Background
Pregnant women are at higher risk of
Plasmodium falciparum infection and disease [
1], frequently manifested as maternal anaemia, pre-term delivery and low birth weight [
2,
3]. Infections during pregnancy need to be eliminated with effective anti-malarials to reduce the burden of disease in mothers and their children. Current guidelines to control malaria in pregnancy in sub-Saharan Africa consist of prompt and effective case management of malaria illness, combined with prevention of infection and/or disease through insecticide-treated nets (ITNs) and intermittent preventive treatment (IPTp) [
4].
Control of malaria in pregnancy remains a challenge due to a number of factors. The low specificity of signs and symptoms [
5,
6] and the limitations of the techniques to detect
P. falciparum infections impose serious difficulties in the identification of malaria episodes. Until recently, malaria diagnosis relied on the microscopic detection of
Plasmodium on Giemsa-stained blood smears. Lately, circulating antigen [
7] and polymerase chain reaction (PCR)-based diagnostic methods [
8‐
12] have been added as malaria diagnostic tools. In malaria endemic areas, PCR allows the identification of a high proportion of pregnant women with
P. falciparum parasitaemia levels below the threshold of microscopy [
8‐
12]. Real-time PCR (RT-PCR), a more sensitive and specific detection methodology [
13,
14], has been shown to be useful in predicting adverse outcomes of malaria in pregnancy [
15,
16].
The declining efficacy of classically recommended anti-malarial drugs due to the increase of anti-malarial-resistant
P. falciparum parasites [
17], results in multiple treatment failures and prolonged parasitization of the foeto-placental unit with the subsequent adverse events on mother and child health. New anti-malarial drug combinations are now being recommended to replace the less effective ones for the general population. However, there is limited or non-existent information on the safety and efficacy of these new drug alternatives in pregnancy. Data obtained from in vivo studies in children may not be appropriated for determining anti-malarial treatment efficacy in pregnant women because of differences in host immunity, as well as pharmacokinetics [
18‐
21]. Critical analysis of the in vivo standard methods to measure drug efficacy is essential to correctly monitor drug resistance and evaluate new anti-malarials to be administered in pregnancy.
In this study, a PCR-based approach to assess the impact of sub-microscopic infections on disease manifestation of pregnant women attending the maternity clinic of a rural hospital in Mozambique was used. Genetic characterization of parasites isolated from consecutive episodes during the same pregnancy allowed to determine the extent of parasite recrudescences after anti-malarial treatment.
Discussion
This study described the prevalence of
P. falciparum infection detected by real time PCR among Mozambican pregnant women attending a rural maternity clinic with clinical complaints suggestive of malaria. These results showed that microscopically detectable
P. falciparum parasitaemia in peripheral blood is a rather poor indicator of the actual presence of infection in pregnancy, as suggested by the low sensitivity of microscopy as compared to RT-PCR (22.7%) and the high prevalence of sub-microscopically infected women (18.9%). In other settings, sub-patent malaria infections have been found in up to 55% of the pregnant women [
8‐
12]. This suggests the existence of a level of host immunity able to restrict parasites to low, microscopically undetectable densities. Alternatively, these low-density infections may represent recently acquired infections which would reach microscopical levels if infection is not treated. Carriage of sub-microscopic infections has been shown to be common also in non-pregnant adults [
30,
31]. However, the occurrence of microscopically undetected infections are likely to be more clinically relevant during pregnancy, since malaria parasitaemia of any density may have a harmful effect on the pregnant women and her developing foetus [
16,
32,
33].
In this study, the majority of women presenting clinical complaints suggestive of malaria were not infected with
P. falciparum, as detected by microscopy (94.7%) and even by RT-PCR (76.8%). Thus, in this context, presumptive malaria treatment would lead to unnecessary exposure to anti-malarial drugs in the majority of pregnant women presenting with "malaria-like" clinical complaints [
6]. On the other hand, treatment based on microscopically confirmed malaria episodes only, might lead to under-treatment because of the significant proportion of infections bellow the detection threshold of microscopy. The ability of RT-PCR to detect this additional population of women at risk for malaria infection that were otherwise microscopically negative would contribute effectively to a better management of malaria in pregnancy. However, PCR-based methods require costly reagents and instrumentation that are not widely available in developing countries. Rapid tests that detect parasite-specific antigens [
7] may overcome the problem of inadequate diagnosis and treatment. In addition, a more sensitive definition of clinical malaria in pregnancy should also be evaluated. Other symptoms and signs could be considered, since those frequently used to define a malaria episode are poor indicators of
P. falciparum infection both in pregnant women [
6] and non-pregnant adults [
31].
In this study, anaemia was the only symptom associated with parasitaemia as detected by RT-PCR. This association was not found for microscopical parasitaemia in this subset of samples, maybe due to limitations of the sample size. Women with sub-microscopic infections were also at higher risk of anaemia compared with women with negative microscopy and RT-PCR test, suggesting a role of low-density infections in the aetiology of maternal anaemia. A similar association has been found in some studies [
31,
34], but not in others [
8,
10,
12,
35], may be due to differences in the sensitivity of the molecular technique used. The results of this study suggest that low-density asymptomatic infections may significantly increase the risk of becoming anaemic. Alternatively, anaemia may be a consequence of recent high density infections controlled at the time of examination.
PCR-confirmed recrudescences were found in 21% of the women experiencing two successive episodes during the same pregnancy. The rate of recrudescences found in this study raises important questions for drug efficacy in pregnancy. First, it suggests that the efficacy of anti-malarial drugs during pregnancy may be reduced. This can be due to inadequate drug levels, limited clearing capacity of placental parasites, or to immunosuppresion and physiological changes during pregnancy which are likely to alter the pharmacokinetics of most anti-malarials [
18‐
21,
36]. Secondly, these results show that recrudescences of
P. falciparum parasites during pregnancy can occur as late as 187 days. In other studies, reappearance of parasites has been shown to occur 35, 49, 85 and 119 days after treatment [
37,
38]. In Thailand, recrudescence was detected up to 62 days post-treatment in non pregnant individuals and up to 121 days in pregnant women [
27]. Thus, the 28 days standard in vivo test [
39] may not be adequate to assess the efficacy of anti-malarial drugs in pregnant women, since it would not detect recrudescences that occur much later. Various alternative assessment times have been used to define treatment failures in anti-malarial drug efficacy studies, ranging from 42 days [
40‐
42] to 63 days [
37,
38] after treatment. This study suggests that even longer follow-up may be needed during pregnancy to capture all treatment failures. It is essential that these studies include molecular genotyping to distinguish recrudescences from reinfections.
Conclusion
These results point out two important issues for malaria control during pregnancy; on the one hand, the need to apply more accurate and sensitive measures to detect malaria infections in pregnancy. On the other hand, it might be necessary to extend the follow-up period for in vivo tests to correctly identify anti-malarial drug failures during pregnancy. Appropriate, standardized genotyping methods should be applied to unequivocally identify recrudescent parasites. This is essential to optimize the drug regimens needed to eliminate the plasmodial biomass in pregnant women, and to correctly measure the efficacy of the existing and new anti-malarials to be used in pregnancy.
Acknowledgements
We thank the women participating in the study; and the staff of the Manhiça District Hospital, for their collaboration in the project; the clinical officers, field supervisors an data manager, whose work was important for the successful completion of the study; and Alfons Jiménez and Diana Barrios for their contribution to the molecular analysis of the samples.
The study received financial support from the Banco de Bilbao, Vizcaya, Argentaria Foundation (grant number BBVA 02-0) and the Spanish Ministry of Health (PI060016). The Manhiça Health Research Center receives core support from the Spanish Agency for International Cooperation. AM was supported by the Spanish Ministry of Health (Program for the Promotion of Biomedical Research and Health Sciences, Instituto de Salud Carlos III, CP-04/00220). The funding source did not have any involvement in study design, collection, analysis and interpretation of data, writing of the report, or in the decision to submit the paper for publication. The researches are independent from the funders.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AM and ES carried out the molecular genotyping study, the analysis and interpretation of data and prepared the manuscript. LP and PC contributed to the molecular genotyping study and the interpretation of data. SS and JA carried the statistical analysis and helped to draft the manuscript. AB and IM carried out the sample collection and contributed to the analysis and interpretation of data. BS contributed to the interpretation of the data and drafting of the paper. CM and PA conceived and coordinated the study, participated in the analysis and interpretation of the data, and contributed to the preparation of the manuscript. All authors read and approved the final manuscript.