Amanda Maestre

Multiple independent introductions of Plasmodium falciparum in South America

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence—archeological and genetic—suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Diagnosis of gestational, congenital, and placental malaria in Colombia: comparison of the efficacy of microscopy, nested polymerase chain reaction, and histopathology.

The technical capability of different methods to diagnose Plasmodium in maternal peripheral blood, placenta, and umbilical cord blood has not been assessed in Colombia and seldom explored in other malaria-endemic regions. We designed a study to compare the technical and the operational-economical performances of light microscopy (LM), nested polymerase chain reaction (nPCR), and histopathology (HP). In maternal blood, LM had 41% sensitivity and 100% specificity and in placental blood, 35% and 100%, respectively, compared with nPCR. In placental tissue, LM had 33% sensitivity and 95% specificity; and nPCR 47% and 77%, respectively; compared with HP. Light microscopy had the best operational-economical qualification. We concluded that nPCR and HP performed better compared with LM, but field implementation of these two techniques remains a problem. Therefore, LM is recommended as the gold standard for diagnosis of gestational malaria and placental blood infection in the field.

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

BackgroundReal-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products.MethodsReagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested.ResultsAmplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA.ConclusionsThe methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings.

Inter-allelic recombination in the Plasmodium vivax merozoite surface protein 1 gene among Indian and Colombian isolates

BackgroundA major concern in malaria vaccine development is the polymorphism observed among different Plasmodium isolates in different geographical areas across the globe. The merozoite surface protein 1 (MSP-1) is a leading vaccine candidate antigen against asexual blood stages of malaria parasite. To date, little is known about the extent of sequence variation in the Plasmodium vivax MSP-1 gene (Pvmsp-1) among Indian isolates. Since P. vivax accounts for >50% of malaria cases in India and in Colombia, it is essential to know the Pvmsp-1 gene variability in these two countries to sustain it as a vaccine candidate. The extent of polymorphism in Pvmsp-1 gene among Indian and Colombian isolates is described.MethodsThe sequence variation in the region encompassing the inter-species conserved blocks (ICBs) five and six of Pvmsp-1 gene was examined. PCR was carried out to amplify the polymorphic region of Pvmsp-1 and the PCR products from twenty (nine Indian and 11 Colombian) isolates were sequenced and aligned with Belem and Salvador-1 sequences.ResultsResults revealed three distinct types of sequences among these isolates, namely, Salvador-like, Belem-like and a third type sequence which was generated due to interallelic recombination between Salvador-like sequences and Belem-like sequences. Existence of the third type in majority (44%) showed that allelic recombinations play an important role in PvMSP1 diversity in natural parasite population. Micro-heterogeneity was also seen in a few of these isolates due to nucleotide substitutions, insertions as well as deletions.ConclusionsIntergenic recombination in the Pvmsp-1 gene was found and suggest that this is the main cause for genetic diversity of the Pvmsp-1 gene.

High genetic polymorphism of relapsing P. vivax isolates in northwest Colombia.

Graphical abstract Highlights ► Microsatellite markers were used to genotype P. vivax in of Colombia. ► A total 54 haplotypes in 58 paired (primary-recurrence/relapse) samples were found. ► Polymorphism in samples from endemic area was high. ► Relapsing and primary isolates had a different genetic conformation. ► The selected markers are useful to study P. vivax populations in the country.

Prevention of Plasmodium vivax malaria recurrence: efficacy of the standard total dose of primaquine administered over 3 days.

BACKGROUND The standard total dose (STD) of primaquine to prevent Plasmodium vivax recurrence is 0.25mg/kg day administered over 14 days (STD-14). We evaluated, in an endemic zone of Colombia, the anti-recurrence efficacy of the STD dose administered over 3 and 14 days, and of sub-STD dose administered over 3 days (71%STD-3, 50%STD-3). METHODS A controlled clinical trial was carried out with 188 subjects allocated into one of four treatment groups: STD-14, STD-3, 71%STD-3, 50%STD-3. RESULTS Recurrences during the 120 days of follow-up were 15% in STD-14, and 57% in STD-3. Treatment with 71%STD-3 and 50%STD-3 resulted in recurrence in >48% subjects within 120 days after the primary episode. High daily doses (1.17 mg/kg day) were well tolerated. CONCLUSIONS (a) The standard dose and regimen (STD-14) of primaquine to prevent P. vivax relapse is recommended. The administration of the same dose over 3 days (STD-3) should be avoided; (b) doses lower than the STD doses administered over 3 days are ineffective in preventing relapse.

Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase

BackgroundIn order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions.MethodsThe P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network.ResultsSynonymous and non-synonymous single nucleotide polymorphisms (SNPs) within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel). SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N) dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T) and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T) mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu.ConclusionIt was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts sharply with the selective sweep of rare antifolate resistant alleles observed in the P. falciparum populations in Asia and Africa. The finding of multiple origins of resistance-conferring mutations has important implications for drug policy.

Molecular Detection of Malaria at Delivery Reveals a High Frequency of Submicroscopic Infections and Associated Placental Damage in Pregnant Women from Northwest Colombia

Plasmodium infection in pregnancy causes substantial maternal and infant morbidity and mortality. In Colombia, both P. falciparum and P. vivax are endemic, but the impact of either species on pregnancy is largely unknown in this country. A cross-sectional study was carried out with 96 pregnant women who delivered at their local hospital. Maternal, placental, and cord blood were tested for malaria infection by microscopy and real-time quantitative polymerase chain reaction (qPCR). A high frequency of infection was detected by qPCR (45%). These infections had low concentrations of parasite DNA, and 79% were submicroscopic. Submicroscopic infections were associated with placental villitis and intervillitis. In conclusion, the overall frequency of Plasmodium infection at delivery in Colombia is much higher than previously reported. These data prompt a re-examination of the local epidemiology of malaria using molecular diagnostics to establish the clinical relevance of submicroscopic infections during pregnancy as well as their consequences for mothers and newborns.

Efecto de la infección submicroscópica o policlonal de Plasmodium falciparum sobre la madre y el producto de la gestación: revisión sistemática

BACKGROUND Malaria in pregnancy causes substantial maternal and infant morbidity-mortality, even at submicroscopic parasite levels. In addition, the presence of polyclonal infections secondary to high parasite genetic diversity is a common finding. OBJECTIVES To determine the frequency of submicroscopic and/or polyclonal plasmodial infection during pregnancy and to establish their impact on clinical presentation, immunity acquisition, and consequences on mother and gestation product. METHODS A search on Medline was performed using key words (MeSH): pregnancy, malaria, PCR, microscopy, genotype, and clones. Studies on plasmodial infection diagnosed by microscopy and PCR were selected. RESULTS A total of 16 studies were included, all carried out in Africa. The weighted mean (WM) of submicroscopic infection was 36%. According to type of infection (microscopic, submicroscopic or negative), the WM of maternal anemia and low birth weight (LBW) were 51%, 42%, 33%, and 19%, 16%, 11%, respectively. Risks (OR), using the negative group as reference, were: a) for maternal anemia 2.12 in microscopic infection and 1.48 in submicroscopic; b) for LBW 1.89 in microscopic and 1.56 in submicroscopic infection. The WM of polyclonal infection was 75% and the mean number of clones by sample was three. CONCLUSIONS Submicroscopic and polyclonal P. falciparum infections during pregnancy are very common, but have been little studied and their impact must be assessed in each specific region because they depend on malaria transmission intensity and stability, maternal age and parity, among other variables, which are influenced by environmental and socio-economic conditions of each region.

Placental Malaria in Colombia: Histopathologic Findings in Plasmodium vivax and P. falciparum Infections

Studies on gestational malaria and placental malaria have been scarce in malaria-endemic areas of the Western Hemisphere. To describe the histopathology of placental malaria in Colombia, a longitudinal descriptive study was conducted. In this study, 179 placentas were studied by histologic analysis (112 with gestational malaria and 67 negative for malaria). Placental malaria was confirmed in 22.35%, 50.0% had previous infections, and 47.5% had acute infections. Typical malaria-associated changes were observed in 37%. The most common changes were villitis, intervillitis, deciduitis, increased fibrin deposition, increased syncytial knots, mononuclear (monocytes/macrophages and lymphocytes), polymorphonuclear cell infiltration, and trophozoites in fetal erythrocytes. No association was found between type of placental changes observed and histopathologic classification of placental malaria. The findings are consistent with those reported for placental malaria in other regions. Plasmodium vivax was the main parasite responsible for placental and gestational malaria, but its role in the pathogenesis of placental malaria was not conclusive.