Structural insights into Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase: oligomeric assembly

The biochemical pathways involved in nicotinamide adenine dinucleotide (NAD) biosynthesis converge at the enzymatic step catalysed by nicotinamide mononucleotide adenylyltransferase (NMNAT, EC: 2.7.7.1). The majority of NMNATs are assembled into homo-oligomeric states that comprise 2-6 subunits. Recently, the NMNAT of Plasmodium falciparum (PfNMNAT) has been identified as a pharmacological target. The enzymatic characterisation, cellular location, and tertiary structure of the PfNMNAT protein have been reported. Nonetheless, its quaternary structure remains to be explored. The present study describes the oligomeric assembly of the 6 x His-PfNMNAT recombinant protein using immobilised metal affinity chromatography coupled with size exclusion chromatography (SEC) and native protein electrophoresis combined with Ferguson plot graphing. These chromatographic approaches resulted in the elution of an active monomer from the SEC column, whereas the Ferguson plot indicated a dimeric assembly of the 6 x His-PfNMNAT protein.

Plasmodium falciparum parasites overexpressing farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase are more resistant to risedronate

Farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains. Risedronate, a bisphosphonate containing nitrogen (N-BP), is a potent inhibitor of blood stage Plasmodium. Here, we show that P. falciparum parasites overexpressing FPPS/GGPPS are more resistant to risedronate, suggesting that this enzyme is an important target, and bisphosphonate analogues can be used as potential antimalarial drugs.

Frequency of antibodies against Sarcocystis neurona and Neospora caninum in domestic cats in the state of Bahia, Brazil / Frequência de anticorpos contra Sarcocystis neurona e Neospora caninum em gatos domésticos do Estado da Bahia, Brasil

Sarcocystis neurona is the major agent of equine protozoal myeloencephalitis. It infects several mammalian species in the Americas, where the definitive hosts, marsupials of the genus Didelphis (D. virginiana and D. albiventris) are found. Domestic cats are one of the confirmed intermediate hosts of the parasite; however, antibodies against S. neurona had never before been demonstrated in Brazilian cats. The aim of this study was to determine whether cats in Bahia, Brazil, are exposed to the parasite. A total of 272 feline serum samples (134 from feral and 138 from house cats) were subjected to an indirect fluorescent antibody test using cultured merozoites of S. neurona as antigen. Positivity was detected in 4.0% (11/272) of the tested samples, with titers ranging from 25 to 800. The feline sera were also tested for antibodies against the protozoan Neospora caninum, with an observed antibody frequency of 2.9%. To the author's knowledge, this is the first study to report antibodies against S. neurona in Brazilian cats. We conclude that cats are exposed to the parasite in the region of this study. Further investigations are needed to confirm the role of cats in the transmission cycle of S. neurona in Brazil.

Sarcocystis neurona é o principal agente da mieloencefalite protozoária equina. Esse parasito infecta várias espécies de mamíferos nas Américas, onde são encontrados os hospedeiros definitivos, os marsupiais do gênero Didelphis (D. virginiana and D. albiventris). O gato doméstico é um dos hospedeiros intermediários do parasito. Contudo, anticorpos contra S. neurona ainda não tinham sido demonstrados em gatos brasileiros. O objetivo deste trabalho foi determinar se gatos da Bahia, Brasil, são expostos ao parasito. Amostras séricas de 272 felinos (134 de gatos errantes e 138 de gatos domiciliados) foram testadas pelo teste de imunofluorescência indireta, utilizando-se como antígeno, merozoítos produzidos em cultura celular. Entre as amostras testadas, 4,0% (11/272) foram positivas com títulos entre 25 e 800. Os soros dos felinos foram também testados para anticorpos contra o protozoário Neospora caninum, cuja frequência de anticorpos foi de 2,9%. Esse é o primeiro relato de anticorpos contra S. neurona em gatos brasileiros. Conclui-se que os gatos da região estudada são expostos a S. neurona. Estudos futuros são necessários, a fim de se confirmar o papel dos gatos no ciclo de transmissão de S. neurona no Brasil.

Estudo do sistema de reparo do DNA tipo“Mismatch Repair” em Plasmodium spp

Cepas de Plasmodium resistentes a diferentes drogas têm sido descritas ao redor do mundo. Embora os mecanismos de desenvolvimento de resistência não sejam bem conhecidos, sabe-se que defeitos nos sistemas de reparo do DNA podem estar envolvidos. Esses defeitos estão relacionados principalmente a mutações nas enzimas do sistema de reparo de mal pareamento do DNA ou mismatch repair(MMR) e já foram descritos em populações naturais de diversos organismos. Devido ao conhecimento limitado sobre o sistema MMR de Plasmodium, faz-se necessário um amplo estudo sobre os genes que codificam as proteínas envolvidas nesse sistema. Neste trabalho, foi realizado um estudo sobre as enzimas envolvidas no sistema de reparo do mal pareamento do DNA em Plasmodium: variabilidade intra e interespecífica em Plasmodium, principalmente nos domínios funcionais, e comparação entre níveis de expressão entre cepas/isolados de P. falciparum. Os parasitos foram também avaliados quanto ao número de cópias e expressão dos genes gch-1 e mdr1. Foram identificadas proteínas pertencentes às classes MSH2,MSH6, MLH1 e PMS1.

As sequências de proteínas mostraram-se muito conservadas, tanto entre o gênero Plasmodium, quanto em relação a outros organismos distantes evolutivamente. Foi encontrada um proteína homóloga a MutSque possui os domínios I e V, mas ainda não identificada quanto à sua classificação.O gene codificador desta proteína teve sua expressão confirmada neste e em outros trabalhos. Alguns SNPs foram encontrados em cepas/isolados depositados no PlasmoDB, no entanto, o sequenciamento da região que compreende os principais domínios funcionais apontou apenas 1 SNP na proteína PMS1. Os genes estudados, em sua maioria, apresentaram-se mais expressos entre 10 e 30 horas após a sincronização. W2 e 3D7 apresentam 2 cópias do genes gch-1 e mdr1. BHZ apresentou apenas 1 cópia do mdr1. Os resultados da análise de expressão desses genes ligados à resistência concordam com os resultados encontrados para o número de cópias gênicas. Este estudo fornece uma análise ampla das principais enzimas do MMR e será importante para estudos futuros do papel funcional destas enzimas e seu envolvimento no desenvolvimento de resistência às drogas

Estudo do sistema de reparo do DNA tipo“Mismatch Repair” em Plasmodium spp / Study of the DNA repair type

Cepas de Plasmodium resistentes a diferentes drogas têm sido descritas ao redor do mundo. Embora os mecanismos de desenvolvimento de resistência não sejam bem conhecidos, sabe-se que defeitos nos sistemas de reparo do DNA podem estar envolvidos. Esses defeitos estão relacionados principalmente a mutações nas enzimas do sistema de reparo de mal pareamento do DNA ou mismatch repair(MMR) e já foram descritos em populações naturais de diversos organismos. Devido ao conhecimento limitado sobre o sistema MMR de Plasmodium, faz-se necessário um amplo estudo sobre os genes que codificam as proteínas envolvidas nesse sistema. Neste trabalho, foi realizado um estudo sobre as enzimas envolvidas no sistema de reparo do mal pareamento do DNA em Plasmodium: variabilidade intra e interespecífica em Plasmodium, principalmente nos domínios funcionais, e comparação entre níveis de expressão entre cepas/isolados de P. falciparum. Os parasitos foram também avaliados quanto ao número de cópias e expressão dos genes gch-1 e mdr1. Foram identificadas proteínas pertencentes às classes MSH2,MSH6, MLH1 e PMS1. As sequências de proteínas mostraram-se muito conservadas, tanto entre o gênero Plasmodium, quanto em relação a outros organismos distantes evolutivamente. Foi encontrada um proteína homóloga a MutSque possui os domínios I e V, mas ainda não identificada quanto à sua classificação.O gene codificador desta proteína teve sua expressão confirmada neste e em outros trabalhos. Alguns SNPs foram encontrados em cepas/isolados depositados no PlasmoDB, no entanto, o sequenciamento da região que compreende os principais domínios funcionais apontou apenas 1 SNP na proteína PMS1. Os genes estudados, em sua maioria, apresentaram-se mais expressos entre 10 e 30 horas após a sincronização. W2 e 3D7 apresentam 2 cópias do genes gch-1 e mdr1. BHZ apresentou apenas 1 cópia do mdr1. Os resultados da análise de expressão desses genes ligados à resistência concordam com os resultados encontrados para o número de cópias gênicas. Este estudo fornece uma análise ampla das principais enzimas do MMR e será importante para estudos futuros do papel funcional destas enzimas e seu envolvimento no desenvolvimento de resistência às drogas.

Diagnosis of Malaria by Detection of Parasite Lactate Dehydrogenase with Advantage Mal Card Test.

Background : Malaria is a global problem. Rapid diagnosis is essential for effective treatment and reducing mortality and morbidity of malaria. Diagnosis of malariaby peripheral smear is labor-intensive and requires considerable expertise for its interpretation. A rapid test , Advantage MAL card test is based on detection of parasite lactate dehydrogenase (pLDH) and has the ability to differentiate the four major Plasmodium species in 20 minutes. Objectives: 1) To evaluate utility of parasite lactate dehydrogenase for diagnosis of malaria with Advantage Mal card test.2) To compare the results of Advantage Mal card test with peripheral smear findings. Materials and Methods: In this retrospective study, total 5242 patients with malaria like symptoms attending OPD and admitted in wards at Acharya Vinoba Bhave Rural Hospital (AVBRH) from January 2008 to August 2011 were studied. Result: The age of patients ranged from < 1 year- >80 years. The commonest age group affected was 21-30 years. Male to female ratio was 1.04: 1. Prevalence rate of malaria was 101/1000 population in AVBRH. Malarial parasites were detected in PS in10.11% patients (P.falciparum 27.73% , P.vivax 71.32% , mixed infection 0.94%) and in 10.07% patients with Advantage Mal test (P. falciparum 28.03%, P.vivax 71.02%, mixed infection 0.95%). 3 cases of P.vivax and 1case of P.falciparum detected by PS were not detected by Advantage Mal test. 2 cases of P.falciparum detected by Advantage Mal and not by PS. Compared to PS, the Advantage Mal had sensitivity 99.24%, specificity 100%, positive predictive value 100%, negative predictive value was 89.92%. Conclusion: Diagnosis of malaria by detection of pLDH with Advantage Mal card test is simple ,rapid, reliable and cheap method. Results are comparable to blood films. It can detect P.flciparum infection when parasites are sequestered.

Localization of heme biosynthesis pathway enzymes in Plasmodium falciparum.

Protein trafficking in the malarial parasite Plasmodium falciparum is dictated by a complex life-cycle that involves a variety of intra-cellular and host cell destinations, such as the mitochondrion, apicoplast, rhoptries and micronemes. Of these, the apicoplast and mitochondrion are believed to account for more than 10% of this traffic. Studies have shown that mechanisms for mitochondrion and apicoplast targeting are distinct, despite their close physical proximity. The heme biosynthesis pathway spans both these organelles, making trafficking studies crucial for the spatial demarcation of the constituent interactions. This minireview highlights the challenges in identifying the possible sub-cellular destinations of the heme pathway enzymes using gleanings from literature survey as well as focussed bioinformatic analysis.

Evaluación de la actividad de extractos marinos sobre Plasmodium falciparum in vitro y aspártico proteasas / Evaluation of the marine extracts action on Plasmodium falciparum in vitro and on aspartic proteases

Introducción: la búsqueda de nuevas drogas o alternativas terapéuticas para el tratamiento de la malaria es una alta prioridad en la lucha por el control de esta enfermedad. En la actualidad, varios estudios se concentran en la evaluación de inhibidores de proteasas de tipo aspártico presentes en la vacuola digestiva de Plasmodium falciparum, las cuales son parte de las enzimas que participan en la degradación de la hemoglobina. Los escasos reportes en la literatura sobre la purificación de inhibidores de proteasas aspárticas a partir de organismos marinos sugieren que constituyen una fuente de este tipo de moléculas prácticamente inexplorada. Métodos: las especies de invertebrados marinos Phallusia nigra, Bugula sp., Lyssodendoryx isodictyalis, Ascidia sydneiensis, Microscosmus goanus, Holothuria mexicana, Lytechinus variegatus y Echinaster sp. fueron colectadas en la localidad de Puerto Esperanza, Pinar del Río, en abril de 2006 y se prepararon extractos etanólicos. Se realizó la evaluación antimalárica in vitro contra Plasmodium falciparum de estos extractos con valores descriptivos de eficacia comparables a los utilizados internacionalmente. Los resultados se relacionaron con los hallazgos de los ensayos de inhibición de la actividad enzimática de pepsina como modelo de proteasa aspártica y con el perfil químico de metabolitos secundarios en estos extractos. Resultados: se encontró una buena reproducibilidad de la actividad antimalárica de los extractos de P. nigra, M. goanus y L. isodictyalis con concentraciones inhibitorias medias menores que 50 µg/mL. El extracto de M. goanus mostró la posible presencia de un inhibidor de pepsina. El perfil químico obtenido para las ascidias se corresponde con los principales compuestos reportados para las familias Pyuridae y Ascidiidae. La actividad antimalárica, así como la actividad inhibidora de pepsina, pudiera ser atribuida a algunos de los grupos de metabolitos secundarios detectados...

Background: the search for new drugs or therapeutic alternatives for malaria treatment is a high priority in the struggle against this disease. At present, several studies are focused on the evaluation of aspartic protease inhibitors present in the digestive vacuole of Plasmodium falciparum, which are part of the enzymes involved in hemoglobin degradation. The few reports in literature on the purification of aspartic proteases inhibitors from marine organisms suggest that they are a practically unexplored source of this type of molecules. Methods: marine invertebrate species Phallusia nigra, Bugula sp., Lyssodendoryx isodictyalis, Ascidia sydneiensis, Microscosmus goanus, Holothuria mexicana, Lytechinus variegatus y Echinaster sp.were detected in Puerto Esperanza area, Pinar del Rio province, on April 2006 and then ethanol extracts were prepared. In vitro antimalarial evaluation of these extracts against Plasmodium falciparum, with descriptive efficacy values being comparable with those used worldwide. The results were associated to the findings of aspartic protease model-like pepsin enzymatic action inhibition tests and to the chemical profile of secondary metabolites in these extracts. RESULTS: good reproducibility of antimalarial action of P. nigra, M. goanus y L. isodictyalis extracts was found, being the average inhibitory concentrations lower than 50 µg/mL. M. goanus extract showed a possible pepsin inhibitor. The chemical profile for ascidians corresponded to the main compounds reported in Pyuridae y Ascidiidae families. The antimalarial activity as well as the pepsin inhibitory activity might be attributed to some of the detected secondary metabolites. Conclusions: the breaking-up of these extracts is recommended in order to isolate the chemical compounds involved in the studied biological activities.

Resistência à sulfadoxina-pirimetamina em Maputo, Moçambique: presença de mutações nos genes dhfr e dhps do Plasmodium falciparum / Sulfadoxine-pyrimethamine resistance in Maputo, Mozambique: presence of mutations in the dhfr and dhps genes of Plasmodium falciparum

Foram analisadas a freqüência e distribuição de mutações nos genes dihidrofolato redutase e dihidropteroato sintetase do Plasmodium falciparum, usando a metodologia de reação em cadeia da polimerase e polimorfismos de hidrólise por enzimas de restrição, em amostras de sangue infectado proveniente de crianças moçambicanas, residentes em Maputo. A análise foi feita antes e 7 dias após o tratamento com sulfadoxina-pirimetamina (S/P). Os resultados mostraram a ocorrência de mutações pontuais nos genes estudados e a presença de combinações de três alelos em dhfr (51Ile, 59Arg e 108Asn) e do quintúplo mutante (dhfr 51Ile, 59Arg, 108Asn e dhps 437Gly, 540Glu), ambas situações associadas à falha terapêutica no sétimo dia após tratamento com S/P. Esses achados mostram a importância de se estudar a resistência à S/P em Moçambique, e como os marcadores moleculares de resistência aos antimaláricos podem fornecer dados importantes para a política nacional de controlo da malária.

The frequency and distribution of mutations in Plasmodium falciparum, dihydrofolate reductase and dihydropteroate synthase genes were analyzed, using the polymerase chain reaction and restriction fragment length polymorphism methodology, in infected blood samples from Mozambican children living in Maputo, before and seven days after treatment with sulfadoxine/pyrimethamine (S/P). The results showed the occurrence of point mutations in the genes studied and the presence of combinations of three alleles in dhfr (51Ile, 59Arg and 108Asn) and "quintuple" mutant (dhfr 51Ile, 59Arg, 108Asn and dhps 437Gly, 540Glu). Both of these situations were associated with seven-day therapeutic failure, following treatment with S/P. These findings show the importance of studying S/P resistance in Mozambique, and how molecular markers for antimalarial resistance can provide important data for national malaria control policy.

'DEAD-box' helicase from Plasmodium falciparum is active at wide pH and is schizont stage-specific.

BACKGROUND & OBJECTIVES: DNA helicases catalyse unwinding of duplex DNA in an ATP-dependent manner and are involved in all the basic genetic processes. In order to study these important enzymes in the human malaria parasite we have recently cloned the first full-length 'DEAD-box' helicase gene from Plasmodium falciparum (3D7). In the present study, we report some of the important activities of the encoded protein. METHODS: We have expressed the P. falciparum helicase in Escherichia coli and characterised the encoded biochemically active helicase protein. The characterisation of the protein was carried out using radioactively labeled substrate and the standard strand displacement assay. The localisation of the enzyme was studied using immunofluorescence assay. RESULTS & CONCLUSION: P. falciparum helicase gene is 1551 bp in length and encodes for a protein consisting of 516 amino acid residues with a predicted molecular mass of 59.8 kDa. The protein is designated as Plasmodium falciparum DEAD-box helicase 60 kDa in size (PfDH60). Purified PfDH60 showed ATP and Mg2+ dependent DNA unwinding, ssDNA-dependent ATPase and ATP-binding activities. Interestingly, this is a unique helicase because it works at a wide pH range (from 5.0-10.0). The peak expression of PfDH60 is mainly in schizont stages of the development of P. falciparum, where DNA replication is active. The cell-cycle dependent expression suggests that PfDH60 may be involved in the process of DNA replication and distinct cellular processes in the parasite and this study should make an important contribution in our better understanding of DNA metabolic pathways in the parasite.

Generation and characterisation of monoclonal antibodies specific to Plasmodium falciparum enolase.

BACKGROUND AND OBJECTIVES: Glycolysis is the sole source of energy for the intraerythrocytic stages of Plasmodium falciparum, making glycolytic enzymes putative therapeutic targets. Enolase, a single copy gene in P. falciparum is one such enzyme whose activity is elevated approximately 10-15 fold in infected RBC's. It holds the possibility of having multiple biological functions in the parasite and hence can be a suitable candidate for diagnostic and chemotherapeutic purposes. METHODS: We have aimed at generating parasite-specific reagents in the form of monoclonal antibodies. We have raised monoclonal antibodies against the recombinant P. falciparum enolase. RESULTS: Two IgG monoclonals were obtained with 1:1000 titre and specific for P. falciparum enolase. Apicomplexan parasites including P. falciparum enolase has a plant like pentapeptide sequence (104EWGWS108) which is uniquely different from the host counterpart. A peptide spanning this pentapeptide region (ELDGSKNEWGWSKSK) coupled to BSA was used to raise parasite-specific antibody. Four monoclonals were obtained with 1:1000 titre and of IgM isotype. INTERPRETATION AND CONCLUSION: All the monoclonals are specific for P. falciparum enolase and one of them display reactivity against native P. falciparum enolase signifying this pentapeptide to be surface exposed and immunogenic.

Markers for population genetic analysis of human plasmodia species, P. falciparum and P. vivax.

Present report deals with the genetic diversity existing among the field isolates of Plasmodium falciparum and P. vivax in India. Isoenzymes and molecular markers were used to analyse field isolates of P. falciparum and P. vivax. High level of length polymorphism was observed in repeat nucleotide sequences of MSP-1, MSP-2 and GLURP in P. falciparum isolates and CSP, GAM-1 and MSP-3 alpha in P. vivax isolates. In study populations a high proportion of isolates (up to 60%) were comprised of more than one genetically distinct parasite type--multiclonal. Presence of identical allelic forms of enzyme and DNA variations in different geographical areas and in different years suggest that isolates belong to a single random mating population of P. vivax and P. falciparum. Observed random combination of alleles in the field isolates suggest the unlinked nature of loci studied. Study supports the feasibility of using molecular markers for the identification of recrudescence in P. falciparum from fresh infection.

Diagnostic and prognostic utility of rapid strip (OptiMal and Paracheck) versus conventional smear microscopy in adult patients of acute, uncomplicated P. falciparum malaria in Mumbai, India.

OBJECTIVES: The present study compared the diagnostic and prognostic utility of two rapid tests the (Paracheck and OptiMal) versus conventional smear microscopy. METHODS: Using two independent microscopists we carried out the three tests in 31 adult cases of smear positive, acute, uncomplicated Plasmodium falciparum malaria. All three tests were done pretreatment, and on Days 8, 15 and 29. RESULTS: Compared to microscopy, the Paracheck had a sensitivity of 100%, while the OptiMal had a sensitivity of 83.7%. The lower sensitivity of OptiMal resulted from misidentification by both microscopists of 6/31 cases as Plasmodium vivax. As a follow up tool, the OptiMal was better than Paracheck, due to the earlier disappearance of the parasite LDH. Also in the Paracheck, between microscopists, there was a significant difference in reading the tests, on Days 8 and 15. CONCLUSION: Our study reiterates, the continued utility of conventional smear microscopy.

Partial purification of mitochondrial DNA topoisomerase II from Plasmodium falciparum and its sensitivity to inhibitors.

Mitochondria of Plasmodium falciparum (K1 strain) were isolated by differential centrifugation. Mitochondrial DNA topoisomerase II from P. falciparum was partially purified using fast protein liquid chromatography(FPLC). Parasite mitochondria contained approximately 8% of DNA topoisomerase II activity compared with its nuclear fraction. The effects of fluoroquinolones, inhibitors of bacterial DNA topoisomerase II or DNA gyrase, against partially purified P. falciparum mitochondrial DNA topoisomerase II were investigated using a decatenation assay. Minimum inhibitory concentrations (MIC) of ofloxacin, ciprofloxacin and norfloxacin were > 1, 10 and 100 mM, compared with that of >0.5 and 10 mM for eukaryotic DNA topoisomerase II inhibitor etoposide (VP-16) and amsacrine, respectively. The results indicate that partially purified mitochondrial DNA topoisomerase II was insensitive to fluoroquinolones and it is suggested that their inhibitory effects on P. falciparum growth may be directed against plastid DNA topoisomerase II.

Comparison of the OptiMAL rapid test with routine microscopic examination of Giemsa-Stained Thick Blood Film for diagnosis of malaria.

The OptiMAL is a rapid immunodiagnostic test developed by Flow Inc., Portland, Oreg. for diagnosis and differentiation of P. falciparum and non P. falciparum malaria infection. It has been based on detection of circulating parasite lactate dehydrogenase enzyme (pLDH), produced by live Plasmodium parasites. The purpose of this study was to compare the efficacy of the OptiMAL test with routine microscopic examination of Giemsa-Stained Thick Blood Film (routine GS-TBF) for the diagnosis of malaria at a local malaria clinic in a hyperendemic area of Thailand by using a standard GS-TBF (standard GS-TBF) as reference. One hundred and seventy five patients attending the clinic were recruited; 50, 42 and 83 were falciparum malaria, vivax malaria and non-malaria patients, respectively. Compared with the reference, the OptiMAL test had sensitivities of 92 per cent and 97.6 per cent, whereas, the routine GS-TBF had sensitivities of 81.3 per cent and 81 per cent for the detection of P. falciparum and P. vivax, respectively. Both tests showed no false positive resulting in 100 per cent specificities. However, the OptiMAL test was able to detect only 20 per cent of infection with less than 200 parasitaemia/microlitre. It was also shown in our study that the OptiMAL test was advantageous in follow-up of the treatment outcome. No false positive occurred among 40 follow-up cases. The OptiMAL test detected malaria infection more accurately than the routine GS-TBF (p < 0.05) and was simple, easy to perform and rapid. It is an alternative tool for the diagnosis of malaria in a hyperendemic area where experienced microscopists are not available.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon Region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60 percent of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.

Detection of glutamate dehydrogenase enzyme activity in Plasmodium falciparum infection.

We describe the separation of an active glutamate dehydrogenase [GDH (NADP+)] enzyme from the plasma of patients with P. falciparum infection using columns of sepharose anti-GDH (NADP+) of Proteus spp. The activity of this enzyme was also detected in P. falciparum culture supernatant. The parasitic origin of this enzyme was suggested by western blot analysis using anti-P. falciparum culture supernatant and anti-whole parasite antibodies. The differential inhibition of the P. falciparum GDH (NADP+) indicates that some epitopes recognised by the antibodies in both preparations may be different. The determination of P. falciparum GDH (NADP+) activity could be developed into a specific technique for the diagnosis of falciparum malaria.

Characterization of Plasmodium falciparum glutamate dehydrogenase-soluble antigen

The major aim of this study was to characterize a soluble Plasmodium falciparum antigen from the plasma of malaria-infected humans and Plasmodium falciparum culture supernatants, using immunoabsorbent techniques and Western blotting. An Mr 60-kDa protein was isolated from the plasma of patients with Plasmodium falciparum malaria by affinity chromatography using rabbit anti-Proteus spp GDH(NADP+) serum as ligand. This protein, present in plasma of patients with acute Plasmodium falciparum infection, in Plasmodium falciparum culture supernatants, and in immune complexes, was tested with Plasmodium falciparum malaria hyperimmune serum from patients living in hyperendemic areas and rabbit anti-Proteus spp GDH(NADP+) serum prepared in the laboratory. In this report, we describe the results of a study showing that parasite GDH(NADP+) can be used to detect the presence of Plasmodium falciparum. It appears that this technique permits the chromatographic detection of a Plasmodium falciparum excretion antigen that may be used in the production of monoclonal antibodies to improve immunodiagnostic assays for the detection of antigenemia, and opens the possibility of its use as a non-microscopic screening method.