protein A was adapted for diagnosis of different clinical manifestations of amoebiasis. The test was sensitive and specific. At a diagnostic antibody titre of 1 : 32, SAT could detect 94°7o amoebic liver abscess, 7207o of amoebic hepatitis and 72070 of intestinal amoebiasis cases. Amongst the controls, no false positive reaction was observed in IA control sera. However, two (4.407o) and three (607o) sera demonstrated false positive reactions from the nonamoebic liver disease controls and healthy controls, respectively. Since the test required only a simple light microscope and common reagents, the simplicity and economy of the method would allow its adaptation at small and primary-health-centre laboratories in the developing countries.
Key words: Amoebiasis; Laboratory diagnosis; SAT; Serodiagnosis
Introduction Demonstration of anti E. histolytica antibodies is reliable for diagnosis of the intestinal and invasive extra-intestinal manifestations (i.e., amoebic liver abscess) of amoebiasis. Indirect haemagglutination (IHA), counter-current immunoelectrophoresis (CIEP), double diffusion in agar, indirect fluorescent antibody (IFA) and enzymelinked immunosorbent assay (ELISA) are the immunoassays most commonly used in serodiagnosis of amoebiasis [1]. In spite of their high sensitivity and specificity, there is still need for the development of a simple yet reliable test for serodiagnosis of amoebiasis at field conditions or small laboratories such as at a primary health centre (PHC). These laboratories, particularly in the developing countries, lack enough tech-
Correspondence to: S.C. Parija, Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry 605006, India.
0167-7012/89/$ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
54 nical expertise as well as expensive equipments and reagents to perform the sophisticated tests. A microscope and simple reagents are often the only diagnostic facilities available at a P H C laboratory in the developing countries. The staphylococci adherence test (SAT) [2] employing simple light microscope and amoebic antigen slides has been developed by us as a simple and reliable method for the diagnosis of amoebic liver abscess at a P H C laboratory. In this communication, we report on the application of this immunoassay in other clinical forms of amoebiasis. Materials and Methods
Sera The study included the sera from clinical cases with different manifestations of amoebiasis and appropriate control groups who attended the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Hospital, Pondicherry, India.
Intestinal amoebiasis (IA) (25 cases). The diagnosis was established by demonstration of trophozoites of Entamoeba histolytica in the stool of symptomatic cases. The group included different forms of intestinal amoebiasis cases such as acute amoebic dysentery, nondysenteric amoebic colitis and recurrent diarrhoea. Intestinal amoebiasis control (25 cases). The group included cases of other nonamoebic parasitic infections of the intestine, such as protozoa and helminths, resulting either in diarrhoea or vague abdominal pain. The repeat stool examination of the above cases revealed either trophozoites, cysts of other protozoa, e.g., Giardia intestinafis and Entamoeba coli, or ova of helminths but did not show evidence of E. histolytica at any time. Nonsuppurative amoebic hepatitis (AH) (25 cases). This included the cases with pain localized to right hypochondrium and tender liver, clinically diagnosed as nonsuppurative amoebic hepatitis. Amoebic liver abscess (ALA) (55 cases). The cases meeting at least three of the following criteria, as recommended by Chuttani et al. [3] with few modifications, were diagnosed as amoebic liver abscess for inclusion in the study: (i) enlarged and tender liver, bacteriologically sterile pus obtained by aspiration and demonstration of trophozoites ofE. histolytica in the pus and/or biopsy specimen; (ii) positive serological test by IHA; (iii) radiological evidence, raised and fixed right cupola of diaphragm (positive evidence by ultrasonography and radioactive scanning); (iv) response to antiamoebic therapy; and (v) confirmation at autopsy. Nonamoebic fiver disease control (45 cases). The group included cases of pyogenic liver abscess, hydatid liver and other pathological lesions of the liver and hepatitis of various aetiology other than the amoebic as nonamoebic liver disease controls. The diagnosis was achieved by either clinical, surgical, microbiological, serological or corn-
55 bination of these studies.
Healthy control (50 cases). This included apparently healthy subjects from staff, students and blood donors who were apparently in good health and had not suffered from symptoms attributable to amoebiasis during the last 6 months. Antigen slides The antigen slides were prepared from axenic cultures ofE. histolytica in Diamond's medium as described by us earlier [2]. 48 - 72-h-old cultures were washed with physiological saline, fixed with 2o7o w/v glutaraldehyde and adjusted to a concentration of 75 - 100 amoebae by means of a low-power field microscope. Drops of 0.025 ml were delivered on clean dry glass slides, air-dried at room temperature. The slides were stored at - 2 0 ° C until use. Staphylococcus aureus (Cowan's strain I) bearing Protein A (SAPA) SAPA cells were grown on Mueller-Hinton agar at 37 °C and fixed with formalin as described by us earlier [2]. After incubation for 18 h under aeration, SAPA ceils were harvested at 3000 × g for 10 min and washed thrice in phosphate-buffered saline (PBS) (pH 7.2) containing 0.05°70 sodium azide (NAN3). The pellet was fixed in 10 vols. with 1.5% formaldehyde in PBS at room temperature for 90 min, washed thrice in buffer, resuspended in 10 vols. of the buffer containing 0.05% N a N 3 and heated at 80 °C for 5 min. The bacteria were again harvested and washed twice. A 10°70 suspension of staphylococci in PBS with 0.1070 NaN 3 was then prepared and stored in small volumes of different aliquots at - 2 0 °C until used.
Test SAT was performed as described by us earlier [2]. The antigen slides were rinsed in PBS just before use. The sera to be tested were used at several dilutions, starting at 1/2. 25 ~1 of serum samples were added to each spot of E. histolytica previously dried out on the glass slides, ensuring it to cover the entire surface of the spot. The slides were incubated at 37 °C for 30 min in a moist chamber, followed by washing thrice with PBS, keeping the duration of each washing up to 5 min. The slide was uniformly covered with the o p t i m u m dilutions of SAPA suspensions and reincubated at 37°C for 30 min. Following incubation, the slides were rinsed gently in PBS as described earlier and fixed in 2°7o v/v formaldehyde for 5 min. The fixed slides were then washed gently with tap-water; followed by staining with the Giemsa method for 30 min. The stained slides were washed and dried at room temperature. The stained smears were examined under a low-power light microscope, followed by high-power and oiMmmersion lens examination without coverslips. Appropriate controls were put for the test as described earlier. Result
In a Giemsa-stained slide, E. histolytica appears blue and the cocci violet on examination by light microscopy. The positive reaction is graded according to the criteria recommended by Parija et al. [2]. In the SAT-positive slide, staphylococci are observed
56 TABLE 1 DISTRIBUTION OF IMMUNOREACTIV1TY OF AMOEBIASIS AND CONTROL SERA IN SAT Clinical diagnosis
Cases (n)
Reciprocal antibody titre
2048 1 Intestinal amoebiasis 2 Intestinal amoebiasis control 3 Nonsuppurative amoebic hepatitis 4 Amoebic liver abscess 5 Nonamoebic liver disease controls 6 Healthy control
1 0 2 4 512
Number positive at
256
128
64
32
16
8
<8
~32
25
0
2
1
2
4
6
3
1
3
3
18
25
0
0
0
0
0
0
0
8
8
9
0
25
1
1
1
4
3
5
3
4
1
2
18
55
2
5
8
6
14
12
5
1
0
2
52
45 50
0 0
0 0
0 0
0 0
0 0
1 0
1 3
1 13
15 11
27 23
2 3
to bind on the wall and entire surface of E. histolytica (strongly positive) or a chain or ring of staphylococci completely encircling the parasite (positive) or clusters of bacteria irregularly scattered on the surface around the parasite (weakly positive). In a SAT-negative slide, bald E. h&tolytica without any staphylococci sticking on its surface or wall were observed. The results of SAT on the sera from different groups of amoebiasis and controls are summarised in Table 1. An antibody titre of 1 : 32 (mean of normal _+ 2 SD) and above was considered to be diagnostic of the amoebic disease [2]. This cut-off titre was obtained after screening the sera from the normal population residing in Pondicherry, endemic for amoebiasis and sera from known amoebic liver abscess cases. At this diagnostic titre, 52 of 55 (94.5°70) A L A sera, 18 of 25 (7207o) A H sera and 18 of 25 (7207o) IA sera were positive by the assay with antibody titres ranging from 32 to 2048. Amongst the controls, two of 45 (4.4o7o) nonamoebic liver disease controls and three of 50 (6%) healthy controls showed positive reactions. None of the sera from intestinal amoebiasis control showed any false positive reactions. Discussion The binding of intact strain of S. aureus (Cowan's strain I) (SAPA) bearing protein A to surface o f E . histolytica coated with specific amoebic antibodies, forms the basis of SAT [2]. The use of SAPA to detect virus in tissue culture [4] and demonstration of specific antibodies reacting with surface of trophozoites of free-living amoebae [5] and Trypanosoma cruzi [6] have been demonstrated earlier in similar studies. SAT described by us is comparable in its sensitivity and specificity with that of the I H A though less sensitive than the ELISA to demonstrate anti E. histolytica antibodies in serodiagnosis of amoebic liver abscess in our earlier study [2]. The reagents (antigen slides, SAPA suspension) needed for the assay are stable on long period of storage
57
either at - 2 0 _+ 4 °C or at room temperature; inexpensive and are easily available [2]. Results of the present study show that specific amoebic antibodies could be demonstrated in all the groups of amoebiasis and their control sera by SAT (Table 1). The test could discriminate between the geometrical mean titre of antibodies in sera of different groups of amoebiasis cases and controls. The assay showed a good level of sensitivity and specificity in diagnosis of different groups of amoebiasis in the present study. SAT is reproducible and requires inexpensive and easily available reagents, minimal technical expertise and ordinary light microscope which could be made available in any PHC laboratory. This test is simple as well as reliable, hence has the potential for its wide application in serodiagnosis of amoebiasis at a PHC laboratory in the developing countries. References 1 WHO (1985) Amoebiasis and its control. Bull. W.H.O. 63, 417-426. 2 Parija, S. C., Kasinathan, S. and Rao, R. S. (1988) Staphylococci adherence test (SAT): a novel immunoassay for serodiagnosis of amoebic liver abscess at primary health centre level. Ann Natl. Acad. Med. Sci. (India) 23, 191-203. 3 Chunani, P.N., Pathania, N.S. and Sharma, I.D. (1963) Amoebic abscess of the liver. An analysis of 135 cases. J. Ind. Med. Assoc. 40, 489-492. 4 Huang, A. S. and Okarie, G. (1978) Rapid diagnosis using surface analysis by bacterial adherence. Lancet 2, 1146-1149. 5 Culbertson, C.G. and Harper, K. (1980) Surface coagglutination with formalised, stained protein A staphylococci in the immunological study of three pathogenic amoebae. Am. J. Trop. Med. Hyg. 29, 785- 794. 6 Camargo, E. P., Manei, D. M., Yoshida, N. and Caulada, L. (1983) Staphylococci adherence to trypanosomas exposed to immune sera as a method for the diagnosis of Chagas disease. Trans. R. Soc. Trop. Med. Hyg. 77, 825-827.
53
Elsevier MIMET 00322
Staphylococci adherence test for serodiagnosis of amoebiasis S.C. Parija l, S. K a s i n a t h a n 2 a n d R.S. R a o 1 1Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India," 2Department of Biology, Pondicherry University, Pondicherry, India (Received 21 December 1988; revision received 8 April 1989; accepted l0 April 1989)
Summary The staphylococci adherence test (SAT) using axenic amoebic antigen slides and Staphylococcus aureus (Cowan's strain I) bearing Protein A was adapted for diagnosis of different clinical manifestations of amoebiasis. The test was sensitive and specific. At a diagnostic antibody titre of 1 : 32, SAT could detect 94°7o amoebic liver abscess, 7207o of amoebic hepatitis and 72070 of intestinal amoebiasis cases. Amongst the controls, no false positive reaction was observed in IA control sera. However, two (4.407o) and three (607o) sera demonstrated false positive reactions from the nonamoebic liver disease controls and healthy controls, respectively. Since the test required only a simple light microscope and common reagents, the simplicity and economy of the method would allow its adaptation at small and primary-health-centre laboratories in the developing countries.
Key words: Amoebiasis; Laboratory diagnosis; SAT; Serodiagnosis
Introduction Demonstration of anti E. histolytica antibodies is reliable for diagnosis of the intestinal and invasive extra-intestinal manifestations (i.e., amoebic liver abscess) of amoebiasis. Indirect haemagglutination (IHA), counter-current immunoelectrophoresis (CIEP), double diffusion in agar, indirect fluorescent antibody (IFA) and enzymelinked immunosorbent assay (ELISA) are the immunoassays most commonly used in serodiagnosis of amoebiasis [1]. In spite of their high sensitivity and specificity, there is still need for the development of a simple yet reliable test for serodiagnosis of amoebiasis at field conditions or small laboratories such as at a primary health centre (PHC). These laboratories, particularly in the developing countries, lack enough tech-
Correspondence to: S.C. Parija, Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry 605006, India.
0167-7012/89/$ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
54 nical expertise as well as expensive equipments and reagents to perform the sophisticated tests. A microscope and simple reagents are often the only diagnostic facilities available at a P H C laboratory in the developing countries. The staphylococci adherence test (SAT) [2] employing simple light microscope and amoebic antigen slides has been developed by us as a simple and reliable method for the diagnosis of amoebic liver abscess at a P H C laboratory. In this communication, we report on the application of this immunoassay in other clinical forms of amoebiasis. Materials and Methods
Sera The study included the sera from clinical cases with different manifestations of amoebiasis and appropriate control groups who attended the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Hospital, Pondicherry, India.
Intestinal amoebiasis (IA) (25 cases). The diagnosis was established by demonstration of trophozoites of Entamoeba histolytica in the stool of symptomatic cases. The group included different forms of intestinal amoebiasis cases such as acute amoebic dysentery, nondysenteric amoebic colitis and recurrent diarrhoea. Intestinal amoebiasis control (25 cases). The group included cases of other nonamoebic parasitic infections of the intestine, such as protozoa and helminths, resulting either in diarrhoea or vague abdominal pain. The repeat stool examination of the above cases revealed either trophozoites, cysts of other protozoa, e.g., Giardia intestinafis and Entamoeba coli, or ova of helminths but did not show evidence of E. histolytica at any time. Nonsuppurative amoebic hepatitis (AH) (25 cases). This included the cases with pain localized to right hypochondrium and tender liver, clinically diagnosed as nonsuppurative amoebic hepatitis. Amoebic liver abscess (ALA) (55 cases). The cases meeting at least three of the following criteria, as recommended by Chuttani et al. [3] with few modifications, were diagnosed as amoebic liver abscess for inclusion in the study: (i) enlarged and tender liver, bacteriologically sterile pus obtained by aspiration and demonstration of trophozoites ofE. histolytica in the pus and/or biopsy specimen; (ii) positive serological test by IHA; (iii) radiological evidence, raised and fixed right cupola of diaphragm (positive evidence by ultrasonography and radioactive scanning); (iv) response to antiamoebic therapy; and (v) confirmation at autopsy. Nonamoebic fiver disease control (45 cases). The group included cases of pyogenic liver abscess, hydatid liver and other pathological lesions of the liver and hepatitis of various aetiology other than the amoebic as nonamoebic liver disease controls. The diagnosis was achieved by either clinical, surgical, microbiological, serological or corn-
55 bination of these studies.
Healthy control (50 cases). This included apparently healthy subjects from staff, students and blood donors who were apparently in good health and had not suffered from symptoms attributable to amoebiasis during the last 6 months. Antigen slides The antigen slides were prepared from axenic cultures ofE. histolytica in Diamond's medium as described by us earlier [2]. 48 - 72-h-old cultures were washed with physiological saline, fixed with 2o7o w/v glutaraldehyde and adjusted to a concentration of 75 - 100 amoebae by means of a low-power field microscope. Drops of 0.025 ml were delivered on clean dry glass slides, air-dried at room temperature. The slides were stored at - 2 0 ° C until use. Staphylococcus aureus (Cowan's strain I) bearing Protein A (SAPA) SAPA cells were grown on Mueller-Hinton agar at 37 °C and fixed with formalin as described by us earlier [2]. After incubation for 18 h under aeration, SAPA ceils were harvested at 3000 × g for 10 min and washed thrice in phosphate-buffered saline (PBS) (pH 7.2) containing 0.05°70 sodium azide (NAN3). The pellet was fixed in 10 vols. with 1.5% formaldehyde in PBS at room temperature for 90 min, washed thrice in buffer, resuspended in 10 vols. of the buffer containing 0.05% N a N 3 and heated at 80 °C for 5 min. The bacteria were again harvested and washed twice. A 10°70 suspension of staphylococci in PBS with 0.1070 NaN 3 was then prepared and stored in small volumes of different aliquots at - 2 0 °C until used.
Test SAT was performed as described by us earlier [2]. The antigen slides were rinsed in PBS just before use. The sera to be tested were used at several dilutions, starting at 1/2. 25 ~1 of serum samples were added to each spot of E. histolytica previously dried out on the glass slides, ensuring it to cover the entire surface of the spot. The slides were incubated at 37 °C for 30 min in a moist chamber, followed by washing thrice with PBS, keeping the duration of each washing up to 5 min. The slide was uniformly covered with the o p t i m u m dilutions of SAPA suspensions and reincubated at 37°C for 30 min. Following incubation, the slides were rinsed gently in PBS as described earlier and fixed in 2°7o v/v formaldehyde for 5 min. The fixed slides were then washed gently with tap-water; followed by staining with the Giemsa method for 30 min. The stained slides were washed and dried at room temperature. The stained smears were examined under a low-power light microscope, followed by high-power and oiMmmersion lens examination without coverslips. Appropriate controls were put for the test as described earlier. Result
In a Giemsa-stained slide, E. histolytica appears blue and the cocci violet on examination by light microscopy. The positive reaction is graded according to the criteria recommended by Parija et al. [2]. In the SAT-positive slide, staphylococci are observed
56 TABLE 1 DISTRIBUTION OF IMMUNOREACTIV1TY OF AMOEBIASIS AND CONTROL SERA IN SAT Clinical diagnosis
Cases (n)
Reciprocal antibody titre
2048 1 Intestinal amoebiasis 2 Intestinal amoebiasis control 3 Nonsuppurative amoebic hepatitis 4 Amoebic liver abscess 5 Nonamoebic liver disease controls 6 Healthy control
1 0 2 4 512
Number positive at
256
128
64
32
16
8
<8
~32
25
0
2
1
2
4
6
3
1
3
3
18
25
0
0
0
0
0
0
0
8
8
9
0
25
1
1
1
4
3
5
3
4
1
2
18
55
2
5
8
6
14
12
5
1
0
2
52
45 50
0 0
0 0
0 0
0 0
0 0
1 0
1 3
1 13
15 11
27 23
2 3
to bind on the wall and entire surface of E. histolytica (strongly positive) or a chain or ring of staphylococci completely encircling the parasite (positive) or clusters of bacteria irregularly scattered on the surface around the parasite (weakly positive). In a SAT-negative slide, bald E. h&tolytica without any staphylococci sticking on its surface or wall were observed. The results of SAT on the sera from different groups of amoebiasis and controls are summarised in Table 1. An antibody titre of 1 : 32 (mean of normal _+ 2 SD) and above was considered to be diagnostic of the amoebic disease [2]. This cut-off titre was obtained after screening the sera from the normal population residing in Pondicherry, endemic for amoebiasis and sera from known amoebic liver abscess cases. At this diagnostic titre, 52 of 55 (94.5°70) A L A sera, 18 of 25 (7207o) A H sera and 18 of 25 (7207o) IA sera were positive by the assay with antibody titres ranging from 32 to 2048. Amongst the controls, two of 45 (4.4o7o) nonamoebic liver disease controls and three of 50 (6%) healthy controls showed positive reactions. None of the sera from intestinal amoebiasis control showed any false positive reactions. Discussion The binding of intact strain of S. aureus (Cowan's strain I) (SAPA) bearing protein A to surface o f E . histolytica coated with specific amoebic antibodies, forms the basis of SAT [2]. The use of SAPA to detect virus in tissue culture [4] and demonstration of specific antibodies reacting with surface of trophozoites of free-living amoebae [5] and Trypanosoma cruzi [6] have been demonstrated earlier in similar studies. SAT described by us is comparable in its sensitivity and specificity with that of the I H A though less sensitive than the ELISA to demonstrate anti E. histolytica antibodies in serodiagnosis of amoebic liver abscess in our earlier study [2]. The reagents (antigen slides, SAPA suspension) needed for the assay are stable on long period of storage
57
either at - 2 0 _+ 4 °C or at room temperature; inexpensive and are easily available [2]. Results of the present study show that specific amoebic antibodies could be demonstrated in all the groups of amoebiasis and their control sera by SAT (Table 1). The test could discriminate between the geometrical mean titre of antibodies in sera of different groups of amoebiasis cases and controls. The assay showed a good level of sensitivity and specificity in diagnosis of different groups of amoebiasis in the present study. SAT is reproducible and requires inexpensive and easily available reagents, minimal technical expertise and ordinary light microscope which could be made available in any PHC laboratory. This test is simple as well as reliable, hence has the potential for its wide application in serodiagnosis of amoebiasis at a PHC laboratory in the developing countries. References 1 WHO (1985) Amoebiasis and its control. Bull. W.H.O. 63, 417-426. 2 Parija, S. C., Kasinathan, S. and Rao, R. S. (1988) Staphylococci adherence test (SAT): a novel immunoassay for serodiagnosis of amoebic liver abscess at primary health centre level. Ann Natl. Acad. Med. Sci. (India) 23, 191-203. 3 Chunani, P.N., Pathania, N.S. and Sharma, I.D. (1963) Amoebic abscess of the liver. An analysis of 135 cases. J. Ind. Med. Assoc. 40, 489-492. 4 Huang, A. S. and Okarie, G. (1978) Rapid diagnosis using surface analysis by bacterial adherence. Lancet 2, 1146-1149. 5 Culbertson, C.G. and Harper, K. (1980) Surface coagglutination with formalised, stained protein A staphylococci in the immunological study of three pathogenic amoebae. Am. J. Trop. Med. Hyg. 29, 785- 794. 6 Camargo, E. P., Manei, D. M., Yoshida, N. and Caulada, L. (1983) Staphylococci adherence to trypanosomas exposed to immune sera as a method for the diagnosis of Chagas disease. Trans. R. Soc. Trop. Med. Hyg. 77, 825-827.