Association between Epstein-Barr virus reactivation and severe malaria in pregnant women living in a malaria-endemic region of Cameroon

Ide Armelle Djuidje Chatue; Maximilienne Ascension Nyegue; Severin Donald Kamdem; Franklin Maloba; Iqbal Taliy Junaid; Pawan Malhotra; Palmer Masumbe Netongo

doi:10.1371/journal.pgph.0003556

. 2024 Aug 12;4(8):e0003556. doi: 10.1371/journal.pgph.0003556

Association between Epstein-Barr virus reactivation and severe malaria in pregnant women living in a malaria-endemic region of Cameroon

Ide Armelle Djuidje Chatue ^1,^2,³, Maximilienne Ascension Nyegue ¹, Severin Donald Kamdem ^2,⁴, Franklin Maloba ^2,^5,⁶, Iqbal Taliy Junaid ³, Pawan Malhotra ^3,^*, Palmer Masumbe Netongo ^2,^6,^7,^*

Editor: Julia Robinson⁸

PMCID: PMC11318859 PMID: 39133703

Abstract

Malaria kills nearly 619,000 people each year. Despite the natural immunity acquired to malaria, pregnant women and children under five die from severe forms of the disease in sub-Saharan Africa. Co-infection with acute Epstein-Barr Virus (EBV) infection has been shown to suppress the anti-malarial humoral responses, but little is known about the impact of EBV reactivation on malaria-associated morbidity. This study investigated the association between EBV reactivation and malaria severity in pregnant women living in a malaria-endemic region in Cameroon. A cross-sectional study was conducted on 220 pregnant women attending antenatal consultations in three health facilities in the West region of Cameroon. Malaria was diagnosed by microscopy, and Plasmodium species were identified by Nested PCR. Plasma samples were analyzed by ELISA for the presence of EBV nuclear antigen, EBV viral capsid antigen, and EBV early antigen to determine EBV reactivation. All statistics were performed using GraphPad Prism and SPSS software. The prevalence of malaria among pregnant women was 23.2%, of which 18.6% were P. falciparum mono-infections and 4.5% mixed infections (3.6% P. falciparum and P. malariae; 0.9% P. falciparum and P. ovale). 99.5% of the women were EBV seropositive, and 13.2% had EBV reactivation. Pregnant women with reactivated EBV were more likely to develop severe malaria than pregnant women with latent EBV (OR 4.33, 95% CI 1.08–17.25, p = 0.03). The median parasitemia in pregnant women with latent EBV was lower than in those with EBV reactivation (2816 vs. 19002 parasites/μL, p = 0.02). Our study revealed that lytic reactivation of EBV may be associated with the severity of malaria in pregnant women. Suggesting that, like acute infection, EBV reactivation should be considered a risk factor for severe malaria in pregnant women in malaria-endemic regions or could serve as a hallmark of malaria severity during pregnancy. Further detailed studies are needed.

Introduction

Malaria is a deadly tropical disease caused by an obligate intracellular protozoan of the genus Plasmodium that infects human hosts through the bites of infected female Anopheles mosquitoes [1]. In 2021, the World Health Organization estimated that around 247 million people were infected worldwide, and 619,000 died [2] due to malaria; ~34% rise in deaths compared to pre-Covid-19 pandemic in 2019 [3]. Malaria accounts for 29.9% of consultations and 64% of hospitalizations in Cameroon. According to the Cameroon National Malaria Control Programme, around 6 million malaria cases and 4,000 deaths were recorded in 2022 [4, 5]. Malaria during pregnancy is a major public health concern for developing countries, especially in sub-Saharan Africa. In 2021, approximately 13.3 million pregnant women living in African regions were exposed to malaria infection during pregnancy, and 961,000 infants born from these women had a low birth weight [2]. The consequences of malaria during pregnancy include severe maternal anemia, low birth weight, premature delivery, and maternal and infant mortality [6–8].

In sub-Saharan Africa, where malaria is endemic, co-infections with one or more pathogens are common due to poor health and socio-economic conditions [9, 10]. One of the most common pathogens is the Epstein-Barr virus, a human herpes virus that infects more than 95% of the world’s population and persists throughout life [11, 12]. The virus is transmitted mainly orally, by direct contact with contaminated saliva, or indirectly through blood transfusion, organ or tissue transplantation, sexual intercourse, and breast milk [13–15]. EBV infection in children usually doesn’t cause symptoms. However, the most common primary symptoms of EBV-infected children include fever, cough, skin eruption, lymphadenopathy, eyelid edema, and pharyngalgia [16]. In adolescents and young adults, the primary infection clinically manifests as acute infectious mononucleosis [11, 17] or can cause autoimmune diseases and lymphoproliferative malignancies in immunocompromised individuals [18, 19]. During pregnancy, 35% of pregnant women show EBV reactivation due to the decrease in cellular immunity [20]. The consequences of EBV reactivation in pregnancy include severe symmetrical fetal growth retardation, low birth weight, stillbirth, congenital malformations, and a shorter duration of pregnancy [21–23].

There is compelling evidence that co-infections with the Epstein-Barr virus worsen the health conditions of people infected with P. falciparum in malaria-endemic areas [24–26]. Previous studies have shown that acute infection with a gammaherpesvirus 68 (MHV68), such as EBV, negatively influences the development of the humoral response to secondary P. falciparum infection, transforming a non-lethal infection into a lethal one [27, 28]. Recently, a study revealed that persistent EBV DNA in the peripheral blood of adults in a P. vivax semi-immune population alters the antibody response to major malaria vaccine candidates (DEKnull-2) [29]. It has also been suggested that the reactivation of EBV infection may facilitate the development of cerebral malaria. Indeed, Indari et al. demonstrated that during malaria, EBV reactivation increased red blood cells adhesion to the human brain endothelial cells and significantly elevated inflammatory markers, contributing to the exacerbation of cerebral malaria [30].

However, the impact of the reactivation of EBV on the severity of malaria during pregnancy has not been studied yet. Although higher EBV viral loads and EBV-specific antibody levels have been observed in pregnant women with malaria than in pregnant women without malaria [31], the association between EBV reactivation and malaria severity during pregnancy remains unclear. Besides this, it is still unclear that some pregnant women in P. falciparum-endemic areas naturally clear the parasites [32, 33], while others are more likely to develop severe forms of the disease. This study aimed to investigate the association between EBV reactivation and severe malaria in pregnant women living in a malaria-endemic region of Cameroon and to assess if EBV reactivation increases the severity of malaria attacks in pregnant women.

Materials and methods

Study design

A cross-sectional study was conducted between January and September 2022 on pregnant women of all gravida attending antenatal consultations or hospitalized in the maternity or gynecology services of the different hospitals. Data were collected during the dry and rainy seasons when transmission of Plasmodium is high [34]. A well-structured questionnaire was administered to participants who voluntarily agreed to participate in the study to collect socio-demographic data.

Study site

The study was conducted in two health facilities in the Nde division (Bangangte District Hospital (BDH) and Universite des Montagnes Teaching Hospital (CUM)) and one health facility in the Mifi division [Mifi District Hospital of Bafoussam (MDHB)] in the West Region of Cameroon (Fig 1). These hospitals provide health services that are accessible to the public with high-quality and low-cost antenatal care for pregnant women. Bafoussam and Bangangte are the heads of the Mifi and Nde divisions. Bangangte covers an area of 829 km² with an estimated population of 200,000, while Bafoussam covers an area of 402 km² with a population of 347,517. These two semi-urban towns are located in the West region of Cameroon (5°30′N and 10°30′E), an area made up of mountains, plains, and plateaux at altitudes of between 1,000 and 1,500 meters. The climate is humid tropical, with heavy rainfall favoring the proliferation of Anopheles mosquitoes because of a rainy season lasting around seven months (from late March to October) and a dry season lasting five months (from late October to early March) [35]. Malaria transmission is relatively stable in the region. Entomological inoculation rates vary from 62.8 to 90.5 infectious bites/per person/year [36, 37].

Fig 1 — BDH: Bangangte District Hospital, MDHB: Mifi District Hospital of Bafoussam; CUM: Universite des Montagnes Teaching Hospital of Bangangte. Source: Own elaboration. The map was created using the software QGIS version 3.32.3-Lima. *https*:*//qgis*.org/. QGIS is licensed under the GNU General Public License version 2, *https*:*//www*.gnu.org/licenses. The base layer of the Cameroon map was obtained on the website *http*:*//www*.diva-gis.org/gdata and data were extracted from the GADM database (*www*.gadm.org), version 2.5.

Study population

The study population consisted of pregnant women of all gestational ages and gravida attending antenatal consultation or hospitalized at the three selected hospitals. Participants approached the hospital with at least one of the following symptoms: fever (temperature ≥ 38°C) accompanied by chills, asthenia, fatigue, headache, sore throat, sore muscle, sweat and thrill, pallor, jaundice, dizziness, abdominal pain, and respiratory distress. The inclusion criteria were as follows: consenting pregnant women of all gestational ages with at least one of the above symptoms and seronegative for Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV). We excluded all pregnant women seropositive for cytomegalovirus, intestinal worms, toxoplasmosis (IgM), or any infections mentioned in the inclusion criteria; pregnant women on corticosteroids, acyclovir, antihistamines, antibiotics, and antimalarial; and those with confirmed depression because these factors may constitute a bias for the rest of this research work.

Ethical statement

This study has been approved by the National Ethics Committee for Human Health Research of Yaounde (N°2021/12/106/CE/CNERSH/SP). Administrative authorizations were obtained from all health establishments. Written consent was obtained from each participant or their parents/guardians (for minors) before registration in the study. Positive malaria cases were referred to clinicians at the hospital for appropriate therapeutic management.

Sample size determination

Participants were recruited by random sampling during the antennal consultation. The sample size (N) was calculated using Cochran’s formula for large population proportion [38]: [N = Z² _α/2 P(1-P) / E²]. Where Z is the area under the acceptance region in a normal distribution (1 –α), it is 1.96 for a 95% confidence interval. P is the prevalence of EBV and malaria coinfection among pregnant women in Kenya, which is 64% [31]. Using a tolerated error of 5% (E), the sample size required for this study was 354 participants.

Data collection

A well-structured questionnaire in both official languages (English and French) was used to collect socio-demographic data (sex, age, residence, weight, environment, employment status, marital status, level of education, ethnicity, and maternal income); obstetric factors (gravidity, parity, trimester of pregnancy); clinical symptoms; malaria prevention measures (use of insecticide-treated mosquito nets, Intermittent Preventive Treatment); and risk factors for EBV reactivation in participants (alcohol, stress, anxiety, illicit substances [39–42]). Insecticide-treated net use was defined as Yes for those who had or used a net and No for those who did not have or use a net. IPT use was defined as follows: Yes, for those who took at least one dose of IPT during pregnancy and No for those who did not take IPT.

Blood sample collection

Approximately 5 mL of blood was collected from each participant by venipuncture into an ethylenediaminetetraacetic acid (EDTA) tube. All samples were transported using a medical cooler box and sent to the Laboratory of Microbiology of “Universite des Montagnes” Teaching Hospital in Bangangte for analysis. The blood collected was used to diagnose malaria and determine hemoglobin levels. The plasma extracted after centrifugation was used to diagnose EBV infection. Up to 50 μL blood spotted on Whatman 903TM filter paper (Lasec, Cape Town, South Africa) was stored at room temperature for the molecular identification of Plasmodium species.

Diagnosis of malaria

Microscopy of malaria

A thick blood smear was prepared to detect malaria parasites as described by WHO [43]. The number of parasites was counted per 200 leukocytes, and the parasite density was calculated assuming a total WBC count of 8,000 cells/μL of blood [44]. Parasite density was classified into three categories: Low (1–999 parasites/μL), Moderate (1000–9999 parasites/μL), and High (≥10000 parasites /μL) [45]. Pregnant women with high parasitemia associated with one or more of the manifestations of severe malaria, such as were classified as severe malaria cases according to WHO [46]. Pregnant women with a parasitemia of less than 10,000 parasites/μL and who did not have these characteristics were considered uncomplicated malaria cases.

Nested PCR

DNA was extracted from Whatman 903 TM cards using the phenol-chloroform method as described previously [47, 48]. The identification of Plasmodium species was performed using the nested PCR technique. Specific sequences of P. falciparum, P. ovale, P. vivax, and P. malariae 18S rRNA small subunit genes were amplified as described by Snounou et al. [49] with minor modification of the amplification conditions. PCR products were purified using the QIAquick PCR Purification Kit (Cat N˚28106, Hilden, Germany) and sent for sequencing to Macrogen in South Korea to confirm the species identified.

Diagnosis of EBV infection

Patient plasma samples were analyzed for the presence and level of viral capsid antigen immunoglobulin IgM (VCA), nuclear antigen immunoglobulin IgG (NA-1), and early antigen IgG (EA-D) using commercial ELISA kits (TestLine Clinical Diagnostics s.r.o, Czech Republic) according to the manufacturer’s instructions. Quantitative results were interpreted as follows: antibody levels >22 U/ml were considered positive; antibody levels <18 U/ml were considered negative, and antibody levels between 18 to 22 were considered borderline area. For semi-quantitative results, the positivity index (PI) was calculated as follows: PI = Absorbance of the sample / mean absorbance of the cut-off. The results were interpreted based on the following index value thresholds: Negative if IP <0.9, positive if IP > 1.1, and borderline if 0.9<IP <1.1, following the manufacturer’s instructions.

Epstein-Barr virus reactivation was defined by the presence of IgG EBNA-1 along with IgG EA or IgG EBNA-1 with IgM VCA or both IgG EA and IgM VCA (Table 1) [50–52].

Table 1. Clinical status of Epstein-Barr infection according to viral serology results.

Clinical Status	VCA IgM	EA IgG	EBNA-1 IgG
No previous infection	-	-	-
Acute infection	+	+	-
Past infection	-	-	+
Reactivation	+/-	+	+/-

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(-) antibody absence; (+) antibody presence

Statistical analysis

The data collected was entered into an Excel spreadsheet and then analyzed using GraphPad Prism 9.0 software and SPSS version 23.0. Quantitative variables were represented by the median (IQR), while frequency and percentage were used to summarise qualitative data. The Mann-Whitney U test was used for comparison between two groups, and the Kruskal-Wallis test was used for comparisons across multiple groups. Multivariate logistic regression was used to determine factors associated with malaria, and univariate logistic regression was used to assess the association between EBV reactivation and severe malaria. Simple linear regression was used to determine the correlation between parasitemia and the level of EBV antibodies. Statistically significance was considered as p-value <0.05 at the 95% confidence interval.

Results

Study flow diagram

A total of 371 symptomatic pregnant women attending antenatal clinics were interviewed in three hospitals in the West region of Cameroon. Of these, 296 voluntarily agreed to participate in the study. Serological diagnosis for HIV, HBV, HCV, and toxoplasmosis (IgM) was performed on consenting participants (Fig 2).

Fig 2 — Ethical clearance and administrative authorizations were obtained from the relevant authorities. Based on EBV and malaria infection status, study participants were classified into 8 groups (EBV: Epstein-Barr virus, M: malaria, R: reactivation, UM: uncomplicated malaria, SM: severe malaria, L: latent, N: Negative and A: Acute, (+): presence, (-): absence).

Sociodemographic and obstetric characteristics of the study population

Using a well-structured questionnaire, sociodemographic data were collected and presented in Table 2. The median age of the participants was 26 years (IQR: 23–31 years), and the median gestational age was 21 weeks (IQR: 16–27 weeks). Ninety participants (40.9%) were married, 151 (68.6%) had secondary education, 146 (66.4%) lived in urban areas, and 69 (31.4%) were unemployed. In addition, 141 participants (64.1%) were multiparous, and 123 (55.9%) were in the second trimester of pregnancy. The study population was diverse, with a predominance of the Bamileke ethnic group (92.7%).

Table 2. Socio-demographic characteristics of study participants from three health centers in the West region of Cameroon, January to September 2022.

Variables	Frequency^* (n = 220)
Median age in years	26 years, IQR: 23–31
Marital status
Single	59 (26.8%)
Married	90 (40.9%)
In relationship	71 (32.3%)
Education
Primary	8 (3.6%)
Secondary	151 (68.6%)
High Education	57 (25.9%)
Illiterate	4 (1.8%)
Environment
Rural	74 (33.6%)
Urban	146 (66.4%)
Occupation
Student	47 (21.4%)
Employed	63 (28.6%)
Farmer	41 (18.6%)
Unemployed	69 (31.4%)
Maternal income
Low	77 (35%)
Middle	143 (65%)
Parity
Primigravidae	79 (35.90%)
Multigravidae	141 (64.1%)
Gestational age (21 Weeks, IQR: 16–27)
1^rst trimester (<14 weeks)	44 (20%)
2^nd trimester (14–27 weeks)	123 (55.9%)
3^rd trimester (≥ 28 weeks)	53 (24.1%)
Ethnic group
Bamileke	204 (92.7%)
Bororo	5 (2.3%)
Foulbe	3 (1.4%)
Others ^†	8 (3.6%)

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* Categorical data were summarised by frequencies/percentages and continuous data such as age and gestational age by the median (IQR)

† People from the North West and South West regions of Cameroon.

Prevalence of malaria among pregnant women and risk factors

Of the 220 pregnant women, 51 (23.2%) had symptomatic malaria with positive microscopy and PCR. Multivariable logistic regression analysis of associated factors for malaria is presented in Table 3. Pregnant women living in urban areas were less likely to be infected with Plasmodium compared with those living in rural areas (AOR 0.12, 95% CI 0.07–0.27). The use of Long-lasting insecticidal nets (LLINs) and Intermittent Preventive Treatment (IPT) had a protective effect against malaria (AOR 0.35, 95% CI 0.14–0.84, and AOR 0.23, 95% CI 0.09–0.55 respectively). Furthermore, severe anemia was strongly associated with malaria during pregnancy (AOR 8.35, 95% CI 1.68–50.06). Malaria cases were more frequent in primiparous women than multiparous women (62.7% vs. 37.3%). However, maternal age, study site, and gestational age were not statistically associated with malaria.

Table 3. Multivariable analysis of associated factors for malaria among pregnant women in the West region of Cameroon.

Variables	Malaria Status		Crude Analysis^*			Adjusted Analysis^†
Variables	Negative (n = 169)	Positive (n = 51)	OR	95% CI	p-value	OR	95% CI	p-value
Environment
Rural	39 (23.1%)	35 (68.6%)	-			-		-
Urban	130 (76.9%)	16 (31.4%)	0.14	0.07–0.27	<0.001	0.12	0.05–0.28	<0.001
Use of LLINs ^*
No	54 (32%)	33 (64.7%)	-			-		-
Yes	115 (68%)	18 (35.3%)	0.26	0.13–0.49	<0.001	0.35	0.14–0.84	0.021
IPT
No	48 (28.4%)	31 (60.8%)	-			-		-
Yes	121 (71.6%)	20 (39.2%)	0.26	0.13–0.49	<0.001	0.23	0.09–0.55	0.001
Severe anemia^**(<7g/dl)
No	165 (97.6%)	40 (78.4%)	-			-		-
Yes	4 (2.4%)	11 (21.6%)	11.34	3.67–42.65	<0.001	8.35	1.68–50.06	0.013
No anemia (≥11g/dl)
No	60 (35.5%)	34 (66.7%)	-			-		-
Yes	109 (64.5%)	17 (33.3%)	0.28	0.14–0.53	<0.001	0.34	0.13–0.83	0.019
Parity
Multigravidae	122 (72.2%)	19 (37.3%)	-			-		-
Primigravidae	47 (27.8%)	32 (62.7%)	4.37	2.28–8.59	<0.001	6.14	2.55–15.91	<0.001

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* Estimated by univariate logistic regression

† Estimated by multivariate logistic regression including all above variables

*Long-lasting insecticidal nets.

** The anemia cut-offs for pregnant women as defined by the World Health Organization [53]

Plasmodium species identification by Nested PCR

The 18S rRNA genes specific to Plasmodium species were amplified by Nested PCR. Three Plasmodium species were identified among the 51 pregnant women, namely P. falciparum, P. malariae, and P. ovale. P. falciparum was the most frequent species (23.2%, 51/220), followed by P. malariae (3.6%, 8/220) and P. ovale (0.9%, 2/220). P. falciparum mono-infections were more common (18.6%, 41/220) than mixed infections (4.5%, 10/220) (Fig 3). No non-falciparum mono-infections were reported in this study.

Fig 3 — P.f: *Plasmodium falciparum*, P.m: *Plasmodium malariae*, and P.o: *Plasmodium ovale*.

Parasitemia in pregnant women infected with Plasmodium

Based on clinical complications and parasitemia results, twenty pregnant women had severe malaria, and thirty-one pregnant women had uncomplicated malaria. The level of parasitemia ranged from 764 to 70020 parasite/μl. The median parasitemia was 4057 parasites/μl (IQR: 1110–25371 parasites/μl). 19.6% (10/51) had a parasitemia <1000 parasites/μl, 41.2% (21/51) had a parasitemia between 1000–9999 parasites/μl, and 39.2% (20/51) had a parasitemia ≥10000 parasites/μl. The median parasitemia among pregnant women with severe malaria was 32742 parasites/μl (IQR: 23196–44393 parasites/μl) and 2062 parasites/μl (IQR: 933–3161 parasites/μl) among pregnant women with uncomplicated malaria. Pregnant women with mixed P. falciparum and P. malariae infections had significantly higher median parasitemia than pregnant women with P. falciparum mono-infections (31604 parasites/μl, IQR: 16899–49572 parasites/μl vs. 2578 parasites/μl, IQR: 1028–15889 parasites/μl; p = 0.0007) (Fig 4). However, most severe malaria cases were due to P. falciparum mono-infections (60%), while 40% were due to mixed infections.

Fig 4 — Individual data points represent the parasite density. The scatter dot plots with lines show the median with interquartile range (IQR). The Mann-Whitney U test was used for statistical analysis.

We compared malaria symptoms in patients with P. falciparum mono-infections and those with mixed infections (S1 Fig). We found that anemia (70.6%), fever (71.4%), fatigue (75.6%), sore muscle (78.8%), nausea (85.7%), headache (74.3%), and respiratory distress (80%) were more frequent in patients with P. falciparum mono-infections than those with mixed infections (p = 0.001).

Prevalence of Epstein-Barr virus reactivation in pregnant women

The overall prevalence of EBV in pregnant women was 99.5% (219/220). Thirteen-point two percent (13.2%, 29/220) had EBV reactivation, of whom 5.5% (12/220) were coinfected with malaria, and 7.7% (17/220) had no malaria. Eighty-five-point nine percent (85.9%, 189/220) had a latent infection, 0.5% (1/220) had an acute infection, and 0.5% (1/220) had no previous infection (Fig 5A). The serological pattern of EBV infection is presented in Fig 5B below.

Fig 5 — Prevalence of different stages of EBV infection in all participants (A) and Frequency of EBV serological pattern observed in the study population (B).

No association was found between stress, anxiety, alcohol consumption, age, parity, and gestational age with EBV reactivation. Symptoms of EBV reactivation appeared to be statistically similar to those of latent infection. No significant differences were found between these groups.

Association between EBV reactivation and malaria severity in pregnant women

Fig 6 shows the distribution of malaria in the two EBV groups, one latent and the other reactivated. A total of 29 pregnant women (29/220) had reactivated EBV, and 189 (189/220) had latent EBV. Among the group with reactivated EBV, 12 (12/29) had malaria, including 8 (8/12) with severe malaria and 4 (4/12) with uncomplicated malaria. In the latent EBV group, 38 had malaria, of whom 12 (12/38) were severe malaria and 26 (26/38) uncomplicated cases. A high proportion of severe malaria was observed in pregnant women with reactivated EBV (66.7% vs. 31.6%), while most pregnant women with latent EBV were more affected by uncomplicated malaria (68.4% vs. 33.3%). Pregnant women with reactivated EBV were more likely to develop severe malaria than women with latent EBV (OR 4.33, 95% CI 1.08–17.25, p = 0.03).

The Link between parasitemia and EBV status

The level of parasitemia in patients with malaria was plotted against EBV infection status, as shown in Fig 7. We found that pregnant women with latent EBV had lower levels of parasitemia than pregnant women with EBV reactivation (median, 2816 vs. 19002 parasites/μl; p = 0.02). EBV reactivation was not associated with malaria symptoms.

We compared the parasitemia of malaria patients infected with different Plasmodium species with the levels of IgM VCA, IgG EBNA, and IgG EA (D) antibodies. No significant association was found between parasitemia and the level of EBV antibodies (p = 0.57, p = 0.79, p = 0.17, respectively) (S2 Fig).

Finally, we assessed the EBV antibody responses in participants with severe malaria, uncomplicated, and without malaria. The level of EBV antibody ranged from 0.8 to 114.4 U/ml for IgM VCA, from 16.9 to 213.1 U/ml for IgG EBNA, and from 0.05 to 3.6 (IP) for IgG EA (Fig 8). The response of VCA IgM was significantly higher in patients with severe malaria compared to patients with uncomplicated malaria or without malaria (median, 10.32 U/ml, p = 0.006). IgG EBNA was high in all groups, but increased concentrations were reported in patients who did not have malaria (median, 155.4U/ml, p<0.0001). The response of IgG EA was comparable in all groups; no significant differences were found (p = 0.54).

Fig 8 — For each graph, the column bars represent the median of the different EBV antibody concentrations (A and B) and the median of the positivity index (C), respectively. The data at the bottom represent median and IQR values. Statistical analysis was performed by the Mann-Whitney U test.

Discussion

Despite compelling evidence that acute Epstein-Barr virus (EBV) infection negatively affects the development of humoral immunity during Plasmodium infection [27–29], the impact of EBV reactivation on malaria severity is not elucidated. The present study aimed to investigate the association between EBV reactivation and malaria severity in women living in a malaria-endemic region of Cameroon and to estimate the burden of EBV reactivation during malaria infection. The major finding is that pregnant women with EBV reactivation were more likely to develop severe malaria than those with latent EBV. This result was supported with lower median parasitemia in pregnant women with latent EBV compared to pregnant women with EBV reactivation. Our study is the first of its kind that revealed that pregnant women with a reactivated EBV had a higher chance of manifesting severe malaria than pregnant women with a latent EBV. This may be explained by the ability of EBV to disrupt the functional capacity of immune cells that it infects when it reactivates, namely monocytes, macrophages, dendritic Cells (DCs), NK cells, and neutrophils [54–57]. These cells are essential in controlling P. falciparum parasitemia thanks to their phagocytic activity, cytokine production, and antigen presentation [58, 59]. Therefore, a dysregulation of these functions could negatively affect the anti-malarial immune response, leading to severe forms of the disease. A recent study conducted by Hem Chandra Jha’s team also supports that lytic reactivation of EBV infection during malaria may facilitate the development of cerebral malaria [30]. Here, we sought to investigate whether the lytic reactivation of EBV infection could influence the parasitemia level in pregnant women infected with Plasmodium. We found that pregnant women with latent EBV had lower levels of parasitemia than pregnant women with EBV reactivation (median, 2816 vs. 19002 parasites/μl). This suggests that EBV reactivation may impair the control of the parasitemia in individuals infected with Plasmodium. This finding is in accordance with a previous study that reported high and prolonged parasitemia in marmoset mice co-infected with P. brasilianum and EBV [60]. It has also been documented that acute EBV infection suppresses anti-malarial humoral responses in C57BL/6 mice infected with Plasmodium yoelii XNL and causes a defect in antibody production, leading to a loss of control of peripheral parasitemia [27, 28]. In this context, it is possible to speculate that during EBV reactivation, the virus induces high secretion of cytokines such as TNF-α, IL-12p40, IL-10, and IFN-γ, which could exacerbate the immune response to malaria and contribute to uncontrolled proliferation of the parasite. Nevertheless, it is also possible that EBV reactivation could serve as a hallmark of malaria severity during pregnancy or a hallmark of an unfavorable pregnancy outcome.

The overall prevalence of symptomatic malaria at the enrolment was 23.2%. This prevalence was similar to the prevalence reported in Bafang (25%) [61] and Dschang (25.3%) [62] in West region of Cameroon, suggesting that the occurrence of malaria infection in this region is moderate and can be considered mesoendemic [63]. Indeed, malaria transmission in this region has been previously described as relatively stable, with entomological inoculation rates Dvarying from 62.8 to 90.5 infectious bites/per person/year [36, 37]. It is important to mention that the study was carried out during the dry and rainy seasons when transmission of Plasmodium is high to better estimate malaria prevalence. Furthermore, 64.1% of pregnant women had taken IPT, and 60.5% were using LLINs at the time of the study, which may have also contributed to the decrease in the burden of malaria. Primiparous pregnant women living in rural areas were more susceptible to Plasmodium infection than multiparous women. This result is consistent with previous studies showing that malaria is associated with parity and residence in rural areas [64–66]. However, no statistically significant association was found between malaria status in pregnant women according to their age, study site, and gestational age (p>0. 05), although some studies found that young women (<25 years) were more at risk of malaria than older women and women in the first and second trimester were more vulnerable to malaria [67–70].

P. falciparum was the most common, followed by P. malariae and P. ovale. P. falciparum mono-infections were more common than mixed infections. About 60% of severe malaria cases were due to P. falciparum mono-infections. This result is not surprising as it has been reported that P. falciparum is the most widespread and pathogenic malaria parasite associated with severe forms of illness, particularly in the WHO African region [71, 72]. However, our study underlines the urgency of strengthening measures to control the transmission of P. falciparum malaria and the need to develop effective therapeutic strategies. Indeed, previous studies revealed that most P. ovale and P. malariae infections are associated with P. falciparum infections [73–75].

The median parasitemia in infected pregnant women was slightly lower than the median parasitemia reported in Colombian pregnant women (4057 vs. 4400 parasites/μl) [76]. This is probably due to the use of intermittent preventive treatment and early malaria diagnosis. Women with mixed P. falciparum and P. malariae infections had significantly higher parasitemia than women with P. falciparum mono-infections and women with mixed P. falciparum and P. ovale infections (p = 0.01). This result may be explained by the predominance of P. falciparum parasite density in mixed infections. Indeed, P. falciparum is the only Plasmodium species that is responsible for high levels of parasitemia due to its ability to infect all red blood cells [77, 78], unlike P. ovale, which only infects young erythrocytes [79, 80] and P. malariae, older erythrocytes [81, 82] leading to a generally lower parasite density [73, 83]. No significant difference was found between malaria symptoms in patients with P. falciparum mono-infection and those with mixed infection (p = 0.88).

Almost all participants (99.5%) had been in contact with EBV in their lifetime, which is in line with previous studies that have shown a high rate of EBV seroprevalence in pregnant women in Africa (100%) [31]. The prevalence of EBV reactivation in pregnancy was relatively low in our study (13.2%) compared to the study conducted by Haeri S. et al., which showed a 35% EBV reactivation rate during pregnancy [20]. This could be due to the difference in methods used to diagnose EBV reactivation, the heterogeneity of the study population, and differences in the inclusion criteria. Indeed, in our study, ELISA method was used to detect IgM VCA, IgG EA, and IgG EBNA, three important markers in serological diagnosis of EBV infection. However, the detection of IgG VCA would also have been useful to have serological patterns that could indicate reactivation [52, 84]. We observed that only 5.5% of pregnant women were coinfected with malaria and had a reactivated EBV. This is inconsistent with a previous study showing elevated EBV DNA loads in pregnant women with malaria [31]. It is plausible that EBV reactivation in individuals with malaria depends on other factors, such as the severity of malaria, the number of malaria attacks, and the intensity of malaria transmission in the area [85].

Our study found no significant correlation between parasitemia and the level of EBV antibodies. In addition, EBV reactivation was not associated with malaria symptoms (p>0.05). A similar result was reported by Budiningsih et al. in Indonesia [86]. However, we observed that patients with severe malaria had significantly higher IgM VCA response compared to patients with uncomplicated malaria or without malaria, but IgG EBNA was relatively high in patients who did not have malaria. High levels of IgM VCA usually indicate primary infection but might also reappear in EBV reactivation [51]. In our study, the simultaneous presence of IgM VCA and IgG EBNA in patients with severe malaria reflects EBV reactivation. On the other hand, the isolated presence of EBNA IgG in patients without malaria would indicate the virus latency. This is consistent with some previous studies that have shown high EBV-specific antibody levels in children and pregnant women with acute malaria in hyperendemic regions [31, 87].

The first limitations of this study were the small sample of EBV reactivation/malaria (12/220), the inability to follow up with women with severe malaria and EBV reactivation to assess the outcome of malaria, the efficacy of antimalarial treatment, and their ability to recover during and after treatment. Secondly, the prevalence of reactivation during pregnancy may have been underestimated due to the lower sensitivity of the ELISA compared to the quantitative PCR. It is also interesting to highlight that, the detection of EBV antibodies by ELISA has limitations, such as the lack of specificity, the difficulty in staging EBV infection, and the equivocal interpretation of some antibody patterns [88]. The measurement of EBV DNA load should be used complementarily with antibody detection to diagnose EBV reactivation (viral replication) in patients.

Conclusion

This study conducted in the Mifi and Nde divisions, a malaria endemic area in Cameroon showed that pregnant women who had EBV reactivation were more likely to develop severe malaria. This result was supported with lower median parasitemia in pregnant women with latent EBV compared to those with EBV reactivation. The response of VCA IgM was higher in patients with SM than those with uncomplicated malaria or without malaria, while IgG EBNA increased in patients without malaria. This suggests that EBV reactivation should be considered a risk factor for severe malaria in pregnant women living in malaria-endemic regions. Further studies are needed to understand how EBV reactivation disrupts the control of parasitemia and the anti-malarial immune response. It will also be interesting to assess the effect of EBV reactivation on placental malaria and investigate whether EBV reactivation increases the sequestration of P. falciparum parasites in the placenta.

Supporting information

S1 Fig. Frequency of malaria symptoms among pregnant women with a P. falciparum mono-infection (n = 41) and mixed infection (n = 10).

The Mann-Whitney U test was used for statistical analysis.

(TIF)

pgph.0003556.s001.tif^{(1MB, tif)}

S2 Fig. Correlation between the parasitemia in malaria patients infected with P. falciparum mono-infection (n = 41) and mixed infection (n = 10) with EBV antibodies responses (IgM VCA, IgG EA, and IgG EBNA.

Statistical analysis was done by linear regression (R²).

(TIF)

pgph.0003556.s002.tif^{(617.8KB, tif)}

S1 Appendix. Questionnaire.

(PDF)

pgph.0003556.s003.pdf^{(100.7KB, pdf)}

Acknowledgments

We are very grateful to Dr. Pawan Malhotra, a recipient of JC Bose Fellowship awarded by SERB, govt Of India, for hosting and supervising this work in the Malaria Biology Laboratory at the ICGEB. We also extend our gratitude to Dr. Emmanuel Haddison, Director of the Bangangte District Hospital, Dr. Ambassa Elime Gregoire, Director of the Mifi District Hospital of Bafoussam, Dr. Tchoukoua Serge, Assistant General Administrator of Universite des Montagnes Teaching Hospital, Dr. Yawat Djogang Anselme Michel, Prof. Pierre Rene Fotsing Kwetche, Dr. Simo Louokdom Josue and all the pregnant women who voluntarily agreed to take part in this study. We also express our sincere appreciation to all the nurses, midwives, and laboratory technicians who assisted us during registration, sample collection, and microscopic analysis and to all the members of the Malaria Biology Laboratory at the International Centre for Genetic Engineering and Biotechnology in New Delhi for their constructrive suggestions and overall support during this research work.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The first phase of this work was supported by funds from the “The African coaLition for Epidemic Research, Response and Training, (ALERRT). “ALERRT” is part of the European and Developing Clinical Trial Partnership (EDCTP2) Programme 2 supported by the European Union under Grant Agreement RIA2016E-1612 (https://www.alerrt.global/content/alerrt-african-coalition-epidemic-research-response-and-training). This second phase of the work was supported by the Italian Ministry of Foreign Affairs and International Cooperation, and the International Centre For Genetic Engineering and Biotechnology (ICGEB)(https://www.icgeb.org/) through the WE-STAR - WS/CMR22-01 mobility fellowship awarded to Ide Armelle DJUIDJE CHATUE (IADC) and by JC Bose fellowship grant (DST/20/015). PM is an Arturo Falaschi Emeritus Scientist (AFES) of ICGEB and J.C Bose Fellow of SERB Govt of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLOS Glob Public Health. doi: 10.1371/journal.pgph.0003556.r001

Decision Letter 0

Mathieu Nacher

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

20 Dec 2023

PGPH-D-23-01980

Association between Epstein-Barr virus reactivation and severe malaria in pregnant women living in a Malaria-endemic region of Cameroon

PLOS Global Public Health

Dear Dr. Djuidje Chatue,

Thank you for submitting your manuscript to PLOS Global Public Health. After careful consideration, we feel that it has merit but does not fully meet PLOS Global Public Health’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

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Mathieu Nacher

Academic Editor

PLOS Global Public Health

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Reviewers' comments:

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1. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This article raises an important question on which there is very little data, that of the relation between severity of malaria during pregnancy and reactivation of EBV.

The work is well conducted, original and well written.

I congratulate the authors for the quality of the study and of the paper, which will be very useful for the malarialogist community.

However, the article would benefit from lighter coverage of the secondary issues and stronger emphasis on the main question - the link between severity and EBV reactivation - which is given little attention in the results and discussion.

Thus, the number of patients with EBV reactivation and severe malaria is not clearly presented. The main result is presented in reverse, even in the abstract, presenting the link between EBV reactivation and uncomplicated malaria. It would be much clearer to present it the other way round: link between EBV reactivation and severe malaria, presenting the numbers, which are likely to be small.

In detail, here are my comments:

Major corrections

Abstract

Present the main result with regards to severe malaria.

Introduction

L93 : and children ?

L94 : “can” instead of “cause”

L96: “decrease” instead of suppression

L120-121: not clear: what do you mean with “naturally clear the parasite”? without treatment? Are there situation when a pregnant woman with malaria is not treated? Even asympto? Please clarify.

L124: what do you mean with “possible outcome”? please specify

Material and method

L138: low-cost: care are not free for pregnant women in Cameroun?

L154-155: please clarify what you mean with “participants approached the hospitals”: this refers to reason of consultation? Or is it an inclusion criteria? If yes modify. Did you included only women consulting for fever?

L157-162: combine non-inclusion and exclusion criteria to avoid repetition, or explain the difference (timing ? it the serological results are known before consent?)

Why did you excluded women with antibiotics?

How did you defined depression?

L166: “all” instead of “various”?

L170: how did you proceed to the random sampling? Please specify

L173: P” is the prevalence of EBV and malaria coinfection among pregnant women in Kenya, which is 64% [31].”: it seems very high, it is among women consulting for fever? If yes specify

L177: please provide the questionnaire as a supplementary material

How did you defined the bednet use? In your results it is presented yes/no: what was the question? The night before the inclusion? Usually? Everyday?

Idem for IPT: how is defined yes or no? according to the trimester of pregnancy? At least one dose? What if very early pregnancy?

How did you measure anxiety?

L192: In my opinion, all the details concerning microscopy and PCR are too detailed. They should be grouped together in a paragraph of no more than 10 lines, with references to the methods used and the detection threshold.

Table 1 should be deleted.

However, please explain why you did the PCR on filter paper when you have whole blood? Was it sent to another laboratory? It would have been easier to perform the PCR directly on the blood and increase sensitivity. Please explain why.

L254-268: idem too detailed in my opinion, to be summarized

Please add the definition of severe malaria++

Results

L287 : and CMV ? inclusion criteria

L288-293: redundant with flow chart, could be deleted

Fig2: remove “ethical clearance…” from the title

The last line is very interesting but should be presented as a tree

Table 3: please define “maternal income”

L312: you did not presented gestational age as a continuous variable, and you did not present BMI, please correct

Table : please correct the table footer calls

As mentioned in the method, the use of IPT and LLIN must be defined, either in the method or here in the form of a footnote.

L328: must be deleted

Fig3: in my opinion, should be deleted as it does not provide any useful information

Fig4: would be more interesting to describe these % in the study population and not % of plasmodium positive

L343: first sentence to be removed

L347 and above: the number at the beginning of a sentence must be in letters, otherwise it must be rephrased.

Parasitemia in pregnant women: could provide the median parasitemia among women with severe malaria vs uncomplicated malaria?

L361-364: the % presented concern which population?

L367: provide the numbers N++

L379: “Symptoms of EBV infection were similar in patients with EBV reactivation or latent infection.” What do you mean? Please clarify

“Association between EBV reactivation and malaria severity in pregnant women”: this is the main result of your study but the results are not clear. Please provide the n/N for each category (severe/non severe malaria, EBV reactivation y/n, what did you do with the woman who had no EBV?)

And please calculate the OR with “severe malaria” as the outcome: it would be much clearer and help the reader understand your main result. This should also be included in your abstract.

Discussion

Start the discussion with a brief summary of your results (this comes too late, L514).

And point out that this is a first investigation of this question (which you say L519 > to move).

Then present the limitations/biases: your sample of EBV reactivation/malaria is small (N not clearly presented, 12?).

But even with this limitation, you have important results; you just need to specify the bias so that the reader can interpret the relevance of your work.

L441: not relevant

L459-460: not relevant

L467 and above: I don't think it's relevant to this article. There's no link with the objective of the study, so it's difficult for the reader to see the point.

L472-477: idem: in your study they were all infected by Pf, at least; so not relevant

L501-503: to be placed in the limitation

L510-513: It's a complex question that is not related to your main objective. I suggest you delete it to simplify your text. Otherwise, it would deserve much more explanation.

Reviewer #2: The authors evaluated the proportion of cases of severe and non-severe malaria in pregnant women randomly selected in 3 centres in Cameroon according to the presence of EBV reactivation. They showed that women with EBV reactivation were more often affected by severe malaria. A link was also established between EBV reactivation profile and parasitaemia.

The diagnostic method seems robust and the work useful for confirming the hypothesis of a link between reactivation and severe malaria in pregnant women.

The study initially included 220 patients, but in the end only 51 had malaria and the number of patients with reactivation and malaria was only 12, which is a very small number for further analysis especially a multivariate analysis was not done here which is somechat problematic considering the confoundig factors of either having positive EBV serology or severe malaria. Besides only serological analyses of EBV were done and no EBV viral load was obtained which could have been informative to ascertain the EBV reactivation profile and avoid possible false positive serological reactivati

PLOS Glob Public Health. 2024 Aug 12;4(8):e0003556. doi: 10.1371/journal.pgph.0003556.r002

Author response to Decision Letter 0

15 Feb 2024

Attachment

Submitted filename: Response to Reviewers.docx

pgph.0003556.s004.docx^{(47.2KB, docx)}

PLOS Glob Public Health. doi: 10.1371/journal.pgph.0003556.r003

Decision Letter 1

Hugh Cowley

20 May 2024

PGPH-D-23-01980R1

Association between Epstein-Barr virus reactivation and severe malaria in pregnant women living in a Malaria-endemic region of Cameroon

PLOS Global Public Health

Dear Dr. Djuidje Chatue,

Your manuscript has been evaluated by one new reviewer, and their comments are appended below.

The reviewer has requested clarification of how severe malaria was diagnosed in this study, as well as an explanation of the relatively low incidence of severe malaria. The reviewer has also identified a few grammatical corrections for you to address. Please ensure you address each of the reviewer's comments when revising your manuscript.

Please submit your revised manuscript by Jun 15 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at globalpubhealth@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgph/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Hugh Cowley

Staff Editor

PLOS Global Public Health

Journal Requirements:

1. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #3: The authors set out to determine whether there is an association between the reactivation of EBV and the occurrence of severe malaria in pregnant women residing in a meso-endemic malaria zone of Cameroon. To answer this research question they conducted a cross-sectional hospital-based study in three locations in the west region of Cameroon. Using a structured questionnaire they recruited 220 consenting pregnant women, from whom they collected blood for diagnosis of malaria by microscopy and PCR and for preparation of plasma for serology of EBV. Reactivation of EBV was done by measuring IgM specific antibodies whereas IgG antibodies detection signified exposure. As expected more than 95 % of the participants were exposed to EBV. Severe malaria was significantly more prevalent in the subgroup of participants with reactivated EBV as compared to those exposed to latent EBV infection.

Severe malaria is usually more prevalent in patients with mega-parasitaemia in a hyper-endemic area. The authors should explain how severe malaria was diagnosed and discuss the relatively low incidence of severe malaria in their study.

There were a few grammatical slips which could be corrected by careful proof reading eg:

line 145 : Write 'study sites.' instead of 'study site'.

p228:Give the key of (+), (-), (+/-) in a legend under Table 1.

line 269:There is no ethnic group in Cameroon called Bamenda. Do you mean people from the North West region?

line 365: Write "We assessed.." instead of "we assess..."

line 388 : Write, "First of its kind.." instead of "first of its.."

line 390: Do not capitalize 'dendritic'

line 423: Write "However..."instead of "On the other hand..."

In conclusion , I find that the study was well planned and well executed. Its significance lies in the fact that it was conducted in an endemic area not previously studied for this kind of association. The article should be accepted after proofreading and correcting the errors.

**********

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Reviewer #3: No

**********

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PLOS Glob Public Health. 2024 Aug 12;4(8):e0003556. doi: 10.1371/journal.pgph.0003556.r004

Author response to Decision Letter 1

4 Jun 2024

Attachment

Submitted filename: Response to Reviewers.docx

pgph.0003556.s005.docx^{(24.7KB, docx)}

PLOS Glob Public Health. doi: 10.1371/journal.pgph.0003556.r005

Decision Letter 2

Julia Robinson

12 Jul 2024

Association between Epstein-Barr virus reactivation and severe malaria in pregnant women living in a Malaria-endemic region of Cameroon

PGPH-D-23-01980R2

Dear Mrs. Djuidje Chatue,

We are pleased to inform you that your manuscript 'Association between Epstein-Barr virus reactivation and severe malaria in pregnant women living in a Malaria-endemic region of Cameroon' has been provisionally accepted for publication in PLOS Global Public Health.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they'll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact globalpubhealth@plos.org.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Global Public Health.

Best regards,

Julia Robinson

Executive Editor

PLOS Global Public Health

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #3: I have reviewed this article before and foubd it acceptable, subject to minor concerns which have been addresse.....

d satisfactorily. Tge article shoukd ve accepted.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.

For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

**********

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Frequency of malaria symptoms among pregnant women with a P. falciparum mono-infection (n = 41) and mixed infection (n = 10).

The Mann-Whitney U test was used for statistical analysis.

(TIF)

pgph.0003556.s001.tif^{(1MB, tif)}

Statistical analysis was done by linear regression (R²).

(TIF)

pgph.0003556.s002.tif^{(617.8KB, tif)}

S1 Appendix. Questionnaire.

(PDF)

pgph.0003556.s003.pdf^{(100.7KB, pdf)}

Attachment

Submitted filename: Response to Reviewers.docx

pgph.0003556.s004.docx^{(47.2KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pgph.0003556.s005.docx^{(24.7KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Association between Epstein-Barr virus reactivation and severe malaria in pregnant women living in a malaria-endemic region of Cameroon

Ide Armelle Djuidje Chatue

Maximilienne Ascension Nyegue

Severin Donald Kamdem

Franklin Maloba

Iqbal Taliy Junaid

Pawan Malhotra

Palmer Masumbe Netongo

Roles

Abstract

Introduction

Materials and methods

Study design

Study site

Fig 1. Illustrative map showing the West region of Cameroon with different study sites.

Study population

Ethical statement

Sample size determination

Data collection

Blood sample collection

Diagnosis of malaria

Microscopy of malaria

Nested PCR

Diagnosis of EBV infection

Table 1. Clinical status of Epstein-Barr infection according to viral serology results.

Statistical analysis

Results

Study flow diagram

Fig 2. Flow chart of the study describing the strategy for recruiting participants in 03 hospitals in the West region of Cameroon.

Sociodemographic and obstetric characteristics of the study population

Table 2. Socio-demographic characteristics of study participants from three health centers in the West region of Cameroon, January to September 2022.

Prevalence of malaria among pregnant women and risk factors

Table 3. Multivariable analysis of associated factors for malaria among pregnant women in the West region of Cameroon.

Plasmodium species identification by Nested PCR

Fig 3. Frequency of Plasmodium species, mono-infection, and mixed infection among infected women.

Parasitemia in pregnant women infected with Plasmodium

Fig 4. Level of parasitemia in malaria patients with P. f mono-infection (n = 41), mixed P. falciparum and P. malariae infection (n = 8); and mixed P. falciparum and P. ovale infection (n = 2).

Prevalence of Epstein-Barr virus reactivation in pregnant women

Fig 5.

Association between EBV reactivation and malaria severity in pregnant women

Fig 6. Distribution of malaria among pregnant women coinfected with a latent EBV and a reactivated EBV.

The Link between parasitemia and EBV status

Fig 7. Comparison of parasitemia level in malaria patients with latent infection and EBV reactivation.

Fig 8. Comparison of EBV-specific antibodies responses in patients with severe malaria (SM), uncomplicated malaria (UM), and patients without malaria (M (-).

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Mathieu Nacher

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Decision Letter 1

Hugh Cowley

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Julia Robinson

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Associated Data

Supplementary Materials

Data Availability Statement

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