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The efficacy of meningococcal polysaccharide vaccine in preventing group A meningococcal disease in The Gambia, West Africa
1006
TRANSACTIONS OF THE ROYAL SOCIETYOF TROPUXL MEDICXNHAND HYO,ENE(1986) 80, CORRESPONDENCE
Rapid serodiagnosis ELISA
of parasitic infections using “dipsticks”
by Dot-
The Dot-ELISA is a rapid, solid phase enzyme immunoassay using minute volumes of parasite antigen adsorbed to nitrocellulose filter paper and a precipitable, chromogenic substrate; positive reactions are characterized by the formation of a distinct colored dot on the white nitrocellulose (PAPPASet al., 1983).
The Dot-ELISA has since been further refined into a simple screening method for the laboratory and field utilizing “dipsticks”. Nitrocellulose filter discs dotted with a ;arieG of parasite antigens were attached to flat ulastic sticks (7 cm lone X 0.6 cm wide X ~0.1 cm -&ck) using a‘water-inssluble glue. After drying and blocking with 5% BSA-PBS, dipsticks were successively incubated with optimally diluted patient serum, wash solution, horseradish peroxidase-conjugated second antibody, wash solution, and then with precipitable chromogenic substrate (Cchloro-l-naphthol). At a reciprocal dilution of 32, 23 of 25 (92%) visceral leishmaniasis patient sera were positive. Control serafrom geographically matched, apparently healthy Africans gave four of 25 (16%) positive reactions and none of 10 serafrom regional trypanosomiasis patients reacted. The dipstick Dot-ELISA was then tested using sera from patients with amoebiasis, hydatidosis and trichinosis, and canines experimentally infected with Leishntunia donovani. Positive reactalons using sera and specific antigen from these oatients were: (i1 amoebiasis eiaht of nine (89%). (ii) hydatidosis all bi seven, (iii) t&hinosis all bf tei&d (iv) canine visceral leishmaniasis all of six. Only one of 32 control sera from these experiments gave a false positive reaction. In additio?, multi-antigen dipsticks with antigen for amoebiasls, hydatidosis, visceral leishmaniasis and trichinosis showed 100% reactivity using serafrom infected patients and minimal reactivity with sera from patients with other diseasesand with pooled normal human serum. The dipstick Dot-ELISA is a novel modification which allows one patient’s serum sample to be simultaneously assayed for a number of diseases. MICHAEL G. PAPPAS Covalent Technology Corporation, P.O. Box 1868, Ann Arbor, Michigan 48106, USA. Reference Pappas, M. G., Hajkowski, R. & Hockmeyer, W. T. (1983). Dot enzyme-linked immunosorlxnt assay(Dot-ELBA): a micro technique for the rapid diagnosis of visceral leishmaniasis. Joumai of Immunological Methods, 64, 205-214.
Accepted
for publication
17th June, 1986.
The efficacy of meningococcal polysaccharide vaccine in preventing gro;p A menmiococcal disease in The Gambia, West Africa
Meningococcal polysacc&aride vaccines have been used to control epidemics of meningococcal diseasein several African countries with apparent success (GREENWOOD, 1984). However, because African
epidemics tend to subside spontaneously after two or three years (LAPEYSSONIE, 1963), caution is needed in assessingthe response to vaccination unless controls are used. There have been few formal studies on the degree and duration of protection provided by meningococcal polysaccharide vaccines in Africa. The most detailed, a case-control study undertaken in Burkina Faso (REINGOLD et al., 1985), showed an overall vaccine efficacy of 87% during the first year after vaccination which declined to 70% and 54% two and three years after vaccination, respectively. Vaccine efficacy declined much more rapidly in children under the age of four years at the time of immunization than it did in adults. During the dry seasonsof 198111982The Gambia was affected by an outbreak of group A meningococcal diseaseduring which 191 caseswere reported to the Epidemiology Unit of the Medical and Health Department. The epidemic subsided during the 1982 rainy seasonbut it broke out again in several parts of the country during the following dry season when 1,166 caseswere recorded. As a third outbreak during the 1983/1984dry seasonseemedlikely a nation-wide vaccination programme was carried out in November and December 1983in which all subjects one year old or older were offered vaccination with a combined group A + group C meningococcal polysaccharide vaccine (Mencevax, Smith Kline RIT). The namesof vaccinated subjects were entered in a register held by each vaccination team and each vaccinated person was given a card on which the date of immunization was recorded. The target population for vaccination was 666,810. Figures provided by team leaders, basedon the number of dosesof vaccine used, gave a vaccine coverage of 92%. Review of vaccmation registers suggested a vaccine coverage of 86%. There were no major regional variations in the level of vaccine coverage achieved. Vaccination appearedto be effective in aborting the epidemic as during the 19830984 dry seasononly 76 casesof epidemic meningitis were reported. However, this fall could have been unrelated to the vaccination programme. To confirm the efficacy of the vaccination campaign we have, therefore, investigated cases of meningococcal diseaseoccurring after the vaccination programme and determined their vaccination status. During the period December 1981November 1985, 41 casesof meningococcal disease were identified at The Royal Victoria Hospital, Banjul, the MRC Hospital, Fajara, the WEC clinic at Sibanor or at the MRC field station at Farafenni. These patients probably represented about one quarter to one third of all casesof meningococcal diseaseoccurring in the country during that period. 32 caseswere caused by group A meningococci, three by group W135 meningococci (recently reported to be a significant cause of meningococcal diseasein other parts of West Africa (DENIS et al., 1982) and one by a group B meningococcus. The remaining five meninaococci were not serogrouped. A vaccina‘iion history Las obtained for 27 of 32 with LOUD A menineococcal disease (the remaining patie:ts died or left h&pital before they or their relatives could be interviewed). 15 of these patients had been vaccinated and 12 had not. Five of the non-vaccinated patients were under the age of three years and were thus too young to have been
TRANSACTIONS OF THE ROYAL SOCEI-Y OF TROPICAL MEDICINE AND HYGIENE (1986) 80, CORRESMNDENCE
immunized. Among patients aged three years or more 14 had been vaccinated and seven had not. Assuming an overall vaccine coverage of about 90% the protective efficacy of vaccination during the first two years after immunization among those old enough to have been immunized is 78% (95% confidence limits 35%-92%). Thus, our findings support the view that the meningococcal vaccination campaign in The Gambia was effective. The degree of protection achieved during the first two years after vaccination (78%) is very similar to that found in Burkina Faso. In Burkina Faso, age at the time of vaccination was found to have an important influence on vaccine efficacy. Our data are insufficient to study the effect of age but only three of the 15 vaccine failures were under the age of four years at the time of immunization. A high proportion of unvaccinated caseshad not been vaccinated because they were too young. Although meningococcal polysaccharide vaccines are poorly immunogenic in the very young they may produce a transient rise in antibody levels and there is no evidence that immunization at this age is harmful. It may, therefore, be wise to include children under the age of one year in future mass immunization campaigns aimed at the control of an endemic. We thank Mrs. Ramou Njie and Dr. G. Schneider for providing isolates of menningococci and Dr. P. G. Smith for statistical help. B. A. M. H.
M. GREENWOOD W. SMITH HASSAN-KING A. BIILMER F. C. SHENTON Medical Research Council Laboratories Fajara, The Gambia. A. S. B. HUGHES P. P. NUNN The Royal Victoria Hospital, Banjul, The Gambia. A. D. JACK P. R. S. GOWERS Epidemiology Unit, Medical and Health Department, Banjul, The Gambia.
Phillips, C., Tiendrebeogo, H. & Yada, A. (1985). Age-specific differences in duration of clinical protection after vaccination with meningococcal polysaccharide A vaccine. Lacer, ii, 114-118.
Accepted for publication 20th June, 1986.
Long term culture of Plasmodium falciparum in human erythrocytes with reduced K+ content We have recently demonstrated that Plasmodium falciparum grows well in human erythrocytes whose K+ levels were reduced by treatment with ouabain, an inhibitor of the human erythrocyte membrane Na+ K+-ATPase (TANABE et aE., 1986). Here, we report the continuous culture of the parasite in such K+reduced erythrocytes. The FCRl3 and GGG strains of P. falciparum were cultured as described by IZUMO & TANABE (1986). Erythrocytes were pre-incubated with 0.2 mM ouabain (or without ouabain for controls) for three days in culture conditions to have low K+ and high Na+ levels (TANABE et al., 1986). The pre-incubated erythrocytes were used for cultures of P. falciparum (FCR3) for another three days with daily changes of medium with or without ouabain. The cultured parasites were successively transferred at three-day intervals into new erythrocytes previously pre-incubated with or without ouabain for three days. Parasitaemiasof cultures with or without ouabain ranged from 0.1 to 0.2% at the start of each culture and from 2.6 to 3.6 on day 3. Both cultures were transferred over 60 times (six months). The parasite growth rate did not differ between the two cultures during and after the transfers. No difference in the growth rate was detected in the GGG strain (three months). Thus, it follows that reduction of the human erythrocyte K+ level does not affect the continuous culture of P. falciparum. Since the parasite is not exposed to the host cell cytoplasm but is surrounded by a parasitophorous vacuole membrane, this finding suggestsan important role of the vacuole membrane in regulating permeabilities to K+ and Na+. Consistent with this suggestion is our observation that the cytoplasm of mouseerythrocytes infected with trophozoite to schizont stages of P. yoelii has elevated Na+ and decreasedK+ levels (unpublished data). We thank Dr K. Kageyama for providing his facilities. This work was supported by the Grant-inAid for Scientific Researchof the JapaneseMinistry of Education, Science and Culture.
References Denis, F., Rey, J. L., Amadou, A., Saliou, P., Prince-
A. IZUMO K. TANABE
David, M., M’Boup, S., Cadoz,M., Diop Mar, I. &
Etienne, J. (1982). Emergence of meningococcal meningitis caused by WI35 serogroup in Africa. Lancer, ii, 1335-1336. Greenwood, B. M. (1984). Selective primary health care: strategies for contiol of diseasein the developing world. XII. Acute bacterial meningitis. Reviews of l&dnu
S. TAKADA Department of Medical Zoology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka, 545, Japan
Diseases. 6. 374-389.
Lapeyssonnie,’ L. (1963). La meningite cerebrospinal en Afrique. Bulletin of the Worth Health Organization, 28, (supplement), 3-114. Reingold, A. L., Broome, C. V., Higbtower, A. W., Ajello, G. W., Bolan, G. A., Adamsbaum, C., Jones, E. E.,
1007
References
Izumo, A. & Tanabe,K. (1986). Inhibition of in virro growth
of Plasmodium falciparm by a brief exposure of the cationic rhodamine dyes. Annals of TropicabMedicine and
Parasitology,
80, 299-305.
TRANSACTIONS OF THE ROYAL SOCIETYOF TROPUXL MEDICXNHAND HYO,ENE(1986) 80, CORRESPONDENCE
Rapid serodiagnosis ELISA
of parasitic infections using “dipsticks”
by Dot-
The Dot-ELISA is a rapid, solid phase enzyme immunoassay using minute volumes of parasite antigen adsorbed to nitrocellulose filter paper and a precipitable, chromogenic substrate; positive reactions are characterized by the formation of a distinct colored dot on the white nitrocellulose (PAPPASet al., 1983).
The Dot-ELISA has since been further refined into a simple screening method for the laboratory and field utilizing “dipsticks”. Nitrocellulose filter discs dotted with a ;arieG of parasite antigens were attached to flat ulastic sticks (7 cm lone X 0.6 cm wide X ~0.1 cm -&ck) using a‘water-inssluble glue. After drying and blocking with 5% BSA-PBS, dipsticks were successively incubated with optimally diluted patient serum, wash solution, horseradish peroxidase-conjugated second antibody, wash solution, and then with precipitable chromogenic substrate (Cchloro-l-naphthol). At a reciprocal dilution of 32, 23 of 25 (92%) visceral leishmaniasis patient sera were positive. Control serafrom geographically matched, apparently healthy Africans gave four of 25 (16%) positive reactions and none of 10 serafrom regional trypanosomiasis patients reacted. The dipstick Dot-ELISA was then tested using sera from patients with amoebiasis, hydatidosis and trichinosis, and canines experimentally infected with Leishntunia donovani. Positive reactalons using sera and specific antigen from these oatients were: (i1 amoebiasis eiaht of nine (89%). (ii) hydatidosis all bi seven, (iii) t&hinosis all bf tei&d (iv) canine visceral leishmaniasis all of six. Only one of 32 control sera from these experiments gave a false positive reaction. In additio?, multi-antigen dipsticks with antigen for amoebiasls, hydatidosis, visceral leishmaniasis and trichinosis showed 100% reactivity using serafrom infected patients and minimal reactivity with sera from patients with other diseasesand with pooled normal human serum. The dipstick Dot-ELISA is a novel modification which allows one patient’s serum sample to be simultaneously assayed for a number of diseases. MICHAEL G. PAPPAS Covalent Technology Corporation, P.O. Box 1868, Ann Arbor, Michigan 48106, USA. Reference Pappas, M. G., Hajkowski, R. & Hockmeyer, W. T. (1983). Dot enzyme-linked immunosorlxnt assay(Dot-ELBA): a micro technique for the rapid diagnosis of visceral leishmaniasis. Joumai of Immunological Methods, 64, 205-214.
Accepted
for publication
17th June, 1986.
The efficacy of meningococcal polysaccharide vaccine in preventing gro;p A menmiococcal disease in The Gambia, West Africa
Meningococcal polysacc&aride vaccines have been used to control epidemics of meningococcal diseasein several African countries with apparent success (GREENWOOD, 1984). However, because African
epidemics tend to subside spontaneously after two or three years (LAPEYSSONIE, 1963), caution is needed in assessingthe response to vaccination unless controls are used. There have been few formal studies on the degree and duration of protection provided by meningococcal polysaccharide vaccines in Africa. The most detailed, a case-control study undertaken in Burkina Faso (REINGOLD et al., 1985), showed an overall vaccine efficacy of 87% during the first year after vaccination which declined to 70% and 54% two and three years after vaccination, respectively. Vaccine efficacy declined much more rapidly in children under the age of four years at the time of immunization than it did in adults. During the dry seasonsof 198111982The Gambia was affected by an outbreak of group A meningococcal diseaseduring which 191 caseswere reported to the Epidemiology Unit of the Medical and Health Department. The epidemic subsided during the 1982 rainy seasonbut it broke out again in several parts of the country during the following dry season when 1,166 caseswere recorded. As a third outbreak during the 1983/1984dry seasonseemedlikely a nation-wide vaccination programme was carried out in November and December 1983in which all subjects one year old or older were offered vaccination with a combined group A + group C meningococcal polysaccharide vaccine (Mencevax, Smith Kline RIT). The namesof vaccinated subjects were entered in a register held by each vaccination team and each vaccinated person was given a card on which the date of immunization was recorded. The target population for vaccination was 666,810. Figures provided by team leaders, basedon the number of dosesof vaccine used, gave a vaccine coverage of 92%. Review of vaccmation registers suggested a vaccine coverage of 86%. There were no major regional variations in the level of vaccine coverage achieved. Vaccination appearedto be effective in aborting the epidemic as during the 19830984 dry seasononly 76 casesof epidemic meningitis were reported. However, this fall could have been unrelated to the vaccination programme. To confirm the efficacy of the vaccination campaign we have, therefore, investigated cases of meningococcal diseaseoccurring after the vaccination programme and determined their vaccination status. During the period December 1981November 1985, 41 casesof meningococcal disease were identified at The Royal Victoria Hospital, Banjul, the MRC Hospital, Fajara, the WEC clinic at Sibanor or at the MRC field station at Farafenni. These patients probably represented about one quarter to one third of all casesof meningococcal diseaseoccurring in the country during that period. 32 caseswere caused by group A meningococci, three by group W135 meningococci (recently reported to be a significant cause of meningococcal diseasein other parts of West Africa (DENIS et al., 1982) and one by a group B meningococcus. The remaining five meninaococci were not serogrouped. A vaccina‘iion history Las obtained for 27 of 32 with LOUD A menineococcal disease (the remaining patie:ts died or left h&pital before they or their relatives could be interviewed). 15 of these patients had been vaccinated and 12 had not. Five of the non-vaccinated patients were under the age of three years and were thus too young to have been
TRANSACTIONS OF THE ROYAL SOCEI-Y OF TROPICAL MEDICINE AND HYGIENE (1986) 80, CORRESMNDENCE
immunized. Among patients aged three years or more 14 had been vaccinated and seven had not. Assuming an overall vaccine coverage of about 90% the protective efficacy of vaccination during the first two years after immunization among those old enough to have been immunized is 78% (95% confidence limits 35%-92%). Thus, our findings support the view that the meningococcal vaccination campaign in The Gambia was effective. The degree of protection achieved during the first two years after vaccination (78%) is very similar to that found in Burkina Faso. In Burkina Faso, age at the time of vaccination was found to have an important influence on vaccine efficacy. Our data are insufficient to study the effect of age but only three of the 15 vaccine failures were under the age of four years at the time of immunization. A high proportion of unvaccinated caseshad not been vaccinated because they were too young. Although meningococcal polysaccharide vaccines are poorly immunogenic in the very young they may produce a transient rise in antibody levels and there is no evidence that immunization at this age is harmful. It may, therefore, be wise to include children under the age of one year in future mass immunization campaigns aimed at the control of an endemic. We thank Mrs. Ramou Njie and Dr. G. Schneider for providing isolates of menningococci and Dr. P. G. Smith for statistical help. B. A. M. H.
M. GREENWOOD W. SMITH HASSAN-KING A. BIILMER F. C. SHENTON Medical Research Council Laboratories Fajara, The Gambia. A. S. B. HUGHES P. P. NUNN The Royal Victoria Hospital, Banjul, The Gambia. A. D. JACK P. R. S. GOWERS Epidemiology Unit, Medical and Health Department, Banjul, The Gambia.
Phillips, C., Tiendrebeogo, H. & Yada, A. (1985). Age-specific differences in duration of clinical protection after vaccination with meningococcal polysaccharide A vaccine. Lacer, ii, 114-118.
Accepted for publication 20th June, 1986.
Long term culture of Plasmodium falciparum in human erythrocytes with reduced K+ content We have recently demonstrated that Plasmodium falciparum grows well in human erythrocytes whose K+ levels were reduced by treatment with ouabain, an inhibitor of the human erythrocyte membrane Na+ K+-ATPase (TANABE et aE., 1986). Here, we report the continuous culture of the parasite in such K+reduced erythrocytes. The FCRl3 and GGG strains of P. falciparum were cultured as described by IZUMO & TANABE (1986). Erythrocytes were pre-incubated with 0.2 mM ouabain (or without ouabain for controls) for three days in culture conditions to have low K+ and high Na+ levels (TANABE et al., 1986). The pre-incubated erythrocytes were used for cultures of P. falciparum (FCR3) for another three days with daily changes of medium with or without ouabain. The cultured parasites were successively transferred at three-day intervals into new erythrocytes previously pre-incubated with or without ouabain for three days. Parasitaemiasof cultures with or without ouabain ranged from 0.1 to 0.2% at the start of each culture and from 2.6 to 3.6 on day 3. Both cultures were transferred over 60 times (six months). The parasite growth rate did not differ between the two cultures during and after the transfers. No difference in the growth rate was detected in the GGG strain (three months). Thus, it follows that reduction of the human erythrocyte K+ level does not affect the continuous culture of P. falciparum. Since the parasite is not exposed to the host cell cytoplasm but is surrounded by a parasitophorous vacuole membrane, this finding suggestsan important role of the vacuole membrane in regulating permeabilities to K+ and Na+. Consistent with this suggestion is our observation that the cytoplasm of mouseerythrocytes infected with trophozoite to schizont stages of P. yoelii has elevated Na+ and decreasedK+ levels (unpublished data). We thank Dr K. Kageyama for providing his facilities. This work was supported by the Grant-inAid for Scientific Researchof the JapaneseMinistry of Education, Science and Culture.
References Denis, F., Rey, J. L., Amadou, A., Saliou, P., Prince-
A. IZUMO K. TANABE
David, M., M’Boup, S., Cadoz,M., Diop Mar, I. &
Etienne, J. (1982). Emergence of meningococcal meningitis caused by WI35 serogroup in Africa. Lancer, ii, 1335-1336. Greenwood, B. M. (1984). Selective primary health care: strategies for contiol of diseasein the developing world. XII. Acute bacterial meningitis. Reviews of l&dnu
S. TAKADA Department of Medical Zoology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka, 545, Japan
Diseases. 6. 374-389.
Lapeyssonnie,’ L. (1963). La meningite cerebrospinal en Afrique. Bulletin of the Worth Health Organization, 28, (supplement), 3-114. Reingold, A. L., Broome, C. V., Higbtower, A. W., Ajello, G. W., Bolan, G. A., Adamsbaum, C., Jones, E. E.,
1007
References
Izumo, A. & Tanabe,K. (1986). Inhibition of in virro growth
of Plasmodium falciparm by a brief exposure of the cationic rhodamine dyes. Annals of TropicabMedicine and
Parasitology,
80, 299-305.