A conserved epitope in VAR2CSA is targeted by a cross-reactive antibody originating from Plasmodium vivax Duffy binding protein.

During Plasmodium falciparum infection in pregnancy, VAR2CSA is expressed on the surface of infected erythrocytes (IEs) and mediates their sequestration in the placenta. As a result, antibodies to VAR2CSA are largely restricted to women who were infected during pregnancy. However, we discovered that VAR2CSA antibodies can also be elicited by P. vivax Duffy binding protein (PvDBP). We proposed that infection with P. vivax in non-pregnant individuals can generate antibodies that cross-react with VAR2CSA. To better understand the specificity of these antibodies, we took advantage of a mouse monoclonal antibody (3D10) raised against PvDBP that cross-reacts with VAR2CSA and identified the epitopes targeted by this antibody. We screened two peptide arrays that span the ectodomain of VAR2CSA from the FCR3 and NF54 alleles. Based on the top epitope recognized by 3D10, we designed a 34-amino acid synthetic peptide, which we call CRP1, that maps to a highly conserved region in DBL3X. Specific lysine residues are critical for 3D10 recognition, and these same amino acids are within a previously defined chondroitin sulfate A (CSA) binding site in DBL3X. We showed by isothermal titration calorimetry that the CRP1 peptide can bind directly to CSA, and antibodies to CRP1 raised in rats significantly blocked the binding of IEs to CSA in vitro. In our Colombian cohorts of pregnant and non-pregnant individuals, at least 45% were seroreactive to CRP1. Antibody reactivities to CRP1 and the 3D10 natural epitope in PvDBP region II, subdomain 1 (SD1), were strongly correlated in both cohorts. These findings suggest that antibodies arising from PvDBP may cross-react with VAR2CSA through the epitope in CRP1 and that CRP1 could be a potential vaccine candidate to target a distinct CSA binding site in VAR2CSA.

Efficacy of Single-Dose Primaquine With Artemisinin Combination Therapy on Plasmodium falciparum Gametocytes and Transmission: An Individual Patient Meta-Analysis.

BACKGROUND: Since the World Health Organization recommended single low-dose (0.25 mg/kg) primaquine (PQ) in combination with artemisinin-based combination therapies (ACTs) in areas of low transmission or artemisinin-resistant Plasmodium falciparum, several single-site studies have been conducted to assess efficacy. METHODS: An individual patient meta-analysis to assess gametocytocidal and transmission-blocking efficacy of PQ in combination with different ACTs was conducted. Random effects logistic regression was used to quantify PQ effect on (1) gametocyte carriage in the first 2 weeks post treatment; and (2) the probability of infecting at least 1 mosquito or of a mosquito becoming infected. RESULTS: In 2574 participants from 14 studies, PQ reduced PCR-determined gametocyte carriage on days 7 and 14, most apparently in patients presenting with gametocytemia on day 0 (odds ratio [OR], 0.22; 95% confidence interval [CI], .17-.28 and OR, 0.12; 95% CI, .08-.16, respectively). Rate of decline in gametocyte carriage was faster when PQ was combined with artemether-lumefantrine (AL) compared to dihydroartemisinin-piperaquine (DP) (P = .010 for day 7). Addition of 0.25 mg/kg PQ was associated with near complete prevention of transmission to mosquitoes. CONCLUSIONS: Transmission blocking is achieved with 0.25 mg/kg PQ. Gametocyte persistence and infectivity are lower when PQ is combined with AL compared to DP.

Antibodies to Cryptic Epitopes in Distant Homologues Underpin a Mechanism of Heterologous Immunity between Plasmodium vivax PvDBP and Plasmodium falciparum VAR2CSA.

Many pathogens evolve extensive genetic variation in virulence proteins as a strategy to evade host immunity. This poses a significant challenge for the host to develop broadly neutralizing antibodies. In Plasmodium falciparum, we show that a mechanism to circumvent this challenge is to elicit antibodies to cryptic epitopes that are not under immune pressure. We previously discovered that antibodies to the Plasmodium vivax invasion protein, PvDBP, cross-react with P. falciparum VAR2CSA, a distantly related virulence factor that mediates placental malaria. Here, we describe the molecular mechanism underlying this cross-species immunity. We identified an epitope in subdomain 1 (SD1) within the Duffy binding-like (DBL) domain of PvDBP that gives rise to cross-reactive antibodies to VAR2CSA and show that human antibodies affinity purified against a synthetic SD1 peptide block parasite adhesion to chondroitin sulfate A (CSA) in vitro The epitope in SD1 is subdominant and highly conserved in PvDBP, and in turn, SD1 antibodies target cryptic epitopes in P. falciparum VAR2CSA. The epitopes in VAR2CSA recognized by vivax-derived SD1 antibodies (of human and mouse origin) are distinct from those recognized by VAR2CSA immune serum. We mapped two peptides in the DBL5ε domain of VAR2CSA that are recognized by SD1 antibodies. Both peptides map to regions outside the immunodominant sites, and antibodies to these peptides are not elicited following immunization with VAR2CSA or natural infection with P. falciparum in pregnancy, consistent with the cryptic nature of these target epitopes.IMPORTANCE In this work, we describe a molecular mechanism of heterologous immunity between two distant species of Plasmodium Our results suggest a mechanism that subverts the classic parasite strategy of presenting highly polymorphic epitopes in surface antigens to evade immunity to that parasite. This alternative immune pathway can be exploited to protect pregnant women from falciparum placental malaria by designing vaccines to cryptic epitopes that elicit broadly inhibitory antibodies against variant parasite strains.

Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA.

Background: In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies. Methods: We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA. Results: Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria. Conclusions: These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.

Clinical Outcomes of Submicroscopic Infections and Correlates of Protection of VAR2CSA Antibodies in a Longitudinal Study of Pregnant Women in Colombia.

Malaria in pregnancy can cause serious adverse outcomes for the mother and the fetus. However, little is known about the effects of submicroscopic infections (SMIs) in pregnancy, particularly in areas where Plasmodium falciparum and Plasmodium vivax cocirculate. A cohort of 187 pregnant women living in Puerto Libertador in northwest Colombia was followed longitudinally from recruitment to delivery. Malaria was diagnosed by microscopy, reverse transcription-quantitative PCR (RT-qPCR), and placental histopathology. Gestational age, hemoglobin concentration, VAR2CSA-specific IgG levels, and adhesion-blocking antibodies were measured during pregnancy. Statistical analyses were performed to evaluate the impact of SMIs on birth weight and other delivery outcomes. Twenty-five percent of women (45/180) were positive for SMIs during pregnancy. Forty-seven percent of infections (21/45) were caused by P. falciparum, 33% were caused by P. vivax, and 20% were caused by mixed Plasmodium spp. Mixed infections of P. falciparum and P. vivax were associated with lower gestational age at delivery (P = 0.0033), while other outcomes were normal. Over 60% of women had antibodies to VAR2CSA, and there was no difference in antibody levels between those with and without SMIs. The anti-adhesion function of these antibodies was associated with protection from SMI-related anemia at delivery (P = 0.0086). SMIs occur frequently during pregnancy, and while mixed infections of both P. falciparum and P. vivax were not associated with a decrease in birth weight, they were associated with significant risk of preterm birth. We propose that the lack of adverse delivery outcomes is due to functional VAR2CSA antibodies that can protect pregnant women from SMI-related anemia.

Evolutionary structure of Plasmodium falciparum major variant surface antigen genes in South America: Implications for epidemic transmission and surveillance.

Strong founder effects resulting from human migration out of Africa have led to geographic variation in single nucleotide polymorphisms (SNPs) and microsatellites (MS) of the malaria parasite, Plasmodium falciparum. This is particularly striking in South America where two major founder populations of P. falciparum have been identified that are presumed to have arisen from the transatlantic slave trade. Given the importance of the major variant surface antigen of the blood stages of P. falciparum as both a virulence factor and target of immunity, we decided to investigate the population genetics of the genes encoding "Plasmodium falciparum Erythrocyte Membrane Protein 1" (Pf EMP1) among several countries in South America, in order to evaluate the transmission patterns of malaria in this continent. Deep sequencing of the DBLα domain of var genes from 128 P. falciparum isolates from five locations in South America was completed using a 454 high throughput sequencing protocol. Striking geographic variation in var DBLα sequences, similar to that seen for SNPs and MS markers, was observed. Colombia and French Guiana had distinct var DBLα sequences, whereas Peru and Venezuela showed an admixture. The importance of such geographic variation to herd immunity and malaria vaccination is discussed.

Efficacy of Different Primaquine Regimens to Control Plasmodium falciparum Gametocytemia in Colombia.

Treatment against Plasmodium falciparum malaria includes blood schizonticides to clear asexual parasites responsible for disease. The addition of gametocytocidal drugs can eliminate infectious sexual stages with potential for transmission and the World Health Organization recommends a single dose (SD) of primaquine (PQ) to this end. The efficacy of PQ at 0.75 mg/kg to suppress gametocytemia when administered in single or fractionated doses was evaluated. A clinical controlled study with an open-label design was executed; three groups of 20 subjects were studied sequentially. All subjects were treated with the standard dose of artemether-lumefantrine plus the total dose of 0.75 mg/kg of PQ administered (without previous G6PD testing) in three different ways: Group "0.75d-3" received 0.75 mg/kg on day 3; Group "0.50d-1 + 0.25d-3" received 0.50 mg/kg on day 1 and 0.25 mg/kg on day 3; Group "0.25d-1,2,3" received 0.25 mg/kg on days 1, 2, and 3. Subjects were evaluated on days 1, 4, and 7 by thick smear microscopy and quantitative polymerase chain reaction to determine the carriage of immature and mature gametocytes. There were no adverse events. The three schemes caused a marked reduction (75-85%) in prevalence of gametocytes on day 4 compared with day 1, but only the group that received 0.75 mg/kg on day 3 maintained the reduced gametocyte burden until day 7. None of the three treatments were able to clear gametocyte carriage on days 4 or 7, but the group that received the SD had the lowest prevalence of gametocytes (15%). Further studies are needed to establish a PQ regimen with complete efficacy against gametocytes.

A sensitive species-specific reverse transcription real-time PCR method for detection of Plasmodium falciparum and Plasmodium vivax.

As the global burden of malaria decreases and countries strive towards disease elimination, there is a greater demand for sensitive diagnostics to target the submicroscopic reservoir of infection. We describe here a sensitive species-specific RT-qPCR method to differentiate between Plasmodium falciparum and P. vivax infections at the submicroscopic level. With amplification of the 18S rRNA genes from total nucleic acids (both DNA and RNA), we discern P. falciparum and P. vivax with a limit of detection of 10 parasites/mL and 18 copies/µL, respectively. This assay was validated with 519 blood samples, negative by thick-smear, from febrile and asymptomatic cohorts from Colombia. These results were directly compared to a qPCR-based method (DNA only) as the gold standard. Of the samples from patients who presented with fever (n = 274), 34 infections were identified by RT-qPCR (16 P. falciparum, 15 P. vivax, and 3 mixed), of which only 10 infections were identified at the species level by qPCR. Within the asymptomatic cohort (n = 245), 13 infections were identified by RT-qPCR (3 P. falciparum, 3 P. vivax, and 7 mixed), whereas the species for only one infection was determined by qPCR. We conclude that this species-specific RT-qPCR method provides a more sensitive tool for species identification compared to DNA based qPCR methods.

Microscopic detection of hemozoin in peripheral leukocytes fails to indicate plasmodial placental infection in pregnant women.

INTRODUCTION: Malaria in pregnancy very often includes gestational (parasites in maternal peripheral blood) and placental (parasites in placental blood) infection, but the later condition can only be detected after delivery. High frequency of placental plasmodial infection has been confirmed in many countries and is associated with negative birth outcomes. With the hypothesis that placental infection is accompanied by hemozoin circulation in maternal peripheral blood, an exploratory study was conducted to evaluate the association between peripheral leukocytes with hemozoin and placental infection by Plasmodium vivax or Plasmodium falciparum in parturient women. METHODOLOGY: A descriptive, transversal and exploratory (pilot type) study was carried out with women from two malaria-endemic localities of northwest Colombia. A total of 25 parturient women with confirmed placental infection and 25 without placental infection were included. Two independent readers measured the number of leukocytes with hemozoin in thick smears of maternal peripheral blood. Plasmodial infection in maternal peripheral blood and placental blood was detected by thick smear and quantitative polymerase chain reaction (qPCR). RESULTS: Four parturient women had leukocytes with hemozoin in peripheral blood; three of them had placental plasmodial infection and one was negative for placental infection. No statistically significant association between leukocytes with hemozoin in peripheral blood and placental infection was observed. CONCLUSIONS: With this limited sample size, detection of leukocytes with hemozoin by thick smear of maternal peripheral blood did not indicate presence of placental infection.

Impact of Malaria in Pregnancy as Latin America Approaches Elimination.

In Latin America, four million pregnancies are at risk of malaria annually, but malaria in pregnancy is largely overlooked. As countries progress toward malaria elimination, targeting reservoirs of transmission is a priority. Pregnant women are an important risk group because they harbor asymptomatic infections and dormant liver stages of Plasmodium vivax that cause relapses. Of significant concern is the discovery that most infections in pregnant women fail to be detected by routine diagnostics. We review here recent findings on malaria in pregnancy within Latin America. We focus on the Amazon basin and Northwest Colombia, areas that harbor the greatest burden of malaria, and propose that more sensitive diagnostics and active surveillance at antenatal clinics will be necessary to eliminate malaria from these final frontiers.

Detección de casos submicroscópicos de Plasmodium spp, utilizando técnicas clásicas y moleculares en pacientes gestantes de Córdoba, Colombia / Detection of submicroscopic infection with Plasmodium spp, using classical and molecular techniques in pregnant patients from Córdoba, Colombia

Introducción: la malaria gestacional afecta a las madres y al embrión o feto en desarrollo; requiere diagnóstico rápido y tratamiento oportuno y efectivo para evitar las complicaciones y muertes. Objetivo: comparar las técnicas de gota gruesa, PCR anidada y PCR en tiempo real (qRT-PCR), para diagnosticar infecciones submicroscópicas por Plasmodium falciparum y P. vivax. Metodología: se estudiaron 21 mujeres con manifestaciones clínicas de malaria, incluyendo gestantes y no gestantes, en Puerto Libertador, Córdoba, Colombia; de todas se obtuvieron muestras de sangre periférica y, en las gestantes, de placenta y cordón umbilical. Se extrajo el ADN y se lo amplificó por PCR anidada y cuantitativa (qRT-PCR). Para el análisis estadístico se usaron los programas Graphpad PRISM y EPIDAT. Resultados: las tres técnicas diagnosticaron satisfactoriamente la presencia de P. falciparum y P. vivax en sangre periférica, cordón y placenta. Las pruebas moleculares presentaron sensibilidad y especificidad del 100%; dos casos de infección por P. falciparum no identificados por gota gruesa (submicroscópicos) se diagnosticaron con las dos técnicas de PCR. Conclusión: la qRT-PCR es ventajosa en comparación con la PCR anidada porque su estandarización es más corta, requiere menos infraestructura y permite cuantificar el ADN.

Introduction: Gestational malaria affects both the mother and the development of her embryo or fetus. Rapid diagnosis and timely and effective treatment are required to prevent complications and deaths. Objective: To compare thick blood smear with nested PCR and real-time PCR (qRT-PCR) for the diagnosis of submicroscopic infections with Plasmodium falciparum and P. vivax. Methodology: 21 women with clinical manifestations of malaria, including both pregnant and non-pregnant, were studied in Puerto Libertador, Córdoba, Colombia. Peripheral blood specimens were obtained from all of them; umbilical cord and placenta blood specimens were taken in the pregnant ones. DNA was extracted and amplified for nested PCR or qRT-PCR. Statistical analysis was done using Graphpad PRISM and EPIDAT softwares. Results: The three techniques were satisfactory for the detection of Plasmodium falciparum and P. vivax in peripheral blood and in the umbilical cord and placenta specimens. Molecular tests were 100% sensitive and specific. Two submicroscopic cases of P. falciparum infection were detected with the two PCR techniques. Conclusion: qRT-PCR is advantageous over nested PCR because its standardization is shorter, it requires lesser infrastructure and it allows the quantification of DNA.

Patterns of selection on Plasmodium falciparum erythrocyte-binding antigens after the colonization of the New World.

Pathogens, which have recently colonized a new host species or new populations of the same host, are interesting models for understanding how populations may evolve in response to novel environments. During its colonization of South America from Africa, Plasmodium falciparum, the main agent of malaria, has been exposed to new conditions in distinctive new human populations (Amerindian and populations of mixed origins) that likely exerted new selective pressures on the parasite's genome. Among the genes that might have experienced strong selective pressures in response to these environmental changes, the eba genes (erythrocyte-binding antigens genes), which are involved in the invasion of the human red blood cells, constitute good candidates. In this study, we analysed, in South America, the polymorphism of three eba genes (eba-140, eba-175, eba-181) and compared it to the polymorphism observed in African populations. The aim was to determine whether these genes faced selective pressures in South America distinct from what they experienced in Africa. Patterns of genetic variability of these genes were compared to the patterns observed at two housekeeping genes (adsl and serca) and 272 SNPs to separate adaptive effects from demographic effects. We show that, conversely to Africa, eba-140 seemed to be under stronger diversifying selection in South America than eba-175. In contrast, eba-181 did not show any sign of departure from neutrality. These changes in the patterns of selection on the eba genes could be the consequence of changes in the host immune response, the host receptor polymorphisms and/or the ability of the parasite to silence or express differentially its invasion proteins.

Functional antibodies against VAR2CSA in nonpregnant populations from colombia exposed to Plasmodium falciparum and Plasmodium vivax.

In pregnancy, parity-dependent immunity is observed in response to placental infection with Plasmodium falciparum. Antibodies recognize the surface antigen, VAR2CSA, expressed on infected red blood cells and inhibit cytoadherence to the placental tissue. In most settings of malaria endemicity, antibodies against VAR2CSA are predominantly observed in multigravid women and infrequently in men, children, and nulligravid women. However, in Colombia, we detected antibodies against multiple constructs of VAR2CSA among men and children with acute P. falciparum and Plasmodium vivax infection. The majority of men and children (>60%) had high levels of IgGs against three recombinant domains of VAR2CSA: DBL5ε, DBL3X, and ID1-ID2. Surprisingly, these antibodies were observed only in pregnant women, men, and children exposed either to P. falciparum or to P. vivax. Moreover, the anti-VAR2CSA antibodies are of high avidity and efficiently inhibit adherence of infected red blood cells to chondroitin sulfate A in vitro, suggesting that they are specific and functional. These unexpected results suggest that there may be genotypic or phenotypic differences in the parasites of this region or in the host response to either P. falciparum or P. vivax infection outside pregnancy. These findings may hold significant clinical relevance to the pathophysiology and outcome of malaria infections in this region.

Immune responses during gestational malaria: a review of the current knowledge and future trend of research.

Women pregnant with their first child are susceptible to severe P. falciparum disease from placental malaria because they lack immunity to placenta-specific cytoadherence proteins. In subsequent pregnancies, as immunity against placental parasites is acquired, there is a reduced risk of adverse effects of malaria on the mother and fetus and asymptomatic parasitaemia is common. In the case of vivax malaria, with increasing reports of severe cases in Asia and South America, the effects of infection by this species during pregnancy remain to be elucidated. This review summarized the main aspects involved in the acquisition of specific antimalarial immune responses during pregnancy with emphasis in research carried out in America and Asia, in order to offer a framework of interpretation for studies on pregnant women with malaria which are recently being produced in these regions. The authors conclude that (1) Effective humoral responses during gestational malaria are mainly directed against variant surface antigens codified by genes of the var2Csa family of P. falciparum; (2) Acquisition of immunity against these variant antigens depends on the degree and intensity of transmission, and the chance increases with age and successive pregnancies; (3) Antibody development is guided by specific cellular immune responses in cases of placental and maternal infection, and (4) The study of the significance of acquisition of specific immunity against both P. falciparum and P. vivax in America, should be performed.

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.

Status of allele frequency and diversity of Plasmodium falciparum msp1, msp2 and glurp before implementation of an artemisinin-based combined therapy in Northwestern Colombia / Estado de la frecuencia alélica y la diversidad de Plasmodium falciparum msp1, msp2 y glurp antes de la implementación de terapia combinada con artemisina en el Noroccidente de Colombia

Introduction: The status of msp1, msp2 and glurp allele frequency and the diversity of Plasmodium falciparum in Northwestern Colombia before the implementation of an artemisinin-combined therapy have been explored only by a few authors and in a relatively small number of samples from this highly endemic region. Objective: To evaluate the frequency of msp1, msp2, and glurp alleles and the diversity of P. falciparum in two Colombian regions before the use of an artemisinin-combined therapy. Methods: This study was part of a major anti-malarial efficacy trial designed as a random, clinically-controlled study for which 224 subjects were recruited. Region 2 of msp1 and msp2 (central region) were amplified by a nested PCR; glurp (region R2) was amplified by a semi-nested PCR. Results: For msp1, five genotypes were observed, representing the K1, MAD20, and RO33 allelic families. All samples corresponded to a MAD20 150 bp allele. For msp2 (IC family), two alleles were detected and for glurp, eight were observed. A total 33 haplotypes were detected. Conclusions: Analysis of glurp can be used to successfully genotype parasite populations in the new studies in Colombia aimed at exploring Plasmodium spp population dynamics. In addition, analysis of msp1 and msp2 can also be of value for comparisons with past studies, but not when the objective is to study parasites obtained from the same patient in a reduced period of time; for instance, during treatment efficacy studies.

Introducción: La frecuencia alélica y la diversidad de msp1, msp2 y glurp de Plasmodium falciparum en el Noroccidente de Colombia antes de la introducción de la terapia combinada con artemisinina, han sido exploradas por pocos autores y en números relativamente bajos de muestras clínicas de esta región de alta endemicidad. Objetivo: Evaluar la frecuencia alélica y la diversidad de msp1, msp2 y glurp de P. falciparum en dos regiones colombianas antes del uso de la terapia combinada con artemisinina. Métodos: El estudio fue parte de un ensayo mayor sobre la eficacia de la terapia antimalárica con un diseño clínico aleatorio controlado, en el cual 224 sujetos fueron reclutados. La región 2 de msp1 y msp2 (región central) se amplificaron por medio de una PCR anidada; glurp (región RII) fue amplificada con una PCR semi-anidada. Resultados: En msp1, se observaron 5 genotipos que representaron las familias alélicas K1, MAD20, y RO33. Todas las muestras correspondieron al alelo 150 bp de MAD20. En msp2 (familia IC), se detectaron 2 alelos y en glurp, se observaron 8. Un total de 33 haplotipos fueron detectados. Conclusiones: El análisis de glurp puede ser utilizado para genotipificar exitosamente poblaciones parasitarias en nuevos estudios en Colombia con el objeto de explorar la dinámica de las poblaciones de Plasmodium spp. Adicionalmente, el análisis de msp1 y msp2 puede también ser de valor para comparar con estudios pasados, pero no cuando el objetivo es estudiar parasitos obtenidos de un mismo paciente en un periodo reducido de tiempo, por ejemplo, durante estudios de eficacia terapéutica.

Prevalence of gestational, placental and congenital malaria in north-west Colombia.

BACKGROUND: The frequency of pregnancy-associated malaria is increasingly being documented in American countries. In Colombia, with higher frequency of Plasmodium vivax over Plasmodium falciparum infection, recent reports confirmed gestational malaria as a serious public health problem. Thick smear examination is the gold standard to diagnose malaria in endemic settings, but in recent years, molecular diagnostic methods have contributed to elucidate the dimension of the problem of gestational malaria. The study was aimed at exploring the prevalence of gestational, placental and congenital malaria in women who delivered at the local hospitals of north-west Colombia, between June 2008 and April 2011. METHODS: A group of 129 parturient women was selected to explore the prevalence of gestational, placental and congenital malaria in a descriptive, prospective and transversal (prevalence) design. Diagnosis was based on the simultaneous application of two independent diagnostic tests: microscopy of thick blood smears and a polymerase chain reaction assay (PCR). RESULTS: The prevalence of gestational malaria (thick smear /PCR) was 9.1%/14.0%; placental malaria was 3.3%/16.5% and congenital malaria was absent. A history of gestational malaria during the current pregnancy was significantly associated with gestational malaria at delivery. Plasmodium vivax caused 65% of cases of gestational malaria, whereas P. falciparum caused most cases of placental malaria. CONCLUSIONS: Gestational and placental malaria are a serious problem in the region, but the risk of congenital malaria is low. A history of malaria during pregnancy may be a practical indicator of infection at delivery.

The effects of Plasmodium vivax gestational malaria on the clinical and immune status of pregnant women in Northwestern Colombia / Efectos de la malaria gestacional por Plasmodium vivax sobre el estado clínico e inmune en gestantes del Noroccidente de Colombia

Abstract Objetive: The study explored the effects of Plasmodium vivax infection on the balance of pro- versus anti- inflammatory cytokines and chemokines and their relationship with some clinical and epidemiology outcomes. Methods: Thirty-five pregnant women were recruited. Of these, 15 subjects had malaria at delivery (GM+), and 20 had no exposition to infection throughout the pregnancy (GM-) and at delivery. Epidemiological and clinical data were recorded after reviewing the clinical records. At delivery, whole blood from the mother as well as placental tissue was collected. Diagnosis of infection was performed by thick smear and a polymerase chain reaction (PCR). Expression of pro-inflammatory and anti-inflammatory cytokines and chemokines was measured by a real time PCR. Results: The clinical and epidemiological variables explored were similar in both groups, with the exception of gestational age. When comparing the GM+ group with the GM- group, it is clear that although the differences generally are not significant, pro- inflammatory cytokines are elevated in both maternal blood and placental; anti-inflammatory ones are elevated in the mother and reduced in the placenta, and the chemokines are reduced in both compartments, except for MCP-1 which is elevated in all. Conclusion: The results appear to be strongly affected by the small number of women with GM by P. vivax at childbirth. Additional studies are needed with larger groups in this and other regions of the country.

Resumen Objetivo: En este estudio se determinó el efecto de la infección por Plasmodium vivax en el balance de citoquinas pro-inflamatorias/anti-inflamatorias y quemoquinas y su relación con algunas variables epidemiológicas y clínicas. Métodos: Se reclutaron 35 gestantes, 15 con malaria en el momento del parto (GM+) y 20 sin malaria en ningún momento de la gestación (GM-) Los datos epidemiológicos y clínicos fueron colectados a partir de la historia clínica. En el momento del parto fueron tomadas muestras de sangre periférica materna y tejido placentario. El diagnóstico fue realizado mediante gota gruesa y reaccion en cadena de la polimerasa (PCR). La expresión de citoquinas pro-inflamatorias/anti-inflamatorias y quimioquinas, fueron medidas por PCR en tiempo real. La expresión de citoquinas pro-inflamatorias/anti-inflamatorias y quemoquinas, fueron medidas por PCR en tiempo real. Resultados: En las variables epidemiológicas y clínicas, los datos fueron similares en ambos grupos. Al comparar el grupo GM+ con el grupo GM-, resulta claro que, aunque las diferencias, en general, no son significativas, las citoquinas proinflamatorias están elevadas tanto en sangre materna como placentaria, las antiinflamatorias están elevadas en la madre y reducidas en la placenta, y las quimioquinas están reducidas en ambos compartimentos, excepto la MCP-1 que está elevada en ambos. Conclusión. Los resultados parecen estar fuertemente afectados por la cantidad pequeña de mujeres con MG por P. vivax en el parto. Es necesario adelantar estudios adicionales con más mujeres tanto en esta región como en otros lugares.

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.