- Email: [email protected]
Bednets, Size and Culture on the Web
Comment 18 Wagatsuma, Y. et al. (1999) Resolution and resurgence of Schistosoma haematobiuminduced pathology after communitybased chemotherapy in Ghana, as detected by ultrasound. J. Infect. Dis. 179, 1515–1522 19 Frenzel, K. et al. (1999) Evidence for a long term effect of a single dose of praziquantel on Schistosoma mansoni-induced hepatosplenic lesions in northern Uganda. Am. J. Trop. Med. Hyg. 60, 927–931 20 Brunet, L.R. et al. (1998) Cytokine interaction and immune responses during Schistosoma
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mansoni infection. Parasitol. Today 14, 422–427 King, M. (1990) Health is a sustainable state. Lancet 336, 664–667 Jefferson, T. et al. (1998) Evidence-based vaccinology: the work of the Cochrane Vaccines Field. J. Epidemiol. Community Health 52, 207–208 World Health Organization (1992) The control of Schistosomiasis. WHO Technical Series 830 Gryseels, B. (1989) The relevance of schistosomiasis for public health. Trop. Med. Parasitol. 40, 134–142
25 Cioli, D. (1998) Chemotherapy of schistosomiasis: an update. Parasitol. Today 14, 418–421 26 Geerts, S. and Gryseels, B. Drug Resistance in Helminths of Humans: Current situation and lessons from Livestock. Clin. Microbiol. Rev. (in press)
Bruno Gryseels is at the Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. Tel: +32 3 247 6200, Fax: +32 3 237 6731, e-mail: [email protected]
ParaSite Bednets, Size and Culture on the Web Malaria Discussion Group Bednets and history Torben Vestergaard (Disease Control Textiles, Kolding, Denmark) was keen to know why people in Africa are reluctant to buy bednets, when formerly they were in common use. Is it just the cost? They were usually imported from Europe and costs have risen and incomes in Africa have fallen. Import duties of 60–100% are prohibitive. Furthermore, in India, it is illegal to import them ready-made, and 85% duty is charged on importing raw materials to make them. But are there other factors? He had heard that, in Zambia, improved sanitation and drainage combined with the use of DDT had changed peoples’ habits, and that now the mosquito is taking its revenge. Christian Lengeler (Swiss Tropical Institute, Basel) thought that Europeans in Africa in the late 19th and early 20th century had reduced their mortality considerably by using bednets, but unfortunately ‘history has not often mentioned the fate of non-Europeans (especially in rural areas)’. Jo Lines (London School of Hygiene & Tropical Medicine, UK), from his experience there in the early 1980s, believed that, in Tanzania, poverty is the answer, because of the state of its economy. So, economists and historians: ‘is it true that the average African household has been poorer in the 1980s and 1990s than in the 1960s and early 1970s, when many African countries were becoming independent?’ Reza Alemi (Zahedan University, Iran) said that in Iran 25–30 years ago ‘everybody slept under bednets, now, nobody does’. Then no insecticides or repellents were available, but following industrialization ‘spraying gardens and houses became popular, the decrease in mosquito population led to decreased use of bednets, until they were abandoned all together’. However, Marina Chua (Philippine General Hospital) said bednets are still commonly used in the Philippines. Made of nylon, without insecticide impregnation, they are ‘quite affordable for even the lowest income family’ and it is the rich who use them less, mainly because they have screens on windows and doorways. Newspapers again Colin Sutherland (London School of Hygiene & Tropical Medicine, UK), without comment, posted an extract from The Observer of Sunday, 17 October 1999: ‘Every 20 seconds a child somewhere in the world dies of malaria. Scientists are racing to find a vaccine as deadlier strains threaten even Europe. Every six months, Dr Stephen Hoffman, a captain in 48
the US navy, enters an insectary swarming with irradiated mosquitoes and allows himself to be bitten repeatedly on the arm. A few days later he repeats the torture until he is sure he has received more than 1000 bites. Then, and only then, is it safe for him to travel to the front zone of the navy’s war against malaria – countries such as Kenya and Ghana where Plasmodium falciparum, the deadliest form of the disease, is rampant’. Some desultory discussion followed, mainly wondering if the mosquitoes were infected (not clear!) and if Steve was boosting his immunity to mosquito saliva. Rob Anderson (Simon Fraser University, Canada) presumed the mosquitoes carried sporozoites of a chloroquine-sensitive strain of P. falciparum and hoped immunity was induced to resistant strains too. How many gametocytes can fit into one red blood cell? Roger Jovani (Universitat de Barcelona, Spain) wanted papers on multiple infections of erythrocytes by P. falciparum gametocytes, and even subjective impressions. He received more of the latter than the former, and not all P. falciparum gametocytes either! Chimanuka Bantuzeko (Vrije Universiteit, Brussels, Belgium), working with P. chabaudi, believed that multiply infected red blood cells (RBCs) generally lyse even before schizonts develop, but Lisa Ranford-Cartwright (University of Edinburgh, UK) sometimes sees RBCs containing two gametocytes of P. falciparum in cultures, though never more. They appear to survive up to stage V and express normal gametocyte antigens by IFA. Mark Wiser (Tulane University, New Orleans, LA, USA) agreed about P. chabaudi, suggesting that perhaps the RBCs can accommodate only a limited amount of parasite growth or that the spleen efficiently removes multiply infected cells – or both. Jack Williams (Walter Reed Army Institute of Research, USA) thought a single RBC capable of supporting two gametocytes but more destroyed the cell: ‘Occasionally there will be three young gametocytes … but we only see stage IVs in doubly infected cells’. He quoted J.B. Jensen, Observations on gametocytogenesis in Plasmodium falciparum from continuous cultures. J. Protozool. 26, 129–132, 1979, which shows two stage IV gametocytes in one RBC. Shenyi He (Kobe University, Japan) then did some counts on cultures of P. falciparum (K1): 20% of infected cells contained more than one parasite, sometimes five ring forms in one cell, and sometimes even a ring, a trophozoite and a
0169-4758/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0169-4758(99)01598-7
Parasitology Today, vol. 16, no. 2, 2000
ParaSite schizont within the same cell. Of the total multiply infected: 63% contained two rings; 31%, three rings; and 6%, four rings. In vitro, one RBC could support maturation of three schizonts without rupturing the cell. ‘For 3D7 … multiple-infection of gametocyte was rarely found … but I do find two gametocytes within one RBC (stage II)’. Michael Duffy (Eijkman Institute for Molecular Biology, Jakarta, Indonesia) found that 20% of ring-infected RBCs contained more than one parasite in stationary cultures of P. falciparum (ITG2), but the number fell to 6% on an orbital shaker. In fact, David Warhurst (London School of Hygiene & Tropical Medicine, UK) has used a wrist action shaker routinely for 15 years to avoid multiple infections (A.H. Fairlamb et al., An improved technique for the cultivation of Plasmodium falciparum in vitro without daily medium change. Ann. Trop. Med. Parasitol. 79, 379–384, 1985). By contrast, Mark Wiser remarked that antibodies against merozoite surface protein (MSP)-2 actually promote multiply infected erythrocytes: R. Ramasamy et al., Antibodies to a merozoite surface protein promote multiple invasion of red blood cells by malaria parasites. Parasite Immunol. 21, 397–407, 1999. Clonal diversity in P. falciparum infections While this has been studied in the past by the number of MSP-1, MSP-2, and GLURP alleles present, said Jos Schall (University of Vermont, USA), why not use microsatellites for the purpose? Hundreds of such loci are known, and they should be ‘more satisfying’ than using surface proteins, which are highly variable and change rapidly. Alfried Vogler (The Natural History Museum, London, UK) replied that they have indeed been used, and quoted a recent paper: T.J. Anderson et al. Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples. Parasitology 119, 113–125, 1999. But ‘multiple strains are not present in equal frequency, and if you try to amplify from blood samples you may overlook the rare strains (which of course could be the predominant forms just a few days later in the infection cycle). Second, because the alleles are unlinked, you can only assess the level of polymorphism in each microsatellite locus, but you won’t know how many clones (ie. haploid genomes) they represent’. Better to analyse microsatellite diversity in single oocysts in the mosquito. ‘Looking at several oocysts from a single mosquito midgut you get a pretty good idea how many clones were taken up with the bloodmeal. Because the mosquitoes feed only once (or, rarely, twice) this tells you how many potentially infectious clones there are in a single human being … looking at strain diversity in P. falciparum using these markers is a lot more meaningful for testing clonal diversity than surface-protein encoding genes’. However, Ross Coppel (Monash University, Australia) pointed out ‘which is more meaningful depends on what you are trying to determine’. Antigens may give a biased view in terms of parasite populations but provide information about the effect of immune responses and immune evasion on the host–parasite relationship. Ruth Sponsler, an entomologist, was worried by the fact that older female Anopheles spp are more likely to carry sporozoites, and they may have fed 3–5 times. Graham White concurred: females usually feed on blood from successive (different) hosts at intervals of 2–4 days at tropical temperatures, but ‘zygote formation would normally involve only the Plasmodium population in one bloodmeal, ie. from a single host’. However, in villages in Sri Lanka, 34% of Anopheles culicifacies and 30% of An. subpictus had bloodmeals ingested from multiple hosts, 90–93% apparently from two hosts and 7–11% from three Parasitology Today, vol. 16, no. 2, 2000
hosts, indicating that, in those circumstances, ‘it is invalid to assume that Plasmodium oocysts come from a single host individual’. (P.H. Amerasinghe and F.P. Amerasinghe, Multiple host feeding in field populations of Anopheles culicifacies and An. subpictus in Sri Lanka. Med. Vet. Entomol. 13, 124–131, 1999.) David Walliker (University of Edinburgh, UK) explained that the most well-studied polymorphic loci-containing repeat sequences, and, thus, easily studied by PCR methodology, had indeed been those encoding antigens such as MSP-1 until the pioneering work of Su and Wellems (see X. Su et al., A Genetic Map and Recombination Parameters of the Human Malaria Parasite Plasmodium falciparum. Science 286, 1351–1353, 1999 for their most recent paper). He agreed that oocyst genotypes provide the best estimates of the number of gametocyte-producing clones in an infection at the time the bloodmeal was taken, but there could be other nongametocyte-producing clones that would escape detection. ‘Also, as Alfried points out, it is indeed difficult to quantify the precise number of multilocus haplotypes in mixed clone infections simply from blood-form PCR methodology’. He recommended a paper (W.G. Hill and H.A. Babiker, Estimation of numbers of malaria clones in blood samples. Proc. R. Soc. London Ser. B 262, 249–257, 1995) for a method of estimating the mean number of clones per individual using blood data and two polymorphic loci (MSP-1 and -2), which agreed well with estimates from oocyst data from the same Tanzanian community. Mark Wiser asked him ‘Is there any evidence that tandem repeat size or composition can change during the asexual stages, especially the blood stage?’ The answer was not in natural infections, though clearly documented for both cultures of P. falciparum and after multiple blood passage of P. berghei through rodents, in terms of chromosomal deletions, especially of sub-telomeric regions and involving repeat sequences (see C. Janse, Chromosome size polymorphism and DNA rearrangements in Plasmodium. Parasitol. Today 9, 19–22, 1993 for a review). Wiser added that there was also indirect evidence that the epigenetic regulation of var gene expression may depend on local chromatin structure (K.W. Deitsch et al., Intra-cluster recombination and var transcription switches in the antigenic variation of Plasmodium falciparum. Mol. Biochem. Parasitol. 101, 107–116, 1999; T.E. Wellems et al., Genome projects, genetic analysis, and the changing landscape of malaria research. Curr. Opin. Microbiol. 2, 415–419, 1999, is also a review to be recommended). Haemozoin-free cell lysate Kabututu Zakayi (Osaka University, Japan) wanted to eliminate haemozoin from a P. falciparum lysate. Lars Hviid (Rigshospitalet, Copenhagen, Denmark) had used powerful magnets to purify late-stage infected erythrocytes, through their content of paramagnetic haemozoin, and thought the method worth a try (T. Staalsoe et al., Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry, 35, 329–336, 1999). Amit Pandey (International Centre for Genetic Engineering and Biotechnology, New Delhi, India) suggested sonication in 2.5% Triton 3 100 and centrifugation.
ParaSite was compiled, from the Internet, by Janice Taverne ( [email protected]) 49
21 22
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mansoni infection. Parasitol. Today 14, 422–427 King, M. (1990) Health is a sustainable state. Lancet 336, 664–667 Jefferson, T. et al. (1998) Evidence-based vaccinology: the work of the Cochrane Vaccines Field. J. Epidemiol. Community Health 52, 207–208 World Health Organization (1992) The control of Schistosomiasis. WHO Technical Series 830 Gryseels, B. (1989) The relevance of schistosomiasis for public health. Trop. Med. Parasitol. 40, 134–142
25 Cioli, D. (1998) Chemotherapy of schistosomiasis: an update. Parasitol. Today 14, 418–421 26 Geerts, S. and Gryseels, B. Drug Resistance in Helminths of Humans: Current situation and lessons from Livestock. Clin. Microbiol. Rev. (in press)
Bruno Gryseels is at the Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. Tel: +32 3 247 6200, Fax: +32 3 237 6731, e-mail: [email protected]
ParaSite Bednets, Size and Culture on the Web Malaria Discussion Group Bednets and history Torben Vestergaard (Disease Control Textiles, Kolding, Denmark) was keen to know why people in Africa are reluctant to buy bednets, when formerly they were in common use. Is it just the cost? They were usually imported from Europe and costs have risen and incomes in Africa have fallen. Import duties of 60–100% are prohibitive. Furthermore, in India, it is illegal to import them ready-made, and 85% duty is charged on importing raw materials to make them. But are there other factors? He had heard that, in Zambia, improved sanitation and drainage combined with the use of DDT had changed peoples’ habits, and that now the mosquito is taking its revenge. Christian Lengeler (Swiss Tropical Institute, Basel) thought that Europeans in Africa in the late 19th and early 20th century had reduced their mortality considerably by using bednets, but unfortunately ‘history has not often mentioned the fate of non-Europeans (especially in rural areas)’. Jo Lines (London School of Hygiene & Tropical Medicine, UK), from his experience there in the early 1980s, believed that, in Tanzania, poverty is the answer, because of the state of its economy. So, economists and historians: ‘is it true that the average African household has been poorer in the 1980s and 1990s than in the 1960s and early 1970s, when many African countries were becoming independent?’ Reza Alemi (Zahedan University, Iran) said that in Iran 25–30 years ago ‘everybody slept under bednets, now, nobody does’. Then no insecticides or repellents were available, but following industrialization ‘spraying gardens and houses became popular, the decrease in mosquito population led to decreased use of bednets, until they were abandoned all together’. However, Marina Chua (Philippine General Hospital) said bednets are still commonly used in the Philippines. Made of nylon, without insecticide impregnation, they are ‘quite affordable for even the lowest income family’ and it is the rich who use them less, mainly because they have screens on windows and doorways. Newspapers again Colin Sutherland (London School of Hygiene & Tropical Medicine, UK), without comment, posted an extract from The Observer of Sunday, 17 October 1999: ‘Every 20 seconds a child somewhere in the world dies of malaria. Scientists are racing to find a vaccine as deadlier strains threaten even Europe. Every six months, Dr Stephen Hoffman, a captain in 48
the US navy, enters an insectary swarming with irradiated mosquitoes and allows himself to be bitten repeatedly on the arm. A few days later he repeats the torture until he is sure he has received more than 1000 bites. Then, and only then, is it safe for him to travel to the front zone of the navy’s war against malaria – countries such as Kenya and Ghana where Plasmodium falciparum, the deadliest form of the disease, is rampant’. Some desultory discussion followed, mainly wondering if the mosquitoes were infected (not clear!) and if Steve was boosting his immunity to mosquito saliva. Rob Anderson (Simon Fraser University, Canada) presumed the mosquitoes carried sporozoites of a chloroquine-sensitive strain of P. falciparum and hoped immunity was induced to resistant strains too. How many gametocytes can fit into one red blood cell? Roger Jovani (Universitat de Barcelona, Spain) wanted papers on multiple infections of erythrocytes by P. falciparum gametocytes, and even subjective impressions. He received more of the latter than the former, and not all P. falciparum gametocytes either! Chimanuka Bantuzeko (Vrije Universiteit, Brussels, Belgium), working with P. chabaudi, believed that multiply infected red blood cells (RBCs) generally lyse even before schizonts develop, but Lisa Ranford-Cartwright (University of Edinburgh, UK) sometimes sees RBCs containing two gametocytes of P. falciparum in cultures, though never more. They appear to survive up to stage V and express normal gametocyte antigens by IFA. Mark Wiser (Tulane University, New Orleans, LA, USA) agreed about P. chabaudi, suggesting that perhaps the RBCs can accommodate only a limited amount of parasite growth or that the spleen efficiently removes multiply infected cells – or both. Jack Williams (Walter Reed Army Institute of Research, USA) thought a single RBC capable of supporting two gametocytes but more destroyed the cell: ‘Occasionally there will be three young gametocytes … but we only see stage IVs in doubly infected cells’. He quoted J.B. Jensen, Observations on gametocytogenesis in Plasmodium falciparum from continuous cultures. J. Protozool. 26, 129–132, 1979, which shows two stage IV gametocytes in one RBC. Shenyi He (Kobe University, Japan) then did some counts on cultures of P. falciparum (K1): 20% of infected cells contained more than one parasite, sometimes five ring forms in one cell, and sometimes even a ring, a trophozoite and a
0169-4758/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0169-4758(99)01598-7
Parasitology Today, vol. 16, no. 2, 2000
ParaSite schizont within the same cell. Of the total multiply infected: 63% contained two rings; 31%, three rings; and 6%, four rings. In vitro, one RBC could support maturation of three schizonts without rupturing the cell. ‘For 3D7 … multiple-infection of gametocyte was rarely found … but I do find two gametocytes within one RBC (stage II)’. Michael Duffy (Eijkman Institute for Molecular Biology, Jakarta, Indonesia) found that 20% of ring-infected RBCs contained more than one parasite in stationary cultures of P. falciparum (ITG2), but the number fell to 6% on an orbital shaker. In fact, David Warhurst (London School of Hygiene & Tropical Medicine, UK) has used a wrist action shaker routinely for 15 years to avoid multiple infections (A.H. Fairlamb et al., An improved technique for the cultivation of Plasmodium falciparum in vitro without daily medium change. Ann. Trop. Med. Parasitol. 79, 379–384, 1985). By contrast, Mark Wiser remarked that antibodies against merozoite surface protein (MSP)-2 actually promote multiply infected erythrocytes: R. Ramasamy et al., Antibodies to a merozoite surface protein promote multiple invasion of red blood cells by malaria parasites. Parasite Immunol. 21, 397–407, 1999. Clonal diversity in P. falciparum infections While this has been studied in the past by the number of MSP-1, MSP-2, and GLURP alleles present, said Jos Schall (University of Vermont, USA), why not use microsatellites for the purpose? Hundreds of such loci are known, and they should be ‘more satisfying’ than using surface proteins, which are highly variable and change rapidly. Alfried Vogler (The Natural History Museum, London, UK) replied that they have indeed been used, and quoted a recent paper: T.J. Anderson et al. Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples. Parasitology 119, 113–125, 1999. But ‘multiple strains are not present in equal frequency, and if you try to amplify from blood samples you may overlook the rare strains (which of course could be the predominant forms just a few days later in the infection cycle). Second, because the alleles are unlinked, you can only assess the level of polymorphism in each microsatellite locus, but you won’t know how many clones (ie. haploid genomes) they represent’. Better to analyse microsatellite diversity in single oocysts in the mosquito. ‘Looking at several oocysts from a single mosquito midgut you get a pretty good idea how many clones were taken up with the bloodmeal. Because the mosquitoes feed only once (or, rarely, twice) this tells you how many potentially infectious clones there are in a single human being … looking at strain diversity in P. falciparum using these markers is a lot more meaningful for testing clonal diversity than surface-protein encoding genes’. However, Ross Coppel (Monash University, Australia) pointed out ‘which is more meaningful depends on what you are trying to determine’. Antigens may give a biased view in terms of parasite populations but provide information about the effect of immune responses and immune evasion on the host–parasite relationship. Ruth Sponsler, an entomologist, was worried by the fact that older female Anopheles spp are more likely to carry sporozoites, and they may have fed 3–5 times. Graham White concurred: females usually feed on blood from successive (different) hosts at intervals of 2–4 days at tropical temperatures, but ‘zygote formation would normally involve only the Plasmodium population in one bloodmeal, ie. from a single host’. However, in villages in Sri Lanka, 34% of Anopheles culicifacies and 30% of An. subpictus had bloodmeals ingested from multiple hosts, 90–93% apparently from two hosts and 7–11% from three Parasitology Today, vol. 16, no. 2, 2000
hosts, indicating that, in those circumstances, ‘it is invalid to assume that Plasmodium oocysts come from a single host individual’. (P.H. Amerasinghe and F.P. Amerasinghe, Multiple host feeding in field populations of Anopheles culicifacies and An. subpictus in Sri Lanka. Med. Vet. Entomol. 13, 124–131, 1999.) David Walliker (University of Edinburgh, UK) explained that the most well-studied polymorphic loci-containing repeat sequences, and, thus, easily studied by PCR methodology, had indeed been those encoding antigens such as MSP-1 until the pioneering work of Su and Wellems (see X. Su et al., A Genetic Map and Recombination Parameters of the Human Malaria Parasite Plasmodium falciparum. Science 286, 1351–1353, 1999 for their most recent paper). He agreed that oocyst genotypes provide the best estimates of the number of gametocyte-producing clones in an infection at the time the bloodmeal was taken, but there could be other nongametocyte-producing clones that would escape detection. ‘Also, as Alfried points out, it is indeed difficult to quantify the precise number of multilocus haplotypes in mixed clone infections simply from blood-form PCR methodology’. He recommended a paper (W.G. Hill and H.A. Babiker, Estimation of numbers of malaria clones in blood samples. Proc. R. Soc. London Ser. B 262, 249–257, 1995) for a method of estimating the mean number of clones per individual using blood data and two polymorphic loci (MSP-1 and -2), which agreed well with estimates from oocyst data from the same Tanzanian community. Mark Wiser asked him ‘Is there any evidence that tandem repeat size or composition can change during the asexual stages, especially the blood stage?’ The answer was not in natural infections, though clearly documented for both cultures of P. falciparum and after multiple blood passage of P. berghei through rodents, in terms of chromosomal deletions, especially of sub-telomeric regions and involving repeat sequences (see C. Janse, Chromosome size polymorphism and DNA rearrangements in Plasmodium. Parasitol. Today 9, 19–22, 1993 for a review). Wiser added that there was also indirect evidence that the epigenetic regulation of var gene expression may depend on local chromatin structure (K.W. Deitsch et al., Intra-cluster recombination and var transcription switches in the antigenic variation of Plasmodium falciparum. Mol. Biochem. Parasitol. 101, 107–116, 1999; T.E. Wellems et al., Genome projects, genetic analysis, and the changing landscape of malaria research. Curr. Opin. Microbiol. 2, 415–419, 1999, is also a review to be recommended). Haemozoin-free cell lysate Kabututu Zakayi (Osaka University, Japan) wanted to eliminate haemozoin from a P. falciparum lysate. Lars Hviid (Rigshospitalet, Copenhagen, Denmark) had used powerful magnets to purify late-stage infected erythrocytes, through their content of paramagnetic haemozoin, and thought the method worth a try (T. Staalsoe et al., Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry, 35, 329–336, 1999). Amit Pandey (International Centre for Genetic Engineering and Biotechnology, New Delhi, India) suggested sonication in 2.5% Triton 3 100 and centrifugation.
ParaSite was compiled, from the Internet, by Janice Taverne ( [email protected]) 49