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Workshop 2C: Advances in diagnosis
Post Congress Scientific Report WORKSHOP 2C: ADVANCES IN DIAGNOSIS
J. ECKERT*and A. M. JonNsoNt * University of Zurich, Institute of Parasitology, Zurich, Switzerland -~ Department of Clinical Microbiology, Flinders Medical Centre, South Australia The contributions submitted to this workshop can be subdivided in three main categories: (a) direct parasite identification, (b) indirect parasite identification and (c) other techniques. (a) Direct parasite identification A new staining technique for the rapid diagnosis of protozoa was described by F. Kawamoto and N. Kumada. By double-staining with DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodine) the nuclei of several species of protozoa stained reddish to blue whereas the cytoplasm appeared pink to red. In the dark field Entamoeba invadens, E. coK, Giardia lamblia, Balantidium coli, other intestinal
protozoa, Plasmodium yoelii, and Trypanosoma gambiense could be easily identified. According to the authors this method has a sensitivity comparable to the merthiolate-iodine-concentration technique. The potential of the new technique for routine application has to be further evaluated. The use of DNA probes for the direct identification of parasites was described in two contributions. S. A. Williams et al., reported on the synthesis of speciesspecific oligonucleotide probes of Brugia species. A 29mer probe specific for B. malayi and a 23mer probe specific for B. pahangi were shown to be extremely species-specific and sensitive. Both probes can detect less than 0.5 ng of parasite DNA. The new technique may be applied for the identification of third-stage larvae from mosquitoes or of microfilariae in blood samples. A new technique for the detection of Plasmodium falciparum using DNA probes was described by J. W. Zolg and E. Andrade. With the system in its present form, 2-10 pg of parasite D N A can be detected. The sensitivity should allow detection of a single parasite in 106 red cells using a sample volume of 0.02 ml. (b) Indirect parasite identification Methods for indirect parasite identification included antigen, antibody and immunecomplex determination. E.L. Green et al. examined 139 human faecal specimens for Giardia antigens using ELISA. The assay was found to have a sensitivity of 96%. According to V. k. Vinayak et al., Entamoeba histolytica antigen could be detected by ELISA in polyethylene glycol-precipitated circulating immune complexes in 93% of patients with amoebic fiver abscess while antigen was undetectable in healthy subjects and non-amoebic hepatic diseases. The question whether this method is superior to antibody detection for the diagnosis of amoebic liver abscess has to be evaluated.
1017
In endemic areas, accurate detection of light infections of malaria parasites still remains a problem. Therefore, the description of a solid-phase antibody binding-inhibition test for the detection of Plasmodium-falciparum soluble antigen by V. Thomas and S. F. Yap is of special interest. It was possible to detect 15 parasites per 106 red blood cells. False negative results were minimal and false positives did not occur. The early detection of human African trypanosomiasis may be improved by the application of a card agglutination test (CAT) based on antibody detection using whole blood samples. J. M. Bafort and C. H. J. Schutte demonstrated a high sensitivity and an acceptable degree of specificity for this test. The indirect immunofluorescent test was employed for antibody detection in patients with Trichomonas vaginalis infection by G. De Carli and P. Saraiva. In a group of 40 cases with parasitological confirmed diagnosis, 85% had serum antibodies against T. vaginalis. Cross-reactions with sera of patients infected with Treponema pallidum, Toxoplasma gondii and Trypanosoma cruzi were not observed. Problems of immunodiagnosis of human "larva migrans" were discussed in two contributions. T. Arulthilakan and W. L. Nicholas presented data on excretory-secretory antigens of Toxocara canis, T. cati, other ascarids and Angio-strongylus cantonensis, and on surface antigens of adult T. canis and other nematode species. Stage and species specific peptides could be identified which will be further examined for their antigenic properties. J.L. Guillen, C. Cuellar and C. del Aguilla described a sandwich ELISA using monoclonal antibodies for the detection of circulating immune complexes in human cases of "larva migrans". G.R. Rajasekariak et al., were successful in the production of a monoclonal antibody (designated as F 32.128.16.34) highly specific to antigens of microfilariae and infective larvae of Wuchereria bancrofti. This antibody has been employed in a sandwich of ELISA for detecting circulating antigens in sera of patients. In epidemiological studies on echinococcosis/ hydatidosis the impossibility of differentiating eggs of Echinococcus from those of Taenia species represented a considerable problem. P.S. Craig, T. McMullen and C. N. L. Macpherson employed a species-specific immunofluorescence test using monoclonal antibodies for the identification of Echinococcus oncospheres hatched from eggs obtained from soil and water samples in endemic areas of Kenya. In this way Echinococcus eggs could be identified in dog faeces and in soil and water samples. P. Marty and Y. Le Fichoux used an immunosorbent agglutination assay to detect Toxoplasmaspecific IgA and IgM in cases of human congenital toxoplasmosis and adult acute infection.
1018
R. C. A. THOMPSON
(c) Othertechniques The important problem of in vitro evaluation of drug sensitivity of Plasmodium falciparum was discussed by A. Sabchareon et al. WORKSHOP 2E: LUMEN-DWELLING HELMINTHS
K. YOSHIMURA*and H. J. S. DAWKXNst * Department of Parasitology, Akita University School of Medicine, Hondo, Akita, Japan t Regional Veterinary Laboratory, Department of Agriculture and Rural Affairs, Benalla, Victoria, Australia Only two posters and two oral and poster presentations were reviewed in this session due to late withdrawals. The first oral presentation by A. Kobayashi, K. Katakura and A. Hamada was entitled "Fate of Ascaris Eggs applied to the Soil under Natural Conditions". Ascaris suum eggs were placed in two locations and at two depths within the soil at monthly intervals for 1 year. At the end of the experimental period, the eggs were assessed for their viability. There was a clear relationship between egg degeneration and exposure to strong sunlight. Thus eggs not shaded or close to the soil surface degenerated very quickly. Ascaris e~gs in deeper soil or in shaded regions survived with the percentage viable after 12 months being between 54 and 99%. The implications of this study and the importance of similar studies prior to embarking on large and extensive epidemiological or transmission studies are obvious. Without a sound understanding of the basic parasitology elaborate studies have no basis for interpretation. Basic research is all too readily dismissed for more expensive and sophisticated research. The second presentation was entitled "Large Trichuris Eggs in Aboriginal Communities in Queensland" by A.J. Stewart and P. Prociv. Trichuris trichiuria eggs in the faeces of Aboriginals were observed to be of two different sizes. These eggs were present simultaneously in the uterus of single female Trichuris worms. The large eggs have been reported over a long period, however, since the commencement of a mebendazole treatment programme the large eggs have increased in incidence. It was suggested, as one possible scenario, that the large eggs were more resistant to mebendazole and their increased incidence was a measure of their selection by the treatment programme. If this should prove to be the situation, then the extensive treatment programme of trichuriasis will require modifying if it is to be effective. Irrespective of the outcome, this study further represented the need for small studies to be carried out prior to embarking on long costly programmes. Without such information all large scale projects have to be considered ill-advised and their success will depend on serendipitous events rather than informed decisions. The poster entitled "Light and Electron Microscopical Examination of the Location of Strongyloides stercoralis in the jejunum of the immunosuppressed dog" by D. I. Grove et al., represented a long overdue
description of the precise location of S. stercoralis adults. Understanding of the location of parasites is paramount to being able to correctly interpret the host response and its effect on the parasite. The second poster "Taiwan Taenia may be Considered as a New Species of Taenia" by P. C. Fan et al., concerned a small epidemiological investigation with a question on the taxonomy of the species. It is significant that this disease entity has been described for the past 70 years but is only now receiving some of the attention due to it. All the papers and posters in this session exemplified the need to continue basic parasitological research. It is only through basic parasitological investigations that the new technologies and sophisticated studies will have the solid grounding on which to base an interpretation of the results and arrive at the correct implications of the research. WORKSHOP 2F: CHEMOTHERAPY-EXPERIMENTAL AND CLINICAL
W. E. GUTTERIDGE* and D. I. GROVEt * Biochemical Microbiology, Wellcome Research Laboratories, Beckenham, Kent, U.K. t Department of Medicine, University of Western Australia, Nedlands, Western Australia In the area of anti-protozoal chemotherapy, further experimental work on D F M O has shown that whereas the drug is quite effective in controlling Trypanosoma gambiense infections, it is only suppressive against T. brucei and T. rhodesiense (abstract number 750). An adherence inhibition assay for Giardia sp. was described which was claimed to be more sensitive than growth inhibition assays (166). However, such growth inhibition assays, utilizing 3H-thymidine incorporation, have been used to demonstrate the variable sensitivity of laboratory stocks of Giardia. This may provide an explanation for treatment failures experienced with 5-nitroimidazoles (512). A clinical study with such drugs found that a single dose of ornidazole or tinidazole is usually effective in giardiasis; in amoebiasis, ornidazole seemed as effective but less toxic than metronidazole (46). CibaGeigy's new 5-nitroimidazole, Cibemid, which is shortly to be registered in India, was also reported to be active in giardiasis in a single dose (516); 3 days of treatment was required for the therapy of amoebic liver abscess (517). Presentations on existing antimalarial drugs indicated that the mutagenic effects of NTG and EMS could lead to chloroquine resistance in Plasmodiurn berghei infections (725), described the ultrastructural effects of chloroquine and mefloquine (119) and reported on the effects of pyrimethamine and proguanil upon the liver stages of Plasmodium cynomolgi (326). The need for new antimalarial drugs was emphasized yet again by a report of decreased sensitivity to chloroquine and quinine of some Plasmodium falciparum isolates from Senegal (91); plants could well serve as the source of such compounds (95).
J. ECKERT*and A. M. JonNsoNt * University of Zurich, Institute of Parasitology, Zurich, Switzerland -~ Department of Clinical Microbiology, Flinders Medical Centre, South Australia The contributions submitted to this workshop can be subdivided in three main categories: (a) direct parasite identification, (b) indirect parasite identification and (c) other techniques. (a) Direct parasite identification A new staining technique for the rapid diagnosis of protozoa was described by F. Kawamoto and N. Kumada. By double-staining with DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodine) the nuclei of several species of protozoa stained reddish to blue whereas the cytoplasm appeared pink to red. In the dark field Entamoeba invadens, E. coK, Giardia lamblia, Balantidium coli, other intestinal
protozoa, Plasmodium yoelii, and Trypanosoma gambiense could be easily identified. According to the authors this method has a sensitivity comparable to the merthiolate-iodine-concentration technique. The potential of the new technique for routine application has to be further evaluated. The use of DNA probes for the direct identification of parasites was described in two contributions. S. A. Williams et al., reported on the synthesis of speciesspecific oligonucleotide probes of Brugia species. A 29mer probe specific for B. malayi and a 23mer probe specific for B. pahangi were shown to be extremely species-specific and sensitive. Both probes can detect less than 0.5 ng of parasite DNA. The new technique may be applied for the identification of third-stage larvae from mosquitoes or of microfilariae in blood samples. A new technique for the detection of Plasmodium falciparum using DNA probes was described by J. W. Zolg and E. Andrade. With the system in its present form, 2-10 pg of parasite D N A can be detected. The sensitivity should allow detection of a single parasite in 106 red cells using a sample volume of 0.02 ml. (b) Indirect parasite identification Methods for indirect parasite identification included antigen, antibody and immunecomplex determination. E.L. Green et al. examined 139 human faecal specimens for Giardia antigens using ELISA. The assay was found to have a sensitivity of 96%. According to V. k. Vinayak et al., Entamoeba histolytica antigen could be detected by ELISA in polyethylene glycol-precipitated circulating immune complexes in 93% of patients with amoebic fiver abscess while antigen was undetectable in healthy subjects and non-amoebic hepatic diseases. The question whether this method is superior to antibody detection for the diagnosis of amoebic liver abscess has to be evaluated.
1017
In endemic areas, accurate detection of light infections of malaria parasites still remains a problem. Therefore, the description of a solid-phase antibody binding-inhibition test for the detection of Plasmodium-falciparum soluble antigen by V. Thomas and S. F. Yap is of special interest. It was possible to detect 15 parasites per 106 red blood cells. False negative results were minimal and false positives did not occur. The early detection of human African trypanosomiasis may be improved by the application of a card agglutination test (CAT) based on antibody detection using whole blood samples. J. M. Bafort and C. H. J. Schutte demonstrated a high sensitivity and an acceptable degree of specificity for this test. The indirect immunofluorescent test was employed for antibody detection in patients with Trichomonas vaginalis infection by G. De Carli and P. Saraiva. In a group of 40 cases with parasitological confirmed diagnosis, 85% had serum antibodies against T. vaginalis. Cross-reactions with sera of patients infected with Treponema pallidum, Toxoplasma gondii and Trypanosoma cruzi were not observed. Problems of immunodiagnosis of human "larva migrans" were discussed in two contributions. T. Arulthilakan and W. L. Nicholas presented data on excretory-secretory antigens of Toxocara canis, T. cati, other ascarids and Angio-strongylus cantonensis, and on surface antigens of adult T. canis and other nematode species. Stage and species specific peptides could be identified which will be further examined for their antigenic properties. J.L. Guillen, C. Cuellar and C. del Aguilla described a sandwich ELISA using monoclonal antibodies for the detection of circulating immune complexes in human cases of "larva migrans". G.R. Rajasekariak et al., were successful in the production of a monoclonal antibody (designated as F 32.128.16.34) highly specific to antigens of microfilariae and infective larvae of Wuchereria bancrofti. This antibody has been employed in a sandwich of ELISA for detecting circulating antigens in sera of patients. In epidemiological studies on echinococcosis/ hydatidosis the impossibility of differentiating eggs of Echinococcus from those of Taenia species represented a considerable problem. P.S. Craig, T. McMullen and C. N. L. Macpherson employed a species-specific immunofluorescence test using monoclonal antibodies for the identification of Echinococcus oncospheres hatched from eggs obtained from soil and water samples in endemic areas of Kenya. In this way Echinococcus eggs could be identified in dog faeces and in soil and water samples. P. Marty and Y. Le Fichoux used an immunosorbent agglutination assay to detect Toxoplasmaspecific IgA and IgM in cases of human congenital toxoplasmosis and adult acute infection.
1018
R. C. A. THOMPSON
(c) Othertechniques The important problem of in vitro evaluation of drug sensitivity of Plasmodium falciparum was discussed by A. Sabchareon et al. WORKSHOP 2E: LUMEN-DWELLING HELMINTHS
K. YOSHIMURA*and H. J. S. DAWKXNst * Department of Parasitology, Akita University School of Medicine, Hondo, Akita, Japan t Regional Veterinary Laboratory, Department of Agriculture and Rural Affairs, Benalla, Victoria, Australia Only two posters and two oral and poster presentations were reviewed in this session due to late withdrawals. The first oral presentation by A. Kobayashi, K. Katakura and A. Hamada was entitled "Fate of Ascaris Eggs applied to the Soil under Natural Conditions". Ascaris suum eggs were placed in two locations and at two depths within the soil at monthly intervals for 1 year. At the end of the experimental period, the eggs were assessed for their viability. There was a clear relationship between egg degeneration and exposure to strong sunlight. Thus eggs not shaded or close to the soil surface degenerated very quickly. Ascaris e~gs in deeper soil or in shaded regions survived with the percentage viable after 12 months being between 54 and 99%. The implications of this study and the importance of similar studies prior to embarking on large and extensive epidemiological or transmission studies are obvious. Without a sound understanding of the basic parasitology elaborate studies have no basis for interpretation. Basic research is all too readily dismissed for more expensive and sophisticated research. The second presentation was entitled "Large Trichuris Eggs in Aboriginal Communities in Queensland" by A.J. Stewart and P. Prociv. Trichuris trichiuria eggs in the faeces of Aboriginals were observed to be of two different sizes. These eggs were present simultaneously in the uterus of single female Trichuris worms. The large eggs have been reported over a long period, however, since the commencement of a mebendazole treatment programme the large eggs have increased in incidence. It was suggested, as one possible scenario, that the large eggs were more resistant to mebendazole and their increased incidence was a measure of their selection by the treatment programme. If this should prove to be the situation, then the extensive treatment programme of trichuriasis will require modifying if it is to be effective. Irrespective of the outcome, this study further represented the need for small studies to be carried out prior to embarking on long costly programmes. Without such information all large scale projects have to be considered ill-advised and their success will depend on serendipitous events rather than informed decisions. The poster entitled "Light and Electron Microscopical Examination of the Location of Strongyloides stercoralis in the jejunum of the immunosuppressed dog" by D. I. Grove et al., represented a long overdue
description of the precise location of S. stercoralis adults. Understanding of the location of parasites is paramount to being able to correctly interpret the host response and its effect on the parasite. The second poster "Taiwan Taenia may be Considered as a New Species of Taenia" by P. C. Fan et al., concerned a small epidemiological investigation with a question on the taxonomy of the species. It is significant that this disease entity has been described for the past 70 years but is only now receiving some of the attention due to it. All the papers and posters in this session exemplified the need to continue basic parasitological research. It is only through basic parasitological investigations that the new technologies and sophisticated studies will have the solid grounding on which to base an interpretation of the results and arrive at the correct implications of the research. WORKSHOP 2F: CHEMOTHERAPY-EXPERIMENTAL AND CLINICAL
W. E. GUTTERIDGE* and D. I. GROVEt * Biochemical Microbiology, Wellcome Research Laboratories, Beckenham, Kent, U.K. t Department of Medicine, University of Western Australia, Nedlands, Western Australia In the area of anti-protozoal chemotherapy, further experimental work on D F M O has shown that whereas the drug is quite effective in controlling Trypanosoma gambiense infections, it is only suppressive against T. brucei and T. rhodesiense (abstract number 750). An adherence inhibition assay for Giardia sp. was described which was claimed to be more sensitive than growth inhibition assays (166). However, such growth inhibition assays, utilizing 3H-thymidine incorporation, have been used to demonstrate the variable sensitivity of laboratory stocks of Giardia. This may provide an explanation for treatment failures experienced with 5-nitroimidazoles (512). A clinical study with such drugs found that a single dose of ornidazole or tinidazole is usually effective in giardiasis; in amoebiasis, ornidazole seemed as effective but less toxic than metronidazole (46). CibaGeigy's new 5-nitroimidazole, Cibemid, which is shortly to be registered in India, was also reported to be active in giardiasis in a single dose (516); 3 days of treatment was required for the therapy of amoebic liver abscess (517). Presentations on existing antimalarial drugs indicated that the mutagenic effects of NTG and EMS could lead to chloroquine resistance in Plasmodiurn berghei infections (725), described the ultrastructural effects of chloroquine and mefloquine (119) and reported on the effects of pyrimethamine and proguanil upon the liver stages of Plasmodium cynomolgi (326). The need for new antimalarial drugs was emphasized yet again by a report of decreased sensitivity to chloroquine and quinine of some Plasmodium falciparum isolates from Senegal (91); plants could well serve as the source of such compounds (95).