Perinatal listeriosis patients at a maternity hospital in Beijing, China, during 2013 – 2018

Background: Listeriosis is a rare but severe foodborne infectious disease. Perinatal listeriosis is extremely prone to causing septicaemia, central nervous system (CNS) infection, and serious adverse pregnancy outcomes (abortion and neonate death). This research reports perinatal listeriosis cases over the course of six years at Beijing Obstetrics and Gynecology Hospital (BOGH), which is the largest maternity hospital in China. Methods: We retrospectively searched all the laboratory conrmed pregnancy-associated listeriosis cases during Jan 1, 2013 to Dec 31, 2018. The clinical manifestations, laboratory results, perinatal complications and outcomes (follow-up for half a year after birth) were investigated. Results: In BOGH, 12 perinatal listeriosis cases were diagnosed based on Listeria monocytogenes positive culture, including 10 single pregnancies and 2 twin pregnancies, and the incidence of which is 13.7/100,000 deliveries. Among those cases, four pregnant women and four newborns had septicaemia, and two of the neonatal septicaemia also got CNS infection. All the maternal patients recovered. For the fourteen offsprings, there were 8 newborns and 6 aborted fetuses, with two newborns died and 6 survived among the 8 newborns, and none of survivors had sequelae. The feto-neonatal fatality ratio is 57.1%. Conclusions: Perinatal listeriosis is associated with high mortality, which has become a public health concern. Subsequently, further researches about perinatal listeriosis among a wider range of population are needed to strengthen the epidemiological understanding of listeriosis in China.


Background
Listeria monocytogenes (L. monocytogenes) is a facultative anaerobic Gram-positive bacterium that causes severe foodborne illnesses carrying substantial mortality (20% to 30%) [1] . This pathogen, a ubiquitous bacterium in nature, can be isolated from soil, stream water, vegetables, fruits, raw meat, milk products, the ready-to-eat products, and even processed foods stored at refrigerator, because Listeria can survive and grow at a wide range of pH and temperatures, and high salt concentrations [2][3][4] .
L. monocytogenes is mainly transmitted through the consumption of contaminated food. After crossing the intestinal mucosal barrier, L. monocytogenes can spread through blood, and shows a preference for central nervous system (CNS) and the placenta [5] . The increased progesterone during pregnancy weakens the cellular immunity, which makes the expectantmothers particularly susceptible to microorganisms like L. monocytogenes [6,7] . The infection risk of pregnant women is 12-to 20-times higher than that of the general population [8,9] . It is reported that incidence of pregnancy-related listeriosis was about 4.3-25 cases per 100,000 births [10][11][12][13] . As shown in systematic reviews, the pregnancy-associated cases accounted for 20.7%-43% of all listeriosis cases worldwide [14,15] . In China, human listeriosis surveillance system was established in 2013. But as yet, listeriosis is not a noti able disease in China. Two recent reports showed that in China, 41.1%-52% of clinical cases were perinatal listeriosis, which means the burden of pregnancy-related listeriosis in the country is not light [16,17] . Pregnant women infected with L. monocytogenes is usually asymptomatic or with nonspeci c clinical symptoms such as gastrointestinal and u-like symptoms. However, perinatal listeriosis patients often have adverse pregnant outcomes, including fetal loss, preterm birth and neonatal listeriosis.
This report retrospectively reviews all the laboratory-con rmed pregnancy-associated listeriosis cases at Beijing obstetrics and gynecology hospital (BOGH) during January 2013 to December 2018, which is a high-level maternal and child health care hospital with 660 beds in Beijing, China [1]. Further, we detail the clinical characteristics and outcomes of these L. monocytogenes infected perinatal patients.

Methods
The number of births in BOGH exceeds 14,000 every year. We retrospectively analyze the clinical data of all the laboratorycon rmed pregnancy-associated L. monocytogenes infections during January 1, 2013 to December 31, 2018, based on a list generated from the department of Disease Prevention and Control and Nosocomial Infection of BOGH. The clinical information of all patients are stored in the electronic database since 2013.
Pregnancy-associated listeriosis cases includes illness with an onset during pregnancy or within the rst 2 weeks of the postpartum period and illness in the neonate between birth and 4 weeks [18] . All the con rmed cases were based on the isolation of L. monocytogenes from a normally sterile site (eg, blood or cerebrospinal uid (CSF)) or products of conception (eg, placental or fetal tissue), with the presence of compatible clinical symptoms. A perinatal listeriosis case was de ned based on isolation of L. monocytogenes from a clinical sterile sample of pregnant woman or foetus, stillborn, and newborn aged < 4 weeks [9] . Con rmed cases with isolation of L. monocytogenes from both mother and neonate were counted as single cases. Neonatal cases were divided to early onset (diagnosed between birth and day 6) and late onset (diagnosed between 7 and 28 days) [18,19] .L. monocytogenes CNS infection were diagnosed in the case that L. monocytogenes was isolated from a patient's CSF or when a patient had neurological symptoms (altered consciousness, seizures, nuchal rigidity, or focal neurological symptoms, and an increased white blood cell (WBC) count in the CSF) and blood cultured L. monocytogenes. When the patient did not meet the criteria of CNS infection diagnosis but L. monocytogenes was cultured from the blood, this patient was considered to have septicaemia [20] .
The culturing, isolation and identi cation of L. monocytogenes rely on the traditional blood agar plating followed by automated biochemical con rmation (bioMérieux VITEK 2 COMPACT, France) with visible colonies [20] .
We de ne 'stillbirth' as death in the fetus between 24 and 41 weeks of gestation, and a fetus loss before 24 weeks is de ned as an inevitable miscarriage. Furthermore, we calculate the overall fatality of pregnancy-associated listeriosis, including miscarriages, stillbirths and newborn deaths.
We used descriptive statistics in this study. Where appropriate, data were expressed as mean ± SD (standard deviation).

Annual and seasonal number of perinatal listeriosis cases in the considered period
We identi ed 12 cases of pregnancy-associated listeriosis from the overall 87,644 deliveries, and the incidence was 13.7/100,000 births. In detail, the annual and seasonal number of pregnancy-associated listeriosis cases were shown in Fig. 1a and Fig. 1b, respectively. We found ten cases (10/12 83.33%) occurred during summer and fall months (i.e. June to November) during 2013 to 2018.

Characteristics of pregnancy-associated listeriosis cases
The characteristics of the pregnancy-associated cases are summarized in Table 1. There were 12 maternal, and 14 neonatal infections with L. monocytogenes.

Clinical characteristics of maternal listeriosis cases
The median age of these women was 29 years (range, 25-41 years). The clinical characteristics of 12 maternal listeriosis patients are described in Table 2. Among them, 10 were singleton pregnancy and 2 were twin pregnancy (i.e. Case 2 and 9). Six cases were infected with L. monocytogenes in the middle trimester pregnancy (between 14 and 27 weeks) and the other six in the third trimester pregnancy (between 28 and 41 weeks), and their median gestation was 29.3 weeks (range, 20.0-38.1 weeks). 11 of 12 maternal listeriosis patients had prenatal fever (38-39.3°C), and 3 of 12 had Flu-like symptoms. Five patients had gastrointestinal symptoms (diarrhea and abdominal pain). Various obstetrical symptoms were presented among the 12 pregnant women, including decreased fetal movement (n = 5), intrauterine fetal death (n = 2), premature rupture of the membranes (PROM, n = 2), and vaginal bleeding (n = 1). All the symptomatic women received antibiotic therapy, and eight patients received only cephalosporin antibiotics at their beginnings.
Several adverse complications were presented during their pregnant periods. Among the 12 maternal patients, apart from one normal labor, ve pregnant women progressed to fetal loss and six result in premature deliveries. Three pregnant women had postpartum hemorrhage, ve progressed to amniotic uid contamination, and three received induce labor. Four cases carried out Caesarean section (C-section) due to the abnormal fetal heart rates. No mother had CNS infection because there were neither neurological symptoms nor positive cultured L. monocytogenes from CSF. One third (4/12) had septicaemia. All the maternal patients nally recovered after delivery with no sequelae.

Clinical characteristics and outcomesof the offsprings
The clinical characteristics and outcomes of all the 14 offsprings born from the 12 maternal listeriosis patients are summarized in Table 3, and we found no late-onset cases of newborn-infant listeriosis. Among the offsprings, 9 out of 14 (64.29%) were girls.
The median birth weight is 1,305g (range, 380-3,565g). Except two inevitable miscarriages and four fetal stillbirths, eight babies were presented with fetal distress. Four newborns received intubation. 6 out of 14 babies were con rmed L. monocytogenes infection based on microbiological methods (laryngeal swab: 2; blood + laryngeal swab: 2; blood + CSF + laryngeal swab: 2). Two neonates were diagnosed with CNS infection and four had septicaemia. Six infants survived, and two newborns were dead within 2 days after birth. None of the survivors had neurological sequelae through a six-month follow-up.

Discussion And Conclusions
The incidence of perinatal listeriosis in BOGH was 13.7/100,000 deliveries, which was consistent with the reports [10][11][12][13] . In our study, half of the pregnancy-associated listeriosis cases (6/12, Case 2, 3, 5, 8, 10, and 12) suffered from listeriosis in the middle trimester pregnancy, and all of their 7 offsprings were dead. While among the other six perinatal cases (Case 1, 4, 6, 7, 9, and 11; 7 newborns in total), except that the infant of Case 1 (31.3 week) deceased day 2 after birth, the remaining 6 livebirths survived without sequelae (including 4 septicaemia newborns and 2 of them also had CNS infection). This was consistent with the reports [13,21] that the prognosis of neonate from maternal listeriosis patients occurring during late pregnancy were quite excellent even the infants were infected with L. monocytogenes, while the outcomes of perinatal cases infected before 28 weeks were very bad. The overall cases-fatality rate of the offsprings was 57.1%, which was close to 32.68%-50.7% reported in the two recent systematic reviews in the mainland of China [16,22] . From these numbers, the feto-neonatal listeriosis mortality rate in China was very high, which was in contrast to the low child mortality estimated by de Noordhout CM and colleagues [14] during 2010.
We found that 66.67% (8/12) of maternal patients had received only cephalosporins antibiotics at their beginnings since cephalosporins are the preferred empirical therapy for the obstetric infections with nonspeci c clinical symptoms in China. However, L. monocytogenes strains were susceptible to most antibiotics (e.g. ampicillin and penicillin G (PNG)) while not sensitive to cephalosporins [23][24][25][26] . Delayed diagnosis and inappropriate antibiotic administration could decrease the probability of a favorable outcome among L. monocytogenes affection cases [21,27] . In China, there are still no national guidelines for the treatment of listeriosis. According to Hof et al [24] , when listeriosis is a likely diagnosis, the use of ampicillin, PNG, or vancomycin provides empiric coverage for L. monocytogenes. But empiric therapy for bacterial meningitis with ampicillin may not be necessary for children beyond the neonatal period [28] . Ampicillin or PNG, with or without aminoglycoside or gentamicin, is recommended for all forms of listeriosis. Trimethoprim-sulfamethoxazole can be used as an alternative treatment. Two to three weeks of therapy is su cient for most forms of listeriosis. Rhombencephalitis with abscess formation in the CNS may require 4-week therapy [27,29] .
However, researchers recently found high resistant levels of L. monocytogenes to many antibiotics (ampicillin, PNG, tetracycline, cefotaxime, etc.) among the clinical and food isolates, which was considered as a serious problem for the treatment of listeriosis [30,31] .
Listeriosis is a typical foodborne disease which means food contamination is the major source of infection. Data from Beijing centers for disease control and prevention (CDC) showed that among the 12 maternal patients, only Case 5 had positive cultured L. monocytogenes in the samples obtained from her kitchen and had the same strain with the patient. While various food sources of L. monocytogenes made it di cult to trace the pathogens and identify the food source.
The inspection reports [32,33] showed the average prevalence of L. monocytogenes in Chinese food products was about 4.42% [32] , and in the retail markets of Beijing 15.20% of the raw pork was contaminated [33] . That is, food safety of L. monocytogenes contamination is still a problem in China. Besides, processed food stored and pasteurized dairy products in the refrigerator are also vulnerable to recontamination because that L. monocytogene can grow very well at 4°C [34,35] . There are no diet guidelines for the prevention of L. monocytogenes infection in China. Therefore, improving the awareness of food safety among the people at higher risk of L. monocytogenes infection, especially the pregnant women and immune compromised population, can facilitate prevention. Due to that doctors are the most credible source of health information for patients, campaigns among doctors about healthy diet habits education (such as, refusing to direct eat pasteurized dairy products and cooked food stored in the refrigerator without recooking, reducing the frequency of eating out eat out less often, etc.) are recommended for pregnant women.
There are several limitations in this study. First, it is a retrospective research over a protracted timespan of six years that there is no fresh sample available for further research, such as microbial typing. Second, the lack of epidemiological investigations is also a limitation of the study which does not allow to identify the food source and make clear recommendations. Third, it consists of a relatively small sample size in this study. Big data investigation of pregnancy-associated listeriosis is conducive to further understanding the demographic distribution in China.

Declarations
Ethics approval and consent to participate The raw data is not publicly available, but as the medical staff at the hospital we have access to it. The need for ethics approval of Obstetrics and Gynecology Hospital. The data used in this study was anonymised before its use.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
Not applicable.
Authors' contributions CH Yin and RX Liu conceived and designed the study. CY Li, RX Liu, HH Zeng, X Ding, Y Chen, XW Liu, L Zhou, X Wang, SS Hu, YM Cheng, Z Cao collected the data. CY Li, RX Liu nished literature search, data interpretation, and writing. All the authors critically reviewed this report and approved the nal version.