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Latex agglutination test (LAT) for antigen detection in the cystic fluid for the diagnosis of cystic echinococcosis
Diagnostic Microbiology and Infectious Disease 45 (2003) 123–126
www.elsevier.com/locate/diagmicrobio
Parasitology
Latex agglutination test (LAT) for antigen detection in the cystic fluid for the diagnosis of cystic echinococcosis Sheela Devi Chandrakesan, Subhash Chandra Parija* Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry, India
Abstract Latex agglutination test (LAT) was standardized and evaluated to detect hydatid antigen in fluid samples aspirated from 6 surgically proved human cases of cystic echinococcosis (CE), 4 suspected human cases of CE (2 cases of cysts in the liver which were not confirmed surgically and 2 cases of pelvic cysts later confirmed as abscesses) and 7 cases of hydatid cysts of liver in cattle. Echinococcus granulosus scolices and hook lets were seen in aspirated fluid by microscopy and the characteristic germinal layer of the cyst wall was demonstrated by histopathology in 6 human hydatid cysts operated and removed by surgery. In case of cattle hydatid liver cysts no scolices or hook lets were seen in aspirated fluid as they were sterile cysts but characteristic laminated layer of the cyst wall was demonstrated by histopathology of these cysts. The LAT could detect antigen in fluid samples collected from all 6 human cases of surgically proved CE and 7 cases of hydatid cyst liver in cattle, thus showing sensitivity of 100%. The LAT could detect antigen in fluid samples collected from 2 suspected cases of CE liver in humans, which were not operated. The LAT was found to be specific .No cross reactivity was observed .The results of the study showed that LAT could be employed as a simple and rapid diagnostic procedure, as an alternate to microscopy, to confirm the hydatid etiology of a suspected cyst. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Hydatid cyst, the larval stage of Echinococcus granulosus, is the main pathology of cystic echinococcosis (CE) in humans. The hydatid cysts can be found in any organ or tissue of the infected human host but are found most commonly in the lungs or liver. The specific aetiological diagnosis of a hydatid cyst helps in the management of the cases either by surgery or chemotherapy. PAIR (percutaneousaspiration-injection-reaspiration) is the method recommended for the management of inoperable and relatively small hydatid cysts (Bret et al., 1988; Mueller et al., 1985; Pelaez et al., 2000). In such situations hydatid antigen detection in the cystic fluid appears to be very useful for specific diagnosis of hydatid cyst. The detection of hydatid antigen in fluid is an approach, frequently followed as an alternate to the cystic fluid microscopy for scolices and hook lets. The enzyme-linked immunosorbent assay (ELISA) was the first immunoassay to be used for the detection of hydatid antigen in the aspirated cyst fluid (Craig et al., 1986) followed by bacterial Co-agglutination
* Corresponding author. E-mail address: [email protected] (S.C. Parija). Tel.: 0091-413253016; Fax: 0091-413-272067
(Co-A) and counter-current immunoelectrophoresis (CIEP), both of which have been developed and evaluated for the first time in this laboratory for detection of hydatid antigen in the cystic fluid. (Parija et al., 1996; Ravinder et al., 1997). In this communication we standardize and evaluate, and report for the first time the application of latex agglutination test (LAT) as a simple and rapid slide agglutination test for the detection of hydatid antigen in cystic fluid samples.
2. Materials and Methods 2.1. Cases The present study included 6 surgically operated cases of human CE, 4 clinically suspected cases of human CE admitted and managed in the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Hospital, Pondicherry, India and 7 cases of hydatid cysts of liver in cattle obtained from slaughter house. Hydatid cysts were removed from the liver during surgery, from 6 human cases of CE. The hydatid etiology of the cysts was confirmed by histopathological study of the cyst, which showed characteristic germinal layer, and the presence of scolices and hook lets in the aspirated fluid by microscopy. 4 other
0732-8893/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 2 ) 0 0 4 7 9 - 0
124
Sheela Devi C, SC Parija / Diagnostic Microbiology and Infectious Disease 45 (2003) 123–126
human cases were clinically diagnosed as CE. Of these, 2 cysts in the liver were not surgically operated and other two in the pelvis were operated but were found to be as abscess. Hydatid cysts were removed from the liver of 7 cattle from a slaughter house .The fluid aspirated from the hydatid cysts did not show any scolices or hook lets, but histopathological examination of the cyst showed characteristic laminated layer. 2.2. Cystic fluid 5-10 mL of fluid were collected aseptically from 6 cysts of the liver removed by surgery, and also from 4 suspected cases of human CE collected by percutaneous aspiration. Fluid was also collected from 7 cattle hydatid cysts .The fluids were collected in sterile glass vials and stored at -20°C until use. 2.3. Hyper immune hydatid antiserum Hyper immune hydatid antiserum was raised in rabbits as per the procedure described by Shariff et al (1993). Briefly, the human hydatid cyst fluid (HHCF) antigen obtained from a case of CE of liver at surgery was emulsified with equal volume of Freund’s complete adjuvant. Two adult male rabbits (3 to 4kgs) were given 0.5 ml of this emulsion in all four limbs intramuscularly (IM). After six weeks, they were re-injected IM with 0.5 ml each in all the limbs with the same antigen but emulsified in the incomplete adjuvant. After 10days blood samples were taken by ear vein bleeding and monitored for the antibodies to HHCF antigen by indirect hemagglutination (IHA) test (Parija et al., 1986). The animal was bled by heart puncture when the titer of the antibodies was 1 in 1024 or more. The antiserum was purified as per the procedure described by Gottstein (1984). Briefly 1 ml of the cold serum was mixed with 1 ml of cold saline at pH 7. The serumsaline mixture (2 mL) was added drop wise to 2 mL of cold saturated ammonium sulfate (pH 7) with stirring for 30 min on ice and then centrifuged at 3000⫻ g at 0°C. The supernatant was then discarded and the precipitate was suspended in 2 ml of saline and the procedure was repeated until the supernatant was colorless. The final precipitate was suspended in 1 ml and dialyzed against PBS (pH 7.2) to remove all the residual ammonium sulfate. Titer of the purified antiserum was 2048 by the IHA test. 2.4. Examination of cystic fluid The aspirated cystic fluid was centrifuged at 2000⫻ g for 3 min. The centrifuged deposit was examined for scolices and hook lets by light microscopy, while the supernatant was examined for the presence of specific hydatid antigen by LAT and Co-A as described herein.
2.5. Microscopy of cystic fluid A drop of the centrifuged deposit was placed on a clean microscopic glass slide and a cover slip was put on it. The wet mount was examined for scolices and hook lets, first under low power (10x) and then under high power (40x) objective of a compound light microscope. A lacto-phenol cotton blue (LPCB) preparation of centrifuged deposit of the cystic fluid was prepared as per the method described of us earlier (Parija et al., 1997). In this method a drop of LPCB was put on a microscopic glass slide, to which a drop of centrifuged deposit was added and mixed well. The cover slip was put on it and examined by a light microscope as described earlier for direct wet mount of centrifuged deposit. Histopathological examination of the surgically removed cyst from humans and cysts obtained from cattle was also performed. 2.6. Detection of hydatid antigen in cystic fluid The cystic fluids were for the hydatid antigen in parallel by LAT and Co-A. 2.7. LAT The procedure consists of the following steps: (i)
Preparation of latex particle suspension: The polystyrene latex suspension 0.81 m (Sigma) was used in the test. A 1% standardized polysterene latex suspension was prepared by mixing 0.1 ml latex particle suspension with 9.9 ml glycine buffered saline (GBS) (pH 8.4). This was stored at 4°C until used. (ii) Sensitization of latex particles: 1 ml of 1% latex particle suspension was mixed with 1 ml of purified hyper immune antisera raised against HHCF in the rabbits. The mixture was incubated at 37°C for 2 h in the water bath. After incubation, antibody-sensitized latex particles were washed two times with GBS (pH8.4) and centrifuged at 3000⫻ g for 5 min. Then the centrifuged pellet consisting of antibody-sensitized latex particles were emulsified with GBS (pH 8.4) with 1% bovine serum albumin (BSA) to make a 2% suspension. The reagents were stored at 4°C until used. (iii) The test procedure: The LAT was performed on a clean slide which was divided with a glass marking pen into two halves. A drop of cystic fluid to be tested was placed on each half of the slide. An equal volume of sensitized latex reagent was added to the cystic fluid on one half. The same volume of latex particles coated with normal rabbit serum was used as a control and
Sheela Devi C, SC Parija / Diagnostic Microbiology and Infectious Disease 45 (2003) 123–126
125
Table 1 Comparison of fluid microscopy and fluid antigen detection methods in the diagnosis of cystic echinococcosis Subject groups
Surgically confirmed human CE Clincially suspected human CE Cattle hydatid cyst
No. of cases
6 4 7
Histopathology confirmed hydatid cysts
6 Not done 7
Microscopy of cyst fluid positive for scolices and hook lets by
Cystic fluid positive for hydatid antigen by
Direct wet mount
LPCB wet mount
LAT
Co-A
6 0 0
6 0 0
6 2* 7
6 2* 7
* Clinically suspected cases of CE liver. LPCB ⫽ Lacto-phenol cotton blue stain; LAT ⫽ latex agglutination test; Co-A ⫽ Co-agglutination test; CE ⫽ Cystic echinococcosis.
added to the cystic fluid on other half of the slide. The slide was then manually rotated for 2 min and inspected. Agglutination with sensitized latex reagent but not with latex particles coated with normal rabbit serum was considered to be positive. With every batch, a known positive control (known hydatid fluid from a histologically confirmed hydatid cyst) and negative control (soluble antigens of Entamoeba histolytica and Setarria digitata) were included. 2.8. Co-A test The Co-A was used to detect specific hydatid antigen in the cystic fluid as per the method described by us earlier (Parija et al., 1996). Briefly, it consists of preparation of bacterial cells, Staphylococcus aureus (Cowan’s strain1) bearing protein A (SAPA). The SAPA cells were grown on Muller Hinton agar at 37°C for 18 h and then harvested, centrifuged at 3000⫻ g for 10 min and washed 3 times in PBS pH 7.2 containing 0.05% sodium azide. The pellet was fixed in 10 volumes of 1.5% formaldehyde in PBS pH 7.2 at room temperature for 90 min, washed in PBS and re-suspended to 10 volumes of buffer containing 0.05% sodium azide and heated for 5 min at 80°C.The SAPA cells were sensitized with purified hyper immune hydatid antiserum immediately after their preparation .The hydatid antibody sensitized-SAPA cells were stored at 4°C in small aliquots for use in the test. The test was performed on clean glass slide divided with a glass marking pencil into two halves. A drop of cyst fluid to be tested was placed on each half of a glass slide. An equal volume of 2% sensitized SAPA cell suspension was added to the cyst fluid on one half. The same volume of 2% un-sensitized SAPA cell suspension were added to the cyst fluid on the other half as a cell control. The slide was rotated manually for 2 min and inspected. Agglutination with the sensitized SAPA cell suspension and not with the un-sensitized SAPA cell suspension was considered as positive test. Appropriate controls were examined in parallel with each test.
3. Results The characteristic germinal layer was demonstrated in 6 human liver cysts and 7 cattle liver cysts by histopathology thus confirming hydatid etiology of these cysts. Microscopy of wet mount of cyst fluid by direct wet mount as well as by LPCB showed scolices and hook lets in fluid samples collected from all the 6 human hydatid cysts removed by surgery. But no scolices or hook lets were detected in the aspirated fluid of 4 clinically suspected human cases of CE or in the fluid of 7 cattle hydatid cysts. The LAT detected hydatid antigen in all the 6 cases of surgically confirmed human CE cases, in 2 of clinically suspected human CE cases and in all the 7 cases of cattle cysts. The Co-A also detected hydatid antigen in all 6 human cases of CE, in 2 cases of clinically suspected human cases of CE and in all the 7 cases of cattle cysts. Two cases of clinically suspected human CE cases had cysts in the liver. The comparative evaluation of fluid microscopy and antigen detection in CE is summarized in Table 1.
4. Discussion We have evaluated and standardized for the first time the LAT for detection of circulating antigen in the serum for diagnosis of the cases of CE (Sheela Devi and Parija, unpublished report). In the present study the LAT using polyclonal hydatid antibody-sensitized latex particles were employed for the detection of hydatid antigen in the cystic fluid. The LAT was found to be 100% sensitive for detection of antigen in the cystic fluid aspirated from cysts confirmed to be of hydatid etiology by histopathology .The antigen could also be detected in 2 suspected human cases of CE which were negative for scolices and hook lets by microscopy (Table 1). The results of the study compares well with that of the Co-A as demonstrated in the present study (Table 1) and also in the study reported by us earlier (Parija et al., 1996); and with that of ELISA reported by Craig et al (1986). In case of cattle cysts, the cyst fluid was negative for
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Sheela Devi C, SC Parija / Diagnostic Microbiology and Infectious Disease 45 (2003) 123–126
scolices and hook lets by microscopy, as it is well known that more than 90% of the cattle cysts are always sterile. All these 7 cyst which were confirmed to be of hydatid by histopathology were positive for fluid hydatid antigen by the LAT. The LAT in the present study was found to be specific. Fluid aspirated from 2 non-hydatid cysts were negative for hydatid antigen by the test. Antigen detection in cystic fluid to confirm the hydatid etiology of a suspected cyst offers many advantages. First, antigen detection is a useful procedure in cystic fluids in which scolices or hook lets cannot be demonstrated by microscopy. Second it is useful particularly in sterile acephalocysts in humans or in cattle, fluid of which are negative for scolices or hook lets. Finally, antigen detection is more useful when a small volume of fluid is aspirated from suspected hydatid cysts. Although, the diagnostic aspiration is not always advisable because of the associated risk of leak of fluid during aspiration resulting in anaphylaxis or secondary echinococcosis, nevertheless the percutaneous drainage of hydatid cysts (Bastid et al., 1994) or fine needle aspiration cytology (FNAC) of cysts under ultrasonography (Das et al., 1995) are known to have least risk of leakage. In conclusion, detection of hydatid antigen in the cystic fluid by LAT offers many advantages. First, it is a simple and rapid slide agglutination test, the results of which can be obtained within minutes of testing. Second, the test unlike ELISA does not require any expensive reagents, technical expertise or highly equipped laboratory, hence can be carried out as a bed side test in the side clinic, at small health centers and also at field level. This test does not require high technical skill to perform and even can be performed by a paramedical staff. The test is also economical.
References Bastid, C., Aazar, C., Doyer, M., & Sahal, J. (1994). Percutaneous treatment of hydatid cyst under sonographic guidance. Dig Dis Sci, 39, 1576 –1580. Bret, P. M., Fond, A., Bretagnolle, M., Valette, P. J., Thiesse, P., Lambert, R., & Labadie, M. (1988). Percutaneous aspiration and drainage of hydatid cysts in the liver. Radiology, 168, 617– 620. Craig, P. S., Bailey, W., & Nelson, G. S. (1986). Specific test for the identification of cyst fluid samples from suspected human hydatid infections. Trans R Soc Trop Med Hyg, 80, 256 –257. Das, D. K., Bhambani, S., & Pant, C. S. (1995). Ultrasound guided fine needle aspiration cytology: Diagnosis of hydatid disease of abdomen and thorax. Dign Cytopath, 12, 173–176. Gottstein, B. (1984). An immunoassay for the detection of circulating antigen in human echinococcosis. Am J Trop Med Hyg, 33, 1105– 1111. Mueller, P. R., Dawson, S. C., Ferruci, I. T., & Nordi, G. L. (1985). Hepatic echinococcal cyst successful percutaneous drainage. Radiology, 155, 627– 628. Parija, S. C., Mishra, S. R., & Rao, R. S. (1986). Sensitised chick cells in indirect haemagglutination test for echinococcosis. J Med Microb, 38, 231–234. Parija, S. C., Ravinder, P. T., & Shariff, M. (1996). Detection of hydatid antigen by Coagglutination in fluid samples from hydatid cysts. Trans R Soc Trop Med Hyg, 90, 255–256. Parija, S. C., & Ravinder, P. T. (1997). Lactophenol cotton blue stain for the wet mount preparation of hydatid fluid. Trop Doc, 17, 108. Pelaez, V., Kugler, C., Correa, D., Delcarpio, M., Guangir Oli, M., Molina, J., Marcos, B., & Lopez, E. (2000). PAIR as percutaneous treatment of hydatid liver cysts. Acta Trop, 75, 197–202. Ravinder, P. T., & Parija, S. C. (1997). CIEP test for detection of hydatid antigen in fluid samples from hydatid cysts, a preliminary report. Acta Trop, 66, 169 –173. Shariff, G. M., & Parija, S. C. (1993). Coagglutination (Co-A) test for circulating antigen in hydatid disease. J Med Microb, 38, 231–234.
www.elsevier.com/locate/diagmicrobio
Parasitology
Latex agglutination test (LAT) for antigen detection in the cystic fluid for the diagnosis of cystic echinococcosis Sheela Devi Chandrakesan, Subhash Chandra Parija* Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry, India
Abstract Latex agglutination test (LAT) was standardized and evaluated to detect hydatid antigen in fluid samples aspirated from 6 surgically proved human cases of cystic echinococcosis (CE), 4 suspected human cases of CE (2 cases of cysts in the liver which were not confirmed surgically and 2 cases of pelvic cysts later confirmed as abscesses) and 7 cases of hydatid cysts of liver in cattle. Echinococcus granulosus scolices and hook lets were seen in aspirated fluid by microscopy and the characteristic germinal layer of the cyst wall was demonstrated by histopathology in 6 human hydatid cysts operated and removed by surgery. In case of cattle hydatid liver cysts no scolices or hook lets were seen in aspirated fluid as they were sterile cysts but characteristic laminated layer of the cyst wall was demonstrated by histopathology of these cysts. The LAT could detect antigen in fluid samples collected from all 6 human cases of surgically proved CE and 7 cases of hydatid cyst liver in cattle, thus showing sensitivity of 100%. The LAT could detect antigen in fluid samples collected from 2 suspected cases of CE liver in humans, which were not operated. The LAT was found to be specific .No cross reactivity was observed .The results of the study showed that LAT could be employed as a simple and rapid diagnostic procedure, as an alternate to microscopy, to confirm the hydatid etiology of a suspected cyst. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Hydatid cyst, the larval stage of Echinococcus granulosus, is the main pathology of cystic echinococcosis (CE) in humans. The hydatid cysts can be found in any organ or tissue of the infected human host but are found most commonly in the lungs or liver. The specific aetiological diagnosis of a hydatid cyst helps in the management of the cases either by surgery or chemotherapy. PAIR (percutaneousaspiration-injection-reaspiration) is the method recommended for the management of inoperable and relatively small hydatid cysts (Bret et al., 1988; Mueller et al., 1985; Pelaez et al., 2000). In such situations hydatid antigen detection in the cystic fluid appears to be very useful for specific diagnosis of hydatid cyst. The detection of hydatid antigen in fluid is an approach, frequently followed as an alternate to the cystic fluid microscopy for scolices and hook lets. The enzyme-linked immunosorbent assay (ELISA) was the first immunoassay to be used for the detection of hydatid antigen in the aspirated cyst fluid (Craig et al., 1986) followed by bacterial Co-agglutination
* Corresponding author. E-mail address: [email protected] (S.C. Parija). Tel.: 0091-413253016; Fax: 0091-413-272067
(Co-A) and counter-current immunoelectrophoresis (CIEP), both of which have been developed and evaluated for the first time in this laboratory for detection of hydatid antigen in the cystic fluid. (Parija et al., 1996; Ravinder et al., 1997). In this communication we standardize and evaluate, and report for the first time the application of latex agglutination test (LAT) as a simple and rapid slide agglutination test for the detection of hydatid antigen in cystic fluid samples.
2. Materials and Methods 2.1. Cases The present study included 6 surgically operated cases of human CE, 4 clinically suspected cases of human CE admitted and managed in the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Hospital, Pondicherry, India and 7 cases of hydatid cysts of liver in cattle obtained from slaughter house. Hydatid cysts were removed from the liver during surgery, from 6 human cases of CE. The hydatid etiology of the cysts was confirmed by histopathological study of the cyst, which showed characteristic germinal layer, and the presence of scolices and hook lets in the aspirated fluid by microscopy. 4 other
0732-8893/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 2 ) 0 0 4 7 9 - 0
124
Sheela Devi C, SC Parija / Diagnostic Microbiology and Infectious Disease 45 (2003) 123–126
human cases were clinically diagnosed as CE. Of these, 2 cysts in the liver were not surgically operated and other two in the pelvis were operated but were found to be as abscess. Hydatid cysts were removed from the liver of 7 cattle from a slaughter house .The fluid aspirated from the hydatid cysts did not show any scolices or hook lets, but histopathological examination of the cyst showed characteristic laminated layer. 2.2. Cystic fluid 5-10 mL of fluid were collected aseptically from 6 cysts of the liver removed by surgery, and also from 4 suspected cases of human CE collected by percutaneous aspiration. Fluid was also collected from 7 cattle hydatid cysts .The fluids were collected in sterile glass vials and stored at -20°C until use. 2.3. Hyper immune hydatid antiserum Hyper immune hydatid antiserum was raised in rabbits as per the procedure described by Shariff et al (1993). Briefly, the human hydatid cyst fluid (HHCF) antigen obtained from a case of CE of liver at surgery was emulsified with equal volume of Freund’s complete adjuvant. Two adult male rabbits (3 to 4kgs) were given 0.5 ml of this emulsion in all four limbs intramuscularly (IM). After six weeks, they were re-injected IM with 0.5 ml each in all the limbs with the same antigen but emulsified in the incomplete adjuvant. After 10days blood samples were taken by ear vein bleeding and monitored for the antibodies to HHCF antigen by indirect hemagglutination (IHA) test (Parija et al., 1986). The animal was bled by heart puncture when the titer of the antibodies was 1 in 1024 or more. The antiserum was purified as per the procedure described by Gottstein (1984). Briefly 1 ml of the cold serum was mixed with 1 ml of cold saline at pH 7. The serumsaline mixture (2 mL) was added drop wise to 2 mL of cold saturated ammonium sulfate (pH 7) with stirring for 30 min on ice and then centrifuged at 3000⫻ g at 0°C. The supernatant was then discarded and the precipitate was suspended in 2 ml of saline and the procedure was repeated until the supernatant was colorless. The final precipitate was suspended in 1 ml and dialyzed against PBS (pH 7.2) to remove all the residual ammonium sulfate. Titer of the purified antiserum was 2048 by the IHA test. 2.4. Examination of cystic fluid The aspirated cystic fluid was centrifuged at 2000⫻ g for 3 min. The centrifuged deposit was examined for scolices and hook lets by light microscopy, while the supernatant was examined for the presence of specific hydatid antigen by LAT and Co-A as described herein.
2.5. Microscopy of cystic fluid A drop of the centrifuged deposit was placed on a clean microscopic glass slide and a cover slip was put on it. The wet mount was examined for scolices and hook lets, first under low power (10x) and then under high power (40x) objective of a compound light microscope. A lacto-phenol cotton blue (LPCB) preparation of centrifuged deposit of the cystic fluid was prepared as per the method described of us earlier (Parija et al., 1997). In this method a drop of LPCB was put on a microscopic glass slide, to which a drop of centrifuged deposit was added and mixed well. The cover slip was put on it and examined by a light microscope as described earlier for direct wet mount of centrifuged deposit. Histopathological examination of the surgically removed cyst from humans and cysts obtained from cattle was also performed. 2.6. Detection of hydatid antigen in cystic fluid The cystic fluids were for the hydatid antigen in parallel by LAT and Co-A. 2.7. LAT The procedure consists of the following steps: (i)
Preparation of latex particle suspension: The polystyrene latex suspension 0.81 m (Sigma) was used in the test. A 1% standardized polysterene latex suspension was prepared by mixing 0.1 ml latex particle suspension with 9.9 ml glycine buffered saline (GBS) (pH 8.4). This was stored at 4°C until used. (ii) Sensitization of latex particles: 1 ml of 1% latex particle suspension was mixed with 1 ml of purified hyper immune antisera raised against HHCF in the rabbits. The mixture was incubated at 37°C for 2 h in the water bath. After incubation, antibody-sensitized latex particles were washed two times with GBS (pH8.4) and centrifuged at 3000⫻ g for 5 min. Then the centrifuged pellet consisting of antibody-sensitized latex particles were emulsified with GBS (pH 8.4) with 1% bovine serum albumin (BSA) to make a 2% suspension. The reagents were stored at 4°C until used. (iii) The test procedure: The LAT was performed on a clean slide which was divided with a glass marking pen into two halves. A drop of cystic fluid to be tested was placed on each half of the slide. An equal volume of sensitized latex reagent was added to the cystic fluid on one half. The same volume of latex particles coated with normal rabbit serum was used as a control and
Sheela Devi C, SC Parija / Diagnostic Microbiology and Infectious Disease 45 (2003) 123–126
125
Table 1 Comparison of fluid microscopy and fluid antigen detection methods in the diagnosis of cystic echinococcosis Subject groups
Surgically confirmed human CE Clincially suspected human CE Cattle hydatid cyst
No. of cases
6 4 7
Histopathology confirmed hydatid cysts
6 Not done 7
Microscopy of cyst fluid positive for scolices and hook lets by
Cystic fluid positive for hydatid antigen by
Direct wet mount
LPCB wet mount
LAT
Co-A
6 0 0
6 0 0
6 2* 7
6 2* 7
* Clinically suspected cases of CE liver. LPCB ⫽ Lacto-phenol cotton blue stain; LAT ⫽ latex agglutination test; Co-A ⫽ Co-agglutination test; CE ⫽ Cystic echinococcosis.
added to the cystic fluid on other half of the slide. The slide was then manually rotated for 2 min and inspected. Agglutination with sensitized latex reagent but not with latex particles coated with normal rabbit serum was considered to be positive. With every batch, a known positive control (known hydatid fluid from a histologically confirmed hydatid cyst) and negative control (soluble antigens of Entamoeba histolytica and Setarria digitata) were included. 2.8. Co-A test The Co-A was used to detect specific hydatid antigen in the cystic fluid as per the method described by us earlier (Parija et al., 1996). Briefly, it consists of preparation of bacterial cells, Staphylococcus aureus (Cowan’s strain1) bearing protein A (SAPA). The SAPA cells were grown on Muller Hinton agar at 37°C for 18 h and then harvested, centrifuged at 3000⫻ g for 10 min and washed 3 times in PBS pH 7.2 containing 0.05% sodium azide. The pellet was fixed in 10 volumes of 1.5% formaldehyde in PBS pH 7.2 at room temperature for 90 min, washed in PBS and re-suspended to 10 volumes of buffer containing 0.05% sodium azide and heated for 5 min at 80°C.The SAPA cells were sensitized with purified hyper immune hydatid antiserum immediately after their preparation .The hydatid antibody sensitized-SAPA cells were stored at 4°C in small aliquots for use in the test. The test was performed on clean glass slide divided with a glass marking pencil into two halves. A drop of cyst fluid to be tested was placed on each half of a glass slide. An equal volume of 2% sensitized SAPA cell suspension was added to the cyst fluid on one half. The same volume of 2% un-sensitized SAPA cell suspension were added to the cyst fluid on the other half as a cell control. The slide was rotated manually for 2 min and inspected. Agglutination with the sensitized SAPA cell suspension and not with the un-sensitized SAPA cell suspension was considered as positive test. Appropriate controls were examined in parallel with each test.
3. Results The characteristic germinal layer was demonstrated in 6 human liver cysts and 7 cattle liver cysts by histopathology thus confirming hydatid etiology of these cysts. Microscopy of wet mount of cyst fluid by direct wet mount as well as by LPCB showed scolices and hook lets in fluid samples collected from all the 6 human hydatid cysts removed by surgery. But no scolices or hook lets were detected in the aspirated fluid of 4 clinically suspected human cases of CE or in the fluid of 7 cattle hydatid cysts. The LAT detected hydatid antigen in all the 6 cases of surgically confirmed human CE cases, in 2 of clinically suspected human CE cases and in all the 7 cases of cattle cysts. The Co-A also detected hydatid antigen in all 6 human cases of CE, in 2 cases of clinically suspected human cases of CE and in all the 7 cases of cattle cysts. Two cases of clinically suspected human CE cases had cysts in the liver. The comparative evaluation of fluid microscopy and antigen detection in CE is summarized in Table 1.
4. Discussion We have evaluated and standardized for the first time the LAT for detection of circulating antigen in the serum for diagnosis of the cases of CE (Sheela Devi and Parija, unpublished report). In the present study the LAT using polyclonal hydatid antibody-sensitized latex particles were employed for the detection of hydatid antigen in the cystic fluid. The LAT was found to be 100% sensitive for detection of antigen in the cystic fluid aspirated from cysts confirmed to be of hydatid etiology by histopathology .The antigen could also be detected in 2 suspected human cases of CE which were negative for scolices and hook lets by microscopy (Table 1). The results of the study compares well with that of the Co-A as demonstrated in the present study (Table 1) and also in the study reported by us earlier (Parija et al., 1996); and with that of ELISA reported by Craig et al (1986). In case of cattle cysts, the cyst fluid was negative for
126
Sheela Devi C, SC Parija / Diagnostic Microbiology and Infectious Disease 45 (2003) 123–126
scolices and hook lets by microscopy, as it is well known that more than 90% of the cattle cysts are always sterile. All these 7 cyst which were confirmed to be of hydatid by histopathology were positive for fluid hydatid antigen by the LAT. The LAT in the present study was found to be specific. Fluid aspirated from 2 non-hydatid cysts were negative for hydatid antigen by the test. Antigen detection in cystic fluid to confirm the hydatid etiology of a suspected cyst offers many advantages. First, antigen detection is a useful procedure in cystic fluids in which scolices or hook lets cannot be demonstrated by microscopy. Second it is useful particularly in sterile acephalocysts in humans or in cattle, fluid of which are negative for scolices or hook lets. Finally, antigen detection is more useful when a small volume of fluid is aspirated from suspected hydatid cysts. Although, the diagnostic aspiration is not always advisable because of the associated risk of leak of fluid during aspiration resulting in anaphylaxis or secondary echinococcosis, nevertheless the percutaneous drainage of hydatid cysts (Bastid et al., 1994) or fine needle aspiration cytology (FNAC) of cysts under ultrasonography (Das et al., 1995) are known to have least risk of leakage. In conclusion, detection of hydatid antigen in the cystic fluid by LAT offers many advantages. First, it is a simple and rapid slide agglutination test, the results of which can be obtained within minutes of testing. Second, the test unlike ELISA does not require any expensive reagents, technical expertise or highly equipped laboratory, hence can be carried out as a bed side test in the side clinic, at small health centers and also at field level. This test does not require high technical skill to perform and even can be performed by a paramedical staff. The test is also economical.
References Bastid, C., Aazar, C., Doyer, M., & Sahal, J. (1994). Percutaneous treatment of hydatid cyst under sonographic guidance. Dig Dis Sci, 39, 1576 –1580. Bret, P. M., Fond, A., Bretagnolle, M., Valette, P. J., Thiesse, P., Lambert, R., & Labadie, M. (1988). Percutaneous aspiration and drainage of hydatid cysts in the liver. Radiology, 168, 617– 620. Craig, P. S., Bailey, W., & Nelson, G. S. (1986). Specific test for the identification of cyst fluid samples from suspected human hydatid infections. Trans R Soc Trop Med Hyg, 80, 256 –257. Das, D. K., Bhambani, S., & Pant, C. S. (1995). Ultrasound guided fine needle aspiration cytology: Diagnosis of hydatid disease of abdomen and thorax. Dign Cytopath, 12, 173–176. Gottstein, B. (1984). An immunoassay for the detection of circulating antigen in human echinococcosis. Am J Trop Med Hyg, 33, 1105– 1111. Mueller, P. R., Dawson, S. C., Ferruci, I. T., & Nordi, G. L. (1985). Hepatic echinococcal cyst successful percutaneous drainage. Radiology, 155, 627– 628. Parija, S. C., Mishra, S. R., & Rao, R. S. (1986). Sensitised chick cells in indirect haemagglutination test for echinococcosis. J Med Microb, 38, 231–234. Parija, S. C., Ravinder, P. T., & Shariff, M. (1996). Detection of hydatid antigen by Coagglutination in fluid samples from hydatid cysts. Trans R Soc Trop Med Hyg, 90, 255–256. Parija, S. C., & Ravinder, P. T. (1997). Lactophenol cotton blue stain for the wet mount preparation of hydatid fluid. Trop Doc, 17, 108. Pelaez, V., Kugler, C., Correa, D., Delcarpio, M., Guangir Oli, M., Molina, J., Marcos, B., & Lopez, E. (2000). PAIR as percutaneous treatment of hydatid liver cysts. Acta Trop, 75, 197–202. Ravinder, P. T., & Parija, S. C. (1997). CIEP test for detection of hydatid antigen in fluid samples from hydatid cysts, a preliminary report. Acta Trop, 66, 169 –173. Shariff, G. M., & Parija, S. C. (1993). Coagglutination (Co-A) test for circulating antigen in hydatid disease. J Med Microb, 38, 231–234.