- Email: [email protected]
Spontaneous healing of Leishmama braziliensis braziliensis skin ulcers
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICIKE AND HYGIENE (1984) 78, CORRESPONXYCE
Improvements centrifugation
in the miniature anion exchange technique for detecting trypanosomes in domestic pigs Despite considerable advances over the last few years, the visual detection of low grade parasitaemias of trypanosomes, especially Trypanosoma brucei gambiense, is still difficult. Therefore during present studies on human and animal trypanosomiasis in West Africa, three methods are simultaneously employed in order to detect infection and to isolate the parasites, namely the Haematocrit Centrifugation Technique (HCT; WOO, 1970)! the miniature anion exchange centrifugation techmque (mAECT; LUMSDEN et aZ., 1979) and the sub-inoculation of blood into Mastomys natalensis (MEHLITZ, 1978). Though the mAEC-technique was very efficient in the search for patients with sleeping- sickness in Liberia as well as for the demonstration of low parasitaemias in the circulating blood of antelopes experimentally infected with T. b. gambiense (SACHS, 1983), considerable difficulties were experienced with its application for detecting trypanosomes in domestic pigs, even when parasites were clearly shown by the HCT. The problems persisted when fresh solutions were prepared, the pH checked and the ionic strength appropriate for pig blood (LANHAM & GODFREY, 1970) was used for both equilibration and elution. A significant improvement was brought about by replacing the cylindrical cellulose sponge (Spontex “Moppets”; LUMSDEN et al., 1977) which holds the DEAE-cellulose in the syringe by a cylindrical disc cut from swabs of Pur-Zellin (Hartmann A. G., Heidenheim, Germany), a highly-bleached surgical cellulose. The trypanosomes from identical blood samples now passed through the Pur-Zellin whereas relatively few or no parasites passed through the sponge plug. The experimental evidence suggests that there are inconsistencies in the properties of the “Spontex”sponge material which may be associated with the low ionic strength buffers used for separating trypanosomes from pig blood. Therefore, the replacement of the sponge with a surgical cellulose pad is to be recommended. R. Bernhard-Nocht-Institute, Deaartment of Veterinarv Medicine. Be=khard-Nocht-Strasse 74, ’ D-2000-Hamburg-#, Federal Republic of Germany.
SACHS
References
Lanham, S. M. & Godfrey, D. G. (1970). Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Experimental Parasitology, 28, 521-534. Lumsden, W. H. R., Kimber, C. D., Evans, D. A. & Doig, S. J. (1979). Typanosoma brucei: miniature anionexchange centrifugation technique for detection of low parasitaemias: adaptation for field use. Transactions of the Royal Society of Tropical Medicine 312-317.
and Hygiene, 73,
Lumsden, W. H. R., Kimber, C. D. & Strange, M. (1977). Typanosoma brucei: detection of low parasitaemias in mice by a miniature anion-exchangeicentrifugation technique. Transactions of the Royal Society of Tropical Medicine and Hygiene, 71, 421-424.
561
Mehlitz. D. (1978). Untersuchuneen zur Emufanelichkeit von ‘Mastdmys natalensis Mr T&panosoma (i’ypkozoon) bmei gambiense. Tropenmedizin und Parasitologic, 29,
101-107. Sachs. R. (1983). A comoarison between the sensitivitv of the Bui‘fy Coat Microscopy and the DEAE-Celluiose Column Technique for the detection of low grade parasitaemia in four antelopes experimentally infected with TryDanosoma brucei gambiense. Abstracts of the 4th Japanes-e-/German Cooperative Symposium on Protozoan Diseases.DiisseldorfiGermany, 14.18.6.1983,pp. 21-27. Woo, P. T. K. (1970). The haematocrit centrifuge technique for the diagnosis of African trypanosomiasis. Acta Tropica, 27, 384-386.
Accepted for publication Spontaneous
10th February,
1984.
healing of Leishmania braziliensis braziliensis skin ulcers DE BELDER (1978) studied retrospectively a group of patients in Tres Braces, Bahia, Brazil, an endemic area of Leishmania braziliensis brazilknsis transmission, who had never had an injection or pills for their leishmanial skin ulcers. He found 50% healed in six months and 80% in one year. NETTO et al. (in press) in a recent study of local concepts relating to mucocutaneous leishmaniasis list five local powders, four infusions of leaves, seven other chemicals and various forms of heat all used as local remedies none of which are of proven efficacy. The reason for such a variety is that many leishmanial ulcers do heal spontaneously in spite of whatever you put on them. We briefly describe below three patients with active leishmanial lesions in TrCs Braces who refused any effective drug treatment but healed. Although no parasite was isolated by hamster inoculation all had compatible histology and positive leishmanin skin tests and circulating anti-leishmanial fluorescent antibodies. A 16-year-old boy (LTB 001) had a 10 cm circular buttock lesion which had grown slowly over 10 years. He had taken a course of terramycin initially. Although healed in the centre the border was of an active verrucose type. Within eight months this border healed to form a uniform scar and his IFA titre of l/40 became negative. He has had no recurrence in six years. The father of an g-year-old girl (LTB 040) refused further iniections for his daughter after nenicillin had failed to heal a 1.5 cm ulcer-below the ieft knee. Six months later the ulcer had healed spontaneously. There has been no recurrence of clinical lesion or positive serology over five years. LTB 0175 is the reference number of a 17-year-old woman who had two ulcers on her right leg of three weeks duration (1.3 to 2.4 cm in diameter) during the ninth month of her pregnancy. Treatment was advised after delivery but when seen three months later the lesions had completely healed and during a year of follow up there was no recurrence. While most patients in T&s Braces will persist with chronic large ulcers for months or years unless antimonials are given, as these short case histories indicate, healing can- occur without specific drug theraov and antibodv titres fall. The mechanism of sponianeous remiss& is unknown, we have no way
562
TRANSACTIONS OFTHE ROYALSOCIETY OF TROPICAL MEDICINEAND HYGIENE(1984)78, CORRESPONDENCE
of predicting its occurrence and as the three patients illustrate it takes place over a varying time scale. It becomesvery difficult to evaluate drug effect although we find lesions close rapidly with antimonials (CUENTAS, 1984).
P. D. MARSDEN
The cellulose acetate zymograms were run at 220~ for 20 min using a cooled system. Tank and membrane buffer were as described for GPl (KREUTZER& CHRISTENSEN,1980). Development was carried out using the substrate mixture in agar gel and the bands on the developed membrane were revealed using fast violet B. ASAT zymograms on all of 19 strains of N.
M. S. TADA A. C. BARRETO C. C. CUBA Ntkleo Tropical, University of Brasilia, Brasilia, Brazil
References Cuentas, E. A. L. (1984). Estudo clinic0 evolutivo da leishmanioseem area endemicade Leishmania braziliensis brazihensis, T&s BraGos, Bahia. Master’s Thesis, University of Brasilia. De Belder, M. et al. (1978). Report of the Cambridge Medical Expedition to Brasil 1977. Mimeographed dot:ument. Netto, E. M., Tada, M. S. Lago, E., Golightly, L., Kalter, D. C., .Barreto, A. C. & Marsden, P. D. (1984). Conceitos de uma popula@o local a respeito de leishmaniose mucocutinea em uma area endemica. Revista da Sociedade Brasileira
Accepted
for publication
de Medic&a
Tropical,
(in press).
16th March, 1983.
Differentiation between Naegleria fowleri and N. lovaniensis using isoenzyme electrophoresis of aspartate aminotransferase
Primary amoebic meningoencephalitis (PAM) is a rapidly fatal disease which follows invasion of the brain by the small free-living amoeba Naegleria fowleri. The disease usually follows inhalation of contaminated
water during swimming
(MARTINEZ,
1983). To confirm the occurrence of the pathogen in recreational waters, it is necessary to differentiate it from the non-pathogenic species N. lovaniensis (STEVENS et al., 1980)which is serologically, morphologically and culturally similar (DE JONCKHEERE et al., 1975). Enzyme electrophoresis patterns are invaluable for this purpose (DAGGET & NERAD, 1983). Although its use in trypanosome characterization is well established (LANHAM et aE., 1981) aspartate aminotransferase(ASAT) has not before been investigated in NaegZeria. The enzyme is extensively studied in relation to liver disorders and coronary heart diseasein all departments of clinical chemistry. We wish to report from a study of environmental isolates of free-living amoebaefrom local hot springs, following a death due to primary amoebic meningoencephalitis (PAM), that cellulose acetatezymograms in respect of ASAT (KOHN, 1976; KREUTZER & CHRISTENSEN,1980; LANHAM et al., 1981) will distinguish clearly and rapidly between N. fowZeri and N. Zovaniensis. Isolates were made by primary culture at 43°C and were identified as Naegleria spp. by their morphology and the production of amoebo-flagellates
at 43°C. Further characterization was by electron microscopy (STEVENSet al., 1980) and by cellulose acetate zymograrns in respect of glucose phosphate
isomerase (GPI) (MANN & WARHURST,1983)
Migration --f Origin Densitometer scans from ASAT zymograms lovaniensis ------N. fowleri; -N. Zovaniensis showed two clearly separate bands. In sharp contrast all of eight strains of mouse-pathogenic N. fowZeri produced a single band with an electrophoretic mobility intermediate between the isoenzymes from N. Zovaniensis.These differential features are seen clearly in densitometer scans of the zymograms (representative diagram attached). As well as being rapid, zymography offers the advantage of greatly reducing the number of mice which must be sacrificed when searching for environmental N. fowleri. This may be important as the basis for instituting preventive measures against further cases of PAM occuring in a locality where an index case has been identified.
SIMON KILVINGTON PAUL MANN Public Health Laboratory, Bath, UK
DAVID WARHURST Amoebiasis Unit, Hospital for Tropical Diseases, London, NW1 OPE References Dagger, P. M. & Nerad, T. A. (1983). The biochemical identification of Vahlkampiiid amoebae.Journal of Protozoology, 30, 126-128. De Jonckheere, J., Van Dijk, P. & Van der Voorde, H. (1975). The effect of thermal pollution on’the distribution of Naegleria fowlek Journal of Hygiene, 75, 7-13. Kohn, J. (1976). In: Chromatographic and Electrophoretic Techniques, Vol. II. Smith I. (Editor). London: Heinemann, pp. 90-137.
Improvements centrifugation
in the miniature anion exchange technique for detecting trypanosomes in domestic pigs Despite considerable advances over the last few years, the visual detection of low grade parasitaemias of trypanosomes, especially Trypanosoma brucei gambiense, is still difficult. Therefore during present studies on human and animal trypanosomiasis in West Africa, three methods are simultaneously employed in order to detect infection and to isolate the parasites, namely the Haematocrit Centrifugation Technique (HCT; WOO, 1970)! the miniature anion exchange centrifugation techmque (mAECT; LUMSDEN et aZ., 1979) and the sub-inoculation of blood into Mastomys natalensis (MEHLITZ, 1978). Though the mAEC-technique was very efficient in the search for patients with sleeping- sickness in Liberia as well as for the demonstration of low parasitaemias in the circulating blood of antelopes experimentally infected with T. b. gambiense (SACHS, 1983), considerable difficulties were experienced with its application for detecting trypanosomes in domestic pigs, even when parasites were clearly shown by the HCT. The problems persisted when fresh solutions were prepared, the pH checked and the ionic strength appropriate for pig blood (LANHAM & GODFREY, 1970) was used for both equilibration and elution. A significant improvement was brought about by replacing the cylindrical cellulose sponge (Spontex “Moppets”; LUMSDEN et al., 1977) which holds the DEAE-cellulose in the syringe by a cylindrical disc cut from swabs of Pur-Zellin (Hartmann A. G., Heidenheim, Germany), a highly-bleached surgical cellulose. The trypanosomes from identical blood samples now passed through the Pur-Zellin whereas relatively few or no parasites passed through the sponge plug. The experimental evidence suggests that there are inconsistencies in the properties of the “Spontex”sponge material which may be associated with the low ionic strength buffers used for separating trypanosomes from pig blood. Therefore, the replacement of the sponge with a surgical cellulose pad is to be recommended. R. Bernhard-Nocht-Institute, Deaartment of Veterinarv Medicine. Be=khard-Nocht-Strasse 74, ’ D-2000-Hamburg-#, Federal Republic of Germany.
SACHS
References
Lanham, S. M. & Godfrey, D. G. (1970). Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Experimental Parasitology, 28, 521-534. Lumsden, W. H. R., Kimber, C. D., Evans, D. A. & Doig, S. J. (1979). Typanosoma brucei: miniature anionexchange centrifugation technique for detection of low parasitaemias: adaptation for field use. Transactions of the Royal Society of Tropical Medicine 312-317.
and Hygiene, 73,
Lumsden, W. H. R., Kimber, C. D. & Strange, M. (1977). Typanosoma brucei: detection of low parasitaemias in mice by a miniature anion-exchangeicentrifugation technique. Transactions of the Royal Society of Tropical Medicine and Hygiene, 71, 421-424.
561
Mehlitz. D. (1978). Untersuchuneen zur Emufanelichkeit von ‘Mastdmys natalensis Mr T&panosoma (i’ypkozoon) bmei gambiense. Tropenmedizin und Parasitologic, 29,
101-107. Sachs. R. (1983). A comoarison between the sensitivitv of the Bui‘fy Coat Microscopy and the DEAE-Celluiose Column Technique for the detection of low grade parasitaemia in four antelopes experimentally infected with TryDanosoma brucei gambiense. Abstracts of the 4th Japanes-e-/German Cooperative Symposium on Protozoan Diseases.DiisseldorfiGermany, 14.18.6.1983,pp. 21-27. Woo, P. T. K. (1970). The haematocrit centrifuge technique for the diagnosis of African trypanosomiasis. Acta Tropica, 27, 384-386.
Accepted for publication Spontaneous
10th February,
1984.
healing of Leishmania braziliensis braziliensis skin ulcers DE BELDER (1978) studied retrospectively a group of patients in Tres Braces, Bahia, Brazil, an endemic area of Leishmania braziliensis brazilknsis transmission, who had never had an injection or pills for their leishmanial skin ulcers. He found 50% healed in six months and 80% in one year. NETTO et al. (in press) in a recent study of local concepts relating to mucocutaneous leishmaniasis list five local powders, four infusions of leaves, seven other chemicals and various forms of heat all used as local remedies none of which are of proven efficacy. The reason for such a variety is that many leishmanial ulcers do heal spontaneously in spite of whatever you put on them. We briefly describe below three patients with active leishmanial lesions in TrCs Braces who refused any effective drug treatment but healed. Although no parasite was isolated by hamster inoculation all had compatible histology and positive leishmanin skin tests and circulating anti-leishmanial fluorescent antibodies. A 16-year-old boy (LTB 001) had a 10 cm circular buttock lesion which had grown slowly over 10 years. He had taken a course of terramycin initially. Although healed in the centre the border was of an active verrucose type. Within eight months this border healed to form a uniform scar and his IFA titre of l/40 became negative. He has had no recurrence in six years. The father of an g-year-old girl (LTB 040) refused further iniections for his daughter after nenicillin had failed to heal a 1.5 cm ulcer-below the ieft knee. Six months later the ulcer had healed spontaneously. There has been no recurrence of clinical lesion or positive serology over five years. LTB 0175 is the reference number of a 17-year-old woman who had two ulcers on her right leg of three weeks duration (1.3 to 2.4 cm in diameter) during the ninth month of her pregnancy. Treatment was advised after delivery but when seen three months later the lesions had completely healed and during a year of follow up there was no recurrence. While most patients in T&s Braces will persist with chronic large ulcers for months or years unless antimonials are given, as these short case histories indicate, healing can- occur without specific drug theraov and antibodv titres fall. The mechanism of sponianeous remiss& is unknown, we have no way
562
TRANSACTIONS OFTHE ROYALSOCIETY OF TROPICAL MEDICINEAND HYGIENE(1984)78, CORRESPONDENCE
of predicting its occurrence and as the three patients illustrate it takes place over a varying time scale. It becomesvery difficult to evaluate drug effect although we find lesions close rapidly with antimonials (CUENTAS, 1984).
P. D. MARSDEN
The cellulose acetate zymograms were run at 220~ for 20 min using a cooled system. Tank and membrane buffer were as described for GPl (KREUTZER& CHRISTENSEN,1980). Development was carried out using the substrate mixture in agar gel and the bands on the developed membrane were revealed using fast violet B. ASAT zymograms on all of 19 strains of N.
M. S. TADA A. C. BARRETO C. C. CUBA Ntkleo Tropical, University of Brasilia, Brasilia, Brazil
References Cuentas, E. A. L. (1984). Estudo clinic0 evolutivo da leishmanioseem area endemicade Leishmania braziliensis brazihensis, T&s BraGos, Bahia. Master’s Thesis, University of Brasilia. De Belder, M. et al. (1978). Report of the Cambridge Medical Expedition to Brasil 1977. Mimeographed dot:ument. Netto, E. M., Tada, M. S. Lago, E., Golightly, L., Kalter, D. C., .Barreto, A. C. & Marsden, P. D. (1984). Conceitos de uma popula@o local a respeito de leishmaniose mucocutinea em uma area endemica. Revista da Sociedade Brasileira
Accepted
for publication
de Medic&a
Tropical,
(in press).
16th March, 1983.
Differentiation between Naegleria fowleri and N. lovaniensis using isoenzyme electrophoresis of aspartate aminotransferase
Primary amoebic meningoencephalitis (PAM) is a rapidly fatal disease which follows invasion of the brain by the small free-living amoeba Naegleria fowleri. The disease usually follows inhalation of contaminated
water during swimming
(MARTINEZ,
1983). To confirm the occurrence of the pathogen in recreational waters, it is necessary to differentiate it from the non-pathogenic species N. lovaniensis (STEVENS et al., 1980)which is serologically, morphologically and culturally similar (DE JONCKHEERE et al., 1975). Enzyme electrophoresis patterns are invaluable for this purpose (DAGGET & NERAD, 1983). Although its use in trypanosome characterization is well established (LANHAM et aE., 1981) aspartate aminotransferase(ASAT) has not before been investigated in NaegZeria. The enzyme is extensively studied in relation to liver disorders and coronary heart diseasein all departments of clinical chemistry. We wish to report from a study of environmental isolates of free-living amoebaefrom local hot springs, following a death due to primary amoebic meningoencephalitis (PAM), that cellulose acetatezymograms in respect of ASAT (KOHN, 1976; KREUTZER & CHRISTENSEN,1980; LANHAM et al., 1981) will distinguish clearly and rapidly between N. fowZeri and N. Zovaniensis. Isolates were made by primary culture at 43°C and were identified as Naegleria spp. by their morphology and the production of amoebo-flagellates
at 43°C. Further characterization was by electron microscopy (STEVENSet al., 1980) and by cellulose acetate zymograrns in respect of glucose phosphate
isomerase (GPI) (MANN & WARHURST,1983)
Migration --f Origin Densitometer scans from ASAT zymograms lovaniensis ------N. fowleri; -N. Zovaniensis showed two clearly separate bands. In sharp contrast all of eight strains of mouse-pathogenic N. fowZeri produced a single band with an electrophoretic mobility intermediate between the isoenzymes from N. Zovaniensis.These differential features are seen clearly in densitometer scans of the zymograms (representative diagram attached). As well as being rapid, zymography offers the advantage of greatly reducing the number of mice which must be sacrificed when searching for environmental N. fowleri. This may be important as the basis for instituting preventive measures against further cases of PAM occuring in a locality where an index case has been identified.
SIMON KILVINGTON PAUL MANN Public Health Laboratory, Bath, UK
DAVID WARHURST Amoebiasis Unit, Hospital for Tropical Diseases, London, NW1 OPE References Dagger, P. M. & Nerad, T. A. (1983). The biochemical identification of Vahlkampiiid amoebae.Journal of Protozoology, 30, 126-128. De Jonckheere, J., Van Dijk, P. & Van der Voorde, H. (1975). The effect of thermal pollution on’the distribution of Naegleria fowlek Journal of Hygiene, 75, 7-13. Kohn, J. (1976). In: Chromatographic and Electrophoretic Techniques, Vol. II. Smith I. (Editor). London: Heinemann, pp. 90-137.