Metagenomic next-generation sequencing for efficient detection of human parvovirus B19 in amniotic fluid: a case study of diagnosis and prenatal management of fetal infection

Abstract

Objective

Human parvovirus B19 (B19V) infection during pregnancy can lead to a range of adverse outcomes such as miscarriage, premature delivery, fetal hydrops, severe anemia, myocarditis, heart failure, and even fetal demise, posing significant risks to maternal and fetal health. The aim of this study was to establish a more efficient method for detecting B19V in amniotic fluid and to explore and optimize early diagnosis and treatment strategies for fetal B19V infection.

Methods

Intrauterine transfusion (IUT) was performed due to the occurrence of severe fetal anemia and hydrops. Amniotic fluid was obtained for genetic detection. Metagenomic next-generation sequencing (mNGS) and bioinformatic analysis were performed on the amniotic cells to identify the viral genome.

Results

In this study, the B19V genome was identified in the amniotic cells of the suspected case, with three viral coding sequences mapped. The coverage density reached 99.9% of the viral sequences. No other pathogen sequences, including bacteria, fungi, parasites, chlamydia, mycoplasma, rickettsia and other viruses, were identified.

Conclusion

Our study confirmed the diagnosis of fetal B19V infection in a suspected case via amniotic fluid virus genome detection. It is the first time to exhibit the clinical application of mNGS to systematically detect the B19V genome in amniotic fluid in prenatal practice, and to achieve good results in combination with clinical management. The study highlighted the importance of comprehensive management of B19V fetal infection and demonstrated the advantages and wide application prospects of mNGS in intrauterine infection diagnosis.

Introduction

Human parvovirus B19 (B19V), which was first described in 1975 by Cossart et al., is a single-stranded DNA virus that is especially cytotoxic to erythroid progenitor cells and cardiomyocytes [1, 2]. It belongs to the family Parvoviridae and the genus Erythrovirus. B19V infection is prevalent worldwide, with the epidemic peaks from late winter through early summer. A primary infection is common in young children, and other susceptible groups include immunocompromised people, organ transplantation patients and women at childbearing age, especially during pregnancy.

B19V mainly spreads through the respiratory droplets and blood, and can also be transmitted vertically from the mother to the fetus [3]. Although most fetuses are asymptomatic or mildly symptomatic with B19V intrauterine infection, it has been confirmed to increase the risk of adverse perinatal outcomes compared with uninfected women, such as spontaneous abortion, fetal death and stillbirth caused by severe infections [4]. Since pregnant women without antibodies are generally susceptible, and the vaccine for B19V is not yet available, strengthening health education and avoiding exposure to the high-risk population are the major ways to prevent infection. Meanwhile, early diagnosis of B19V intrauterine infection and timely intrauterine treatment are particularly important to avoid adverse perinatal outcomes. As the currently recommended diagnostic methods for B19V infections during pregnancy, maternal serological testing and polymerase chain reaction (PCR) assay facilitate the diagnosis of infection, yet both methods still exhibit inherent limitations.

In this paper, based on a suspected case of B19V intrauterine infection in a Chinese pregnant woman, we described the method of Metagenomic next-generation sequencing (mNGS) performed on the amniotic cells, by which it is the first time to identify B19V genome in a real context of severe fetal anemia and hydrops. And meanwhile, our overall monitoring and management including timely intrauterine transfusion therapy obtained success and a good pregnancy outcome.

Materials and methods

Case histories

A 29-year-old woman, with her first pregnancy, was referred to our fetal medicine center at the 12th week of gestation because of the positive result for serological determination of anti-B19V IgM (19.0) and IgG (1.9) in maternal serum (cut-off: 1.0). No symptoms were noted for her, and the medical history was absent except for the cholecystectomy for gallbladder stones. The rest of the routine laboratory investigations were all within the normal range except for maternal vaginal inflammation. Ultrasound examination of the fetus was normal with the nuchal translucency (NT) 1.7 mm.

Four weeks later, serum samples were obtained for further serological detection. Both IgM (12.0) and IgG (35.0) antibodies remained positive with the increasing titer of IgG from 1.9 to 35.0. In addition, several ultrasonographic soft markers in fetus were found at ultrasound screening, including bilateral choroid plexus cysts, hyperechogenic bowel (less than Grade 2, 20 × 17 × 13 mm) and both kidneys appeared hyperechoic in normal size. Doppler of middle cerebral artery peak systolic velocity (MCA-PSV) valued 58.9 cm/sec, representing 2.77 multiples of the median (MoM). The pregnant woman remained asymptomatic with a low-risk genetic screening result for non-invasive prenatal testing (NIPT).

Subsequently, Doppler ultrasound was carried out once a week, during which fetal hydrops was observed and getting worse with increased nuchal fold (NF), subcutaneous edema of head, pericardial effusion and fetal ascites, although amniotic fluid and placenta both appeared normal (Fig. 1A-E). No pathological malformation or cardiac disease was observed. Serological tests for other possible fetal infections, including cytomegalovirus, rubella, toxoplasmosis, syphilis and herpes simplex were all negative. The MCA-PSV at the 18th and 19th weeks were 50.6 cm/sec and 50.0 cm/sec (Fig. 1F), respectively. Based on all the facts, it was highly indicative of a recent fetal infection for B19V, and moderate to severe fetal anemia was suggested for the presence of the MCA-PSV greater than 1.5 MoM.

Fig. 1.

Main ultrasonographic findings of the fetus at the 18 and 19 weeks of gestation. A Increased nuchal fold. B Pericardial effusion. C Subcutaneous edema of head. D and E Fetal ascites. F Doppler measurement of the peak systolic velocity of the middle cerebral artery at 19 weeks of gestation

According to the less encouraging condition of the fetus and the gestational age, intrauterine fetal transfusion was indicated. The parents accepted the management after detailed genetic counseling and multidisciplinary evaluation. Intrauterine transfusion (IUT) was performed at 19 weeks and 2 days of gestation without any complications. Effective treatment was observed the next day with the recovery of MCA-PSV (27.8 cm/sec), representing 1.12 MoM (Fig. 2A). Fetal movement was normal, and the mother subsequently discharged 2 days after the operation without any complications.

Fig. 2.

Ultrasound image of the fetus after intrauterine transfusion. A The peak systolic velocity of the middle cerebral artery recovered to 27.8 cm/sec the next day. B No significant fetal ascites was observed on the 5th day after IUT. C Normal MCA-PSV on the 5th day after IUT

B19V IgM and IgG serology

Maternal and neonatal serum samples were collected for routine B19V serological testing using chemiluminescence immunoassay (CLIA) tests to capture antibodies. The detection tests were performed with LIAISON ^® Biotrion Parvovirus B19 IgG and LIAISON ^® Biotrion Parvovirus B19 IgM kits (DiaSorin Italia S.P.A., Saluggia (VC), Italy).

IUT and sample collection

Following the Society for Maternal-Fetal Medicine (SMFM) clinical guideline for the management of fetal severe anemia [5], IUT by ultrasound-monitored transabdominal cordocentesis was performed after counseling the parents. During the procedure, 10 ml of irradiated, leukocyte-free, type O Rh (D) negative fresh blood was transfused slowly, which was watched flowing through umbilical vein. After the transfusion was completed, fetal heart rate was normal (150 bpm) with regular heart rhythm.

At the same time, ultrasound-guided transabdominal amniocentesis was done at 19 weeks and 2 days of gestation. About 35 ml of amniotic fluid was aspirated and subsequently prepared for genetic disease and intrauterine infection detection. Remaining amniotic fluid was stored at -80 °C immediately.

Viral mNGS and bioinformatic analysis

1.5 ml of the original sample was added into a 2 mL microcentrifuge tube and centrifuged at 13,000 g for 5 min for cell collection. The remaining sample (about 450 µL) was fully vortexed after removal of supernatant. Then transfer 450 µL of the sample to a new 1.5 mL microcentrifuge tube and add 11.5 µL of saponin, 65 µL of digestion buffer, 10 µL of nuclease to the tube. The sample was vortexed thoroughly for 15 s and then incubated at 37℃ for 10 min. Add 3 µL of dithiothreitol (DTT) to the tube, followed by shaking mix, and then incubate at 37℃ for another 10 min to stop the reaction. After the incubation, the sample was centrifuged briefly, then was transferred to a 2mL custom lysis tube containing 250µL glass beads. The tube was attached to the instrument of FastPrep-24™ 5G grinder for cell disruption and sample lysis, with the reaction condition setting at 45 s for 2 cycles, with a 2-minute interval. Then the sample was centrifuged at 2000 rpm for 20 s and DNA was extracted using the VAMNE Magnetic Pathogen DNA/RNA Kit (RM601-01, Vazyme, Nanjing, China) according to the manufacturer’s recommendation.

DNA libraries were then constructed following the steps DNA-fragmentation, end-repair and A-tailing, adapter ligation, and subsequently PCR amplification. Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used for quality control to ensure the fragments of DNA libraries were approximately 300 base pairs (bp) in size. And the concentration of DNA library was measured using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific Inc.). Quality qualified libraries were pooled and converted into single-stranded circular structure. Through rolling-circle amplification (RCA), DNA Nanoball (DNB) was generated from the circularized DNA. The prepared DNBs were loaded onto the sequencing chip and sequenced using the MGISEQ-2000 platform.

High-quality sequencing data were generated by removing low-quality reads, followed by exclusion of human sequences mapped to the human reference genome (hg19) using Burrows-Wheeler Alignment (BWA) (http://bio-bwa.sourceforge.net/). The remaining sequences were further processed to remove low-complexity reads to eliminate potential noise and enhance the signal from pathogen-derived sequences. The cleaned data were then aligned against the Pathogens Metagenomics Database (PMDB, v7.0 [6]), consisting of bacteria, fungi, viruses and parasites, to identify sequences that match known pathogens. PMDB is a commercial pathogen genome catalog developed and owned by BGI-Shenzhen, China. The microbial classification reference genomes were downloaded from National Center for Biotechnology Information (NCBI) database (ftp://ftp.ncbi.nlm.nih.gov/genomes/).

Results

The results of chromosome analysis and chromosomal microarray analysis (CMA) for amniotic fluid were both normal. Thanks to the early check for specific IgM and IgG antibodies that showing positive in IgM, the pregnancy was well arranged for the regular follow-up and the serial monitoring by Doppler ultrasound to evaluate fetal complications. With our timely diagnosis and effective IUT procedure, the fetus recovered gradually. In the following days, continuous outpatient visits and routine Doppler scans were recommended to assess the fetal condition. Ascites disappeared 5 days later, and complete recovery reached at 22 weeks (Fig. 2B and C). Throughout the rest of the pregnancy, no abnormal sign was observed in her prenatal care except for several soft sonographic markers. Fetal magnetic resonance imaging (MRI) was conducted twice (22 weeks and 32 weeks) both with negative changes. The clinical and ultrasound parameters found in the fetus were presented in Table 1.

Table 1.

The relevant clinical and ultrasound parameters of the fetus from the 12 weeks of gestation to the delivery

Weeks of gestation	Maternal serology	Ultrasound image	MCA-PSV (cm/sec), MOM	MVP / AFI (mm)	EFW (g)
12 weeks	B19V IgM (+): 19.0 B19V IgG (+): 1.9	N	-	31	-
16 weeks	B19V IgM (+): 12.0 B19V IgG (+): 35.0	Choroid plexus cysts Hyperechogenic bowel Kidneys appeared hyperechoic	58.9, 2.77	41	-
18 weeks	-	Subcutaneous edema of head (4.9 mm) Pericardial effusion (2.9 mm) Ascites (12 mm) CTR 0.37	50.6, 2.15	41	288
19 weeks	-	increased NF (6.8 mm) Pericardial effusion (4.8 mm) Ascites (17 mm) CTR 0.36	50.0, 2.06	51	307
19+ 2 weeks	IUT treatment
19+ 3 weeks	-	Pericardial effusion (4.4 mm) Ascites (15 mm) CTR 0.38	27.8, 1.12	44	350
20 weeks	-	Pericardial effusion (3.1 mm) CTR 0.36	29.1, 1.14	61	342
22 weeks	-	Hyperechogenic bowel	-	38	475
24 weeks	-	Hyperechogenic bowel	30.4, 0.99	47	655
28 weeks	-	N	41.7, 1.12	53	1167
32 weeks	-	N	-	117	1924
36 weeks	-	N	56.5, 1.05	124	2850
38 weeks	-	N	66.7, 1.13	114	3158
39 weeks	-	N	72.7, 1.17	140	3528
39+ 4 weeks	Cesarean delivery

IUT intrauterine transfusion, NF nuchal fold, CTR cardio-thoracic ratio, MCA-PSV middle cerebral artery peak systolic velocity, MVP maximum vertical pocket, AFI amniotic fluid index, EFW estimating fetal weight, N Normal, - not detected

Cesarean section was performed at 39 weeks and 4 days on account of fetal distress. A female baby was born with 3370 g and 50 cm in length, and Apgar scores is 9/10. Laboratory tests of the newborn revealed no obvious anemia for hemoglobin (Hb) 16.1 g/dl and hematocrit (HCT) 47.9%. IgG against B19V was positive (higher than 46.0) and IgM was negative (lower than 0.1) in serologic test. Over our follow-up of 24 months, the baby was normal without any neurodevelopmental abnormalities.

The total DNA of amniotic cells was extracted and used for mNGS analysis. The estimated sequencing depth was 127×, and 115,226,912 reads were generated totally. After removing human sequences (about 95% of sequencing data mapped to the human genome), 14,264 sequences showed similarity with B19V sequences. The relative abundance of the virus was 97.29%. The remaining cleaned reads were assembled by SPAdes (v3.15.5) software to generate scaffolds of B19V genome [7]. Then genome map of the virus isolated from the amniotic fluid of patient (Fig. 3) was drawn with CGView (http://cgview.ca) and Proksee (https://proksee.ca). The coverage density plot in B19V genome for NGS sequence of the sample was also analyzed showing in Fig. 4.

Fig. 3.

Genome map of human parvovirus B19 virus. The outermost circle represents the genome sequence position coordinates. Circles from outside to center are coding gene (CDS, including NS1, VP1 and VP2), genome GC content (the inward and outward part indicate that the higher or lower GC content of the region, compared to the average GC content of the whole genome), and genomic GC skew value (the inward purple part demonstrates that GC content of G in the region is lower than that of C, the outward green part indicates that GC content of G in the region is higher than that of C ). Three coding sequences of the virus including NS1 (non-structural protein), VP1 and VP2 were predicted with Prokka and NCBI database

Fig. 4.

Pathogen coverage of sequencing data in NGS platforms.

The coverage rate in B19V genome reached 99.9%

Discussion

A successful management for B19V intrauterine infection characterized by severe fetal anemia and non-immune hydrops was achieved in our research. Meanwhile, to the best of our knowledge, this is the first clinical practice in the detection of B19V genome in amniotic fluid employing the approach of mNGS.

Usually, B19V infection is mild and self-limited, and even many infections are asymptomatic. Whereas in some cases, the virus can lead to a variety of clinical manifestations. The possible symptoms associated with B19V infection include childhood infectious erythema, acute aplastic crisis, chronic anemia, cytopenia, thrombocytopenia, arthropathy and inflammatory cardiomyopathies [3, 8–10]. When During pregnancy, B19V can infect fetus transplacentally from the pregnant mother. It was reported that B19V could infect placental endothelial cells causing structural and functional damage, which played a critical role in virus spreading from the mother to fetus [11]. Compared to the last months of pregnancy, the virus is more likely to trigger greater complications in the first and second trimesters, manifested as severe fetal anemia, non-immune hydrops, myocarditis, high output heart failure, disorders of physical and neurological development, and even intrauterine death [10, 12]. The patient associated with severe fetal anemia and hydrops in our case also acquired the infection before the 12th week. After the discovery of positive IgM and IgG antibodies, MCA-PSV was first observed significantly high at the 16 weeks, followed by extensive fetal hydrops over the next two weeks, while the pregnant woman remaining asymptomatic from the beginning to the end.

The reasons for that mainly lie in the extensive expression of P antigen in placental trophoblast and different fetal tissues. As the major cell surface receptor for B19V infection, P antigen is permissive for virus transmission into the fetal circulations across the placenta, whereas the expression of it in the placenta declines significantly within the third trimester [13, 14]. On the other hand, the fetal liver full of hematopoietic cells is the mainly hematopoietic organ from the 6th to 8th weeks of the embryo till about 24 weeks of gestation. During this period, fetal blood cells are produced and renewed actively with a relatively short lifespan compared to the later bone marrow stage of hematopoiesis [10, 15]. Once B19V is attracted to the liver hematopoietic cells, fetal red blood cell production will be disturbed and suppressed, leading to severe fetal anemia and non-immune hydrops [14, 16]. Besides, P antigen receptor has been also demonstrated existing in fetal myocardial cells, erythroblasts, megakaryocytes, platelets, endothelial cells and fibroblasts, explaining the other clinical features of the fetus [17]. Thus, the fetus is more vulnerable to B19V infection during the first half of pregnancy.

According to previous literatures, B19V infection can be asymptomatic in almost 30–50% of pregnant women, and transplacental transmission will occur in 33–51% among them, following by fetal demise in about 10% of affected fetuses [18–20]. The mortality was higher in those presenting both severe fetal anemia and hydrops. What causes anemia and hydrops occurring simultaneously are possible including high-output heart failure caused by severe anemia, fetal viral myocarditis and increased capillary leakage due to the endothelial damage [21]. A meta-analysis conducted by Bascietto et al. [22] showed that, in fetuses affected by B19V infection, spontaneous resolution of the infection was observed in about 50% of cases without hydrops, while the rate was only about 5% in the presence of fetal hydrops. Consistent with several other research, the risk of abnormal fetal brain imaging and neurodevelopmental complications in survivors with severe fetal hydrops was also indicated higher compared with that in the non-hydropic cases [16, 20, 22]. That means hydrops may be the main predictor of perinatal death and adverse outcome in B19V-infected fetuses.

In view of the inconspicuously infections of B19V in pregnant women and the increased susceptibility of fetuses during the early stages of pregnancy, more attention should be paid for B19V infection during pregnancy. Asymptomatic infections make it difficult for the timely detection until any change in fetal ultrasonic imaging. At present, serologic screening for B19V antibodies is the most essential and effective means of laboratory diagnosis for early infection. Whereas in most cases without symptoms of infection or abnormal ultrasound findings, it may not be recommended in routine prenatal care except for the pregnant women with suspicion of recent exposure to the virus [23]. For example, contacting with an infected individual, or with school-age children at home or workplace.

In our case, the pregnant woman was not working in educational institutions, so there was no remarkable epidemiological risk factor along with her as it was her first pregnancy. Nonetheless, the positive result for both B19V IgM and IgG of the initial test reminded us to initiate the fetal ultrasound surveillance. Combined with the result of serologic testing repeated after 4 weeks, the raised peak velocity in the MCA and progressive abnormal ultrasonic changes, an acute fetal infection was confirmed. The development of fetal hydrops and the persistent MCA-PSV value above 1.5 MoM were suggestive of severe fetal anemia. So, we immediately performed IUT treatment prior to 20 weeks’ gestation, which was recognized as the most effective treatment for severe intrauterine anemia and was reported to improve fetal survival rates from 55% to 82% [5, 24]. Usually, intraperitoneal transfusion is suggested to be a safer approach for early gestation (less than 20 weeks), given the circumstance of the technical difficulty of successful access to fetal circulation by percutaneous puncture and the relatively higher risks at this gestational age [25]. In our study, we carried out an intravascular transfusion for the fetus with blood volume 10 ml on the condition of unknown fetal HCT. Fortunately, satisfactory curative effect was obtained after only one transfusion, and the fetus had a favorable outcome.

For some other cases described in literature, adverse pregnancy outcomes associated with B19V infection were reported. Bertoldi A et al. [26] described a pregnant patient without any appreciable signs and symptoms. An acute B19V infection and fetal hydrops were confirmed unexpected until the patient consulted for inappropriate contractions and fluid loss at 22 weeks of gestation. The fetal Hb was 3 g/dl and intrauterine blood transfusion was performed instantly. But unfortunately, the fetus did not survive during the second transfusion. Kielaite D et al. [3] presented a case of severe congenital B19V fetal infection with fetal subdural hematoma and ventriculomegaly. The pregnant woman experienced a sudden change in fetal movement at 27 weeks of gestation and pathological changes were observed in the fetal brain by ultrasound examination and MRI imaging. A planned cesarean section was performed at 31 weeks of gestation. The premature infant was born with hydrocephalus, severe progressive encephalopathy and incurable complications related to congenital B19V infection. By contrast, the favorable outcome of our patient largely benefitted from our routine screening at the beginning of gestation. All the above highlight the significance of the early detection and diagnosis of B19V infection, for which will be helpful to manage the further fetal monitor and prevent the risk of serious complications and poor outcomes.

For the maternal B19V infection, serologic testing and viral nucleic acid detection in blood serum are the two basic methods used to assess the status of infection. The seroconversion of B19V antibodies is thought to be the most sensitive and reliable marker for recent infection. However, it also has limitations such as the confusingly persistent positive, the false-negative result due to the detectable window after exposure. B19V IgM antibody was reported appearing early toward the end of the first week of infection, whereas possibly being undetectable in maternal serum when the fetal symptoms appeared [27, 28]. The sensitivity and specificity of B19V DNA assay in maternal blood are high for identifying acute infection, but coinciding with IgM antibody, peak viremia occurs early and persists short, and most ceased prior to the onset of symptoms and even before the positivity of IgM and IgG [16, 29]. So, timely detection in asymptomatic patients is therefore challenging.

For the diagnosis of fetal infection, PCR for B19V performed on amniotic fluid sample is preferred for the moment, with the sensitivity greater than 97% and a specificity of 79–99%. Fetal cord blood sample is another choice for B19V PCR test but without widely used because of the higher risk of associated complications than amniotic fluid sampling [30, 31]. But the limitation of PCR should be noted, and the viral DNA may only be detected during the viremic stage [29, 32]. In this scenario, an optimal diagnostic approach may be required. In our research, viral metagenomic based on NGS was performed on the amniotic cells and by which fragments of B19V genome were identified. A 4,560 bp-contig was obtained and three coding sequences of the virus including NS1 (non-structural protein, 2,016 bp), VP1 (2,346 bp) and VP2 (1,665 bp) were predicted with Prokka and NCBI database [33], which further confirmed the diagnosis of fetal infection in our patient. The sequence reads covered 99.9% of the virus nucleotide sequences. In the analyses, none of other pathogen sequence was identified in the sample, including bacteria, fungi, parasites, chlamydia, mycoplasma, rickettsia and other viruses.

The feasibility of direct pathogens identification and even antimicrobial resistance (AMR) detection in different clinical samples of infectious diseases by NGS platforms has been demonstrated in several previous literatures [34–36], including peripheral blood, skin and soft tissue, respiratory sample, cerebrospinal fluid, vitreous humor and some intraoperative resected samples. Whereas the application in amniotic fluid of pregnant women was rarely reported. The research of comprehensive human amniotic fluid metagenomics from Patel MS et al. [37] suggested that amniotic fluid was sterile in mid-gestation, supporting the sterile womb hypothesis. Another prospective study from China found that most amniotic fluid exhibited low microbial diversity and abundance [38]. Calvet G et al. [39] was the first to detect the whole genome of Zika virus directly from the amniotic fluid of pregnant women with microcephalic fetuses. The viral metagenomics analysis performed on 10 samples of amniotic fluid that had been found positive by qPCR also showed a good efficiency of this approach, with almost 100% agreement with qPCR results [40].

Compared with conventional serology detection, mNGS has the advantages of high accuracy rate, abbreviated time consumption and high sensitivity in pathogens identification, especially for the capacity to detect rare and previously unknown pathogens, which does not apply to qPCR. Although qPCR in amniotic fluid is currently the primary diagnostic tool for B19V fetal infection, yet other less common or rare pathogens associated with fetal abnormalities may be missed by the application of PCR assays. For the ability to detect multiple microorganisms simultaneously and the decided advantage in the detection of unexpected pathogens, mNGS may be used as a supplemental or exploratory tool for clinical diagnosis for the pregnant women that are suspected of intrauterine infection. Based on detection results, the patient may have an opportunity to receive timely intervention and even pre-symptomatic treatment, for which of great significance to improve short or long-term outcomes.

Yet, it is worth noting that our study is not without limitations. Only a single case from a single-center was assessed in this study, and B19V-specific qPCR in amniotic fluid was not performed in our study. Thus, it is impossible to conduct a controlled study comparing the two approaches for qPCR and mNGS on the diagnosis of intrauterine infections. Of course, such a comparison requires a larger sample size in the future. Additionally, RNA virus in amniotic fluid could not be completely excluded for the fact that it was not sequenced in the study. Currently, the higher cost and more advanced bioinformatics requirment of mNGS were also limitations in its clinical application.

Conclusion

In conclusion, management and timely intervention of intrauterine infectious diseases relies on the accurate identification and early diagnosis of pathogens. Since there is no specific antiviral treatment for B19V infection, and no reliable way to predict whether fetuses will develop life-threatening outcomes, early detection and diagnosis of B19V infection in fetuses are significantly important, for which will contribute to effective surveillance and timely intervention in pregnancy. For the woman of childbearing age, especially whose immune status for B19V is unknown, serological tests for IgM and IgG are recommended before pregnancy or in early maternal serum samples. Meanwhile, our results also provide insight into the application of mNGS in rapid diagnosis of intrauterine infections with amniotic fluid samples. Of course, further studies and more data will be needed in the future to explore the recognized interpretation criteria, and to evaluate the clinical value of mNGS in the identification of etiological agents for intrauterine infections.

Abbreviations

B19V

Human parvovirus B19

IUT

Intrauterine transfusion

mNGS

Metagenomic next-generation sequencing

NT

Nuchal translucency

MCA-PSV

Middle cerebral artery peak systolic velocity

MoM

Multiples of the median

NIPT

Non-invasive prenatal testing

NF

Nuchal fold

SMFM

Society for Maternal-Fetal Medicine

RCA

Rolling-circle amplification

CMA

Chromosomal microarray analysis

MRI

Magnetic resonance imaging

PCR

Polymerase chain reaction

AMR

Antimicrobial resistance

Authors’ contributions

RY M drafted the manuscript. RY M, RY H and YL W conceptualized and designed the study. JL S and J Z performed ultrasound scans and provided imaging data. Y W, YR S, YF Y and S W were responsible for the comprehensive clinical management, monitoring and surgical care of the patient. X H and SY L assisted in the testing of the samples and performed the data analysis for the laboratory testing. L G and XR Z developed the consent forms for the study, ensured compliance with ethical standards and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.

Funding

This work was supported by the Key Research and Development Program of the Ministry of Science and Technology (grant number 2023YFC2705901); Shanghai Municipal Science and Technology Commission (grant numbers 22Y11902300, 23DZ2300300); Shanghai Municipal Health Commission (grant number 202440131); Shanghai Jiao Tong University STAR Grant (grant numbers YG2023ZD26); and Medical Science and Technology Talent Promotion Project of The International Peace Maternity and Child Health Hospital (grant number HHJH2412).

Data availability

The datasets generated during the current study are available in the Genome Sequence Archive repository (Genomics, Proteomics & Bioinformatics 2025) in National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA032232) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

Declarations

Ethics approval and consent to participate

This study was approved by the Institutional Review Board of The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University (9th February, 2025, IRB number: GKLW-A-2025-017-01). Written informed consent was obtained from the patient. The study followed the ethics standards recommended by the Declaration of Helsinki.

Consent for publication

We provide consent for the publication of the manuscript detailed above.

Competing interests

The authors declare no competing interests.