- Email: [email protected]
The Montenegro and MIF tests in three cured and one chronic case of human leishmaniasis
536
CORRESPONDENCE
L. donovani (isolated in 1966 and maintained on NNN medium since that date). The cultures were incubated at 24°C in a CO2 incubator and examined at regular intervals. The promastigote to amastigote transformation began at the sixth hour of incubation and was practically complete in 48 hours. The amastigotes were localized in the cytoplasm of the cells, but were also found extracellularly. The number of amastigotes increased tenfold between the 48th and 96th hour. Throughout the development cycle of the Leishmania, the parasitized cells of A. albopicrus represented only a restricted and relatively constant number of the total cells (20 to 40 %). Obtaining the amastigote form of L. donovani in an invertebrate cell line is in itself an important achievement. Although temperature was previously considered a critical factor for determining the morphological stage of the parasite (promastigote at 24”, amastigote at 36”), the amastigote form has now been obtained at 24”. Our results open up interesting practical possibilities: in particular, the easy maintenance of the cell line of A. albopictus will allow immunological studies using the amastigotes, which correspond to the naturally occurring parasite in vertebrates, instead of the promastigotes which rapidly lose their pathogenicity when propagated on NNN medium. Grateful acknowledgements are due to Dr. Y. Robin, Director of the Pasteur Institute, Dakar, for providing us with the cells of A. albopictus, and to Dr. M. S. Ben Rachid of the Pasteur Institute, Tunis, for isolating the strain of L. donovani. We also thank Mrs. J. El Kolli for her technical assistance. We are, etc., JEAN-PIERREDEDET ODETTE-GERMAINE GAUDIN Department of Epidemiology, Department of Virology, Pasteur Institute, Algiers, Algeria References
Lupascu, G., Bossie, A., Dincoulesco, M., Epurean, E. & Profeta, A. (1968). Contributions g 1’Btude de la culture et de l’action cyto-pathogbne de L. a’onovani sur cultures de tissus. Archives Roumaines de Pathologie Exp&imentale et de Microbiologic, 27, 641-650. Mitsuhashi, J. & Maramorosch, K. (1964). Leafhopper tissue culture: embryonic, nymphal and imaginal Boyce tissues from aseptic insects. Contributions. Thompson Institute for Plant Research, 22,435460.
Pipkin, A. C. (1960). Avian embryos and tissue culture in the study of parasitic Protozoa. II. Protozoa other Experimental Parasitology, 9, than Plasmodium. 167-203. Singh, K. R. P. (1967). Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.). Current Science, 36, 506-508. Bancroftian
filariasis
in the Dominican
Republic
SIR-Blood examination of a man, 24 years of age, was carried out in September 1975 on the subject’s arrival in Moscow from the Dominican Republic. On two separate investigations, a thick film of this blood, stained with Giemsa revealed the presence of a number of sheathed microfilariae, with nuclei seemingly not extending to the tip of the tail. Our identification of
the parasite as Wuchereria bancrofti was confirmed by Dr. F. Hawking, to whom we are grateful for his advice. The subject, who had no subjective symptoms of the infection, was adamant in stating that neither he nor any member of his family ever travelled outside the Dominican Republic. Since Dr. F. Hawking stated in one of his reviews on the distribution of filariasis in the Americas, that there is no recent information from the Dominican Republic, we believe that the present note may be of some interest to the readers of the Transactions. We are, etc., A.J. LYSENKO E. A. PAVLOVA Central Institute for Advanced Medical Barrikadnaja, 2, Moscow, U.S.S.R.
Z.I. GLAZIJNOVA Training,
The Montenegro and MIF tests in three cured and one chronic case of human leishmaniasis
%+-Correlation of the Montenegro skin test for delayed hypersensitivity and the production of macrophage migration inhibition factor (MIF) by lymphocytes was determined in four leishmaniasis patients. Two of these patients originally presented cutaneous lesions, one (aged 59) with several ulcers in different areas of the body, and the other (aged 40) with only one lesion on the arm; both had been positive for Leishmania. No parasites or lesions have appeared during the two years since treatment. The other two cases had a history of mucocutaneous leishmaniasis (20 and 27 years of evolution with perforations of the nasal septum). One case, a man 62 years old, had responded to antimonial compounds with disappearance of the parasites and clinical cure. The other, a 42-year-old woman, had not responded to full treatments with either Repodral or Glucantime on four different occasions. This patient recently came again to the hospital and a positive culture for Leishmania braziliensis s.1.was obtained from a nasal septum biopsy. For the MIF test, the techniques of THOR (1971) and of ROCKLIN & DAVID (1971) were combined to obtain macrophages from guinea-pigs and for the production of MIF from the patient’s lymphocytes. Two types of antigens were prepared from culture forms of Costa Rica O-CR strain of Leishmania grown in Senekiie’s medium: a soluble fraction (supemat&t after freezing and thawing and several centrifugations) and an insoluble fraction (residue of the same preparation). Both antigens were adjusted to 50 gamma of nitrogen per ml. The Montenegro tests were performed in the forearm with the insoluble antigen adjusted to 4 gamma of nitrogen per ml. The soluble antigen, in a concentration of 75 gamma of nitrogen per ml, was also used for skin tests performed in the shoulder. In all four patients the Montenegro delayed skin test was strongly positive. The soluble antigen produced only an immediate skin reaction in all patients, in the form of a wheal with pseudopods and erythema, within 10 to 15 minutes. Nevertheless, in the two cases with mucocutaneous leishmaniasis this reaction persisted as a soft infiltrated area for at least 48 hours. With the soluble antigen the degree of migration ranged from 44 to 71% in the three cured cases, whereas in the presence of the insoluble antigen there was complete inhibition
CORRESPONDENCE of migration. On the other hand, the mucocutaneous case which had not responded to treatment presented a migration of 62% (average of three tests) with the insoluble antigen, indicating partial absence of the inhibitory factor and an apparent dichotomy of cellular immune mechanisms. We are, etc., RODRIGOZELED~N ELISA DE PONCE CARLOSPONCE Louisiana State University, International Center for Medical Research and Training, Apartado 10155 San Jose, Costa Rica and Department of Parasitology, University of Costa Rica References
Rocklin, R. E. & David, J. R. (1971). Method of the production of MIP by human blood lymphocytes. In: B. R. Bloom & P. R. Glade (eds.), In vitro methods in cell-mediated immunity. New York: Academic Press, pp. 281-287. Thor, D. E. (1971). The capillary tube migration inhibition technique applied to human peripheral lymphocytes using the guinea pig peritoneal exudate as the indicator cell population. In: B. R. Bloom & P. R. Glade (eds.), In vitro methods in cell-mediated immunity. New York: Academic Press, pp. 273-280.
Comparison
of counting chamber and measured lllms in microlilaria estimation
blood
SIR-The counting chamber was introduced by DENHAM et al. (1971) and given field trials by SOUTHGATE& DESOW~TZ(1971) DE~OWITZ et al. (1973) SOUTHGATE (1973) and DENNIS et al. (1976), who showed this technique- to be more sensitive than the conventional blood film. If the same quantity of blood was examined, then more microfilariae were found in the counting chamber than the conventional measured blood slide. During the course of a survey conducted between the hours of 20.00-01.00 in villages on Choiseul in the Solomon Islands, 60 mm3 of blood were examined from each person (finger prick) by the counting chamber method, as described by DENHAM et al. (1971). When a positive was found, a measured conventional blood slide was taken (finger prick) from the person as soon as possible (within one hour), making three circular films of 20 mm3 each on the same slide. These slides were allowed to dry and were stained with Giemsa in the method adopted by the Malaria Eradication Programme in the Solomons. This is to filter on 3 ‘A Giemsa (in pH 7.2 buffered water), without prior de-haemoglobinization, leave for the particular batch time (about 30 min), briefly blue in distilled water and leave to dry. The slides were then read with a Beck compound microscope at x 60 magnification, examining the whole field. The same microscope was used to count the motile microfilariae in the whole of the counting chamber. Twenty positives were found, so a direct comparison of the two techniques using equal quantities of blood from the same person, could be made. The individual results are given in the table.
Slide Number
537 Microfilaria counts
Difference count. chamber minus blood slide
Counting chamber
Measured blood slide 16 4 3 3
-8
7::
-12 +1
10 11 12 13 14 15 16 17 18 19
8 4 2 8 3 65 4 3 12 5 3 2 3 4 9 3 1 3 2
20
1 2 3 4 5 6 7 8 9
0
-1 +5 0
3 1 5 6 2 6 9 2 3 2
+2 +7 -1 +1 +1 -3 -5 +7 0
19
-1 -16
8
26 3
-24 +5
152
194
-42
There was no significant difference between the two techniques; this result differs from those obtained by SOIJTHGATE& Dssowrrz (1971), Dasowrrz et al. (1973) and SOUTHGATE(1973). It is appreciated that in this region of nocturnally-periodic Wucheria bancrofti, there will be a slight increase in microfilariae with taking the blood slide later than the counting chamber sample. Periodicity tables have been worked out for this area (WEBBER,1975) and, even allowing the maximum period (one hour) between taking the two samples, there is a 12 % discrepancy, which makes no appreciable difference to the results. It would seem that the similar results obtained by the two techniques were due to omitting de-haemoglobinization in staining. DENHAM et al. (1971) thought that microfilariae were lost in this way and examined the washings with a membrane filter, from which they recovered them. There is no loss of definition in reading the slides and species definition can easily be made. These results were obtained during surveys to test the most suitable technique in the conditions of the Solomons, i.e., scattered island communities with nocturnally periodic W. bancrofti and very limited resources. Night time conditions made the counting chamber and Millipore technique difficult, and the further lack of facilities, cost and resistance to venepuncture precluded the latter’s use, except in special circumstances (WEBBER, 1975). Lack of resources and finance also prevented a proper prevalence survey from being made comparing the counting chamber and measured blood slide, but it is still felt that the results are sufficiently valid to recommend the measured blood slide in the prevailing conditions. This technique is still the most widely used and practical method in filariasis surveys and it is suggested here that if the blood films are stained without de-haemoglobinizing them, then greater sensitivity is obtained. I am, etc., R. H. WEBBER Ministry of Health & Welfare, Honiara, Solomon Islands
CORRESPONDENCE
L. donovani (isolated in 1966 and maintained on NNN medium since that date). The cultures were incubated at 24°C in a CO2 incubator and examined at regular intervals. The promastigote to amastigote transformation began at the sixth hour of incubation and was practically complete in 48 hours. The amastigotes were localized in the cytoplasm of the cells, but were also found extracellularly. The number of amastigotes increased tenfold between the 48th and 96th hour. Throughout the development cycle of the Leishmania, the parasitized cells of A. albopicrus represented only a restricted and relatively constant number of the total cells (20 to 40 %). Obtaining the amastigote form of L. donovani in an invertebrate cell line is in itself an important achievement. Although temperature was previously considered a critical factor for determining the morphological stage of the parasite (promastigote at 24”, amastigote at 36”), the amastigote form has now been obtained at 24”. Our results open up interesting practical possibilities: in particular, the easy maintenance of the cell line of A. albopictus will allow immunological studies using the amastigotes, which correspond to the naturally occurring parasite in vertebrates, instead of the promastigotes which rapidly lose their pathogenicity when propagated on NNN medium. Grateful acknowledgements are due to Dr. Y. Robin, Director of the Pasteur Institute, Dakar, for providing us with the cells of A. albopictus, and to Dr. M. S. Ben Rachid of the Pasteur Institute, Tunis, for isolating the strain of L. donovani. We also thank Mrs. J. El Kolli for her technical assistance. We are, etc., JEAN-PIERREDEDET ODETTE-GERMAINE GAUDIN Department of Epidemiology, Department of Virology, Pasteur Institute, Algiers, Algeria References
Lupascu, G., Bossie, A., Dincoulesco, M., Epurean, E. & Profeta, A. (1968). Contributions g 1’Btude de la culture et de l’action cyto-pathogbne de L. a’onovani sur cultures de tissus. Archives Roumaines de Pathologie Exp&imentale et de Microbiologic, 27, 641-650. Mitsuhashi, J. & Maramorosch, K. (1964). Leafhopper tissue culture: embryonic, nymphal and imaginal Boyce tissues from aseptic insects. Contributions. Thompson Institute for Plant Research, 22,435460.
Pipkin, A. C. (1960). Avian embryos and tissue culture in the study of parasitic Protozoa. II. Protozoa other Experimental Parasitology, 9, than Plasmodium. 167-203. Singh, K. R. P. (1967). Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.). Current Science, 36, 506-508. Bancroftian
filariasis
in the Dominican
Republic
SIR-Blood examination of a man, 24 years of age, was carried out in September 1975 on the subject’s arrival in Moscow from the Dominican Republic. On two separate investigations, a thick film of this blood, stained with Giemsa revealed the presence of a number of sheathed microfilariae, with nuclei seemingly not extending to the tip of the tail. Our identification of
the parasite as Wuchereria bancrofti was confirmed by Dr. F. Hawking, to whom we are grateful for his advice. The subject, who had no subjective symptoms of the infection, was adamant in stating that neither he nor any member of his family ever travelled outside the Dominican Republic. Since Dr. F. Hawking stated in one of his reviews on the distribution of filariasis in the Americas, that there is no recent information from the Dominican Republic, we believe that the present note may be of some interest to the readers of the Transactions. We are, etc., A.J. LYSENKO E. A. PAVLOVA Central Institute for Advanced Medical Barrikadnaja, 2, Moscow, U.S.S.R.
Z.I. GLAZIJNOVA Training,
The Montenegro and MIF tests in three cured and one chronic case of human leishmaniasis
%+-Correlation of the Montenegro skin test for delayed hypersensitivity and the production of macrophage migration inhibition factor (MIF) by lymphocytes was determined in four leishmaniasis patients. Two of these patients originally presented cutaneous lesions, one (aged 59) with several ulcers in different areas of the body, and the other (aged 40) with only one lesion on the arm; both had been positive for Leishmania. No parasites or lesions have appeared during the two years since treatment. The other two cases had a history of mucocutaneous leishmaniasis (20 and 27 years of evolution with perforations of the nasal septum). One case, a man 62 years old, had responded to antimonial compounds with disappearance of the parasites and clinical cure. The other, a 42-year-old woman, had not responded to full treatments with either Repodral or Glucantime on four different occasions. This patient recently came again to the hospital and a positive culture for Leishmania braziliensis s.1.was obtained from a nasal septum biopsy. For the MIF test, the techniques of THOR (1971) and of ROCKLIN & DAVID (1971) were combined to obtain macrophages from guinea-pigs and for the production of MIF from the patient’s lymphocytes. Two types of antigens were prepared from culture forms of Costa Rica O-CR strain of Leishmania grown in Senekiie’s medium: a soluble fraction (supemat&t after freezing and thawing and several centrifugations) and an insoluble fraction (residue of the same preparation). Both antigens were adjusted to 50 gamma of nitrogen per ml. The Montenegro tests were performed in the forearm with the insoluble antigen adjusted to 4 gamma of nitrogen per ml. The soluble antigen, in a concentration of 75 gamma of nitrogen per ml, was also used for skin tests performed in the shoulder. In all four patients the Montenegro delayed skin test was strongly positive. The soluble antigen produced only an immediate skin reaction in all patients, in the form of a wheal with pseudopods and erythema, within 10 to 15 minutes. Nevertheless, in the two cases with mucocutaneous leishmaniasis this reaction persisted as a soft infiltrated area for at least 48 hours. With the soluble antigen the degree of migration ranged from 44 to 71% in the three cured cases, whereas in the presence of the insoluble antigen there was complete inhibition
CORRESPONDENCE of migration. On the other hand, the mucocutaneous case which had not responded to treatment presented a migration of 62% (average of three tests) with the insoluble antigen, indicating partial absence of the inhibitory factor and an apparent dichotomy of cellular immune mechanisms. We are, etc., RODRIGOZELED~N ELISA DE PONCE CARLOSPONCE Louisiana State University, International Center for Medical Research and Training, Apartado 10155 San Jose, Costa Rica and Department of Parasitology, University of Costa Rica References
Rocklin, R. E. & David, J. R. (1971). Method of the production of MIP by human blood lymphocytes. In: B. R. Bloom & P. R. Glade (eds.), In vitro methods in cell-mediated immunity. New York: Academic Press, pp. 281-287. Thor, D. E. (1971). The capillary tube migration inhibition technique applied to human peripheral lymphocytes using the guinea pig peritoneal exudate as the indicator cell population. In: B. R. Bloom & P. R. Glade (eds.), In vitro methods in cell-mediated immunity. New York: Academic Press, pp. 273-280.
Comparison
of counting chamber and measured lllms in microlilaria estimation
blood
SIR-The counting chamber was introduced by DENHAM et al. (1971) and given field trials by SOUTHGATE& DESOW~TZ(1971) DE~OWITZ et al. (1973) SOUTHGATE (1973) and DENNIS et al. (1976), who showed this technique- to be more sensitive than the conventional blood film. If the same quantity of blood was examined, then more microfilariae were found in the counting chamber than the conventional measured blood slide. During the course of a survey conducted between the hours of 20.00-01.00 in villages on Choiseul in the Solomon Islands, 60 mm3 of blood were examined from each person (finger prick) by the counting chamber method, as described by DENHAM et al. (1971). When a positive was found, a measured conventional blood slide was taken (finger prick) from the person as soon as possible (within one hour), making three circular films of 20 mm3 each on the same slide. These slides were allowed to dry and were stained with Giemsa in the method adopted by the Malaria Eradication Programme in the Solomons. This is to filter on 3 ‘A Giemsa (in pH 7.2 buffered water), without prior de-haemoglobinization, leave for the particular batch time (about 30 min), briefly blue in distilled water and leave to dry. The slides were then read with a Beck compound microscope at x 60 magnification, examining the whole field. The same microscope was used to count the motile microfilariae in the whole of the counting chamber. Twenty positives were found, so a direct comparison of the two techniques using equal quantities of blood from the same person, could be made. The individual results are given in the table.
Slide Number
537 Microfilaria counts
Difference count. chamber minus blood slide
Counting chamber
Measured blood slide 16 4 3 3
-8
7::
-12 +1
10 11 12 13 14 15 16 17 18 19
8 4 2 8 3 65 4 3 12 5 3 2 3 4 9 3 1 3 2
20
1 2 3 4 5 6 7 8 9
0
-1 +5 0
3 1 5 6 2 6 9 2 3 2
+2 +7 -1 +1 +1 -3 -5 +7 0
19
-1 -16
8
26 3
-24 +5
152
194
-42
There was no significant difference between the two techniques; this result differs from those obtained by SOIJTHGATE& Dssowrrz (1971), Dasowrrz et al. (1973) and SOUTHGATE(1973). It is appreciated that in this region of nocturnally-periodic Wucheria bancrofti, there will be a slight increase in microfilariae with taking the blood slide later than the counting chamber sample. Periodicity tables have been worked out for this area (WEBBER,1975) and, even allowing the maximum period (one hour) between taking the two samples, there is a 12 % discrepancy, which makes no appreciable difference to the results. It would seem that the similar results obtained by the two techniques were due to omitting de-haemoglobinization in staining. DENHAM et al. (1971) thought that microfilariae were lost in this way and examined the washings with a membrane filter, from which they recovered them. There is no loss of definition in reading the slides and species definition can easily be made. These results were obtained during surveys to test the most suitable technique in the conditions of the Solomons, i.e., scattered island communities with nocturnally periodic W. bancrofti and very limited resources. Night time conditions made the counting chamber and Millipore technique difficult, and the further lack of facilities, cost and resistance to venepuncture precluded the latter’s use, except in special circumstances (WEBBER, 1975). Lack of resources and finance also prevented a proper prevalence survey from being made comparing the counting chamber and measured blood slide, but it is still felt that the results are sufficiently valid to recommend the measured blood slide in the prevailing conditions. This technique is still the most widely used and practical method in filariasis surveys and it is suggested here that if the blood films are stained without de-haemoglobinizing them, then greater sensitivity is obtained. I am, etc., R. H. WEBBER Ministry of Health & Welfare, Honiara, Solomon Islands