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On the reliability of the in vitro and in vivo determinations for testing anti-amoebic compounds
598
CORRESPONDENCE
dose blood examinations were made. T h e children possessed the usual advanced degree of premtmition common to this area of holoendemicity. T h e last dose, sufficient for parasite clearance in other holoendemic malarious districts, was in all cases given by myself with the usual strict regard of technique. T h e preceding doses, in the case of proguanil inadvertently at too low a rate, had been given by the teachers. T h e results follow :
Croup 1.
Group 2.
Group 3.
26 initially infected children. Lapudrlne 20 mg. once weekly for 5 weeks. Lapudrine 20 rag. one dose at 6th week. Final examination : 11 asexual P. falcipamm infections. 43 initially infected children. Proguanil 100 mg. twice weekly for 5 weeks. Proguanil 300 mg. one dose at 6th week. Final examination : 16 asexual P. falciparum infections. 20 initially infected children. Proguanil 100 mg. twice weekly for 5 weeks. Lapudrine 20 rag. one dose at 6th week. Final examination : 7 asexual P. falciparum infections.
T h e presence of cross-resistance to pyrimethamine, proguanil and Lapudrine refutes my observation made in 1957. T h e only explanation that can be given for this is that the cross-testing doses of proguanil given in 1957 were greater than those given in the present investigation. I am, etc., D. F. CLYDE. Malaria Service, Ministry of Health, Tanganyika. 13th jYuly, 1960.
ON THE RELIABILITY OF THE in vitro AND in ~i~o DETERMINATIONS FOR TESTING ANTI-AMOEBIC COMPOUNDS SIR,--It is possible to a v o i d / n vitro the appearance of a secondary anti-emoehic activity of antibacterial substances, seeding the test tubes containing 4 ml. of monophasic medium used for screening, with approximately 10,000 amoeba per ml., associated with at least 400,000,000 co-biont bacteria per ml. (de Carneri, 1957, 1958). However, it has recently been noted that substances which exert an extremely high direct anti° amoebic activity in vitro may be completely inactive when tested on laboratory animals. F o r example, on the one hand chlorophenoxamide (NO2-C6H4-O-C6H~-CH2-N-CH2-CH2-OH) inactive against
l
CO-CHC12 bacteria, shows great anti-amoebic activity both in vitro (0.6 ~tg./ml.) and in rat and man ; while, on the other hand, other derivatives of the same series equally inactive against bacteria but extremely active against E. histolytica in vitro (up to 0 . 2 ~g./ml.) are completely inactive if administered orally or endoperitoneally to rats infected endocaecally or to hamsters with hepatic amoebiasis (data in print). We wish now to mention a new series of dichloroacetamide derivatives of rather different structure, of which one of the most interesting members is N'-dichloroacetyl-N"-p-chlorobenzyl-piperazine (I)
CI--~--CH,--N
~-~.N--CO-
CHCI,
It is inactive up to a concentration of 500 ~tg./ml. as regards the many bacteria, mycetes and other protozoa tested, but has a sterilizing activity on the trophozoites of E. histolytica up to a concentration of 0.3 ~tg./ml.--i.e., it is 30 times more active than emetine and fumagillin, under the same experimental conditions (de Carneri, 1938).
CORRESPONDENCE
599
We studied the activity of (1) in intestinal amoebiasis, on groups of 12 rats each weighing 25 g., infected endocaecally with the virulent Meah strain of E. histolytica. T h e compound, administered five times in 3 days by forced feeding up to a total of 500 mg./kg., was completely inactive. In fact, the average degree of infection (ADI) of the control on the 5th day was 4.25 (maximum value of the scale = 5), and that of the group treated with (I) was 4.01. A group treated with chlorophenoxamide for a total of 300 mg./kg, over 3 days, showed an A D I of 0.8. Taking into consideration the possibility that (I) is absorbed or inactivated in the first part of the digestive tract, we repeated the test by anal administration. On using this route of administration, Deschiens and Lamy (1947) demonstrated that the drugs reach the small intestine, so that it was possible to obtain an effect against murine hymenolepiasis. T h e above-mentioned doses of (I), suspended in 1 ml. of 10 per cent. gum arabic, were forcibly introduced per anum into the intestine of the rats, using a syringe with a protected needle. At the 5th day of infection, the A D I of the control group was 4.07, while that of the treated group was 3.6. T h e A D I of a group treated with chlorophenoxamide by the anal route for comparison (total 300 mg./kg, over 3 days) was 0.9. Also the activity on hepatic amoebiasis in the golden hamster protected against the associated bacteria with penicillin and streptomycin (de Carneri, 1958), was found to be nil after both oral and endoperitoneal administration. These results are difficult to explain. In fact, while (I) may be inactivated on oral or endoperitoneal administration, it is not possible to understand how it remains inactive on anal administration, as it is active in vitro. However, these results show that although the in vitro anti-amoebic test may be generally useful for screening, it must always be supplemented by suitable in vivo tests. A further limitation to the reliability of the former is that some substances showing little in vitro activity (chloroquine, glaucarubin) have, in contrast, been observed to be very active in laboratory animals and in man. We are, etc., Ivo DE CARNERI, LUIGI ALMIRANTE. Instituto Carlo Erba per Ricerche Terapeutiche, Milan. 19th ffuly, 1960. REFERENCES :
de Carneri, I. (1957). Riv. Parassit., 18, 133. (1958). Arch. Int. Pharmacodyn. Ther., 113, 273. (1958). Z. Tropenmed. Parasit., 9, 32. Deschiens, R. & Lamy, L. (1947). Ann. Soc. beige M~d. trop., Liber ffubilaris J. Rodhain (reprint).
USE OF FLUORESCENT ANTIBODY TECHNIQUE FOR DETECTION OF Pasteurella pestis IN RODENTS SlR,--Fluorescent antibody techniques recently have been used in the rapid identification of plague bacilli from laboratory cultures and in tissue smears from experimentally infected white laboratory mice (Winter and Moody, M o o d y and Winter, ft. infect. Dis., 1959, 104, 281, 288). In studies of the epidemiology of sylvatic plague it would be an advantage to demonstrate the suitability of this technique in detecting Pasteurella pestis in the tissues of wild rodents. A number of laboratory and field-trapped wild rodents were inoculated with virulent P. pestis (Alexander strain). Tissue impression smears of each animal, obtained at necropsy, were stained with fluorescent antibody preparations. Routes of inoculation and dosage of inocula depended upon previous information relating to susceptibility of the species. F o r example, highly susceptible laboratory mice were given 10~-10 e organisms intracutaneously, whereas resistant wood rats (Neotoma) and white footed mice (Peromyscus) were inoculated with 50 x 106 bacilli subcutaneously or intraperitoneally. Tissue impression smears were heat-fixed and stained with fluorescein isothiocyanate conjugate, using the one-step inhibition technique of Goldman (ft. exp. Med., 1957, 105, 557). All examinations were checked by bacteriological culture, animal inoculation of tissues, and bipolar staining with Wayson's stain. Results are summarized in the Table. All animals tested were found suitable for fluorescent antibody examination, although results varied. No differences in non-specific staining between
CORRESPONDENCE
dose blood examinations were made. T h e children possessed the usual advanced degree of premtmition common to this area of holoendemicity. T h e last dose, sufficient for parasite clearance in other holoendemic malarious districts, was in all cases given by myself with the usual strict regard of technique. T h e preceding doses, in the case of proguanil inadvertently at too low a rate, had been given by the teachers. T h e results follow :
Croup 1.
Group 2.
Group 3.
26 initially infected children. Lapudrlne 20 mg. once weekly for 5 weeks. Lapudrine 20 rag. one dose at 6th week. Final examination : 11 asexual P. falcipamm infections. 43 initially infected children. Proguanil 100 mg. twice weekly for 5 weeks. Proguanil 300 mg. one dose at 6th week. Final examination : 16 asexual P. falciparum infections. 20 initially infected children. Proguanil 100 mg. twice weekly for 5 weeks. Lapudrine 20 rag. one dose at 6th week. Final examination : 7 asexual P. falciparum infections.
T h e presence of cross-resistance to pyrimethamine, proguanil and Lapudrine refutes my observation made in 1957. T h e only explanation that can be given for this is that the cross-testing doses of proguanil given in 1957 were greater than those given in the present investigation. I am, etc., D. F. CLYDE. Malaria Service, Ministry of Health, Tanganyika. 13th jYuly, 1960.
ON THE RELIABILITY OF THE in vitro AND in ~i~o DETERMINATIONS FOR TESTING ANTI-AMOEBIC COMPOUNDS SIR,--It is possible to a v o i d / n vitro the appearance of a secondary anti-emoehic activity of antibacterial substances, seeding the test tubes containing 4 ml. of monophasic medium used for screening, with approximately 10,000 amoeba per ml., associated with at least 400,000,000 co-biont bacteria per ml. (de Carneri, 1957, 1958). However, it has recently been noted that substances which exert an extremely high direct anti° amoebic activity in vitro may be completely inactive when tested on laboratory animals. F o r example, on the one hand chlorophenoxamide (NO2-C6H4-O-C6H~-CH2-N-CH2-CH2-OH) inactive against
l
CO-CHC12 bacteria, shows great anti-amoebic activity both in vitro (0.6 ~tg./ml.) and in rat and man ; while, on the other hand, other derivatives of the same series equally inactive against bacteria but extremely active against E. histolytica in vitro (up to 0 . 2 ~g./ml.) are completely inactive if administered orally or endoperitoneally to rats infected endocaecally or to hamsters with hepatic amoebiasis (data in print). We wish now to mention a new series of dichloroacetamide derivatives of rather different structure, of which one of the most interesting members is N'-dichloroacetyl-N"-p-chlorobenzyl-piperazine (I)
CI--~--CH,--N
~-~.N--CO-
CHCI,
It is inactive up to a concentration of 500 ~tg./ml. as regards the many bacteria, mycetes and other protozoa tested, but has a sterilizing activity on the trophozoites of E. histolytica up to a concentration of 0.3 ~tg./ml.--i.e., it is 30 times more active than emetine and fumagillin, under the same experimental conditions (de Carneri, 1938).
CORRESPONDENCE
599
We studied the activity of (1) in intestinal amoebiasis, on groups of 12 rats each weighing 25 g., infected endocaecally with the virulent Meah strain of E. histolytica. T h e compound, administered five times in 3 days by forced feeding up to a total of 500 mg./kg., was completely inactive. In fact, the average degree of infection (ADI) of the control on the 5th day was 4.25 (maximum value of the scale = 5), and that of the group treated with (I) was 4.01. A group treated with chlorophenoxamide for a total of 300 mg./kg, over 3 days, showed an A D I of 0.8. Taking into consideration the possibility that (I) is absorbed or inactivated in the first part of the digestive tract, we repeated the test by anal administration. On using this route of administration, Deschiens and Lamy (1947) demonstrated that the drugs reach the small intestine, so that it was possible to obtain an effect against murine hymenolepiasis. T h e above-mentioned doses of (I), suspended in 1 ml. of 10 per cent. gum arabic, were forcibly introduced per anum into the intestine of the rats, using a syringe with a protected needle. At the 5th day of infection, the A D I of the control group was 4.07, while that of the treated group was 3.6. T h e A D I of a group treated with chlorophenoxamide by the anal route for comparison (total 300 mg./kg, over 3 days) was 0.9. Also the activity on hepatic amoebiasis in the golden hamster protected against the associated bacteria with penicillin and streptomycin (de Carneri, 1958), was found to be nil after both oral and endoperitoneal administration. These results are difficult to explain. In fact, while (I) may be inactivated on oral or endoperitoneal administration, it is not possible to understand how it remains inactive on anal administration, as it is active in vitro. However, these results show that although the in vitro anti-amoebic test may be generally useful for screening, it must always be supplemented by suitable in vivo tests. A further limitation to the reliability of the former is that some substances showing little in vitro activity (chloroquine, glaucarubin) have, in contrast, been observed to be very active in laboratory animals and in man. We are, etc., Ivo DE CARNERI, LUIGI ALMIRANTE. Instituto Carlo Erba per Ricerche Terapeutiche, Milan. 19th ffuly, 1960. REFERENCES :
de Carneri, I. (1957). Riv. Parassit., 18, 133. (1958). Arch. Int. Pharmacodyn. Ther., 113, 273. (1958). Z. Tropenmed. Parasit., 9, 32. Deschiens, R. & Lamy, L. (1947). Ann. Soc. beige M~d. trop., Liber ffubilaris J. Rodhain (reprint).
USE OF FLUORESCENT ANTIBODY TECHNIQUE FOR DETECTION OF Pasteurella pestis IN RODENTS SlR,--Fluorescent antibody techniques recently have been used in the rapid identification of plague bacilli from laboratory cultures and in tissue smears from experimentally infected white laboratory mice (Winter and Moody, M o o d y and Winter, ft. infect. Dis., 1959, 104, 281, 288). In studies of the epidemiology of sylvatic plague it would be an advantage to demonstrate the suitability of this technique in detecting Pasteurella pestis in the tissues of wild rodents. A number of laboratory and field-trapped wild rodents were inoculated with virulent P. pestis (Alexander strain). Tissue impression smears of each animal, obtained at necropsy, were stained with fluorescent antibody preparations. Routes of inoculation and dosage of inocula depended upon previous information relating to susceptibility of the species. F o r example, highly susceptible laboratory mice were given 10~-10 e organisms intracutaneously, whereas resistant wood rats (Neotoma) and white footed mice (Peromyscus) were inoculated with 50 x 106 bacilli subcutaneously or intraperitoneally. Tissue impression smears were heat-fixed and stained with fluorescein isothiocyanate conjugate, using the one-step inhibition technique of Goldman (ft. exp. Med., 1957, 105, 557). All examinations were checked by bacteriological culture, animal inoculation of tissues, and bipolar staining with Wayson's stain. Results are summarized in the Table. All animals tested were found suitable for fluorescent antibody examination, although results varied. No differences in non-specific staining between