Study population and design
Patients recruited in this study resided in the municipalities of Turbo (08°05′N,76°44′W) and Necoclí (08°25′N,76°47′W) of the Antioquia department, and Puerto Libertador (07°54′N,75°40′W) of the Córdoba department. Together, these departments comprise a high malaria transmission area of Colombia, termed Urabá-Altos Sinú-San Jorge-Bajo Cauca. This region has an estimated area of 43506 km2, with 35 municipalities and a population of 2.5 million at risk of malaria. The epidemiological characteristics of this region have been described elsewhere
[17, 22, 23]. The mean annual parasitic index (malaria cases/1000 inhabitants) during 2000–2009 was 46.6 in Turbo, 74.4 in Necoclí, and 23.4 in Puerto Libertador. Plasmodium vivax was reported in about 70% of malaria cases in the region by microscopic diagnosis
This study included 57 pregnant women at delivery (“delivery group”) with a malaria infection confirmed by quantitative real-time PCR (qPCR) in peripheral and/or placental blood. For comparison, another group of 50 pregnant women with a positive thick smear for P. vivax (n = 30) or P. falciparum (n = 20) during antenatal care (“antenatal group”) were included. A third group included 80 men or non-pregnant women (“non-pregnant group”) with a positive thick smear for P. vivax (n = 20 men and 20 women) or P. falciparum (n = 20 men and 20 women). All subjects resided in the same three municipalities.
Inclusion and exclusion criteria
The general inclusion criteria for the study population were permanent residency in a malaria-endemic community of Turbo, Necoclí or Puerto Libertador, absence of serious general disease, complicated pregnancy or complicated malaria, and informed consent. The only exclusion criterion was consent withdrawal.
Blood samples in the delivery group were collected in EDTA tubes within 8 hours of delivery. Maternal peripheral blood (delivery-periphery group) was obtained by venipuncture. Placental blood (delivery-placenta group) was collected from a pool formed on the maternal side when small sections of placenta were removed (approximate 1 cm3) after cleaning with saline. Peripheral blood from subjects in groups “antenatal” and “non-pregnant” was collected by venipuncture, prior to antimalarial treatment.
Thick smears were made for microscopic examination and dried blood spots were prepared on filter paper (Whatman 3MM) for DNA extraction. Blood spots were sealed in plastic bags, stored at 4°C and transported to the laboratory in Medellín.
Malaria diagnostic tests
Field-stained thick films were read by an experienced microscopist in the local research laboratory. Parasite density was measured by counting the number of parasites per 200 leukocytes, based on a mean count of 8,000 leukocytes/μL of blood. All subjects with a peripheral blood thick smear positive for malaria received anti-malarial treatment according to the guidelines of the Colombian health authorities
For diagnosis by qPCR, an alcohol-sterilized hole punch was used to cut a circle (approximately 6 mm) from each filter paper and DNA was extracted using the Saponin-Chelex method described by Plowe et al. 1995
. The qPCR was performed as described by Shokoples et al. 2009
. Samples were first tested for Plasmodium using a genus-specific set of primers and hydrolysis probe (Plasprobe). Real-time PCR was performed on the ABI 7500 FAST platform, under universal cycling conditions. Samples with a Cycle Threshold (Ct) value under 45 were tested in a duplex species-specific real-time PCR reaction for P. falciparum and P. vivax. Parasite DNA concentration was quantified in the genus-specific screening reaction against a plasmid standard curve of known copy number included in each run. Concentrations are reported as the number of copies of the 18S rRNA gene per microliter of purified DNA.
All molecular markers were amplified by nested or semi-nested PCR, using 3 μL of extracted DNA as template in the first amplification step and 1 μL of the first PCR product for the second amplification. For samples positive for P. vivax, the microsatellites 1.501, 3.502, 3.27, and MS16, as well as the msp3α gene were analyzed based on the protocol described by Koepfli et al. 2009
. The PCR reaction was performed in a final volume of 20 μL containing 1× PCR buffer (Qiagen), 2 mM of MgCL2 (Qiagen), 200 μM of each dNTP (Takara Bio), 0.25 μM of each primer, and 1.5 units of HotStar Taq DNA polymerase (Qiagen).The cycling program was as follows: initial denaturation for 5 min at 95°C; 30 cycles of 1 min at 95°C, 1 min at 56°C–60°C (according to the marker analysed
), 1 min at 72°C; and a final step for 5 min at 72°C.
Samples positive for P. falciparum were genotyped based on the microsatellites ARA2, TA1, Polyα and PFPK2 and the gene msp2. Amplification of microsatellites was according to the protocol described by Anderson et al.. The reaction volume was 15 μL containing 1× PCR buffer, 2 mM of MgCL2, 200 μM of each dNTP, 0.25 μM of each primer, and 0.2 units of HotStar Taq DNA polymerase. The cycling conditions were as follows: initial denaturation for 2 min at 94°C; 45 cycles of 30 sec at 94°C, 30 sec at 42°C, 30 sec at 40°C, and 40 sec at 65°C for the first reaction. The nested reaction consisted of 40 cycles of: 30 sec at 94°C, 30 sec at 45°C and 30 sec at 65°C. The final elongation step in both reactions was 5 min at 65°C. The msp2 gene was amplified based on the protocol described by Felger and Beck
. The reaction volume was 20 μL containing 1× PCR buffer, 1.5 mM of MgCL2, 200 μM of each dNTP, 0.25 μM of each primer for the first reaction and 0.4 μM for the nested reaction, and 0.6 units of HotStar Taq DNA polymerase. The cycling program was: initial denaturation for 2.5 min at 94°C; 40 cycles of 30 sec at 94°C, 45 sec at 42°C for the first reaction or 50°C for the nested reaction, and elongation for 1.5 min at 70°C with a final elongation step of 10 min at 70°C.
All PCR analyses were performed in an Applied Biosystems 2720 Thermal Cycler. Amplification was confirmed by visualization in 2% agarose gels and PCR products were stored at 4°C in the dark. Product sizes were resolved by capillary electrophoresis in an ABI Prism 3100 Genetic Analyzer (Perkin Elmer Applied Biosystems), using GS500 LIZ as internal size standard and the microsatellite conditions as the default settings. The results were analysed using GeneMapper software (version 3.5; Applied Biosystems). All electropherograms were visually inspected; peaks above a cut-off of 300 relative fluorescent units (RFU) were considered true amplification products. Alleles were grouped manually based on their repeat length: 3-bp bins for all the P. falciparum microsatellites, PvMS16 and Pvmsp3α; 4-bp bins for Pv3.27; 7-bp bins for Pv1.501 or 8-bp bins for Pv3.502. Multiple alleles per locus were scored if minor peaks were >33% of the height of the predominant allele present for each locus. All mixed infections were genotyped with molecular makers of both species.
Microsatellite analyzer version 4.05
 was used for calculating allele frequency, expected heterozygosity (He) and the FST index. He was defined as the probability that two clones selected from the population at random carry different alleles, and was calculated with the formula He = [n/(n − 1)] [1 − Σpi
2, where n is the number of isolates analyzed and pi is the frequency of the ith allele in the population. The FST index
 with pairwise comparisons was used to evaluate the genetic differentiation between subpopulations of parasites isolated from the different groups of patients (delivery-periphery vs. delivery-placenta vs. antenatal vs. non-pregnant). Each FST value was tested to determine whether it was statistically different from 0, involving 10000 random permutations of the data. Kruskal-Wallis and Chi-squared tests were used for comparison of continuous and categorical variables, respectively. Significance was set at p <0.05.
Patients or guardians, in case of <18 years of age, signed a voluntary consent form. The study involved minor risk and approval was granted by the Comité de Ética of Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia (Approval Certificate: IIM889ADV) and the Health Research Ethics Board of the University of Alberta.