Links to Full-text Journals & Reports

• Malaria Journal:  an online journal published by BioMed Central 
• USAID NetMark baseline survey on insecticide treated materials (ITMs) : cross-national summary of findings, 2001.
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• USAID NetMark baseline survey on insecticide treated materials (ITMs) in Zambia, 2001. 
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• USAID NetMark baseline survey on insecticide treated materials (ITMs) in Uganda, 2001.
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Social Sciences and Malaria

Tropical Medicine and International Health 2001, 6/11 (845-848) 

History and importance of antimalarial drug resistance

D’Alessandro U.; Buttiens H.
Prince Leopold Inst. Tropical Med., Nationalestraat 155, 2000 Antwerp  Belgium  

The emergence of Plasmodium falciparum resistance to widely used antimalarial drugs such as chloroquine (CQ) has made malaria control and treatment much more difficult. This is particularly dramatic for Africa, as few affordable alternatives are available. Drug pressure has been identified as one of the key factors for the emergence and spread of resistance. The contribution of the extensive use and misuse of antimalarial drugs to the selection of resistant parasites became particularly evident during the Global Malaria Eradication campaign, launched by World Health Organization (WHO) in 1955. The first reports confirming P. falciparum resistance to CQ came almost simultaneously in the early 1960s from South America and South-East Asia, where direct or indirect (through use of medicated cooking salt) mass drug administration (MDA) had been implemented. Similar approaches were very limited in Africa, where P. falciparum resistance to CQ was first reported from the eastern region in the late 1970s and spread progressively west. Most African countries still rely heavily on CQ as first-line treatment despite various levels of resistance, although some states have changed to sulphadoxine-pyrimethamine (SP) as the first-line drug. Unfortunately, the predicted SP useful therapeutic life might be very short, probably because of its prolonged half-life, causing a higher probability of selecting resistant strains and a consequent fast development of resistance. CQ resistance is not evenly distributed and important differences can be found within and between countries. It seems to have spread more rapidly in East than in West Africa. Considering the high level of CQ use in West Africa, other factors such as intensity of transmission, population immunity or population movements should be considered when explaining the different levels of resistance. Understanding such factors may help us in devising strategies to contain the spread of drug resistance. 

Bull World Health Organ 2001;79(11):1014-1023

Malaria control in Bungoma District, Kenya: a survey of home treatment of children with fever, bednet use and attendance at antenatal clinics.

Hamel MJ, Odhacha A, Roberts JM, Deming MS.

International Child Survival and Emerging Infections Program Support Office, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, United States Department of Health and Human Services, Atlanta, Georgia, USA.

OBJECTIVE: To lay the basis for planning an improved malaria control programme in Bungoma District, Kenya. METHODS: By means of a cluster sample household survey an investigation was conducted into the home management of febrile children, the use of bednets, and attendance at antenatal clinics. FINDINGS: Female carers provided information on 314 recently febrile children under 5 years of age, of whom 43% received care at a health facility, 47% received an antimalarial drug at home, and 25% received neither. Of the antimalarial treatments given at home, 91% were started by the second day of fever and 92% were with chloroquine, the nationally recommended antimalarial at the time. The recommended dosage of chloroquine to be administered over three days was 25 mg/kg but the median chloroquine tablet or syrup dosage given over the first three days of treatment was 15 mg/kg. The total dosages ranged from 2.5 mg/kg to 82 mg/kg, administered over one to five days. The dosages were lower when syrup was administered than when tablets were used. Only 5% of children under 5 years of age slept under a bednet. No bednets had been treated with insecticide since purchase. At least two antenatal visits were made by 91% of pregnant women. CONCLUSIONS: Carers are major and prompt providers of antimalarial treatment. Home treatment practices should be strengthened and endorsed when prompt treatment at a health facility is impossible. The administration of incorrect dosages, which proved common with chloroquine, may occur less frequently with sulfadoxine-pyrimethamine, as its dosage regimen is simpler. High levels of utilization of antenatal clinics afford the opportunity to achieve good coverage with presumptive intermittent malaria treatments during pregnancy, and to reach the goal of widespread bednet use by pregnant women and children by distributing nets during antenatal clinic visits.

PubMed

Protoplasma 2001;217(1-3):43-9

Thioredoxin and glutathione system of malaria parasite Plasmodium falciparum.

Muller S, Gilberger TW, Krnajski Z, Luersen K, Meierjohann S, Walter RD.

Department of Parasite Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Federal Republic of Germany.

Plasmodium falciparum is the causative agent of malaria tropica. Due to the increasing resistance towards the commonly used plasmodicidal drugs there is an urgent need to identify and assess new targets for the chemotherapeutic intervention of parasite development in the human host. It is established that P. falciparum-infected erythrocytes are vulnerable to oxidative stress, and therefore efficient antioxidative systems are required to ensure parasite development within the host cell. The thioredoxin and glutathione redox systems represent two powerful means to detoxify reactive oxygen species and this article summarizes some of the recent work which has led to a better understanding of these systems in the parasite and will help to assess them as potential targets for the development of new chemotherapeutics of malaria. 

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Ann Trop Paediatr 2001 Dec: 21(4): 307-312

Treatment of falciparum malaria in Vietnamese children: the need for combination therapy and optimized dosage regimens.

Trung TN, Davis TM, Hewitt S, Thuan LK, Quang HH, Anh CV, Thuy PT, Thoa NT, Tuan NT, Hang NT, Giang LT.

Institute of Malariology, Parasitology and Entomology, Quy Nhon City, Binh Dinh Province, Viet Nam.

To assess the in vivo sensitivity of Plasmodium falciparum to mefloquine and artesunate in a hyperendemic area of southern Viet Nam, we studied 41 children and 21 adults from a remote commune who had uncomplicated falciparum malaria without previous treatment. Patients were randomly allocated to artesunate (4 mg/kg on day 0 and 2 mg/kg on days 1-4) or mefloquine (10 mg/kg followed by 5 mg/kg at 6 h). Serial assessments were performed over 28 days. Of 31 patients allocated artesunate, nine (29%) redeveloped parasitaemia during follow-up compared with 23% (seven of 30) who received mefloquine. Of the 41 children, 15 (37%) had recrudescence/re-infection compared with only one of 20 adults (5%; p < 0.001). Significantly more children than adults failed on mefloquine treatment (37% vs 0%; p = 0.021) and one case showed RIII resistance. There was no significant difference in the case of artesunate. In regression analysis, parasitaemia was an independent predictor of recrudescence/re-infection after mefloquine (p = 0.02). These data support the use of combination therapy such as artesunate plus mefloquine for falciparum malaria in a hyperendemic area of Viet Nam. Primarily because of their greater parasite densities, children should be given higher doses of mefloquine (e.g. 25 mg/kg). 

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Med J Malaysia 2001 Sep;56(3):271-4

Clinical features of malaria in Orang Asli population in Pos Piah, Malaysia.

Norhayati M, Rohani AK, Hayati MI, Halimah AS, Sharom MY, Abidin AH, Fatmah MS.

Department of Parasitology and Medical Entomology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur.

A malaria survey was conducted to examine the presence of common clinical features of malaria in individuals living in an endemic area of malaria. The overall infection rate was 11.0% with 7.5% and 3.5% infected with Plasmodium vivax and Plasmodium falciparum respectively. The mean parasitaemia level of both species was 2905.9 parasites/microliter blood, with the mean parasitaemia level of P. vivax and P. falciparum at 682.7 parasites/microliter blood and 6981.7 parasites/microliter blood respectively. The infection rates were higher in the younger age group. Hepatomegaly, hepatosplenomegaly and clinical anaemia were significantly associated with malaria. None of the patients were febrile. In conclusion, in low endemic areas, the presence of clinical anaemia, hepatomegaly and hepatosplenomegaly in afebrile individuals could be considered as useful criteria for the presence of asymptomatic parasitaemia. It is important to carry out laboratory diagnostic investigations, to ensure all the asymptomatic parasitaemia which act as reservoirs are detected and treated. 

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J Biol Chem 2001 Nov 29;  Free full-text article at http://www.jbc.org

A single, bi-functional aquaglyceroporin in blood-stage Plasmodium falciparum malaria parasites.

Hansen M, Kun J, Beitz E.

Department of Pharmaceutical Chemistry, University of Tbingen, Tbingen 72076.

The malaria parasite Plasmodium falciparum faces drastic osmotic changes during kidney passages and is engaged in the massive biosynthesis of glycerolipids during its development in the blood-stage. We identified a single aquaglyceroporin (PfAQP) in the nearly finished genome of P. falciparum with highest similarity to the E. coli glycerol facilitator (50.4%), but both canonical asn-pro-ala (NPA) motifs in the pore region are changed to asn-leu-ala (NLA) and asn-pro-ser (NPS), respectively. Expression in Xenopus oocytes renders them highly permeable for both, water and glycerol. Sugar alcohols up to five carbons and urea pass the pore. Mutation analyses of the NLA / NPS motifs showed their structural importance but the symmetrical pore properties were maintained. PfAQP is expressed in blood-stage parasites throughout the development from rings via trophozoites to schizonts and is localized to the parasite but not to the erythrocyte cytoplasm or membrane. Its unique bi-functionality indicates functions in the protection from osmotic stress and efficiently provides access to the serum glycerol pool for the use in ATP generation and primarily in the phospholipid synthesis. 

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Scand J Infect Dis 2001;33(10):752-4

Evaluation of a dipstick test for the rapid diagnosis of imported malaria among patients presenting within the network TropNetEurop.

Jelinek T, Grobusch MP, Harms G.

Department of Infectious Diseases and Tropical Medicine, University of Munich, Germany. [email protected]

Lack of experience on the part of involved laboratory personnel frequently complicates swift diagnosis of imported falciparum malaria in non-endemic areas. Diagnostic tools based on the dipstick principle for the detection of plasmodial histidine-rich protein 2 have been marketed for several years and have been extensively evaluated. Recently, a test kit capable of detecting antigen of Plasmodium falciparum and P. vivax has been introduced. In order to evaluate this newly available tool, specimens from 664 patients were screened during the course of a prospective multicentre study within the European Network on Imported Infectious Disease Surveillance (TropNetEurop). Among the screened specimens, samples from 82 patients (12.3%) were positive for falciparum malaria using expert microscopy. A further 17 samples were positive for vivax malaria. The evaluated test kit performed with a sensitivity of 87.8% and a specificity of 99% for detection of falciparum malaria. Respective values for vivax malaria were 76.5% and 100%. Dipstick tests have the potential of improving the speed and accuracy of the diagnosis of falciparum malaria, especially if non-specialized laboratories are involved. However, decreased values of sensitivity and specificity, in comparison with expert microscopy, still impose a clear limit on the usefulness of the currently available kits. 

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Expert Opin Biol Ther 2001 Jul;1(4):619-28

Current developments in malaria transmission-blocking vaccines.

Stowers A, Carter R.

Malaria Vaccine Development Unit, Laboratory of Parasitic Diseases, NIAID/NIH, 5640 Fishers Lane, Rockville, MD 20852, USA.

Malaria is still a leading cause of morbidity and mortality in human populations. Problems, including drug-resistant parasites and insecticide resistant mosquitoes, ensure the continued hold of malaria in the tropics and sub-tropics. Each year around 100 million cases of malaria result in at least 50,000 deaths outside of sub-Saharan Africa; within sub-Saharan Africa itself, malaria causes around one million child deaths per year. New approaches for malaria control are badly needed and much effort has gone to develop malaria vaccines. In addition to giving personal protection, most such vaccines would also tend to reduce the transmission of malaria. One class of vaccine is being developed specifically for this purpose–the malaria transmission-blocking vaccines (TBV). TBVs are based upon antigens expressed on the surface of the sexual and mosquito mid-gut stages of malaria parasites. These antigens are the targets of antibodies induced by vaccination of the host and ingested with the parasites in a mosquito blood meal. The antibodies act by inhibiting the parasite’s development within the mosquito itself and they thereby prevent the onward transmission of the parasites. TBVs could contribute to the total interruption of malaria transmission in many locations with relatively low transmission rates, mostly outside sub-Saharan Africa. Under almost all transmission rates, however, TBVs would help reduce malaria incidence and malaria-related morbidity and mortality. Promising recombinant TBV candidate antigens for the two main human malaria parasite species, Plasmodium falciparum and Plasmodium vivax, have been produced and tested in the laboratory; one has undergone early clinical trials. 

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Proc Natl Acad Sci U S A 2001 Dec 4;98(25):14693-14697

Candidate odorant receptors from the malaria vector mosquito Anopheles gambiae and evidence of down-regulation in response to blood feeding.

Fox AN, Pitts RJ, Robertson HM, Carlson JR, Zwiebel LJ.

Department of Biological Sciences, Program in Developmental Biology and Center for Molecular Neuroscience, Vanderbilt University, Nashville, TN 37235; Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; and Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520.

Olfaction plays a major role in host preference and blood feeding, integral behaviors for disease transmission by the malaria vector mosquito Anopheles gambiae sensu stricto (henceforth A. gambiae). We have identified four genes encoding candidate odorant receptors from A. gambiae that are selectively expressed in olfactory organs, contain approximately seven transmembrane domains, and show significant similarity to several putative odorant receptors in Drosophila melanogaster. Furthermore, one of the putative A. gambiae odorant receptors exhibits female-specific antennal expression and is down-regulated 12 h after blood feeding, a period during which substantial reduction in olfactory responses to human odorants has been observed. Taken together, these data suggest these genes encode a family of odorant receptors in A. gambiae, whose further study may aid in the design of novel antimalarial programs. 

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Am J Clin Nutr 2001 Dec;74(6):776-82

Prevalence of iron deficiency with and without concurrent anemia in population groups with high prevalences of malaria and other infections: a study in Cote d’Ivoire.

Asobayire FS, Adou P, Davidsson L, Cook JD, Hurrell RF.

Laboratory of Human Nutrition, the Institute of Food Science, Swiss Federal Institute of Technology, Ruschlikon, Switzerland.

BACKGROUND: Iron deficiency is highly prevalent in most developing countries. However, its detection is often obscured by infections and inflammatory disorders that are common in the same populations. OBJECTIVE: The aim of this study was to estimate the prevalence of iron deficiency with or without concurrent anemia in different population groups from Cote d’Ivoire and to evaluate the influence of infectious and inflammatory disorders on iron-status indexes. DESIGN: Blood samples from 1573 children, women, and men were analyzed for hemoglobin, serum ferritin, zinc protoporphyrin, and serum transferrin receptor. C-reactive protein was used as the indicator of inflammation or infection, and samples were screened for malarial parasites and hemoglobinopathies. Iron deficiency was defined as 2 of 3 iron-status indexes outside the cutoff values, and iron deficiency anemia (IDA) was defined as iron deficiency with concurrent anemia. Pearson’s correlation coefficients were used to evaluate the influence of malaria and inflammation on iron-status indexes. RESULTS: The prevalence of iron deficiency was 41-63% in the women and children and 13% in the men, whereas the prevalence of IDA was 20-39% in the women and children and 4% in the men. The detection of iron deficiency and IDA was obscured by the high prevalence of inflammatory disorders. CONCLUSIONS: Iron deficiency and IDA are highly prevalent in the women and children in Cote d’Ivoire. Iron deficiency was detected in approximately 50% of anemic women and children, which indicates that hemoglobin alone is not a good indicator of iron status when inflammatory disorders are highly prevalent. The serum transferrin receptor is the most useful single indicator of iron deficiency because it was the only iron-status index unaffected by malaria or inflammation. 

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Am J Clin Nutr 2001 Dec;74(6):767-75

Serum transferrin receptor concentration indicates increased erythropoiesis in Kenyan children with asymptomatic malaria.

Verhoef H, West CE, Ndeto P, Burema J, Beguin Y, Kok FJ.

Division of Human Nutrition and Epidemiology, Wageningen University, Wageningen, The Netherlands.

BACKGROUND: Serum transferrin receptor concentrations indicate both erythropoietic activity and the deficit of functional iron in the erythron. In contrast with serum ferritin concentrations, serum transferrin receptor concentrations are not or are only marginally influenced by the inflammatory response to infection. OBJECTIVE: We assessed iron status and examined the relation between serum transferrin receptor concentrations and malaria in children aged 2-36 mo who were asymptomatic for malaria. DESIGN: This was a community-based cluster survey (n = 318). RESULTS: Prevalences of malaria, anemia (hemoglobin concentration <110 g/L), iron deficiency (serum ferritin concentration <12 microg/L), and iron deficiency anemia were 18%, 69%, 53%, and 46%, respectively. Malaria was associated with lower mean hemoglobin concentrations (92.7 compared with 104.1 g/L; P = 0.0001) and higher geometric mean serum concentrations of transferrin receptor (11.4 compared with 7.8 mg/L; P = 0.005), ferritin (21.6 compared with 11.9 microg/L; P = 0.05), and C-reactive protein (12.5 compared with 6.8 mg/L; P = 0.004). There was no evidence for an association between serum concentrations of C-reactive protein and transferrin receptor. Children with malaria had higher serum transferrin receptor concentrations than expected for the degree of anemia, even after adjustment for inflammation indicated by serum C-reactive protein concentration quartiles (P = 0.02). CONCLUSIONS: Our findings are consistent with the notion that malaria-induced hemolysis is accompanied by increased erythropoiesis. Serum transferrin receptor concentration is not useful for detecting iron deficiency in individuals with malaria. Individuals with high concentrations of serum C-reactive protein or similar acute phase reactants should be excluded from analysis if serum ferritin concentrations <12 microg/L are to be used to measure iron deficiency in malaria-endemic areas. 

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Gene 2001 Nov 14;279(1):41-8

Limited recombination events in merozoite surface protein-1 alleles of Plasmodium falciparum on islands.

Sakihama N, Kaneko A, Hattori T, Tanabe K.

Laboratory of Biology, Osaka Institute of Technology, Ohmiya, Asahi-ku, 535-8585, Osaka, Japan

Intragenic recombination is a principal mechanism for the generation of allelic variation in the merozoite surface protein-1 gene (Msp-1) of the human malaria parasite Plasmodium falciparum. In the present study, linkage disequilibrium between the 5′- and 3′-polymorphic sites was analyzed to determine the frequency of recombination events in Msp-1 in parasite populations on four islands in Vanuatu, the southwestern Pacific, where malaria transmission is moderate and comparable to other mesoendemic areas. Of 141 isolates, whose 5′-haplotypes (Msp-1 blocks 2-6) were determined by PCR-based typing, 138 were successfully sequenced for the 3′-polymorphism (block 17). A total of four distinct 5′-haplotypes and three distinct 3′-sequence types were identified with apparently different frequency distribution among islands. The number of 5′-haplotypes in each island was one to four, far smaller than in other previously studied geographic areas (ten to 21). Associations between the 5′- and 3′-polymorphisms (here termed Msp-1 gene types) were subjected to the R(2) linkage disequilibrium test. The test revealed complete or very strong linkage disequilibrium in all four islands. Mixed infection was unusually rare (2.1%) and the mean number of Msp-1 alleles per person was nearly 1.0. The heterozygosity of the Msp-1 gene type calculated for each island (h=0.41-0.65) was significantly lower than that in other areas of comparable endemicity (h=0.81-0.89) (P<0.01). These results indicate that recombination events in Msp-1 would be extremely limited in Vanuatu, and stress that the frequency of recombination in Msp-1 is determined by not only the intensity of malaria transmission but the frequency of mixed clone infections, the mean number of clones per person and a repertoire of clones in a local area.

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Life Sci 2001 Oct 26;69(23):2725-33

In vitro beta-hematin formation assays with plasma of mice infected with Plasmodium yoelii and other parasite preparations: comparative inhibition with quinoline and endoperoxide antimalarials.

Tripathi AK, Gupta A, Garg SK, Tekwani BL.

Division of Biochemistry, Central Drug Research Institute, Lucknow, India.

Formation of beta-hematin in vitro could be catalyzed in the presence of various preparations related to the malaria parasite viz., the cell free homogenate of Plasmodium yoelii, lipid extract of the parasite homogenate, purified malarial hemozoin and synthetic beta-hematin. Plasma from mice infected with P. yoelii also catalyzed in vitro beta-hematin formation with highly significant efficiency. The plasma based beta-hematin formation assay was highly sensitive, as the background absorbance was almost negligible due to absence of any preformed hemozoin. The plasma beta-hematin synthesizing activity was recovered in the lipid extract. The quinoline and endoperoxide antimalarials act by inhibiting hemozoin biosynthesis in the malaria parasite. Therefore, the in vitro beta-hematin formation assay is useful for the screening and identification of blood schizontocidal antimalarials acting through interruption of heme detoxification in the parasite. Quinoline and endoperoxide antimalarials showed about three fold greater inhibition of beta-hematin synthesizing activity in the plasma-based assays as compared to that of P. yoelii homogenate-based assays. The specificity of the inhibition was similar in both preparations. The plasma-based assay therefore provides a better alternative than the parasite homogenate-based assay for in vitro screening and identification of novel inhibitors of hemozoin biosynthesis as potential blood schizontocidal antimalarials. 

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Blood 2001 Dec 1;98(12):3489-91

The association of the glycophorin C exon 3 deletion with ovalocytosis and malaria susceptibility in the Wosera, Papua New Guinea.

Patel SS, Mehlotra RK, Kastens W, Mgone CS, Kazura JW, Zimmerman PA.

Division of Geographic Medicine, Case Western Reserve University, and University Hospitals of Cleveland, Cleveland, OH; and Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea.

Erythrocyte polymorphisms, including ovalocytosis, have been associated with protection against malaria. This study in the Wosera, a malaria holoendemic region of Papua New Guinea, examined the genetic basis of ovalocytosis and its influence on susceptibility to malaria infection. Whereas previous studies showed significant associations between Southeast Asian ovalocytosis (caused by a 27- base pair deletion in the anion exchanger 1 protein gene) and protection from cerebral malaria, this mutation was observed in only 1 of 1019 individuals in the Wosera. Polymerase chain reaction strategies were developed to genotype individuals for the glycophorin C exon 3 deletion associated with Melanesian Gerbich negativity (GPCDeltaex3). This polymorphism was commonly observed in the study population (GPCDeltaex3 frequency = 0.465, n = 742). Although GPCDeltaex3 was significantly associated with increased ovalocytosis, it was not associated with differences in either Plasmodium falciparum or P vivax infection measured over the 7-month study period. Future case-control studies will determine if GPCDeltaex3 reduces susceptibility to malaria morbidity. (Blood. 2001;98:3489-3491) 

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Parasitol Int 2001 Nov;50(4):235-9

Association of adhesion molecule PECAM-1/CD31 polymorphism with susceptibility to cerebral malaria in Thais.

Kikuchi M, Looareesuwan S, Ubalee R, Tasanor O, Suzuki F, Wattanagoon Y, Na-Bangchang K, Kimura A, Aikawa M, Hirayama K.

Department of Medical Zoology, Saitama Medical School, Moroyrma, Iruma, 350-0495, Saitama, Japan

Adhesion molecules on endothelial cells are known to be important ligands for malaria infected red blood cells (PRBC) [Mol Biochem Parasitol, 76, (1996) 1], and may be involved in the pathogenic process of cerebral malaria (CM) which is the most serious complication of falciparum malaria, through enhancing micro embolism or sequestration in the capillaries of the brain. PECAM-1/CD31 is one of these candidate ligands and is coded by a polymorphic gene. Two hundred and ten Thai malaria patients (43 cerebral, 89 severe and 78 uncomplicated) were analyzed for their genetic polymorphism of CD31 to examine the clinical relationship between the disease and specific genotypes. Four alleles were defined 125 valine (V)-563 asparagine (N); 125V-563 serine (S); 125 leucine (L)-563N; and 125L-563S. We found that the frequency of the 125 V/V 563 N/N genotype was significantly high in CM patients as compared with severe cases without CM (P<0.01, OR=2.92), suggesting that this genotype is one of the risk factors for CM. 

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Can J Microbiol 2001 Oct;47(10):903-7

Comparison of a nested polymerase chain reaction–restriction fragment length polymorphism method, the PATH antigen detection method, and microscopy for the detection and identification of malaria parasites.

Parkes R, Lo T, Wong Q, Isaac-Renton JL, Byrne SK.

British Columbia Centre for Disease Control, Vancouver, Canada.

A nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, the PATH antigen detection method, and light microscopy were compared for their capacity to detect and identify Plasmodium species. One hundred and thirty-six blood specimens obtained from patients suspected of having malaria were examined by each of the three methods. Forty-four specimens were positive for malaria using microscopy as the “gold standard”. The sensitivity for nested PCR was 100%, and the specificity was 98%. For the detection of Plasmodium falciparum, the antigen detection method had a sensitivity of 100% and a specificity of 97%. Species identification obtained using PCR-RFLP was identical or superior to light microscopy in 42 cases (96%). Although the nested PCR-RFLP method was more sensitive and specific, the rapid turnaround time and high sensitivity of the antigen detection method makes it a useful adjunct to standard microscopy.

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Am J Trop Med Hyg 2001 Nov;65(5):644-7

Association of helminth infections with increased gametocyte carriage during mild falciparum malaria in Thailand.

Nacher M, Singhasivanon P, Silachamroon U, Treeprasertsu S, Krudsood S, Gay F, Mazier D, Looareesuwan S.

Unite Institut National de la Sante et de la Recherche Medicale 511, Immunobiologie Cellulaire et Moleculaire des Infections Parasitaires, Faculte de Medecine Pitie-Salpetriere, Paris, France.

The objective of this study was to determine whether pre-existing helminth infections could affect sexual forms of Plasmodium falciparum. A cross-sectional case record study compared 120 mild P. falciparum malaria cases with patent gametocyte carriage and 187 without gametocytes for helminth exposure. Relevant crude odds ratios and potential confounders were included in a logistic regression model. Helminth infections were associated with the presence of gametocytes with a crude odds ratio of 1.9 (95% confidence interval = 1.1-3.3) (P = 0.01). A positive linear trend was observed between the odds of having patent gametocytemia and the number of different helminth species (P = 0.003). However, when adjusting for hemoglobin concentration the significance of the association between helminths and gametocytes disappeared (P = 0.15). Pre-existing helminth infections may increase the severity of malarial anemia and therefore increase the likelihood of carrying gametocytes. At a population level, helminth infections may thus have a significant influence on malaria transmission. 

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Am J Trop Med Hyg 2001 Nov;65(5):639-43

Association of splenomegaly with cerebral malaria and decreased concentrations of reactive nitrogen intermediates in Thailand.

Nacher M, Singhasivanon P, Treeprasertsuk S, Chantachum Y, Vannaphan S, Traore B, Gay F, Looareesuwan S.

Institut National de la Sante et de la Recherche Medicale Unite 511, Immunobiologie Cellulaire et Moleculaire des Infections Parasitaires, Faculte de Medecine Pitie-Salpetriere, Paris, France. [email protected]

The role of the spleen during Plasmodium falciparum malaria in humans is unclear. In Thailand, malaria transmission is low and splenomegaly is rarer than in high transmission areas. We compared the prevalence of splenomegaly between 52 cerebral malaria patients and 191 patients without complications despite a high parasite biomass. We also measured concentrations of reactive nitrogen intermediates (RNIs) in a fraction of these cases recruited in 1998 (24 cerebral malaria and 56 controls). Splenomegaly was significantly associated with cerebral malaria (adjusted odds ratio = 2.07 [95% confidence interval = 1-4.2]; P = 0.048). There was a linear trend for this association (P = 0.0003). After adjusting for potential confounders, concentrations of RNIs were significantly lower in the presence of splenomegaly (P = 0.01). These results suggest that in humans, as in animal models, the spleen may be involved in the pathogenesis of cerebral malaria. The relationship between RNI concentrations and the spleen suggest that nitric oxide may have a regulating role in the complex physiology of the spleen during malaria. 

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Am J Trop Med Hyg 2001 Nov;65(5):637-8

Case report: Retinopathy after malaria prophylaxis with chloroquine.

Bertagnolio S, Tacconelli E, Camilli G, Tumbarello M.

Department of Infectious Diseases, Catholic University, Rome, Italy.

Chloroquine retinopathy (CR) is a major complication of long-term malaria prophylaxis (LTMP) causing permanent visual dysfunction and occasionally blindness. After an extensive review of the published accounts of CR, we concluded that the risk of retinopathy in subjects receiving LTMP is limited to a cumulative dose that does not exceed 140 g. We present a case of CR that occurred after 8 years of malaria prophylaxis with chloroquine at a cumulative dose of 125 g. Because a threshold dose of chloroquine for retinal toxicity has not been established, careful, ongoing screening is required, especially as the cumulative dose increases. 

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Am J Trop Med Hyg 2001 Nov;65(5):631-6

Multiplicity of Plasmodium falciparum infection in pregnancy.

Beck S, Mockenhaupt FP, Bienzle U, Eggelte TA, Thompson WN, Stark K.

Institute of Tropical Medicine, Charite, Humboldt University, Berlin, Germany.

Little is known about the distribution and disease association of multiple Plasmodium falciparum infections in pregnant women. Genotyping of the merozoite surface protein-1 region was performed in 332 P. falciparum infected pregnant women in Ghana, and clinical and epidemiologic data were obtained. Overall, 68% of the women were infected with more than one strain (mean number of strains per carrier = 2.9). The multiplicity of infection decreased significantly with an increasing number of pregnancies, and infection with multiple P. falciparum strains was significantly associated with anemia. In logistic regression, women infected with four or more strains were 2.3 times more likely to be anemic than women harboring fewer strains. This association, however, was only observed in women with up to three pregnancies. The results suggest that with increasing gravidity and subsequent infections with multiple strains effective immune mechanisms against more and more strains develop. In pregnant women, the multiplicity of infection may be an important factor for the acquisition and maintenance of immunity against malaria. 

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Am J Trop Med Hyg 2001 Nov;65(5):623-30

Human immunodeficiency virus seropositivity and malaria as risk factors for third-trimester anemia in asymptomatic pregnant women in western Kenya.

van Eijk AM, Ayisi JG, ter Kuile FO, Misore A, Otieno JA, Kolczak MS, Kager PA, Steketee RW, Nahlen BL.

Kenya Medical Research Institute, Center for Vector Biology and Control Research, Kisumu.

To assess risk factors for anemia in late pregnancy, we studied healthy pregnant women with a singleton uncomplicated pregnancy of > or = 32 weeks attending the prenatal clinic in the Provincial Hospital in Kisumu, Kenya. Between June 1996 and December 1998, 4,608 pregnant women had a blood sample collected for hemoglobin (Hb) measurement, malaria smear, and testing for human immunodeficiency virus (HIV). The mean +/- standard deviation of Hb was 9.58 +/- 1.8 g/dL; 21% had malaria in their blood; and 25% of the women were HIV seropositive. Plasmodium falciparum parasitemia was more common among HIV-seropositive women in all gravidities compared with HIV-seronegative women (risk ratio, 1.71; 95% confidence interval, 1.53-1.92). In a multivariate analysis, for primi- and secundigravidae women, the factors malaria, belonging to the Luo tribe, and HIV seropositivity were significantly associated with any anemia (Hb < 11 g/dL), and HIV seropositivity and documented fever were associated with severe anemia (Hb < 7 g/dL). In women of higher gravidities, HIV seropositivity was the only statistically significant factor associated with any anemia or with severe anemia. Asymptomatic HIV seropositivity is an important risk factor to be considered in the differential diagnosis of maternal anemia, independent of P. falciparum parasitemia. 

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Am J Trop Med Hyg 2001 Nov;65(5):614-22

Factors contributing to anemia after uncomplicated falciparum malaria.

Price RN, Simpson JA, Nosten F, Luxemburger C, Hkirjaroen L, ter Kuile F, Chongsuphajaisiddhi T, White NJ.

Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand. [email protected]

The factors contributing to anemia in falciparum malaria were characterized in 4,007 prospectively studied patients on the western border of Thailand. Of these, 727 patients (18%) presented with anemia (haematocrit < 30%), and 1% (55 of 5,253) required blood transfusion. The following were found to be independent risk factors for anemia at admission: age < 5 years, a palpable spleen, a palpable liver, recrudescent infections, being female, a prolonged history of illness (> 2 days) before admission, and pure Plasmodium falciparum infections rather than mixed P. falciparum and Plasmodium vivax infections. The mean maximum fractional fall in hematocrit after antimalarial treatment was 14.1% of the baseline value (95% confidence interval [CI], 13.6-14.6). This reduction was significantly greater in young children (aged < 5 years) and in patients with a prolonged illness, high parasitemia, or delayed parasite clearance. Loss of parasitized erythrocytes accounted for < 10% of overall red blood cell loss. Hematological recovery was usually complete within 6 weeks, but it was slower in patients who were anemic at admission (adjusted hazards ratio [AHR], 1.9, 95% CI, 1.5-2.3), and those whose infections recrudesced (AHR, 1.2, 95% CI, 1.01-1.5). Half the patients with treatment failure were anemic at 6 weeks compared with 19% of successfully treated patients (relative risk, 2.8, 95% CI, 2.0-3.8). Patients coinfected with P. vivax (16% of the total) were 1.8 (95% CI, 1.2-2.6) times less likely to become anemic and recovered 1.3 (95% CI, 1.0-1.5) times faster than those with P. falciparum only. Anemia is related to drug resistance and treatment failure in uncomplicated malaria. Children aged < 5 years of age were more likely than older children or adults to become anemic. Coinfection with P. vivax attenuates the anemia of falciparum malaria, presumably by modifying the severity of the infection. 

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Am J Trop Med Hyg 2001 Nov;65(5):599-602

Comparison of methods for the rapid laboratory assessment of children with malaria.

Planche T, Krishna S, Kombila M, Engel K, Faucher JF, Ngou-Milama E, Kremsner PG.

Department of Infectious Diseases, St. George’s Hospital Medical School, London, United Kingdom.

Rapid diagnosis and accurate quantification of Plasmodium falciparum parasitemia are important for the management of malaria. The assessment of disease severity also depends on evaluation of metabolic indexes such as blood glucose and lactate concentrations. Here we describe an accurate and rapid alternative to conventional thick film examination (Lambarene method). We also assess near-patient methods for measuring blood glucose (OneTouch) and lactate (Accusport). The accuracy of the Lambarene method is similar to that of thin films. Results from the OneTouch glucose meter also are in good agreement with a YSI 2300 reference meter. Overall, the Accusport lactate meter agrees poorly with the YSI 2300 reference meter. However, the sensitivity and specificity to detect hyperlactatemia (blood lactate > or = 5 mmol/L) are 0.94 and 0.98, respectively. 

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Am J Trop Med Hyg 2001 Nov;65(5):593-8

Detection of histidine rich protein 2 and panmalarial ICT Malaria Pf/Pv test antigens after chloroquine treatment of uncomplicated falciparum malaria does not reliably predict treatment outcome in eastern Indonesia.

Tjitra E, Suprianto S, Dyer ME, Currie BJ, Anstey NM.

National Institute of Health Research and Development, Ministry of Health, Jakarta, Indonesia.

In regions with drug-resistant malaria, the ability to rapidly detect or predict treatment failure (TF) soon after a course of standard therapy for Plasmodium falciparum malaria would facilitate the prompt institution of second-line therapy. We thus evaluated longitudinally the ability of the ICT Malaria Pf/Pv immunochromatographic test to predict treatment outcome. Sixty-six Sumbanese Indonesians with uncomplicated falciparum malaria were treated with chloroquine and followed for 28 days by use of 1997 World Health Organization criteria for assessment of therapeutic efficacy of antimalarial drugs. The ICT Pf/Pv testing could be compared with microscopy in approximately half of the patients on each day of follow-up. Although strongly positive histidine rich protein 2 (HRP2) line intensities (equal to or greater than the control band) in convalescence were highly predictive of TF, any degree of positivity for the HRP2 and panmalarial antigens in convalescence was only moderately predictive of TE Positive predictive values of the HRP2 and panmalarial antigens for TF were 76.9% and 87.0%, respectively, on Day 3, 82.4% and 87.5% on Day 7, and 78.9% and 78.9% on Day 14. Negative HRP2 and panmalarial antigen results in convalescence were even less predictive of an adequate clinical response, and false-negative HRP2 and panmalarial antigen test results were found in one-sixth (6 of 37) of recrudescent infections diagnosed by microscopy among patients with late treatment failure. To reliably predict treatment outcome with rapid antigen tests, further development appears necessary to improve sensitivity for viable asexual parasites while avoiding detection of both gametocytes and persistent antigen in convalescence. 

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Am J Trop Med Hyg 2001 Nov;65(5):588-92

Identification of cryptic coinfection with Plasmodium falciparum in patients presenting with vivax malaria.

Mayxay M, Pukritrayakamee S, Chotivanich K, Imwong M, Looareesuwan S, White NJ.

Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

In Thailand, approximately 8% of patients treated for vivax malaria are found subsequently to have coinfection with Plasmodium falciparum. A P. falciparum histidine rich protein 2 (PfHRP-2) dipstick test was evaluated as a predictor of mixed infections with subpatent P. falciparum in a prospective study of 238 patients admitted to the hospital with acute vivax malaria. Of these, 23 (10%) had subsequent development of falciparum malaria without reexposure. Patients with cryptic P. falciparum infection had a significantly lower mean (standard deviation) hematocrit than those with P. vivax alone: 29.6 (7.6%) versus 37.2 (6.4%) (P < 0.0001). Using microscopic appearance of P. falciparum after the start of treatment as the reference standard, the PfHRP-2 test was 74% sensitive and 99% specific in predicting mixed infections with subpatent P. falciparum parasitemia at presentation. The PfHRP-2 dipstick test may be a useful adjunct to microscopy in areas where mixed infections are common. 

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Am J Trop Med Hyg 2001 Nov;65(5):523-7

Risk factors for gametocyte carriage in Gambian children.

von Seidlein L, Drakeley C, Greenwood B, Walraven G, Targett G.

Medical Research Council Laboratories, Fajara, The Gambia. [email protected]

A widespread reduction in Plasmodium falciparum gametocyte prevalence could reduce malaria transmission. After infection with P. falciparum, a variable proportion of people are found to be gametocytemic. We analyzed risk factors associated with gametocytemia at presentation and 7 days later. We enrolled 1,198 children in 2 antimalarial drug trials between September and December 1998. The children were assigned to 1 of 4 treatment groups: chloroquine only; pyrimethamine-sulfadoxine (PSD) only; PSD combined with 1 dose of artesunate; and PSD combined with 3 doses of artesunate. By the time of enrollment, 200 (17%) of 1,198 children were gametocyte carriers. Three independent risk factors were associated with gametocytemia at enrollment. Children with anemia were more likely to carry gametocytes, whereas children with fever (> 37.4 degrees C) or high parasite densities (> 100,000 parasites/microL) were less frequently gametocyte carriers. Children with at least 2 of the risk factors were 4 times more likely to be gametocytemic than children with < 2 risk factors (odds ratio [OR], 4.4; 95% confidence interval [CI], 2.7-7.1). Seven days after the start of treatment, 355 (37%) of 466 assessable children were found to be gametocyte carriers. Children treated with PSD alone had a significantly higher risk of being gametocytemic by Day 7 compared with children in the other 3 treatment groups. In the subgroup of children who had no detectable gametocytes on enrollment, the effect of treatment with PSD + 3 doses of artesunate was most marked. Nineteen (10%) of 198 children treated with PSD + 3 doses of artesunate became gametocytemic, in contrast to 184 (57%) of 321 children treated with PSD alone (OR, 12.7; 95% CI, 7.3-22.1). Early treatment with highly effective antimalarial therapy has the greatest chance of preventing gametocytemia. The choice of a first-line antimalarial drug for uncomplicated malaria should not only take into consideration the ablation asexual parasitemia but also the suppression of gametocytemia. 

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Am J Trop Med Hyg 2001 Nov;65(5):471-6

Association of subtherapeutic dosages of a standard drug regimen with failures in preventing relapses of vivax malaria.

Duarte EC, Pang LW, Ribeiro LC, Fontes CJ.

National Health Foundation, Ministry of Health, Brazil. [email protected]

This study evaluated the cure rate of the standard recommended regimen for Plasmodium vivax malaria in Brazil and assessed risk factors for failures. Fifty patients with vivax malaria given supervised medical treatment (standard dose of chloroquine: total dose = 1.5 g over a three-day period plus primaquine: total dose = 210 mg over a 14-day period) were followed for six months in a non-endemic area. Cox’s regression was used to identify predictors of relapses. Among the 289 patient-months of follow-up, seven relapses were identified (2.4 relapses per 100 person-months) between 33 and 137 days after treatment initiation. Risk factors for relapses (P < or = 0.05) were female sex, higher parasitemia at baseline, shorter number of days with symptoms prior to baseline, and lower mg/kg dose of primaquine. Relapses following supervised vivax treatment is in principle a necessary, but not sufficient, component of in vivo parasite resistance. Results indicate that other factors, principally sub-therapeutic primaquine doses, may explain the occurrence of vivax treatment failures.

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J Immunol 2001 Dec 1;167(11):6510-7

Cytoadherence of Plasmodium falciparum-infected erythrocytes is mediated by a redox-dependent conformational fraction of CD36.

Gruarin P, Primo L, Ferrandi C, Bussolino F, Tandon NN, Arese P, Ulliers D, Alessio M.

DIBIT, San Raffaele Scientific Institute, Milan, Italy.

The adherence of Plasmodium falciparum-infected RBC (IRBC) to postcapillary venular endothelium is an important determinant of the pathogenesis of severe malaria complications. Cytoadherence of IRBC to endothelial cells involves specific receptor/ligand interactions. The glycoprotein CD36 expressed on endothelial cells is the major receptor involved in this interaction. Treatment of CD36-expressing cells with reducing agents, such as DTT and N-acetylcysteine, was followed by CD36 conformational change monitorable by the appearance of the Mo91 mAb epitope. Only a fraction of the surface expressed CD36 molecules became Mo91 positive, suggesting the presence of two subpopulations of molecules with different sensitivities to reduction. The Mo91 epitope has been localized on a peptide (residues 260-279) of the C-terminal, cysteine-rich region of CD36. Treatment with reducing agents inhibited the CD36-dependent cytoadherence of IRBC to CD36-expressing cells and dissolved pre-existent CD36-mediated IRBC/CD36-expressing cell aggregates. CD36 reduction did not impair the functionality of CD36, since the reactivity of other anti-CD36 mAbs as well as the binding of oxidized low density lipoprotein, a CD36 ligand, were maintained. The modifications induced by reduction were reversible. After 14 h CD36 was reoxidized, the cells did not express the Mo91 epitope, and cytoadherence to IRBC was restored. The results indicate that IRBCs bind only to a redox-modulated fraction of CD36 molecules expressed on the cell surface. The present data indicate the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or treat malaria complications due to IRBC cytoadhesion. 

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 J Immunol 2001 Dec 1;167(11):6421-30

Functional and Structural Similarity of Vgamma9Vdelta2 T Cells in Humans and Aotus Monkeys, a Primate Infection Model for Plasmodium falciparum Malaria.

Daubenberger CA, Salomon M, Vecino W, Hubner B, Troll H, Rodriques R, Patarroyo ME, Pluschke G.

Department of Molecular Immunology, Swiss Tropical Institute, Basel, Switzerland. Instituto de Inmunologia, Universidad Nacional de Colombia, Santafe de Bogota, Leticia Colombia. Solvias Aktiengesellschaft, Basel, Switzerland.

gammadelta T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human gammadelta T cells carrying the Vgamma9Vdelta2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating Vgamma9Vdelta2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the Vgamma9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human gammadelta T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR gene segments used, and 3) the diversity of the CDR3 regions. Phylogenetic analysis of human, Pan troglodytes, and A. nancymaae TRGV gene segments showed that the interspecies differences are smaller than the intraspecies differences with TRGV9 gene segments located on a distinct clade of the phylogenetic tree. The structural and functional conservation of Vgamma9Vdelta2 T cells in A. nancymaae and humans implicates a functionally important and evolutionary conserved mechanism of recognition of phosphorylated microbial metabolites. 

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Afr J Med Med Sci 2000 Sep-Dec;29(3-4):211-3

Congenital malaria in a hyperendemic area: a revisit.

Olowu JA, Sowunmi A, Abohweyere AE.

Department of Paediatrics, College of Medicine, University College Hospital, Ibadan, Nigeria.

Screening of 104 mother-baby pairs for P. falciparum malaria revealed that 29% of mothers from low socio-economic group and 11% of their babies had malaria parasitaemia. The corresponding figures for middle and high socio-economic groups were 15% and 7%, respectively. The parasite densities in the babies were not proportional to maternal load and were generally low, although higher in the low socio-economic group. Maternal pyrimethamine prophylaxis did not appear to protect babies from parasitisation and there was no demonstrable beneficial effect on the babies’ birtth-weights. 

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Dev Biol (Basel) 2000;104:171-9

DNA-based vaccines for malaria: a heterologous prime-boost immunisation strategy.

Hill AV, Reece W, Gothard P, Moorthy V, Roberts M, Flanagan K, Plebanski M, Hannan C, Hu JT, Anderson R, Degano P, Schneider J, Prieur E, Sheu E, Gilbert SC.

Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, UK.

A generic approach to inducing high level CD8+ T cell responses would be of value for prophylactic and therapeutic immunisation against several infectious diseases. However, it has been very difficult to achieve such immune responses using available vaccination strategies. Malaria is one of several diseases against which a new generation of better CD8+ T cell-inducing vaccines might be useful and is unusual in that it allows assessment of vaccine efficacy in small numbers of volunteers in carefully controlled challenge studies. Here we review the identification of a heterologous prime-boost regime using DNA priming and recombinant modified vaccinia Ankara (MVA) boosting that induces high level T cell responses in both mice and non-human primates. Clinical trials to determine whether this prime-boost approach is immunogenic in humans are in progress. 

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Dev Biol (Basel) 2000;104:121-32

Can malaria DNA vaccines on their own be as immunogenic and protective as prime-boost approaches to immunization?

Hoffman SL, Doolan DL.

Malaria Program, Naval Medical Research Center, Silver Spring, MD 20910-7500, USA.

To develop a multi-stage, multi-antigen, multi-immune response-inducing vaccine against malaria we have focused on DNA vaccines because of their simplicity of construction and modification, ease of mixing, and effectiveness in inducing CD8+ T cell responses. DNA malaria vaccines induce CD8+ T cell dependent protection in mice and CD8+ CTL in rhesus monkeys and humans after intramuscular needle administration. Clinical trials in normal, healthy humans are in progress or planned, assessing alternative methods and routes of administration, and the capacity of a plasmid expressing human GM-CSF to enhance the protective efficacy of a five-gene liver-stage malaria vaccine. In mice, we have demonstrated that priming with the combination of DNA plasmids encoding a Plasmodium yoelii protein and murine GM-CSF and boosting with recombinant poxvirus expressing the same P. yoelii protein induces a 30-fold increase in antigen-specific antibodies, a 10-fold increase in antigen-specific IFN-gamma spot forming cells, a significant (p<0.05) increase in protection, and the capacity to reduce the dosage of DNA by 10-100 fold, compared to immunizing with DNA alone. In Aotus monkeys priming with DNA and boosting with recombinant protein in adjuvant is more protective than homologous priming and boosting with either DNA or recombinant protein in adjuvant. Clinical trials are now planned using these immunization strategies. Because of the complexity and cost of the heterologous regimens, we are working to make DNA vaccination alone as immunogenic and protective as the prime-boost approach. Our most encouraging findings have resulted from altering codon usage from the highly A+T rich P. falciparum native sequence to that more closely resembling mammalian sequences. Although much progress is required for the development of a vaccine that provides sustainable protective immunity against malaria, a strategy using DNA vaccine technology as a core component of such a vaccine is promising. 

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Nature 2001 Nov 15;414(6861):305-8

Haemoglobin C protects against clinical Plasmodium falciparum malaria.

Modiano D, Luoni G, Sirima BS, Simpore J, Verra F, Konate A, Rastrelli E, Olivieri A, Calissano C, Paganotti GM, D’Urbano L, Sanou I, Sawadogo A, Modiano G, Coluzzi M.

[1] Dipartimento di Scienze di Sanita Pubblica, Sezione di Parassitologia, WHO Collaborating Centre for Malaria Epidemiology and Control; and [2] Istituto Pasteur Fondazione Cenci Bolognetti, University of Rome “La Sapienza”, 00185, Rome, Italy.

Haemoglobin C (HbC; beta6Glu –> Lys) is common in malarious areas of West Africa, especially in Burkina Faso. Conclusive evidence exists on the protective role against severe malaria of haemoglobin S (HbS; beta6Glu –> Val) heterozygosity, whereas conflicting results for the HbC trait have been reported and no epidemiological data exist on the possible role of the HbCC genotype. In vitro studies suggested that HbCC erythrocytes fail to support the growth of P. falciparum but HbC homozygotes with high P. falciparum parasitaemias have been observed. Here we show, in a large case-control study performed in Burkina Faso on 4,348 Mossi subjects, that HbC is associated with a 29% reduction in risk of clinical malaria in HbAC heterozygotes (P = 0.0008) and of 93% in HbCC homozygotes (P = 0.0011). These findings, together with the limited pathology of HbAC and HbCC compared to the severely disadvantaged HbSS and HbSC genotypes and the low betaS gene frequency in the geographic epicentre of betaC, support the hypothesis that, in the long term and in the absence of malaria control, HbC would replace HbS in central West Africa. 

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Mol Med 2001 Oct;7(10):698-710

The Impact of Whole Genome Sequence Data on Drug Discovery–A Malaria Case Study.

Joachimiak MP, Chang C, Rosenthal PJ, Cohen FE.

Background: Identification and validation of a drug discovery target is a prominent step in drug development. In the post-genomic era it is possible to reevaluate the association of a gene with a specific biological function to see if a homologous gene can subsume this role. This concept has special relevance to drug discovery in human infectious diseases, like malaria. A trophozoite cysteine protease (falcipain-1) from the papain family, thought to be responsible for the degradation of erythrocyte hemoglobin, has been considered a promising target for drug discovery efforts owing to the antimalarial activity of peptide based covalent cysteine protease inhibitors. This led to the development of non-peptidic non-covalent inhibitors of falcipain-1 and their characterization as antimalarials. It is now clear from sequencing efforts that the malaria genome contains more than one cysteine protease and that falcipain-1 is not the most important contributor to hemoglobin degradation. Rather, falcipain-2 and falcipain-3 appear to account for the majority of cysteine hemoglobinase activity in the plasmodium trophozoite. Materials and Methods: We have modeled the falcipain-2 cysteine protease from one of the major human malaria species, Plasmodium falciparum and compared it to our original work on falcipain-1. As with falcipain-1, computa-tional screening of the falcipain-2 active site was conducted using DOCK. Using structural superpositions within the protease family and evolutionary analysis of substrate specificity sites, we focused on the commonalities and the protein specific features to direct our drug discovery effort. Results: Since 1993, the size of the Available Chemicals Directory had increased from 55313 to 195419 unique chemical structures. For falcipain-2, eight inhibitors were identified with IC(50)’s against the enzyme between 1 and 7 &mgr;M. Application of three of these inhibitors to infected erythrocytes cured malaria in culture, but parasite death did not correlate with food vacuole abnormalities associated with the activity of mechanistic inhibitors of cysteine proteases like the epoxide E64. Conclusions: Using plasmodial falcipain proteases, we show how a protein family perspective can influence target discovery and inhibitor design. We suspect that parallel drug discovery programs where a family of targets is considered, rather than serial programs built on a single therapeutic focus, will become the dominant industrial paradigm. Economies of scale in assay development and in compound synthesis are expected owing to the functional and structural features of individual family members. One of the remaining challenges in post-genomic drug discovery is that inhibitors of one target are likely to show some activity against other family members. This lack of specificity may lead to difficulties in functional assignments and target validation as well as a complex side effect profile. 

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Clin Infect Dis 2001 Dec 15;33(12):2009-16

Artemisinin Antimalarials in Pregnancy: A Prospective Treatment Study of 539 Episodes of Multidrug-Resistant Plasmodium falciparum.

McGready R, Cho T, Keo NK, Thwai KL, Villegas L, Looareesuwan S, White NJ, Nosten F.

Shoklo Malaria Research Unit, Mae Sot, and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; and Centre for Tropical Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom.

The emergence and spread of multidrug-resistant Plasmodium falciparum compromises the treatment of malaria, especially during pregnancy, where the choice of antimalarials is already limited. Artesunate (n=528) or artemether (n=11) was used to treat 539 episodes of acute P. falciparum malaria in 461 pregnant women, including 44 first-trimester episodes. Most patients (310 [57.5%]) received re-treatments after earlier treatment with quinine or mefloquine. By use of survival analysis, the cumulative artemisinin failure rate for primary infections was 6.6% (95% confidence interval, 1.0-12.3), compared with the re-treatment failure rate of 21.7% (95% confidence interval, 15.4-28.0; P=.004). The artemisinins were well tolerated with no evidence of adverse effects. Birth outcomes did not differ significantly to community rates for abortion, stillbirth, congenital abnormality, and mean gestation at delivery. These results are reassuring, but further information about the safety of these valuable antimalarials in pregnancy is needed. 

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Clin Infect Dis 2001 Dec 15;33(12):1990-7

Randomized, parallel placebo-controlled trial of primaquine for malaria prophylaxis in papua, indonesia.

Baird JK, Lacy MD, Basri H, Barcus MJ, Maguire JD, Bangs MJ, Gramzinski R, Sismadi P, Krisin, Ling J, Wiady I, Kusumaningsih M, Jones TR, Fryauff DJ, Hoffman SL.

Parasitic Diseases Program, US Naval Medical Research Unit 2, American Embassy Jakarta, FPO AP 96520-8132, USA. [email protected]

Malaria causes illness or death in unprotected travelers. Primaquine prevents malaria by attacking liver-stage parasites, a property distinguishing it from most chemoprophylactics and obviating 4-week postexposure dosing. A daily adult regimen of 30 mg primaquine prevented malaria caused by Plasmodium falciparum and P. vivax for 20 weeks in 95 of 97 glucose-6-phosphate dehydrogenase (G6PD)-normal Javanese transmigrants in Papua, Indonesia. In comparison, 37 of 149 subjects taking placebo in a parallel trial became parasitemic. The protective efficacy of primaquine against malaria was 93% (95% confidence interval [CI] 71%-98%); against P. falciparum it was 88% (95% CI 48%-97%), and >92% for P. vivax (95% CI >37%-99%). Primaquine was as well tolerated as placebo. Mild methemoglobinemia (mean of 3.4%) returned to normal within 2 weeks. Blood chemistry and hematological parameters revealed no evidence of toxicity. Good safety, tolerance, and efficacy, along with key advantages in dosing requirements, make primaquine an excellent drug for preventing malaria in nonpregnant, G6PD-normal travelers. 

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Science 2001 Nov 16;294(5546):1439

GENES AND DISEASE: Genetic Change Wards Off Malaria.

Pennisi E.

Researchers have determined that a genetic alteration can provide almost complete protection against malaria, which in Africa kills 3000 children each day. The alteration produces a form of the oxygen-transport molecule called hemoglobin C that seems to have no adverse effects. Understanding how hemoglobin C works could help guide the development of vaccines and drugs, malaria researchers say. 

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J Med Chem 2001 Nov 22;44(24):4268-76

A prodrug form of a Plasmodium falciparum glutathione reductase inhibitor conjugated with a 4-anilinoquinoline.

Davioud-Charvet E, Delarue S, Biot C, Schwobel B, Boehme CC, Mussigbrodt A, Maes L, Sergheraert C, Grellier P, Schirmer RH, Becker K.

UMR 8525 CNRS-Universite Lille II-Institut Pasteur de Lille, Institut de Biologie de Lille, 1 rue du Professeur Calmette, BP447 59021 Lille Cedex, France. [email protected]

Glutathione (GSH), which is known to guard Plasmodium falciparum from oxidative damage, may have an additional protective role by promoting heme catabolism. An elevation of GSH content in parasites leads to increased resistance to chloroquine (CQ), while GSH depletion in resistant P. falciparum strains is expected to restore the sensitivity to CQ. High intracellular GSH levels depend inter alia on the efficient reduction of GSSG by glutathione reductase (GR). On the basis of this hypothesis, we have developed a new strategy for overcoming glutathione-dependent 4-aminoquinoline resistance. To direct both a 4-aminoquinoline and a GR inhibitor to the parasite, double-drugs were designed and synthesized. Quinoline-based alcohols (with known antimalarial activity) were combined with a GR inhibitor via a metabolically labile ester bond to give double-headed prodrugs. The biochemically most active double-drug 7 of this series was then evaluated as a growth inhibitor against six Plasmodium falciparum strains that differed in their degree of resistance to CQ; the ED(50) values for CQ ranged from 14 to 183 nM. While the inhibitory activity of the original 4-aminoquinoline-based alcohol followed that of CQ in these tests, the double-drug exhibited similar efficiency against all strains, the ED(50) being as low as 28 nM. For the ester 7, a dose-dependent decrease in glutathione content and GR activity and an increase in glutathione-S-transferase activity were determined in treated parasites. The drug was subsequently tested for its antimalarial action in vivo using murine malaria models infected with P. berghei. A 178% excess mean survival time was determined for the animals treated with 40 mg/kg 7 for 4 days. No cytotoxicity due to this compound was observed. Work is in progress to extend and validate the strategy outlined here.

Ann N Y Acad Sci 2001 Sep;945:234-8

Detection of Plasmodium falciparum DNA in plasma.

Gal S, Fidler C, Turner S, Lo YM, Roberts DJ, Wainscoat JS.

Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Headington, United Kingdom. [email protected]

We present a new application in the field of circulating nucleic acids in plasma: the detection of parasite DNA in plasma. We have investigated the presence of Plasmodium falciparum (P. falciparum) DNA in the blood cells and plasma of patients with malaria. Ten blood samples from malaria patients and 9 blood samples from healthy volunteers were collected. Plasma was separated and DNA was extracted from both plasma and blood cells. DNA samples were subjected to a nested polymerase chain reaction (PCR) for the small subunit rRNA gene of P. falciparum and a control amplification of the human rRNA gene. All 6 cases positive by microscopy for P. falciparum were positive by PCR on DNA extracted from blood cells and plasma. Two cases negative by microscopy for malaria were positive by PCR–the blood samples were taken from one of the cases at 7 days after successful treatment of malaria and the other case at the onset of the malaria illness. Two other cases were negative by microscopy and by PCR on blood cells and plasma, both after successful treatment of malaria. The quantitation of Plasmodium DNA in plasma may prove to be a useful prognostic measurement in malaria, and the detection of P. falciparum DNA in archival stored plasma samples may allow the reconstruction of the recent historic evolution of the parasite genome.