RESEARCH ARTICLE
Impact of malaria and hepatitis B co-infection
on clinical and cytokine profiles among
pregnant women
Nsoh Godwin Anabire 1,2,3ID , Paul Armah Aryee
4
ID , Abass Abdul-Karim
5,
Osbourne Quaye1,2, Gordon Akanzuwine Awandare1,2, Gideon Kofi Helegbe1,3*
1 West African Centre for Cell Biology of Infectious Pathogens (WACCBIP), University of Ghana, Legon-
Accra, Ghana, 2 Department of Biochemistry, Cell & Molecular Biology, University of Ghana, Legon- Accra,
Ghana, 3 Department of Biochemistry & Molecular Medicine, School of Medicine and Health Sciences,
University for Development Studies, amale- Ghana, 4 Department of Nutritional Sciences, School of Allied
a1111111111 Health Sciences, University for Development Studies, Tamale- Ghana, 5 Zonal Public Health Laboratory,
a1111111111 Tamale Teaching Hospital, Tamale- Ghana
a1111111111
a1111111111 * ghelegbe@uds.edu.gh
a1111111111
Abstract
OPEN ACCESS Background
Citation: Anabire NG, Aryee PA, Abdul-Karim A, The overlap of malaria and chronic hepatitis B (CHB) is common in endemic regions, how-
Quaye O, Awandare GA, Helegbe GK (2019) Impact
ever, it is not known if this co-infection could adversely influence clinical and immunological
of malaria and hepatitis B co-infection on clinical
and cytokine profiles among pregnant women. responses. This study investigated these interactions in pregnant women reporting to ante-
PLoS ONE 14(4): e0215550. https://doi.org/ natal clinics in Ghana.
10.1371/journal.pone.0215550
Editor: Jason Blackard, University of Cincinnati Methods
College of Medicine, UNITED STATES
Clinical parameters (hemoglobin, liver function biomarker, peripheral malaria parasitemia,
Received: January 5, 2019 and hepatitis B viremia) and cytokine profiles were assayed and compared across four cate-
Accepted: April 3, 2019 gories of pregnant women: un-infected, mono-infected with Plasmodium falciparum (Malaria
Published: April 19, 2019 group), mono-infected with chronic hepatitis B virus (CHB group) and co-infected (Malaria
+CHB group).
Copyright: © 2019 Anabire et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which Results
permits unrestricted use, distribution, and Women with Malaria+CHB maintained appreciably normal hemoglobin levels (mean±SEM
reproduction in any medium, provided the original
= 10.3±0.3 g/dL). That notwithstanding, Liver function test showed significantly elevated lev-
author and source are credited.
els of alanine aminotransferase, aspartate aminotransferase and total bilirubin [P<0.001 for
Data Availability Statement: All relevant data are
all comparisons]. Similarly, the Malaria+CHB group had significantly elevated pro-inflamma-
within the manuscript and its Supporting
Information files. tory cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6
[P<0.05 for all comparisons]. In women with Malaria+CHB, correlation analysis showed sig-
Funding: This work was supported by Nsoh
Godwin Anabire’s MPhil fellowship from a World nificant negative association of the pro-inflammatory cytokines responses with malaria para-
Bank African Centres of Excellence Grant (ACE02- sitemia [IL-1β (P<0.001; r = -0.645), IL-6 (P = 0.046; r = -0.394) and IL-12 (P = 0.011; r =
WACCBIP: Awandare) and funds from the
-0.49)]. On the other hand, the pro-inflammatory cytokine levels positively correlated with
University for Development Studies, Tamale. Views
expressed in this publication are those of the HBV viremia [TNF-α (P = 0.004; r = 0.549), IL-1β (P<0.001; r = 0.920), IL-6 (P<0.001; r =
author(s) and not necessarily the funders. 0.777), IFN-γ (P = 0.002; r = 0.579), IL-2 (P = 0.008; r = 0.512) and IL-12 (P<0.001; r =
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 1 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
Competing interests: The authors have declared 0.655)]. Also, for women in the Malaria+CHB group, parasitemia was observed to diminish
that no competing interests exist. HBV viremia [P = 0.003, r = -0.489].
Conclusion
Put together the findings suggests that Malaria+CHB could exacerbate inflammatory cyto-
kine responses and increase susceptibility to liver injury among pregnant women in endemic
settings.
Background
Coinfections are increasingly being recognized as common risk factors that may contribute to
the increased burden of morbidity in pregnancy. In many endemic setting, the overlap of
chronic hepatitis virus (HBV) and P. falciparum infections is common, and an increased prev-
alence, from 0.7% to 1.7%, of this co-infection has been reported among pregnant women in
Ghana [1, 2]. The disease causing pathogens share a common intra-hepatic niche, and each
may independently cause liver function test abnormalities [3–9]. Immunologically, both path-
ogen may also overlap, as each is observed to mainly trigger T helper type 1 (Th1) cytokine
responses [10–14]. P. falciparum causes anemia by reducing red cell counts, while HBV is pos-
tulated to increase hemoglobin (Hb) levels by increasing the release of erythropoietin from
regenerating hepatic tissues [15–17]. Thus, it may be logical to postulate that HBV could com-
pensate for the effect of P. falciparum on Hb levels in co-infection state.
Anemia, liver dysfunctions and cytokine imbalance (towards Th1) in pregnancy are associ-
ated with significant morbidity and mortality for both the mother and the newborn [18–20].
Though physiologic and biochemical (high serum alkaline phosphatase) changes in pregnancy
are often mistaken for signs of liver disease, levels of aminotransferases remain normal, while
bilirubin is below normal range [21, 22]. Therefore, elevated levels of aminotransferases and
bilirubin in pregnancy gives evidence of liver dysfunction and damage. In normal pregnancy,
Th1 and T helper type 2 (Th2) responses are tightly regulated throughout the trimesters [23],
but excess of Th1 responses are observed to affect pregnancy outcomes [24].
Although P. falciparum and HBV are routinely diagnosed in pregnant women on antenatal
visits, the hematological and hepatological impact of co-infection has received little attention
mainly because: falciparum malaria is curable and does not cause chronic infections, and liver
functions tests are not conducted. This study therefore evaluates the influence of co-infection
on clinical (Hb levels and liver function) and cytokine profiles in pregnant women. The results
of the study provide important information that would be useful in guiding policy/decisions
on P. falciparum/HBV diagnosis, treatment and management in pregnant women in endemic
countries.
Methods
Study sites and participants
Pregnant women were recruited on their first antenatal visit at various hospitals and health
centres in the Northern Region of Ghana, from October 2016 to February 2017. The study
areas were Tamale metropolis and Central Gonja District. Recruitments were done at Tamale
Teaching Hospital, Tamale Central Hospital, Tamale West Hospital and Bilpella Health Cen-
tre, all located in Tamale metropolis. In the Central Gonja District, the participants were
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 2 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
recruited from Sankpala and Kosawgu Health Centres. A total of 2071 pregnant women were
screened for P. falciparum and HBV infections. Following the exclusion of women with docu-
mented chronic alcoholism, diagnosed eclampsia/preeclampsia, chronic degenerative diseases,
use of hepatotoxic or immunosuppressant drugs, sickle cell trait, and other viral (HIV, hepati-
tis C) and parasitic (amebiasis, hydatid cyst, ascaris and schistosomiasis) infections, 257 were
enrolled and used for this study. Also, demographic and obstetric data were obtained from the
participants’ antenatal care log books.
Ethical consideration
Informed consent was obtained from each study participant after an explanation of the pur-
pose, benefits, and risks of the study was provided. The ethics committee of the Tamale Teach-
ing Hospital approved the study (Approval ID: TTHERC/21/04/16/02).
Clinical and DNA samples
Stool and urine samples were collected and screened for the other parasitic infections that are
listed in the exclusion criteria. Venous blood samples (5 mL) were collected and a portion (4
mL) was processed for serum. The serum was used for the diagnosis of CHB, liver function
test and quantification of cytokines. The remaining blood (1 mL) was used for diagnoses of
malaria, and screening of sickle cell trait and the other viral infections captured in the exclu-
sion criteria. DNA was extracted, from both whole blood and serum, with the QIAamp DNA
mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The extracted
DNA samples were used in the PCR assays for plasmodium species detection and HBV
quantification.
Clinical laboratory analyses
CareStart histidine-rich protein 2 (HRP-2) test cassettes (Access Bio Inc., New Jersey, USA)
and hepatitis B surface antigen (HbsAg) test strips (Ark Biotech., Shanghai, China) were used
for rapid diagnoses of malaria and hepatitis B respectively. Both tests were performed in accor-
dance with the manufacturer’s recommendations.
Malaria parasite densities were determined by microscopy, performed by two independent
World Health Organization (WHO) certified microscopists based on WBC count of 8,000
cells/μL [25].
Hemoglobin was quantified by the cyanomethemoglobin (Drabkin’s) method, using BS-
3000 Chemistry Analyzer (Sinnowa Medical Science and Technology Co. Ltd, Nanjing,
China). Five (5) uL of EDTA blood and standards were thoroughly mixed in separate plain
tubes containing 5 ml Drabkin’s solution (Sigma-Aldrich, St. Louis, USA) and allowed to incu-
bate at room temperature for 30 minutes. Absorbances were read at 540 nm and the hemoglo-
bin concentrations were obtained using a calibration curve. Samples were measured in
duplicates, and those with standard deviations <15% were used in the data analysis.
Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline
phosphatase (ALP) and total bilirubin (Tbil) were measured using the Selectra Pro X5 auto-
mated chemistry analyzer (Elitech Group of Companies, Puteaux, France). The instrument
was calibrated and standardized daily before the measurements were obtained. Samples were
quantified in duplicates and duplicate samples with<15% percentage coefficient of variation
were considered.
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 3 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
PCR for plasmodium species detection
Plasmodium species were detected using a previously described nested PCR method [26, 27].
In the first PCR, the small sub-unit ribosomal genes of plasmodium were amplified using
genus-specific forward and reverse primers. In the second PCR, species-specific forward and
reverse flanking primers were used for detection of P. falciparum, P. vivax, P. malariae, and P.
ovale. For both rounds of PCR, the total reaction volume was 20 μL, containing 2.5 μL of 10X
PCR buffer, 1.5 μL of 25 mM MgCl2, 0.5 μL of 10 μM primers, 0.5 μL of 10 μM deoxynucleo-
tides and 0.2 μL of 1 U Taq DNA polymerase (Qiagen, Hilden, Germany). The PCR products
were visualized on 2% agarose gel containing 1.5 μL ethidium bromide, and the plasmodium
species were identified by respective band sizes.
PCR for quantification of HBV DNA
A PCR assay that was described previously was used to amplify a 98 base pair product of the S-
gene of HBV genome [28]. The total reaction volume of 10 μL consisted of 0.5 μL each of
10 μM forward and reverse primers, 5 μL Low ROX PerfeCTa syber green SuperMix (Quanta
Biosciences Inc., Gaithersburg, USA), 2 μL nuclease free water and 2 μL DNA. Thermal cycling
was performed on QuanStudio 5 TaqMan quantitative real-time PCR system (Thermofisher
Scientific, New Jersey, USA). Cycling conditions were: initial denaturation at 95˚C for 15 min-
utes, followed by 40 cycles of denaturation at 94˚C for 15 seconds, annealing at 55˚C for 30
seconds, and elongation at 68˚C for 30 seconds. The AcroMetrix HBV High Control (Thermo-
fisher Scientific, New Jersey, USA) with viral load of 7.43 log (10) IU/mL was serially diluted
with nuclease free water to four different dilutions of 1/5, 1/25, 1/125, and 1/625 with final
concentrations of 6.82, 6.17, 5.52 and 4.87 log (10) IU/mL, respectively. Before assaying the
samples, the study assessed the performance of the assay by comparing it with the Roche
Molecular Systems for detection of HBV DNA. The co-efficient of variation between both
assays was <5%.
Levels of human anti-HBV virus core antibody (HBcAb)
Sera of pregnant women who tested positive for HBV infection by PCR were each tested for
the presence of HBcAb-IgG and HBcAb-IgM using IgG-HBcAb ELISA kit and IgM- HBcAb
ELISA kit (MyBioSource Inc., San Diego, USA), respectively, following the manufacturer’s
protocols. Absorbances were read at 450 nm using a Varioscan lux plate reader (Thermofisher
Scientific, New Jersey, USA). Each sample was tested in duplicate, and those with standard
deviations <15% were considered. The results obtained were interpreted in accordance with
the manufacturer’s recommendations, and all samples tested were IgG positive and IgM
negative.
Cytokine analysis
Serum samples were thawed and clarified by centrifugation at 14, 000 rpm for 10 minutes and
used to determine the levels of 10 cytokines; interferon gamma (IFN-γ), Tumor necrosis factor
alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-12, IL-2, IL-10, IL-4, IL-5 and IL-13. The cyto-
kines were measured on the xMAP Technology platform (Luminex Corporation, Austin,
USA) using the magnetic beaded Milliplex MAP 13-plex kit (Merck, Darmstadt, Germany),
and following the manufacturer’s protocol. Twenty-six samples of each category of the study
participants were randomly selected for the assay. The samples were assayed in duplicates and
those with<15% percentage coefficient of variation were included in the data analysis.
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 4 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
Statistical analysis
Categorical variables were presented as frequencies and percentages, and compared by Pear-
son’s chi-square test. Continuous parametric data were described as mean and standard devia-
tion (SD) or standard error of mean (SEM), and compared by one-way analysis of variance
(ANOVA) or Tukey’s multiple comparisons test with Tukey’s correction. Continuous non-
parametric data were presented as median and interquartile range (IQR), and compared by
Dunn’s multiple comparison tests with Dunn’s correction. Correlation analyses were done by
Spearman’s test.
Results
Infection statuses and obstetric information
Of the 257 women enrolled, 73 were uninfected, 80 were mono-infected with P. falciparum
(Malaria group), 68 were mono-infected with HBV (CHB group), and 36 were co-infected with
both pathogens (Malaria+CHB group). The infection statuses of the pregnant women were
independent of age, gestation and gravidity (Table 1). All the women in the CHB and Malaria
+CHB groups were IgG positive and IgM negative, which suggests that they had chronic HBV
infections.
Hemoglobin levels
The mean Hb ± SEM was significantly higher in the CHB group (11.0 ± 0.2 g/dL) than the un-
infected (10.2 ± 0.2 g/dL) and the Malaria group (10.0 ± 0.2 g/dL) (Fig 1). However, Hb levels
were statistically similar between the CHB and Malaria+CHB groups (10.3 ± 0.3 g/dL),
P = 0.129. In addition, a higher proportion of women with CHB had normal Hb levels (Hb�
11g/dL) (χ2 = 12.47, P< 0.006, S1 Table).
Serum levels of liver and inflammatory biomarkers
Compared with the other groups, the Malaria+CHB group had significantly elevated levels of
the liver biochemical parameters including ALT (Fig 2A), AST (Fig 2B) and Tbil (Fig 2C). Sim-
ilarly, the group had elevated serum levels of pro-inflammatory cytokines including TNF-α
Table 1. Distribution of demographic and obstetric characteristics in the different groups of pregnant women.
Parameters un-infected Malaria group CHB group Malaria+CHB group α/βP
(n = 73) (n = 80) (n = 68) (n = 36)
Age (years), mean ± SD 27.6 ± 4.9 25.5 ± 5.3 26.7 ± 5.1 26.7 ± 5.7 α0.103
Gravidity, n (%)
Primigravida 12 (16.4%) 26 (32.5%) 16 (23.5%) 9 (25.0%) β0.147
Multigravida 61 (83.6%) 54 (67.5%) 52 (76.5%) 27 (75.0%)
Gestation, n (%)
First trimester 26 (35.6%) 24 (30.0%) 31 (45.6%) 12 (33.3%) β0.257
Second trimester 34 (46.6%) 46 (57.5%) 32 (47.1%) 21 (58.3%)
Third trimester 13 (17.8%) 10 (12.5%) 5 (7.4%) 3 (8.3%)
α: analyzed by one-way ANOVA
β: analyzed by Pearson’s chi-square or Fisher’s exact test
SD: standard deviation
n = number of samples
Statistical significance was considered at P <0.05.
https://doi.org/10.1371/journal.pone.0215550.t001
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 5 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
Fig 1. Comparison of levels of hemoglobin (Hb) amongst the groups of pregnant women. Pair-wise differences
between the groups was tested by the Tukey’s multiple comparisons test with Tukey’s correction, �P = 0.043,
��P = 0.004. Horizontal bar with error bars represents the mean and standard error of mean respectively.
https://doi.org/10.1371/journal.pone.0215550.g001
(Fig 3A), IL-1β (Fig 3B), and IL-6 (Fig 3C). However, compared with the other groups, anti-
inflammatory cytokines including IL-10 (Fig 4A) and IL-4 (Fig 4B), were lower in women
with Malaria+CHB. Women with Malaria and CHB had similar levels of the liver biomarkers
and cytokines, except for significantly higher levels of IL-10 in the Malaria group (Fig 4A).
Correlation of pro-inflammatory cytokine responses with malaria or HBV
intensity in co-infected cohorts
In women with Malaria+CHB, Levels of 3 out of the 6 pro-inflammatory cytokines negatively
correlated with malaria parasitemia [IL-1β (P<0.001; r = -0.645), IL-6 (P = 0.046; r = -0.394)
and IL-12 (P = 0.011; r = -0.49)]. That notwithstanding, parasitemia was similar between the
Malaria+CHB and CHB groups (P = 0.304, Table 2). On the other hand, levels of all the pro-
inflammatory cytokines positively correlated with HBV viremia [TNF-α (P = 0.004; r = 0.549),
IL-1β (P<0.001; r = 0.920), IL-6 (P<0.001; r = 0.777), IFN-γ (P = 0.002; r = 0.579), IL-2 (P =
0.008; r = 0.512) and IL-12 (P<0.001; r = 0.655)], and viremia was significantly lower in the
Malaria+CHB compared to the CHB group (P = 0.016, Table 2). Further analysis showed that
malaria parasitemia negatively correlated with hepatitis B viremia in women with Malaria
+CHB (P = 0.003, r = -0.489).
Discussion
Maternal anemia is an issue of important public health relevance. During pregnancy, women
with Hb <11g/dL are diagnosed as anemic while those with Hb� 11g/dL are considered to
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 6 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
Fig 2. Differences in levels of liver biochemical parameters across the different categories of pregnant women. Dunn’s multiple comparison tests for
pair-wise differences in serum levels of (A) alanine aminotransferase (ALT), (B) aspartate amino transferase (AST), (C) total bilirubin (Tbil) and (D) alkaline
phosphatase (ALP). Significant P-values were observed at �P< 0.05, ��P< 0.01 and ����P< 0.0001. Horizontal bar with error bars represents the median
and the interquartile range respectively.
https://doi.org/10.1371/journal.pone.0215550.g002
have normal level [29]. P. falciparum causes anemia by reducing red cell counts, HBV on the
other hand is postulated to increase Hb levels by increasing the release of erythropoietin
from regenerating hepatic tissues [15–17]. It is therefore logical that pregnant women with
Malaria+CHB maintained intermediate levels of hemoglobin (above the Malaria group and
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 7 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
Fig 3. Differences in levels of pro-inflammatory cytokines amongst the groups of pregnant women. Dunn’s multiple comparison tests for differences in
serum levels of (A) tumour necrosis factor alpha (TNF-α), (B) interleukin (IL)-1β, (C) IL-6, (D) interferon gamma (IFN-γ), (E) IL-2 and (F) IL-12. Statistical
significance was observed at �P< 0.05, ��P< 0.01 and ���� P< 0.0001. Horizontal bar with error bars represents the median and the interquartile range
respectively. n = number of samples of each category of the study participants assayed.
https://doi.org/10.1371/journal.pone.0215550.g003
un-infected, and below CHB group). This observation may suggest that in women with
Malaria+CHB, activities of the plasmodium parasites in reducing red cell count may be com-
pensated for by the activities of the virus, thus maintaining an appreciable hemoglobin concen-
tration. The significant association of CHB with Hb�11g/dL further justifies the possible
impact of HBV in increasing hemoglobin levels among the pregnant women. However, this
might not necessarily translate into improved pregnancy outcomes, since cases of gestational
diabetes, antepartum hemorrhage and preterm delivery are observed to be more frequent in
pregnant women with CHB [30–32].
Relative to the un-infected (Figs 2 and 3), the increased serum levels of the liver function
biomarkers and pro-inflammatory cytokines in the Malaria or CHB groups have been previ-
ously reported [3–6, 8–14]. In particular, elevated levels of IL-10 among the Malaria group is a
common finding amongst African multigravidae mothers with P. falciparum infection [14,
33–35], and as such, the cytokine has been implicated in the immunopathology of placental
malaria [36].
Similarity in levels of the liver biomarkers between women with Malaria and CHB may
lend credence that the elevated levels in those with Malaria+CHB resulted from additive activi-
ties of both pathogens. The synergistic elevation in levels of the liver biomarker in the Malaria
+CHB group may be attributed to immune factors that limit the infections at the liver-stage.
Particularly, natural killer (NK) and natural killer T (NKT) cells which are abundantly
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 8 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
Fig 4. Comparison of serum levels of anti-inflammatory cytokines in the different categories of pregnant women. Dunn’s multiple comparison tests for significant P-
values in levels of (A) IL-10, (B) IL-4 (C) IL-5 and (D) IL-13. Statistical significance was observed at �P< 0.05, �� P< 0.01 and ���P< 0.001. Horizontal bar with error
bars represents the median and the interquartile range respectively. n = number of samples of each category of the study participants assayed.
https://doi.org/10.1371/journal.pone.0215550.g004
available in the liver, interact with pathogens and initiate liver-stage cell-mediated immunity
[37, 38]. For HBV infections, NK cells contribute to liver inflammation by tumor necrosis fac-
tor–related apoptosis-inducing ligand (TRAIL)-mediated death of hepatocytes, which is non-
antigen–specific, and can be switched on by a milieu of cytokines [39]. Therefore, it is possible
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 9 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
Table 2. Differences in malaria parasitemia and hepatitis B viremia amongst the different categories of pregnant women.
Parameters Malaria group CHB group Malaria+CHB group P
(n = 80) (n = 68) (n = 36)
Malaria parasite count 1310 860 0.304
(parasites/μL), (490–2560) (420–1870)
Median (IQR)
Hepatitis B viremia 4.65 4.05 0.016
[Log10 mean (IU/mL)], (4.09–5.24) (3.54–5.09)
Median (IQR)
P: analyzed by Mann Whitney test, considered statistically significant at <0.05
IQR: interquartile range.
n = number of samples
https://doi.org/10.1371/journal.pone.0215550.t002
that, in women with Malaria+CHB, cytokines released in response to P. falciparum could fur-
ther activate the apoptosis of HBV-infected hepatocytes, and exacerbate liver damage.
While peripheral levels of TNF-α, IL-1β and IL-6 were similar in women with Malaria and
CHB, the pro-inflammatory cytokines were significantly increased in those with Malaria
+CHB, which further suggests a possible additive effect of the infections. The correlation analy-
sis with the Malaria+CHB group suggests that increased pro-inflammatory cytokine levels as a
necessary immune response against malaria helped in reducing HBV intensity. This corrobo-
rates with studies indicating that P. falciparum malaria modulates viremia in chronic hepatitis
B virus infection [40, 41]. IL-10 and IL-4 are key anti-inflammatory cytokines that regulate the
activities of pro-inflammatory cytokines responses. Thus, the diminished peripheral levels of
IL-10 and IL-4 in the women with Malaria+CHB may suggest susceptibility to cytokine imbal-
ance (towards Th1). In particular, placental cytokine imbalance is noted to be associated with
threatened abortion, recurrent spontaneous miscarriage, and preterm delivery [18, 42]. That
notwithstanding, biased peripheral pro-inflammatory cytokine responses has been implicated
with pregnancy complications [43, 44].
Limitations
A longitudinal approach on changes of Hb levels in the co-infected cohorts after clearance of
P. falciparum would have been paramount in substantiating the compensatory effect of the
viral infection. Again such an approach would have enabled us to see the kinetics of Malaria
+CHB co-infection and to identify times when the markers of liver injury and inflammatory
responses are enhanced. In addition, such an approach would have enabled us to substantiate
our findings by profiling placental cytokines responses, malaria parasitemia and HBV viremia,
and associating the results with pregnancy complications. Also profiling of innate immune
cells and T-cell populations would have given a better insight of the effect of the co-infection
on the immune response. Nonetheless, the current study provides evidence on the need to
take into cognizance the possible deleterious impact of Malaria+CHB on pregnant women. In
this regard, liver functions test which is important in guiding diagnosis and treatment of liver
diseases in pregnancy must be readily accessible to pregnant women on ANC clinics.
Conclusion
Put together the findings suggests that Malaria+CHB could exacerbate inflammatory cytokine
responses and increase susceptibility to liver injury among pregnant women in endemic
settings.
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 10 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
Supporting information
S1 Table. Association between infection type and normal hemoglobin levels during preg-
nancy.
(DOCX)
Acknowledgments
We thank the study participants, and the numerous health personnel at Tamale Metropolis
and Central Gonja for their immense support.
Author Contributions
Conceptualization: Gideon Kofi Helegbe.
Data curation: Nsoh Godwin Anabire, Paul Armah Aryee, Abass Abdul-Karim, Osbourne
Quaye, Gordon Akanzuwine Awandare, Gideon Kofi Helegbe.
Formal analysis: Nsoh Godwin Anabire, Paul Armah Aryee, Abass Abdul-Karim, Osbourne
Quaye, Gordon Akanzuwine Awandare, Gideon Kofi Helegbe.
Funding acquisition: Nsoh Godwin Anabire, Gordon Akanzuwine Awandare.
Investigation: Nsoh Godwin Anabire.
Methodology: Nsoh Godwin Anabire, Paul Armah Aryee, Abass Abdul-Karim, Osbourne
Quaye.
Project administration: Gideon Kofi Helegbe.
Supervision: Gordon Akanzuwine Awandare, Gideon Kofi Helegbe.
Validation: Nsoh Godwin Anabire, Paul Armah Aryee, Osbourne Quaye, Gordon Akanzu-
wine Awandare, Gideon Kofi Helegbe.
Visualization: Nsoh Godwin Anabire, Paul Armah Aryee, Abass Abdul-Karim, Osbourne
Quaye, Gordon Akanzuwine Awandare, Gideon Kofi Helegbe.
Writing – original draft: Nsoh Godwin Anabire.
Writing – review & editing: Paul Armah Aryee, Osbourne Quaye, Gordon Akanzuwine
Awandare, Gideon Kofi Helegbe.
References
1. Helegbe GK, Aryee PA, Mohammed BS, Wemakor A, Kolbila D, Abubakari AW, Askanda S, Alhassan
R, Barnie C, Donkoh AA, and Ofosu E. Seroprevalence of Malaria and Hepatitis B Coinfection among
Pregnant Women in Tamale Metropolis of Ghana: A Cross-Sectional Study. Canadian Journal of Infec-
tious Diseases and Medical Microbiology. 2018;2018.
2. Anabire NG, Aryee PA, Abdul-Karim A, Abdulai IB, Quaye O, Awandare GA, and Helegbe GK. Preva-
lence of malaria and hepatitis B among pregnant women in Northern Ghana: Comparing RDTs with
PCR. PloS one. 2019 Feb 6; 14(2):e0210365. https://doi.org/10.1371/journal.pone.0210365 PMID:
30726218
3. Mackillop L, and Williamson C. Liver disease in pregnancy. Postgrad Med J. 2010; 86(1013):160–4.
https://doi.org/10.1136/pgmj.2009.089631 PMID: 20237010
4. Nwosu DC, Nwanjo HU, Obeagu EI, Ibebuike JE, Ezeama MC, and Ihekireh. Changes in liver enzymes
and lipid profile of pregnant women with malaria in Owerri, Nigeria. International Journal of Current
Research and Academic Review. 2015; 3(5):376–83.
5. Eldaim N, and Elbadawi E. The effect of malaria on biochemical liver function parameters in Sudanese
pregnant women. Journal of Physiobiochemical Metabolism. 2012.
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 11 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
6. Woodford J, Shanks GD, Griffin P, Chalon S, and McCarthy J. The Dynamics of Liver Function Test
Abnormalities after Malaria Infection: A Retrospective Observational Study. Am J Trop Med Hyg. 2018.
7. Mahtab M-A, Rahman S, Khan M, Mamun A, and Afroz S. Etiology of fulminant hepatic failure: experi-
ence from a tertiary hospital in Bangladesh. Hepatobiliary Pancreat Dis Int. 2008; 7(2):161–4. PMID:
18397851
8. Tan H-H, Lui H-F, and Chow W-C. Chronic hepatitis B virus (HBV) infection in pregnancy. Hepatology
international. 2008; 2(3):370–5. https://doi.org/10.1007/s12072-008-9063-4 PMID: 19669267
9. Elefsiniotisa IS, Brokalakia H, Argyropoulosa E, Magaziotou I, Derdemezib A, Mihasa C, and Tsouma-
kasb K. Evaluation of liver enzymes in asymptomatic chronic hepatitis B virus infected pregnant
women. Annals of Gastroenterology: Quarterly Publication of the Hellenic Society of Gastroenterology.
2013; 26(1):59.
10. Sarin SK, Kumar M, Shrivastava S, Sinha S, and Pati NT. Influence of chronic HBV infection on preg-
nancy: a human model of maternofetal virus host interactions. Gastroenterology. 2011; 141(4):1522–5.
https://doi.org/10.1053/j.gastro.2011.08.016 PMID: 21867707
11. Saxena R, Chawla YK, Verma I, and Kaur J. Association of interleukin-10 with hepatitis B virus (HBV)
mediated disease progression in Indian population. The Indian journal of medical research. 2014; 139
(5):737. PMID: 25027084
12. Kfutwah A, Mary JY, Lemen B, Leke R, Rousset D, Barré-Sinoussi F, Nerrienet E, Menu E, Ayouba A,
and ANRS 1267 study team. Plasmodium falciparum infection significantly impairs placental cytokine
profile in HIV infected Cameroonian women. PloS one. 2009; 4(12):e8114. https://doi.org/10.1371/
journal.pone.0008114 PMID: 19956547
13. Ifeanyichukwu M, Okamgba O, Amilo G, and Nwokorie E. Peripheral parasitaemia and its association
with plasma cytokines levels in malaria-infected pregnant women in Aba, Abia State, Nigeria. African
Journal of Infectious Diseases. 2017; 11(2):54–61. https://doi.org/10.21010/ajid.v11i2.7 PMID:
28670640
14. Nmorsi O, Isaac C, Ohaneme B, and Obiazi H. Pro–inflammatory cytokines profiles in Nigerian preg-
nant women infected with Plasmodium falciparum malaria. Asian Pacific Journal of Tropical Medicine.
2010; 3(9):731–3.
15. Ifudu O, and Fowler A. Hepatitis B virus infection and the response to erythropoietin in end-stage renal
disease. ASAIO journal. 2001; 47(5):569–72. PMID: 11575840
16. Simon P, Boffa G, Ang K, and Menault M. Polycythaemia in a haemodialyzed anephric patient with hep-
atitis. Demonstration of erythropoietin secretion (author’s transl). La Nouvelle presse medicale. 1982;
11(18):1401–3. PMID: 7079159
17. Klassen DK, and Spivak JL. Hepatitis-related hepatic erythropoietin production. The American journal
of medicine. 1990; 89(5):684–6. PMID: 2239989
18. Raghupathy R, and Kalinka J. Cytokine imbalance in pregnancy complications and its modulation.
Front Biosci. 2008; 13(1):985–94.
19. Joshi D, James A, Quaglia A, Westbrook RH, and Heneghan MA. Liver disease in pregnancy. The Lan-
cet. 2010; 375(9714):594–605.
20. Westbrook RH, Yeoman AD, Joshi D, Heaton ND, Quaglia A, O’Grady JG, Auzinger G, Bernal W,
Heneghan MA, and Wendon JA. Outcomes of Severe Pregnancy-Related Liver Disease: Refining the
Role of Transplantation. American Journal of Transplantation. 2010; 10(11):2520–6. https://doi.org/10.
1111/j.1600-6143.2010.03301.x PMID: 20977643
21. Baheti R, Laddha P, and Gehlot R. Liver involvement in falciparum malaria–A Histo-pathological analy-
sis. J Indian Acad Clin Med. 2003; 4(1):34–8.
22. Harinasuta T, and Bunnag D. The clinical features of malaria. Malaria: principles and practice of malari-
ology Edited by Wernsdorfer WH, McGregor I London: Churchill Livingstone. 1988:709–34.
23. Mor G, Cardenas I, Abrahams V, and Guller S. Inflammation and pregnancy: the role of the immune
system at the implantation site. Annals of the New York Academy of Sciences. 2011; 1221(1):80–7.
24. Saito S, Nakashima A, Shima T, and Ito M. REVIEW ARTICLE: Th1/Th2/Th17 and Regulatory T-Cell
Paradigm in Pregnancy. American journal of reproductive immunology. 2010; 63(6):601–10. https://doi.
org/10.1111/j.1600-0897.2010.00852.x PMID: 20455873
25. World Health Organization. Routine examination of blood films formalaria parasites. In: World Health
Organization, editor Basic Mala´riaMicroscopy, Learner’s guide Switzerland: WHO. 2010:69–76.
26. Snounou G, Viriyakosol S, Jarra W, Thaithong S, and Brown KN. Identification of the four human
malaria parasite species in field samples by the polymerase chain reaction and detection of a high prev-
alence of mixed infections. Molecular and biochemical parasitology. 1993; 58(2):283–92. PMID:
8479452
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 12 / 13
Liver function and cytokine responses in pregnant women co-infected with malaria and hepatitis B
27. Nsobya SL, Parikh S, Kironde F, Lubega G, Kamya MR, Rosenthal PJ, and Dorsey G. Molecular evalu-
ation of the natural history of asymptomatic parasitemia in Ugandan children. Journal of Infectious Dis-
eases. 2004; 189(12):2220–6. https://doi.org/10.1086/421281 PMID: 15181569
28. Garson J, Grant P, Ayliffe U, Ferns R, and Tedder R. Real-time PCR quantitation of hepatitis B virus
DNA using automated sample preparation and murine cytomegalovirus internal control. Journal of viro-
logical methods. 2005; 126(1):207–13.
29. World Health Organization. Haemoglobin concentrations for the diagnosis of anaemia and assessment
of severity. 2011.
30. Jonas MM. Hepatitis B and pregnancy: an underestimated issue. Liver international: official journal of
the International Association for the Study of the Liver. 2009; 29 Suppl 1:133–9.
31. Tse KY, Ho LF, and Lao T. The impact of maternal HBsAg carrier status on pregnancy outcomes: a
case-control study. Journal of hepatology. 2005; 43(5):771–5. https://doi.org/10.1016/j.jhep.2005.05.
023 PMID: 16139923
32. Safir A, Levy A, Sikuler E, and Sheiner E. Maternal hepatitis B virus or hepatitis C virus carrier status as
an independent risk factor for adverse perinatal outcome. Liver international: official journal of the Inter-
national Association for the Study of the Liver. 2010; 30(5):765–70.
33. Boström S, Ibitokou S, Oesterholt M, Schmiegelow C, Persson JO, Minja D, Lusingu J, Lemnge M, Fie-
vet N, Deloron P, and Luty AJ. Biomarkers of Plasmodium falciparum infection during pregnancy in
women living in northeastern Tanzania. PLoS One. 2012; 7(11):e48763. https://doi.org/10.1371/
journal.pone.0048763 PMID: 23155405
34. Kabyemela ER, Fried M, Kurtis JD, Mutabingwa TK, and Duffy PE. Decreased susceptibility to Plasmo-
dium falciparum infection in pregnant women with iron deficiency. The Journal of infectious diseases.
2008; 198(2):163–6. https://doi.org/10.1086/589512 PMID: 18500927
35. Boeuf P, Aitken EH, Chandrasiri U, Chua CL, McInerney B, McQuade L, Duffy M, Molyneux M, Brown
G, Glazier J, and Rogerson SJ. Plasmodium falciparum malaria elicits inflammatory responses that dys-
regulate placental amino acid transport. PLoS pathogens. 2013 Feb 7; 9(2):e1003153. https://doi.org/
10.1371/journal.ppat.1003153 PMID: 23408887
36. Suguitan AL Jr, Cadigan TJ, Nguyen TA, Zhou A, Leke RJ, Metenou S, Thuita L, Megnekou R, Fogako
J, Leke RG, and Taylor DW. Malaria-associated cytokine changes in the placenta of women with pre-
term deliveries in Yaounde, Cameroon. The American journal of tropical medicine and hygiene. 2003
Dec 1; 69(6):574–81. PMID: 14740871
37. Gao B, Radaeva S, and Park OJJolb. Liver natural killer and natural killer T cells: immunobiology and
emerging roles in liver diseases. 2009; 86(3):513–28. https://doi.org/10.1189/JLB.0309135 PMID:
19542050
38. Holz LE, Fernandez-Ruiz D, and Heath WR. Protective immunity to liver-stage malaria. Clinical & trans-
lational immunology. 2016 Oct; 5(10):e105
39. Dunn C, Brunetto M, Reynolds G, Christophides T, Kennedy PT, Lampertico P, Das A, Lopes AR, Bor-
row P, Williams K, and Humphreys E. Cytokines induced during chronic hepatitis B virus infection pro-
mote a pathway for NK cell–mediated liver damage. 2007; 204(3):667–80. https://doi.org/10.1084/jem.
20061287 PMID: 17353365
40. Pasquetto V, Guidotti LG, Kakimi K, Tsuji M, and Chisari FV. Host–virus interactions during malaria
infection in hepatitis B virus transgenic mice. The Journal of experimental medicine. 2000; 192(4):529–
36. PMID: 10952722
41. Brown AE, Mongkolsirichaikul D, Innis B, Snitbhan R, and Webster HK. Falciparum malaria modulates
viremia in chronic hepatitis B virus infection. Journal of Infectious Diseases. 1992; 166(6):1465–6.
PMID: 1431272
42. Sharma L, and Shukla G. Placental Malaria: A new insight into the Pathophysiology. Frontiers in medi-
cine. 2017 Jul 25; 4:117. https://doi.org/10.3389/fmed.2017.00117 PMID: 28791290
43. Azizieh FY, and Raghupathy RG. Tumor necrosis factor-α and pregnancy complications: a prospective
study. Medical Principles and Practice. 2015; 24(2):165–70. https://doi.org/10.1159/000369363 PMID:
25501617
44. Azizieh F, Dingle K, Raghupathy R, Johnson K, VanderPlas J, and Ansari A. Multivariate analysis of
cytokine profiles in pregnancy complications. American Journal of Reproductive Immunology. 2018
Mar; 79(3):e12818.
PLOS ONE | https://doi.org/10.1371/journal.pone.0215550 April 19, 2019 13 / 13