Effect of Plasmodium Infection during Pregnancy on Passive Neonatal Immunity against Tetanus Toxoid and Rotavirus.

Passive immunity acquired through transplacental IgG transport is essential to protect infants against pathogens as childhood vaccination programs begins. Diarrhea caused by rotavirus and neonatal tetanus are common and potentially fatal childhood infections that can be prevented by transplacental IgG. However, it is not known whether maternal infections in pregnancy can reduce the transfer of these antibodies to the fetus. This study evaluated the effect of submicroscopic Plasmodium infection during pregnancy on the transfer of maternal IgG antibodies against rotavirus (anti-RV) and tetanus toxoid (anti-TT) to newborns of pregnant women residing in Puerto Libertador and Tierralta, Colombia. Expression of different immune mediators and levels of IgG against rotavirus and tetanus toxoid were quantified in pregnant women with and without Plasmodium infection during pregnancy. Submicroscopic infection at the time of delivery was associated with a cord-to-maternal ratio (CMR) > 1 for anti-RV and < 1 for anti-TT IgG, as well as with an increase in the expression of immune mediators of inflammation (IFN-γ), anti-inflammation (IL-10, TGF-ß), and regulation (FoxP3, CTLA-4). When compared by species, these findings (CMR > 1 for anti-RV and < 1 for anti-TT IgG) were conserved in submicroscopic Plasmodium vivax infections at delivery. The impact of Plasmodium infections on neonatal susceptibility to other infections warrants further exploration.

Asymptomatic plasmodial infection in Colombian pregnant women.

Information about asymptomatic plasmodial infection is scarce in the world, and the current antimalarial program goals (control, elimination, and eradication) demand this evidence to be well documented in different populations and malaria transmission settings. This study aimed to measure the prevalence of API in Colombian pregnant women at delivery. A retrospective prevalence survey was used. Women were recruited at hospital obstetric facility in each of the municipalities of Turbo, Necoclí in Antioquia department, and Puerto Libertador in Córdoba department. Malaria infection was tested by thick blood smear (TBS) and real-time quantitative PCR (qPCR). Ninety-six pregnant women at delivery were studied: 95% were asymptomatic (91/96), 45% had asymptomatic plasmodial infection (API) by qPCR (41/91), and only 8% (7/91) had API by microscopy. The prevalence of submicroscopic infections (TBS negative and qPCR positive) was very high, 37% (34/91) in asymptomatic women and 41% (39/96) in total women studied (91 asymptomatic and 5 symptomatic). The prevalence of API in Colombian pregnant women is much higher than which is expected for a country that does not have the level of malaria transmission as Sub-Saharan African countries.

Submicroscopic infection of placenta by Plasmodium produces Th1/Th2 cytokine imbalance, inflammation and hypoxia in women from north-west Colombia.

BACKGROUND: A large-scale study was set up in order to study the epidemiology, clinical aspects, and immunopathology of gestational and placental malaria in north-west Colombia. In this region, recent reports using a qPCR technique, confirmed frequencies of infection, by Plasmodium falciparum or Plasmodium vivax, up to 45%. Given the high rates of infection observed both in mother and placenta, a first exploratory study was proposed in order to characterize the effect on the inflammation status, tissue damage and hypoxia in Plasmodium spp. infected placentas. METHODS: A descriptive, prospective, cross-sectional design was applied to pregnant women with (PM+) and without (PM-) placental malaria. Messenger RNA expression of Fas, FasL; COX-1, COX-2, HIF, VEGF, and the cytokines IL-2, IL-4, IL-10, IFN-γ and TNF, were measured in peripheral and placental blood using a quantitative PCR. The percentage of apoptotic cells was determined with a TUNEL assay. RESULTS: In total 50 placentas were studied: 25 were positive for submicroscopic infection and 25 were negative for Plasmodium infection. Expression of IL-4 and IL-10 was observed high in placental tissue of PM+, while IL-2 was high in peripheral blood of the same group. Expression of TNF and IFNγ in peripheral blood of the PM + group was high. Similarly, the apoptotic index and Fas expression were significantly high in PM+. However, FasL expression was observed low in PM + compared to PM-. Inflammation markers (HIF, VEGF) and hypoxia markers (COX-1, COX-2) were high in the PM + group. CONCLUSION: During placental malaria expression of some pro-inflammatory cytokines is up-regulated and markers of hypoxia and tissue damage are increased in cases of submicroscopic infection.

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.

Genotype comparison of Plasmodium vivax and Plasmodium falciparum clones from pregnant and non-pregnant populations in North-west Colombia.

BACKGROUND: Placental malaria is the predominant pathology secondary to malaria in pregnancy, causing substantial maternal and infant morbidity and mortality in tropical areas. While it is clear that placental parasites are phenotypically different from those in the peripheral circulation, it is not known whether unique genotypes are associated specifically with placental infection or perhaps more generally with pregnancy. In this study, genetic analysis was performed on Plasmodium vivax and Plasmodium falciparum parasites isolated from peripheral and placental blood in pregnant women living in North-west Colombia, and compared with parasites causing acute malaria in non-pregnant populations. METHODS: A total of 57 pregnant women at delivery with malaria infection confirmed by real-time PCR in peripheral or placental blood were included, as well as 50 pregnant women in antenatal care and 80 men or non-pregnant women with acute malaria confirmed by a positive thick smear for P. vivax or P. falciparum. Five molecular markers per species were genotyped by nested PCR and capillary electrophoresis. Genetic diversity and the fixation index FST per species and study group were calculated and compared. RESULTS: Almost all infections at delivery were asymptomatic with significantly lower levels of infection compared with the groups with acute malaria. Expected heterozygosity for P. vivax molecular markers ranged from 0.765 to 0.928 and for P. falciparum markers ranged from 0.331 to 0.604. For P. vivax infections, the genetic diversity was similar amongst the four study groups and the fixation index from each pairwise comparison failed to show significant genetic differentiation. For P. falciparum, no genetic differentiation was observed between placental and peripheral parasites from the same woman at delivery, but the parasites isolated at delivery showed significant genetic differentiation compared with parasites isolated from subjects with acute malaria. CONCLUSIONS: In North-west Colombia, P. vivax parasites have high genetic diversity that is equivalent in pregnant and non-pregnant populations as well as in symptomatic and asymptomatic infections. For P. falciparum, the overall genetic diversity is lower, with specific genotypes associated with asymptomatic infections at delivery.

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

BACKGROUND: Real-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. METHODS: Reagents 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. RESULTS: Amplification 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. CONCLUSIONS: The 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.