Historical Analysis of the Risk of Hepatitis E and Its Complications in Pregnant Women in Nepal, 1996–1998

ABSTRACT.

Hepatitis E (HE) during pregnancy can be fatal; there are no prospective risk estimates for HE and its complications during pregnancy. We followed 2,404 pregnant women for HE and pregnancy outcomes from 1996 to 1998. Subjects from Nepal were enrolled at an antenatal clinic with pregnancy of ≤ 24 weeks. Most women (65.1%) were anti-HE virus negative. There were 16 cases of HE (6.7 per 1,000); three mothers died (18.8%) having had intrauterine fetal death (IUFD). Thirteen mothers survived: five preterm and seven full-term deliveries, one IUFD. HE among seronegative women was the sole cause of maternal death and increased the risk of IUFD (relative risk [RR]: 10.6; 95% confidence interval [CI]: 4.29–26.3) and preterm delivery (RR: 17.1, 95% CI 7.56–38.5). HE vaccination of females in at-risk regions before or as they attain reproductive age would reduce their risk for preterm delivery, IUFD, and maternal death.

INTRODUCTION

Hepatitis E virus (HEV; family Hepeviridae) is the etiologic agent of hepatitis E (HE), causing considerable morbidity and mortality, especially in pregnant women. Rein et al. estimated that in 2005, HEV genotypes 1 and 2 in Asia and Africa accounted for 20.1 million incident HEV infections, 3.4 million cases of symptomatic disease, 70,000 deaths, and 3,000 stillbirths.¹ HE is usually self-limited and often occurs where laboratory diagnosis is unavailable. Consequently, the true HE disease burden is unknown.

HE is endemic in Nepal, Pakistan, Afghanistan, India, and Bangladesh. Within Nepal’s Kathmandu Valley, there is year-round transmission of HEV with attack rates as high as 45 people per 1,000 annually.^2,3 In Nepal, HEV genotype 1 is most prevalent, which of the four HEV genotypes most commonly infects humans and is strongly associated with serious and life-threatening disease in South Asia.¹

HE during pregnancy leads to poor maternal and fetal outcomes.^4–9 A tertiary hospital in India found that 60% of acute hepatic injuries were HE; the maternal case fatality and intrauterine fetal death (IUFD) ratios were 41% and 58%, respectively. Pregnant women with HE had a 6-fold increased risk of mortality relative to pregnant women with hepatitis from other causes, a 1.9 relative risk (RR) of IUFD, and a 1.2 RR of preterm delivery.⁶

There is uncertainty regarding the burden of HE during pregnancy because few studies are designed to prospectively measure the risks. This limits assessment of the value of vaccination against HE, which is highly effective in adults.^10,11 We present prospective estimates of maternal and fetal death associated with HEV infection and other complications of pregnancy occurring in a cohort of 2,404 women attending an antenatal clinic in Nepal’s Kathmandu Valley from 1996 to 1998. This study was undertaken to prospectively estimate the HE disease burden among pregnant women, recognizing that women of child-bearing age would likely be an important risk group for immunization. Subsequently, we evaluated an HE vaccine and established its protective efficacy.¹⁰ Nevertheless, development of that vaccine candidate ended due to uncertain market demand. In 2012, another HEV vaccine was approved in China.^11,12 The International Vaccine Institute worked with the WHO to highlight the potential public health value of an HEV vaccine¹³ and with the Chinese vaccine’s manufacturer to ready the vaccine for Prequalification by WHO, which would allow its purchase by United Nations agencies such as UNICEF.¹⁴ In light of this renewed interest in prevention and control of HEV, we now disclose the prospective data we generated more than 20 years ago because they offer uniquely valuable information concerning the historical risk of HE and maternal-fetal fatalities. We think they are pertinent to inform contemporary health policy because our review of recent evidence concerning the epidemiology of HEV suggests that it is unchanged in low-resource settings. For instance, during the 2015 earthquake in Nepal that displaced 2.6 million people,¹⁵ HEV was the leading health concern with a projected estimate of 510 deaths in pregnant women due to HEV infection.¹⁶ A study of blood donors from earthquake-affected areas conducted several months thereafter found 3.2% (95% confidence interval [CI] 2.7–4.0%) were HEV IgM positive, indicative of a recent infection.¹⁷ As we review and discuss herein, our data remain relevant to millions of women and represent the burden of HE on women of reproductive age in South Asia, as well as highlight the lifesaving potential for vaccination against HE in women of reproductive age.

METHODS

Study design.

This was a historical look at previously unpublished data of a prospective cohort study of healthy pregnant women using serology to define baseline and the subsequent occurrence of HEV infections. Baseline anti-HEV status was not provided to investigators until after study completion.

Setting.

The study was performed at Patan Hospital, a 138-bed community hospital at the time with approximately 400 deliveries per month in Lalitpur District but serving the wider Kathmandu Valley.

Cohort participants and outcomes.

From August 9, 1996 through March 31, 1998, consecutive, consenting women presenting to Patan Hospital’s antenatal clinics with a pregnancy of ≤ 24 weeks’ duration were enrolled and followed until delivery and/or approximately 6 weeks postpartum. A structured interview covering demographics, obstetric, and medical history was administered and a blood sample obtained. Each participant was seen at least once more at a medical visit, parturition, or postpartum for an interval history and serum collection. Cohort members were instructed to comply with the hospital’s antenatal clinic schedule and return to the clinic for any untoward events. Any medically attended illness or complication of pregnancy was recorded, including all causes of hepatic injury, HE, and events culminating in maternal or fetal death.

All clinic attendees and inpatient admissions were reviewed daily to identify cohort members. If a cohort member came to the clinic or hospital for illness or a complication of pregnancy, she was evaluated, diagnosed, and followed with additional interviews, along with blood and stool samples. Clinical laboratory evaluations were obtained according to standard hospital procedures, including complete blood count, platelet count, erythrocyte sedimentation rate, serum biochemistries (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, direct and total bilirubin), and prothrombin time.

A preterm delivery was a delivery before 37 weeks of gestation, calculated from the date of the last menstrual period with the exception that births occurring at 35 to 36 weeks were considered at term if the birth weight was at least 2,500 g. A spontaneous abortion was fetal loss before 24 weeks of gestation. A still birth was fetal loss at 24 weeks of gestation or later. IUFD comprised spontaneous abortions and still births.

Planned sample size and duration.

We wished to describe the outcomes for at least 20 cases of HE; the target enrollment into the cohort was 4,000, based on an expected annual HE attack rate of 2%, an expected average duration of pregnancy of 20 weeks at entry and 25% attrition. Because enrollment proceeded more slowly than expected, we capped enrollment at approximately 3,000.

Hepatitis surveillance in other institutions.

To provide context, an indicator of HE activity in the Kathmandu Valley was obtained from jaundice admission data gathered from Patan Hospital and five other sentinel hospitals in the Kathmandu Valley (Sukraraj Tropical and Infectious Diseases Hospital at Teku, Ayurvedic Hospital, Birendra Police Hospital, Birendra Military Hospital, and Teaching Hospital). HEV is the most common cause of jaundice, accounting for 56% of cases in a 1997 series from the Bir Hosital¹⁸ and 88% of cases in a 1993 series from the Sukraraj Tropical and Infectious Diseases Hospital.¹⁹

Case series of hepatitis.

During the period of surveillance, any consenting pregnant woman not enrolled in the cohort but presenting to Patan Hospital with hepatic injury consistent with HE was included in a separate case series. Women in the case series had blood specimens collected for etiologic characterization of hepatitis, underwent structured interviews, and were followed for pregnancy outcomes to either parturition or 6 weeks postpartum.

Acute viral hepatitis definition.

Hepatic injury consistent with acute viral hepatitis was defined as an acute-onset illness, serum alanine aminotransferase level > 2.5 times the upper limit of normal, and either history of recent jaundice or total bilirubin > 2 mg/dL plus one of the following: weakness/lassitude, fever, anorexia, nausea or vomiting, or right upper quadrant abdominal pain.

Laboratory analysis.

Specimens were centrifuged within 2 hours of collection and held at –70°C, including during shipment to the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand. All serum specimens were tested for anti-HEV by a qualified ELISA with adequate sensitivity and specificity.²⁰ HE susceptibility was established by anti-HEV < 20 Walter Reed antibody units (WR U)/mL, equivalent to < 2.5 WHO U/L.²⁰ Subjects with anti-HEV ≥ 20 WR U/mL were considered seropositive, and those with anti-HEV ≥ 250 WR U/mL were considered to have sustained an HEV infection within the past year.²¹ Serum specimens collected at parturition and the postpartum visit were also tested for anti-HEV IgM to identify incident HEV infections using a qualified ELISA.²² HEV infection was defined as a minimum 4-fold rise in anti-HEV in a postenrollment serum specimen relative to baseline.

Serum specimens from patients meeting the definition of hepatic injury consistent with acute viral hepatitis were evaluated for markers of hepatitis A (anti-HAV-IgM), hepatitis B (anti-HBc-IgM and HBsAg), and hepatitis C (anti-HCV-Ig) (Abbott Laboratories, Abbott Park, IL). HE was diagnosed by detection of HEV RNA in serum or stool by reverse transcriptase polymerase chain reaction and/or by detection of anti-HEV-IgM ≥ 100 WR U/mL.²² These analytic methods were the same that were deployed in the vaccine efficacy trial reported by Shrestha et al.¹⁰

Statistical methods.

Chi-square or Fisher’s exact tests were used to compare categorical variables, as appropriate. Student’s t-tests were used to compare continuous variables. Exact 95% CIs for proportions were calculated using the binomial distribution or Poisson distribution of the risk of maternal-fetal death. CIs for RRs were calculated using the methods described by Armitage and Berry.²³

Ethical approval.

All participants provided written informed consent, including married women less than 18 years old, considered emancipated minors. The institutional review boards of the Nepal Health Research Council and the U.S. Army approved the protocol.

RESULTS

Per protocol cohort.

The cohort included 3,113 enrolled women (Figure 1). Of the 3,049 women with baseline anti-HEV status characterized, 603 (19.8%) did not return to the hospital for parturition or a postpartum visit and were lost to follow-up. Forty-two (1.4%) women were not included due to missing data on demographic or other characteristics. The per-protocol cohort comprised 2,404 women (77.2% of those enrolled) who were followed through pregnancy or longer with at least two assessments and whose pregnancy outcome was known without missing data.

Figure 1.

Cohort of pregnant women followed for HE in Kathmandu, Nepal, 1996–1998.

The median age at enrollment was 23.5 years (interquartile range [IQR] 20–26), 64.6% were nulliparous, and 87.2% were literate. At enrollment, 839 (34.9%, 95% CI: 33.0–36.8) women were seropositive for anti-HEV antibodies. The prevalence of antibodies appeared to increase slightly with age, from 33.2% in those aged 15 to 19 years to a peak of 37.5% in those aged 25 to 29 years (Figure 2). Approximately 2% to 4% had anti-HEV ≥ 250 WR U/mL, suggesting exposure to HEV during the past year (Figure 2). In contrast, 1,565 women (65.1%, 95% CI: 63.2–67.0) were seronegative for anti-HEV. Of these, 1,209 (77.3%, 95% CI: 75.1–79.3) had an anti-HEV level < 10 WR U/mL, indicating that they were definitely negative. Therefore, approximately half of all women of child-bearing age were seronegative and likely susceptible to HEV infection. Three women who were incubating HEV at the time of enrollment were included in the HEV seronegative group; they had baseline anti-HEV levels of 690, 351, and 38 WR U/mL and developed overt HE 7, 8, and 28 days after enrollment, respectively.

Figure 2.

Age-specific prevalence of anti-HEV pregnant women in the per-protocol cohort in Kathmandu, Nepal, 1996–1998 (n = 2,401, three seropositive women ≥ 40 years of age were excluded).

A history of jaundice, as measured by participant recall, was only weakly predictive of antibodies to HEV; 10.5% in the seropositive group versus 5.9% among the seronegatives (P < 0.0001). The relatively low positive predictive value (48.4%) and negative predictive value (66.8%) suggests that neither a history of jaundice nor its absence can reliably identify women who might be immune or, conversely, benefit from HEV vaccination.

Illnesses or complications of pregnancy prompting medical attention, including all pregnancy outcomes different from term delivery of a healthy child, occurred in 351 (14.6%; 95% CI: 13.2–16.1) women. There were 20 complications that occurred at a rate exceeding 1 case per 1,000, affecting 334 (13.9%, 95% CI: 12.5–15.3) women (Table 1). The most common complication, illness manifesting as hepatic injury consistent with acute viral hepatitis, occurred in 52 (2.2%, 95% CI: 1.6–2.8; 21.6 per 1000) women. None of these 52 women had markers for hepatitis A, B, or C. Sixteen of theses 52 women developed HE, representing 30.8% (95% CI: 19.9–44.3) of hepatic injury cases. All 16 women who developed HE were seronegative at baseline. The incidence of hepatic injury not due to hepatitis A, B, C, or E appeared to be similar between seronegative and seropositive subjects (17.9 and 9.5 cases per 1,000 respectively, P = 0.157). HE was the only medically attended complication of pregnancy, occurring at a rate that exceeded 1 per 1,000 and also differed between HEV seronegative and seropositive groups (P = 0.002). The HE incidence was 10.2 (95% CI: 5.9–16.5) cases per 1,000 among the seronegative women and 6.7 (95% CI: 3.8–10.8) cases per 1,000 in the overall per-protocol cohort, placing it among the top seven complications of pregnancy (Table 1). Notably, HE was the only complication leading to maternal death.

Table 1.

Complications of pregnancy occurring > 1/1,000 (per-protocol cohort) in Kathmandu, Nepal, 1996–1998 (N = 2,404).

	Anti-HEV positive (N = 839)		Anti-HEV negative (N = 1,565)		All subjects (N = 2,404)
Diagnosis	N	Rate per 1,000	N	Rate per 1,000	N	Rate per 1,000
Preterm delivery	16	19.1	33	21.1	49	20.4
Hepatic injury (not hepatitis A, B, C, or E)	8	9.5	28	17.9	36	15.0
Urinary tract infection	17	20.3	17	10.9	34	14.1
Still birth	10	11.9	20	12.8	30	12.5
Spontaneous abortion	8	9.5	20	12.8	28	11.6
Puerperal sepsis	5	6.0	16	10.2	21	8.7
HE	0	0.0	16	10.2	16	6.7
Hypertension (all causes)	5	6.0	11	7.0	16	6.7
Itching	2	2.4	13	8.3	15	6.2
Neonatal death	8	9.5	6	3.8	14	5.8
Postpartum hemorrhage	2	2.4	12	7.7	14	5.8
Intrauterine growth retardation (term birth weight ≤ 2,500 g)	2	2.4	9	5.8	11	4.6
Threatened abortion	5	6.0	6	3.8	11	4.6
Cholestasis	3	3.6	4	2.6	7	2.9
Antepartum hemorrhage	2	2.4	4	2.6	6	2.5
Enteric fever	2	2.4	4	2.6	6	2.5
Gastroenteritis	0	0.0	6	3.8	6	2.5
Respiratory infection	1	1.2	5	3.2	6	2.5
Premature contraction	1	1.2	3	1.9	4	1.7

Of the 16 mothers with HE, 11 of the 15 (73.3%, 95% CI: 44.9–92.2) with recorded illness onset were in their third trimester (Supplemental Table 1). Three mothers (18.8%, 95% CI: 4.0–45.6), all in their third trimester, developed acute liver failure with hepatic encephalopathy and died; their infants were stillborn. Thus, the maternal fatality rate due to HE was 1.9 (95% CI: 0.4–5.6) per 1,000 among the seronegative women and 1.2 (95% CI: 0.3–3.6) per 1,000 in the overall per-protocol cohort. Among the 13 mothers who survived HE, five had preterm deliveries and one had IUFD. Among the mothers with HE, the risk of IUFD (25.0%, 95% CI: 7.3–52.4) was increased 10.6-fold (95% CI: 4.29–26.3), and the risk of preterm delivery (31.2%, 95% CI: 11.0–58.7) was increased 17.1-fold (95% CI: 7.56–38.5) relative to seronegative mothers without HEV infection (Table 2).

Table 2.

Comparison of maternal and infant outcomes in the per-protocol cohort and case series in Kathmandu, Nepal, 1996–1998.

	Per-protocol cohort (N = 2,404)											HE case series (N = 34**)
	Anti-HEV positive (N = 839)		Anti-HEV negative
			No HEV infection (n = 1530)			Sub. HEV infection (N = 19)			HE (N = 16*)
Outcomes	N	% (95% CI)	N	% (95% CI)	RR (ref. anti-HEV positive) (95% CI)	N	% (95% CI)	RR (ref. anti-HEV negative, no infection). (95% CI)	N	% (95% CI)	RR (ref. anti-HEV negative, no infection (95% CI)	N	% (95% CI)	RR (ref. anti-HEV negative, HE) . (95% CI)
Maternal death	0	0.0% (0.0–0.4)†	0	0.0% (0.0–0.2)†	–	0	0.0% (0.0–17.7)†	–	3	18.8% (4.5–45.7)	–	5	14.7% (5.0–31.1)	0.78 (0.21–2.88)
Live birth, full term	797	95.0% (93.2–96.4)	1,460	95.4% (94.4–96.5)	1.01 (0.99–1.02)	19	100.0% (82.4–100)†	1.05 (1.04–1.06)	7	43.8% (19.8–70.1)	0.46 (0.26–0.80)	17	50.0% (32.4–67.6)	1.14 (0.60–2.19)
Live birth, preterm	16	1.9% (1.1–3.1)	28	1.8% (1.2–2.6)	0.96 (0.52–1.76)	0	0.0% (0.0–17.7)†	–	5	31.2% (11.0–58.7)	17.1 (7.56–38.5)	5	14.7% (5.0–31.1)	0.47 (0.16–1.40)
Intrauterine death	18	2.1% (1.3–3.4)	36	2.3% (1.7–3.2)	1.10 (0.63–1.92)	0	0.0% (0.0–17.7)†	–	4	25.0% (7.3–52.4)	10.6 (4.29–26.3)	10	29.4% (15.1–47.5)	1.18 (0.43–3.18)
Live birth– neonatal death	8	1.0% (0.4–1.9)	6	0.4% (0.1–0.9)	0.41 (0.14–1.18)	0	0.0% (0.0–17.7)†	–	0	0.0% (0.0–20.6)†	–	2	5.9% (0.7–19.7)	–

ref. = reference; RR = relative risk; Sub. = subclinical.

^* 30.7% of cases with acute hepatocellular injury.

^** 58.6% of cases with acute hepatocellular injury.

^† One-sided 97.5% CI.

In addition to the 16 cases of overt HE, there were 19 cases of subclinical HEV infection, all in the seronegative group (Supplemental Table 1). Overall, the incidence of HEV infection among seronegative women was 2.2% (35/1,565; 95% CI: 1.6–3.1), and the ratio of overt to subclinical infections was approximately 1.1. There were no IUFDs and no preterm deliveries among the subclinical cases (Table 2). Two of the 19 cases of subclinical HEV infection had peripartum complications, but the causal association of HEV infection is unclear. A 30-year-old nulliparous female pregnant with twins presented with preeclampsia 16 days before delivery; she had HEV RNA in blood but no acute antibody response and a normal ALT (14 U/L). At parturition, she still had a negative test for Ig and IgM to HEV. She delivered twins, both with intrauterine growth retardation, and then developed severe postpartum hemorrhage, but neither hepatic transaminases nor a prothrombin time were assessed. Six weeks postpartum, she had recovered and also seroconverted (anti-HEV 2,152 WR U/mL); the twins were in stable condition. A 21-year-old nulliparous female delivered a healthy infant at term, but 7 days later, she developed postpartum hemorrhage and sepsis from which she recovered.

Case series.

During the study, 62 pregnant women outside of the cohort were admitted to Patan Hospital for evaluation of suspected hepatic injury. Of 58 cases tested, 34 were confirmed to be HE (58.6%, 95% CI: 44.9–71.4). The demographic characteristics of the 34 women in the HE case series were generally similar to those in the per-protocol cohort who developed HE, suggesting they were drawn from a similar population. The median age was 22 years (IQR: 20–25; P = 0.670); 38.2% were nulliparous (a lower proportion than in the prospective cohort, P = 0.001); 73.5% were literate (P = 0.034).

The outcomes were similar to those observed in the cohort (Table 2, Supplemental Table 2). Five women from the case series died (maternal case fatality rate of 14.7%): one became ill with acute hepatitis in her first trimester, one with acute liver failure in her second trimester, and three with acute liver failure (two with hepatic encephalopathy) in their third trimester. One survivor recovered from acute liver failure with hepatic encephalopathy in her third trimester, although her infant was stillborn at 30 weeks. There were 17 term births (50.0%), five preterm births (14.7%), 10 IUFDs (29.4%), and two neonatal deaths (5.9%). Three survivors who had stillbirths had a low level of IgM to HEV in their acute illness serum specimens (range: 7–139 WR U/mL) despite a high level of total Ig to HEV (range: 5,199–14,317 WR U/mL). This pattern could result from reinfection with HEV, but no confirmation of this speculation is possible without preillness specimens.

Timing and intensity of HEV transmission.

The 16 HE cases in the per-protocol cohort occurred sporadically, at intervals never exceeding several months. We conducted surveillance for jaundice admissions, as a surrogate for HE, in the six main hospitals treating hepatitis for the entire Kathmandu Valley (∼500,000 people).²⁴ The epidemic curve illustrates the typical seasonality of HE in Nepal, with a 4-fold seasonal increase from December to April nadir to the annual August peak that coincides with the mid-rainy season (Figure 3). During the period of this study, the mean number of jaundice cases found by hospital surveillance per month was increased 1.7-fold (95% CI: 1.0–42.0) relative to the 10-year average for 1994 to 2003 (Supplemental Figure 1).

Figure 3.

Timing of jaundice admissions to sentinel hospitals (N = 2,399) (line plot) and of HE onset in the per-protocol cohort (N = 15, one HE with unknown onset was excluded) (vertical bars) in Kathmandu, Nepal, 1996–1998.

DISCUSSION

We present a historical analysis of a prospective cohort to enroll healthy pregnant women to examine systematically the risk of HEV infection and its associated health outcomes during pregnancy. The epidemiology of HEV in Nepal has not changed drastically since this study was completed. HE remains endemic in Nepal and its neighboring countries.^25–29 HEV is still a major cause of acute hepatocellular injury in both pregnant women and the general population, with significant maternal mortality.^{3,9,18,26,27,30–33} Our estimates of population seroprevalence in pregnant women for anti-HEV and the proportion of HE cases among women with clinically overt acute liver injury among the case series are in line with more recent studies from Nepal and other HEV-endemic countries in Southeast Asia (Table 3). However, those studies were unable to estimate a prospective risk and death from HE due with their design. Recent systematic reviews and meta-analyses found the case-fatality rate of HE in pregnancy to be 20.8% (95% CI: 16.6–25.3) and 26% (IQR: 17–41%),^9,33 similar to the 16% found here. Therefore, the estimates of prospective risk and death from HE found here are still relevant today.

Table 3.

Comparison of HE studies from HEV-endemic countries of South Asia

Epidemiological characteristic	Study	Population	Years of data collection	Location	Result
Seroprevalence
	This analysis	Pregnant women	1996–1998	Kathmandu, Nepal	34.9% (95% CI: 33.0–36.8)
	Shrestha et al., 201646	All women	2015	Various cities, Nepal	35.6% (95% CI: 30.3–41.0)
	Izopet et al., 201529	Adults 16–30 years old	2010–2012	Kathmandu, Nepal	43.0%
	Kmush et al., 201647	Pregnant women	2008–2010	Gaibandha, Bangladesh	10.0%
	Rahman et al., 201448	Adults 18–67 years	Not reported	Dhaka, Bangladesh	30.0%
	Labrique et al., 200949	General population	2003–2004	Matlab, Bangladesh	22.5% (95% CI: 20.1–25.0)
	Tripathy et al., 201850	Blood donors	2017	Pune, India	17.7% (95% CI: 16.2–19.3)
	Katiyar et al., 201851	Blood donors	2016	Lucknow, India	60.5%
	Kotwal et al., 201452	Young adults	2010–2011	Various locations, India	17.0% (95% CI: 15.9–18.3)
	Vivek et al., 201053	Adults 16–40 years old	1999–2000	Vellore, India	29.5%
Proportion of clinically overt liver injury with confirmed HE
	This analysis	Pregnant women	1996–1998	Kathmandu, Nepal	58.6% (95% CI: 44.9–71.4)
	Shrestha et al., 200930	Pregnant women	2001–2007	Kathmandu, Nepal	20.6%
	Gupta et al., 201826	General population	2014	Kathmandu, Nepal	69.2%
	Shrestha et al., 200318	General population	1997	Kathmandu, Nepal	56.0%
	Paul et al., 202054	Pregnant women	2014–2017	Sentinel Hospitals, Bangladesh	39.0% (95% CI: 32.0–46.0)
	Kumar et al., 201755	Pregnant women	2016	Lucknow, India	18.0%
	Solanke et al., 201641	Pregnant women	2013–2015	Western India	27.1%
	Singla et al., 201642	Pregnant women	2011	Delhi, India	43.9%
	Patra et al., 20076	Pregnant women	2003–2005	New Delhi, India	60.0%
	Kumar et al., 200756	Pregnant women	2002–2006	Chandigarh, India	58.3%
	Rathi et al., 200757	Pregnant women	2002–2004	Mumbai, India	15.0%
	Kumar et al., 20044	Pregnant women	2001–2002	New Delhi, India	45.2%
	Beniwal et al., 200358	Pregnant women	2000–2002	New Delhi, India	47.4%
	Bhatia et al., 200859	Pregnant women	1986–2006	New Delhi, India	59.4%
	Javed et al., 201760	Pregnant women	2015	Various Locations, Pakistan	15.5%
	Sultana and Humayun, 201461	Pregnant women	2012–2013	Lahore, Pakistan	65.8%
	Hossain et al., 200962	Pregnant women	2006	Karachi, Pakistan	63.0%

Most women in the per-protocol cohort (65.1%) were seronegative for antibodies against HEV despite living in a region where HEV is endemic. Among seropositive cohort members, 2.5% had an anti-HEV level consistent with exposure to HEV within the previous year. This proportion is in line with other estimates of an average 1% to 2% annual incidence of HE among young adults in Nepal’s Kathmandu Valley.^2,10

In this cohort, HE was the only cause of death. HEV caused 58% of cases with hepatic injury consistent with acute viral hepatitis. The observed rate of HE was 6.7 per 1,000 and of HEV-caused maternal or fetal death was 2.9 per 1,000. HE was the seventh most common cause of pregnancy complications and the only one leading to maternal death, with a maternal case-fatality rate of 16%. The risks of IUFD and preterm delivery were greatly increased among women with HE. These observations are consistent with other studies of the adverse impact of HE on maternal and fetal outcomes.^3,9,30–33 However, none of these other studies were able to estimate the risks of these complications compared with healthy pregnant women. Nutritional status and immune function have been suggested to be predictive of worse outcomes among pregnant women with HE but were not examined.^34,35 None of the demographic factors examined seemed to be related to HE mortality.

Subclinical infections, despite being responsible for 54% of HEV infections, did not increase risk of death in this cohort. In one woman, subclinical infection may have contributed to postpartum hemorrhage, but this could not be verified. Few studies have been able to examine subclinical HEV infections during pregnancy. Among the case series, approximately 10% of the HE cases may have been reinfections. Reinfection with HEV has been well documented in China where genotype 4 is endemic.³⁶ A study from Nepal suggests that reinfection presenting as disease is uncommon, but more studies are needed.²² This evidence suggests a beneficial role of vaccination even in women who have been exposed to HEV to boost waning immunity.

This cohort represented a selected population with increased access to prenatal and delivery health care, which may have resulted in overall better maternal and neonatal outcomes. For Nepal, the proportion of pregnant women receiving such services during 2003–2008 as reported by UNICEF was 44% and 18%, respectively.³⁷ However, due to the nature of HE, it is unlikely that enhanced medical attention or hospitalization improved outcomes. During the time of the study, most women did not seek antenatal care before 24 weeks’ gestation, which was the major factor limiting cohort entrants. Conversely, this study was conducted during a period of increased HEV activity, which likely inflated the absolute risk estimates. Approximately 20% of the initially enrolled women were unable to be reached for a second visit. The reasons for attrition are complex, and it is not possible to know the pregnancy outcomes. A limitation of this study is that it was conducted more than 20 years ago. Because of the amount of time that has passed, there is the potential that transmission patterns have changed and health care practices and availability have improved. However, the results found here are consistent with more recent studies about HEV infections and deaths in Nepal (Table 3). Therefore, the evidence from this study, despite its generation 20 years earlier, remains relevant and supportive of the benefit of vaccinating women of reproductive age against HEV to reduce maternal and fetal mortality in areas where HEV is endemic, even if adequate obstetrical care is prevalent.

Hepatitis E is not reported as a cause of death in national surveys of pregnancy outcomes in Nepal. The illness tends to be nonspecific, and unless prospective viral testing is available, HE cannot be diagnosed. However, the national surveys report that hemorrhage is the leading cause of maternal death in Nepal.^38,39 HE causes coagulopathy and is a major contributor to both antepartum and postpartum hemorrhage deaths.^40–44 Indeed, all of the women in this study who died had prolonged prothrombin times suggestive of coagulopathy (Supplemental Tables 1 and 2).

There are approximately 500,000 and 35 million births per year in Nepal and in the HEV-endemic countries of South Asia (Afghanistan, Bangladesh, India, Nepal, and Pakistan), respectively.⁴⁵ Our results suggest that prevention of HE would prevent approximately 1,500 (95% CI: 698–3,490) maternal-fetal deaths in Nepal and 100,000 (95% CI: 44,939–224,693) maternal-fetal deaths in South Asia each year. Because this study was conducted during a period of increased transmission of HEV, reducing the estimated risk of maternal-fetal death to 1.7 per 1,000 (2.9 deaths per 1,000 divided by 1.7-fold increased jaundice cases during the study compared with the 10-year average) allows for a more conservative calculation of disease impact. By the reduced estimate, HEV leads to 550 (95% CI: 262–2501) preventable deaths in Nepal and 60,000 (95% CI: 16,852–161,030) preventable deaths in the HEV-endemic countries in South Asia per year. This study documented the large burden of HE in pregnant women and strongly supports programs for immunization against HE. Future studies are needed to determine the best methods to implement immunization to prevent 60,000 maternal-fetal deaths per year in South Asia.

Note: Supplemental figure and tables appear at www.ajtmh.org.