Skip to content

Advertisement

Open Access

Hyperparasitaemia during clinical malaria episodes in infants aged 0–24 months and its association with in utero exposure to Plasmodium falciparum

BMC Research Notes201811:232

https://doi.org/10.1186/s13104-018-3339-0

Received: 2 February 2018

Accepted: 30 March 2018

Published: 4 April 2018

Abstract

Objective

Existing information has shown that infants who are prenatally exposed to P. falciparum are susceptible to subsequent malaria infections. However, the effect of prenatal exposure to P. falciparum on parasite density during clinical malaria episodes has not been fully elucidated. This study is a component of a prospective cohort study for which initial results have been published. This component was designed to determine the effect of prenatal exposure to P. falciparum on parasite density during clinical malaria episodes in the first 24 months of life. A total of 215 infants were involved and monitored for clinical malaria episodes defined by fever (≥ 37 °C) and parasitaemia. The geometric mean parasite counts between exposed and unexposed infants were compared using independent samples t test. The effect of in utero exposure to P. falciparum on parasite density was assessed using binary logistic regression.

Results

The geometric mean parasite count per µl of blood during clinical malaria episodes in exposed infants was 24,889 (95% CI 18,286–31,490) while in unexposed infants it was 14,035 (95% CI 12,111–15,960), P < 0.05. Prenatal exposure to P. falciparum was associated with hyperparasitaemia during clinical malaria episodes (OR 7.04, 95% CI 2.31–21.74), while other factors were not significantly associated (P > 0.05).

Keywords

Parasite density P. falciparum In uteroMalariaInfantsHyperparasitaemia

Introduction

Plasmodium falciparum malaria is a major cause of death in children in sub-Saharan Africa [1, 2]. In endemic regions, infants born to mothers with placental malaria are exposed in utero to P. falciparum antigens, like P. falciparum merozoite surface protein 1–19 (PfMSP1–19) and P. falciparum merozoite surface protein 2 (PfMSP2) [38]. Prenatal exposure to P. falciparum antigens affects the development of fetal immune cells and infant susceptibility to malaria infections [35]. Furthermore, prenatal exposure to P. falciparum is also associated with poor pregnancy outcomes [9, 10].

During childhood, on average children experience hyperparasitaemia in severe malaria [11]; however, hyperparasitaemia may be encountered both in severe and mild malaria infections [1]. This implies that severity of malaria disease may not only be attributable to high parasite density but also other factors like transmission season, use of long lasting insecticide treated bed nets (LLINs), parasite virulence and host immunity.

Although prenatal exposure has been demonstrated to influence susceptibility to malaria infection [12], the paucity of information on its effect on susceptibility to infection and disease severity requires further investigation. This study was designed to assess the effect of prenatal exposure to P. falciparum on parasite density during clinical malaria episodes in infants aged zero up to 24 months.

Main text

Methods

Study area

The detailed description of the study area was reported earlier [12]. Briefly, the study was conducted in Rufiji District Tanzania, characterized by perennial malaria transmission.

Study design and recruitment research participants

The study design, sample population, recruitment and follow up procedures were reported earlier [12]. Briefly, 215 infant mother pairs were recruited after parturition and placed into two categories, after establishing the mothers’ placental malaria status. Fifty mothers had placental malaria and their infants were categorized as exposed in utero to malaria and the remaining (n = 165) were unexposed. Recruited infants were followed from birth to 24 months, for 3 monthly visits. During visits, exposure to malaria infection was monitored, and clinical malaria episodes were diagnosed.

Diagnosis of placental malaria

Procedures for collection of placental tissues, storage and processing, for histological examinations were reported earlier [12]. Briefly, placental tissues were stored in 10% neutral buffered formalin, processed overnight on automatic processor, sectioned, mounted, stained using hematoxylin and eosin, dried and examined with a light microscope to establish placental malaria status.

Determination of parasite density

Capillary blood samples were collected at each visit for determination of parasite density and species identification. Thick and thin blood smears were prepared, the thin smears were fixed in methyl alcohol, dried, stained with 10% giemsa and examined using a light microscope at 100× objective for diagnosis and parasite quantification. Two laboratory technicians examined the slides, discrepancies in slide reading, were resolved by a third technologist and P. falciparum was the only parasite species identified. Parasite counts were expressed per µl of blood, assuming a leukocyte count of 8000 per µl. Levels of parasitaemia were categorized into low parasitaemia (< 1000 parasites/µl blood), intermediate parasitaemia (1000–9999 parasites/µl blood) and hyperparasitaemia (> 10,000 parasites/µl blood).

Determination of prenatal sensitization to Plasmodium falciparum antigens

Cord blood sera was collected following delivery and stored at − 20 °C at field site, then transported in ice parked cool box to Muhimbili University and transferred to – 80 °C freezers until use. Detailed procedures for determination of seropositivity and seronegativity for IgM against recombinant P. falciparum antigens (PfMSP1–19, PfMSP2) were described elsewhere [13]. Briefly, 100 μl of serum diluted to 1:1000 was added to duplicate wells and incubated overnight at 4 °C; washed and incubated with horseradish peroxidase conjugated goat anti-human IgM antibodies diluted at 1:6000 (Mabtech) for 3 h at room temperature. The test was developed with a substrate Tetramethylbenzidine at 4 °C for 10 min and reactions were stopped using 20 μl of 2 M H2SO4 per well. The optical density (OD) was measured at 450 nm. Control samples were used in each run and tested sera were defined as positive if they gave OD above the cut-off point (mean + 3SD) of negative control group born and living in Sweden. Based on the test results infants were categorized as, in utero sensitized (seropositivity for IgM) and non sensitized to P. falciparum antigens (seronegativity for IgM).

Data analysis

Data were analyzed using Statistical Package for Social Sciences (SPSS), IBM version 20. In order to use parametric tests data were log transformed. Assessment of effect of prenatal exposure to P. falciparum and other factors (live birth weight, gravidity, season of birth, IPTp-SP and use of LLINs) on hyperparasitaemia were assessed using binary logistic regression. The difference in the geometric mean parasite counts between exposed and unexposed was assessed using independent samples t test and the difference was judged at P < 0.05.

Results

Socio-demographic characteristics of the study population

Detailed description of socio-demographic characteristics of the study population was reported earlier [12]. Briefly, the social demographic characteristics of the mothers were not statistically different. The social demographic characteristics of exposed and unexposed infants were not statistically different except birth weight which was statistically significant P < 0.05.

Parasite density during clinical malaria episodes in recruited infants and prenatal immunosensitization to PfMSP1–19 and PfMSP2

The geometric mean parasite count per µl of blood during clinical malaria episodes in exposed and unexposed infants were 24,889 (95% CI 18,286–31,490) 14,035 (95% CI 12,111–15,960) respectively and the difference was statistically significant (P < 0.05). Ninety percent (n = 45) of infants born to mothers with placental malaria were not sensitized to P. falciparum specific antigens while 10% (n = 5), had specific IgM for PfMSP1–19 and PfMSP2 in cord blood sera and were therefore sensitized to P. falciparum antigens. All infants born to mothers without placental malaria (unexposed) had no detectable IgM against P. falciparum antigens, mean parasite count during clinical malaria episodes in exposed sensitized infants was 12,731 (95% CI 9364–16,098) and in exposed-non sensitized it was 28,087 (95% CI 20,333–35,842) and the difference was statistically significant, P < 0.01 (Fig. 1).
Figure 1
Fig. 1

Mean parasite counts during clinical malaria episodes in exposed sensitized, exposed non sensitized and unexposed infants. The figure with standard error bars shows the geometric mean counts/µl of blood in the infants born to mothers without placental malaria (no exposure group), infants born to mother with placental malaria (pm+) and were seropositive for IgM against Pfmsp1–19 and Pfmsp2 in their cord blood sera (exposed sensitized group) and infants born to mothers with placental malaria (pm+) and were seronegative for IgM against Pfmsp1–19 and Pfmsp2 in their cord blood sera (exposed non-sensitized)

Effect of prenatal exposure to P. falciparum on parasite density

Prenatal exposure to P. falciparum was significantly associated with hyperparasitaemia (P < 0.01, OR 7.04, 95% CI 2.31–21.74) during clinical malaria episodes in the first 2 years of life. Other factors; gravidity, season of birth, gender of infant, birth weight and IPT-SP were not significantly associated with hyperparasitaemia in a univariate analysis (Table 1); and these factors did not statistically influence the effect of prenatal exposure to P. falciparum on parasite density in a multivariate analysis.
Table 1

Effect of prenatal exposure to P. falciparum, gravidity, season of birth, birth weights and IPTp-SP on hyperparasitaemia: binary logistic regression (BLR)

Factors

OR (CI 95%)

P value

Non exposure to P. falciparum

1 (–)

Exposure to P. falciparum

7.04 (2.31–21.74)

< 0.01

Gravidity

 Primigravida

1 (–)

 Multigravida

1.97 (0.717–5.125)

0.195

Season of birth

 Dry season

1 (–)

 Wet season

0.994 (0.280–1.803)

0.992

Gender

 Males

1 (–)

 Female

1.803 (0.673–4.831)

0.241

Birth weight

 (≥ 2.5 kg)

1 (–)

 (≤ 2.5 kg)

2.267 (0.637–8.072)

0.207

IPTp-sp

 Use

1 (–)

 Non use

0.711 (0.043–11.865)

0.812

The weight ≤ 2.5 kg was considered underweight while ≥ 2.5 was considered normal weight

OR odds ratio, CI confidence interval, IPTp-SP intermittent preventive treatment with sulfadoxine pyrimethamine. BLR binary logistic regression

Discussion

Results of this study have shown that prenatal exposure to P. falciparum is significantly associated with hyperparasitaemia during clinical malaria episodes in the first 24 months of life in a malaria endemic area.

The findings of this study of hyperparasitaemia in infants prenatally exposed to P. falciparum may suggest inappropriate anti parasitic protective immunity. This suggestion is supported by observations from previous studies indicating that prenatal exposure to P. falciparum affected development of fetal regulatory and effector T cell responses [14, 15]. The effect of prenatal exposure to P. falciparum on fetal immune cells may persist to childhood; leading to inappropriate anti-parasitic immunity and failure of effective immune response to control parasite replication during clinical malaria episodes, with subsequent hyperparasitaemia. A study conducted in Kenya, demonstrated that prenatal exposure to P. falciparum affected the fetal immune cells and leads to immunosensitization or immunotolerance to malaria parasite antigens [4]. Although the present study did not directly assess the effect of prenatal exposure to P. falciparum on subsequent development of protective immunity; implicitly the findings of the current study corroborate the study in Kenya, in that 90% of the infants born to mothers with placental malaria were non sensitized to P. falciparum specific antigens (PfMSP1–19 and PfMSP2). In this study, a small proportion of 10% of the exposed infants were immunosensitized as demonstrated by IgM seropositivity, and had relatively lower parasitaemia compared to the 90% who were non sensitized. This observation indicates that early exposure to the parasite antigen may have implications on their capacity to mount appropriate immune response when naturally challenged with similar parasite antigens.

The vulnerability of exposed infants to high parasite density observed in this study is further corroborated by previous studies, which demonstrated that prenatal exposure to malaria may negatively affect the acquisition of antibodies against P. falciparum [5], rendering exposed infants more susceptible to malaria infection [5, 1620].

Furthermore, the findings in this study, that prenatal exposure to P. falciparum predisposes the exposed infants to high parasite density during clinical malaria episodes, may be supported by observations from another study which indicated that; prenatal exposure to P. falciparum is associated with immaturity of fetal/neonatal antigen presenting cells and provide insufficient co-stimulatory signals to T cells which are important in combating parasitaemia [21].

In the pathogenesis of malaria infection, parasite density has been demonstrated to be associated with onset of severe malaria [22]. However, high parasite density has also been observed in infants with mild malaria infection. This implies that high parasite density may not be a sole indicator for the severity of malaria infection since other factors like gravidity; use of long lasting insecticide treated bed nets (LLINs) and season of birth may influence the severity of malaria and parasite density. Gravidity has been demonstrated to influence the acquisition of placental malaria, and primigravida mothers lack acquired antibodies against parasites binding chondroitin sulfate A that would protect them against placental malaria infection unlike multigravida mothers. Implicitly, infants born to primigravida mothers are more likely to be exposed to placental malaria which may subsequently influence their susceptibility to malaria infection. Similarly, the use of LLINs is likely to influence vector abundance and risk of malaria infection. Therefore, in situations where LLINs are not used, there may be increased exposure to mosquito bites and risk of malaria infection during high transmission season.

Hyperparasitaemia leads to destruction of host erythrocytes, consumption of host glucose, stimulation of lactate formation through hypoxia and alteration of erythrocyte membrane transport systems [23]. Thus infants exposed in utero to parasite antigens may be at higher risk of serious complications associated with malaria infection despite the fact that other factors may be at play in influencing levels of parasitaemia. However, the findings in this study of a high proportion of exposed non-sensitized infants (immunotolerant) may indicate that prenatal exposure to the parasite antigen plays a significant role in influencing hyperparasitaemia in malaria infection.

In this study, gravidity, use of LLINs, birth weight, age of mother at delivery, season of birth and use of IPTp-SP were not significantly associated with hyperparasitaemia during clinical malaria episodes. However, in another study conducted in Tanzania, season and use of LLINs were significantly associated with parasite density [1]. The observed inconsistencies in the role of other factors in influencing parasite density may be attributable to the differences in the study designs.

Prospectively, the findings of this study further emphasize the importance of preventing malaria in women of reproductive age in order to mitigate the exposure of the fetus to P. falciparum antigens.

Conclusion

Prenatal exposure to P. falciparum is associated with hyperparasitaemia during clinical malaria episodes in the first 24 months of life.

Limitation

Anti malaria campaigns were going on in the study area.

Abbreviations

OR: 

odds ratio

BLR: 

binary logistic regression

BSA: 

bovine serum albumin

CI: 

confidence interval

ELISA: 

enzyme linked immunosorbant assay

GST: 

glutathione s-transferase

LLINs: 

long lasting Insecticide treated bed nets

PfMSP1: 

Plasmodium falciparum merozoite surface protein 1

PfMSP2: 

Plasmodium falciparum merozoite surface protein 2

PBS: 

phosphate buffer saline

pm+: 

placental malaria positive

pm−: 

placental malaria negative

TMB: 

tetra methyl benzidine

Declarations

Authors’ contributions

DBG, SM, SA, and DT conceived the study, participated in the study protocol development, coordination, and manuscript writing. RM participated in the data analysis and manuscript writing. SG participated in the data generation, coordination and manuscript writing. BMS participated in study protocol development, data collection, data management, data analysis, and in the preparation and writing of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We acknowledge the participation of mothers and infants in the study area and much appreciate the assistance of nurses, laboratory technicians and medical doctors at the health facilities of Utete, Kibiti, Ikwiriri, and Mohoro.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Consent for publication

Not applicable.

Ethical approval and consent to participate

Ethical clearance to conduct the study was granted by the Senate Research and Publications Committee of Muhimbili University of Health and Allied Sciences in Tanzania. Permission to conduct the study in Rufiji District health facilities was granted by the Rufiji District Medical Officer. Written informed consent was obtained from mothers after explanation of the aims and nature of study prior to recruitment and participation in the study. All infants found to have clinical malaria were managed in accordance with the existing national treatment guidelines.

Funding

The study was funded by The Swedish International Development and Cooperation Agency (Sida).

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Parasitology and Medical Entomology, School of Public Health and Social Sciences, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania
(2)
Department of Microbiology and Immunology, School of Medicine, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania
(3)
Department of Obstetrics and Gynaecology, School of Medicine, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania
(4)
Department of Epidemiology and Biostatistics, School of Public Health and Social Sciences, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania
(5)
Department of Medical Biochemistry and Microbiology, Biomedical Centre, Uppsala University, Uppsala, Sweden

References

  1. Goncalves BP, Huang CY, Morrison R, Holte S, Kabyemela E, Prevots DR, et al. Parasite burden and severity of malaria in Tanzanian children. N Engl J Med. 2014;370(19):1799–808.View ArticlePubMedPubMed CentralGoogle Scholar
  2. Bardaji A, Sigauque B, Sanz S, Maixenchs M, Ordi J, Aponte JJ, et al. Impact of malaria at the end of pregnancy on infant mortality and morbidity. J Infect Dis. 2011;203:691–9.View ArticlePubMedPubMed CentralGoogle Scholar
  3. King CL, Malhotra I, Wamachi A, Kioko J, Mungai P, Wahab SA, et al. Acquired immune responses to Plasmodium falciparum merozoite surface protein-1 in the human fetus. J Immunol. 2002;168:356–64.View ArticlePubMedGoogle Scholar
  4. Malhotra I, Dent A, Mungai P, Wamachi A, Ouma JH, Narum DL, et al. Can prenatal malaria exposure produce an immune tolerant phenotype? A prospective birth cohort study in Kenya. PLoS Med. 2009;6:e1000116.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Dent A, Malhotra I, Mungai P, Muchiri E, Crabb BS, Kazura JW, et al. Prenatal malaria immune experience affects acquisition of Plasmodium falciparum merozoite surface protein-1 invasion inhibitory antibodies during infancy. J Immunol. 2006;177:7139–45.View ArticlePubMedGoogle Scholar
  6. Steiner K, Myrie L, Malhotra I, Mungai P, Muchiri E, Dent A, et al. In utero activation of fetal memory T cells alters host regulatory gene expression and affects HIV susceptibility. Virology. 2012;425(1):23–30.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Mayor A, Rovira-Vallbona E, Machevo S, Bassat Q, Aguilar R, Quinto L, et al. Parity and placental infection affect antibody responses against Plasmodium falciparum during pregnancy. Infect Immun. 2011;79(4):1654–9.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Mackroth MS, Malhotra I, Mungai P, Koech D, Muchiri E, King CL. Human cord blood CD4+ CD25hi regulatory T cells suppress prenatally acquired T cell responses to Plasmodium falciparum antigens. J Immunol. 2011;186:2780–91.View ArticlePubMedGoogle Scholar
  9. Desai M, ter Kuile FO, Nosten F, McGready R, Asamoa K, Brabin B, et al. Epidemiology and burden of malaria in pregnancy. Lancet Infect Dis. 2007;7(2):93–104.View ArticlePubMedGoogle Scholar
  10. Adam I, Elhassan EM, Haggaz AE, Ali AA, Adam GK. A perspective of the epidemiology of malaria and anaemia and their impact on maternal and perinatal outcomes in Sudan. J Infect Dev Ctries. 2011;5(2):83–7.View ArticlePubMedGoogle Scholar
  11. Argy N, Kendjo E, Auge-Courtoi C, Cojean S, Clain J, Houze P, et al. Influence of host factors and parasite biomass on the severity of imported Plasmodium falciparum malaria. PLoS ONE. 2017;12(4):e0175328.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Sylvester B, Gasarasi DB, Aboud S, Tarimo D, Massawe S, Mpembeni R, et al. Prenatal exposure to Plasmodium falciparum increases frequency and shortens time from birth to first clinical malaria episodes during the first two years of life: prospective birth cohort study. Malar J. 2016;15(1):379.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Metzger WG, Okenu DM, Cavanagh DR, Robinson JV, Bojang KA, Weiss HA, et al. Serum IgG3 to the Plasmodium falciparum merozoite surface protein 2 is strongly associated with a reduced prospective risk of malaria. Parasite Immunol. 2003;25(6):307–12.View ArticlePubMedGoogle Scholar
  14. Odorizzi PM, Feeney ME. Impact of in utero exposure to malaria on fetal T cell immunity. Trends Mol Med. 2016;22(10):877–88.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Prahl M, Jagannathan P, McIntyre TI, Auma A, Farrington L, Wamala S, et al. Timing of in utero malaria exposure influences fetal CD4 T cell regulatory versus effector differentiation. Malar J. 2016;15(1):497.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Le Hesran JY, Personne M, Cot P, Fievet N, Dubois B, Beyeme M, et al. Maternal placental infection with Plasmodium falciparum and malaria morbidity during the first 2 years of life. Am J Epidemiol. 1997;146(10):826–31.View ArticlePubMedGoogle Scholar
  17. Ismaili J, van der Sande M, Holland MJ, Sambou I, Keita S, Allsopp C, et al. Plasmodium falciparum infection of the placenta affects newborn immune responses. Clin Exp Immunol. 2003;133(3):414–21.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Le Port A, Watier L, Cottrell G, Ouedraogo S, Dechavanne C, Pierrat C, et al. Infections in infants during the first 12 months of life: role of placental malaria and environmental factors. PLoS ONE. 2011;6(11):e27516.View ArticlePubMedPubMed CentralGoogle Scholar
  19. McDermott JM, Wirima JJ, Steketee RW, Breman JG, Heymann DL. The effect of placental malaria infection on perinatal mortality in rural Malawi. Am J Trop Med Hyg. 1996;55(1 Suppl):61–5.View ArticlePubMedGoogle Scholar
  20. N’Dao CT, N’Diaye JL, Gaye A, Le Hesran JY. Placental malaria and pregnancy outcome in a peri urban area in Senegal. Rev Epidemiol Sante Publique. 2006;54(2):149–56.PubMedGoogle Scholar
  21. Broen K, Brustoski K, Engelmann I, Luty AJ. Placental Plasmodium falciparum infection: causes and consequences of in utero sensitization to parasite antigens. Mol Biochem Parasitol. 2007;151(1):1–8.View ArticlePubMedGoogle Scholar
  22. Mangal P, Mittal S, Kachhawa K, Agrawal D, Rath B, Kumar S. Analysis of the clinical profile in patients with Plasmodium falciparum malaria and its association with parasite density. J Glob Infect Dis. 2017;9(2):60–5.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Arevalo-Herrera M, Rengifo L, Lopez-Perez M, Arce-Plata MI, Garcia J, Herrera S. Complicated malaria in children and adults from three settings of the Colombian Pacific Coast: a prospective study. PLoS ONE. 2017;12(9):e0185435.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© The Author(s) 2018

Advertisement