EHP Library Malaria Bulletin No. 33: March 16-28, 2002

Social Sciences and MalariaTrop Med Int Health 2002 Mar;7(3):240-8

Community factors associated with malaria prevention by mosquito nets: an exploratory study in rural Burkina Faso.

Okrah J, Traore C, Pale A, Sommerfeld J, Muller O.

Ministry of Health, Public Health Division, Accra, Ghana, Centre de Recherche en Sante de Nouna, Nouna, Burkina Faso, Department of Tropical Hygiene and Public Health, Ruprecht-Karls-University, Heidelberg, Germany.

Malaria-related knowledge, attitudes and practices (KAP) were examined in a rural and partly urban multiethnic population of Kossi province in north-western Burkina Faso prior to the establishment of a local insecticide-treated bednet (ITN) programme. Various individual and group interviews were conducted, and a structured questionnaire was administered to a random sample of 210 heads of households in selected villages and the provincial capital of Nouna. Soumaya, the local illness concept closest to the biomedical term malaria, covers a broad range of recognized signs and symptoms. Aetiologically, soumaya is associated with mosquito bites but also with a number of other perceived causes. The disease entity is perceived as a major burden to the community and is usually treated by both traditional and western methods. Malaria preventive practices are restricted to limited chloroquine prophylaxis in pregnant women. Protective measures against mosquitoes are, however, widespread through the use of mosquito nets, mosquito coils, insecticide sprays and traditional repellents. Mosquito nets are mainly used during the rainy season and most of the existing nets are used by adults, particularly heads of households. Mosquito nets treated with insecticide (ITN) are known to the population through various information channels. People are willing to treat existing nets and to buy ITNs, but only if such services would be offered at reduced prices and in closer proximity to the households. These findings have practical implications for the design of ITN programmes in rural areas of sub-Saharan Africa (SSA).

---

Trop Med Int Health 2002 Mar;7(3):220-30

Insecticide-treated bednet use, anaemia, and malaria parasitaemia in Blantyre District, Malawi.

Holtz TH, Marum LH, Mkandala C, Chizani N, Roberts JM, Macheso A, Parise ME, Kachur SP.

Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA. 
Email: [email protected]

OBJECTIVE: To evaluate the use of insecticide-treated bednets and the effectiveness of social marketing for their distribution. METHODS: Systematic cluster sample survey of 1080 households in 36 census enumeration areas across Blantyre district, Malawi, in February 2000. RESULTS: A total of 672 households had one or more children under 5. Bednet ownership was low (20.5% of households) overall, and significantly lower in rural areas than urban areas (6.4 vs. 29.8%, P=0.001). Only 3.3% of rural children under 5 had slept under a net the previous night, compared with 24.0% of urban children (P < 0.001). When asked why they did not own a net, nearly all (94.9%) caretakers in households without nets stated they had no money to buy them. In multivariate statistical models that controlled for the influence of house structure, urban vs. rural location, gender of the head of household, and the primary caretaker’s education, rural children under 5 in households without nets experienced a statistically significant higher prevalence of malaria parasitaemia [RR (risk ratio) 4.9, 95% CI (confidence interval) 2.3-10.5] than children in households with at least one bednet. This was also true for urban children under 5 (RR 2.1, 95% CI 1.0-4.2, P=0.04). CONCLUSION: Social marketing approaches to promoting insecticide-treated nets in Blantyre District may have produced measurable health benefits for children in those households in which residents bought and used the products. Market-based approaches may take years to achieve high levels of coverage and may exaggerate inequities between urban and rural populations.

---

Trop Med Int Health 2002 Mar;7(3):201-9

Case management of malaria in under-fives at primary health care facilities in a Tanzanian district.

Nsimba SE, Massele AY, Eriksen J, Gustafsson LL, Tomson G, Warsame M.

Department of Clinical Pharmacology, Muhimbili University College of Health Sciences, Dar-es-Salaam, Tanzania.

OBJECTIVE: To study case management of malaria in children under 5 years of age at primary health care facilities in Kibaha district, Tanzania and to evaluate the accuracy of self-reported mothers’/guardians’ information on chloroquine use in children. METHOD: A random sample of 652 mothers/guardians with sick children under 5 years of age attending 10 primary health care facilities was observed and interviewed. Blood samples for determination of chloroquine levels were taken from all children and thick smears for detection of malaria parasites were taken from the children who were prescribed chloroquine. Information on diagnoses and prescriptions was collected from recording books. RESULTS: Fever and respiratory problems were the most common complaints and accounted for 75% and 46% of the presenting conditions, respectively (some complained of both). Fifty-four per cent of the children received medication at home, most commonly antipyretics and chloroquine, 20% had been taken to another health facility and 3% to traditional healers before coming to the health facilities. There was a significantly higher use of antipyretics among home treated children compared with those taken previously to health facilities (P <or= 0.001). Use of antibiotics was higher among children who had been taken to health facilities previously (P < 0.0001). Nine per cent had received injections. The average consultation time was 3.8 min. Thirty-nine per cent of the children were physically examined, with large interfacility variations. In 71% of the children malaria was diagnosed, either as a single condition or in combination with others, and with respiratory problems as the leading overlapping condition (29%). Malaria parasites were found in 38% of the cases given a malaria diagnosis. A total of 81% of the health facility prescriptions included analgesics, 71% chloroquine and 54% antibiotics. A fourth of all prescriptions were injections. The proportions of chloroquine and antibiotic injections in relation to the total number of prescriptions varied between the facilities. Of the 529 blood samples successfully analysed for chloroquine, 98% had detectable blood drug levels. Ninety-seven per cent of the children without history of prior chloroquine treatment had detectable drug levels in the blood, 11% had high levels (>or= 1000 nmol/l). Of those prescribed chloroquine, 16% already had high blood concentrations of the drug. CONCLUSION: Health care services, i.e. presumptive malaria diagnosis, consultation time and procedure for physical examination need to be improved.

---

PUBMEDCell Mol Life Sci 2002 Feb;59(2):258-71

Pathogenesis of Plasmodium falciparum malaria: the roles of parasite adhesion and antigenic variation.

Beeson JG, Brown GV.

Department of Medicine, University of Melbourne, Post Office Royal Melbourne Hospital, Victoria, Australia.

Malaria results in up to 2.5 million deaths annually, with young children and pregnant women at greatest risk. The great majority of severe disease is caused by Plasmodium falciparum. A characteristic feature of infection with Pfalciparum is the accumulation or sequestration of parasite-infected red blood cells (RBCs) in various organs, such as the brain, lung and placenta, and together with other factors is important in the pathogenesis of severe forms of malaria. Sequestration results from adhesive interactions between parasite-derived proteins expressed on the surface of infected RBCs and a number of host molecules on the surface of endothelial cells, placental cells and uninfected RBCs. Some receptors for parasite adhesion have been implicated in particular malaria syndromes, such as intercellular adhesion molecule 1 in cerebral malaria and chondroitin sulfate A and hyaluronic acid in placental infection. The principal parasite ligand and antigen on the RBC surface, P falciparum erythrocyte membrane protein 1 encoded by a multigene family termed var, is clonally variant, enabling evasion of specific immune responses. An understanding of these host-parasite interactions in the context of clinical disease and immunity may reveal potential targets to prevent or treat severe forms of malaria.

---

Int J Epidemiol 2002 Feb;31(1):175-180

Usefulness of a dispensary-based case-control study for assessing morbidity impact of a treated net programme.

Abdulla S, Schellenberg JA, Mukasa O, Lengeler C.

Ifakara Health Research and Development Centre (IHRDC), PO Box 53, Ifakara, Tanzania. Swiss Tropical Institute, PO Box, 4002 Basel, Switzerland.

Background Case-control studies have been proposed as an appropriate tool for health impact evaluation of insecticide-treated nets (ITN) programmes. Methods A dispensary-based case-control study was carried out in one village in Tanzania. Each case of fever and parasitaemia in a child under 5 years was paired with one community and one dispensary control without fever and parasitaemia. Cases and controls were compared with regard to ITN ownership and other factors assessed by a questionnaire. A cross-sectional survey of factors associated with parasitaemia, including ITN use, was carried out during the study. Dispensary attendance rates of the study children were calculated using passive case detection data. Results Cases and dispensary controls had higher dispensary attendance rates compared to community controls and children with nets attended more for most of the illness events. A comparison of cases and community controls showed a strong and statistically significant association between untreated net use and being a case (odds ratio [OR] = 2.1, 95% CI : 1.3-3.4). For those with ITN there was a smaller and weaker association between risk of being a case and ITN use (OR =1.4, 95% CI : 0.9-2.2). Comparison of cases and dispensary controls showed no association between untreated or treated nets and the risk of being a case (for treated nets OR = 0.9, 95% CI : 0.5-1.4 and for untreated nets OR = 1.2, 95% CI : 0.7-2.0). These results are contrary to those from the cross-sectional assessment, where children with ITN had a lower prevalence of parasitaemia than those with no nets (OR = 0.5, 95% CI : 0.3-0.9), and also contrary to other assessments of the health impact of ITN in this population. Conclusion The positive association between mild malaria and net ownership is counter-intuitive and best explained by attendance bias, since children with nets attended more frequently for all curative and preventive services at the dispensary than those without nets. Dispensary-based case-control studies may not be appropriate for assessing impact of treated nets on clinical malaria, while cross-sectional surveys might represent an attractive alternative.

---

Biochem Pharmacol 2002 Mar 1;63(5):833-42

Novel phenothiazine antimalarials: synthesis, antimalarial activity, and inhibition of the formation of beta-haematin.

Kalkanidis M, Klonis N, Tilley L, Deady LW.

Department of Chemistry, La Trobe University, 3086, Victoria, Australia

We report the synthesis of a series of novel phenothiazine compounds that inhibit the growth of both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. We found that the antimalarial activity of these phenothiazines increased with an increase in the number of basic groups in the alkylamino side chain, which may reflect increased uptake into the parasite food vacuole or differences in the toxicities of individual FP-drug complexes. We have examined the ability of the parent phenothiazine, chlorpromazine, and some novel phenothiazines to inhibit the formation of beta-haematin. The degree of antimalarial potency was loosely correlated with the efficacy of inhibition of beta-haematin formation, suggesting that these phenothiazines exert their antimalarial activities in a manner similar to that of chloroquine, i.e. by antagonizing the sequestration of toxic haem (ferriprotoporphyrin IX) moieties within the malaria parasite. Chlorpromazine is an effective modulator of chloroquine resistance; however, the more potent phenothiazine derivatives were more active against chloroquine-sensitive parasites than against chloroquine-resistant parasites and showed little synergy of action when used in combination with chloroquine. These studies point to structural features that may determine the antimalarial activity and resistance modulating potential of weakly basic amphipaths.

---

Microbes Infect 2002 Mar;4(3):291-300

Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis.

Brian de Souza J, Riley EM.

Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Sciences, Royal Free and University College London Medical School, 46 Cleveland Street, W1T 4JF, London, UK

Cerebral malaria is a serious and often fatal complication of Plasmodium falciparum infections. The precise mechanisms involved in the onset of neuropathology remain unknown, but parasite sequestration in the brain, metabolic disturbances and host immune responses are all thought to be involved. This review outlines the current state of knowledge of cerebral disease in humans, and discusses the contribution of studies of animal models to elucidation of the underlying mechanisms.

---

J Immunol 2002 Apr 1;168(7):3444-50

Plasmodium falciparum Variant Surface Antigen Expression Varies Between Isolates Causing Severe and Nonsevere Malaria and Is Modified by Acquired Immunity.

Nielsen MA, Staalsoe T, Kurtzhals JA, Goka BQ, Dodoo D, Alifrangis M, Theander TG, Akanmori BD, Hviid L.

Center for Medical Parasitology, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark. Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana. Immunology Unit, Noguchi Memorial Institute for Medical Research, Legon, Ghana.

In areas of endemic parasite transmission, protective immunity to Plasmodium falciparum malaria is acquired over several years with numerous disease episodes. Acquisition of Abs to parasite-encoded variant surface Ags (VSA) on the infected erythrocyte membrane is important in the development of immunity, as disease-causing parasites appear to be those not controlled by preexisting VSA-specific Abs. In this work we report that VSA expressed by parasites from young Ghanaian children with P. falciparum malaria were commonly and strongly recognized by plasma Abs from healthy children in the same area, whereas recognition of VSA expressed by parasites from older children was weaker and less frequent. Independent of this, parasites isolated from children with severe malaria (cerebral malaria and severe anemia) were better recognized by VSA-specific plasma Abs than parasites obtained from children with nonsevere disease. This was not due to a higher infection multiplicity in younger patients or in patients with severe disease. Our data suggest that acquisition of VSA-specific Ab responses gradually restricts the VSA repertoire that is compatible with parasite survival in the semi-immune host. This appears to limit the risk of severe disease by discriminating against the expression of VSA likely to cause life-threatening complications, such as cerebral malaria and severe anemia. Such VSA seem to be preferred by parasites infecting a nonimmune host, suggesting that VSA expression and switching are not random, and that the VSA expression pattern is modulated by immunity. This opens the possibility of developing morbidity-reducing vaccines targeting a limited subset of common and particularly virulent VSA.

---

Vaccine 2002 Mar 15;20(13):1707-10

Remarkably high antibody levels and protection against P. falciparum malaria in Aotus monkeys after a single immunisation of SPf66 encapsulated in PLGA microspheres.

Rosas JE, Pedraz JL, Hernandez RM, Gascon AR, Igartua M, Guzman F, Rodriguez R, Cortes J, Patarroyo ME.

Fundacion Instituto de Inmunologia de Colombia (FIDIC), Carrera 50, No. 2600, Santafe de Bogota, Colombia

Single dose immunisation is a major goal in vaccine design. The purpose of this study was the development of a single dose delivery system for the SPf66 malaria vaccine, based on this antigen’s microencapsulation in PLGA microspheres by double emulsion method.Results indicate that a single immunisation in mice and monkeys with the SPf66 malaria vaccine, encapsulated in a mixture of two formulations of PLGA microspheres, induced a remarkably high and long-lasting immune response as assessed by ELISA and Western Blott. This immune response was associated with a good protective capacity in Aotus monkeys, after experimental challenge, indicating that antigen integrity lasted following the microencapsulation process. PLGA biodegradable microspheres thus serve as an effective delivery system for the design of a single dose immunisation vaccine, such as the SPf66 synthetic malaria vaccine.

---

J Pept Res 2002 Feb;59(2):62-70

Double dimer peptide constructs are immunogenic and protective against Plasmodium falciparum in the experimental Aotus monkey model.

Rivera Z, Granados G, Pinto M, Varon D, Carvajal C, Chaves F, Calvo J, Rodriguez R, Guzman F, Patarroyo ME.

Fundacion Instituto de Inmunologia de Colombia (FIDIC), Bogota, Colombia.

Multiple antigen peptide constructs (MAPs) have been used to obtain defined multimeric peptide molecules useful in the development of possible synthetic malaria vaccines. In this context, a method was developed, named double dimer constructs (DDCs), involving the direct synthesis of a dimeric peptide with a C-terminal cysteine. A tetrameric molecule was then obtained by oxidation of sulfhydryl groups. Dimer synthesis was optimized using a Fmoc/tBu strategy, dimers were purified by HPLC, oxidized with DMSO and characterized by HPLC and MALDI-TOF-MS. The tetramers or DDCs obtained by this method were used as immunogens in the search for a possible malaria vaccine. It was found that they were immunogenic in the experimental Aotus monkey model, and were able to induce protective immunity when challenged experimentally with a highly infective Plasmodium falciparum malaria strain.

---

Nature Rev Immunol 2001 Nov;1(2):117-25

Towards a blood-stage vaccine for malaria: are we following all the leads?

Good M F.

Cooperative Research Centre for Vaccine Technology, The Queensland Institute of Medical Research, Herston, Australia. 
Email: [email protected]

Although the malaria parasite was discovered more than 120 years ago, it is only during the past 20 years, following the cloning of malaria genes, that we have been able to think rationally about vaccine design and development. Effective vaccines for malaria could interrupt the life cycle of the parasite at different stages in the human host or in the mosquito. The purpose of this review is to outline the challenges we face in developing a vaccine that will limit growth of the parasite during the stage within red blood cells–the stage responsible for all the symptoms and pathology of malaria. More than 15 vaccine trials have either been completed or are in progress, and many more are planned. Success in current trials could lead to a vaccine capable of saving more than 2 million lives per year.

---

Crit Rev Biochem Mol Biol 2002;37(1):29-54

DNA vaccine against malaria: a long way to go.

Tuteja R.

International Centre for Genetic Engineering and Biotechnology, New Delhi. 
Email: [email protected]

Vaccination is the attempt to mimic certain aspects of an infection for the purpose of causing an immune response that will protect the individual from that infection. Malaria, a disease responsible for immense human suffering, is caused by infection with Plasmodium spp. parasites, which have a very complex life cycle–antigenically unique stages infect different tissues of the body. It is a parasitic disease for which no successful vaccine has been developed so far, despite considerable efforts to develop a subunit vaccine that offers protective immunity. Due to the spread of drug-resistant malaria, efforts to develop an effective vaccine have become increasingly critical. DNA vaccination provides a stable and long-lived source of protein vaccine capable of inducing both antibody- and cell-mediated immune responses to a wide variety of antigens. Injected DNA enters the cells of the host and makes the protein, which triggers the immune response. According to present needs, the flexibility of DNA vaccine technology permits the combination of multiple antigens from both the preerythrocytic and erythrocytic stages of malaria parasite. DNA vaccines with genes coding for different antigenic parts of malaria proteins have been created and presently some of these are undergoing field trials. The results from these trials will help to determine the likelihood of success of this technology in humans. This review presents an update of the studies carried out in malaria using DNA vaccine approach, the challenges, and the future prospects.

---

Proc Natl Acad Sci U S A 2002 Mar 19;99(6):3860-3865

Immunization of Aotus monkeys with a functional domain of the Plasmodium falciparum variant antigen induces protection against a lethal parasite line.

Baruch DI, Gamain B, Barnwell JW, Sullivan JS, Stowers A, Galland GG, Miller LH, Collins WE.

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341; Malaria Vaccine Development Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; and Scientific Resources Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333.

Immunity to Plasmodium falciparum in African children has been correlated with antibodies to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) variant gene family expressed on the surface of infected red cells. We immunized Aotus monkeys with a subregion of the Malayan Camp variant antigen (MCvar1) that mediates adhesion to the host receptor CD36 on the endothelial surface and present data that PfEMP1 is an important target for vaccine development. The immunization induced a high level of protection against the homologous strain. Protection correlated with the titer of agglutinating antibodies and occurred despite the expression of variant copies of the gene during recurrent waves of parasitemia. A second challenge with a different P. falciparum strain, to which there was no agglutinating activity, showed no protection but boosted the immune response to this region during the infection. The level of protection and the evidence of boosting during infection encourage further exploration of this concept for malaria vaccine development.

---

FEBS Lett 2002 Feb 27;513(2-3):159-62

Activity and inhibition of plasmepsin IV, a new aspartic proteinase from the malaria parasite, Plasmodium falciparum.

Wyatt DM, Berry C.

Cardiff School of Biosciences, Cardiff University, P.O. Box 911, CF1 3US, Wales, Cardiff, UK

A new aspartic proteinase from the human malaria parasite Plasmodium falciparum is able to hydrolyse human haemoglobin at a site known to be the essential primary cleavage site in the haemoglobin degradation pathway. Thus, plasmepsin IV may play a crucial role in this critical process which yields nutrients for parasite growth. Furthermore, synthetic inhibitors known to inhibit parasite growth in red cells in culture are able to inhibit the activity of this enzyme in vitro. As a result, plasmepsin IV appears to be a potential target for the development of new antiparasitic drugs.

---

Acta Trop 2002 Apr;82(1):51-9

A comparison of two rapid field immunochromatographic tests to expert microscopy in the diagnosis of malaria.

Mason DP, Kawamoto F, Lin K, Laoboonchai A, Wongsrichanalai C.

University of California School of Medicine, San Francisco, CA, USA

In Myanmar, we tested two rapid malaria immunochromatographic kits: the OptiMAL assay for the detection of parasite lactate dehydrogenase (pLDH), and the ICT Malaria P.f./P.v. test for histidine-rich protein 2 (PfHRP2) and panmalarial antigens. A total of 229 patients were examined, of whom 133 were found to be malaria positive by Giemsa microscopy. Both OptiMAL and ICT gave lower sensitivities than previously reported. ICT sensitivity for Plasmodium falciparum and non-falciparum parasites were 86.2 and 2.9%, respectively; specificity was 76.9 and 100%, respectively. OptiMAL sensitivity for P. falciparum and non-falciparum parasites were 42.6 and 47.1%, respectively; specificity was 97.0 and 96.9%, respectively. The sensitivity of both tests for the detection of both P. falciparum and non-falciparum parasites increased with parasite density. Several explanations for these results are explored. Our results raise particular concern over batch quality variations of malaria rapid diagnostic devices (MRDDs).

---

Acta Trop 2002 Apr;82(1):11-23

A sero-epidemiological study of malaria in human and monkey populations in French Guiana.

Volney B, Pouliquen JF, De Thoisy B, Fandeur T.

Laboratoire de Parasitologie Moleculaire, Institut Pasteur de Guyane, 97306 Cedex, Cayenne, French Guiana

This paper describes a sero-epidemiological study of malaria prevalence in French Guiana. An immunofluorescence assay and an enzyme-linked immunosorbent assay were used to detect antibodies against blood-stage antigens and synthetic peptides mimicking the repetitive epitope of the sporozoites of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae/brasilianum, in 218 human sera and 113 non-human primate sera collected in French Guiana. Almost all the monkey sera tested had antibodies against malaria blood-stages (98%) and a large majority (73%) also tested positive with the P. malariae/brasilianum circumsporozoite peptide. A number of primate samples also reacted positively with P. falciparum NANP repeats in a very specific manner, suggesting that monkeys in the rainforest are bitten by mosquitoes infected with human malaria parasites. Seroprevalences were lower in the humans tested but Indian tribes on the borders with Suriname and Brazil were clearly more exposed to malaria than other ethnic groups, with a prevalence of nearly 70% seropositivity. P. vivax infections accounted for much of the observed pattern of reactivity, but there was also a high frequency of positive reactions to the P. brasilianum/malariae peptide. Similarly, a large proportion of the sera obtained from Bush Negro populations tested positive for P. malariae/brasilianum repeats. These data add to the emerging evidence that non-human primates might constitute a natural reservoir, not only for simian, but also for human malaria, and therefore suggest that they might be responsible for the maintenance of foci of P. malariae, and possibly of other malaria species, in isolated areas of the Amazonian rainforest.

---

Trop Med Int Health 2002 Mar;7(3):265-70

In vitro activities of ferrochloroquine against 55 Senegalese isolates of Plasmodium falciparum in comparison with those of standard antimalarial drugs.

Pradines B, Tall A, Rogier C, Spiegel A, Mosnier J, Marrama L, Fusai T, Millet P, Panconi E, Trape JF, Parzy D.

Unite de Parasitologie, Institut de Medecine Tropicale du Service de Sante des Armees, Le Pharo, Marseille, France. 
Email: [email protected]

The in vitro activities of ferrochloroquine, chloroquine, quinine, mefloquine, halofantrine, amodiaquine, artesunate, atovaquone, cycloguanil and pyrimethamine were evaluated against Plasmodium falciparum isolates from Senegal (Dielmo, Ndiop), using an isotopic micro-drug susceptibility test. The IC50 values for ferrochloroquine ranged from 0.55 to 28.2 nM and the geometric mean IC50 for the 55 isolates was 7.9 nM (95% CI, 6.5-9.7 nM). Ferrochloroquine was 35 times more active than chloroquine (35-fold greater against chloroquine-resistant isolates), quinine, mefloquine, amodiaquine, cycloguanil and pyrimethamine. Weak positive correlations were observed between the responses to ferrochloroquine and that to chloroquine, quinine, and amodiaquine, but not compulsorily predictive of cross-resistance. There was no significant correlation between the response to ferrochloroquine and that to mefloquine, halofantrine, artesunate, atovaquone, cycloguanil and pyrimethamine. Ferrochloroquine may be an important alternative drug for the treatment of chloroquine-resistant malaria.

---

Trop Med Int Health 2002 Mar;7(3):249-56

Level and dynamics of malaria transmission and morbidity in an equatorial area of South Cameroon.

Bonnet S, Paul RE, Gouagna C, Safeukui I, Meunier JY, Gounoue R, Boudin C.

OCEAC, Yaounde, Cameroon. 
Email: [email protected]

We conducted parasitological and entomological malaria surveys among the population of Mengang district in southern Cameroon to analyse the relationship between malaria transmission intensity and malaria morbidity. We investigated two adjacent areas which differ 10-fold in transmission intensity [annual entomological inoculation rate (EIR) 17 vs. 170], but have very similar Plasmodium falciparum malariometric profiles with parasite prevalences of 58 vs. 64%, high parasitaemia prevalences (> 1000 parasites/microl) of 15 vs. 16% and the same morbidity of 0.17-0.5 attacks/person/year. Plasmodium malariae prevalence was 14 vs. 16%. One possible explanation is that the similarity of the duration of the short and high transmission seasons in both areas is equally, if not more, significant for parasitological and clinical profiles as the annual EIR. We discuss the relationships between variations in transmission levels, parasitaemia and clinical incidence, and draw parallels to similar situations elsewhere.

---

Trop Med Int Health 2002 Mar;7(3):231-9

Wide distribution of Plasmodium ovale in Myanmar.

Win TT, Lin K, Mizuno S, Zhou M, Liu Q, Ferreira MU, Tantular IS, Kojima S, Ishii A, Kawamoto F.

Department of International Health, Nagoya University School of Medicine, Nagoya, Japan.

The presence of Plasmodium ovale has never been previously reported in Myanmar. Using blood samples obtained in many villages across the country between 1996 and 2000, molecular diagnosis of Plasmodium species was made with semi- or full-nested polymerase chain reaction (PCR) with species-specific primers, followed by agarose gel electrophoresis to detect amplification products. The presence of P. ovale was also confirmed with the another PCR-based diagnosis, the microtiterplate hybridization (MPH) method using species-specific probes. Both methods target the A type of the small subunit ribosomal RNA gene of the four human malaria parasites. Plasmodium ovale DNA was amplified in samples from 65 (4.9%) of 1323 PCR-positive patients, with perfect agreement between results obtained by nested PCR and MPH. Only four P. ovale-infected patients had single-species infection; all others were coinfected with P. falciparum, P. vivax and/or P. malariae. Quadruple infections were observed in six subjects. Parasites with typical P. ovale morphology were found in only 19 patients by conventional microscopy of Giemsa-stained thin smears or fluorescence microscopy of acridine orange-stained thin smears. Plasmodium ovale infections were found in villages situated in the southern, central and western regions of Myanmar, suggesting that P. ovale may be widely distributed in this country.

---

Neuropathol Appl Neurobiol 2001 Dec;27(6):421-33

Cellular stress and injury responses in the brains of adult Vietnamese patients with fatal Plasmodium falciparum malaria.

Medana IM, Mai NT, Day NP, Hien TT, Bethell D, Phu NH, Farrar J, White NJ, Turner GD.

Nuffield Department of Clinical Laboratory Sciences, Nuffield Department of Medicine, The John Radcliffe Hospital, Oxford University, UK, Centre for Tropical Diseases and Wellcome Trust Research Unit, Cho Quan Hospital, Ho Chi Minh City, Vietnam.

Immunohistochemical techniques have been used to investigate specific patterns of potentially reversible cellular injury, DNA damage, and apoptosis in the brainstems of Vietnamese patients who died of severe Plasmodium falciparum malaria. The degree and pattern of neuronal and glial stress responses were compared between patients with cerebral and non-cerebral malaria (CM), and appropriate non-malaria infected controls. The following markers were examined: (i) heat shock protein 70 (HSP70), for reversible injury; (ii) heme oxygenase-1, for oxidative stress; (iii & iv) two DNA-repair proteins, poly(ADP) ribose polymerase (PARP) and DNA-dependent protein kinase catalytic subunit; (v) poly(ADP) ribose, an end-product of PARP activity; and (vi) caspase-3-active, for apoptosis. Stress responses were found in a range of cell types as reflected by the widespread expression of HSP70. Oxidative stress predominated in the vicinity of vessels and haemorrhages. Some degree of DNA damage was found in the majority of malaria patients, but the distribution and frequency of the damage was much less than that observed in controls with irreversible neuronal injury. Similarly, caspase-3-active expression, as a measure of apoptosis, was no higher in the majority of malaria patients than the negative control cases, although 40% of CM cases expressed caspase-3-active in a small number of neurones of the pontine nuclei or within swollen axons of the pontocerebellar and corticospinal tracts. In conclusion, cells within the brainstem of all patients who died from severe malaria showed staining patterns indicative of considerable stress response and reversible neuronal injury. There was no evidence for a specific pattern of widespread irreversible cell damage in those patients with cerebral malaria.

---

Insect Mol Biol 2001 Dec;10(6):619-28

Developmental variation in epidermal growth factor receptor size and localization in the malaria mosquito, Anopheles gambiae.

Lycett G, Blass C, Louis C.

Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Crete, Greece; European Molecular Biology Laboratory, Heidelberg, Germany; Department of Biology, University of Crete, Heraklion, Crete, Greece.

The AGER gene encoding the epidermal growth factor receptor (EGFR) of the malaria mosquito Anopheles gambiae was cloned and sequenced. It represents a canonical member of this family of tyrosine kinase proteins exhibiting many similarities to orthologues from other species, both on the level of genomic organization and protein structure. The mRNA can be detected throughout development. Western analysis with an antibody raised against the extracellular domain of the mosquito protein suggests developmental variation in protein size and location that may be involved in the function of EGFR in the mosquito.

---

Insect Mol Biol 2001 Dec;10(6):597-604

Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element.

Grossman GL, Rafferty CS, Clayton JR, Stevens TK, Mukabayire O, Benedict MQ.

Centers for Disease Control and Prevention (CDC), Division of Parasitic Diseases, Entomology Branch, 4770 Buford Highway, Mailstop F-22, Atlanta, GA 30341, USA.

Germline transformation of the major African malaria vector, Anopheles gambiae, was achieved using the piggyBac transposable element marked with the enhanced green fluorescent protein (EGFP) injected into mosquito embryos. Two G1 generation male mosquitoes expressing EGFP were identified among 34 143 larvae screened. Genomic Southern data and sequencing of the piggyBac insertion boundaries showed that these two males arose from one piggyBac insertion event in the injected G0 embryos. Genetic cross data suggest that the insertion site of the element either resulted in, or is tightly linked to, a recessive lethal. This was demonstrated by a deficiency in the number of EGFP-expressing offspring from inbred crosses but expected ratios in outcrosses to non-transformed individuals and failure to establish a pure-breeding line. The insertion was weakly linked to the collarless locus on chromosome 2 and was shown by in situ hybridization to be located in division 28D of that chromosome. Particularly high levels of expression were observed uniformly in salivary glands and, in most individuals, in the anterior stomach. An improvement in the injection technique at the end of the studies resulted in increased G0 hatching, transient expression and EGFP-expression rates among G1 progeny.

---

Heredity 2001 Dec;87(Pt 6):647-58

Genetic differentiation of the malaria vector Anopheles gambiae across Nigeria suggests that selection limits gene flow.

Onyabe DY, Conn JE.

Department of Biology, The University of Vermont, 321 Marsh Life Science Building, Burlington, VT 05405, U.S.A.

Gene flow was investigated in Anopheles gambiae from eight localities that span the ecological zones of Nigeria (arid savanna zones in the north gradually turn into humid forest zones in the south). Genetic differentiation was measured over 10 microsatellite loci and, to determine any effects of selection, five loci were located within chromosome inversions and the other five were outside inversions. Over all loci, the largest estimates of differentiation were in comparisons between localities in the savanna vs. forest zones (range FST 0.024-0.087, Nm 2.6-10.1; RST 0.014-0.100, Nm 2.2-16.4). However, three loci located within inversions on chromosome II, whose frequencies varied clinally from north to south, were responsible for virtually all of the differentiation. When the three loci were removed, genetic distances across the remaining seven loci were markedly reduced even between localities in the forest and savanna zones (range FST 0.001-0.019, Nm 12.7-226.1) or no longer significant (P > 0.05) in the case of RST. Although tests of isolation by distance gave seemingly equivocal results, geographical distance does not appear to limit gene flow. These observations suggest that gene flow is extensive across the country but that selection on genes located within some inversions on chromosome II counters the homogenizing effects of gene flow.

---

Curr Mol Med 2001 Sep;1(4):437-46

Biology of gammadelta T cells in tuberculosis and malaria.

Dieli F, Troye-Blomberg M, Farouk S E, Sirecil G, Salerno A.

Department of Biopathology, University of Palermo, Italy. 
Email: [email protected]

Tuberculosis and malaria remain the leading causes of mortality among human infectious diseases in the world. It is estimated that 3 to 5 million people die from tuberculosis and malaria each year. Although it is traditionally believed that CD4 and CD8 alphabeta T lymphocytes are mandatory for protective immune responses against Mycobacterium tuberculosis and Plasmodium falciparum (the ethiologic agents of tuberculosis and the most severe form of malaria, respectively), there is still incomplete understanding of the mechanisms of immune protection and of the causes of its failure in the affected patients. Several studies in humans and animal models have suggested that Vgamma9/Vdelta2 T cells may play an important role in the immune responses against Mycobacterium tuberculosis and Plasmodium falciparum. Vgamma9/Vdelta2 T cells represent about 75% of all circulating gammadelta T cells while they can be greatly expanded during the acute phase of Mycobacterium tuberculosis and Plasmodium falciparum malaria. Vgamma9/Vdelta2 T recognize a new class of antigenic molecules which are nonpeptidic in nature and contain critical phosphate moieties (phosphoantigens). Interestingly, phosphoantigens isolated from Mycobacterium tuberculosis and Plasmodium falciparum share strong structural homology and are probably identical. However, despite a large body of data reported in the literature, it is not yet clear whether Vgamma9/Vdelta2 T cells play a protective or pathogenic role in immune responses against Mycobacterium tuberculosis and Plasmodium falciparum. In this review we summarize our current knowledge of the biology of Vgamma9/Vdelta2 T cells in response to the two pathogens, Mycobacterium tuberculosis and Plasmodium falciparum, and provide evidence suggesting definition of a novel and important protective role through which Vgamma9/Vdelta2 T cells can contribute to the killing of microorganisms residing in intracellular compartments.

---

J Commun Dis 2001 Mar;33(1):1-6

Eco-epidemiological characteristics of an unstable peri-urban focus of falciparum malaria.

Saxena VK.

National Institute of Communicable Diseases, 22-Sham Nath Marg, Delhi-110054, India.

Two fever surveys were carried out in Shahbad dairy, Delhi in the post-monsoon months of October and November, 1996. Shahbad dairy is a peri-urban locality in the northern periphery of Delhi. The prevalence of fever was found to be 11.6% in October and 2% in November. In the two surveys a total of 21 (28%) fever cases were found infected with malaria (17 with P. falciparum and 4 with P. vivax). The prevalence of malaria in the surveyed population was lower (25%) in October than in November (36.8%). More adults and males suffered from malaria than other age groups and the females. In November the mean asexual parasitaemia, for P. falciparum infection, sharply declined among 5-14-years old children by 68%, but among adults it increased by 32%. Collection of adult mosquitoes, from human houses, revealed the presence of 5 species of anopheline mosquitoes comprising Anopheles culicifacies, An. stephensi, An. annularis, An. subpictus, and An. nigerrimus, and a culicine mosquito Culex vishnui complex. Extensive breeding of these mosquitoes was detected in the vicinity of Shahbad dairy. Characteristic ecological support system (ESS) for malaria transmission was identified at the peri-urban focus of Shahbad dairy.

---

Antimicrob Agents Chemother 2002 Apr;46(4):1059-1066

In Vitro Antiplasmodium Effects of Dermaseptin S4 Derivatives.

Dagan A, Efron L, Gaidukov L, Mor A, Ginsburg H.

Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram 91904, Jerusalem, Israel.

The 13-residue dermaseptin S4 derivative K(4)S4(1-13)a (P) was previously shown to kill intraerythrocytic malaria parasites through the lysis of the host cells. In this study, we have sought peptides that will kill the parasite without lysing the erythrocyte. To produce such peptides, 26 compounds of variable structure and size were attached to the N terminus of P and screened for antiplasmodium and hemolytic activities in cultures of Plasmodium falciparum. Results from this screen indicated that increased hydrophobicity results in amplified antiplasmodium effect, irrespective of the linearity or bulkiness of the additive. However, increased hydrophobicity also was generally associated with increased hemolysis, with the exception of two derivatives: propionyl-P (C3-P) and isobutyryl-P (iC4-P). Both acyl-peptides were more effective than P, with 50% growth inhibition at 3.8, 4.3, and 7.7 &mgr;M, respectively. The antiparasitic effect was time dependent and totally irreversible, implying a cytotoxic effect. The peptides were also investigated in parallel for their ability to inhibit parasite growth and to induce hemolysis in infected and uninfected erythrocytes. Whereas the dose dependence of growth inhibition and hemolysis of infected cells overlapped when cells were treated with P, the acyl-peptides exerted 50% growth inhibition at concentrations that did not cause hemolysis. Noticeably, the acyl derivatives, but not P, were able to dissipate the parasite plasma membrane potential and cause depletion of intraparasite potassium under nonhemolytic conditions. These results clearly demonstrate that the acyl-peptides can affect parasite viability in a manner that is dissociated from lysis of the host cell. Overall, the data indicate the potential usefulness of this strategy for development of selective peptides as investigative tools and eventually as antimalarial agents.

---

Antimicrob Agents Chemother 2002 Apr;46(4):1026-31

Artemisinin pharmacokinetics and efficacy in uncomplicated-malaria patients treated with two different dosage regimens.

Gordi T, Huong DX, Hai TN, Nieu NT, Ashton M.

Division of Pharmacokinetics and Drug Therapy, Faculty of Pharmacy Uppsala University, Uppsala, Sweden. National Institute of Malariology, Parasitology, and Entomology, Hanoi, Vietnam.

The immediate efficacies of two oral dosage regimens of artemisinin were investigated in 77 male and female adult Vietnamese falciparum malaria patients randomly assigned to treatment with either 500 mg of artemisinin daily for 5 days (group A; n = 40) or artemisinin at a dose of 100 mg per day for 2 days, with the dose increased to 250 mg per day for 2 consecutive days and with a final dose of 500 mg on the fifth day (group B; n = 37). Parasitemia was monitored every 4 h. The average parasite clearance time was longer in group B than in group A (means +/- standard deviations, 50 +/- 23 and 34 +/- 14 h, respectively; P < 0.01). Artemisinin concentrations in saliva samples obtained on days 1 and 5 were quantified by high-performance liquid chromatography. The average oral clearance, based on saliva drug concentrations in group B patients, was twofold higher than that in group A patients on day 1 (P < 0.01), with no differences in drug half-lives (P = 0.40), indicating a saturable first-pass metabolism. Female patients had higher oral clearance values on day 1. Artemisinin’s pharmacokinetic parameters were similar on day 5 in both groups, although a significant increase in oral clearance from day 1 to day 5 was evident. Thus, artemisinin exhibited both dose- and time-dependent pharmacokinetics. The escalating dose studied did not result in higher artemisinin concentrations toward the end of the treatment period.

---

Antimicrob Agents Chemother 2002 Apr;46(4):958-65

Malaria parasites giving rise to recrudescence in vitro.

Nakazawa S, Maoka T, Uemura H, Ito Y, Kanbara H.

Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523. Japan Foundation of Research Institute for Production Development, Shimogamo-Morimoto 15, Kyoto 606-0805, Japan.

Recrudescences were simulated in vitro with drug treatment to examine how drug-sensitive parasites survive the treatment. Various numbers of cultured parasites were treated with lethal doses of pyrimethamine or mefloquine for various lengths of time. Recrudescences were observed in parasite populations with larger initial numbers of parasites when the treatment duration was prolonged. Equal numbers of parasitized erythrocytes were treated with various concentrations of pyrimethamine or mefloquine. There was no clear linear relationship between the incidence of recrudescence and the drug concentration. Parasites that had recrudesced were continuously allowed to recrudesce in the succeeding recrudescence experiments. Both the duration from the cessation of treatment to the time at which the recrudescent parasitemia level reached 1% and the growth rate of recrudescent parasites were equal among these recrudescences. The recrudescent parasites in these experiments were as sensitive to the drugs as the parasites tested before treatment were. These results suggest that a parasite culture may contain parasites in some phases that are not killed by drug for up to 10 days, which explains the recrudescences that occur even after treatment.

---

Mol Biochem Parasitol 2002 Apr 9;120(2):215-24

The Plasmodium vivax homologues of merozoite surface proteins 4 and 5 from Plasmodium falciparum are expressed at different locations in the merozoite.

Black CG, Barnwell JW, Huber CS, Galinski MR, Coppel RL.

Department of Microbiology, Monash University, PO Box 53, 3800 Vic., Calyton, Australia

Merozoite surface proteins of Plasmodium falciparum are one major group of antigens currently being investigated and tested as malaria vaccine candidates. Two recently described P. falciparum merozoite surface antigens, MSP4 and MSP5, are GPI-anchored proteins that each contain a single EGF-like domain and appear to have arisen by an ancient gene duplication event. The genes are found in tandem on chromosome 2 of P. falciparum and the syntenic region of the genome was identified in the rodent malarias P. chabaudi, P. yoelii and P. berghei. In these species, there is only a single gene, designated MSP4/5 encoding a single EGF-like domain similar to the EGF-like domain in both PfMSP4 and PfMSP5. Immunization of mice with PyMSP4/5 provides mice with high levels of protection against lethal challenge with blood stage P. yoelii. In this study, we show that in P. vivax, which is quite phylogenetically distant from P. falciparum, both MSP4 and MSP5 homologues can be found with their relative arrangements with respect to the surrounding genes mostly preserved. However, the gene for MSP2, found between MSP5 and adenylosuccinate lyase (ASL) in P. falciparum, is absent from P. vivax. The PvMSP4 and PvMSP5 genes have a two-exon structure and encode proteins with potential signal and GPI anchor sequences and a single EGF-like domain near the carboxyl-terminus. Rabbit antisera raised against purified recombinant proteins show that each of the antisera react with distinct proteins of 62 kDa for PvMSP4 and 86 kDa for PvMSP5 in parasite lysates. Indirect immunofluorescence assays (IFA) localized PvMSP4 over the entire surface of P. vivax merozoites, as expected, whereas, the MSP5 homologue was found to be associated with an apical organellar location consistent with micronemes or over the polar prominence.

---

Mol Biochem Parasitol 2002 Apr 9;120(2):177-86

Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA).

Li J, Matsuoka H, Mitamura T, Horii T.

Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita, 565-0871, Osaka, Japan

The Plasmodium falciparum serine repeat antigen (SERA), a malaria vaccine candidate, is processed into several fragments (P73, P47, P56, P50, and P18) at the late schizont stage prior to schizont rupture in the erythrocytic cycle of the parasite. We have established an in vitro cell-free system using a baculovirus-expressed recombinant SERA (bvSERA) that mimics the SERA processing that occurs in parasitized erythrocytes. SERA processing was mediated by parasite-derived trans-acting proteases, but not an autocatalytic event. The processing activities appeared at late schizont stage. The proteases are membrane associated, correlating with the secretion and accumulation of SERA within the parasitophorous vacuole membrane (PVM). The activity responsible for the primary processing step of SERA to P47 and P73 was inhibited by serine protease inhibitor DFP. In contrast, the activity responsible for the conversion of P56 into P50 was inhibited by each of the cysteine protease inhibitors E-64, leupeptin and iodoacetoamide. Moreover, addition of DFP, E-64 or leupeptin to the cultures of schizont-stage parasites blocked schizont rupture and release of merozoites from PVM. These results indicate that SERA processing correlates to schizont rupture and the processing is mediated by at least three distinct proteases.

---

Infect Immun 2002 Apr;70(4):2049-205

Susceptibility to Experimental Cerebral Malaria Induced by Plasmodium berghei ANKA in Inbred Mouse Strains Recently Derived from Wild Stock.

Bagot S, Idrissa Boubou M, Campino S, Behrschmidt C, Gorgette O, Guenet JL, Penha-Goncalves C, Mazier D, Pied S, Cazenave PA.

Unite d’Immunophysiopathologie Infectieuse, CNRS URA 1961, LEA14C, and Universite Pierre et Marie Curie. Unite de Genetique des Mammiferes, Institut Pasteur, 75724 Paris Cedex 15. Unite 511, INSERM, Immunobiologie Cellulaire et Moleculaire des Infections Parasitaires, CHU Pitie-Salpetriere, 75643 Paris Cedex 13, France and. Instituto Gulbenkian de Ciencia, 2780-156 Oeiras, Portugal.

The neurological syndrome caused by Plasmodium berghei ANKA in rodents partially mimics the human disease. Several rodent models of cerebral malaria (CM) exist for the study of the mechanisms that cause the disease. However, since common laboratory mouse strains have limited gene pools, the role of their phenotypic variations causing CM is restricted. This constitutes an obstacle for efficient genetic analysis relating to the pathogenesis of malaria. Most common laboratory mouse strains are susceptible to CM, and the same major histocompatibility complex (MHC) haplotype may exhibit different levels of susceptibility. We analyzed the influence of the MHC haplotype on overcoming CM by using MHC congenic mice with C57BL/10 and C3H backgrounds. No correlation was found between MHC molecules and the development of CM. New wild-derived mouse strains with wide genetic polymorphisms were then used to find new models of resistance to CM. Six of the twelve strains tested were resistant to CM. For two of them, F(1) progeny and backcrosses performed with the reference strain C57BL/6 showed a high level of heterogeneity in the number and characteristics of the genetic factors associated with resistance to CM.

---

IUBMB Life 2001 Dec;52(6):285-9

Novel approaches to tackling malarial drug resistance using yeast.

Sibley C H, Macreadie I.

Department of Genetics, University of Washington, Seattle, USA.

Yeasts have a justified reputation as one of the world’s most versatile organisms. Baker’s yeast continues to live up to this recognition by joining the war against malaria. Yeast can now be used to study antifolate drug resistance patterns that depend on the dihydrofolate reductase enzyme (DHFR) from the malaria parasite.