Approximately 50 million pregnancies occur each year in countries where malaria is endemic, half of which are in Africa (20
); but our tools for diagnosing placental malaria—peripheral and placental microscopy, placental histology, and more recently, antigen detection tests and PCR—have rarely been evaluated in a systematic manner. We compared microscopy (including different techniques for preparation of placental blood films) and histology as tools to detect P
infection and for their associations with maternal hemoglobin concentrations and infant birth weight.
Placental histology was considerably more sensitive than microscopy with peripheral or placental blood in detecting malaria, and microscopy with placental blood was more sensitive than that with peripheral blood (Table ), consistent with previous observations (10
infection of peripheral blood in the absence of placental infection was rare and was detected in 1.4% of women. Pregnancy-associated malaria is believed to be due to the presence of parasite variants capable of sequestering in the placenta through adhesion to ligands such as chondroitin sulfate A and hyaluronic acid (4
), explaining the high prevalence of placental infection seen relative to the prevalence of peripheral blood infection. A minority of women may be infected with variants that also circulate in the wider population; these variants adhere to CD36 cells and form erythrocyte rosettes (4
) and may sequester elsewhere in the body. These variants may not be detected in placental smears. Placental pathological changes associated with malaria are reported to be uniformly spread throughout the placenta, although no data were presented (24
). Our samples for placental histology were collected after initial examination of the placental film, sometimes from a different paracentric site. Systematic comparison of the detection and the density of parasitized erythrocytes at different sites in single placentas would be of interest.
Microscopically detectable P
infection was more common in women with their first and second pregnancies than in multigravid women; but infection detected by histology only and past infection did not differ with gravidity, and similar numbers of women were uninfected. Previous studies with Malawian women show parasite prevalences and densities as determined by microscopy to be higher in women with their first or second pregnancies than in multigravid women (13
). Gravidity appears to influence the detection of infection by microscopy but not the prevalence of low-grade P
infection in pregnancy.
Birth weights differed significantly with a diagnosis of malaria, and the prevalence of LBW babies was significantly higher for women with microscopically detectable infection (Table ). Recent analysis suggests that malaria in pregnancy predisposes the infant to death through its effects on birth weight and may be responsible for 75,000 to 200,000 infant deaths each year (20
). Women with microscopically detectable malaria were more likely to be anemic and had a mean hemoglobin concentration 0.7 g/dl lower than that for uninfected women. Malaria in pregnancy is responsible for an estimated 400,000 cases of severe anemia and 10,000 maternal deaths each year (6
). Successful antimalarial drug interventions with increased birth weight and the maternal hemoglobin concentration as the key endpoints have been reported to result in changes in birth weight of 100 to 200 g and changes in hemoglobin concentration of about 0.5 g/dl (9
). The differences in infant birth weights and maternal hemoglobin concentrations between women with present or past infection with P
identified by histology alone and uninfected women are within this range. While microscopy identified the women at highest risk, women with malaria identified by histology alone form a significant, and often neglected, proportion of those with malaria-related morbidity in pregnancy.
Apart from histology and microscopy of peripheral and placental blood, other methods for detection of placental malaria have recently been evaluated. Antigen detection tests (rapid immunochromatographic tests) measure parasite-derived histidine-rich protein 2 or parasite-specific lactate dehydrogenase secreted into blood. In our hands, the Optimal test for measurement of lactate dehydrogenase was no better than microscopy for detection of placental parasitemia (8
), whereas others reported that the test for measurement of parasite-derived histidine-rich protein 2 detected more placental infections than peripheral blood microscopy (7
). PCR amplifies P. falciparum
-specific DNA for detection of low-level parasitemia or circulating genetic material (19
) and was significantly more sensitive than microscopy with peripheral blood for detection of placental parasitemia (8
). Women in whom malaria was detected by PCR but not microscopy were more likely to be anemic than uninfected women (10
) but did not have babies with LBWs (8
). In each of these studies microscopy with placental blood rather than histology was used as the comparator, and further evaluation of these techniques in comparison to histology is indicated.
HIV infection has been associated with an increased prevalence and density of malaria in pregnancy in a number of studies (21
). In our study, HIV infection was not significantly related to the likelihood of malaria as detected by microscopy or placental histology. This may reflect our relatively small sample size compared to those in the epidemiological studies cited.
Microscopy performed with blood from a placental incision had a relatively low sensitivity for detection of malaria in comparison to that of histology. To determine whether the method of preparation of placental blood films affected the likelihood of a positive film, we compared three different methods for preparation of placental blood films. The placental incision is commonly used. The blood welling into the incised placenta is collected. Cells which are free in the intervillous space or fetal vessels are preferentially sampled. Incision gave the lowest parasite densities, suggesting that parasitized erythrocytes may be preferentially retained in the intervillous space through adhesion to host receptors or other processes. The washing approach is analogous to the methods that we have used to extract parasitized erythrocytes from the intervillous space (3
). In the present series, by using small pieces of tissue, the washing approach gave a modest increase in the level of parasitemia, but it may not greatly facilitate the detachment of cells from the syncytiotrophoblast (5
) or from masses in the intervillous space (1
). Scraping may detach some of these adherent cells and gave the highest parasite densities of any approach tested. Interestingly, all approaches were very similar in their specificities, sensitivities, and predictive values compared to the results of histology. Over 20% of samples positive by histology were negative by use of blood films prepared by all three methods. The mode of preparation of placental blood films appears to influence the level of parasitemia but not the likelihood of detection of malaria.
Blood film microscopy identifies a subset of women with malaria who have a high risk of anemia and of having LBW infants, but malaria in pregnancy is most sensitively diagnosed by placental histology. The diagnosis of present and past infections by histology alone is also associated with decreased infant birth weight and decreased maternal hemoglobin concentration. Use of histological diagnosis of placental malaria in epidemiological studies will allow more detailed characterization of the burden of morbidity attributable to malaria in pregnancy.