Few studies have described placental malaria associated histopathology outside sub-Saharan Africa. Histological changes associated with P. falciparum and P. vivax infections and the corresponding impact on adverse birth outcome and maternal anaemia were assessed in an area of low malaria transmission in central India. This was a pregnant population at low risk of malaria and despite the low parasitaemia seen by the low maternal blood geometric mean parasite density and placental parasite percentage, the impact of infection was considerable and independent of the presence of documented of fever.
Based on placental incision microscopy, the overall prevalence of malaria at delivery was 1.0%, and comparable to recent studies in other regions in India
[31, 32]. As expected, placental histology was more sensitive to detect P. falciparum malaria than placental or peripheral smear microscopy alone. Approximately one in 40 women (2.4%) that were negative by conventional microscopy (either placental or peripheral) or pLDH-based RDT were found to have active placental P. falciparum infections (acute or chronic) by histology. When this estimate of ‘subpatent’ infections was extrapolated to the overall survey sample of 2282 women, the prevalence of P. falciparum placental malaria increased from 2.2% (50/2282) to 4.5%, doubling the estimate. Placental incision microscopy alone missed about two out of every three infections which could be explained by the largely low parasitaemia. Women with subpatent infections had lower maternal haemoglobin, mean birth weight and gestational age, although the difference did not reach statistical significance, likely because of the small number of women with submicroscopic infections.
Most (89.3%, 25/28) of the women with patent maternal blood P. falciparum infections detected by conventional microscopy also had evidence of an active placental histopathology infection. In contrast, no placental parasites could be detected by histology among the 11 women with patent peripheral P. vivax infections and none of the women who were smear negative for peripheral P. vivax had evidence of infection with histopathology. This highlights the importance of using sensitive methods such as placental histology or PCR to detect placental P. falciparum infection as conventional peripheral and placental microscopy smear results alone may markedly underestimate the true burden of P. falciparum malaria in this region.
Histopathology is a useful method to grade the chronology of placental P. falciparum infections
[3, 4]. Compared to observations in areas of high transmission, a relatively large proportion (38%) of histopathological infections with P. falciparum were acute rather than chronic or past infections. This might be explained by the low malaria exposure and low host immunity in this population triggering an inflammatory response and early delivery when the placenta becomes infected, although the adverse impact of infection was independent of the occurrence of fever in this study. The low exposure is also reflected by the low parasitaemia within the placenta and the corresponding mild inflammatory response. This contrasts with the dense parasitaemia and the occasionally massive chronic intervillositis and monocyte infiltration reported from highly endemic areas in sub-Saharan Africa
, but is similar to observations from the Thai-Burmese border
 and Colombia
[16–18]. Similar to previous reports, most parasites were found in the intervillous space without adhering to the syncytial layer and none were observed within the villous structure
[4, 10, 33].
Chronic P. falciparum infection was associated with an increase in syncytial knots, focal fibrinoid necrosis and calcification. These changes were also evident in past infections, suggesting they persist after parasites are cleared
. Alternatively, there might have been insufficient time for their resolution since time between infection and delivery might have been short, similar to observations on the Thai-Burmese border
Placental P. falciparum malaria was associated with maternal anaemia. Acute infection was associated with lower levels of mean haemoglobin (1.4 g/dL), comparable to observations in a study from Malawi
. Chronic infections were not associated with lower haemoglobin level. The proportion of low birth weight in this population is high and is consistent with previous reports from India
[34, 35]. Adding to it, the impact of placental P. falciparum infection on birth weight was considerable, despite the scarcity of parasites within the placenta and the mild degree of placental inflammation. Of all the histological measures, only the presence of parasites and pigmented monocytic infiltration were consistently associated with reduced birth weight and shorter gestation in multivariate analysis. Further analysis showed that the effect on birth weight and gestation was particularly evident among the active infections with pigmented monocyte infiltration, a reduction in birth weight of 356 grams compared to 42 grams in women with presence of parasites alone, especially if this monocyte infiltration was moderate to severe. While mononuclear cells as a source of inflammatory cytokines have been suggested to have a role in poor foetal growth
[11, 36][37–39] the infection level of 1–5 parasites per 100x magnification field approximating to <1% parasitaemia in 60% of cases in this study also suggests that mechanisms other than the presence of placental parasitaemia per se may play a role.
The observed shortening of the duration of pregnancy with active infections was an important contributor to the reduction in birth weight. It is possible that with the increased risk of acute infections in women with history of fever, the inflammatory response to infections might induce early labour. Thus infections may not last to lead to chronicity. Alternatively clinical symptoms may lead women to seek treatment and prevent chronicity. The average duration of pregnancy was 4 days shorter among women with an active infection and mild monocyte infiltration, and 8 days shorter in women with moderate to severe infiltration. Although this may appear a relatively small difference, the average increase in birth weight between 37 to 40 weeks gestation is estimated at 169 grams per week
. Additionally, chronic or past infections were not associated with low birth weight or shorter gestation, which supports the notion that malaria associated low birth weight results, at least in part, from a shortening of gestation triggered by acute infections in women with low protective immunity
[4, 7, 9, 41].
There was no evidence of P. vivax sequestration in the placenta. Pigment in the fibrin was observed in two peripherally patent P. vivax cases, which is consistent with reports from Thailand and Peru
[10, 24], although previous infections with P. falciparum cannot be excluded. Recent studies on immune aspect of placental malaria in Colombia indicate a possible pathogenic role of P. vivax[16, 18] A noteworthy observation in Plasmodium vivax cases was the significantly higher presence of polymorphonuclear leukocytes and syncytial knots compared to P. falciparum infected placentas, suggesting a possible placental inflammatory response that might be different from that observed with P. falciparum. In addition, decreased fibrin deposition was seen compared to P. falciparum or uninfected placentas. There was no association, however, between polymorphonuclear cells or increased syncytial knots and birth weight or preterm births, although numbers may have been too small.
The study was limited by its cross-sectional design, which did not allow assessing the impact of earlier pregnancy infections via peripheral microscopy. If such infections resolve and are no longer evident as ‘past’ infections but still impact on maternal anaemia and birth outcome, the burden of malaria may have been underestimated. The number of women with P. vivax mono-infections was too low to draw firm conclusions. Although a single reader examined the sections and she was not blinded to the peripheral, placental microscopy and RDT results, a random sample of positive slides and those corresponding to patent P. vivax cases were reviewed blinded by a senior placental histopathologist in the US and confirmed the accuracy of the results of the first reading. PCR conducted in a subset of samples, provided further confirmation of species and the accuracy of the histological examination. The Ballard score that was used has not been validated in India. It was beyond the scope of this study to assess conditions other than malaria that may affect birth weight, haemoglobin level and prematurity (e.g. iron deficiency, other infections).