- Email: [email protected]
Poster meeting, London School of Hygiene and Tropical Medicine 26 November 1992
377
TRANSACTIONS OF THEROYALSOCIETY OF TROPICAL MEDICINE AND HYGIENE (1993) 87, 377-382
Royal Society
of Tropical
Poster Meeting, London 26 November 1992 Enhancement ization
of selective
toxicity
Medicine
School
of brusatol
of Hygiene
by derivat-
D. Allen’, G. C. Kirby’, J. D. Phillipson’, I. Toth3, D. C. Departments of ‘Pharmacognosy Warhurst and C. W. Wright’ and 3Chemisny, The School of Pharmacy, University of London, 29-39 Brunswick Square, London, WCIN IAX, UK; 2Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
Plant-derived quassinoids, many of which inhibit protein synthesis by Plasmodium falciparum (KIRBY et al., 1989: Biochemical Pharmacology, 38, 4367-4374), often possess gross toxicity against mammalian cells. Derivatization of quassinoids to promote selective toxicity may prove to be a feasible method for obtaining novel antimalarial drugs. We previously reported an increased antimalarial effect with concomitant reduced cytotoxicity for 3-methoxyethoxymethyl brusatol, a semisynthetic derivative of the natural quassinoid brusatol (PATEL et al., 1991: Transactions of the Royal Society of Tropical Medicine and Hygiene, 85, 316). Two novel 3-acylated derivatives, brusatol-3-yl-2-(tertbutoxycarbonylamino) decanoate and brusatol-3-yl-acetate were prepared and evaluated: both had high antimalarial activity in vitro, comparable to that of the parent brusatol, but both were significantly less cytotoxic in vitro against KB tumour cells, suggesting that substituents at the C3 position may radically affect cytotoxic, whilst sparing antimalarial, properties. In contrast, conversion of the Cl3 methoxycarbonyl of brusatol and brusatol-3-yl-2-(tertbutoxycarbonylamino) decanoate to an amide (13-amido-brusatol; 13-amido-brusatol-3-yl-2-(tertbutoxycarbonylamino) decanoate) significantly decreased both antimalarial activity and cytotoxicity suggesting that this ester moiety is important for biological activity. (Supported by the Wellcome Trust, Science and Engineering ResearchCouncil, and the Medical ResearchCouncil.) Splenic lymphoma with villous mon lymphoma in Ghana
lymphocytes
and Hygiene
is a com-
G. Bedu-Addo’, T. R. Rutherford2, D. H. Bevan’ and I. Bates2 ‘Komfo Anokye Teaching Hospital, Kumasi, Ghana; 2St George’s Hospital Medical School, London, UK
Splenic lymphoma with villous lymphocytes (SLVL) is a B-cell lymphoma which in the West predominantly affects elderly men (MULLIGAN, S. 81 CATOVSKY, D., 1992: Leukaemia and Lymphoma, 6, 97-105). We have diagnosed SLVL in 10 Ghanaian patients with massive splenomegaly, no lymphadenopathy but with lymphocytosis. Characteristic villous lymphocytes with polar cytoplasmic projections formed over 30% of circulating lymet al., 1989: Journal of Clinical phocytes (BENNET Pathology, 42, 567-584). The lymphocytes were positive for the pan-B surface marker CD19 but negative for CD5 and CDllc, which are positive in chronic lymphocytic leukaemia (CLL) and hairy cell leukaemia respectively. 20% of patients had a monoclonal gammopathy without immune paresis and all of the 5 examined had rearrangements of the immunoglobulin gene. Compared to countries in the West, SLVL is more common in Ghana and predominantly affects middleaged women. It is clinically indistinguishable from the African variant of CLL, which presents with splenomegaly rather than lymphadenopathy (ESSIEN, 1976: East African Medical Journal, 53, 9&103). As African CLL is generally diagnosed without evidence of CD5
and Tropical
Medicine
positive surface markers, it is likely that SLVL has previously been misdiagnosed as CLL. Some patients with hyper-reactive malarial splenomegaly have significant numbers of villous lymphocytes. This fact, combined with demographic features and lower than expected inatients, leads cidence of haemoglobin AS among SLVL to speculation about the involvement oF malaria and other co-factors in the aetiology of SLVL. Construction of a vector suitable for cloning major outer membrane protein into vaccinia
chlamydial
Tom J. Blanchard, Jacquie Keer, N. G. Stoker and D. C. W. Mabey Department of Clinical Sciences, London School of Hygieneand Tropical Medicine, Keppel Street, London, WClE 7HT, UK
Chlamydiae are the cause of much human disease in the form of trachoma, sexually transmitted disease and pneumonia. It is hoped that recombinant vaccinia may serve as a vehicle to express isolated chlamydial proteins in an immunogenic form. This would enable cytotoxicity assays to be performed using recombinant-infected target cells and histocompatibility-matched peripheral blood lymphocytes (PBL). By taking PBL from epidemiologitally defined groups exposed to trachoma it is hoped that any protective role of specific cytotoxicity may be defined. The recombinant vaccinia (or other poxvirus recombinants) mav also serve as vaccine candidates in the future. ’ Construction of vaccinia recombinants by a method similar to that of MACKETT. M. & SMITH. G. L., (1986: Journal of Virology, 67,206?-2082) first requires &at the coding sequence for the foreign gene be inserted into a vector plasmid site in the correct orientation. A successful method for the precise and rapid cloning of appropriately modified chlamydial MOMP B sequence into pSCl1 is described. The method uses T:A cloning of polymerase chain reaction (PCR) products incorporating a microwave/chemiluminescent hybridization screen. The precise nature of the ligation is confirmed by altered restriction site analysis. The PCR primers used are suitable for cloning all serotypes of chlamydial major outer membrane protein. The use of recombinant mycobacterial antigens in functional assays with human peripheral blood mononuclear cells K. Britton, S. K. Young, A. Alvi, M. F. R. Waters, K. P. W. J. McAdam and H. M. Dockrell Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
An increasing number of recombinant Mycobacterium leprae and M. tuberculosis antigens is becoming available for use in functional assays through the World Health Organization Mycobacteria Antigen Bank. We have tested these antigens in T cell proliferation assays and found that all are suitable for use in immunological assays. T cell proliferation confirms that the M. tuberculosis 70 kDa, M. bovis 65 kDa, M. leprae 65 kDa and M. leprae 18 kDa antigens can stimulate T cells in BCG vaccinated normal donors and leprosy patients. The presence of endotoxin in some of the preparations does not appear to affect T cell proliferation. Thus these antigens can be used to probe antigen recognition and T cell function in mycobacterial disease.
378 Cryptosporidiumin
wild rodents
R. M. Chalmed, A. Miller’. D. P. Casemore* and A. Cm3 ‘School of Natural and Envimkmental Sciences, Covenhy University, Covens, UK; ‘Rhyl Public Health Laboratory; 3Manchester Public Health Laboratoly
The protozoan parasite Cryptosporidium causes diarrhoeal disease in humans and livestock. There has been increasing evidence of the importance of environmental routes of transmission. The genus has at least 4 named, well characterized species, of which C. muris, the first species to be described, and C. parvurn are found in mammals. It is believed that most human and livestock infections are with C. parzwn. Since the first description in 1907 in mice in the USA, C. muris has been described only occasionally. It is not known how frequently cryptosporidia are present in wild mammals, although they were found in a small survey in north Wales in 1984. This 2-year project was set up to look for cryptosporidia in livestock and wild animal species on an agricultural college farm in Warwickshire, and to explore the role of wild mammals in the natural history of this parasite. Mammals were trapped in live mammal traps and their faeces collected, together with those of associated livestock. Smears were examined using standard methods (MZN? auramine-phenol, IFAT) to detect oocysts. Prehminary results confirmed the presence of cryptosporidia in a high proportion of the mice trapped, mostly typical of C:- pawum but sometimes with the larger oocysts typical of C. murk. Mice excreting putative C. muris oocysts were killed and the enteric tract examined by light and electron microscopy. This confiied typical morphology and localization of endogenous stages restricted to the peptic glands of the stomach. Studies are continuing on the parasite and its ecology in the farm setting. lmmunodisgnostic kDa larval antigens
potential of 41 kDa, 31 kDa, and 28 of Strongyloidesstercoralis
D. J. Conway’, J. W. Baile$, J. F. Lmdo3, R. D. Robinson3, D. A. P. Bandy’ and A. E. Bianco’l’ ‘Wellcome Research Centre for Parask Infections, Department of Biology, Imperial College, Lo&m, SW7 2BB, UK; ‘Liverpool School of Tropical Medicine, Liverpool, L3 SQA, UK; 3Department of Zoology, University of The West Indies, Kingston 7,Jamaica
Proteins from a deoxycholate-soluble extract of Strungyloidesstercoralis infective larvae were separatedby SDSPAGE, at 20 @cm width of gel, and Western blotted on
to nitrocellulose membrane. Sera from individuals with confirmed S. stercoralis infections (a= lOO),long-standing S. stercoralis infections in whom no larva could be detected at time of serum sampling (n=27), and other nematode infections (n= 100; 40 with Wuchereria bancrofti, 20 with Onchocerca volvulus, 20 with Necator americanus, and 20 with mixed Ascaris lumbricoides and Trichuris trichiura), were tested at a l/100 dilution against strips of the membrane-bound proteins. Immunodominant proteins of approximately 41 kDa, 31 kDa, and 28 kDa were respectively recognized by IgG in 91/100, 88/100, and 90/100 of the confvmed strongyloidiasis sera, in 27127, 27/27, and 25127of sera from those with parasitologically undetectable strongyloidiasis, and in 9/100, 12/100, and 14/100 of sera from other nematode infections. Each of these 3 proteins was well represented in a larval fraction soluble in phosphate-buffered saline, and did not require deoxycholate solubilization for extraction. These data suggest that any of the 3 proteins could be used to develop a sensitive and specific immunodiagnostic test for S. stercoralis infection.
The threat of pyrethroid impregnated bednetr
resistance
to the effectiveness
of
C. F. Curtis and 0. Aina
London School of Hygiene and Tropical Medicine, London, WCZE 7HT, UK
The precedents in agricultural pests and house flies might suggest that there is an imminent risk of pyrethroid resistance interfering with the successful use of bed net impregnation against malaria vectors. However, in Sichuan, China, where up to 2.25 million nets have been treated with deltamethrin annually for 5 years, 100% mortality was found in preliminary tests with the World Health Organization (WHO) recommended discriminating dosage of deltamethrin on both the vector species: Anopheles anthropophagus and An. sinensis. An artificially selected strain of An. stephensi of Dubai origin showed unequivocal resistance to permethrin in WHO test kits. As a more realistic test it was given the opportunity of feeding on blood through impregnated netting. There was a higher rate of feeding and lower death rate in the resistant-strain than in a susceptible strain when any of 3 pyrethroids, or the related but non-pyrethroid compound Trebon@, were used. However, comparison with results using untreated netting showed that the resistant strain was not fully protected against any of these insecticides under realistic testing conditions. The killing of bedbugs has been one of the attractive features of impregnated bed nets of tropical villagers. Bedbugs showed rapid evolution of DDT resistance and it is important to monitor these insects for pyrethroid susceptibility. The morphogenesis
of Hantaan
virus
D. S. Ellis’, D. G. Tovey’, P. Skidaria and D. I. H. Simpson2 ‘London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; ‘The Queen’s University ofBelfast, Grosoenor Road, Belfast, BT12 6BN, UK
Hantaan virus is the causative agent of Korean haemorrhagic fever which, in the Korean war, caused 3000 caseswith 5% mortalitv. It has since been recognized in many European and A&an countries. It is a pleomorphic RNA virus. 80 to 140 mn in diameter. covered with 10 nm spikes and containing ‘ribosomes’. ’ When grown in Vero cells, by 36 h the Golgi apparatus had become enlarged and 36 h later the cytoplasm contained increased numbers of ribosomes together with numbers of multivesicular bodies containing 50 nm particles apparentlv not dire& related to the maturation procesc -Aggregations of ‘pie-viral stroma’ were seen, which bv 96 h had become laree massesof soiral material (similar to the RNA spirals se& previously in developing Marburg virus), some lying within vacuoles. At 120 h, the first mature Hantaan particles were seen budding into large vacuoles. The liberation of these virions occurred during the rupture of the vacuoles or following the death of the host cell. While a considerable degree of asynchronicity was found in the development of Hantaan virus, a clearlv recognizable ‘second’ generation of virions was found maturing between 204 and 216 h, the ‘RNA spirals’ appearing again at 196h. Species-specific
serodiagnosis
of schistosomiaris
M. El-Tahawy’, M. J. Doeohoff2, A. J. Probert=, E. Ahari’, K. LaUoo3 and P. L. Chiodioi3 ‘Department of Clinical Pathology, Ain Shams University, Cairo, Egypt; 2School of Biological Sciences, University College of Wales, Bangor, Gwynedd, LL57 2UW, UK; 3Department of Clinical Parasitology, Hospital for Tropical Diseases, London, NW1 OPE, UK
The immunological sensitivity and specificity of Schistosoma mansoni crude soluble egg antigen (SEA), 2 cationic egg antigens (CEF6), and S. margrebowiei egg antigen were studied by ELISA (MCLAREN et al., 1981:
379 Transactions of the Royal SO&Y of Tropical Medicine and Hygiene, 75, 72-79) in 221 patients with schistosomal, other parasitic and viral diseases.S. margrebowieiis a sibling species of S. haematobiumwhich is easier to maintain. Since it is difficult to get S. huematobium antigens, S. margrebowieiprovides a good, readily available source of antigens. Use of SEA in ELBA is a sensitive epidemiological screening method for identifying schistosomiasis. SEA and S. margrebowiei egg antigens cross reacted significantly with sera from patients infected with Trichinella spiralis and Clonorchis sinensis. CEF6 showed no cross reaction with non-schistosomal patients’ sera. S. margrebowieiegg antigen was more specific to S. haemotobium natients’ sera. while CEF6 was more snecific to S. mansom natients’ sem. Serodiagnosis of sctistosomiasis using the 3 antigens provided-a serological profile capable of differentiating parasitologically-negative patients into those infected with S. man.& or S. haematobium. By comparing the pre-treatment and post-treatment ELISA results from 30 S. munsoni patients, CEF6 was k$;Abe a better predictor of chemotherapy success Characterization of mepacrine uptake by erythrocytes infected with ~/asmodium fakipsrum using fluorometric techniques
E. I. Elueze’, L.-J. Wu*, S. L. Cro!? and D. C. Warhurst’ ‘Department of Medical Parasitoloav, London School of Hwiene and-Tropical kedicine, Keppel Stree~,iondon, WClE, iHT;UK; 2hstitute of Parasitic Diseases,Shanghai, Chino
Mepacrine uptake into erythrocytes infected with chloroquine-sensitive (T.996 clone) and chloroquine-resistant (Kl strain) Phmodium fakipancm was evaluated using flow cytometry and fluorescence spectrometry. In both fluorometric techniques, uptake was seen to be rapid (steady state reached in 10 min) and both glucose and temperature dependent. Preincubation with N-ethyhnaleimide (NEM), dicyclohexylcarbodiimide (DCCD), oligomycin and carbonylcyanide-chlorophenylhydrazone (CCCP) resulted in a decreasein total drug uptake, presumably by causing a reduction in the acidity of the lysosomes. Ouabain and vanadate had no effect on mepacrine uptake. The rise in apparent drug concentration in the cytoplasmic compartment detected by flow cytometry in the presence of NEM, DCCD and oligomycin suggeststhat an additional drug accumulation system is operating at the parasite cytoplasmic membrane or that the drug is being exported from the lysosome into the cytoplasm. Electron microsconv revealed changes in the food vacuole after 30 min exposure of erythrocytes infected with P. falcibarum (Kl strain) to menacrine (2X10-’ M). Changes included enlargement of-the central food vacuole, formation of intravacuolar membrane whorls, and a decreasein the number of pigment granules. Acknowledgements. E.I.E. and L.-J.W. are sponsoredby the Associationof CommonwealthUniversities, UK and the &o-British FellowshipTrust respectively. In vitro antimalarial
N. Hi,
effects
of some insecticides
G. C. Kirby and D. C. Warhurst
Department of Medical Parasitology, London Schoolof Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK
Recent research has shown that a number of pyrethroid insecticides inhibit oocyst development in the mosquito gut (CARLE et al:, 1986: ComptesRendus de I’Academie ah Sciences,She 3, 303, 565-568; HILL et a&, 1989: Transactions of the Royal Society of Tropical Medtcine and Hygiene, 83, 42-26); in the earlier of these works it was also reported that the pyrethroid deltamethrin exhibited activity against erythrocytic Plasmodium fakiparum in vitro comparable to that of chloroquine. We have attempted to demonstrate the antiplasmodial activ-
ity of this and of some other insecticides using a standard in vine antimalarial screening test; compounds tested were deltamethrin (highest concentration O-5mg/mL), hcyhalothrin, DDT, permethrin (1 mg/mL) and malathion (10 mg/mL). None of these 5 agents (representing 3 distinct classesof insecticide) showed sianiticant activity against erythrocytic P. fakiparum; ona molar basis, h-cyhalothrin and permethrin, with the highest degrees of antiplasmodial activity, were approximately 25 and 32 thousand times less active than chloroquine in the same tests. Despite inhibitory effects of pyrethroids upon oocyst development during the sporogonic cycle of Plasmediumin Anophelesstephensi,direct toxic action of these insecticides upon the erythrocytic stages of P. falciparum appearsnot to occur. (Supportedby the WellcomeTrust.) Plasmodium in Anopheles insecticides
falciparum and P. yoeliioocyst development mosquitoes exposed to sublethal doses of
Nigel Hill’ and Lisa Ranford-Csrhvright2 ‘Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; 2Depamnentof ProtozoanGenetics,University of Edinburgh, Edinburgh, UK
It has been reported that the pyrethroid deltamethrin has the ability to suppress oocyst development in mosquitoes at doseswhich are not lethal to the insect. Our studies on this and two other pyrethroids, h-cyhalothrin and permethrin, involved feeding Anopheles stephensi mosquitoes, through treated or control netting, on a mouse infected with P. yoelii. Exposure to any of these pyrethroids reduced the total number of individuals that becameinfected as well as the number of oocysts per gut in those that were infected. Investigations using a range of other insecticide classes failed to show any similar effect on infection rates or oocyst numbers. In an attempt to ascertain how important this phenomenon could be in terms of human malaria transmission, we have conducted experiments using Pkmwdium falciparum. Mosquitoes were membrane-fed on cultured P. faZciDaramgametocvtes of strain 3D7 (a clone of NF54) and then exposed to &25% permethrinpapers for 1 h. On every occasion infection rates were on average20% lower in treated females compared to controls. Having confiied that sublethal contact with pyrethroids does reduce subsequent infection levels with P. falciparum, it is suggested that this phenomenon may have an effect on diseasetransmission, particularly in relation to intervention using pyrethrotd impregnated bed nets. Metabolism and malaria
of antipyrine infection
and metronidazole
during fever
Gilbert 0. Kokwaro, Sabariab Ismail, Anthony P. Glazier, Stephen A. Ward and Geoffrey Edwards Department of
Pharmacologyand Therapeutics,University of Liverpool, P.O.Box 147, Liverpool, L69 3BX, UK
Malaria infection and fever impair drug metabolism in rats (MANSOR, S. et al., 1990: 3ouwwl of Pharmacy and Pharmacology, 42, 428-$38; MANSOR, S. et al:,. 1990: Transactionsof the Royal Society of Tropical Medtane and Hygiene, 84,460). Impaired drug metabolism could lead to dru toxicity or altered efficacy. We have used a ‘cocktail’ oBantipyrme and metronidazole to investigate the effeet of malaria infection and fever on the activities of different isozymes in the rat. Rats (12 for each treatment) were inoculated with Plasmodium berghei, bacterial lipopolysaccharide, or saline before administration of antipyrine and metronidazole at an appropriate time. Clearance of antipyrine and metronidazole was determined from concentrations in a 4 h saliva sample.
380 Malaria infection and fever reduced clearance of metronidazole by 20% and 23% respectively. Clearance of antipyrine was unaffected by malaria infection, but fever reduced clearance of antipyrine by 36%. These results indicate that malaria infection and fever have different effects on different isozymes. The clinical relevance of these observations is being investigated. Host proteins hangi
on the surface of microfilariae
of Brugia pa-
M. N. Malecula, C. I. Baldwin and D. A. Denham Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK
[No abstract submitted.] Cryptosporidium murk in SCID mice: adoptive immunity with spleen or mesenteric lymph from infected BALE/C mice
transfer of node cells
V McDonald and G. J. Bancroft Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK Cryptospcnidium muris infection is readily obtained in adult immunocompetent mice, and this model system should be valuable for the study of the mammalian immune response to cryptosporidial infection. We previously found that immunocompetent mice recovered from C. muris infection but nude and SCID mice developed unresolved chronic infections. Recovery was observed in SCID mice if they were injected with uninfected BALB/C spleen or mesenteric lymph node (MLN) cells. In the present study an attempt was made to transfer immunity adoptively to KID mice from infected BALB/C mice by intraperitoneal injection of lx 10’ spleen or MLN cells taken either when oocyst production was just past its peak (day 21), when the production had just ceased (day 29), or several days after infections had become subpatent (day 43). Control groups were also included. of SCID mice given MLN cells from uninfected mice or no lymphoid cells. The reconstituted SCID mice were infected with 2X lo5 oocysts of C. muris and the protective effect of the donor cells was measured by oocyst production. Immunity could be transferred adoptiveiy to SCID mice with MLN or spleen cells taken from infected donor mice on davs 43 or 29, but not on day 21. Recipients of primed MLN cells from day 43 produced more than 8 times fewer oocysts than recipients of unprimed MLN cells. The results indicated clearly that immunity could be transferred adoptively if the donor animals had developed resistance to infection. Resistance of SCID mice to Cryptosporidium parvum infection and evidence for a protective mechanism involving interferon y V. McDonald
and G. J. Bancroft
Department of Clinical
Sciences,London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
Most mammalian
species are normally susceptible to infection only in the first few weeks of life. Adult mice are comnletelv resistant to infection unless deprived of T cells (e.g., infections can be obtained in nude or SCID mice). The immunological basis for this resistance was examined further in SCID mice. Infection in these animals is chronic: in the first 4-8 weeks small numbers of oocysts are excreted, but subsequently there is a sharp rise in oocyst production and the animals demonstrate signs of CrvDtosnoridiosis. Treatment of mice during theearly weeks ofinfection with an anti-interferon Y hamster monoclonal antibodv (H22) resulted in a significant increase in oocyst excretion and’an earlier onset of illness. When administration of the antibody was stopped, oocyst excretion decreased almost to the level
Cyptosporidium
paruim
observed in untreated SCID mice. These results suggested that SCID mice showed non specific immunological resistance to C. parvum during the early weeks of the chronic infection, and that a T cell-independent source of interferon y was involved in this resistance. Using a chemiluminesent in a ‘low tech’ laboratory
DNA probe to detect
parasites
R. McNerney’, S. M. Wilson’, M. A. Miles2, J. A. Fonseca de Castro3 and A. Vexenat3 ‘Department of Clinical Sciences and 2Applied Molecular Biology and Diagnosrics Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK; ‘Deparnento de Parasitologia e Microbiologia, Universidade Federal do Piaui, Teresina, Piaui, Brasil
DNA probe technology has proved to be an invaluable tool in biomedical research laboratories. The apnlication of this technology has, however, been restricted by the necessity to use radiolabels which are inherently unstable and require specialized handling. The development of sensitive chemiluminescent alternatives has enabled us to adapt a DNA probe assay for use in less sophisticated or ‘low tech’ laboratories such as those found in developing g;nries (WILSON, S. M., 1992: Parasitology, 104,421-
.--,.
The Lmet2 DNA probe (HOWMID, M. K., 1991: MoleParasitolow, 44. 63-72’1 was used to show the presence of parasites ~f’the~Leis&znia donovani complex in ear scrapes from dogs in Teresina, an area of Brazil where visceral leishmaniasis is endemic. The result was obtained within 24 h of the samples being taken and was compared to microscopical examination and the direct agglutination test (DAT). The DNA assay was performed without the use of sophisticated equipment or facilities such as hybridizing ovens or darkrooms. The spread of other new techniques for parasite diagnosis to laboratories in developing countries has in the nast been limited by the requirement for snecialized reagents such as monoclonal antibodies. It is hoped that the relative ease with which labelled DNA nrobes can be produced and the ready availability of the other components required will make the DNA probe technique appropriate for transfer to those countries where parasitic diseases are a public health problem.
cular and Biochemical
An age-stratified study of total sponses to Necator americanus in two endemic communities
IgE and specific IgE readult cuticular antigens
D. R. Palmer’. M. Bradlev* and D. A. P. Bundv’ ‘Wellcome Research Centre, Department of Biology, Imperial College, Prince Consort Road, London, SW7 2BB, UK; ‘Blair Research Institute, Harare, Zimbabwe
Epidemiological studies show lower rates of egg production by Necator americanus in adults living in rural communities in the Charara estates, Kariba, Zimbabwe and in Marakissa, The Gambia. The overall prevalence of infection in Zimbabwe was about 60% with moderate egg counts (O-2600 eggs/g). In contrast, in The Gambia, infection was light (O-650 eggs/g) with an overall prevalence of 29%. As part of a continuing project to identify possible immunological mechanisms, we measured both total and parasite specific IgE levels in individuals living in these communities. Total IgE levels in both communities were elevated above those of healthy European controls and correlated oositivelv with age. However. snecilic IgE resnonses to ihe parasite wer
381 and antibody responses.The lack of specificity in the IgE response in the Marakissa study may have been due to the lower level of antigenic challenge in infected individuals. These observations support the view that the elevation of IgE antibody levels reflects exposure to infection and may have a limited anti-fecundity role. Haptoglobin in children in adjacent malaria meso-endemic and non-endemic villages in Amapl State, Brazil M. M. P&oa’>* and D. C. Warhurst ‘Setor de Parasitologti, Institute Evandro Chagas-FNS, BeUrn, Par& Brasil; ‘AMBDU, Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK
A study of the relations between ahaptoglobinaemia and malaria was carried out in 100 children aged 2-14 years from the Serra do Navio region, Amap State, Brazil including Serra do Navio. Coloma Aeua Branca. Porto Terezinhi, Arrependido &d an indigeious group: Their serum haptoglobin levels were measured by-a radial immunodiffusion assav. The mean hautoglobin level in Serra do Navio and Port0 Terezinha, &hi;h are non-endemic and hypo-endemic areas for malaria, resnectivelv, was similar (normal level) while for the other areas, ali meso-endemic areas for n&aria, it was lower. There was a higher correlation @‘<0X)01) between ahaotoglobinaemia &d current ma&a than between ahaptbglobinaemia and a history of past malaria (PcO.01). Financial support was provided by the UNDlYWorld Bank/WHO/SpecialProgrammefor Researchand Training in Tropical Diseases(grant no. 870284),ICOMI, and Instituto EvandroChagas-FNS. Malaria endemicity Amap& State, Brazil
in the
Serra
do Navio
region
of
albitarsis, An. nuneztovari, An. braziliensis, An. triannulatus and An. rangeli. All mosquitoes positive for P. ma-
luriue were collected in the forest adjacent to the study areas(i.e., they may have been infected with P. brasilianum from non-human primates). This work was supported by the UNDP/World Bank/ WHO/SpecialProgrammefor Researchand Training in Tropical Diseases(grant no. 870284),ICOMI, and Instituto Evandro Chagas-FNS. A Schistosoma mansoni esterase mune dependence of chemotherapy
implicated
in the im-
B. A. Ripley, P. G. Fallon, A. J. Probert and M. J. Doenhoff School of Biological Sciences, University of Wales, Bangor, UK
[No abstract submitted.]
M. M. Pbvoa’,‘, P. Amorim’, V. Calvosa’, E. Santa Rosa’, J. M. Nascimento’, V. Ros6rio3, M. A. Miles4 and D. C. Warhurst4 ‘Setor de Parositologia, Institute Evandro Chagas-FNS, Belt%, Par& Brasil; *ICOMI-Sewa do Navio, Amapci, Brasil; 31nstiko de kfigiene e Medicina Tropical, Urtiversidade Nova de Lisboa. Lisbon. Portueal; 4AMBDU. Debartment of Medical Parasitology, L&don &h&l of Hygiek a& Tropical-Medicine, Keppel Street, London, WCIE 7HT, UK
To determine the malaria endemicitv in 4 adjacent villages of Serra do Navio region (Serra ho Navid, Colonia Agua Branca, Porto Terezinha and Arrependido), Amapfi State, Brazil, we carried out a blood film survey and measured the spleen of individuals aged 2-9 years. Based on these observations. Serra do Navio village (mevalence and spleen rate=O) &as classified as non-endemic for malaria, Porto Terezinha (prevalence= 1% and spleen rate=7%) as hypo-endemic, and the other 2 villages, Colonia Agua Branca (prevalence=21% and spleen rate= 18%) and Arrependido (prevalence= 17% and spleen rate= 11%) as meso-endemic areas for malaria. Reasonsfor the difference in endemicity in these villages in the samearea were discussed. This work was supported by the UNDPiWorld Bank/ WHO/SpecialProgrammefor Researchand Training in Tropical Diseases(grant no. 870284),ICOMI, and Instituto Evandro Chagas-FNS. Anophelines infected with human Plasmodium Serra do Navio region of AmapL State, Brazil
An epidemiological study to determine the distribution of anopheline speciesand their potentiality as vectors of malaria was carried out in 4 villages of the Serra do Navio region, Amap State in the north of Brazil. Fifteen anopheline species were identified among 3053 mosquitoes collected as human biting catches in the 4 study areas from February 1990 to October 1991. 77.2% of the mosquitoes were collected during the dry season. ELISA, basedon the use of species-specificanti-sporozoite monoclonal antibodies, was used to analyse mosquitoes collected for human Plasmodium species. The positivity rate of the mosquitoes of all 15 species tested was 0.799% (23/2876): 15 Arwpheles albitarsis, 4 An. nuneztovari, and one each of An. braziliensis, An. triannulatus, An. oswaldoi and An. rangeli. Plasmodium falciparum sporozoite antigen was detected in An. albitarsis and An. oswaldoi; P. vivax and P. vivax variant VIZ247 were detected in An. albitarsis and An. nuneztovari; and P. malariae in An.
in the
M. M. Pbvoa’, N. Segura’, R. Lacerda’, 0. Vaz da Silva’, R. N. Almeida’, R. Lessa3, V. RosPrio4, M. A. Miles5 andD. C. Warhurst ‘Institute Evandro Chagas-FNS, Be&, Parch, Brasil; ‘Funda@o National de Satide, Belkm, Parti, Brasil; 3Hospita1 de Serra do Navio, Amapd State, Brasil; 41nstituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal; ‘AMBDU, Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK
[This presentation was awarded a prize as the best poster at the meeting.] Poliomyelitis in Punjab, muscular injections
India:
role of unnecessary
intra-
A. S. Sethi Department of Virology, United Medical and Dental Schools of Guy’s and St Thomas’s Hospital, St Thomas’s Campus, London, UK
This report represents the results of experience at the Oberoi Hospital, Jallandhar in Punjab during October to December 1991 conducted whilst on a student elective. Fifty patients with poliomyelitis-induced paralysis (28 male, 22 female) under 5 years of age were examined. 76% of children who acquired infection had completed a 3 dose course of orally-administered live attenuated vaccine. 64% had been injected intramuscularly by either local doctors or unqualified practitioners for a variety of febrile illnesses within 14 d or less of patients developing poliomyelitis. Invariably, the paralysed limbs were those injected, mostly the lower limbs following gluteal injections. Our results indicate that the administration of 3 doses of oral polio vaccine, which is manufactured in Bombay and transported to Punjab, is often ineffective, perhaps because of inadequate maintenance of the ‘cold chain’. Efforts must therefore be directed towards improving vaccination strategies, including giving further doses of vaccine, as has been shown to be successful in South India. Although details of intramuscular injections rely on parental recall, the high incidence of paralysis preceded by intramuscular injections emphasizes the importance of directing the attention of local practitioners to the risks of unnecessary intramuscular injections in polio endemic areas. [See WYATT, H. V. et al., 1992: Transactions of the Royal Society of Tropical Medicine and Hygiene, 86, 546549.-Editor.]
382
Trypanothione reductase apy of trypanosomiasis
as a target
for the chemother-
‘Department K. Smith’, W. H. Hunted and A. H. Fairlamb’ of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; 2Department of Chemise, Vniversi’ry of Manchester, Manchester, UK
[No abstract submitted.] The effect of axenic versus xenic culture the total and secreted proteolytic activity histoolyticastrains
conditions on of Entamoeba
Department of Medical ParasitoW. M. Spice and J. P. Ackers logy, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT
Isolates of N. gonorrhoeaewere collected from patients attending a sexually transmitted disease clinic in Mwanza, a large industrial town in northern Tanzania; 71 strains were collected and tested against 10 antimicrobial agents. 59% of strains were penicillin resistant; of these resistance was plasmid mediated in 48% and chromosomal in 11%. 80% of strains were resistant to tetracycline; of these 30% had high level, plasmid mediated resistance. This level of resistance is higher than that reported in recent studies from Zaire (VAN DYKE, E. et uE., 1992: Genitourinary Medicine, 68, 11 I-116). However, in southern Tanzania the resistance level remains low (unpublished observations). Only 1% of the strains were resistant to co-trimoxazole at the recommended treatment levels. All strains remained sensitive to spectinomycin, erythromycin, ciprofloxacin, norfloxacin, cefotaxime and cefuroxime.
[No abstract submitted.] Impregnated rural Zanzibar
bed nets
reduce
malaria
transmission
in
‘London A. H. R. Stich’, C. A. Maxwell’>l and C. F. Curtis* School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; 2Zanzibar Minishy of Health, P.O. Box 236, Zanzibar, Tanzania
Several recent reports have suggested that impregnated bed nets may not be sufficiently powerful to control malaria when transmission is very intense. However, we report encouraging results in a trial which was carried out in the seasonof the highest malaria transmission in a holoendemic area of rural Zanzibar. The initial parasitaemia in all age groups of almost 70% was cleared with Fansida@ from a cohort in 2 villages, one of which was provided with permethrin treated bed nets. The subsequent rate of reinfection was 0.9% per week in the netted village and 9% in the control. The mean haemoglobin level rose significantly in the former but not in the latter. Reports of fever with shivering, headacheand exhaustion while working all declined significantly in the 3 months after nets were introduced into the intervention village, but not in the control village. Anopheks mosquitoes caught in the netted village showed a significantly lower sporozoite rate than in the control. The study clearly indicates a considerable impact of impregnated bed nets on malaria transmission in rural Zanzibar during the time of the highest infective pressure. Differentiation of Pseudomonas restriction profiles
pseudomallei
by rDNA
S. Trakulsomboon, D. A. B. Dance and T. L. Pitt Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
[No abstract submitted.] Antimicrobial from northern
susceptibility Tanzania
of
Neisseria
gonorrhoeae
B. West’, J. Changalucha2, H. Grosskurth3, P. Mayaud3, D. ‘London School of Hygiene and TropiMabey’ and R. Hayes’ cal Medicine, Keppel Street, London, WCIE 7HT, UK; 2National Institute for Medical Research, Mwanza, Tanzania; 3~~~~~, P.O. Box 1482, Mwanza, Tanzania
In Tanzania most laboratories do not have the facilities to isolate or sensitivity test Neisseriagonorrhoeae.It is important, therefore, to look at any trends or patterns in the susceptibility of N. gonorrhoeueisolated in the area to assesscurrent antibiotic treatment regimes. The present recommendations for treating genital discharge syndrome is a rrimethoprtisulphonamide combination and tetracycline, to cover the possibility of gonorrhoeae or chlamydial mfection.
Progress towards a simplified polymerase assay for the detection of tuberculosis
chain reaction
S. M. Wilson, R. McNerney, P. Godfrey-Faussett and N. G. Stoker Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
Problems of expense, contamination and. the logistics of the reaction preclude the simultaneous PCR of large numbers of samples and lead critics to question whether PCR could ever be used in routine diagnosis particularly in developing countries. We have been working to address these problems and have devised a simplified protocol for the detection of Mycobucterium tuberculosis by PCR. Sputum collected on filter paper disks was used for PCR and the results agreed with microscopy. This greatly simplified sample collection and storage as samplescan be readily posted and remain stable for up to 4 years. A one-tube nested PCR was devised which combines 2 successivePCRs with different primer sets into a single reaction. This reduces expense and risk of contamination. A microtitre plate-based calorimetric method has been devised for detection of PCR product which greatly simplifies detection and enables large numbers of sample to be processedsimultaneously. Combined, these measures reduce the reagent cost of PCR to less than CO.5(sterling) per reaction and should promote the more widespread application of the PCR technique. lmmunocytochemical localization (hsp-70) in leprosy lesions
of heat shock
protein
S. K. Young’, D. B. You& M. J. Colston, J. N. A. S. StanlDepartment of Clinical Sciences, ley4 and D. N. J. Lockwood’ London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; ‘MRC Tuberculosis Unit, Hammersmith Hospital, Du Cane Road, London, W12 OHS, UK; 3National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 lAA, UK; 4Dhoolpet Leprosy Research Centre, Hyderabad, India
Leprosy is regularly complicated by the development of reversal reactions when peripheral nerve and skin lesions become acutely inflamed. This inflammation frequently results in nerve damage. One of the immunodominant Mycobucttium leprue proteins, of molecular mass70 kDa, has been shown to belong to the heat shock protein family (hsp-70). We have studied a group of patients with acute reversal reactions attending Dhoolpet Leprosy Research Centre. Skin and nerve biopsies were taken from these patients for histological and immunological studies. Mouse monoclonal antibody specific for the host-inducible form of hsp-70 has been used in a twostep immunoperoxidase technique. The study demonstrated a significant increase of the patients’ own inducible hsp-70 in skin and nerve biopsies and its distribution in granulomas.
TRANSACTIONS OF THEROYALSOCIETY OF TROPICAL MEDICINE AND HYGIENE (1993) 87, 377-382
Royal Society
of Tropical
Poster Meeting, London 26 November 1992 Enhancement ization
of selective
toxicity
Medicine
School
of brusatol
of Hygiene
by derivat-
D. Allen’, G. C. Kirby’, J. D. Phillipson’, I. Toth3, D. C. Departments of ‘Pharmacognosy Warhurst and C. W. Wright’ and 3Chemisny, The School of Pharmacy, University of London, 29-39 Brunswick Square, London, WCIN IAX, UK; 2Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
Plant-derived quassinoids, many of which inhibit protein synthesis by Plasmodium falciparum (KIRBY et al., 1989: Biochemical Pharmacology, 38, 4367-4374), often possess gross toxicity against mammalian cells. Derivatization of quassinoids to promote selective toxicity may prove to be a feasible method for obtaining novel antimalarial drugs. We previously reported an increased antimalarial effect with concomitant reduced cytotoxicity for 3-methoxyethoxymethyl brusatol, a semisynthetic derivative of the natural quassinoid brusatol (PATEL et al., 1991: Transactions of the Royal Society of Tropical Medicine and Hygiene, 85, 316). Two novel 3-acylated derivatives, brusatol-3-yl-2-(tertbutoxycarbonylamino) decanoate and brusatol-3-yl-acetate were prepared and evaluated: both had high antimalarial activity in vitro, comparable to that of the parent brusatol, but both were significantly less cytotoxic in vitro against KB tumour cells, suggesting that substituents at the C3 position may radically affect cytotoxic, whilst sparing antimalarial, properties. In contrast, conversion of the Cl3 methoxycarbonyl of brusatol and brusatol-3-yl-2-(tertbutoxycarbonylamino) decanoate to an amide (13-amido-brusatol; 13-amido-brusatol-3-yl-2-(tertbutoxycarbonylamino) decanoate) significantly decreased both antimalarial activity and cytotoxicity suggesting that this ester moiety is important for biological activity. (Supported by the Wellcome Trust, Science and Engineering ResearchCouncil, and the Medical ResearchCouncil.) Splenic lymphoma with villous mon lymphoma in Ghana
lymphocytes
and Hygiene
is a com-
G. Bedu-Addo’, T. R. Rutherford2, D. H. Bevan’ and I. Bates2 ‘Komfo Anokye Teaching Hospital, Kumasi, Ghana; 2St George’s Hospital Medical School, London, UK
Splenic lymphoma with villous lymphocytes (SLVL) is a B-cell lymphoma which in the West predominantly affects elderly men (MULLIGAN, S. 81 CATOVSKY, D., 1992: Leukaemia and Lymphoma, 6, 97-105). We have diagnosed SLVL in 10 Ghanaian patients with massive splenomegaly, no lymphadenopathy but with lymphocytosis. Characteristic villous lymphocytes with polar cytoplasmic projections formed over 30% of circulating lymet al., 1989: Journal of Clinical phocytes (BENNET Pathology, 42, 567-584). The lymphocytes were positive for the pan-B surface marker CD19 but negative for CD5 and CDllc, which are positive in chronic lymphocytic leukaemia (CLL) and hairy cell leukaemia respectively. 20% of patients had a monoclonal gammopathy without immune paresis and all of the 5 examined had rearrangements of the immunoglobulin gene. Compared to countries in the West, SLVL is more common in Ghana and predominantly affects middleaged women. It is clinically indistinguishable from the African variant of CLL, which presents with splenomegaly rather than lymphadenopathy (ESSIEN, 1976: East African Medical Journal, 53, 9&103). As African CLL is generally diagnosed without evidence of CD5
and Tropical
Medicine
positive surface markers, it is likely that SLVL has previously been misdiagnosed as CLL. Some patients with hyper-reactive malarial splenomegaly have significant numbers of villous lymphocytes. This fact, combined with demographic features and lower than expected inatients, leads cidence of haemoglobin AS among SLVL to speculation about the involvement oF malaria and other co-factors in the aetiology of SLVL. Construction of a vector suitable for cloning major outer membrane protein into vaccinia
chlamydial
Tom J. Blanchard, Jacquie Keer, N. G. Stoker and D. C. W. Mabey Department of Clinical Sciences, London School of Hygieneand Tropical Medicine, Keppel Street, London, WClE 7HT, UK
Chlamydiae are the cause of much human disease in the form of trachoma, sexually transmitted disease and pneumonia. It is hoped that recombinant vaccinia may serve as a vehicle to express isolated chlamydial proteins in an immunogenic form. This would enable cytotoxicity assays to be performed using recombinant-infected target cells and histocompatibility-matched peripheral blood lymphocytes (PBL). By taking PBL from epidemiologitally defined groups exposed to trachoma it is hoped that any protective role of specific cytotoxicity may be defined. The recombinant vaccinia (or other poxvirus recombinants) mav also serve as vaccine candidates in the future. ’ Construction of vaccinia recombinants by a method similar to that of MACKETT. M. & SMITH. G. L., (1986: Journal of Virology, 67,206?-2082) first requires &at the coding sequence for the foreign gene be inserted into a vector plasmid site in the correct orientation. A successful method for the precise and rapid cloning of appropriately modified chlamydial MOMP B sequence into pSCl1 is described. The method uses T:A cloning of polymerase chain reaction (PCR) products incorporating a microwave/chemiluminescent hybridization screen. The precise nature of the ligation is confirmed by altered restriction site analysis. The PCR primers used are suitable for cloning all serotypes of chlamydial major outer membrane protein. The use of recombinant mycobacterial antigens in functional assays with human peripheral blood mononuclear cells K. Britton, S. K. Young, A. Alvi, M. F. R. Waters, K. P. W. J. McAdam and H. M. Dockrell Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
An increasing number of recombinant Mycobacterium leprae and M. tuberculosis antigens is becoming available for use in functional assays through the World Health Organization Mycobacteria Antigen Bank. We have tested these antigens in T cell proliferation assays and found that all are suitable for use in immunological assays. T cell proliferation confirms that the M. tuberculosis 70 kDa, M. bovis 65 kDa, M. leprae 65 kDa and M. leprae 18 kDa antigens can stimulate T cells in BCG vaccinated normal donors and leprosy patients. The presence of endotoxin in some of the preparations does not appear to affect T cell proliferation. Thus these antigens can be used to probe antigen recognition and T cell function in mycobacterial disease.
378 Cryptosporidiumin
wild rodents
R. M. Chalmed, A. Miller’. D. P. Casemore* and A. Cm3 ‘School of Natural and Envimkmental Sciences, Covenhy University, Covens, UK; ‘Rhyl Public Health Laboratory; 3Manchester Public Health Laboratoly
The protozoan parasite Cryptosporidium causes diarrhoeal disease in humans and livestock. There has been increasing evidence of the importance of environmental routes of transmission. The genus has at least 4 named, well characterized species, of which C. muris, the first species to be described, and C. parvurn are found in mammals. It is believed that most human and livestock infections are with C. parzwn. Since the first description in 1907 in mice in the USA, C. muris has been described only occasionally. It is not known how frequently cryptosporidia are present in wild mammals, although they were found in a small survey in north Wales in 1984. This 2-year project was set up to look for cryptosporidia in livestock and wild animal species on an agricultural college farm in Warwickshire, and to explore the role of wild mammals in the natural history of this parasite. Mammals were trapped in live mammal traps and their faeces collected, together with those of associated livestock. Smears were examined using standard methods (MZN? auramine-phenol, IFAT) to detect oocysts. Prehminary results confirmed the presence of cryptosporidia in a high proportion of the mice trapped, mostly typical of C:- pawum but sometimes with the larger oocysts typical of C. murk. Mice excreting putative C. muris oocysts were killed and the enteric tract examined by light and electron microscopy. This confiied typical morphology and localization of endogenous stages restricted to the peptic glands of the stomach. Studies are continuing on the parasite and its ecology in the farm setting. lmmunodisgnostic kDa larval antigens
potential of 41 kDa, 31 kDa, and 28 of Strongyloidesstercoralis
D. J. Conway’, J. W. Baile$, J. F. Lmdo3, R. D. Robinson3, D. A. P. Bandy’ and A. E. Bianco’l’ ‘Wellcome Research Centre for Parask Infections, Department of Biology, Imperial College, Lo&m, SW7 2BB, UK; ‘Liverpool School of Tropical Medicine, Liverpool, L3 SQA, UK; 3Department of Zoology, University of The West Indies, Kingston 7,Jamaica
Proteins from a deoxycholate-soluble extract of Strungyloidesstercoralis infective larvae were separatedby SDSPAGE, at 20 @cm width of gel, and Western blotted on
to nitrocellulose membrane. Sera from individuals with confirmed S. stercoralis infections (a= lOO),long-standing S. stercoralis infections in whom no larva could be detected at time of serum sampling (n=27), and other nematode infections (n= 100; 40 with Wuchereria bancrofti, 20 with Onchocerca volvulus, 20 with Necator americanus, and 20 with mixed Ascaris lumbricoides and Trichuris trichiura), were tested at a l/100 dilution against strips of the membrane-bound proteins. Immunodominant proteins of approximately 41 kDa, 31 kDa, and 28 kDa were respectively recognized by IgG in 91/100, 88/100, and 90/100 of the confvmed strongyloidiasis sera, in 27127, 27/27, and 25127of sera from those with parasitologically undetectable strongyloidiasis, and in 9/100, 12/100, and 14/100 of sera from other nematode infections. Each of these 3 proteins was well represented in a larval fraction soluble in phosphate-buffered saline, and did not require deoxycholate solubilization for extraction. These data suggest that any of the 3 proteins could be used to develop a sensitive and specific immunodiagnostic test for S. stercoralis infection.
The threat of pyrethroid impregnated bednetr
resistance
to the effectiveness
of
C. F. Curtis and 0. Aina
London School of Hygiene and Tropical Medicine, London, WCZE 7HT, UK
The precedents in agricultural pests and house flies might suggest that there is an imminent risk of pyrethroid resistance interfering with the successful use of bed net impregnation against malaria vectors. However, in Sichuan, China, where up to 2.25 million nets have been treated with deltamethrin annually for 5 years, 100% mortality was found in preliminary tests with the World Health Organization (WHO) recommended discriminating dosage of deltamethrin on both the vector species: Anopheles anthropophagus and An. sinensis. An artificially selected strain of An. stephensi of Dubai origin showed unequivocal resistance to permethrin in WHO test kits. As a more realistic test it was given the opportunity of feeding on blood through impregnated netting. There was a higher rate of feeding and lower death rate in the resistant-strain than in a susceptible strain when any of 3 pyrethroids, or the related but non-pyrethroid compound Trebon@, were used. However, comparison with results using untreated netting showed that the resistant strain was not fully protected against any of these insecticides under realistic testing conditions. The killing of bedbugs has been one of the attractive features of impregnated bed nets of tropical villagers. Bedbugs showed rapid evolution of DDT resistance and it is important to monitor these insects for pyrethroid susceptibility. The morphogenesis
of Hantaan
virus
D. S. Ellis’, D. G. Tovey’, P. Skidaria and D. I. H. Simpson2 ‘London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; ‘The Queen’s University ofBelfast, Grosoenor Road, Belfast, BT12 6BN, UK
Hantaan virus is the causative agent of Korean haemorrhagic fever which, in the Korean war, caused 3000 caseswith 5% mortalitv. It has since been recognized in many European and A&an countries. It is a pleomorphic RNA virus. 80 to 140 mn in diameter. covered with 10 nm spikes and containing ‘ribosomes’. ’ When grown in Vero cells, by 36 h the Golgi apparatus had become enlarged and 36 h later the cytoplasm contained increased numbers of ribosomes together with numbers of multivesicular bodies containing 50 nm particles apparentlv not dire& related to the maturation procesc -Aggregations of ‘pie-viral stroma’ were seen, which bv 96 h had become laree massesof soiral material (similar to the RNA spirals se& previously in developing Marburg virus), some lying within vacuoles. At 120 h, the first mature Hantaan particles were seen budding into large vacuoles. The liberation of these virions occurred during the rupture of the vacuoles or following the death of the host cell. While a considerable degree of asynchronicity was found in the development of Hantaan virus, a clearlv recognizable ‘second’ generation of virions was found maturing between 204 and 216 h, the ‘RNA spirals’ appearing again at 196h. Species-specific
serodiagnosis
of schistosomiaris
M. El-Tahawy’, M. J. Doeohoff2, A. J. Probert=, E. Ahari’, K. LaUoo3 and P. L. Chiodioi3 ‘Department of Clinical Pathology, Ain Shams University, Cairo, Egypt; 2School of Biological Sciences, University College of Wales, Bangor, Gwynedd, LL57 2UW, UK; 3Department of Clinical Parasitology, Hospital for Tropical Diseases, London, NW1 OPE, UK
The immunological sensitivity and specificity of Schistosoma mansoni crude soluble egg antigen (SEA), 2 cationic egg antigens (CEF6), and S. margrebowiei egg antigen were studied by ELISA (MCLAREN et al., 1981:
379 Transactions of the Royal SO&Y of Tropical Medicine and Hygiene, 75, 72-79) in 221 patients with schistosomal, other parasitic and viral diseases.S. margrebowieiis a sibling species of S. haematobiumwhich is easier to maintain. Since it is difficult to get S. huematobium antigens, S. margrebowieiprovides a good, readily available source of antigens. Use of SEA in ELBA is a sensitive epidemiological screening method for identifying schistosomiasis. SEA and S. margrebowiei egg antigens cross reacted significantly with sera from patients infected with Trichinella spiralis and Clonorchis sinensis. CEF6 showed no cross reaction with non-schistosomal patients’ sera. S. margrebowieiegg antigen was more specific to S. haemotobium natients’ sera. while CEF6 was more snecific to S. mansom natients’ sem. Serodiagnosis of sctistosomiasis using the 3 antigens provided-a serological profile capable of differentiating parasitologically-negative patients into those infected with S. man.& or S. haematobium. By comparing the pre-treatment and post-treatment ELISA results from 30 S. munsoni patients, CEF6 was k$;Abe a better predictor of chemotherapy success Characterization of mepacrine uptake by erythrocytes infected with ~/asmodium fakipsrum using fluorometric techniques
E. I. Elueze’, L.-J. Wu*, S. L. Cro!? and D. C. Warhurst’ ‘Department of Medical Parasitoloav, London School of Hwiene and-Tropical kedicine, Keppel Stree~,iondon, WClE, iHT;UK; 2hstitute of Parasitic Diseases,Shanghai, Chino
Mepacrine uptake into erythrocytes infected with chloroquine-sensitive (T.996 clone) and chloroquine-resistant (Kl strain) Phmodium fakipancm was evaluated using flow cytometry and fluorescence spectrometry. In both fluorometric techniques, uptake was seen to be rapid (steady state reached in 10 min) and both glucose and temperature dependent. Preincubation with N-ethyhnaleimide (NEM), dicyclohexylcarbodiimide (DCCD), oligomycin and carbonylcyanide-chlorophenylhydrazone (CCCP) resulted in a decreasein total drug uptake, presumably by causing a reduction in the acidity of the lysosomes. Ouabain and vanadate had no effect on mepacrine uptake. The rise in apparent drug concentration in the cytoplasmic compartment detected by flow cytometry in the presence of NEM, DCCD and oligomycin suggeststhat an additional drug accumulation system is operating at the parasite cytoplasmic membrane or that the drug is being exported from the lysosome into the cytoplasm. Electron microsconv revealed changes in the food vacuole after 30 min exposure of erythrocytes infected with P. falcibarum (Kl strain) to menacrine (2X10-’ M). Changes included enlargement of-the central food vacuole, formation of intravacuolar membrane whorls, and a decreasein the number of pigment granules. Acknowledgements. E.I.E. and L.-J.W. are sponsoredby the Associationof CommonwealthUniversities, UK and the &o-British FellowshipTrust respectively. In vitro antimalarial
N. Hi,
effects
of some insecticides
G. C. Kirby and D. C. Warhurst
Department of Medical Parasitology, London Schoolof Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK
Recent research has shown that a number of pyrethroid insecticides inhibit oocyst development in the mosquito gut (CARLE et al:, 1986: ComptesRendus de I’Academie ah Sciences,She 3, 303, 565-568; HILL et a&, 1989: Transactions of the Royal Society of Tropical Medtcine and Hygiene, 83, 42-26); in the earlier of these works it was also reported that the pyrethroid deltamethrin exhibited activity against erythrocytic Plasmodium fakiparum in vitro comparable to that of chloroquine. We have attempted to demonstrate the antiplasmodial activ-
ity of this and of some other insecticides using a standard in vine antimalarial screening test; compounds tested were deltamethrin (highest concentration O-5mg/mL), hcyhalothrin, DDT, permethrin (1 mg/mL) and malathion (10 mg/mL). None of these 5 agents (representing 3 distinct classesof insecticide) showed sianiticant activity against erythrocytic P. fakiparum; ona molar basis, h-cyhalothrin and permethrin, with the highest degrees of antiplasmodial activity, were approximately 25 and 32 thousand times less active than chloroquine in the same tests. Despite inhibitory effects of pyrethroids upon oocyst development during the sporogonic cycle of Plasmediumin Anophelesstephensi,direct toxic action of these insecticides upon the erythrocytic stages of P. falciparum appearsnot to occur. (Supportedby the WellcomeTrust.) Plasmodium in Anopheles insecticides
falciparum and P. yoeliioocyst development mosquitoes exposed to sublethal doses of
Nigel Hill’ and Lisa Ranford-Csrhvright2 ‘Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; 2Depamnentof ProtozoanGenetics,University of Edinburgh, Edinburgh, UK
It has been reported that the pyrethroid deltamethrin has the ability to suppress oocyst development in mosquitoes at doseswhich are not lethal to the insect. Our studies on this and two other pyrethroids, h-cyhalothrin and permethrin, involved feeding Anopheles stephensi mosquitoes, through treated or control netting, on a mouse infected with P. yoelii. Exposure to any of these pyrethroids reduced the total number of individuals that becameinfected as well as the number of oocysts per gut in those that were infected. Investigations using a range of other insecticide classes failed to show any similar effect on infection rates or oocyst numbers. In an attempt to ascertain how important this phenomenon could be in terms of human malaria transmission, we have conducted experiments using Pkmwdium falciparum. Mosquitoes were membrane-fed on cultured P. faZciDaramgametocvtes of strain 3D7 (a clone of NF54) and then exposed to &25% permethrinpapers for 1 h. On every occasion infection rates were on average20% lower in treated females compared to controls. Having confiied that sublethal contact with pyrethroids does reduce subsequent infection levels with P. falciparum, it is suggested that this phenomenon may have an effect on diseasetransmission, particularly in relation to intervention using pyrethrotd impregnated bed nets. Metabolism and malaria
of antipyrine infection
and metronidazole
during fever
Gilbert 0. Kokwaro, Sabariab Ismail, Anthony P. Glazier, Stephen A. Ward and Geoffrey Edwards Department of
Pharmacologyand Therapeutics,University of Liverpool, P.O.Box 147, Liverpool, L69 3BX, UK
Malaria infection and fever impair drug metabolism in rats (MANSOR, S. et al., 1990: 3ouwwl of Pharmacy and Pharmacology, 42, 428-$38; MANSOR, S. et al:,. 1990: Transactionsof the Royal Society of Tropical Medtane and Hygiene, 84,460). Impaired drug metabolism could lead to dru toxicity or altered efficacy. We have used a ‘cocktail’ oBantipyrme and metronidazole to investigate the effeet of malaria infection and fever on the activities of different isozymes in the rat. Rats (12 for each treatment) were inoculated with Plasmodium berghei, bacterial lipopolysaccharide, or saline before administration of antipyrine and metronidazole at an appropriate time. Clearance of antipyrine and metronidazole was determined from concentrations in a 4 h saliva sample.
380 Malaria infection and fever reduced clearance of metronidazole by 20% and 23% respectively. Clearance of antipyrine was unaffected by malaria infection, but fever reduced clearance of antipyrine by 36%. These results indicate that malaria infection and fever have different effects on different isozymes. The clinical relevance of these observations is being investigated. Host proteins hangi
on the surface of microfilariae
of Brugia pa-
M. N. Malecula, C. I. Baldwin and D. A. Denham Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK
[No abstract submitted.] Cryptosporidium murk in SCID mice: adoptive immunity with spleen or mesenteric lymph from infected BALE/C mice
transfer of node cells
V McDonald and G. J. Bancroft Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK Cryptospcnidium muris infection is readily obtained in adult immunocompetent mice, and this model system should be valuable for the study of the mammalian immune response to cryptosporidial infection. We previously found that immunocompetent mice recovered from C. muris infection but nude and SCID mice developed unresolved chronic infections. Recovery was observed in SCID mice if they were injected with uninfected BALB/C spleen or mesenteric lymph node (MLN) cells. In the present study an attempt was made to transfer immunity adoptively to KID mice from infected BALB/C mice by intraperitoneal injection of lx 10’ spleen or MLN cells taken either when oocyst production was just past its peak (day 21), when the production had just ceased (day 29), or several days after infections had become subpatent (day 43). Control groups were also included. of SCID mice given MLN cells from uninfected mice or no lymphoid cells. The reconstituted SCID mice were infected with 2X lo5 oocysts of C. muris and the protective effect of the donor cells was measured by oocyst production. Immunity could be transferred adoptiveiy to SCID mice with MLN or spleen cells taken from infected donor mice on davs 43 or 29, but not on day 21. Recipients of primed MLN cells from day 43 produced more than 8 times fewer oocysts than recipients of unprimed MLN cells. The results indicated clearly that immunity could be transferred adoptively if the donor animals had developed resistance to infection. Resistance of SCID mice to Cryptosporidium parvum infection and evidence for a protective mechanism involving interferon y V. McDonald
and G. J. Bancroft
Department of Clinical
Sciences,London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
Most mammalian
species are normally susceptible to infection only in the first few weeks of life. Adult mice are comnletelv resistant to infection unless deprived of T cells (e.g., infections can be obtained in nude or SCID mice). The immunological basis for this resistance was examined further in SCID mice. Infection in these animals is chronic: in the first 4-8 weeks small numbers of oocysts are excreted, but subsequently there is a sharp rise in oocyst production and the animals demonstrate signs of CrvDtosnoridiosis. Treatment of mice during theearly weeks ofinfection with an anti-interferon Y hamster monoclonal antibodv (H22) resulted in a significant increase in oocyst excretion and’an earlier onset of illness. When administration of the antibody was stopped, oocyst excretion decreased almost to the level
Cyptosporidium
paruim
observed in untreated SCID mice. These results suggested that SCID mice showed non specific immunological resistance to C. parvum during the early weeks of the chronic infection, and that a T cell-independent source of interferon y was involved in this resistance. Using a chemiluminesent in a ‘low tech’ laboratory
DNA probe to detect
parasites
R. McNerney’, S. M. Wilson’, M. A. Miles2, J. A. Fonseca de Castro3 and A. Vexenat3 ‘Department of Clinical Sciences and 2Applied Molecular Biology and Diagnosrics Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK; ‘Deparnento de Parasitologia e Microbiologia, Universidade Federal do Piaui, Teresina, Piaui, Brasil
DNA probe technology has proved to be an invaluable tool in biomedical research laboratories. The apnlication of this technology has, however, been restricted by the necessity to use radiolabels which are inherently unstable and require specialized handling. The development of sensitive chemiluminescent alternatives has enabled us to adapt a DNA probe assay for use in less sophisticated or ‘low tech’ laboratories such as those found in developing g;nries (WILSON, S. M., 1992: Parasitology, 104,421-
.--,.
The Lmet2 DNA probe (HOWMID, M. K., 1991: MoleParasitolow, 44. 63-72’1 was used to show the presence of parasites ~f’the~Leis&znia donovani complex in ear scrapes from dogs in Teresina, an area of Brazil where visceral leishmaniasis is endemic. The result was obtained within 24 h of the samples being taken and was compared to microscopical examination and the direct agglutination test (DAT). The DNA assay was performed without the use of sophisticated equipment or facilities such as hybridizing ovens or darkrooms. The spread of other new techniques for parasite diagnosis to laboratories in developing countries has in the nast been limited by the requirement for snecialized reagents such as monoclonal antibodies. It is hoped that the relative ease with which labelled DNA nrobes can be produced and the ready availability of the other components required will make the DNA probe technique appropriate for transfer to those countries where parasitic diseases are a public health problem.
cular and Biochemical
An age-stratified study of total sponses to Necator americanus in two endemic communities
IgE and specific IgE readult cuticular antigens
D. R. Palmer’. M. Bradlev* and D. A. P. Bundv’ ‘Wellcome Research Centre, Department of Biology, Imperial College, Prince Consort Road, London, SW7 2BB, UK; ‘Blair Research Institute, Harare, Zimbabwe
Epidemiological studies show lower rates of egg production by Necator americanus in adults living in rural communities in the Charara estates, Kariba, Zimbabwe and in Marakissa, The Gambia. The overall prevalence of infection in Zimbabwe was about 60% with moderate egg counts (O-2600 eggs/g). In contrast, in The Gambia, infection was light (O-650 eggs/g) with an overall prevalence of 29%. As part of a continuing project to identify possible immunological mechanisms, we measured both total and parasite specific IgE levels in individuals living in these communities. Total IgE levels in both communities were elevated above those of healthy European controls and correlated oositivelv with age. However. snecilic IgE resnonses to ihe parasite wer
381 and antibody responses.The lack of specificity in the IgE response in the Marakissa study may have been due to the lower level of antigenic challenge in infected individuals. These observations support the view that the elevation of IgE antibody levels reflects exposure to infection and may have a limited anti-fecundity role. Haptoglobin in children in adjacent malaria meso-endemic and non-endemic villages in Amapl State, Brazil M. M. P&oa’>* and D. C. Warhurst ‘Setor de Parasitologti, Institute Evandro Chagas-FNS, BeUrn, Par& Brasil; ‘AMBDU, Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK
A study of the relations between ahaptoglobinaemia and malaria was carried out in 100 children aged 2-14 years from the Serra do Navio region, Amap State, Brazil including Serra do Navio. Coloma Aeua Branca. Porto Terezinhi, Arrependido &d an indigeious group: Their serum haptoglobin levels were measured by-a radial immunodiffusion assav. The mean hautoglobin level in Serra do Navio and Port0 Terezinha, &hi;h are non-endemic and hypo-endemic areas for malaria, resnectivelv, was similar (normal level) while for the other areas, ali meso-endemic areas for n&aria, it was lower. There was a higher correlation @‘<0X)01) between ahaotoglobinaemia &d current ma&a than between ahaptbglobinaemia and a history of past malaria (PcO.01). Financial support was provided by the UNDlYWorld Bank/WHO/SpecialProgrammefor Researchand Training in Tropical Diseases(grant no. 870284),ICOMI, and Instituto EvandroChagas-FNS. Malaria endemicity Amap& State, Brazil
in the
Serra
do Navio
region
of
albitarsis, An. nuneztovari, An. braziliensis, An. triannulatus and An. rangeli. All mosquitoes positive for P. ma-
luriue were collected in the forest adjacent to the study areas(i.e., they may have been infected with P. brasilianum from non-human primates). This work was supported by the UNDP/World Bank/ WHO/SpecialProgrammefor Researchand Training in Tropical Diseases(grant no. 870284),ICOMI, and Instituto Evandro Chagas-FNS. A Schistosoma mansoni esterase mune dependence of chemotherapy
implicated
in the im-
B. A. Ripley, P. G. Fallon, A. J. Probert and M. J. Doenhoff School of Biological Sciences, University of Wales, Bangor, UK
[No abstract submitted.]
M. M. Pbvoa’,‘, P. Amorim’, V. Calvosa’, E. Santa Rosa’, J. M. Nascimento’, V. Ros6rio3, M. A. Miles4 and D. C. Warhurst4 ‘Setor de Parositologia, Institute Evandro Chagas-FNS, Belt%, Par& Brasil; *ICOMI-Sewa do Navio, Amapci, Brasil; 31nstiko de kfigiene e Medicina Tropical, Urtiversidade Nova de Lisboa. Lisbon. Portueal; 4AMBDU. Debartment of Medical Parasitology, L&don &h&l of Hygiek a& Tropical-Medicine, Keppel Street, London, WCIE 7HT, UK
To determine the malaria endemicitv in 4 adjacent villages of Serra do Navio region (Serra ho Navid, Colonia Agua Branca, Porto Terezinha and Arrependido), Amapfi State, Brazil, we carried out a blood film survey and measured the spleen of individuals aged 2-9 years. Based on these observations. Serra do Navio village (mevalence and spleen rate=O) &as classified as non-endemic for malaria, Porto Terezinha (prevalence= 1% and spleen rate=7%) as hypo-endemic, and the other 2 villages, Colonia Agua Branca (prevalence=21% and spleen rate= 18%) and Arrependido (prevalence= 17% and spleen rate= 11%) as meso-endemic areas for malaria. Reasonsfor the difference in endemicity in these villages in the samearea were discussed. This work was supported by the UNDPiWorld Bank/ WHO/SpecialProgrammefor Researchand Training in Tropical Diseases(grant no. 870284),ICOMI, and Instituto Evandro Chagas-FNS. Anophelines infected with human Plasmodium Serra do Navio region of AmapL State, Brazil
An epidemiological study to determine the distribution of anopheline speciesand their potentiality as vectors of malaria was carried out in 4 villages of the Serra do Navio region, Amap State in the north of Brazil. Fifteen anopheline species were identified among 3053 mosquitoes collected as human biting catches in the 4 study areas from February 1990 to October 1991. 77.2% of the mosquitoes were collected during the dry season. ELISA, basedon the use of species-specificanti-sporozoite monoclonal antibodies, was used to analyse mosquitoes collected for human Plasmodium species. The positivity rate of the mosquitoes of all 15 species tested was 0.799% (23/2876): 15 Arwpheles albitarsis, 4 An. nuneztovari, and one each of An. braziliensis, An. triannulatus, An. oswaldoi and An. rangeli. Plasmodium falciparum sporozoite antigen was detected in An. albitarsis and An. oswaldoi; P. vivax and P. vivax variant VIZ247 were detected in An. albitarsis and An. nuneztovari; and P. malariae in An.
in the
M. M. Pbvoa’, N. Segura’, R. Lacerda’, 0. Vaz da Silva’, R. N. Almeida’, R. Lessa3, V. RosPrio4, M. A. Miles5 andD. C. Warhurst ‘Institute Evandro Chagas-FNS, Be&, Parch, Brasil; ‘Funda@o National de Satide, Belkm, Parti, Brasil; 3Hospita1 de Serra do Navio, Amapd State, Brasil; 41nstituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal; ‘AMBDU, Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK
[This presentation was awarded a prize as the best poster at the meeting.] Poliomyelitis in Punjab, muscular injections
India:
role of unnecessary
intra-
A. S. Sethi Department of Virology, United Medical and Dental Schools of Guy’s and St Thomas’s Hospital, St Thomas’s Campus, London, UK
This report represents the results of experience at the Oberoi Hospital, Jallandhar in Punjab during October to December 1991 conducted whilst on a student elective. Fifty patients with poliomyelitis-induced paralysis (28 male, 22 female) under 5 years of age were examined. 76% of children who acquired infection had completed a 3 dose course of orally-administered live attenuated vaccine. 64% had been injected intramuscularly by either local doctors or unqualified practitioners for a variety of febrile illnesses within 14 d or less of patients developing poliomyelitis. Invariably, the paralysed limbs were those injected, mostly the lower limbs following gluteal injections. Our results indicate that the administration of 3 doses of oral polio vaccine, which is manufactured in Bombay and transported to Punjab, is often ineffective, perhaps because of inadequate maintenance of the ‘cold chain’. Efforts must therefore be directed towards improving vaccination strategies, including giving further doses of vaccine, as has been shown to be successful in South India. Although details of intramuscular injections rely on parental recall, the high incidence of paralysis preceded by intramuscular injections emphasizes the importance of directing the attention of local practitioners to the risks of unnecessary intramuscular injections in polio endemic areas. [See WYATT, H. V. et al., 1992: Transactions of the Royal Society of Tropical Medicine and Hygiene, 86, 546549.-Editor.]
382
Trypanothione reductase apy of trypanosomiasis
as a target
for the chemother-
‘Department K. Smith’, W. H. Hunted and A. H. Fairlamb’ of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; 2Department of Chemise, Vniversi’ry of Manchester, Manchester, UK
[No abstract submitted.] The effect of axenic versus xenic culture the total and secreted proteolytic activity histoolyticastrains
conditions on of Entamoeba
Department of Medical ParasitoW. M. Spice and J. P. Ackers logy, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT
Isolates of N. gonorrhoeaewere collected from patients attending a sexually transmitted disease clinic in Mwanza, a large industrial town in northern Tanzania; 71 strains were collected and tested against 10 antimicrobial agents. 59% of strains were penicillin resistant; of these resistance was plasmid mediated in 48% and chromosomal in 11%. 80% of strains were resistant to tetracycline; of these 30% had high level, plasmid mediated resistance. This level of resistance is higher than that reported in recent studies from Zaire (VAN DYKE, E. et uE., 1992: Genitourinary Medicine, 68, 11 I-116). However, in southern Tanzania the resistance level remains low (unpublished observations). Only 1% of the strains were resistant to co-trimoxazole at the recommended treatment levels. All strains remained sensitive to spectinomycin, erythromycin, ciprofloxacin, norfloxacin, cefotaxime and cefuroxime.
[No abstract submitted.] Impregnated rural Zanzibar
bed nets
reduce
malaria
transmission
in
‘London A. H. R. Stich’, C. A. Maxwell’>l and C. F. Curtis* School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; 2Zanzibar Minishy of Health, P.O. Box 236, Zanzibar, Tanzania
Several recent reports have suggested that impregnated bed nets may not be sufficiently powerful to control malaria when transmission is very intense. However, we report encouraging results in a trial which was carried out in the seasonof the highest malaria transmission in a holoendemic area of rural Zanzibar. The initial parasitaemia in all age groups of almost 70% was cleared with Fansida@ from a cohort in 2 villages, one of which was provided with permethrin treated bed nets. The subsequent rate of reinfection was 0.9% per week in the netted village and 9% in the control. The mean haemoglobin level rose significantly in the former but not in the latter. Reports of fever with shivering, headacheand exhaustion while working all declined significantly in the 3 months after nets were introduced into the intervention village, but not in the control village. Anopheks mosquitoes caught in the netted village showed a significantly lower sporozoite rate than in the control. The study clearly indicates a considerable impact of impregnated bed nets on malaria transmission in rural Zanzibar during the time of the highest infective pressure. Differentiation of Pseudomonas restriction profiles
pseudomallei
by rDNA
S. Trakulsomboon, D. A. B. Dance and T. L. Pitt Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
[No abstract submitted.] Antimicrobial from northern
susceptibility Tanzania
of
Neisseria
gonorrhoeae
B. West’, J. Changalucha2, H. Grosskurth3, P. Mayaud3, D. ‘London School of Hygiene and TropiMabey’ and R. Hayes’ cal Medicine, Keppel Street, London, WCIE 7HT, UK; 2National Institute for Medical Research, Mwanza, Tanzania; 3~~~~~, P.O. Box 1482, Mwanza, Tanzania
In Tanzania most laboratories do not have the facilities to isolate or sensitivity test Neisseriagonorrhoeae.It is important, therefore, to look at any trends or patterns in the susceptibility of N. gonorrhoeueisolated in the area to assesscurrent antibiotic treatment regimes. The present recommendations for treating genital discharge syndrome is a rrimethoprtisulphonamide combination and tetracycline, to cover the possibility of gonorrhoeae or chlamydial mfection.
Progress towards a simplified polymerase assay for the detection of tuberculosis
chain reaction
S. M. Wilson, R. McNerney, P. Godfrey-Faussett and N. G. Stoker Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
Problems of expense, contamination and. the logistics of the reaction preclude the simultaneous PCR of large numbers of samples and lead critics to question whether PCR could ever be used in routine diagnosis particularly in developing countries. We have been working to address these problems and have devised a simplified protocol for the detection of Mycobucterium tuberculosis by PCR. Sputum collected on filter paper disks was used for PCR and the results agreed with microscopy. This greatly simplified sample collection and storage as samplescan be readily posted and remain stable for up to 4 years. A one-tube nested PCR was devised which combines 2 successivePCRs with different primer sets into a single reaction. This reduces expense and risk of contamination. A microtitre plate-based calorimetric method has been devised for detection of PCR product which greatly simplifies detection and enables large numbers of sample to be processedsimultaneously. Combined, these measures reduce the reagent cost of PCR to less than CO.5(sterling) per reaction and should promote the more widespread application of the PCR technique. lmmunocytochemical localization (hsp-70) in leprosy lesions
of heat shock
protein
S. K. Young’, D. B. You& M. J. Colston, J. N. A. S. StanlDepartment of Clinical Sciences, ley4 and D. N. J. Lockwood’ London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; ‘MRC Tuberculosis Unit, Hammersmith Hospital, Du Cane Road, London, W12 OHS, UK; 3National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 lAA, UK; 4Dhoolpet Leprosy Research Centre, Hyderabad, India
Leprosy is regularly complicated by the development of reversal reactions when peripheral nerve and skin lesions become acutely inflamed. This inflammation frequently results in nerve damage. One of the immunodominant Mycobucttium leprue proteins, of molecular mass70 kDa, has been shown to belong to the heat shock protein family (hsp-70). We have studied a group of patients with acute reversal reactions attending Dhoolpet Leprosy Research Centre. Skin and nerve biopsies were taken from these patients for histological and immunological studies. Mouse monoclonal antibody specific for the host-inducible form of hsp-70 has been used in a twostep immunoperoxidase technique. The study demonstrated a significant increase of the patients’ own inducible hsp-70 in skin and nerve biopsies and its distribution in granulomas.