Research in progress: short presentations

TRANSACTIONS

OF THE ROYAL

SOCIETY

OF TROPICAL

MEDICINE

AND HYGIENE

(2002) 96,353-367

Royal Society of Tropical Medicine and Hygiene Meeting at Manson House, London, 13 December Research in progress:

2001

short presentations

Oral presentations Cloning, characterization gene encoding squalene mexicana

and knockout of the synthase of Leishmania

S. A. AlHarthi, P. A. Bates and M. L. Chance Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, UK Squalene synthase is the first dedicated enzyme in the sterol biosynthetic pathway, a chemotherapeutic target in leishmaniasis. A gene encoding squalene synthase was cloned from gDNA of Leishmaniu mexicana and revealed a 1245 nucleotide sequence encoding a polypeptide of 415 amino acids (47.4 kDa). The sequence showed 9 1.3% identity with the putative L. major squalene synthase gene and 36.0% identity with human squalene synthase. RNA analysis idicated that the gene is transcribed at low levels that can only be detected using RT-PCR. To assess the functional importance of this gene to L. mexicana, targeted gene disruption was performed. Two rounds of targeting resulted in replacement of squalene synthase alleles with PUfz and SAT antibiotic resistance markers. However, double knockout clones showed the presence of at least 2 further undisrupted squalene synthase alleles. No intrachromosomal changes were detected during the gene replacement process and restriction digestion indicated that squalene synthase is a single copy gene. One explanation is that L. mexicana may be aneuploid, which means extra small numbers of chromosomes are present in addition to the diploid set. Creation of L. mexicana squalene synthase null mutants by further rounds of gene replacement will address the importance of this enzyme to Leishmania. Childhood tuberculosis: a functional approach towards the identification of mutations and polymorphisms in genes controlling the interferon gamma pathway S. T. Anderson’, K. Wood’, S. M. Pienaati, B. Eley*, D. Beat@ and M. Levitt’ ‘Department of Paediatrics, St May’s at Imperial College, London W2 IPG, UK; ‘School of Child and Adolescent Health, University of Cape Town, South Africa Genetically determined differences in immune response are likely to affect outcome to infection with Mycobacterium tuberculosis. We hypothesized that susceptibility to tuberculosis (TB) in the general population is explained, in part, by functional polymorphisms in genes controlling the IFN-y pathway. Previous studies on children with a mutation in the IFNyRl gene have demonstrated a clear association between genotype and a functional defect in macrophage activation resulting in disseminated mycobacterial infection (NEWPORT, M. J. et al., 1996: New England Journal of Medicine, 335, 1941-1949). Children under 14 years of age, with microbiologitally proven or ‘probable’ TB, and healthy controls were screened for their ability to upregulate key cytokines in the IFN-y pathway. The children screened to date comprised 145 TB cases and 176 controls. Four children have been identified with consistently low TNF-a responses to stimulation with IFN-y and LPS and lymphocytes from 6 children have failed to upregu-

late IFN-y in response to both PHA and PPD, despite normal lymphocyte proliferation. These all require further investigation to identify the likely site of any molecular defect. Correlating immunological phenotype with genotype in well-defined patient groups may be a successful approach to identifying functional polymorphisms in TB susceptibility genes. The emergence and transmission resistant tuberculosis

of multiple

drug

Department 0x4 G. Bahcall and Roy M. Anderson of Infectious Disease Epidemiology, Imperial College School of Medicine, London, UK The resurgence of tuberculosis (TB) in the past 2 decades has led the WHO to declare a global emergency. Recent epidemics of multiple drug resistant (MDR) TB, which are far more di&ult and expensive to treat, have posed a significant world health problem. An epidemiological model is used to explore the development of drug resistance at 2 levels: first, the emergence of resistance during treatment (acquired resistance) and second, its transmission to new susceptible individuals in the population (primary resistance). The dynamics of an MDR-TB epidemic are shown to emerge in a series of linked sub-epidemics. There are 3 possible outcomes for such a TB epidemic: eradication of all TB, persistence of only resistant, or co-existence of both sensitive and resistant strains. Which outcome occurs is predicted in terms of the relative fitness of sensitive and resistant strains, given by the values of the respective effective reproductive number (Rs and RR). Using parameter values estimated from the literature and assuming control strategies as given by the WHO case detection and treatment rates, a prevalence of drug resistance comparable to that currently observed is predicted. Erythrocyte falciparum Africa

invasion phenotypes of Plasmodium field isolates from The Gambia, West

Margaret Pinde3, Adama JoofJake Baum’, Se&a*, Demba S. Jammeh’ and David J. Conway’ ‘Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK; 2Medical Research Council Laboratories, Fajara, I? 0. Box 273, Banjul, The Gambia Experiments on erythrocyte invasion by Plasmodium falciparum have led to the conclusion that there are at least 3 receptor-ligand interactions involved in invasion. These involve (i) a sialic acid-dependermtrypsinsensitive receptor (erythrocyte glycophorin A bound by the I? falciparum erythrocyte binding antigen EBA175), (ii) a sialic acid-dependermtrypsin-insensitive receptor (erythrocyte glycophorin B with an unknown ligand) and (iii) sialic acid-independentitrypsin-sensitive receptor(s) (as yet uncharacterized). These have been described using laboratory maintained isolates of I? falciparum, the majority of which depend to some degree on pathway (i). Recent characterization of invasion phenotypes of field isolates from India (OKEYEH,

354

SHORTPRESENTATIONS

J. N. et al., 1999: Infection and Immunity, 67, 57845791) found that many were able to invade erythrocytes’lacking either sialic acid (neuraminidase treated) or glycophorin A (trypsin treated) raising doubts about how representative laboratory isolates are of parasites in the field. Here we nresent the first descrintion of erythrocyte invasion phenotypes of parasites from Africa, where the burden of malaria mortality is greatest. The sensitivity of field isolates sampled in The Gambia, West Africa, to neuraminidase and trypsin fits well within the distribution seen in laboratory maintained isolates, highlighting the importance of characterizing invasion phenotypes of field isolates from different populations. [This oral presentation was awarded first prize at the meeting.] Gene sequence and transcript of a trypsin-like enzyme (TLl) species Culex quinquefasciatus

expression pattern from the mosquito

J. De Alcarax-Fossoul, N. Hawkes and Department of Parasite and Vector BiolJ. Hemingway ogy, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, UK Mosquitoes are responsible for the transmission of a large number of diseases, including malaria, dengue fever and filariasis. The ingestion of blood bv female mosquitoes alters the activity patterns of several digestive enzymes including trypsins, which are responsible for blood meal digestion (MULLER, H. J. et al., 1993: EMBO Journal, 12,289 l-2900; NORIEGA, F. G. et al., 1996: Insect Biochemistry and Molecular Biology, 2, 119126). The parasites that the mosquitoes transmit are taken in with the blood meal and have to survive the digestive process. The level of midgut trypsin activity correlates with the inhibition of parasite infectivity (SHAHABUDDIN, M. et al., 1995: Experimental Parasitology, 80, 2 12-2 19). Identification and characterization of the activity patterns of these enzymes is important in understanding the physiology of the complex digestive system and as a possible route to disrupting parasite transmission through the insect. We characterized a new Culex trypsin, TLI, which is similar to trypsins from the mosquito Aedes aegypti (NORIEGA, F. G. et al., 1999: Journal of Insect Physiologv, 45, 613-620). The innovative LightCycler technique was used to accurately determine the amount of enzyme expression in different mosquito life stages. TLl was expressed in all life stages except pupae. It was not induced or inhibited by blood ingestion, but its levels decreased gradually after feeding. TLl inhibition in late stages of Culex larvae could lead to the death of the adult insect before it is able to act as a vector for filariasis (for which the adult has to live a minimum of 10 days). VACSEL-a immunity contacts

longitudinal to tuberculosis

study of correlates of in TB patients and their

H. A. Fletcher, G. A. W. Rook, A. Zumla and The Centre for Infectious Diseases, VACSEL Study Group Windeyer Institute of Medical Sciences, 46 Cleveland Street, London WIT 4JF UK The failure of chemotherapy to halt the tuberculosis (TB) epidemic highlights the need for a more effective TB vaccine. The need is particularly acute for poorer nations where TB exerts a significant economic effect. The major obstacle for the development of a new TB vaccine is the size and lenath of clinical trials. Clinical trials could be simplified- by identifying markers of protection and susceptibility to TB. Enrolling susceptible individuals into clinical trials could reduce the over-

all number of subjects recruited and follow-up times could be reduced by using markers of protection as a trial end-point. VACSEL is an INCO-DEV-funded longitudinal study evaluating correlates of immunity in TB patients, and their contacts in The Gambia, Zambia and Ethiopia. Blood samples and clinical data are being collected from patients and their contacts over a 2-year period. Quantitative RT-PCR is being used to detect Type 1 and Type 2 cytokine mRNAs in unstimulated PBMCs (peripheral blood mononuclear cells) and in PBMCs stimulated with a selection of TB antigens. Immunological data are being combined with clinical information to identify clinically useful correlates of susceptibility or protection. Phenotypic characterization of anti-BCG sponses by whole genome statement analysis

re-

C. A. Hemingway’, A. Goodsall’, E. Scholvinck’, ‘Department of PaediaD. Relman2 and M. Levin’ tries, Imperial College School of Medicine, St May’s Campus, London W2, UK; ‘Department of Biochemisty, CCSR, 269 Campus Drive, Stanford University, Stanford, California, USA Socio-economic factors have long been known to play an important role in the development of tuberculous disease. In addition it is now widely accepted that genetic factors too can confer resistance or increase susceptibility to infection with mycobacteria. Microarray technology permits a genome-wide approach in the search to identify the genes and their products responsible for increased resistance or susceptibility. Bacille Calmette-Guerin (BCG) transformed with a luciferase reporter gene was used in a whole blood assay to assess individuals’ ability to restrict growth (KAMPMANN, B. et al., 2000: Journal of Infectious Disease, 182, 895-901). Individuals who showed a restriction in growth, i.e., a fall in luminescence at 96 h, were defined as ‘killers’. Total RNA was extracted from 9 timepoints over a 96-h period from 6 different individuals-3 ‘killers’ and 3 ‘non-killers’. Each timepoint had a corresponding BCG-lux negative control. Messenger RNA was then isolated, amplified and labelled with fluorophores for hybridization on to a cDNA spotted microarray. The results were analysed for changes in gene expression over the timecourse in killers and non-killers. These results were presented. Psychosocial functioning Salaam, Tanzania

of orphans

in Dar

es

V. Makame, C. C. Ani and S. M. GranthamMcGregor Centre for International Child Health, Institute of Child Health, University College London, 30 Guilford Street, London WCIN IEH, UK Forty-one children, whose parents had died from AIDS but were uninfected themselves, were compared with 41 non-orphans matched for age and neighbourhood in Dar es Salaam, Tanzania. They were given an arithmetic test and a structured questionnaire concerning their psychological well-being, attendance at school, experiences of punishment, reward and hunger. Comnared with non-ornhans, ornhans were sign& candy less likely to be in-school (P‘= 0.028), but lhose attending had similar arithmetic scores (P = 0.73). Signilicamly more orphans than non-orphans went to bed hungj (P = 0.02). Orphans had markedly poorer usvcholoaical well-beine P < 0.0001) with more iniernaliz&g problems and less positive’affect. As many as 34% reported they had contemplated suicide in the previous year compared with 12% of non-orphans (P = 0.016). Multiple regression indicated that the independent predictors of poor psychological wellbeing were: sex (female), going to bed hungry, less

SHORT

reward for good behaviour, and no current school attendance and explained 28% of the variance. A further 12% of the variance was explained by being an ornhan. It is concluded that these AIDS ornhans not only have unmet basic needs, but also have-markedly poorer psychological well-being, and without appropriate psychological interventions, their long-term mental health is in serious jeopardy. Clinical sparing

355

PRESENTATIONS

evaluation of azathioprine as a steroidagent for Type 1 reactions in leprosy

S. N. S. Marlowe’, R. A. Hawksworth2, C. R. M. lacob2 and D. N. 1. Lockwood’ ButlW. ‘Department of hfeectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, UK; 2Anandaban Leprosy Hospital, I? 0. Box 151, Kathmandu, Nepal

Type 1 (reversal) reactions (TIR) are important tissue-damaging phenomena responsible for a significant proportion of nerve damage in leprosy. Currently, prednisolone is the treatment for severe reactions but only 40-70% of patients respond. There is a clinical need for new and better immunosuppressant therapy. There are no controlled clinical trials of treatment for TlR and no published reports of the use of azathioprine in leprosy. Consequently this is a pilot study, comparing the treatment of 20 patients taking a 12week course of azathioprine (dose 3 mg/kg/day) plus a reduced S-week course of nrednisolone with 20 control patients taking a 12-week course of prednisolone. The study was carried out in a leprosy referral hospital in Nepal with all patients initially diagnosed with severe TlR and the 2 groups matched for severity. As there are no standard trial designs for TlR, we developed a new methodology in the form of a clinical severity scale (CSS) which was used for treatment monitoring and for comparisons between the 2 groups. All patients were assessedregularly during the 3-month treatment period and at 1 and 3 months post-treatment. [This oral presentation was awarded second prize at the meeting.] A community-based intervention trial to test the effectiveness of deltamethrin-impregnated dog collars to control canine zoonotic visceral leishmaniasis

when using a more sensitive outcome measure, i.e., the level of dog anti-Leishmaniu antibody units/ml (a negative change in antibody units/ml corresponding to ‘improvement in dog health’), collars were shown to confer 43% protection (Yates’ corrected x2, P = 0.02), with 811136 and 42197 dogs having a negative change in antibody units/ml in Dl and D2, respectively. Although of borderline significance, the latter results appear to confirm the findings from previous studies in Italy and Iran that DMICs not only protect dogs from sandfly bites, but from ZVL as well. Synthetic cecropin B, an insect antimicrobial peptide, demonstrates activity against the promastigote stage of Leishmania donovani and Brugia malayi microfilariae C. Strode and H. Townson Parasite and Vector Biology Division, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK

Antimicrobial peptides play an important role in the response of insects to infection but their role in combating parasitic infection remains unclear. A major group of peptides, the cecropins, are found in several groups of insects of medical importance. To study their specificity and mode of action, synthetic cecropin B has been tested in vitro against a range of organisms, including the promastigotes of Leishmania donovani and the microfilariae of Brugia malayi. Activity against promastigotes is dose-dependent within the range 12.5 100 w, whilst anti-filarial activity is seen only at 225 @. In trypanosomatids, apoptosis occurs in response to heat shock, peroxide and lectins, but is not involved in cecropin-mediated killing of promastigotes, as shown by electron microscop< DNA intearitv studies and the use of fluorescent probes. Ce&opin affects the permeability of the plasma membrane of Leishmania in a manner analogous to its effect on the cytoplasmic membrane of Escherichiu coli; in both cases 12.5 w cecropin induces permeability and subsequent death after 5-min exposure. Brugia requires a more lengthy exposure (12 h+) to cause death and the mode of action remains unclear. Investigations are underway to determine more precisely the interaction of cecropin with Leishmania plasma membrane. The malaria scavenger receptor: essential parasite development in the invertebrate host

for

R. Reithinger’ , B. Alexander’ and C. R. ‘Disease Control and VectorBiology Unit, DeDavies’ partment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK; ‘Centro de Pesquisa R&e Rachou, Avda August0 Lima 1715, Caha Postal 1743, CEP 30190-002 Belo Horizonte, Brazil

H. E. Trueman, C. Claudianos, J. T. Dessens, J. Mendoza, M. Arai, G. A. Butcher and R. E. Sir Alexander Fleming Building, Department of Sinden Biological Sciences, Imperial College of Science, Technology and Medicine, London S W7 2AZ, UK

Domestic dogs are the main peridomestic reservoir for zoonotic visceral leishmaniasis (ZVL). a disease transmitted to humans by phlebotomine sandflies. Experimental trials have shown that deltamethrinimpregnated dog collars (DMICs) reduce the sandfly biting rate on dogs by >90% for up to 6 months. We tested the effectiveness of DMICs to reduce the incidence of canine ZVL in a community-based intervention trial in Capitgo Eneas, Brazil. After blood was taken from 441 dogs in 2 different neighbourhoods (Dl and D2), DMICs (Scalibor, Intervet) were attached to surveyed dogs in Dl. In Dl and D2,47/25 1 (18.7%) and 24/190 (12.6%) sampled dogs, respectively, were positive by either PCR or ELISA (or both). After 5 months dogs were surveyed and sampled again in both areas. Although ZVL incidence was 32% lower (13/109 vs 15/85) in collared as compared to uncollared dogs, the difference was not significant (possibly owing to the trial sample size and duration). However,

A novel Plasmodium gene, Plasmodium berghei scavenger receptor (PbSR) has been identified from an ookinete enriched cDNA library. The gene consists of a single reading frame of 39 15 bp encoding a 1304 amino acid protein. A secretion signal has been predicted using the signalP algorithm (NIELSEN, H. et al., 1997: Protein Engineering, 10, l-6) at the N-terminal of the protein. Through homology searches several highly conserved protein domain families have been detected. These domains are the PLAT domain, the LCCL domain, the Pentraxin domain and the SRCR domain. These domains are associated with proteins involved in cell-cell interactions and innate immunity. PbSR is upregulated in the mosquito stagesof the malaria lifecycle at the transcription level and the protein is expressed bv the sporozoite in the salivary glands of the mosquito. Targeted disruntion of this gene in I? berohei shows a phenotype reminiscent of the circumsporoioite protein (CSP) knockout (MENARD, R. et al., 1997: Nature,

356

385, 336-340). Parasite growth is arrested at the oocyst, therefore not producing sporozoites in the mosquito, Anopheles stephensi. Orthologues of PbSR have

SHORTPRESENTATIONS

been identified in l? yoelii, I? falciparum, Cyptospori: dium parvum and Theileria parva.

Poster presentations Genetic diversity in populations of Anopheles arabiensis in Khartoum State, Sudan M. Aboud and H. Townson Parasite and Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, UK

In Sudan, the main vector of malaria is Anopheles arabiensis. Previously it has been postulated that populations of An. arabiensis in Khartoum State undergo aestivation during the dry season (OMER, S. M. & CLOUDSLEY-THOMSON, J. L., 1970: Bulletin of the World Health Organization, 42, 319-330). If aestivating individuals were the main source of populations in the following wet season, we would expect to see an impact on population structure because of bottleneck effects. Alternatively, it is possible that mosquitoes migrate into such villages from more permanent populations in irrigated regions adjoining the Nile. We have studied population structure by examining sequence variation in mitochondrial genes, namely 701 bp of cytochrome oxidase I (COI) and 600 bp of NADH dehydrogenase subunit 5 (ND5). Samples were analysed to assessgenetic variation between populations from dry and irrigated areas. Considerable haplotype diversity was detected within An. arabiensis populations, based on maximum parsimony network analysis (TEMPLETON, A. R. et al., 1992: Genetics, 132, 619-633), but the distribution of haplotypes showed little correspondence with environment. The evidence suggeststhat there are no barriers to gene flow between ;z;;lr from dry or irrigated areas separated by up

ModelIing the effectiveness of syndromic management by pharmacy workers in Lima, Peru E. J. Adams 1*2, P. J. Garcia3, G. P. Garnett4 and ‘HIVISTI Division, PHLS ComW. J. Edmunds1>5 municable Disease Surveillance Centre, 61 Colindale Avenue, London NW9 SEQ, UK; ‘London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK; ‘AIDS and STD Unit, Universidad Peruano Cayetano Heredia, Facultad de Salud Ptiblica, Lima, Peru; ‘Department of Infectious Disease Ep’demiology, Imperial College, St May’s Campus, No$olk Place, London W2 lPG, UK; ‘Department of Economics, City University, London EC1 V OHB, UK

As part of a randomized controlled trial of syndromic management for sexually transmitted diseases (STDs), pharmacy workers were trained to provide management of urethral discharge, genital ulcer, vaginal discharge and pelvic inflammatory disease. To measure programme effectiveness, simulated patients (each with typical ‘symptoms’) randomly visited pharmacies to determine the proportion offered appropriate management. The current study aimed to estimate the costeffectiveness of the intervention. However, it is required that the overall effectiveness (numbers of adequately managed clients) be compared with the overall costs. To achieve this, a model was constructed to extrapolate the results from the simulated patients to the whole population. The model estimated the incidence of sexually transmitted syndromes and the number of clients adequately managed in pharmacies. Results from the study population of 1.3 million showed that approximately 57 000 extra clients were

properly managed in pharmacies annually. Preliminary estimates suggest that the cost per adequately managed case was around USgl.40. Model results were most sensitive to variation in the duration of infections responsible for vaginal discharge, and the proportion of women who visit pharmacies for treatment. Results suggest that this intervention may be both effective and cost-effective in managing STDs in Lima, and sensitivity results may help guide further research. Insecticide usage and pyrethroid populations of Anopheles gambiae licidae) in Accra, Ghana

resistance in (Diptera: Cu-

K . Adasi’, D. A. Boakye2 and M. D. Wilson2 ‘Parasite and Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, UK; ‘Noguchi Memorial Institute for Medical Research, Accra, Ghana

Insecticide resistance due to knockdown resistance (kdr) mutations in Anopheles gambiae has been reported

in West Africa. The hypothesis is that agricultural or household use of insecticides is responsible. To establish baseline information on insecticide use pattern and vector susceptibility levels, a questionnaire survey was conducted in Accra. Adult An. gambiae were tested at diagnostic doses of permethrin (0.75%) and deltamethrin (0.05%) using the WHO test kit. Polymerase chain reaction was used for species identification and detection of gene mutations (SCOTT et al., 1993: American Journal of Tropical Medicine and Hygiene, 49, 520-~~~;MARTINEz-TORRES etal., 1998: InsectMolecular Biology, 7, 179-184). A total of 171 households

surveyed revealed that the frequency of pyrethroid application was high. Bioassays showed mortalities ranging from 68% to 88% (mean 78%). The specimens identified so far were all An. gambiae s.s., with kdr mutations in 52/195 (26.7%). The implications of the findings in the context of malaria control in Ghana are discussed. Hydatid disease of the lung: clinical and outcome

presentation

Y. N. Akhtar’, A. Jan’, A. S. Khokar’, M. A. Bangash’, S. Fatimi’ and A. Afaq’ ’ The Agha Khan University Hospital, Department of Cardiothoracic Surgey, Karachi, Pakistan; ‘Faculty of Medicine, Imperial College, Charing Cross Campus, The Reynolds Building, St Dunstan’s Road, London SW6 8RP, UK Echinococcus granulosus causes hydatid disease and is an increasing public health problem in parts of Asia. The lung is the most common site of cyst formation after the liver. Data-extraction sheets for patients presenting in a Karachi tertiary-care hospital between January 1995 and August 2001 were analysed to ascertain demographics, clinical features, treatment and outcomes of patients with pulmonary hydatid disease. Nineteen patients had pulmonary hydatid cysts out of a total of 100 patients with hydatid disease. Demographics of the 19 patients were: male (68%), female (32%); mean age 21 years; Pakistanis (63%), Afghanis (37%). Clinical features included productive cough (84%), fever (73%), haemoptysis (52%), dyspnoea (42%), chest pain (42%), co-existing liver cysts (11%). Treatment was with albendazole (12%), or with alben-

SHORT

PRESENTATIONS

dazole and surgical removal (88%). Mean length of admission was 1 week. No intra-operative cyst rupture, mortality or recurrence of cysts was documented. Presentation is more common in young males with symptoms of cough, fever and haemoptysis. Surgical removal with albendazole cover is the treatment of choice, and this carries low mortality and recurrence rates. Early clinical detection, sterilization of the source and interruption of transmission of the parasite should be combined with increased awareness as necessary prevention strategies to combat hydatid diseasein Pakistan and other parts of Asia. Identification and contribution of host bloodderived xanthurenic acid to the induction of Plasmodium gametogenesis Y. I. H. Alavi, M. Arai, M. Panico, S. J. North, H. R. Morris and R. E. Sinden Department of Biology and Biochemistry, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, London S W7 2AZ, UK

The transmission of the malaria parasite (Plasmodium) from the mosquito vector to the vertebrate host is dependent on gametogenesis, a process that takes place in the first few minutes of blood meal uptake in the mosquito midgut. Induction of gametogenesis in vivo is by the combined stimulus of a 5°C fall in temperature, the presence of mosquito-derived xanthurenic acid (XA), and an as yet unidentified vertebrate blood factor. This recently reported vertebrate factor(s) was shown to induce 50% of total I? gallinaceum exflagellation in vivo in Aedes aegvpti (ARAI, M., et al., 2001: Molecular and Biochemical Parasitology, 116, 17-24). Here we report that the only gametocyte activating factor (GAF) detectable in chicken serum is XA. Investigations into the relative contributions of serumversus mosquito-derived XA to the induction of gametogenesis in vivo indicate that the mosquito is the predominant source of XA. Work in progress involves the measurement of both mosquito and vertebrate blood XA levels in 3 different malaria models (P. berghei, I? gallinaceum and I? falciparum). In addition to providing new insights into the regulation of parasite infectivity by XA, these data form the basis for targeting XA in the development of transmission-blocking malaria control strategies. Production and characterization of stable amphotericin B-resistant lines of amastigotes and promastigotes of Leishmania mexicana

357

remove the effect of amphotericin B, which exerts its effect by binding to membrane sterols. Amphotericinresistant L. mexicana promastigotes were able to infect BALB/c mice and retained their resistant phenotype upon recovery and culture in vitro. The effect of amphotericin B chemotherapy in mice infected with resistant parasites is currently under investigation. Hut tests of a carbamate and pyrethroid ments of bednets, washed or unwashed, pyrethroid-resistant mosquitoes

treatagainst

A. N. Asidi’, R. N’Guessan’, R. A. Hutchinson3, M. Traor&Lamizana2, P. Carnevale’ and C. F. CurtiS ‘London School of Hygiene and Tropical Medicine, London WClE 7HT, UK; ‘Institut Pierre Richet, 01 BP 1500 Bouake 01, C&e d’lvoire; 3University of Durham, Durham DH13LE, UK

Nets treated with carbosulfan or one of 4 different pyrethroids were tested in experimental huts in C&e d’Ivoire where Anopheles gambiue has a high frequency of knockdown resistance to pyrethroids and Culex quinF., ei al., quefusciatus is pyrethroid resistant (CHANDRE, 1999: Parassitolopia. 41. 319-322). The uersonal vrotections from b&g provided by the nets and iheir insecticidal power were assessedbefore and after the nets were washed. Before washing, all treatments except permethrin caused significantly reduced blood feeding and high insecticidal activity against An. gambiae. Carbosulfan performed significantly better than all except the Permanet in killing An. gambiae. Against Culex spp., carbosulfan gave significantly better protective effect and higher insecticidal power than all other treatments. After 5 washes, all 5 treated nets retained some protective and insecticidal power compared with an untreated net but carbosulfan significantly lost power against both genera of mosquitoes. However, 3 of the pyrethroid insecticides showed unexpected increases in mortality against Culex after washing. Among the pyrethroid treatments was a Permanet, which is sold as a permanently insecticidal product, but, after 5 washes, it performed no better than the other nets. [This poster presentation was awarded joint second prize at the meeting.] Psychosocial characteristics of mothers nourished children in Jamaica

of under-

H. I. Almohammed, M. L. Chance and P. A. Bates Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, UK

H. Baker’, C. Powell’, S. Walketi and S. McGregor’ ‘Centre of International Child Health. Institute of Child Health, University College London, 30 Guilford- Street, London WClN IEH, UK; 2Epidemiology Research Unit, Tropical Medicine Research Institute, University of the West Indies, Jamaica

In this study we investigated potential mechanisms of amphotericin B resistance in Leishmania mexicana. Amphotericin B is a second-line drug for treating leishmaniasis, which is now seeing increased clinical usage in a new liposomal formulation (Ambisome). Amastigote and promastigote cultures of L. mexicana were established and exposed to gradually increasing concentrations of the drug in vitro in successive subpassages.Using this approach, it has proved possible to establish stably resistant lines of both amastigotes and promastigotes, which can grow in concentrations of amphotericin B that are normally lethal, and retain their resistance in the absence of drug pressure. In a morphological study, electron microscopy has revealed several changes in these parasites with &cumulation of membrane-like material in the flaeellar Docket. This suggeststhat the amastigotes and promast[gotes may be responding to drug pressure by over-production of membrane material, possibly in an effort to dilute or

Mothers (n = 139) of children with low weight-forage (WAZ s -1.50 z scores of the NCHS references) aged 9-30 months were compared with 71 mothers of adequately nourished children (WAZ > - 1.0 z scores) matched for age and sex. Children and mothers were recruited from 18 government health centres in Jamaica. The mothers’ verbal IQ was measured and a structured questionnaire was administered concerning maternal depression, parenting self-esteem, social support and stressors. Mothers of undernourished children were more depressed and had lower levels of parenting self-esteem than the mothers of adequately nourished children (both P < 0.01). They also reported higher levels of economic stress-(P < 6001) and had a lower verbal IQ (P < 0.051. There were no differences between the groups in’social support and levels of other stressors including partner stress, domestic violence and community violence.

SHORTPRESENTATIONS

358

Maternal depression and low parenting self-esteem are associated with poorer parenting behaviours and poor child development. These findings indicate that in order to promote good parenting and optimal child development, attention should be paid to the psychosocial status of the mother (in addition to the physical condition of the child) as part of the rehabilitative care of undernourished children. Microsatellite toes

loci in Aedes polynesiensis

mosqui-

A. Behbahani, H. Townson and S. P. Sinkins Parasite and Vector Biology Division, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, UK

We are interested in the effects of the maternally inherited endosymbiont Wolbachia on nuclear and mitochondrial genetic diversity in mosquitoes of the Aedes scutellaris group. In order to characterize mosquito nuclear genome variability, it has been necessary to isolate microsatellite loci. Ae. polynesiensis mosquitoes were collected from Fiji from populations which are positive for Wolbachia. DNA was extracted from single mosquitoes and digested using Sau3A and, after adaptor ligation, the product was enriched using the biotinylated oligonucleotides CCAlo and GTA,,,. Twenty-two clones which were positive with CCAlo were sequenced and primers have been designed for 7 of the loci. The next step is to use the fluorescently labelled primers to assess allelic variation in natural populations of Ae. polynesiensis. Zinc augments

tuberculosis

skin test reactions

V. Bhargavi Rae’, Tom F. Pelly’, Robert H. GilmanL~2~3, Mayuko Saito’, Lilla Cabrera2, Jose Delgado2, Robert E. Black’, Jon S. Friedland’, Barbara E. Golden4, Tom Doherty5 and Carlton A. Evans’p2p3+5 ’ Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Hospital Department of Infectious Diseases, London W12 ONN, UK; 2A. B. PRISMA, Lima, Peru; 3Department of International Health, Johns Hopkins BloombergSchool of Public Health, Mayland, USA; 4Department of Child Health, University of Aberdeen, UK; ‘Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WClE 7HT, UK

The anergy of malnutrition limits the utility of tuberculosis (TB) skin testing in resource-poor countries. Zinc has been implicated in cutaneous hypersensitivity and we therefore investigated the effect of topical zinc cream on TB skin testing in a double-blind, randomized, placebo-controlled study in Peru. Five-unit Mantoux tests were performed simultaneously on both forearms, with zinc cream applied to one side only. In 50 healthy volunteers, the topical zinc caused a 34% (P < 0~001) increase in the diameter of induration. Zinc-deficient individuals had smaller skin tests (P = 0.04), but greater augmentation with topical zinc (P = 0.02). Mantoux test reactions on both arms were 3049% larger than those in healthy individuals randomly selected from the same population who had no cream applied (P < 0.005), implying that systemic zinc absorption affected the contra-lateral arm. Similarly, zinc cream caused a 24% increase in the size of Mantoux test reactions in 50 hospital patients with suspected TB disease (P < 0.0001). The local effect of zinc cream was strongly statistically significant compared with the placebo arm, but the greatest biological effect was the systemic augmentation of skin tests compared with people who had no creams applied. The results demon-

strate that zinc supplementation modifies TB immunity and may have implications for improving TB diagnosis. Anaemia in children using bednets Tanzanian community and its relation cide activity of the nets

in a rural to insecti-

A. Joynson-Hicks2, A. Unwin3, J. J. BoreeP, H. Mponda4, H. Mshmda4, J. Lines2 and C. F. ‘Depanment of the Surgeon General, Admiralty Curtis2 Royal Netherlands Navy, l? 0. Box 20702, 2500 ES The Hague, The Netherlands; ‘London School of Hygiene and Tropical Medicine, London WClE 7HT UK; 3National Malaria Control Programme, Tanzania; 41fakara Health Research and Development Centre, Ifakara, Tanzania

Malaria morbidity and mortality can be reduced through the wide-scale use of insecticide treated nets (ITNs). In the KINET bednet social marketing project, in the Kilombero District in Tanzania, persistent reductions in parasitaemia and anaemia were reported despite low rates of net re-treatment (ARMSTRONG SCHELLENBERG, J. R. M. et al., 2001: Lancer, 357,

124 1- 1247). However, the correlation between duration of insecticidal effectiveness and indicators of malaria morbidity is poorly understood and warrants further investigation. Haemoglobin levels and spleen sizes were measured amongst children who have regularly slept under sampled. ITNs. A multivariate regression analysis on these 2 outcome variables showed no significant association with insecticidal activity remaining on the nets (bio-assays performed in a parallel study reported at this Meeting by A. Joynson-Hicks et al.), bednet age and re-treatment frequency. A strong association was shown between frequency of bednet-washing and mean haemoglobin levels (P < 0*0005) and incidence of anaemia (P < 0*00005), with children using nets reported to be washed at least once a week showing higher levels of anaemia. These findings suggest the importance of encouraging lessfrequent washing of nets and/or more-frequent re-treatment by people who wish to wash nets very frequently. Impact of insecticide-treated and pupae of Aedes aegypti storage jars

covers on larvae in household water-

H. Carneiro’, H. Bugoro’, C. curtis’, P. Coleman’, M. Nathan2, S. Hoyer3, M. Chang, D. Socheat4, N. Chanta4, T. Setha4, C. Bunnavy4, E. Sambath and V. Rien4 ‘London School of Hygiene and Tropical Medicine, London WClE 7HT, UK; 2World Health Organization, Geneva, Switzerland; 3World Health Organization, Cambodia; 4National Malaria Centre, Cambodia

In Cambodian villages, daily counts assessed the effect on immature stages of Aedes aegypti in 400-litre outdoor domestic water-storage jars of covers of supposedly wash-resistant deltamethrin-treated mosquito netting (50 mg/m2). This was compared with untreated netting covers and with netting covers more conventionally treated with the same insecticide. Dead larvae and pupae were seen in jars with insecticidal lids after rainfall and there was rapid disappearance of live late-instar larvae after application of these lids; this was more pronounced under the more conventionally treated covers. A more gradual decline in larval densities was observed in water jars with untreated covers. The leaching of insecticide was confirmed in the laboratory by sprinkling water through the treated netting materials. Mortality was higher with a pyrethroid-susceptible than a -resistant strain of Ae. aegypti. Under laboratory conditions, newly emerged adults and adults attempting to oviposit in jars covered by insecticide-treated

SHORTPRESENTATIONS nets were also killed (BUGORO,H., 2001: MSc thesis, University of London). A preliminary risk-assessment study confirmed that deltamethrin from the supposedly wash-resistant netting material is unlikely to pose a health hazard to people using water from these jars. Neisseria meningiddis DNA blood and cerebrospinal fluid tients: preliminary report

bacterial loads in from Brazilian pa-

J. B. Correia’p*, M. C. M. B. Duarte2, N. B. Correia2, R. J. A. Casado2,3, M. Guive#, E. Kaczmarski4, L. E. Cuevas’ and C. A. Hart’ ‘Department of Medical Microbiology and Genito-Urinay Medicine, University of Liverpool, 8th Floor Duncan Building, Liverpool L69 3GA, UK; ‘Institute Matemo Infantil de Pemambuco, Rua dos Coelhos 300, Recife, 50070-550, Brazil; ‘Hospital Correia Picanco, Rua Padre Roma 149, Recife, 52050-l 50, Brazil; 4Meningococcal Reference Unit, Withington Hospital, Manchester M20 2LR, UK; ‘Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK

Meningococcal diseaseis an important cause of morbidity and mortality worldwide and its main clinical presentations are septicaemia and meningitis. Septicaemia is observed in up to 75% of casesin the UK, but in only <5% of patients in sub-Saharan Africa where most cases occur as meningitis (HART, C. A. & CUEVAS, L. E., 1997: Annals of Tropical Medicine and In other regions of the Parasitology, 91, 777-785). developing world, such as in Brazil, the presentation resembles that observed in industrialized countries (BRYAN, J. P. et al., 1990: Reviews of Infectious Diseases, 12, 128- 135). Whether these differences are due to environmental, bacterial or host factors remains unclear. There is some evidence that bacterial DNA load correlates with disease severity (HACKETT, S. J. et al., 2002: Archives of Diseases in Childhood, 86, 44-46), but no studies from developing countries are yet available. We report Neisseria meningitidis bacterial DNA loads measured by Taqman polymerase chain reaction in cases with confirmed meningococcal disease, who are being followed during their hospital stay in Recife, Brazil. Bacterial DNA loads ranged from 9.7 X lo3 to 3.6 X 1O9DNA copies per mL. Anopheles funestus: an important in southern Mozambique

malaria

vector

Nelson Cuamba and Harold Townson Vector and Parasite Biology, Livelpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, UK

In Mozambique, malaria vector control is based on an assumption that mosquitoes of the Anopheles gambiae complex are the primary malaria vectors during the wet season, with An. funestus maintaining transmission in the dry season, albeit at a lower level. To test this assumption, a study was conducted of the vectors of malaria and their relative role in transmission in southern Mozambique. House-resting mosquitoes were collected by pyrethrum spray collection or on human bait. The sporozoite rate (SR) for Plasmodium falcipantm was determined by ELISA. Both An. gambiae s.1. and An. funestus s.1. were found throughout the year, although the latter was relatively more common during the dry season. During the dry season the mean SR was the same (0.02) in both species groups. In the wet season, An. funestus continued to play an important role with an SR as high as 0.05 in one locality. Species-specific PCR has confirmed that, of the funestus complex, only An. funestus S.S.occurs indoors and transmits malaria, whereas An. arabiensis and An. gambiae S.S.are found in varying proportions and seem equally effective vectors.

These results indicate that An. funestus S.S.is a major vector of malaria in southern Mozambique, regardless of season. Development of a low-cost hearing screen and appropriate service provision for young deaf children in Binga District, Zimbabwe S. Dube, S. Wirz and K. Sullivan Centre for International Child Health, Institute of Child Health (University College London), 30 Guilford Street, London WClN lEH, UK

Evidence for developing a low-cost tool to identify hearing loss (HL) above 50 dBHL averaged across the frequencies 0.5 k, 1 k, 2 k and 4 k in children aged 3-6 was first reported in the UK (SU?TON, G. J. & SCANLON, P. E., 1999: BritishJournal ofAudiology, 33, 145156). In 1998 a pilot study was undertaken in Brazil testing the DUBE (1995) ‘Low Cost Hearing Screen’. It was reported that the tool could identify 97% children with hearing impairment in excess of 66 dBHL averaged across all frequencies (WIRZ, S. & LICHTIG, I., 1998: Disability and Rehabilitation, 20, 189-194). Studies carried out in the UK and USA show that lowcost hearing screening reduces cost, increases efficacy of treatment and management of severe to profound cases of hearing impairment enrolled in community programmes (STEWART,I. F., 1984: Layngoscope, 94, 784-799; TODD, N. W., 1994: International Journal of Paediatric Otorhinola yngology, 29, 1l-2 1). DUBE (1995) tool was refined and used in Zimbabwe. It is expected that this tool will identify 70% correctly children aged 3-6 with hearing loss above 50 dB. Lowcost intervention implemented by existing agencies has been developed. The study should influence policy towards deaf children in Zimbabwe. Isolation of polymorphic from Aedes polynesiensis Wuchereria bancrofti

microsatellite markers and susceptibility to

T. Dutton and S. Sinkins Parasite and Vector Biology Division, Liverpool School of Tropical Medicine, Pembroke Place, Livetpool L3 SQA, UK Aedes polynesiensis, a vector of lilariasis in the Pacific is a member of the Ae. scutellarts complex which consists of approximately 30 species distributed from South-East Asia to the Marquesas Islands. Some of the species are susceptible to filariasis whereas others are refractory. As a prerequisite to determining the mode of inheritance to filariasis, and to the genetic mapping of the loci involved in susceptibility, laboratory colonies of wild-caught species must be established, and interspecies crosses performed. In addition, polymorphic genetic markers are needed. Fieldwork has recently been carried out in the South Pacific in order to collect several species of interest, and laboratory colonies are currently being established. DNA was extracted from Ae. polynesiensis collected from Fiji, and dinucleotide microsatellite sequences were isolated, and primers designed. In total, 12 microsatellite loci have been identified, and are currently being assessedwith respect to their application and polymorphism in Ae. polynesiensis and other members of the scutellaris group. It is intended to use polymorphic genetic markers in order to map the location of loci affecting mosquito susceptibility to tilarial infection. Preliminary employing

studies into rabies virus trafficking in-situ hybridization techniques

C. J. Finnegan, G. Parsons, S. M. Brookes, S. Marsh, K. L. Mansfield, N. Johnson, L. M. McElhinney and A. R. Fooks Rabies Research and

SHORT PRESENTATIONS

360

Diagnostic Group, Department of Virology, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrev KT1.5 3NB, UK

Rabies and rabies-related viruses enter the peripheral nervous system and travel by retrograde transport to the central nervous system (CNS). The time taken for virus to travel from peripheral tissue through to the brain is variable. Once virus has reached the brain it later disseminates to other organs. However, the mechanisms by which these processes occur are still unclear. Studies are underway in our laboratory to investigate whether virus replication occurs at a peripheral bite site. This process may be essential in order to achieve sufficient viral load for subsequent viral entry into the CNS. In order to help elucidate virus trafficking, rabies virus-specific non-radioactive in-situ tools have been developed for host neuronal and non-neuronal tissues. Here we describe the successful employment of digoxigenin-labelled riboprobes for in-situ hybridization (ISH) based on the nucleoprotein of the classical rabies virus. Identification of an immunoprotective B cell epitope in the 27-28 kDa S. mansoni cercarial elastase Katherine Fran&low, Amy Stewart and Michael Doenhoff School of Biological Sciences, University of Wales, Bangor, Deiniol Road, Bangor, Gwynedd LL.57 27JW, UK Schistosoma mansoni cercariae possess a 27-28 kDa serine protease, the ‘cercarial elastase’, that is presumed to facilitate penetration of these larvae into host skin. It was previously shown that the larval protease was a candidate vaccine (DARANI, H. Y. et al., 1997: Parasitology, 115, 237-247) and we have begun to investigate whether a construct incorporating a B epitope from this molecule can be developed into an effective vaccine. Analysis of the protease’s amino acid sequence with the computer program PREDICT7 (CARMENS et al., 1988: Biochemical and Biophysical Research Communications,

159, 687-693) allowed a linear sequence of 16 amino acids in the second of the molecule’s 3 exons to be identified as a potential B cell epitope. The peptide in question (Bl) was synthesized and conjugated to thyronlobulin (TG) as a ‘carrier’ molecule and used to immunize mice. In replicate experiments all mice that were immunized with the Bl-TG conjugate produced antibody specifically reactive with the peptide Bl, but only a minority of the immunized animals produced antibody that also cross-reacted with the native protease. The mice that produced antibody reactive with the native protease were, however, partially protected against S. mansoni infection. A strategy to increase the yield of antibodies that cross-react with native elastase has been developed by transferring lymphocytes from individual immune donor mice to a group of T lymphocyte-depleted recipient mice, the latter mice being also challenged with antigen at the time of cell transfer. This method will be used to continue defining a B cell epitope on the native protein that is suitable for incorporation into a synthetic vaccine. Common antigenicity between allergens schistosomes: involvement of carbohydrate topes

and epi-

Ronald G. Stanley’, Keith Heidi R. Fuller’, ‘School of BioGriffiths* and Michael J. Doenhoff logical Sciences, University of Wales, Bangor, Deiniol Road, Bangor, Gwynedd LL57 2UW, UK; 2Ysbyty Guynedd, Bangor, Gwynedd, UK

Rabbit antibodies specific for antigen Sm480, a high-

molecular weight glycoprotein of Schistosoma mansoni eggs (CURTIS, R. H. et al., 1996: Parasite Immunology, 18, 149-157) have been found to react also with the antigenic constituents of a wide range of organisms associated with allergic reactions. Examples of the latter include aero-allergens such as grass and tree pollens, house dust mites and cockroach, a variety of foods including peanuts, shellfish, fungi and other fruits and vegetables associated with food-related allergies, and venom from bees and wasps. The reactivity of antiSm480 antibodies against the allergen extracts is largely sodium-metaperiodate sensitive, indicating that the antigenic cross-reactivity between schistosomes and allergens is due to their having carbohydrate epitopes in common. This observation may be relevant to the ‘hygiene hypothesis’ which has been formulated to account for the relatively recent, but marked, increase in prevalence of asthma and other allergic disorders in many countries, particularly those with better developed health care systems. Feasibility paediatric

of cord blood transfusions anaemia in under-resourced

for severe countries

G. Bedu-Addo2, M. Adarkwa2, 0. Hassall’, ‘Liverpool School of Tropical K. Danso and I. Bates’ Medicine, Liverpool, UK; 2Komfo Anokye Teaching Hospital, Kumasi, Ghana

In poor, malaria-endemic countries 50-67% of all blood transfusions are for children aged under 5 years. The mortality of children with severe anaemia who do not receive a blood transfusion within 12 h is 4 1%. The need to improve emergency blood supplies, which are often unavailable, is urgent. Umbilical cord blood was collected from 105 uncomplicated, full-term vaginal deliveries in a lame West African hosnital. Blood from suspended placentas was collected ini0 cord donation bags, and haematological parameters, maternal and baby data, and the hospital’s paediatric transfusion needs were recorded. The mean (SD) cord blood volume was 79 mL (27) and was unrelated to maternal age (mean 26 years, SD 7), parity or birthweight. The cord blood was successfully screened, grouped and cross-matched. Bacterial contamination rate was 4.8% but decreased over time. It was calculated that cord blood collected from 67% of the vaainal deliveries would provide all the transfusion requi;ements for the paediatric unit in the same hospital. This study has shown that cord blood can be successfully collected under prevailing conditions in a resource-poor tropical country. After further refinements of the technique this novel source of blood will have the potential to impact significantly on malaria mortality in the developing world. Maternal HIV status, infant nutrition/health knowledge and feeding practices in Muhimbili National Hospital, Dar es Salaam, Tanzania A. Ibrahim’ , A. Massawe2, K. Sullivan’ and ‘Centre for International Child Health. A. Tomkins’ Institute of Child Health,* University College London, 30 Guilford Street, London WClN lEH, UK; 2Department of Paediatrics, Muhimbili National Hospital, Dar es Salaam, Tanzania

A cross-sectional study was carried out to compare infant nutrition/health knowledge and practices between 3 groups of mothers: 42 HIV positive, 58 HIV negative and 64 untested. Tested women knew more about the advantages and disadvantages of breastfeeding (BF) and infant formula feeding (IFF). They also knew more about practices which reduce mother-tochild transmission (MTCT) of HIV: discontinuing BF after 4 months (P < O.OOl), use of IFF alone

361

(P < 0~001) or use of mixed feeding not including breast milk (P < 0.001). Significantly more HIV-positive than HIV-negative mothers or untested mothers gave IFF (40%, 3.8% and 2*9%, respectively). More untested and HIV-negative than HIV-positive mothers gave exclusive BF (91.4%, 885% and 60%, respectively). HIV counselling and testing is associated with considerable differences in feeding patterns and may well decreasepost-partum MTCT of HIV. Towards a standard battery of microsatellite markers for the analysis of the Leishmania donovani complex M. B. Jamjoom’, R. W. Ashford’, P. A. Bates’, S. S. Kemn’ and H. A. Naves’ ‘Liverpool School of Tro&al Midicine, Pembroke place, Live$ool L3 5QA: UK; ‘School of Biological Sciences, University of Liverpool L69 720, UK

Microsatellites were isolated and 40 primers designed and tested against Leishmania donovani (MHOMET/67/HU3;LV9). Primers that reliably generated a single band on agarose gels were then tested against a panel of 19 Leishmania stocks. Twenty-two out of the 40 primers pair tested produced a single band on L. donovani stocks, 9 out of 40 with L. tropica and only 5 with L. major. Of the 22 L. donovani loci, 20 were tested for the presence of polymorphisms in 16 Leishmania stocks by polyacrylamide gel electrophoresis with isotopically labelled primers. Two to 6 alleles were identified at each locus in the strains studied. No alleles were shared between L. donovani (MON18) and L. infantum (MONl) at any locus, indicating a complete absence of gene flow between the limited numbers of strains used. Also, Sudanese stocks had very similar identical alleles, while stocks from different countries were very different. This exemplifies the high level of polymorphism commonly found with microsatellites. This is the first panel of microsatellite markers to be isolated de novo that is large enough to be suitable for high-resolution studies within any Leishmania species. We now plan to use this panel of markers to address taxonomic questions within the L. donovani complex. The longevity of action of insecticide mosquito nets in rural Tanzania

deposits

on

A. Joynson-Hicks’ , A. Unwin’, H. Mponda’, H. Mshinda3, J. Lines’ and C. Curtis’ ‘London School of Hygiene and Tropical Medicine, London WClE 7HT, UK; 2National Malaria Control Programme, Tanzania; ‘Ifakara Health Research and Development Centre, Ifakara, Tanzania

In Tanzania, insecticide re-treatment of bednets (ITNs) every 3 months has been recommended. This study investigated whether ITNs actually remain insecticidally effective for longer than this. ITNs (n = 103) were collected from a village in Tanzania, where a social marketing project is in progress (ARMSTRONG SCHELLENBERG, J. et al., 2001: Lancet, 357, 12411247). Nets covered a range of ages, re-treatment and washing histories. The 24-h mortality of Anopheles gambiae following 3-min exposure to the nets was recorded. Mosquito mortality was significantly higher with nets washed less frequently. The mortality rates for any net age or re-treatment group did not exceed 66% but, even in nets reportedly last re-treated 3-4 years ago, bioassay mortality averaged over 50%. This indicates that ITNs do not need to be re-treated every 3 months, which is costly and could lead to a build-up of excessive insecticide deposits. The relationship of nets from this sample to anaemia in their users is reported by J. Boreel in this Meeting.

Entomological evaluation of the PermaNet K-O Tab-treated nets in Iran

and

M. H. Kayedi’, J. Lines’, A. Haghdoost’, H. Vatandoost2 and Y. Rassi’ ‘Department of Znfectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK; 2Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences,I? 0. Box 6446-14155, Tehran, Iran

Bioassay tests were conducted with Anopheles stephensi to measure the effect of various factors on the residual insecticidal activity of PermaNet (a brand of longlasting net), and on ordinary nets treated with deltamethrin (K-O Tab). The factors investigated were: repeated hand-washing in cold water (0, 5, or 15 washes); exposure to dust, dirt and smoke between washes; and various methods of drvine. Field-collected blood-fed An. stephensi females were exposed to the nets in groups of 11 until all were knocked down, and the time to knockdown for each individual was recorded. Results (as mean or median time to knockdown) were subject to analysis of variance. The insecticidal activity of the PermaNet was significantly greater than that of the K-O Tab-treated nets after both 5 and 15 washes. There was no significant difference between 0 and 5 washes in both nets but the difference between 0 and 15 washes and 5 and 15 washes in K-O Tab was significant. There was significantly greater loss of activity in nets exposed to the sun for 3 days between washes, but no significant effect of exposure to dirt, dust and smoke, and no difference between nets that were dried flat or hanging vertically after washing. Drug-resistance mutations synthetase of Plasmodium from north-east Tanzania

in the dihydropteroate falciparum in samples

S. A. Kiani, J. Curtis and D. C. Warhurst London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK

The extensive use of the combination of anti-folate drugs pyrimethamine-sulfadoxine (Fansidar) against falciparum malaria has led to rapid selection of resistant Plasmodium falciparum populations. Blood samples collected in 2000 from an endemic region in north-east Tanzania were analysed by PCR-RFLP for mutations in residues 436, 437, 520, 581 and 613 of I? falciparum dihydropteroate synthetase (DHPS) which have previously been associated with resistance to sulfa drugs. Mutations were found in 4 of these residues. There was no significant change in the frequency of these mutations compared to data from the same area in 1995 (CURTIS, j. et al., 1998: Journal of Infectious Diseases, 177. 1429-1433). However over this oeriod the uarasite clearance rate with pyrimethamine-sulfadoxine treatment has fallen from 83% (TRIGG, J, K. et al., 1997: Acta Tropica, 63, 185-189) to 14% (MUTABINGWA, T. K. et al., 200 1: Transactions of the Royal Society of Tropical Medicine and Hygiene, 95, 433-438). The latter study also showed little or no change in the frequency of resistance-associated mutations for dihydrofolate reductase, leaving the mechanism of this serious increase of drug resistance in need of urgent investigation. Pyrethroid resistance in Ogun State, Nigeria

in Anopheles gambiae s.1.

M. K&tan’, R. Oyeneye2, G. Biersack and C. Amajoh3 ‘London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK; ‘Sacred Heart Hospital, Lantoro, I? 0. Box 816, Abeoku-

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362

ta, Nigeria; 3National Malaria and Vector Control Division, Federal Ministry of Health, Abuja, Nigeria

A comparison and Russia

Pyrethroid resistance in Anopheles gambiae s.s caused by the kdr gene has been described in West Africa, but not previously in Nigeria. An. gambiae s.1. were collected in urban and rural localities in Ogun State, Nigeria, and exposed to WHO recommended discriminating dosages of deltamethrin, permethrin, or DDT in test kits. Percentage survival was O-20% on permethrin, O-4.1% on deltamethrin, and O-23.5% on DDT. PCR showed that resistance existed in both An. gambiae s.s and An. arabiensis. Some 88-97% of tested mosquitoes in urban localities proved to be An. arabiensis; 85-97% in rural localities were An. gambiae S.S. These results are consistent with those of COLUZZI, M. et al. (1979: Transactions of the Royal Society of Tropical Medicine and Hygiene, 13, 483-497). The results suggest that eradication by the sterile-insect technique of urban ‘island’ populations of An. arabiensis in the rainforest belt of West Africa might be possible. Tests are in progress for the presence of kdr allele (KOLACZINSKI, J. H. et al., 2000: Bulletin of Entomological Research, 90, 125 132).

A. S. Lee’, R. Hutchinson and N. V. Nikolaeva’ 1London School of Hyeiene and Trobical Medicine, London WClE 7HT, UK; 2’&iversiy of Durham, Durham DHl 3LE, UK; ‘Znstitute of Plant and Animal Ecology, Ekaterinburg, Russia

Impact of Plasmodium falciparum malaria on cellular compartments of human immunodeficiency virus type 1 replication in coinfected persons

Religious leaders trained to teach about HIV/ AIDS can reach and educate a large rural/urban Ethiopian population

S. D. Lawn’F2, T. L. Pisell’, I. F. Ho&an3, C. S. Jere4, M. E. Molyneux516 and S. T. Butera’ ‘Hospital for Tropical Diseases, London, UK; ‘HIV and Retrovirology Branch, Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control and PTevention, Public Health Service, US Department of Health and Human Services, Atlanta, GA, USA; ‘Division of Infectious Diseases, University of North Carolina, Chapel Hill, NC, USA; 4Department of Medicine, College of Medicine, University of Malawi, Blantyre, Malawi; ‘Malaria Project and Malawi-LiverpoolWellcome Trust Research Programme, College of Medicine, Blurt yre, Malawi; 6School of Tropical Medicine, University of Liverpool, UK

The aim of the study was to determine the impact of Plasmodium falciparum malaria on cellular reservoirs of

viral replication in HIV-1-coinfected persons and to relate this to changes in systemic immune activation. Plasma samples were obtained from HIV-l-infected individuals (n = 10) at diagnosis of acute malaria, 4 weeks after parasite clearance and from HIV-infected aparasitaemic controls (n = 10). Immunomagnetic virus capture analysis was used to determine the cellular origin of cell-free HIV-l particles present in plasma samples and indices of immune activation were measured using enzyme-linked immunosorbent assays. Compared to controls, the detectable proportion of HIV-l particles derived from CD14+ macrophages and CD26+ lymphocytes was increased in persons with acute malaria coinfection and correlated with markedly increased plasma concentrations of both proinflammatory cytokines and soluble markers of macrophage and lymphocyte activation. Parasite clearance following treatment with antimalarial drugs was associated with decreased detection of HIV-l particles derived from CD14+ macrophages and correlated with a marked diminution in systemic immune activation. In conclusion, acute Z? falciparum malaria coinfection impacts virus-host dynamics in HIV-l-infected persons at the cellular level, notably showing a reversible induction of HIV-l replication in CD14+ macrophages.

of Anopheles

messeae

in the UK

Anopheles messeae, a member of the Palearctic An. maculipennis complex, is considered to have been the

principal vector of malaria in Russia (NIKOLAEVA, N. V., 1996: Society for Vector Ecology Newsletter, 27, 10-l 1). However, when there was malaria transmission in the UK and the Netherlands, An. atroparvus, and not the highly zoophilic An. messeae,was considered to be the vector (HACKETT, L. W., 1937: Malaria in Europe, OUP). An. maculipennis s.1. mosquitoes were collected blood fed from animal sheds on the Somerset Levels in the UK and Palkino, near Ekaterinburg in Russia. Both laid eggs with markings classifiable as An. messeae.In all-night human biting catches outside these sheds, few were collected in Russia and none in the UK. Using PCR (CROFT, J. et al., 1999: Parasitology Research, 85, 837-843) UK and Russian specimens showed identical species-specific ITS2 regions of rDNA corresponding to An. messeae.

S. N. S. Marlowe’, K. Mirgissa2, S. Ferux’ and E. Carlin ‘Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK; ‘Jimma University, Jimma, Ethiopia; ‘Nottingham City Hospital, Hucknull Road, Nottingham, UK

In 1999 the estimated HIV prevalence in Ethiopia was 7.3% but knowledge concerning HIV/AIDS in the general population was poor. Ethiopians are religious and all family members attend religious gatherings regularly. This study assessesthe impact of giving HIV/AIDS training to leaders from both Orthodox Christian (OC) and Muslim communities, in a defined rural/urban population of 25 million. Initial focus group discussions (FGDs) were held with religious leaders and the communities to identify HIV/AIDS knowledge; 183 leaders, who were given HIV/AIDS training, returned to their communities to teach. The impact of training them was assessedafter 9 months and l-year. We confirmed an initiallv noor HIV/AIDS knowledge in leaders and in the community, but found that both the OC and Muslim leaders showed enthusiasm for learning and teaching about HIV/AIDS. OC leaders are more effective at communicating about HIV/AIDS than their Muslim counterparts, with 1 OC religious leader reaching 3000 people and 1 Muslim leader reaching 1500 people. FGDs at 9 months showed that rural and urban people knew basic facts about HIV/AIDS and that they had relinquished their prejudices towards its victims. We conclude that training religious leaders increases HIV/AIDS knowledge in a rural/urban Ethiopian population. Assessment of therapeutic Khartoum, Sudan

efficacy

of Fansidar

in

Abdel-Rahman Medani’, Nawal Osman’ and I Unit of Malaria Research, Z?0. Box Saadia Younis2 12386, National Health Laboratories, Khartoum, Sudan; ‘Department of Zoology, Faculty of Science, University of Khartoum, Khartoum, Sudan

The study aimed to look at the reliability of the

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PRESENTATIONS

363

antimalarial combination of pyrimethamine-sulfadoxine (Fansidar) as a first-line treatment since chloroquine resistance has spread widely in Khartoum State, Sudan. The studv also aimed to investieate the nrevalence of point mutations associated with-FansidaLresistance in the studv area. A cohort study was conducted in Elhaj Yousif area 12 km east north Khartoum State between Februarv and April 1999. Of the 656 patients monitored in the outpatient clinic, 341 patients (52%) were diagnosed as malaria positive and 315 (48%) patients as malaria negative. One patient was reported as RI resistant, 8 as RII, and none as RIII. One hundred patients were recruited for follow-up based on the WHO protocol for assessment of therapeutic efficacy of antimalarial drugs. Seventy-seven patients completed the follow-up while 23 were lost mainly due to leaving the area. The minimum age included in the cohort was 1.2 years and the maximum age was 50 years. There was 32 males and 41 were female. Mutation-specific polymerase chain reaction (MSPCR) was used to investigate the prevalence of point mutations in the Plasmodium falciaarum dihvdrofolate reductase gene (DHFR) in in-&o Fansidar resistance. In codon 108, 3 (3.9%) samples were carrying the wild genotype (Ser), 50 (64.9%) were mutant (Asn), 16 (20.8%) samples were mixed with both genotypes and 8 (10.4%) samples did not give any product. No sample carried the Thr mutant genotype. Sixty-four (83.1%) samples were carrying the wild genotype (Cys) of codon 59, 3 (3.9%) samples were carrying mutant (Arg) genotype and 3 (3.9%) were carrying both genotypes. Twenty-three (29.9%) samples were carrying the wild genotype (Asn) of codon 51, 36 (46.8%) samples were carrying mutant genotype (Ile) and 11 (14.3%) were carrying both genotypes. In sensitive cases, the prevalence of the Asn-108 point mutation was 83*8%, Arg-59 was 7.3%, and Ile-51 was 55.9%. In resistant cases. the urevalence of Asn-108 point mutation was lOO%,< while Arg-59 was 1 I %, and Ile-5 1 was 100%. Results showed that Fansidar is highly effective when used as first-line antimalarial. The use of molecular techniques for assessment of therapeutic efficacy of antimalarials can play a major role in control and management of infectious diseases in developing countries. Mating

competitiveness

in irradiated

tsetse flies

M. Menjeta’, N. J. Seal’, U. Feldman’, A. S. Robinson’ and P. J. McCall’ ‘Parasite and Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liveqool L3 SQA, UK; ‘Entomology Unit, Agriiulture and Biotechnology Laboratory, International Atomic Energy Authority, A-2444 Seibersdofl, Austria The sterile-insect technique (SIT) is an effective and environmentally benign method of vector control. Sterilized males are released into the wild to mate with wild females, rendering them infertile and reducing reproductive potential or possibly eradicating the population. As the technique requires the release of huge numbers of laboratory-reared insects for long periods of time, it is expensive and ensuring released insects are fully competitive with wild counterparts is essential. We examined the effect of irradiation (sterilization) on the mating behaviour of Gloss&a austeni in the laboratory. Normal and irradiated males were equally effective at capturing and copulating with females. Both normal and irradiated males inseminated females with approximately the same volume of sperm but copulation duration was longer in irradiated males. Both groups of males induced female refractoriness to further mating attempts to the same degree. Time to initiation of mating was significantly increased when 2 males were

present, compared to when one male was present with the female. This was a result of male-male interference. The results suggest that, while irradiated males compete well with normal males, mass releases, by increasing the numbers of competing males, could increase interference during mating and reduce mating success. A randomized, double-blind, placebo-controlled trial of atovaquone-proguanil versus sulfadoxine-pyrhnethamine in the treatment of Zambian children with severe Plasmodium fdciparum malarial anaemia Modest Mulenga’3’, Caroline Shulman’, Steve Bennett’, Phllllp Thuma3 and Brian M. Greenwood’ ‘London School of Hygiene and Tropical Medicine, Department of Infectious and Tropical Diseases, Keppel Street, London WClE 7HT, UK; 2Tropical Diseases Research Centre, Department of Clinical Sciences, P.0. Box 71769, Ndola, Zambia; 3Macha Malaria Research InstitutelMacha Mission Hospital, I? 0. Box 63043, Macha, Zambia Treatment of severe malarial anaemia (SMA) requires effective antimalarials and blood transfusion if the condition is complicated. Because resistance to chloroquine, and sulfadoxine-pyrimethamine (SP) is widespread, atovaquone-proguanil (AP) may be a suitable alternative. Resistance to AP has never been reported in Zambia. The value of folic acid in SMA is controversial and it may antagonize SP. Judicious use of blood transfusion in clinically stable cases is advocated to reduce the risk of transmitting HIV. The objective of the study is to determine the impact of AP compared to SP treatment, with or without folic acid, on treatment failure in Zambian children with clinically stable SMA. The design of the study and some emerging results are presented. In a 2-by-2 factorial, double-blind (double-dummy) placebo-controlled trial, 640 children with clinically stable SMA will be randomized to receive either active SP and placebo AP or vice versa. The 2 groups are being stratified for haematocrit (HTC) 9- 15% (A)-no folic acid and HTC 16-20% (B). Both groups are being followed in hospital for the first 3 days, and as outpatients on days 7, 14 and 28. Between December 2000 and June 2001, 192 (66 in A and 126 in B) were enrolled: 162 (58 in A, 97 in B) completed, 10 in A and 15 in B were withdrawn, and 2 in A and 4 in B died [sic]. There was recrudescence or re-infection in 6 and 18 patients in A and B respectively. The statistical difference in HCT and parasitaemia observed between A and B at days 0 and 7 had disappeared at days 14 and 28. Since the study is still blinded and ongoing, SP and AP arms cannot be compared. Population mobility and the spread of HIV rural villages in Mwanza region, Tanzania

in

W. Mwita’, R. White’, J. Chllogani’, K. Orroth’, B. Zaba’. D. Mabev2. R. Haves’. H. Grosskurth*. D. Ross~~~, J. Todd’, A. “Anemona’,2 and F: ‘National Insitute for Medical Research, Mosha’ Mwanza Centre, Tanzania; ‘London School of Hygiene and Tropical Medicine, London WCIE 7HT, UK We measured annual inmigration, outmigration and circulation rates in rural villages and studied the association between population mobility, sexual behaviour and HIV sero-status in rural Tanzania. A cross-sectional population survey in 1320 adults aged 15-54 years in 6 rural communities was carried out in 2001. Data on migration, travel, sexual behaviour and biomedical samples were collected from all subjects present during the survey and proxy information on mobility

SHORT PRESENTATIONS

364

and travel from absent subjects. All sera were tested for HIV. The cohort will be followed-up in 2003 to establish migration rates. The outmigration and inmigration rates into the communities were 84/1000 and 106/1000 per year. Circulation rate (‘temporary’ movement with no change of household membership) varied greatly between community household members (486/1000/ year) and subjects whose household was out of the community ( 1200/ 1OOO/year). The HIV prevalence was 7.4% among inmigrants and 3.8% among nonmigrants (OR 1.96, 95% CI 1.1-3.6). HIV was more prevalent among inmigrants who had higher numbers of and more risky recent sexual partners. The outside sexual contact rates differed greatly between residents and migrants. In this rural population, an increased risk of HIV infection is associated with increased mobility and more risky sexual behaviour among the mobile population. These findings imply that AIDS control programmes should provide specific interventions for migrants and frequent travellers. Use of geographical information systems and remote sensing technologies in the mapping of Loa

loa J. Kamgno2, V. Obsomer’ , M. Boussinesq’, S P. Maya&, Connor’, D. H. Molineux’ and M. C. Thomson * ?- ‘MALSAT Group, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, UK; ‘Centre Pasteur du Cameroon, BP 1274, Yaounde, Cameroon; 3Joint Research Centre, Ispra, Italy

When environmental information is needed quickly over extended areas satellite imagery can provide good approximation of a range of environmental factors. Risk maps can be created from models based on these factors by using geographical information systems. To respond to the urgent need to identify the distribution of Loa Zoa-endemic areas, a preliminary model of distribution has been developed for Central/West Africa (THOMSON, M. C. et al., 2000: Lancet, 356, 10771078). Testing this model in Cameroon using extensive prevalence survey data has revealed insufficient spatial resolution to pick out gallery forests in Savannah areas where the Chrysops vector of this filarial worm may be found. A new model has been created using environmental data from the VEGETATION sensor on board the SPOT satellite and radar image from JERS-1 SAR. This new Loa loa risk map has predictive capacity in both the forest and Savannah areas of Cameroon and enables extrapolation to other countries. [This poster presentation was awarded joint second prize at the meeting.] Genetic diversity among Leishmania bradiensis*

Paraguayan

isolates

Infant-feeding practices known HIV status

of Zambian

mothers

of

A. A. Omari’, C. Kankasa2, G. Bhat’, C. Luo”~ ‘Liverpool School of Tropical and J. E. G. Bunn’ Medicine, Livelpool, UK; ‘Department of Paediatrics, University Teaching Hospital, Lusaka, Zambia

We assessedand compared the infant-feeding practices of HIV-infected mothers with HIV-uninfected mothers. Mothers of infants aged 2-12 months, 55 HIV infected and 85 HIV uninfected, were recruited from an HIV Family Support Unit in Lusaka, Zambia. HIV status was known for 121 of these mothers, who had all received pre- and post-test HIV counselling. Feeding practices were determined by verbal questionnaire. All HIV-infected mothers had breastfed their infant. Only 35% of infants under 4 months were exclusively breastfed and more HIV-infected mothers planned to stop before 1 year (12% vs 2%, P = 0.03). More HIV-infected mothers reported using breast milk substitutes (BMS) before 2 months (28% vs 1I%, P = 0.03), and they introduced weaning foods significantly earlier (P = 0.002). No BMS were appropriately modified with 4 mothers adding salt. Infants of HIVinfected mothers were significantly lighter (WAZ -0.22 vs 0.41, P = 0.004). Infant feeding practices of HIV-infected mothers were significantly different from HIV-uninfected mothers. Paradoxically these mothers’ feeding practices could be putting their infants at an increased risk of both HIV transmission and non-HIV-related morbidity.

of

R. Oddone’32, A. Canese3, M. Jamjoon4, ‘Institute D. Nolder2, M. A. Miles’ and H. Noyes’ de Investigaciones en Ciencias de la Salud, Universidad National de Asuncion, Asuncion, Paraguay; 2Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK; 3Laboratoria Central de Salud Publica, Ministerio de Salud Publica y Bienestar Social, Asuncion, Paraguay; 4Liverpool School of Tropical Medicine, Liverpool L3 SQA, UK; 5School of Biological Sciences, University of Livetpool, Crown Street, Liverpool L69 720, UK

Cutaneous leishmaniasis is endemic in Paraguay but *Supported by the ALFA European Commission Program (Project ALRiB7-301 l/94.04-6.0008.9).

little reported in the international literature. Most of 70 isolates from Paraguayan caseshave been identified by isoenzyme electrophoresis as Leishmania braziliensis (R. Oddone et al., unpublished): some phenotypic polymorphisms have been found but genetic diversity at the DNA level has not been studied. Three microsatellites discriminate stocks within the Leishmania subgenus Viannia (RUSSELL, R. et al., 1999: Molecular and Biochemical Parasitology, 103, 71-77). Using these microsatellites extensive diversity has been found in a single locality endemic for L. braziliensis, L. peruviana and L. braziliensis-L. peruviana hybrids (NOLDER, D., 2000: PhD thesis, University of London). For reliable population genetic analyses more microsatellite loci are required. We have isolated further loci, identified by oligonucleotide (GT,,) screening a microsatelliteenriched library of a WHO L. bra&lien& reference strain (MHOM/BR/84/LTB300) (see BLOOR, P. et al., 2001: http://www.liv.ac.uk~kempsj/genomics.html). So far we have sequenced 24 new microsatellite loci, with dinucleotide or complex repeats ranging in size from 18 to 118 base pairs, and we are assessingtheir suitability for genotyping Viannia.

Exchange

Data-driven models of malaria risk using remote sensing: high-resolution maps for disease control in Kenya JtiAS.toFrnbo

1,2,S. I. Hay*,2, D. J. Rogers’ and R. ‘TALA Research Group, Depanment of Zooloky, Oxford University, South Parks Road, Oxford OX1 3PS, UK; ‘Kenya Medical Research Institute1 Wellcome Trust Collaborative Programme, I? 0. Box 43640, Nairobi, Kenya

Endemicity maps are a useful tool for epidemiologists to understand and model the spatial determinants of disease transmission and for disease control personnel to plan efficient and effective interventions. Currently available malaria maps for sub-Saharan Africa are limited in that most describe endemicity based on only a few of the factors that drive Plasmodium falciparum

SHORT PRESENTATIONS

365

transmission and have focused only on the potential of transmission as opposed to actual transmission. Developments in geographical information systems offer new methods of spatial analysis combining several data sets. Remote sensing provides a wider, improved range of data on meteorological transmission determinants allowing more detailed analyses. This paper explores the use of available remotely sensed meteorological and empirical malariometric data to better define malaria transmission in Kenya. Such simple tools would assist in decision support and thus ease the task of healthcare planners in targeting limited resources. Morphometrics of Rhodnius ecuadoriensis (Triatominae) populations: a tool for Chagas disease vector control surveillance in Ecuador and Peru* James S. Patterson’, Fernando Abad-FranchIt* , H. Marcel0 Aguilar V.2, CCsar A. Cuba Cuba3 and Michael A. Miles’ ‘Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK; ‘Unidad de Medic&a Tropical, Institute yuan G&ar Garcia’, CP 171 106292 Quito, Ecuador; 3Unidade de Parasitologia Midica-Patologia, Faculdade de Medicina, Universidade de Brasilia, DF, 7091 O-900, Brazil Rhodnius ecuadoriensis is an important vector of Trypanosoma cruzi (the causative agent of Chagas disease)

in Ecuador and Peru. Phenetic differences between bugs of sylvatic and domestic origin were characterized by digital morphometrics. Measurements were taken from digital images of the heads of 80 bugs of one sylvatic (Ecuador) and of 3 domestic (2 Ecuadorian and 1 Peruvian) R. ecuadoriensis populations, plus R. pallescens as an outgroup. The data set obtained was subjected to size free multivariate analyses. Results showed a clear separation of bugs from sylvatic and domestic origins. Whilst the sylvatic and domesticperidomestic populations were the most dissimilar (probably reflecting habitat differences), the 3 synanthropic populations were also distinguishable from each other. The Peruvian cluster was initially distorted by a single bug until that specimen alone was traced back to a collection site adjacent to one Ecuadorian population. We conclude that morphometric analysis can provide a key tool for the investigation of reinfestations after residual spraying. Furthermore, the extent of morphometric differences between sylvatic populations (only reported from palm trees in central Ecuador) and synanthropic populations in southern Ecuador and Peru (where palms are absent) suggestsa certain degree of isolation, implying that eradication of the latter domestic populations may be attainable. Tuberculin skin testing tions: anergy, nutritional fee tion

in two Peruvian populastatus and parasitic in-

Tom F. Pelly’ , Carlos Santillan’, Robert H. Gilman2~‘~*, Lilia Cabrera2, Edwin Garcia*, Mayuko Saito’ and Carlton A. Evans2’3g4 ‘University College Hospital and Royal Free Medical School, London, UK; 2A. B. PRISMA, Lima, Peru; ‘Department of International Health, Johns Hopkins Bloomberg School of Public Health, Mayland, USA; 4Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College of Science,

*Supported by the ECLAT network, UNDl?/World Banki WHO TDR (grant 970195), CAPES (MinistCrio de Educa@o e Cultura, Brazil), the Oficina National de Epidemiologia (Instituto National de Salud, Ministerio de Salud Ptiblica, Peru), the University General Hospital of Valencia (Spain), the Sir Halley Stewart Trust, and the Cafiada-Blanch Foundation (Spain).

Technology and Medicine, Hammersmith Hospital, Department of infectious Diseases, London Ml2 ONN, UK

In disadvantaged communities, malnutrition and intestinal parasitism may cause anergy. This results in false-negative tuberculin skin testing (TST) and consequently underestimation of tuberculosis prevalence. We therefore studied parasitic infection, nutrition, socioeconomic status, TST and anergy skin tests in urban and rural adult populations in Peru. One hundred and two randomly selected residents of a village in the Amazon jungle had greater anthropometric muscle mass, more meat in their diets and more helminthic infections than 112 randomly selected shanty-town residents in Lima. However, there were no differences in TST results, 52% of which were positive (>lO mm induration). Income was similar in the 2 populations. Average body mass index was 24.9 (SD 3.4), and midarm muscle circumference, a measure of protein nutrition, was 23.4 (SD 5.3). TST results were associated with diet, mid-arm muscle circumference (P = 0.003) and, in Lima, with past exposure to tuberculosis (P = 0.03), but not with body mass index (P = 0,6), or parasitic infection (P = 0.8). Simultaneous tetanus and Candida skin testing revealed that anergy was rare (0.5%), despite high rates of parasitic infection (62%) and poor nutrition. In conclusion, one half of adults in these populations had positive TST, and neither malnutrition nor intestinal parasitism appeared to confound these results. Gene 5ow and population structure minimus in South-East Asia

of Anopheles

H. K. Phuc and H. Townson Division of Parasite and Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK Anopheles minimus is a major malaria vector in SouthEast Asia and is known to comprise at least 2 sibling species, A and C. In laboratory crossing experiments of species C with species A of differing geographical origin, the Fl progeny show significantly different survival rates. To determine whether these differences are reflected in other population genetic features, we have studied the mitochondrial genome and 12 microsatellite loci. A total of 53 haplotypes of the mtDNA CO1 gene were seen in specimens of species A and C from Laos, Cambodia and Viet Nam. A maximum parsimony tree and Nei’s network analysis show evidence of at least 2 mtDNA haplotype groups within each of species A and C. The average Kimura distance between groups is high (0.0341), inferring possible barriers to reproduction between the groups. Allelic variation was studied at 12 microsatellite loci in samples from 7 populations of haplotype group Al of species A, and 2 populations from each of haplotype groups Cl and C2 within species C. These data provide evidence of population bottlenecks within both species and of population subdivision within species C. However, there is no evidence of barriers to gene flow within groups C 1, C2 and A 1. Locating remotely

potential mosquito sensed data

breeding

sites using

S. E. Rodgers and M. A. Oliver Depanment of Soil Science,The University of Reading, P. 0. Box 233, miteknights, Reading RG6 60 W, UK Accurate

maps

of the risk

of malaria

are vital

to

control malaria adequately. Geostatistical methods have been used to explore the spatial distribution of Normalized Difference Vegetation Index (NDVI), as an indicator of possible mosquito breeding sites. In

SHORT PRESENTATIONS

semi-arid West Africa, the number of breeding sites might be greater in some areas (given the same climatic conditions) because of impeded drainage associated with soil physical properties and the position of the site in the landscape (THOMSON, M. C. et al., 1999: American Journal of Tropical Medicine and Hygiene, 61, 2-8). A nested variogram model was fitted to NDVI. The spatial structures it identified are similar to those of the field variables and to topography and rainfall. Dysjunctive kriging was used to estimate the probabilities that the values of NDVI would exceed a threshold and be likely to support mosquito development. By using different thresholds it is possible to see how the extent of areas needing malaria control changes. The aim is to be able to target control measures objectively. Field bioassays investigating the effectiveness of the fenitrothion house spraying control programme against Rhodnius prolixus in Portuguesa State, Venezuela A. Skelton’, C. R. Davies’ and D. Feliciangeli’ ‘Disease Control and Vector Biology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London, UK; 21nstituito de Entomologica, BIOMED, Universidad de Carabobo, Maracay, Venezuela

There has been no significant reduction in house infestation with triatomine bugs during the past 10 years in Venezuela, despite a sustained house spraying programme with fenitrothion, and bug densities in some states (including Portuguesa) are persisting at well above target levels (D. Campbell-Lendrum & C. R. Davies, unpublished). Field studies were carried out during 2001 to test whether this may be partly due to low lethal effectiveness of the insecticide. The residual effect of fenitrothion against Rhodnius prolixus was tested by field bioassays in houses (with different wall surfaces) sprayed previously by the Chagas disease control programme. Following l-h contact with sprayed walls, mean 72-h mortality rates dropped from 24% to 14% and 10% (at 1, 3, and 5 months postspray, respectively). The lethal effect persisted longest on metal, compared to wood and mud. This poor residual effect was not due to insecticide resistance as the bioassays used laboratory bugs, which had been cultured since 1960 (fenitrothion was first used in Venezuela in the 1980s). Furthermore, 100% bug mortality was achieved using the same bioassay procedure when a mud house was freshly sprayed at the appropriate dose (2 g/m*). While the reason for the apparently poor effectiveness of the house spraying programme is unclear, we conclude that the serious concerns raised by these results should be addressedby larger-scale studies. Ecology of Anopheles S.S. in Malawi

arabiensis

and An. gambiae

A. A. Spiers’ , D. Atkinson2, T. Mzilahowa3 and I’. 1. McCall’ ‘Division of Parasite and Vector Biolow. LiGeerpool School of Tropical Medicine, Pembroke PIaye; Live@ool L3 SQA, UK; ‘Population and Evolutionary Biology Research Group, School of Biological Sciences, The University of Liverpool, Liverpool L69 3GS, UK; 3Department of Community Health, College of Medicine, University of Malawi and Wellcome Trust Research Laboratories, Queen Elizabeth Central Hospital, Chichiri, Blantyre 3, Malawi

Despite the importance of Anopheles arabiensis and An. gumbiae sensu stricto as malaria vectors in Africa,

comparatively little is known of their individual ecolopies. With a view to understanding the distribution and &e effect of environmental fact&s and potential climate change on malaria, we investigated 2 Malawian

vector populations, their breeding sites, climatic influences and ultimately how these factors might affect survival and vectorial capacity. Rainfall affected numbers but not relative abundance of the 2 species: An. arubiensis comprised 80% of both adult and larval catches, while An. gambiae S.S. comprised 11% and 9%, respectively. The remainder were An. quadriannulutus, recorded in Malawi for the first time. Adult numbers of both An. gambiae S.S. and An. arabiensis were reduced, but An. funestus numbers increased, during periods of high rainfall. Minimum temperature affected both vector abundance and adult body size. At the end of the wet season, with the drop in minimum temperature, mosquito numbers fell. Wing length was also negatively correlated with minimum temperature. We are now investigating whether this observed change in body size is caused by slower development rates at lower temperature, or is the result of reduced competition through higher mortality. Resistance

to artesunate

in rodent

malaria

L. B. Stewart and B. L. Robinson Centrefor Tropical Antiprotozoal Chemotherapy, Y Block, Northwick Park Institute for Medical Research, Wat$ord Road, Harrow, Middlesex HA1 3UJ UK

Chloroquine-sensitive Plasmodium berghei N was exposed to selection pressure with the endoperoxide artesunate at 600 mg/kg S.C. using the ‘2% relapse technique’. Resistance was slow to develop during the first 23 passages and the strain, named SAN, was frozen in liquid nitrogen (PETERS, W. & ROBINSON, B. L., 1999: Annals of Tropical Medicine and Parasitology, 93, 325-339). SAN was later retrieved from nitrogen and drug pressure was reapplied at 300 mg/kg S.C. Resistance has been developed during 110 passages with a ‘I-fold increase in resistance compared to the parent strain in the standard ‘4-day test’. Studies to assessthe stability of resistance in the absence of drug pressure continue. The fact that resistance to artesunate is very slow to develop supports the argument that artesunate could be a suitable partner compound in a combination with, for example, pyronaridine or chlorproguanil/dapsone and that such combinations may extend the life of the individual components. Polymorphisms within the macrophage mannose receptor and genetic susceptibility to leprosy Kerrie Tosh’, Sarah Meisner’, Suchismita Roy’, M. Ruby Siddiqui’ , Karuppiah Balakrishnan2, Nallakandy P. Shanker Narayan3, Thiagarajan Sitaraman4, Ramasamy Pitchappan2 and Adrian V. S. Hill’ ’ Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, UK; 2Madurai Kamaraj University, Madurai 625021, Indi?; 3Leprosy Project, Sakthi Nagar, India; 4Hindu Mzssion Hospital, Kumakonam, India

Several lines of evidence indicate an important role for host genetic factors in predisposition towards Ieprosy. These include the presence of familial clustering despite social segregation (SHIELDS, E. D. et al., 1987: Journal of Clinical Investigation, 19, 1139- 1143), a difference in concordance rate between dizygotic and M. et al., 1973: monozygotic twins (CHAKRAVATTI, Topics in Human Genetics, 1, l-123) and comolex segregation analysis (ABEL, L. et al., 1986: Ame&an ‘iournal of Human Genetics. 42. 2562651. More recently, a -major susceotibili& locus for leprosy has been identified on chromosome 10 (1 Op13) by linkage analysis (SIDDIOUI. R. M. et al.. 2001: Nature Genetics. 27. 439’-44 1) .- A* positional candidate gene, the macro: phage mannose receptor (MRCl), lies within this re-

SHORTPRESENTATIONS

367

gion of linkage. This receptor recognizes LAM on the surface of mycobacteria, allowing phagocytosis of the organism (SHLESINGER, L. S. et al., 1994: Journal of Immunology, 152, 4070-4079). We have identified several single nucleotide polymorphisms within the MRC 1 gene and a family-based association study has assessed whether any of these show an association with leprosy susceptibility.

Quantifying the cost-effectiveness of the Community Health Club strategy in rural Zimbabwe J. Waterkeyn

Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT, UK

Much effort to reduce diarrhoeal diseases by the provision of safe water and improved sanitation has been undermined by poor hygiene within the home. Whilst many attempts at health promotion have been made, few published articles have shown enough methodological rigour for real evidence of success (LOEVINSOHN, B. 1990: International

j’ournal of Epidemiology,

19, 788-794). In Zimbabwe a new strategy to achieve safe hygiene practices has been developed using Community Health Clubs as a ‘vehicle for change’. This intervention study demonstrates, using proxy indicators of improved hygiene practices, (FEACHEM, R. G., 1984: Bulletin

of the World Health

Organization,

62,

467-476) that although the participatory PHAST methodology has been disappointing over the past decade (UNDPWSP, 1999), the strategy does succeed in modifying hygiene habits when incorporated into a more structured programme. This is shown within 32 Clubs (2105 members), where a ‘culture of health’ has prompted a 50% hygiene behaviour change and overwhelming demand for sanitation. Using this strategy, family health can be improved at a cost of approximately US$3 per family. [This poster presentation was awarded f;rst prize at the meeting.]

Traditional

herbal medicines for malaria

M. L. Willcox

Research Initiative on Traditional Antimalarial Methods, 9 Croft Road, Oxford OX3 OJY, UK

Malaria is responsible for much morbidity and mortality, especially in Africa. Two of the 3 main classesof antimalarial drugs are derived from traditional herbal remedies for malaria. Many other herbal remedies exist and are used, and have been shown to have antimalarial activity in vitro and/or in vivo. However, there has been little coordination of research in this area. The Research Initiative on Traditional Antimalarial Methods (RITAM) was founded to fill this gap. It aims to collate all existing evidence, and to foster collaboration between researchers working in this area. This paper will summarize progress to date, including a review of studies on treatment-seeking behaviour; a review of ethnobotanical studies; a review of clinical studies; and proposed protocols for further preclinical and clinical studies, on traditional herbal medicines for malaria.

Trypanosoma cruzi: multiclonality, genetic diversity, and experimental proof of genetic hybridization Matthew Yeo, Michael Gaunt, Graham Miles and Michael Miles Depanment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, UK

To assessthe extent of genetic diversity of Trypanosoma cruzi in single triatomine vectors we have successfully plated clonal colonies of T. cruzi on solid medium, directly from the faeces of naturally infected bugs. In Amazonian Brazil, distinct, single genotypes of T. cruzi I could be found in separate specimens of Rhodnius, and occasionally mixed genotypes. When studying the genetic diversity of T. cruzi in Paraguay, we found that several subgroups of T. cruzi II occurred, and a putatively hybrid strain (T. cruzi IId) was common (CHAPMAN et al., 1984: Journal of fiotozoology, 31, 35-44). Using putatively parental isolates of T. cruzi I, from a single locality in the Amazon basin, we have proved experimentally that T. cmzi has an active capacity for genetic hybridization (STOTHARD et al., 1999: Memorias do Instituto Oswald0 Crux, 94, supplement 1, 189193). A combination of parental phenotypes and genotypes was found in experimentally derived hybrid clones. We thank the Wellcome Trust for funding, and Iain Frame, Russell Stothard, Hernan Carrasco, Martin Taylor and Aldo Valente for participating; details will be published elsewhere. Low-birthweight neonates born at term and behavioural response to stressors in Sudan M. Yousif, McGregor

C. C. Ani

and S. M. Grantham-

Centre for International Child Health, Znstitute of Child Health, University College London, 30 Guilford Street, London WClN lEH, UK

Nineteen percent of all births in developing countries are low birthweight (LBW, weight c2.5 kg) and are more likely to be bon at term than LBW children in developed countries. Very little is known of these children’s neonatal behaviour. We compared 65 LBW babies with 67 normal birthweight (NBW) babies matched for age (4-6 h), and mode and place of delivery in Sudan. All babies had a gestational age of 38-42 weeks assessed by a Dubowitz examination. The babies’ crying behaviour was rated before and after physical stressors: a clinical examination and a heel prick. The mean birthweights (mean i SD) were 2.lf 0.3 kg and 3.3 5 0.4 kg for the LBW and NBW babies respectively. Mothers of LBW babies were more likely to have had anaemia in pregnancy (P = 0.031), and their fathers more likely to be unemployed (P = 0.0 18). During clinical examination, the intrauterine growth-retarded neonates cried less vigorously (P = 0.02) and for a shorter duration (P < 0.001). In response to heel prick, the LBW neonates were less likely to cry or to cry immediately (P = 0,03), they cried less vigorously and for a shorter duration (P < 0.0001). It is concluded that these LBW term neonates had inhibited behavioural response to stressors. We hypothesize that alterations in the hypothalamic-pituitary axis may be responsible for this observation.