Triosephosphate isomerase gene promoter variation: -5G/A and -8G/A polymorphisms in clinical malaria groups in two African populations.

TPI1 promoter polymorphisms occur in high prevalence in individuals from African origin. Malaria-patients from Angola and Mozambique were screened for the TPI1 gene promoter variants rs1800200A>G, (-5G>A), rs1800201G>A, (-8G>A), rs1800202T>G, (-24T>G), and for the intron 5 polymorphism rs2071069G>A, (2262G>A). -5G>A and -8G>A variants occur in 47% and 53% in Angola and Mozambique, respectively while -24T>G was monomorphic for the wild-type T allele. Six haplotypes were identified and -8A occurred in 45% of the individuals, especially associated with the GAG haplotype and more frequent in non-severe malaria groups, although not significantly. The arising and dispersion of -5G>A and -8G>A polymorphisms is controversial. Their age was estimated by analyses of two microsatellite loci, CD4 and ATN1, adjacent to TPI1 gene. The -5G>A is older than -8G>A, with an average estimate of approximately 35,000 years. The -8A variant arose in two different backgrounds, suggesting independent mutational events. The first, on the -5G background, may have occurred in East Africa around 20,800 years ago; the second, on the -5A background, may have occurred in West Africa some 7500 years ago. These estimates are within the period of spread of agriculture and the malaria mosquito vector in Africa, which could has been a possible reason for the selection of -8A polymorphism in malaria endemic countries.

Polymorphisms in Plasmodium falciparum K13-propeller in Angola and Mozambique after the introduction of the ACTs.

We report the presence of SNPs in Plasmodium falciparum K13-propeller gene in two African countries, Angola and Mozambique, where malaria is a serious public health problem. Samples were collected before and after ACT introduction as first-line treatment. In each country 50 samples collected before and 50 after ACT introduction were analysed. A total of three different mutations (R471R and R575R in Angola and V494I in Mozambique) were identified in five samples, all collected after the introduction of ACT. The R471R mutation detected in Angola has already been reported in Africa (DR-Congo and Gabon). However, the mutations R575R (Angola) and V494I (Mozambique), have never been reported. V494I is adjacent to the known K13 resistance-associated mutation Y493H, although functional analysis did not predict a deleterious effect on protein function.

Prevalence of pfmdr1 alleles associated with artemether-lumefantrine tolerance/resistance in Maputo before and after the implementation of artemisinin-based combination therapy.

BACKGROUND: Mozambique implemented artemisinin-based combinations therapy (ACT) using artemether-lumefantrine (AL) as the first-line treatment for uncomplicated malaria in 2009. AL remains highly efficacious, but widespread use may soon facilitate emergence of artemisinin tolerance/resistance. The prevalence of pfmdr1 different alleles in Maputo and Mozambique is not known, either after or before the introduction of ACT. Pfmdr1 molecular markers related to Plasmodium falciparum susceptibility were analysed before and after transition to ACT. METHODS: A first group of samples was collected between June 2003 and June 2005 and a second group in the period between March 2010 and March 2012. Three alleles were analysed by PCR-RFLP: N86Y, Y184F and D1246Y, in the pfmdr1 gene. RESULTS: Alleles N86, 184F and D1246 increased from 19.5, 19.6 and 74.4% in 2003-2005 to 73.2, 22.7 and 96.7% in 2010-2012, respectively. After implementation of ACT (2010-2012), pfmdr1 haplotypes, either two- and three-codon basis, were generally less diverse than before the implementation of ACT (2003-2005). The prevalence of haplotypes N86-184Y, N86-D1246 and 184Y-D1246 increased from 12,2, 27.3 and 71.7% in 2003-2005 to 59.4, 84.3 and 78.6% in 2010-2012. The three-codon basis haplotypes NFD and NYD also increased significantly during the same period. CONCLUSION: The alleles N86 and 184 F and the triple haplotype N86-184 F-D1246 showed a significantly increased prevalence after introduction of ACT.

Intestinal parasites in children hospitalized at the Central Hospital in Maputo, Mozambique.

INTRODUCTION: Intestinal parasites are important contributors to the global disease burden, especially in children of low-income countries. The present study determined the frequency of intestinal parasites in children hospitalized at the diarrhea section of the Infectious-Contagious Diseases ward and at the Malnutrition ward of the Department of Pediatrics of the Maputo Central Hospital in Mozambique. METHODOLOGY: This pilot study conducted between February and March 2009 enrolled a total of 93 children between 1.5 and 48.2 months of age; 87.1% were younger than 24 months. Parasite detection in stool samples was achieved using direct microscopic observation and Ritchie's concentration technique. RESULTS: Infection with pathogenic intestinal parasites was detected in 16.1% (15/93) of the children. Giardia duodenalis and Trichuris trichiura were the most common parasites (6.5%, 6/93 each), followed by Ascaris lumbricoides (2.2%, 2/93). One case of mixed infection with A. lumbricoides plus T. trichiura was also detected. CONCLUSION: This study reinforces the importance of routinely examining stool samples for the diagnosis of intestinal parasites (including protozoa) in children hospitalized in endemic areas.

Pyruvate kinase deficiency in sub-Saharan Africa: identification of a highly frequent missense mutation (G829A;Glu277Lys) and association with malaria.

BACKGROUND: Pyruvate kinase (PK) deficiency, causing hemolytic anemia, has been associated to malaria protection and its prevalence in sub-Saharan Africa is not known so far. This work shows the results of a study undertaken to determine PK deficiency occurrence in some sub-Saharan African countries, as well as finding a prevalent PK variant underlying this deficiency. MATERIALS AND METHODS: Blood samples of individuals from four malaria endemic countries (Mozambique, Angola, Equatorial Guinea and Sao Tome and Principe) were analyzed in order to determine PK deficiency occurrence and detect any possible high frequent PK variant mutation. The association between this mutation and malaria was ascertained through association studies involving sample groups from individuals showing different malaria infection and outcome status. RESULTS: The percentage of individuals showing a reduced PK activity in Maputo was 4.1% and the missense mutation G829A (Glu277Lys) in the PKLR gene (only identified in three individuals worldwide to date) was identified in a high frequency. Heterozygous carrier frequency was between 6.7% and 2.6%. A significant association was not detected between either PK reduced activity or allele 829A frequency and malaria infection and outcome, although the variant was more frequent among individuals with uncomplicated malaria. CONCLUSIONS: This was the first study on the occurrence of PK deficiency in several areas of Africa. A common PKLR mutation G829A (Glu277Lys) was identified. A global geographical co-distribution between malaria and high frequency of PK deficiency seems to occur suggesting that malaria may be a selective force raising the frequency of this 277Lys variant.

Modeling the effect of mixing in biodiesel production.

The transesterification reaction models available in the literature are valid only for one particular mixing condition. In this work, a modeling strategy is presented in order to predict the effect of mixing conditions in the transesterification process. The proposed methodology was applied to independent sets of experimental data available in the literature that show the dependency of the transesterification reaction on the frequency of rotation of the stirrer. The accuracy of the developed models corroborates the validity of the proposed modeling approach.

Malaria: looking for selection signatures in the human PKLR gene region.

The genetic component of susceptibility to malaria is both complex and multigenic and the better-known protective polymorphisms are those involving erythrocyte-specific structural proteins and enzymes. In vivo and in vitro data have suggested that pyruvate kinase deficiency, which causes a nonspherocytic haemolytic anaemia, could be protective against malaria severity in humans, but this hypothesis remains to be tested. In the present study, we conducted a combined analysis of Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs) in the pyruvate kinase-encoding gene (PKLR) and adjacent regions (chromosome 1q21) to look for malaria selective signatures in two sub-Saharan African populations from Angola and Mozambique, in several groups with different malaria infection outcome. A European population from Portugal, including a control and a pyruvate kinase-deficient group, was used for comparison. Data from STR and SNP loci spread along the PKLR gene region showed a considerably higher differentiation between African and Portuguese populations than that usually found for neutral markers. In addition, a wider region showing strong linkage disequilibrium was found in an uncomplicated malaria group, and a haplotype was found to be associated with this clinical group. Altogether, this data suggests that malaria selective pressure is acting in this genomic region.

Contribuição para o estudo de resistência aos antimaláricos e análise de marcadores moleculares de Plasmodium falciparum e do hospedeiro humano, em Maputo, Moçambique / Contribution to the study of antimalarial resistance and analysis of molecular markers of Plasmodium falciparum and the human host, in Maputo, Mozambique

A malária é de longe a doença parasitária mais importante em Moçambique, constituindo um grave problema de saúde pública no País. Apesar de se considerar que um diagnóstico atempado e um tratamento correcto são os elementos básicos para um programa de controlo da malária bem sucedido, nas últimas décadas, o controlo e tratamento têm sido bastante dificultados pelo aparecimento e disseminação da resistência parasitária aos antimaláricos mais utilizados. Os mecanismos que conferem ao parasita a capacidade de resistir à maioria dos antimaláricos disponíveis não se encontram completamente elucidados. Deste modo, este trabalho teve como objectivo principal avaliar o envolvimento dos genes pfdhfr, pfdhps, pfcrt, pfmdr1 e pfATPase6 na resistência aos antimaláricos em Moçambique, recorrendo a populações naturais de parasitas em isolados colhidos de doentes com malária em Maputo, Moçambique. Neste contexto, foi efectuada a caracterização clínica dos pacientes com diferentes formas clínicas de malária e os perfis genotípicos relacionados com a resistência em P. falciparum para quatro antimaláricos: cloroquina, sulfadoxina/pirimetamina, amodiaquina e artemisinina, através da técnica de PCR-RFLP, para os polimorfismos pfcrt K76T e N75E, pfdhfr N51I, C59R, S108N e I164L, pfdhps A437G e K540E, pfmdr1 N86Y e N1246Y e pfATPase6, G1916A, G110A, A2694T e G2306A. Os dados genotípicos foram subsequentemente analisados estatísticamente, no intuito de detectar associações significativas entre a presença de um determinado marcador antes e depois do tratamento com os diferentes antimaláricos utilizados. Foram também avaliados os polimorfismos em dois marcadores moleculares (ICAM-1 e CD36) do hospedeiro relacionados com susceptibilidade/resistência à malária, em isolados de pacientes com e sem malária. Os polimorfismos da variante CYP-450 (CYP2C8), relacionada com o metabolismo dos fármacos antimaláricos em pacientes com malária, foram também aqui analisados. Os resultados demonstraram a gravidade do problema de resistência a antimaláricos, evidenciado pelas elevadas prevalências dos alelos mutantes antes e depois dos tratamentos efectuados. Foi aqui observada uma elevação significativa de amostras contendo o alelo mutado pfdhps 437G após do tratamento com Fansidar® e com Fansidar®+Amodiaquina. Verificou-se existir uma correlação positiva entre o quíntuplo mutante e o número de isolados, depois do tratamento com Fansidar®. Estes resultados indicam reservas na utilização do Fansidar ® como uma componente da primeira linha de tratamento antimalárico no País e apontam o polimorfismo pfdhps 437G como um possível marcador para a monitorização da resistência a este fármaco. Apesar de não significativos, os resultados da análise do ICAM-1 mostraram a existência de uma possível associação entre a presença da mutação ICAM-1kilifi e a infecção malárica nas suas formas grave e não grave, enquanto para o CD36 foi notória a ausência da mutação T1264G no grupo controle (sem malária). Os resultados da análise dos polimorfismos no gene CYP2C8 demonstraram alguma inconclusividade, tendo no entanto permitido a obtenção de conhecimentos para estudos que possam ser realizados no futuro.

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

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.

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.

Analysis of sulphadoxine/pyrimethamine resistance-conferring mutations of Plasmodium falciparum from Mozambique reveals the absence of the dihydrofolate reductase 164L mutant.

BACKGROUND: Plasmodium falciparum is the predominant human malaria species in Mozambique and a lead cause of mortality among children and pregnant women nationwide. Sulphadoxine/pyrimethamine (S/P) is used as first line antimalarial treatment as a partner drug in combination with artesunate. METHODS: A total of 92 P. falciparum-infected blood samples, from children with uncomplicated malaria attending the Centro de Saude de Bagamoyo in the Province of Maputo-Mozambique, were screened for S/P resistance-conferring mutations in the pfdhfr and pfdhps genes using a nested mutation-specific polymerase chain reaction and restriction digestion (PCR-RFLP). The panel of genetic polymorphisms analysed included the pfdhfr 164L mutation, previously reported to be absent or rare in Africa. RESULTS: The frequency of the S/P resistance-associated pfdhfr triple mutants (51I/59R/108N) and of pfdhfr/pfdhps quintuple mutants (51I/59R/108N + 437G/540E) was 93% and 47%, respectively. However, no pfdhfr 164L mutants were detected. CONCLUSION: The observation that a considerably high percentage of P. falciparum parasites contained S/P resistance-associated mutations raises concerns about the validity of this drug as first-choice treatment in Mozambique. On the other hand, no pfdhfr 164L mutant was disclosed, corroborating the view that that this allele is still rare in Africa.