Seroprevalence of Dengue Virus Among Pregnant Women in Guangdong,China

Abstract

Guangdong is a main dengue epidemic area in China and undergoes dengue outbreaks frequently. In 2014, Guangdong experienced a serious dengue outbreak with 45,224 confirmed cases and six deaths, which might affect the seroprevalence among the local population. There is evidence that dengue virus (DENV) infection during pregnancy may cause adverse outcomes. Therefore, it is important to assess the seroprevalence of DENV among Guangdong pregnant women. We aimed to survey the seroprevalence of anti-DENV antibodies among pregnant women in Guangdong and to analyze the features of different seroprofiles. We collected a total 951 samples from pregnant women living in Guangdong in 2016. All serum samples were screened for DENV-specific antibodies (IgG and IgM) by indirect enzyme-linked immunosorbent assay (ELISA) and confirmed using capture ELISA. In IgM-positive samples, we performed DENV RNA detection using reverse transcriptase polymerase chain reaction. We collected information of delivery outcomes, neonate features, and clinical laboratory variables of parturients and newborns following delivery. Seroprevalence of DENV among the women in our sample was 4.31%. A total 1.26% and 3.15% of samples were IgM and IgG positive, respectively. In addition, 22.22% of IgM-positive and 9.09% of IgG-positive participants had adverse outcomes. There was no difference with respect to adverse outcomes compared with controls (8.80%) who were IgG and IgM negative. There was no difference in clinical laboratory variables among the different seroprofiles. We found a high seroprevalence of DENV among pregnant women in Guangdong comparing with the overall seroprevalence of local population before 2014. Asymptomatic DENV infection during pregnancy was not found to contribute to adverse outcomes.

Introduction

Dengue is an arthropod-borne viral infection caused by the dengue virus (DENV). DENV has an 11 kb positive-sense RNA genome encoding 10 proteins, including three structural proteins (capsid, membrane, and envelope proteins) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). DENV belongs to the genus Flavivirus and family Flaviviridae and includes four serotypes (DENV1, DENV2, DENV3, and DENV4). These cause human dengue diseases that are often epidemic in Southeast Asia, the Americas, the western Pacific, Africa, and eastern Mediterranean regions (15). It has been estimated that DENV is epidemic in more than 100 countries and regions worldwide (41); there are about 390 million cases of DENV infection and 96 million cases with clinical manifestations each year (6).

The Aedes mosquito, which is present nearly worldwide, is the main vector of DENV. The Ae. aegypti mosquito is increasing its geographic distribution and this expansion is newly putting large human populations at risk (28). In addition, the Ae. albopicus is becoming another important dengue vector and it was discovered in 20 Europe countries, such as Albania, Belgium, France, German, Greece, and Italy (16). As a result, the risk of DENV infection may be increasing globally, due to increased activities among people from different regions owing to economic globalization. Therefore, dengue is an emerging concern not only in tropical and subtropical regions but also in other areas worldwide.

Most patients infected with DENV are asymptomatic or have mild clinical manifestations such as fever, myalgia, and rash, collectively known as dengue fever. However, a few people with DENV infection may develop severe dengue (SD) diseases that can lead to vascular leakage and/or shock, known as dengue hemorrhagic fever and dengue shock syndrome (42). The pathogenesis for SD is multifactorial and is not known as yet. Antibody-dependent enhancement (ADE) owing to nonneutralizing cross-reactive antibodies may have a vital role in the mechanisms of SD diseases, especially in secondary infection, according to previous study findings (7,8,10,39).

For several decades, researchers have sought different types of vaccines to prevent DENV infection. The first licensed recombinant, live-attenuated dengue vaccine (Dengvaxia) recently became clinically available. However, a high risk of adverse outcomes was found among vaccinated individuals who had not been previously exposed to dengue (12,35). Patients with dengue are treated symptomatically, as there are no special treatments or drugs available. The mortality of SD is more than 20% without appropriate and timely treatment, but this rate is <1% if patients receive optimal fluid management in the early stage of infection (22).

Guangdong is located in Southern China and is a global cultural and economic center. Guangdong has a hot, humid subtropical climate, conditions under which mosquitoes can breed easily. Guangdong became a DENV epidemic area after the first outbreak of DENV-4 in Foshan in 1978. Since that time, infections with all four DENV serotypes have been reported in Guangdong (43). Based on a retrospective study from 2005–2011, a gradual change in the epidemic pattern of dengue infection has been observed in the province. Before 2014, the overall reported seroprevalence of dengue IgG antibody was 2.43% (range: 0.28–5.42%) among local populations in Guangdong (13).

Dengue outbreaks have been frequently reported in Guangdong (37), and it experienced a serious outbreak of DENV, with 45,224 confirmed cases and six deaths in 2014 (44). These outbreaks impacted the seroprevalence of anti-DENV antibodies among people living in Guangdong. Therefore, the risk of SD diseases may be increased after the outbreak owing to ADE.

Although DENV is known as a mosquito-borne virus, vertical transmission has been identified in several reports (4,5,9). However, it is unclear how DENV is transmitted from mother to infant. Some studies (11,20,25,32,38) have reported that DENV infection during pregnancy contributes to adverse pregnancy outcomes such as abortion, prematurity, low birth weight, fetal abnormalities, and stillbirth; however, other reports contradict these findings (23,27,45). Yet, there are data indicating that pregnant women are 3.4 times more likely to develop SD diseases compared with nonpregnant women (20). Previous studies have also shown that IgG antibody can be transmitted from mother to infant; this mechanism might contribute to SD occurring in infants (18,24,33,41).

High seroprevalence among pregnant women has been reported in Thailand and other endemic areas (2,17,29,34). It is urgent to investigate the seroprevalence among pregnant women living in Guangdong after the outbreak of DENV in 2014. Thus, we conducted the present seroepidemiological study, to assess the seroprevalence of antibodies against DENV among pregnant women in Guangdong following the 2014 outbreak and to analyze their different seroprofiles.

Materials and Methods

Participant recruitment and clinical data collection

Pregnant women living in Guangdong who underwent a prenatal examination at the First Affiliated Hospital of Guangzhou Medical University (FAHGMU) from May 2016 to November 2016 were enrolled in this study. The characteristics of participants, including age and weeks of gestation, were collected at time blood samples were drawn. We excluded parturients with congenital anemia and other infection diseases, including hepatitis, syphilis, AIDS, and all bacterial infections. For deliveries performed at FAHGMU, we collected the information of parturients and newborns, including the weeks of gestation at delivery, infant birth weight, and clinical laboratory variables (blood cell counts) at the time of delivery. All clinical information of participants and their newborns was collected during physician visits, and clinical laboratory variables were recorded in the hospital and/or laboratory information systems.

Term definition

Early, middle, and late gestation were defined as <14 weeks, between 14 and 27 weeks, and more than 28 weeks' gestation, respectively. Premature birth was defined as delivery occurring between 28 and 36 weeks of gestation; miscarriage was defined as expulsion of the fetus before 28 weeks' gestation. Low birth weight was defined as an infant born after 36 weeks of gestation and weighing <2.5 kg within the first hour of life.

Sample collection and measurement of IgG and IgM

The serum was separated from a 3-mL intravenous blood sample from each participant, by centrifuging at 3,500 rpm for 4 min, and aliquots were frozen at −40° until further analyses. All samples were screened to detect IgG and IgM antibodies against DENV antigen serotypes (DENV1–4) using indirect enzyme-linked immunosorbent assay (ELISA) kits (Zhongshan Biological Engineering Co., Ltd., Guangdong, China). Suspected positive samples were confirmed using Panbio^® Dengue Duo IgM Capture and IgG Capture ELISA kits (Pan Bio, Queensland, Australia). All test procedures were done according to the manufacturer's instructions. Double-positive samples were defined as true positives. We defined participants whose screening results were IgG⁻/IgM⁻ as the control group.

DENV RNA detection

Samples testing positive for IgM antibody against DENV were subjected to reverse transcriptase polymerase chain reaction (RT-PCR). We used commercial kits for total RNA extraction and DENV RNA detection (DaAn Gene, China) and the ABI 7500 Real-Time PCR system (Thermo) for RT-PCR, according to the manufacturer's instructions. In brief, 200 μL of serum was lysed and RNA was purified in a 50-μL volume. A 5 μL RNA solution was used in a 25 μL RT-PCR reaction volume. The reaction was run on the PCR system using a program of 50°C for 15 min, 95°C for 15 min, followed by 40 cycles of 94°C for 15 sec and 55°C for 40 sec. The test was considered valid when positive control samples showed a ≤32 cycle threshold (CT) value. Samples with a ≤38 CT value were identified as positive and those with a >38 CT value were identified as negative.

Statistical analysis

The data were analyzed using Microsoft Office Excel. Values were presented as percentage, frequency, and mean (standard deviation). We evaluated the differences between groups. Categorical data were analyzed using a chi-square test, and measurement data were assessed using one-way analysis of variance (ANOVA). A p-value <0.05 was considered statistically significant.

Ethics statement

The Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University approved the protocol of this study (Reference No. 2018-54). Only sera from participants who had consented to having their residual sera used for further research were included in this study.

Results

Study population

We included a total 951 pregnant women with age range 18–44 (mean, 29.3) years: 525 (55.21%) participants were aged 20–29 years and 402 (42.27%) were aged 30–39 years. Among participants, 124 (13.04%), 592 (62.25%), and 235 (24.72%) were in early, middle, and late gestation, respectively, when their blood samples were drawn.

Antibody detection

A total of 18 and 40 samples were positive for IgM and IgG, respectively, by indirect ELISA. However, only 12 and 30 samples were positive, respectively, by capture ELISA. Therefore, 12 (1.26%) samples were confirmed to be positive for IgM, and 30 (3.15%) were confirmed positive for IgG. Among 41 (4.31%) participants seropositive for either IgM or IgG, one participant was positive for both IgM and IgG. All IgM-positive samples were subjected to RT-PCR for DENV RNA detection, and no samples were positive. According to these results, the three seroprofiles were grouped as follows: recent infections (IgG⁻/IgM⁺ or IgG⁺/IgM⁺), past infections (IgG⁺/IgM⁻), and noninfections (IgG⁻/IgM⁻). There were no differences when compared with IgG or IgM seropositive among different gestation periods (p > 0.05). The overall seroprevalence among studied samples of pregnant women from Guangdong is summarized in Table 1.

Table 1.

Seroprevalence in Different Gestation Periods

Gestation period^a	Noninfections, n (%)	Past infections, n (%)	Recent infections, n (%)
Late	119 (95.97)	3 (2.42)	2 (1.61)
Middle	566 (95.61)	17 (2.87)	9 (1.52)
Early	225 (95.74)	9 (3.83)	1 (0.43)
Total	910 (95.69)	29 (3.05)	11 (1.16)
p ^b	—	0.716	0.422

Recent infections: IgG⁻/IgM⁺ or IgG⁺/IgM⁺; Past infections: IgG⁺/IgM⁻; Noninfections: IgG⁻/IgM⁻.

Blood sample collection performed during this period.

Compared with noninfection among different gestation period.

Clinical laboratory variables

Information and data of 586 parturients and 589 newborns who delivered at FAHGMU were collected at the time of delivery. The average delivery time was 38.39, 39.39, and 38.88 weeks for the past infections, recent infections, and noninfections groups, respectively. We also collected blood cell count variables of newborns and mothers and the weight of neonates. In comparison with the control group (noninfections), we found the variables with similar results among screened participants in the past infections and recent infections groups (p > 0.05). Details are given in Table 2.

Table 2.

Characteristics of Parturients and Neonates

Indexes	Noninfections, n = 586/589^a	Past infections, n = 21	p ^b	Recent infections, n = 8	p^b
Delivery (weeks' gestation)	38.88 ± 1.52	38.39 ± 2.72	0.156	39.39 ± 0.47	0.337
Mothers
WBC ( × 10⁹)	10.4 ± 2.9	9.7 ± 3.6	0.248	10.5 ± 3.1	0.961
NEU (%)	75.2 ± 7.2	73.4 ± 8.9	0.267	75.6 ± 5.2	0.812
LYM (%)	16.9 ± 8.8	18.4 ± 7.5	0.422	16.7 ± 4.9	0.955
MON (%)	6.9 ± 2.0	6.9 ± 1.6	0.954	7.3 ± 2.4	0.491
PLT ( × 10¹²)	213 ± 68	218 ± 56	0.733	227 ± 63	0.557
Hb (g/L)	118 ± 15	121 ± 9	0.391	122 ± 16	0.397
MCV (fL)	88.4 ± 7.9	90.7 ± 8.5	0.176	92.0 ± 4.4	0.201
HCT (%)	35.7 ± 3.7	36.3 ± 2.7	0.439	35.6 ± 4.0	0.956
Neonates
Weight (kg)	3.16 ± 0.44	3.14 ± 0.64	0.848	3.09 ± 0.35	0.655
WBC ( × 10⁹)	22.1 ± 8.1	23.81 ± 8.00	0.337	20.42 ± 9.05	0.551
NEU (%)	66.4 ± 8.6	69.4 ± 8.6	0.111	63.8 ± 8.2	0.435
LYM (%)	22.9 ± 7.3	20.3 ± 7.0	0.100	24.8 ± 6.1	0.462
MON (%)	7.3 ± 2.4	6.8 ± 2.0	0.395	7.7 ± 3.5	0.637
PLT ( × 10¹²)	277 ± 61	285 ± 58	0.577	275 ± 69	0.915
Hb (g/L)	180 ± 25	183 ± 21	0.501	172 ± 24	0.354
MCV (fL)	102.8 ± 5.9	102.7 ± 6.6	0.905	103.7 ± 3.4	0.680
HCT (%)	53.07 ± 6.40	54.89 ± 5.53	0.188	49.72 ± 5.58	0.143

1. Noninfections: IgG⁻/IgM⁻; Past infections: IgG⁺/IgM⁻; Recent infections: IgG⁻/IgM⁺ or IgG⁺/IgM⁺.

2. There were no differences for all factors among parturients and newborns in the recent infections group and the past infections compared with noninfections groups (p > 0.05).

There were a total of 586 mothers and 589 neonates, as there were 3 twin births.

Compared with noninfections group.

Hb, hemoglobin; HCT, hematocrit; LYM, lymphocytes; MCV, mean corpuscular volume; MON, monocytes; NEU, neutrophils; PLT, platelets; WBC, white blood cells.

Adverse outcomes and clinical manifestations

We compared the recent DENV infection, past DENV infection, and no DENV infection groups in our current study. Two participants (22.22%) with a recent DENV infection experienced adverse pregnancy outcomes, one low-birth-weight infant and one abortion. Two participants (9.09%) with a past DENV infection also had adverse outcomes, one miscarriage and the other premature birth and low-birth-weight infant. However, there were no differences when compared with the rate of adverse outcomes (8.80%) among women in the IgG⁻/IgM⁻ control group (p > 0.05). The details of adverse pregnancy outcomes are shown in Table 3. No parturient complained of fever, rash, or other clinical dengue manifestations during monthly physician visits and blood sampling or delivery; all DENV infections were presumable asymptomatic.

Table 3.

Adverse Pregnancy Outcomes According to Different Seroprevalence Profiles

Outcomes	Noninfections	Past infections	Recent infections
Normal	546 (90.70)	20 (86.96)	7 (77.78)
Adverse outcomes	53 (8.80)	2 (9.09)	2 (22.22)
Prematurity	27 (4.49)	1 (4.55)	0 (0.00)
Low birth weight	14 (2.32)	0 (0.00)	1 (12.50)
Miscarriage	3 (0.50)	1 (4.55)	1 (12.50)
Stillbirth	9 (1.50)	0 (0.00)	0 (0.00)
Total^a	602	22	9
p ^b	—	0.86	0.16

1. Noninfections: IgG⁻/IgM⁻; Past infections: IgG⁺/IgM⁻; Recent infections: IgG⁻/IgM⁺ or IgG⁺/IgM⁺.

2. Data are n (%).

Patients who delivered at the First Affiliated Hospital of Guangzhou Medical University (FAHGMU).

Compared with noninfections group.

Discussion

Dengue diseases have become a global problem during the past several decades and preventing DENV transmission is a primary concern. Since 1990, DENV is a class B notifiable disease in China (40), and cases of DENV infection must be notified by all hospitals and health centers via the Centre for Diseases Control and Prevention's public health information system.

Although SD diseases often occur as secondary infections, the risk of SD among people with nonneutralizing cross-reactive antibodies is not yet well understood. In dengue epidemic areas, the actual incidence of DENV may be higher than the reported incidence as people often fail to notice asymptomatic DENV infections. Serological testing of healthy individuals can be used to identify such asymptomatic infections, (14,36) to help clarify the seroprevalence of DENV in epidemic areas.

In the present study, most infectious individuals were IgM seropositive but IgG seronegative and therefore susceptible to primary DENV infection, except one participant who was both IgM and IgG positive; and all IgM-positive samples were DENV RNA negative. The reason might be that IgM antibodies elevated after several months of the dengue infection occurrence. In addition, no participants complained acute febrile illness and other symptoms of dengue infection, thus, all infections were presumable asymptomatic.

Although the seroprevalence in Guangdong is not as high as in those DENV endemic areas, the influence of dengue outbreaks, especially the most serious one in 2014, on the seroprevalence among the local population should be closely monitored. The data in the current study showed a higher seroprevalence of DENV IgG antibody (3.15%) among our sample of pregnant women living in Guangdong after the 2014 dengue outbreak. This was higher than the overall seroprevalence of DENV IgG antibody (2.43%) among the population in Guangdong before the outbreak (13). There are no available seroprevalence data as yet on pregnant women in Guangdong. However, there were 1,699 cases of DENV infection reported in 2015 (37); thus, we suspect that the 2014 dengue outbreak contributed to the high seropositivity found in our study.

In recent years, Guangdong has undergone rapid urbanization, which has increased the density of mosquitoes, leading to an increased risk of dengue infection (46). In addition, international trade and travel has been on the rise in Guangdong, which may also increase the number of local and imported cases of dengue infection. Although lower than in other epidemic areas, increasing DENV infection in the future may be expected in Guangdong. Cross-reactive antibodies exist among the different DENV serotypes in the population, which could lead to an increased number of SD cases in Guangdong.

The average age of the population in this study was 29.3 (18 –44) years, with 55.21% of participants aged 20–29 years and 42.27% aged 30–39 years. The estimated age-specific incidence in a previous study was highest among people 30–39 years old and second highest among those aged 20–29 years (13). In Guangdong, the major mosquito species is Ae. albopictus that originated at the edges of forests and bred in natural habitats (14,30). Young people tend to work and have more activities outdoors more than other age groups and thus have a greater risk of exposure to the mosquito vector. That is why most DENV infections occur among younger populations.

Whether DENV infection can lead to adverse neonatal outcomes remains debated. There are some case reports of vertical transmission in asymptomatic DENV infections, leading to adverse outcomes and even death (3,9,31). However, it remains disputed whether DENV infection during pregnancy can directly cause disease or other adverse outcomes among pregnant women or infants (3,5). Agarwal et al. reported that women with dengue infection would present at the stage of SD if the diagnosis were delayed and SD was associated with maternal morbidity and mortality (1). However, high seropositivity for IgG antibodies was found in one study in Malaysia, but no adverse neonatal outcomes were observed (21).

In our study, pregnant women with recent and past DENV infection accounted for 22.22% and 9.09% of adverse outcomes, respectively. However, there was no difference compared with the rate of adverse outcomes among controls (8.80%) and in other reported data (20.79%) (19). Therefore, our data indicated that asymptomatic DENV infection during pregnancy would not contribute to adverse outcomes. Nevertheless, the risk of SD diseases might be increased in this population because of the unique physiological conditions in women of childbearing age and pregnant women, as well as the contribution of ADE. Because there is no specific therapeutics available for SD diseases, it is vital to take precautions against these diseases, particularly among vulnerable populations.

No predictive factors related to SD diseases have been identified as yet. Research data have shown that some laboratory indicators such as white blood cell count, percent monocytes, platelet count, and hematocrit may be associated with greater risk of developing SD diseases (26). We collected the information of parturients and newborns, including the weeks of gestation at delivery, infant birth weight, and clinical laboratory variables at the time of delivery. All indexes were compared among the different seroprofiles, but no differences were detected. The reason might be that the participants in our study were asymptomatic infections. Based on the current study findings and due to the small sample size in current study, we cannot conclude that DENV infection increases the risk of adverse pregnancy outcomes.

However, the study results will contribute to raising concern about the risks of asymptomatic DENV infection during pregnancy, to enhance dengue control in the future, especially in epidemic areas. Additional research among a larger number of pregnant women with DENV infection, including asymptomatic women, is warranted, to obtain additional clinical data and better elucidate the effect of DENV infection on the population of pregnant women in epidemic areas.

One limitation of the current study is that there was no seroprevalence data for neonates, and so we could not confirm whether there was mother-to-child transmission of antibodies against DENV. Although two different ELISA methods have been used to detect IgG/IgM against DENV in this study, another issue we need to address is that the crossing reaction of the infection and/or vaccination of Japanese encephalitis virus, one of Flavivirus family that is endemic in China. Future large-scale prospective studies are necessary to clarify the seroprevalence of different populations, such as pregnant women, students, elderly adults, and outdoor and indoor workers. Effective methods to detect asymptomatic cases are needed to improve the prevention and control of dengue diseases, especially SD.

Footnotes

Acknowledgments

We thank Dr. Zhihui He for assisting of defining study population and collecting the information of participants and Mrs. Bo Xiang and Mr. Qijian Gao for their assistance in performing ELISAs.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work supported by Guangzhou Science Technology and Innovation Committee (No. 201607010163), Health and Family Planning Commission of Guangdong Province (No. A2016448) and Guangzhou Medical University (No. 2014C24).

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