Parasite-host interaction in malaria: genetic clues and copy number variation

In humans, infections contribute highly to mortality and morbidity rates worldwide. Malaria tropica is one of the major infectious diseases globally and is caused by the protozoan parasite Plasmodium falciparum. Plasmodia have accompanied human beings since the emergence of humankind. Due to its pathogenicity, malaria is a powerful selective force on the human genome. Genetic epidemiology approaches such as family and twin studies, candidate gene studies, and disease-association studies have identified a number of genes that mediate relative protection against the severest forms of the disease. New molecular approaches, including genome-wide association studies, have recently been performed to expand our knowledge on the functional effect of human variation in malaria. For the future, a systematic determination of gene-dosage effects and expression profiles of protective genes might unveil the functional impact of structural alterations in these genes on either side of the host-parasite interaction

Molecular surveillance of mutations in the cytochrome b gene of Plasmodium falciparum in Gabon and Ethiopia

BACKGROUND: Atovaquone is part of the antimalarial drug combination atovaquone-proguanil (Malarone(®)) and inhibits the cytochrome bc(1 )complex of the electron transport chain in Plasmodium spp. Molecular modelling showed that amino acid mutations are clustered around a putative atovaquone-binding site resulting in a reduced binding affinity of atovaquone for plasmodial cytochrome b, thus resulting in drug resistance. METHODS: The prevalence of cytochrome b point mutations possibly conferring atovaquone resistance in Plasmodium falciparum isolates in atovaquone treatment-naïve patient cohorts from Lambaréné, Gabon and from South Western Ethiopia was assessed. RESULTS: Four/40 (10%) mutant types (four different single polymorphisms, one leading to an amino acid change from M to I in a single case) in Gabonese isolates, but all 141/141 isolates were wild type in Ethiopia were found. CONCLUSION: In the absence of drug pressure, spontaneous and possibly resistance-conferring mutations are rare

A globally occurring indel polymorphism in the promoter of the IFNA2 gene is not associated with severity of malaria but with the positivity rate of HCV

<p>Abstract</p> <p>Background</p> <p>Type I Interferons (IFNs) are well known cytokines which exert antiviral activity, antitumor activity and immunomodulatory effects. Single-nucleotide polymorphisms (SNP) and deletions in the gene coding for <it>IFNA2 </it>have been shown to influence the level of expression <it>in vitro</it>. The indel polymorphism -305_-300delAACTTT showed the strongest effect <it>in vitro</it>. To analyse the worldwide distribution of this polymorphism we analyzed five different populations (586 Vietnamese, 199 Central Africans, 265 Brazilians, 108 Kaingang and 98 Guarani). To investigate a possible association with susceptibility to infectious diseases we determined the polymorphism in malaria patients suffering either mild or severe malaria and in a cohort of hepatitis C virus infected individuals.</p> <p>Results</p> <p>We could detect the indel polymorphism in all populations analysed. There was no association with this polymorphism and the outcome of malaria but we found an increase of this indel polymorphism in hepatitis C virus positive individuals compared to healthy controls (p = 0.014).</p> <p>Conclusion</p> <p>Polymorphisms in genes involved in the interferon pathway have been implicated in the resistance or susceptibility against cerebral malaria and HBV. Here we show that an indel polymorphism, which mediates a disadvantageous effect in HBV patients, may also play a disadvantageous role in HCV infections stressing the importance of a fully functional interferon pathway.</p

Rapid detection of Pfcrt and Pfmdr1 mutations in Plasmodium falciparum isolates by FRET and in vivo response to chloroquine among children from Osogbo, Nigeria

BACKGROUND: Chloroquine (CQ) has been in use in Africa for a long time. Because of misuse, this drug has now lost its efficacy due to the emergence of resistance strains in most parts of Africa. Recently, it was shown that after chloroquine has been withdrawn from the market, chloroquine-sensitive Plasmodium falciparum re-emerged and chloroquine could again be used successfully as an antimalarial. Surveillance of parasite populations is, therefore, important to decide whether chloroquine could be re-introduced. METHODS: To estimate the prevalence of the most pivotal polymorphisms, including Pfcrt K76T, Pfmdr1 N86Y and Pfmdr1 Y184F mutations, and their contributions to the outcome of CQ treatment, isolates from Osogbo Western Nigeria were tested using the Fluorescence Resonance Energy Transfer (FRET) method on a real-time PCR instrument. RESULTS: 116 children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of CQ and followed-up for 28 days. Blood samples were collected on filter paper at enrollment and during follow-up for identification of parasite carrying the chloroquine resistant transporter (pfcrt) and P. falciparum-multi drug resistance (pfmdr1) gene mutations. Parasitological assessment of response to treatment showed that 62% of the patients were cured and 38% failed the CQ treatment. The presence of single mutant pfcrt (T76) alleles (P = 0.003) and in combination with mutant pfmdr1 Y86 (P = 0.028) was significantly associated with in vivo CQR. No other mutation on its own or in combinations was significantly associated with treatment outcome. Mutant pfcrt was more prevalent in both pre- and post-treatment isolates. No association was observed between age or initial level of parasitaemia and chloroquine treatment outcome. CONCLUSION: The result established the usefulness and accuracy of real time PCR in pfcrt and pfmdr1 mutation detection and also give further evidence to the reliability of the pfcrt T76 point mutation as a molecular marker for CQ resistance

Haplotype specific-sequencing reveals MBL2 association with asymptomatic Plasmodium falciparum infection

<p>Abstract</p> <p>Background</p> <p>Mannose binding lectin (MBL) has an important role in the activation of the complement system and opsonization of pathogenic microorganisms. Frequent polymorphisms found in the <it>MBL2 </it>gene affect the concentration and functionality of the protein and are associated with enhanced susceptibility to severe malaria in African children. Most <it>MBL2 </it>typing strategies were designed to the analysis of selected variants, the significance of whole haplotypes is poorly known. In this work, a new typing strategy was developed and validated in an association analysis of <it>MBL2 </it>with adult asymptomatic infection.</p> <p>Methods</p> <p><it>MBL2 </it>allele-specific fragments of 144 healthy Gabonese adults were amplified by using haplotype-specific sequencing (HSS), a new strategy that combines sequence-specific PCR and sequence-based typing. The Gabonese were investigated for the presence of <it>Plasmodium falciparum </it>parasitaemia by the amplification of parasite genes, immunochromatographic antigen detection and microscopic analysis. HSS results were also compared with a previously used real-time PCR (RT-PCR) method in 72 Euro-Brazilians.</p> <p>Results</p> <p>Fourteen polymorphisms were identified beside the commonly investigated promoter (<it>H, L</it>; <it>X, Y</it>; <it>P, Q</it>) and exon 1 (<it>A, O</it>; <it>O </it>= <it>B</it>, <it>C </it>or <it>D</it>) variants. The <it>MBL2*LYPA/LYPA </it>genotype was associated with the absence of asymptomatic infection (P = 0.017), whereas the <it>MBL2*LYQC </it>haplotype and <it>YA/YO </it>+ <it>YO/YO </it>genotypes were associated with positive parasite counts in asymptomatic adults (P = 0.033 and 0.018, respectively). The associations were specific to <it>LYPA </it>(identical to the reference sequence Y16577) and <it>LYQC </it>(Y16578) and would not have been revealed by standard genotyping, as there was no association with <it>LYPA </it>and <it>LYQC </it>haplotypes carrying new polymorphisms defined by sequence-based typing. In contrast, HSS and RT-PCR produced very similar results in the less diverse European-derived population.</p> <p>Conclusion</p> <p>In this work, a new typing strategy for a highly polymorphic gene was developed and validated focusing on the asymptomatic status of <it>P. falciparum-</it>infected adults. In populations with high nucleotide diversity, it allowed for the identification of associations with fine-scaled haplotypes that would not have been found using common typing techniques. In this preliminary study, <it>MBL2 </it>haplotypes or SNPs linked to them were found associated with susceptibility to infection and parasitaemia control of asymptomatic adults.</p

Artesunate – amodiaquine combination therapy for falciparum malaria in young Gabonese children

BACKGROUND: Artesunate-amodiaquine combination for the treatment of childhood malaria is one of the artemisinin combination therapies (ACTs) recommended by National authorities in many African countries today. Effectiveness data on this combination in young children is scarce. METHODS: The effectiveness of three daily doses of artesunate plus amodiaquine combination given unsupervised (n = 32), compared with the efficacy when given under full supervision (n = 29) to children with falciparum malaria were assessed in an unrandomized study. RESULTS: 61 patients analysed revealed a PCR-corrected day-28 cure rate of 86 % (25 of 29 patients; CI 69 – 95 %) in the supervised group and 63 % (20 of 32 patients; CI 45 – 77 %) in the unsupervised group. The difference in outcome between both groups was statistically significant (p = 0.04). No severe adverse events were reported. CONCLUSION: The effectiveness of this short course regimen in young children with falciparum malaria could be augmented by increased adherence and improved formulation

Phylogenetic nomenclature and evolution of mannose-binding lectin (MBL2) haplotypes

<p>Abstract</p> <p>Background</p> <p>Polymorphisms of the mannose-binding lectin gene (<it>MBL2</it>) affect the concentration and functional efficiency of the protein. We recently used haplotype-specific sequencing to identify 23 <it>MBL2 </it>haplotypes, associated with enhanced susceptibility to several diseases.</p> <p>Results</p> <p>In this work, we applied the same method in 288 and 470 chromosomes from Gabonese and European adults, respectively, and found three new haplotypes in the last group. We propose a phylogenetic nomenclature to standardize <it>MBL2 </it>studies and found two major phylogenetic branches due to six strongly linked polymorphisms associated with high MBL production. They presented high Fst values and were imbedded in regions with high nucleotide diversity and significant Tajima's D values. Compared to others using small sample sizes and unphased genotypic data, we found differences in haplotyping, frequency estimation, Fu and Li's D* and Fst results.</p> <p>Conclusion</p> <p>Using extensive testing for selective neutrality, we confirmed that stochastic evolutionary factors have had a major role in shaping this polymorphic gene worldwide.</p

High prevalence of dhfr triple mutant and correlation with high rates of sulphadoxine-pyrimethamine treatment failures in vivo in Gabonese children

BACKGROUND: Drug resistance contributes to the global malaria burden. Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) polymorphisms confer resistance to sulphadoxine-pyrimethamine (SP). METHODS: The study assessed the frequency of SP resistance-conferring polymorphisms in Plasmodium falciparum-positive samples from two clinical studies in Lambaréné. Their role on treatment responses and transmission potential was studied in an efficacy open-label clinical trial with a 28-day follow-up in 29 children under five with uncomplicated malaria. RESULTS: SP was well tolerated by all subjects in vivo. Three subjects were excluded from per-protocol analysis. PCR-corrected, 12/26 (46%) achieved an adequate clinical and parasitological response, 13/26 (50%) were late parasitological failures, while 1/26 (4%) had an early treatment failure, resulting in early trial discontinuation. Of 106 isolates, 98 (92%) carried the triple mutant dhfr haplotype. Three point mutations were found in dhps in a variety of haplotypic configurations. The 437G + 540E double mutant allele was found for the first time in Gabon. CONCLUSIONS: There is a high prevalence of dhfr triple mutant with some dhps point mutations in Gabon, in line with treatment failures observed, and molecular markers of SP resistance should be closely monitored

Limited response of NK92 cells to Plasmodium falciparum-infected erythrocytes

<p>Abstract</p> <p>Background</p> <p>Mechanisms by which anti-malarial immune responses occur are still not fully clear. Natural killer (NK) cells are thought to play a pivotal role in innate responses against <it>Plasmodium falciparum</it>. In this study, the suitability of NK92 cells as models for the NK mechanisms involved in the immune response against malaria was investigated.</p> <p>Methods</p> <p>NK92 cells were assessed for several signs of activation and cytotoxicity due to contact to parasites and were as well examined by oligonucleotide microarrays for an insight on the impact <it>P. falciparum</it>-infected erythrocytes have on their transcriptome. To address the parasite side of such interaction, growth inhibition assays were performed including non-NK cells as controls.</p> <p>Results</p> <p>By performing microarrays with NK92 cells, the impact of parasites on a transcriptional level was observed. The findings show that, although not evidently activated by iRBCs, NK92 cells show transcriptional signs of priming and proliferation. In addition, decreased parasitaemia was observed due to co-incubation with NK92 cells. However, such effect might not be NK-specific since irrelevant cells also affected parasite growth <it>in vitro</it>.</p> <p>Conclusions</p> <p>Although NK92 cells are here shown to behave as poor models for the NK immune response against parasites, the results obtained in this study may be of use for future investigations regarding host-parasites interactions in malaria.</p