Pregnancy-Associated Listeriosis in a Tertiary Hospital in Ningbo, Zhejiang, China: A Retrospective Study

Pregnancy-associated listeriosis is a severe infectious disease and potentially leading to fetal/neonatal fatal. Limited information on pregnancy-associated listeriosis is available in China. We performed a retrospective study on maternal and neonatal patients with pregnancy-associated listeriosis. The clinical characteristics of pregnancy-associated listeriosis were studied, and the outcome determinants of neonatal listeriosis were explored.


Background
Listeriosis is a rare but severe disease caused by Listeria monocytogenes (L. monocytogenes), a ubiquitous food-borne bacterial pathogen [1,2]. People principally get listeriosis by the ingestion of contaminated food [3,4]. Especially, it has a predilection to infect pregnant women, which is at approximately 10-100 times greater risk for infection than the general population [5][6][7]. In Europe and North America, it has been observed previously that the proportion of pregnancy-associated listeriosis out of the total listeria infections ranges from 9% to 20% [2,[8][9][10]. Moreover, recent studies performed in China showed that more than half of listeria cases were pregnancy-associated infections [11,12].
The high incidence of listeriosis during pregnancy may due to the increased progesterone levels which would affect immune functions [13,14]. L. monocytogenes can actively cross the intestinal barrier, disseminate within the circulation, cross the placental barrier owing to its speci c placental tropism, and eventually leading to placental and fetal infections [15]. Although maternal illness is usually mild, neonatal illness is frequently severe and potentially fatal. Pregnancy-associated listeriosis can lead to miscarriage, preterm delivery or stillbirth. It was reported that more than 80% of infected mothers would experience major fetal or neonatal complications [9]. Therefore, pregnancy-associated listeriosis is recognized as one of the worst maternal infections.
It has been reported that the incidence of pregnancy-associated listeriosis varied between different minorities [4,9,16]. Recently, a study in Singapore found that the incidence of congenital listeriosis was highest in the infants of Chinese origin [17]. Besides, a study observed that the rate of pregnancy-associated listeriosis in Beijing Obstetrics and Gynecology Hospital was 13.7 per 100,000 births [18], which was higher than that in United States, United Kingdom and New Zealand (3.4, 3.4 and 12.3 per 100,000 births, respectively) [19][20][21]. Listeriosis has not yet been included in the noti able disease in China, the surveillance of listeriosis has not been thoroughly implemented. Moreover, owning to the nonspeci c obstetrical signs and long incubation period, the diagnosis of pregnancy-associated listeriosis would be challenging [22,23].
To better understand the maternal and neonatal listeriosis in Ningbo, Zhejiang Province, China and to provide more information for formulating appropriate therapeutic and controlling strategies, we performed a retrospective study to analyze the clinical characteristics and outcomes of pregnancy-associated listeriosis cases.

Study design and patient selection
This was a retrospective study conducted at Ningbo women and children's hospital, a tertiary maternity and pediatric hospital, where the number of births is about 10,000 every year. This study was approved by the the institutional ethics board (EC2021-028), and the informed consent was waived for this retrospective study.
We conducted a retrospective study to identify all perinatal cases of culture-con rmed L. monocytogenes infections at Ningbo women and children 's hospital, from August 2013 to September 2021. Data on past medical history, characteristics at admission, underlying medical conditions, laboratory results and treatments were collected from medical record database.
A pregnancy-associated listeriosis case was de ned based on isolation of L. monocytogenes from a normally sterile site of the pregnant women and/or newborns aged ≤28 days. Each Mother and her newborn were counted as a single case. Neonatal listeriosis cases were classi ed as early onset (diagnosed between birth and days 6) listeriosis or late onset (diagnosed between days 7 and 28) listeriosis. We de ned "inevitable miscarriage" as fetal loss before 24 weeks of gestation, "stillbirth" as death of the fetus between 24 and 41 weeks of gestation, "maternal leukocytosis" as a white blood cell (WBC) count > 12×10 9 /L, "neonatal leukocytosis" as a WBC count >34×10 9 /L, "neonatal leukopenia" as a WBC count <5×10 9 /L, "neonatal thrombocytopenia" as a platelet count<150×10 9 /L, "neonatal severe thrombocytopenia" as a platelet count <5×10 9 /L, "inborn neonate" as a neonate born in our hospital, and "outborn neonate" as a neonate born in other hospitals and transferred to our hospital after birth.

Microbiological methods
For blood culturing, whole blood for bacterial culture was collected and incubated in an automatic blood culture system (BACT/ALERT 3D) for 5 days or until rated positive. A positive blood culture was incubated on blood agar at 36℃ for 18-24 h. CSF, cervical secretion, eye secretion, gastric aspirate of newborn and products of conception samples were cultured directly on blood agar at 36℃ for 18-48 h, then, bacteria with a positive growth were isolated and cultured onto blood agar at 36℃ for 18-24 h.
The identi cation of bacteria was performed by using the VITEK 2 COMPACT automatic analysis system (BioMérieux, France). Standard strains (ATCC700323, ATCC25922, ATCC700327, ATCC29213) were used to control the microbiological quality.

Statistical analysis
Clinical, laboratory and meteorology data were recorded in a Microsoft Excel database (Microsoft, Richmond, US). For continuous variables, non-normally distributed variables were expressed asmedians and ranges, for categorical variables, data were presented as percentages. The differences between two groups were determined by using the Wilcoxon Manne-Whitney U test for quantitative data and Fisher's exact test for qualitative data, respectively. A P <0.05 was considered statistically signi cant. All statistical analyses were performed by using SPSS statistical 22.0 software (IBM, Armonk, NY, USA).

Epidemiological characteristics of pregnancy-associated listeriosis cases
During the study period, 14 cases of pregnancy-associated listeriosis were identi ed. 12 cases were inborn neonates, the rest (B2 and B3) were outborn neonates. The incidence of pregnancy-associated listeriosis in our hospital was 16.69/100,000 (14/83,875) births during our study period. They were occurred in all seasons, and a half were occurred in summer (50.0%, 7/14).

Characteristics of the neonatal cases
The characteristics of neonatal listeriosis cases were summarized in table 2. There were 11 culture-con rmed cases and 3 probable cases.  Most of the 11 con rmed neonatal listeriosis cases were early onset (90.9%, 10/11) (B2-B10, B13). Among the 10 early onset cases, 8 cases (80.0%, 8/10) had respiratory distress and 7 case (70.0%, 7/10) received resuscitation at birth. The late onset one was a female who was delivered by caesarean section at GA of 38 weeks, and admitted to hospital with fever for one day at 8 days after birth.
Laboratory analysises were performed immediately on admission. Thrombocytopenia was observed in 54.5% (6/11) of culture-con rmed cases. Cerebrospinal uid tap was performed in 81.8% (9/11) of neonates, and 4 of them were diagnosed as purulent meningitis, however, their CSF culture were all negative.
Cranial imaging revealed intracranial hemorrhage in 2 neonates (B4 and B8), and one of them (B4) was diagnosed as brain injure in premature infants (BIPI).
All of the neonates with con rmed listeriosis were treated empirically, and most of them treated with Penicillin or Cephalosporin or Meropenem. Eight newborns received intubation. Three neonates (B2, B3 and B10) died, and two of them were outborn.

Probable neonatal listeriosis cases
Three newborns (B11, B12.1 and B12.2) were identi ed as probable neonatal listeriosis cases, including a pair of twins (B12.1 and B12.2). Both of their mothers were con rmed maternal listeriosis, but the culture results for these three neonates were negative. All of them were preterm infants, with GA of 32, 36 and 36 weeks, and BW of 1600g, 2550g and 2250g, respectively. They all had respiratory distress at birth, and were diagnosed as pneumonia subsequently, their laboratory parameters were mild abnormal. All of them were recovered soon after birth.

Risk factors for mortality in neonates with pregnancy-associated listeriosis
We compared the characteristics of the surviving neonates (n=11) with those of the non-surviving neonates (n=3), trying to identify risk factors for mortality in neonates with pregnancy-associated listeriosis (Table 3).

Discussion
To our knowledge, this is the rst detailed study of pregnancy-associated listeriosis infections among children in Ningbo, Zhejiang province, China. The incidence of pregnancy-associated listeriosis in our hospital was 16.69/100,000 deliveries, most of the neonatal listeriosis cases (92.9%, 13/14) were early onset. 3 cases died, all of them were early onset and culture-con rmed neonates, 2 of them were outborn neonates. All of the maternal patients recovered after delivery shortly with no sequelae.
The diagnosis of maternal listeriosis would be challenging, apart from nonspeci c obstetrical signs, fever or u-like symptoms are the only clinical signs in infected mothers [24], but they were not always present. In our study, only one patient had u-like symptoms before delivery. The di culty of diagnose was associated with adverse fetal or neonatal outcome. First, we and others all observed that most maternal listeriosis cases were appeared during the second and third trimester period of pregnancy, mainly based on the adverse signs of their infants. While, the occurrence of rst trimester maternal listeriosis was likely underestimated as the non-speci c nature of disease presentation, thus may causing early fetal losses. Second, non-speci c signs in infected pregnant women would in uence the antibiotic prophylaxis to prevent neonatal listeriosis [22]. Previous studies have showed that adequate maternal antimicrobial treatment before delivery was associated with a signi cant decrease of infants' severity [24,25]. Unfortunately, in our study and others performed in China[18], none of the maternal listeriosis cases received adequate rst-line antimicrobial treatment [9]. This may partly account for the adverse outcome of L. monocytogenes infected neonates in the present study. In China, there are no national guidelines for the treatment of listeriosis currently. Although rare, listeriosis should be considered in the differential diagnosis of pregnancy-associated infections.
Identi ed risk factors for mortality in neonates with pregnancy-associated listeriosis is important to treat and manage the high-risk neonates. It was found that fatality rates were signi cantly higher in the neonates who were born in other hospitals (P=0.005) in our study, these hospitals were lack of neonatal intensive care unit. It has been observed in a previous study that preterm neonates, who were delivered in hospitals with lower levels of neonatal care, suffered from higher rates of adverse outcomes [26]. The capability to provide timely and optimal resuscitative measures is important to reduce mortality of neonatal listeriosis. trimester, is typically associated with more favorable outcomes than earlier infection. In our study, higher mortality rates also were observed in neonates with lower BW (P=0.038), GA <28 weeks (P=0.056), however, the differences of later one were not statistically signi cant. We did not show it may due to the low sample size. Further studies are needed to explore their relationship.
Some studies suggested that L. monocytogenes was more commonly associated with sporadic episodes and outbreaks rather than being affected by factors such as climate and season [20,27]. However, seasonal trends of pregnancy-associated listeriosis cases have been observed in the Beijing and Taiwan[18, 28], with peaks reported in summer. Similarly, in our study, more than a half pregnancy-associated listeriosis cases were occurred in summer (50.0%, 7/14). As L. monocytogenes was a psychrophilic organism, the ready-to-eat foods and foods stored at refrigeration temperatures are the main sources of L. monocytogenes [29]. And people have more opportunity to eat food that is inadequately cooked and improperly stored in the refrigerator during the warmer seasons. Therefore, they were more likely to be L. monocytogenes infected in summer.
Unfortunately, the incidence of pregnancy-associated listeriosis in our hospital (16.69/100,000) was higher than that in the United States, United Kingdom and New Zealand (3.4, 3.4 and 12.3 per 100,000 births, respectively) [19][20][21], where listeriosis was a noti able disease. The information of listeriosis especially pregnancy-associated cases is limited, and this might due to the lack of a good listeriosis surveillance system in China [30]. Besides, the incidence of pregnancy-associated listeriosis in our hospital was also higher than that in one hospital of Beijing (13.7/100,000), this might because our hospital is the only tertiary maternity and pediatric hospital in this area that admits many critically ill pregnant women and neonates.
There are several limitations in our study. First, because the miscarriage products at the rst trimester GA were usually not available for microbiological investigations, and the blood cultures were not routine examinations for pregnant women without severe performance of infections in our hospital, the occurrence of maternal listeriosis was likely to be underestimated. Second, it was a hospital-based study, the sample size was relatively small, and some information were missing (information of two mothers of outborn listeriosis neonates), so our results could not be generalized to whole population. Third, it was a pity that we did not preserve the strains of L monocytogenes, molecular epidemiology data were unavailable. Therefore, a study with a large sample size and relevant experiments is strongly encouraged.

Conclusion
We nd that there is a neglected burden of pregnancy-associated listeriosis in our hospital. Listeriosis can have a devastating effect on fetus/neonates. The outborn neonates, who born in hospitals with lower levels of neonatal care, were probably associated with the increased severity. Although rare, listeriosis should be considered in the differential diagnosis of pregnancy-associated infections.

Declarations
Ethics approval and consent to participate This study was approved by Ningbo Women and Children's Hospital Ethics Committee and our committee's reference number was EC2021-028. The informed consent was waived for this retrospective study by Ningbo Women and Children's Hospital Ethics Committee. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication:
Not applicable.

Competing interests
The authors declare that they have no con ict of interest. Authors' contributions YK contributed to the study design, data analysis, and manuscript writing and revision. LY, PZ and YS contributed to data collection and management. ZZ contributed to the data analysis and manuscript revision. All authors read and approved the nal manuscript.