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Introduction: placental malaria (PM) is an important predictor of infant morbidity and mortality in sub-Saharan Africa. Although placental histology is the gold standard test to diagnose PM, the placenta impression smears remains widely used in epidemiological studies. This study is set to evaluate the performance of placental impression smears to detect PM in pregnant women in southern Benin. Methods: a cross-sectional analysis was performed on data collected in the framework a multicenter randomized clinical trial (Mal aria in Pregnancy Preventive and Alternative Drugs). Samples from 491 pregnant women were examined in the district of Allada, Southern Benin. Plasmodium falciparum infections have been assessed in placental blood and placental biopsy. Results: placental malaria detected by placenta impression smears and histology were prevalent in 11.4% and 10.8%, respectively. Sensitivity and specificity of placental impression smears were 90.6% and 98.4%. Among 55 pregnant women tested positive by placenta impression smears, 48 were positive by the histology, while 7 were negative (positive predictive value: 87.3%). Four hundred and twenty four (424) of the 429 tested negative by the placenta impression smears, were also negative according to histology whereas the rest (5 of 429) of the women were positive (negative predictive value: 98.8%). Conclusion: placenta impression smear is an accurate and easy method for the diagnosis of placental malaria.
Study design: a cross sectional analysis of data collected on four hundred and ninety-one pregnant women between January 2010 and May 2012 has been performed. Pregnant women were followed-up from the first antenatal clinical (ANC1) visit until the time of delivery. Study site, population and procedures: the study site, population and procedure have been described elsewhere [14,15]. Briefly, the study was conducted in three maternity clinics (Allada, Attogon and Sékou) in the district of Allada, a semi-rural area located 50 km north of Cotonou, the economic capital of Benin. Allada district is characterized by a subtropical climate and malaria is hyperendemic with an average of 20.5 infected anopheles/person/year. Plasmodium falciparum: is the predominant species transmitted (97%). The study population was composed of HIV-negative pregnant women residing in the district of Allada. During the study, socio-demographic (age, parity, marital status, education level), clinical (gestational age, weight, height) and biological (blood smear, haemoglobin level) data were collected. Two doses of IPTp (1500/75 mg SP per dose or 15 mg/kg MQ per dose) were administered throughout ANC visits. The second dose of IPTp was given at least of 1 month apart from the administration of the first dose. At enrolment, each woman received a LLITN and their use was assessed at each ANC visit. Clinical malaria episodes were treated with oral quinine or artemether-lumefantrine in the first and subsequent trimesters, respectively, for uncomplicated malaria, and with parental quinine for severe malaria. At delivery, women’s peripheral blood, cord blood, and placenta (biopsy and impression smears) samples were collected for hematological and parasitological evaluation. Laboratory methods: thick and thin blood films were stained and read for Plasmodium species detection according to standard quality-control procedures [16]. Tissue samples were collected from the maternal side of the placenta and placed into 10% neutral buffered formalin. Biopsies were processed, stained, and examined following standard procedures [17]. Impression smears from the placenta blood were stained with Giemsa and read following a standardized protocol [18,19]. Placental impression smears: a 2.5 x 2.5 cm3 sample from the selected placenta area was cut. The sample included the thickness of tissue from the maternal to the fetal side limited by the fetal membranes. One of the internal faces of sample was carefully put into contact with the slide, on the closest location to the identification area of slide. Then, the placental section was dry with a small piece of filter paper. One of the faces of the dried placental section was put into contact with the slide, on the most distal location to the identification area in the slide. The same procedure was repeated on a second slide. Placental histology: the 2.5 x 2.5 cm3 sample collected for placental impression smears was immediately put in a 50 ml of 10% buffered formalin container. This container was stored in a 4°C fridge until the placental tissue is processed at the department of pathology of the faculty of Medicine of the University of Abomey Calavi. The maximum of fixation was of 5 days. PM was characterized using the classification of Bulmer et al. [20]: uninfected (no parasites or pigment), acute (Figure 1, parasites in intervillous spaces), chronic (Figure 2, parasites in maternal erythrocytes and pigment in fibrin or cells within fibrin and/or chorionic villous syncytiotrophoblast or stroma), past (Figure 3, no parasites and pigment confined to fibrin or cells within fibrin). In this analysis, the chronic and active malaria infections have been taking into account to compared placental impression smears and histology. Each placenta impression smear was independently examined by two technicians. In case of discordances, a third reading was required. Placental histology was examined without knowledge of the placental impression and peripheral blood smears results. In addition, an external quality control was made on 100% of positive slide and 10% of negative slide in reference laboratory at Barcelona Centre for International Health Research (CRESIB), Hospital Clínic Universitat de Barcelona. Placental tissue with active malaria infection †(by histology) Placental tissue with chronic malaria infection †(by histology) Placental tissue with past malaria infection †(By histology) Ethical considerations: this study was approved by the Ethics Committee of the Faculty of Medicine of Cotonou in Benin. Before each inclusion, all participants involved in the study provided their written informed consent. In case the woman could not read, an impartial witness was involved in the process. Statistical analysis: data were double-entered into Microsoft Access 2003 database and analyzed with Stata 12.0 Software for Windows. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive and negative likelihood ratio (LH ±) were calculated to determine the accuracy diagnosis of placental impression smears vs. placental histology. Sensibility was calculated as true positives / (true positives + false negatives), Sp as true negatives / (true negatives + false positives), PPV as true positives / (true positives + false positives), NPV as true negatives / (true negatives + false negatives) [21].
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