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Immunoperoxidase Staining of Leptospires in Blood and Urine
Zbl. Bakt. H yg., I. Abt. Orig. A 254, 534-539 (1983) Royal Tropi cal Institute, Depart ment of Tropical Hygiene, 1092 AD Amsterdam, The Netherlands
Immunoperoxidase Staining of Leptospires in Blood and Urine Irnmunperoxidase-Parbung von Leptospiren in HInt und Harn W.J. T ERPSTRA , J. JABBOURY-POST EMA, and H. KOR VER With 2 Figures' Received November 21, 1982
Abstract Immunoperoxidase staining of leptospircs is a relati vely simple method giving satisfacto ry results. Leptospires can be identified by the specificity of the serological reaction and by their characteristic morphology. The method could contr ibute to a rapid diagnosis by the direct demonstration of leptospires in clinical specimens. Zusammenfassung Die Immunperoxidase-Earbung von Leptospiren ist eine verhaltnisrnatiig einfache Methode und gibt gute Erfolge. Leprospi ren konnen ident ifiziert werden rnirtels der Spezifitat der serologischen Reaktion und der charakteristischen Morpho logie. Der direkte Nachweis von Leprospiren in klinischen Proben konnte zur schnellen Diagnostik beitragen. Introduction Direct dem on strati on of the causative agent in acute leptosp irosis is valu able for a rapid dia gn osis. H owever , cur rent method s for th e detection of leptospires in clini cal spec ime ns such as blood , liquor c. s. and urine ha ve disad vantages: leptospires are poorl y sta ined with con ventional bacteriological sta ins, dark field microscopy is not very reli able, becau se of possible errors by leptosp ira-like artifacts, silver-staining is too complicated and not very reli able either, culturing is too timeconsuming to be of value for a rapid diagnosis (Shotts, 1976). Immunofluorescence stai ning is an imp ro vement (Sheldon, 1953; Whi te and Ristic, 1959). However in many situat ion s immunofluorescence stai n ing is also rather co mp licated as it requires sophisticated optical equipment. Immunop eroxidase sta ining is comp ar able to immuno fluo rescence but ordinar y light mi cro scopy is sufficient for the ex am ination of stained materi al. T he technique h as pr o ved to be va lua ble in histop athology (Taylor, 1978; Heyd erman, 1979; Farr and Nakan e, 1981), also in experimentalleptospira infections (Cox and Twigg, 1981).
Immunoperoxidase Staining of Leptospires
535
Immunoperoxidase statrung of leptospires in clinical material could be an improvement, also in comparison with immunofluorescence. In this study we have tried immunoperoxidase staining of leptospires from a culture which had been mixed with blood or urine. This enabled a quantitative approach to the detection of leptospires in the specimens. In the second place we tried immunoperoxidase staining of leptospires in a blood specimen of an experimentally infected animal and in a urine specimen of a naturally infected animal.
Materials and Methods
Specimens Six blood specimens from seronegative blood donors and ten urine specimens from human patients were mixed with a culture of Leptospira interrogans serovar copenbageni (strain Wijnberg). From the blood a thin smear was made on a microscope slide. Five ml of urine was centrifuged for 5 min at 3000 x g. From the sediment a smear was made on a slide. A golden hamster was experimentally infected with a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae. Five days after inoculation a thin smear was made of blood obtained by heart puncture. A urine specimen was collected from a cow suspected of leptospirosis. The urine sediment was smeared on a slide. Part of the urine was cultured. After approximately one week leptospires were growing in the culture which were subsequently identified as serovar hardjo,
Staining procedure 1. The slide with blood or urine was dried and fixed for 5 min in methanol.
2. The slide was incubated for 30 min in PBS pH 7.2 containing Tween 20 (0.05%) and bovine serum albumin fraction V (BSA) (0.5%) and rabbit anti-Wijnberg serum 1/100 (conventionally prepared by intravenous immunisation) at 37°C. 3. The slide was washed 2 x 5 min in PBS containing Tween 20 (0.05%). 4. The slide was incubated for 30 min at 37 °C in PBS containing Tween and BSA and peroxidase labelled sheep anti-rabbit serum (Institut Pasteur) 1/100. 5. The slide was washed for 2 x 5 min in PBS Tween. 6. The slide was incubated for 30 min with the substrate at room temperature. Substrate: dissolve 4 mg 3-amino-9-ethylcarbazol (Sigma) in 0.5 ml N, Nvdimethyl-formamide. Add aqua dest. ad 10 ml. Bring pH to 5 with HCII N. Prior to use add 0.1 ml of freshly prepared H,O, 1%. (Van Raamsdonk et aI., 1977). 7. The slide was washed for 5 min in running tap water. 8. The slide was dried and mounted in glycerin. The cover slip was sealed with nail polish. The slide was examined under the microscope with magnification 1000 X for a standard period of 15 min before being discarded as negative. The leptospires were clearly visible by the reddish brown stain of the carbazol and their characteristic morphology. To test the specificity of the reaction: a) Strain Wijnberg was also stained by antiserum Poi, Celledoni, Hardjoprajitno, Jez Bratislava and Patoc 1. b) Strains R. G. A., Poi, Celledoni, Hond Utrecht IV, Mus 127, Salinem, Cynopteri, Rachmat, Jez Bratislava, Pomona, Moskva V, Hebdomadis, Sari, van Tienen, Mitis Johnson, CZ 214 K, LT 821, Patoc I, CH 11, ICF Ranarum and Hardjoprajitno were stained with antiserum Wijnberg. c) Strains were incubated with a rabbit negative control serum.
536
W.J. Terpstra, J.Jabbou ry-Postema, and H.Korver
Results
In a dilution of 1/100 rabbit antiseru m against Wijnberg was able to stain the homolo gous strain Wi jnberg as well as several other strai ns including sapro phy tic stra in Pato c I. Vice versa, other antisera were able to stai n in thi s dilution st rain Wijn berg. Apparently in th is dilution cro ss-reactions were st ro ng enough to make th e test genusspecific. N o stai ning wa s ob served after incubation with negative control seru m.
Table 1. Proportion of positive blood smears in 6 samples with different concentrations of leprospires
Leptospires ml- l (1-10) (1-10) (1-10) (1-10) (1-10) (1-10)
X 107 x 10· X 10 5 x 104 x l a' x 102
Blood smear positive 6/6 6/6 6/6 2 /6 0 /6 0/6
Table 2. Proporti on of positive urine sediments in 10 samples with different concentrations of leptospires . Lcpto sp ir es rnl "
Urine sediment positive
(1- 10) X 10 7 (1- 10) x 10· (1-10) x 105 (1-10) X 10 4 (1-1 0) x 103 (1-10) X 102
10/10 10 /10 10/10 10 /10 8/10 4 /10
The results of the quantitative examination are presented in T able 1 and 2. In blood leptospires could be found to a density of approximately 105 per ml. In urine the threshold level was lower, app roximately 103 per ml. Fig. 1 shows leptospires in th e blood of a golden hamster experimentally infected with sero var icterohaemorrhagiae. Fig. 2 shows leptospires in th e urine of a cow naturally infected with serovar bardio, Both serovars we re sta ined with anti-W ijnberg antiseru m which emphasizes th e broad crossreactivity of th e antiseru m. The cow 's urine had been pres erved with formaline, final concentrati on approximately 1%. Several da ys after pr eser vati on th e staining results with pre served leprospires wer e still satisfactory.
Immunoperoxidase Staining of Leptospires
537
Fig. 1. Leptospira interrogans serovar icterohaemorrhagiae in the blood of an experimentally infected golden hamster. Immunoperoxidase staining using carbazol as a substrate.
,
Fig. 2. Leptospira interrogans serovar hardjo in the urine of a cow. Immunoperoxidase staining using carbazol as a substrate. 36 Zbl. Bakt. Hyg., I. Abt. Orig. A 254
538
W. J. Terpstra, J.Jabboury-Postema, and H. Korver
Discussion The indirect immunoperoxidase staining as used in this study is a relatively simple technique which combines the specificity of a serological test with staining properties that reveal the characteristic morphology of leptospires, Especially in blood thread-like filaments may be seen by dark field microscopy or by conventional staining methods which are easily mistaken for leptospires. Kathe and Engelhardt (1951) and Wolff (1954) cite numerous references to the problem of a false positive diagnosis because "pseudospirochetes" as these artifacts are called were observed. Immunoperoxidase staining ensures a reliable differentiation between "pseudospirochetes" and true leptospires. The procedure is in many respects similar to immunofluorescence. Immunoperoxidase staining has the advantage that material can be examined by ordinary light microscopy in (semi-)permanent slides. We have mainly used carbazol as a substrate, making semipermanent slides by mounting in glycerin. 4-chloro-1-naphtol was also a suitable substrate to make semi-permanent slides (Nakane, 1968). The use of the more dangerous to handle diaminobenzidine permitted the preparation of permanent slides. Non-carcinogenous p-phenylenediamine (Hanker et aI., 1977) was an unsatisfactory substrate in our hands. Leptospires stained well but the slides invariably suffered from the presence of nonspecific precipitates which were confusingly similar to leptospires. The possible value of immunoperoxidase staining of blood for a rapid diagnosis was demonstrated by the clear presence of leptospires in the blood of an experimentally infected hamster. This was a case of an overwhelming infection and not necessarily representative of the situation in naturally infected humans and animals. Yet the examination of peripheral blood smears has proved to be valuable in the detection of infecting agents other than leptospires in severe infections (Storm, 1981). Presumably the efficacy of the staining test can be improved by differential centrifuging as described by Wolff (1954) to concentrate leptospires from the blood for dark field examination or by examining the plasma layer just above the buffy coat in centrifuged microhaematocrit tubes (Mackintosh and Thompson, 1979). The higher sensitivity of the method for urine than for blood is doubtlessly attributable to concentration by mild centrifugation according to standard procedure for bacteriological examination of urine. The threshold level is similar to the lowest demonstrable densities found by Staak and Schonberg (1980) with immunofluorescence in artificially infected materials. In studies on immunofluorescence rabbit antisera usually showed preference for the homologous antigen but cross-reactions were common (White and Ristic, 1957; Hodges and Ekdahl, 1973; Staak and Schonberg, 1980). In our study rabbit antisera cross-reacted in the dilution we used to a degree that made the test genusspecific. For practical purposes it may be satisfactory to know whether there are leptospires or not. It is doubtful if rabbit antisera even after absorption will be sufficiently specific to enable reliable serotyping. In this respect more is to be expected from the development of monoclonal antibodies. Staining results with formalinised leptospires in urine were as good as with freshly fixed micro-organisms. Urine is an unsuitable environment for leptospires for long survival. For this reason urine should be cultured as soon as possible after voiding which is not always possible. Preservation of urine on the spot with formalin allows a delay in examination by staining until a convenient moment. A series of examinations of positive urine samples is necessary to evaluate the relative merit of immunoperoxidase staining versus culturing.
Immunoperoxidase Staining of Leptospires
539
It is concluded that leptospires can be stained satisfactorily with the irnmunoperoxidase technique. Immunoperoxidase staining may contribute to a more convenient and rapid diagnosis by the demonstration of Ieptospires in clinical specimens.
References 1. Cox, P.]. and G.l. Twigg: Observations on kidney damage in hamsters following a nonicterohaemorrhagiac form of disease resulting from infection by Leptospira interrogans serotype icterohaemorrhagiae. J. Compo Path. 91 (1981) 153-157 2. Farr, A. G. and P. K. Nakane: Immunohistochemistry with enzyme labeled antibodies: a brief review. J. Immunol. Meth. 47 (1981) 129-144 3. Hanker, ].S., P.E. Yates, C.B.Metz, and A.Rustioni: A new specific, sensitive and noncarcinogenic reagent for the demonstration of horseradish peroxidase. Histochem. J. 9 (1977) 789-792 4. Heyderman, E.: Immunoperoxidase technique in histopathology: applications, methods and controls. J. Clin. Path. 32 (1979) 971-978 5. Hodges, R. T. and M. O. Ekdahl: Use of a fluorescent antibody technique for the serological differentiation of leptospiral serotypes in cultures and in bovine urine. N. Z. Vet. J. 21 (1973) 109-115 6. Kathe, ]. and K. Engelhardt: Leptospiren und Pseudospirochaten, Ein Wort zur rnikroskopischen und kulturellen Blutuntersuchung bei Verdacht auf Leptospirose. Z. arztl. Fortbild. Heft 23/24 (1951) 640-644 7. Mackintosh, C. G. and]. C. Thompson: A rapid method for the detection of leptospiraemia. N. Z. Vet. J. 27 (1979) 224-225 8. Nakane, P. K.: Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J. Histochem. Cytochem. 16 (1968) 557-560 9. Raamsdonk, W. van, C. W.Pool, and C.Heyting: Detection of antigens and antibodies by an immuno-peroxidase method applied on thin longitudinal sections of SDS-polyacrylamide gels. J. Immunol. Meth. 17 (1977) 337-348 10. Sheldon, W. H.: Leptospiral antigen demonstrated by the fluorescent antibody technic in human muscle lesions of Leptospira icterohaemorrhagiae. Proc. Soc. expoBioI. (N. Y.) 84 (1953) 165-167 11. Shotts, E. B.: Laboratory diagnosis of Leptospirosis. In: The biology of parasitic spirochetes, Ed. R. C.]ohnson. Academic Press, New York-San Francisco-London (1976) 12. Staak, C. and A. Schiinberg: Ein Beitrag zur Durchfuhrung der Fluoreszenzserologie bei Leptospirose. Zbl. Bakt. Hyg., I. Abt. Orig. A 247 (1980) 138-141 13. Storm, W.: Early detection of bacteremia by peripheral blood smears in critically ill newborns. Acta Paediat. Scand. 70 (1981) 415-416 14. Taylor, C. R.: Immunoperoxidase Techniques. Arch. Path. Lab. Med. 102 (1978) 113-121 15. White, F.H. and M.Ristic: Detection of Leptospira pomona in Guinea Pig and Bovine Urine with Fluorescein-labeled Antibody. J. infect. Dis. 105 (1959) 118-123 16. Wolff, ]. W.: The laboratory diagnosis of leptospirosis. Charles C. Thomas, Springfield, Ill./U.S.A. (1954)
Dr. W.] Terpstra, Royal Tropical Institute, Department of Tropical Hygiene, Mauritskade 57, NL-iOn AD Amsterdam, The Netherlands
Immunoperoxidase Staining of Leptospires in Blood and Urine Irnmunperoxidase-Parbung von Leptospiren in HInt und Harn W.J. T ERPSTRA , J. JABBOURY-POST EMA, and H. KOR VER With 2 Figures' Received November 21, 1982
Abstract Immunoperoxidase staining of leptospircs is a relati vely simple method giving satisfacto ry results. Leptospires can be identified by the specificity of the serological reaction and by their characteristic morphology. The method could contr ibute to a rapid diagnosis by the direct demonstration of leptospires in clinical specimens. Zusammenfassung Die Immunperoxidase-Earbung von Leptospiren ist eine verhaltnisrnatiig einfache Methode und gibt gute Erfolge. Leprospi ren konnen ident ifiziert werden rnirtels der Spezifitat der serologischen Reaktion und der charakteristischen Morpho logie. Der direkte Nachweis von Leprospiren in klinischen Proben konnte zur schnellen Diagnostik beitragen. Introduction Direct dem on strati on of the causative agent in acute leptosp irosis is valu able for a rapid dia gn osis. H owever , cur rent method s for th e detection of leptospires in clini cal spec ime ns such as blood , liquor c. s. and urine ha ve disad vantages: leptospires are poorl y sta ined with con ventional bacteriological sta ins, dark field microscopy is not very reli able, becau se of possible errors by leptosp ira-like artifacts, silver-staining is too complicated and not very reli able either, culturing is too timeconsuming to be of value for a rapid diagnosis (Shotts, 1976). Immunofluorescence stai ning is an imp ro vement (Sheldon, 1953; Whi te and Ristic, 1959). However in many situat ion s immunofluorescence stai n ing is also rather co mp licated as it requires sophisticated optical equipment. Immunop eroxidase sta ining is comp ar able to immuno fluo rescence but ordinar y light mi cro scopy is sufficient for the ex am ination of stained materi al. T he technique h as pr o ved to be va lua ble in histop athology (Taylor, 1978; Heyd erman, 1979; Farr and Nakan e, 1981), also in experimentalleptospira infections (Cox and Twigg, 1981).
Immunoperoxidase Staining of Leptospires
535
Immunoperoxidase statrung of leptospires in clinical material could be an improvement, also in comparison with immunofluorescence. In this study we have tried immunoperoxidase staining of leptospires from a culture which had been mixed with blood or urine. This enabled a quantitative approach to the detection of leptospires in the specimens. In the second place we tried immunoperoxidase staining of leptospires in a blood specimen of an experimentally infected animal and in a urine specimen of a naturally infected animal.
Materials and Methods
Specimens Six blood specimens from seronegative blood donors and ten urine specimens from human patients were mixed with a culture of Leptospira interrogans serovar copenbageni (strain Wijnberg). From the blood a thin smear was made on a microscope slide. Five ml of urine was centrifuged for 5 min at 3000 x g. From the sediment a smear was made on a slide. A golden hamster was experimentally infected with a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae. Five days after inoculation a thin smear was made of blood obtained by heart puncture. A urine specimen was collected from a cow suspected of leptospirosis. The urine sediment was smeared on a slide. Part of the urine was cultured. After approximately one week leptospires were growing in the culture which were subsequently identified as serovar hardjo,
Staining procedure 1. The slide with blood or urine was dried and fixed for 5 min in methanol.
2. The slide was incubated for 30 min in PBS pH 7.2 containing Tween 20 (0.05%) and bovine serum albumin fraction V (BSA) (0.5%) and rabbit anti-Wijnberg serum 1/100 (conventionally prepared by intravenous immunisation) at 37°C. 3. The slide was washed 2 x 5 min in PBS containing Tween 20 (0.05%). 4. The slide was incubated for 30 min at 37 °C in PBS containing Tween and BSA and peroxidase labelled sheep anti-rabbit serum (Institut Pasteur) 1/100. 5. The slide was washed for 2 x 5 min in PBS Tween. 6. The slide was incubated for 30 min with the substrate at room temperature. Substrate: dissolve 4 mg 3-amino-9-ethylcarbazol (Sigma) in 0.5 ml N, Nvdimethyl-formamide. Add aqua dest. ad 10 ml. Bring pH to 5 with HCII N. Prior to use add 0.1 ml of freshly prepared H,O, 1%. (Van Raamsdonk et aI., 1977). 7. The slide was washed for 5 min in running tap water. 8. The slide was dried and mounted in glycerin. The cover slip was sealed with nail polish. The slide was examined under the microscope with magnification 1000 X for a standard period of 15 min before being discarded as negative. The leptospires were clearly visible by the reddish brown stain of the carbazol and their characteristic morphology. To test the specificity of the reaction: a) Strain Wijnberg was also stained by antiserum Poi, Celledoni, Hardjoprajitno, Jez Bratislava and Patoc 1. b) Strains R. G. A., Poi, Celledoni, Hond Utrecht IV, Mus 127, Salinem, Cynopteri, Rachmat, Jez Bratislava, Pomona, Moskva V, Hebdomadis, Sari, van Tienen, Mitis Johnson, CZ 214 K, LT 821, Patoc I, CH 11, ICF Ranarum and Hardjoprajitno were stained with antiserum Wijnberg. c) Strains were incubated with a rabbit negative control serum.
536
W.J. Terpstra, J.Jabbou ry-Postema, and H.Korver
Results
In a dilution of 1/100 rabbit antiseru m against Wijnberg was able to stain the homolo gous strain Wi jnberg as well as several other strai ns including sapro phy tic stra in Pato c I. Vice versa, other antisera were able to stai n in thi s dilution st rain Wijn berg. Apparently in th is dilution cro ss-reactions were st ro ng enough to make th e test genusspecific. N o stai ning wa s ob served after incubation with negative control seru m.
Table 1. Proportion of positive blood smears in 6 samples with different concentrations of leprospires
Leptospires ml- l (1-10) (1-10) (1-10) (1-10) (1-10) (1-10)
X 107 x 10· X 10 5 x 104 x l a' x 102
Blood smear positive 6/6 6/6 6/6 2 /6 0 /6 0/6
Table 2. Proporti on of positive urine sediments in 10 samples with different concentrations of leptospires . Lcpto sp ir es rnl "
Urine sediment positive
(1- 10) X 10 7 (1- 10) x 10· (1-10) x 105 (1-10) X 10 4 (1-1 0) x 103 (1-10) X 102
10/10 10 /10 10/10 10 /10 8/10 4 /10
The results of the quantitative examination are presented in T able 1 and 2. In blood leptospires could be found to a density of approximately 105 per ml. In urine the threshold level was lower, app roximately 103 per ml. Fig. 1 shows leptospires in th e blood of a golden hamster experimentally infected with sero var icterohaemorrhagiae. Fig. 2 shows leptospires in th e urine of a cow naturally infected with serovar bardio, Both serovars we re sta ined with anti-W ijnberg antiseru m which emphasizes th e broad crossreactivity of th e antiseru m. The cow 's urine had been pres erved with formaline, final concentrati on approximately 1%. Several da ys after pr eser vati on th e staining results with pre served leprospires wer e still satisfactory.
Immunoperoxidase Staining of Leptospires
537
Fig. 1. Leptospira interrogans serovar icterohaemorrhagiae in the blood of an experimentally infected golden hamster. Immunoperoxidase staining using carbazol as a substrate.
,
Fig. 2. Leptospira interrogans serovar hardjo in the urine of a cow. Immunoperoxidase staining using carbazol as a substrate. 36 Zbl. Bakt. Hyg., I. Abt. Orig. A 254
538
W. J. Terpstra, J.Jabboury-Postema, and H. Korver
Discussion The indirect immunoperoxidase staining as used in this study is a relatively simple technique which combines the specificity of a serological test with staining properties that reveal the characteristic morphology of leptospires, Especially in blood thread-like filaments may be seen by dark field microscopy or by conventional staining methods which are easily mistaken for leptospires. Kathe and Engelhardt (1951) and Wolff (1954) cite numerous references to the problem of a false positive diagnosis because "pseudospirochetes" as these artifacts are called were observed. Immunoperoxidase staining ensures a reliable differentiation between "pseudospirochetes" and true leptospires. The procedure is in many respects similar to immunofluorescence. Immunoperoxidase staining has the advantage that material can be examined by ordinary light microscopy in (semi-)permanent slides. We have mainly used carbazol as a substrate, making semipermanent slides by mounting in glycerin. 4-chloro-1-naphtol was also a suitable substrate to make semi-permanent slides (Nakane, 1968). The use of the more dangerous to handle diaminobenzidine permitted the preparation of permanent slides. Non-carcinogenous p-phenylenediamine (Hanker et aI., 1977) was an unsatisfactory substrate in our hands. Leptospires stained well but the slides invariably suffered from the presence of nonspecific precipitates which were confusingly similar to leptospires. The possible value of immunoperoxidase staining of blood for a rapid diagnosis was demonstrated by the clear presence of leptospires in the blood of an experimentally infected hamster. This was a case of an overwhelming infection and not necessarily representative of the situation in naturally infected humans and animals. Yet the examination of peripheral blood smears has proved to be valuable in the detection of infecting agents other than leptospires in severe infections (Storm, 1981). Presumably the efficacy of the staining test can be improved by differential centrifuging as described by Wolff (1954) to concentrate leptospires from the blood for dark field examination or by examining the plasma layer just above the buffy coat in centrifuged microhaematocrit tubes (Mackintosh and Thompson, 1979). The higher sensitivity of the method for urine than for blood is doubtlessly attributable to concentration by mild centrifugation according to standard procedure for bacteriological examination of urine. The threshold level is similar to the lowest demonstrable densities found by Staak and Schonberg (1980) with immunofluorescence in artificially infected materials. In studies on immunofluorescence rabbit antisera usually showed preference for the homologous antigen but cross-reactions were common (White and Ristic, 1957; Hodges and Ekdahl, 1973; Staak and Schonberg, 1980). In our study rabbit antisera cross-reacted in the dilution we used to a degree that made the test genusspecific. For practical purposes it may be satisfactory to know whether there are leptospires or not. It is doubtful if rabbit antisera even after absorption will be sufficiently specific to enable reliable serotyping. In this respect more is to be expected from the development of monoclonal antibodies. Staining results with formalinised leptospires in urine were as good as with freshly fixed micro-organisms. Urine is an unsuitable environment for leptospires for long survival. For this reason urine should be cultured as soon as possible after voiding which is not always possible. Preservation of urine on the spot with formalin allows a delay in examination by staining until a convenient moment. A series of examinations of positive urine samples is necessary to evaluate the relative merit of immunoperoxidase staining versus culturing.
Immunoperoxidase Staining of Leptospires
539
It is concluded that leptospires can be stained satisfactorily with the irnmunoperoxidase technique. Immunoperoxidase staining may contribute to a more convenient and rapid diagnosis by the demonstration of Ieptospires in clinical specimens.
References 1. Cox, P.]. and G.l. Twigg: Observations on kidney damage in hamsters following a nonicterohaemorrhagiac form of disease resulting from infection by Leptospira interrogans serotype icterohaemorrhagiae. J. Compo Path. 91 (1981) 153-157 2. Farr, A. G. and P. K. Nakane: Immunohistochemistry with enzyme labeled antibodies: a brief review. J. Immunol. Meth. 47 (1981) 129-144 3. Hanker, ].S., P.E. Yates, C.B.Metz, and A.Rustioni: A new specific, sensitive and noncarcinogenic reagent for the demonstration of horseradish peroxidase. Histochem. J. 9 (1977) 789-792 4. Heyderman, E.: Immunoperoxidase technique in histopathology: applications, methods and controls. J. Clin. Path. 32 (1979) 971-978 5. Hodges, R. T. and M. O. Ekdahl: Use of a fluorescent antibody technique for the serological differentiation of leptospiral serotypes in cultures and in bovine urine. N. Z. Vet. J. 21 (1973) 109-115 6. Kathe, ]. and K. Engelhardt: Leptospiren und Pseudospirochaten, Ein Wort zur rnikroskopischen und kulturellen Blutuntersuchung bei Verdacht auf Leptospirose. Z. arztl. Fortbild. Heft 23/24 (1951) 640-644 7. Mackintosh, C. G. and]. C. Thompson: A rapid method for the detection of leptospiraemia. N. Z. Vet. J. 27 (1979) 224-225 8. Nakane, P. K.: Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J. Histochem. Cytochem. 16 (1968) 557-560 9. Raamsdonk, W. van, C. W.Pool, and C.Heyting: Detection of antigens and antibodies by an immuno-peroxidase method applied on thin longitudinal sections of SDS-polyacrylamide gels. J. Immunol. Meth. 17 (1977) 337-348 10. Sheldon, W. H.: Leptospiral antigen demonstrated by the fluorescent antibody technic in human muscle lesions of Leptospira icterohaemorrhagiae. Proc. Soc. expoBioI. (N. Y.) 84 (1953) 165-167 11. Shotts, E. B.: Laboratory diagnosis of Leptospirosis. In: The biology of parasitic spirochetes, Ed. R. C.]ohnson. Academic Press, New York-San Francisco-London (1976) 12. Staak, C. and A. Schiinberg: Ein Beitrag zur Durchfuhrung der Fluoreszenzserologie bei Leptospirose. Zbl. Bakt. Hyg., I. Abt. Orig. A 247 (1980) 138-141 13. Storm, W.: Early detection of bacteremia by peripheral blood smears in critically ill newborns. Acta Paediat. Scand. 70 (1981) 415-416 14. Taylor, C. R.: Immunoperoxidase Techniques. Arch. Path. Lab. Med. 102 (1978) 113-121 15. White, F.H. and M.Ristic: Detection of Leptospira pomona in Guinea Pig and Bovine Urine with Fluorescein-labeled Antibody. J. infect. Dis. 105 (1959) 118-123 16. Wolff, ]. W.: The laboratory diagnosis of leptospirosis. Charles C. Thomas, Springfield, Ill./U.S.A. (1954)
Dr. W.] Terpstra, Royal Tropical Institute, Department of Tropical Hygiene, Mauritskade 57, NL-iOn AD Amsterdam, The Netherlands