Cytomegalovirus reactivation in a SARS-CoV-2 infected woman experiencing fetal demise in the first trimester with fetal trisomy 21: A case report

Abstract

Cytomegalovirus (CMV) is the most common cause of congenital viral infections. Women seropositive for CMV prior to pregnancy can develop a non-primary CMV infection. Here, we present a case of first trimester pregnancy loss during active SARS-CoV-2 infection. There was no evidence of SARS-CoV-2 RNA in placenta and fetal tissue, but there was presence of congenital cytomegalovirus infection by nested PCR. To the best of our knowledge, this is the first report demonstrating association of early congenital CMV infection due to reactivation and fetal demise in a SARS-CoV-2 positive woman with fetal trisomy 21.

1. Introduction

Among the vertically transmitted viral infections, cytomegalovirus (CMV) is the most common cause of congenital infection and complicates approximately 1–2% of all live births. Latency following a primary CMV infection may be punctuated by periodic reactivations, and in-utero transmission may occur during either primary or post-primary infections [1]. It remains to be established whether SARS- CoV-2 virus is transmitted vertically during pregnancy. Some studies are indicative of a lack of vertical transmission [[2], [3], [4]]. While others indicate vertical transmission of the virus [5,6]. In this study, we report a case with fetal demise was tested Covid-19 positive by RT-PCR. The case showed no placental and fetal transmission of SARS-CoV-2 but showed the evidence of congenital cytomegalovirus infection (CMV) and fetal trisomy 21.

2. Case details

A 26-year-old primigravida, who was apparently healthy at 8.5 weeks developed mild symptoms of COVID-19 (loss of taste and dry cough) at ∼10.5 weeks. RT-PCR performed for SARS-CoV-2 showed positive results. On routine antenatal follow up, USG performed at 13 weeks (as per LMP) suggested a missed abortion at 10.6 weeks. The case was referred to us for further evaluation. Since the patient had persistent dry cough, repeat RT-PCR for COVID-19 testing was performed which confirmed active infection. The products of conception (POC) were processed for nucleic acid extraction. Extracted RNA and DNA samples were subjected to SARS-CoV-2 PCR and nested PCR for CMV (Fig. 1 ) respectively. Amplicons for CMV were also subjected to Sanger sequencing (mtr-II region) [7]. Maternal blood samples were processed for TORCH, SARS-CoV-2 serology and immunological markers. A split sample of fetal tissue was processed for genetic studies by microarray to rule out genetic factor. An additional sample was used for histopathology studies. Results of the investigations carried out in maternal and fetal tissues are listed in Table 1 . There was no evidence of placental and fetal transmissions of SARS-CoV-2 virus. These samples were however found to be positive for CMV DNA by nested PCR. Sanger sequencing of maternal and fetal amplicons showed high homology (>99%) with CMV reference sequences. The microarray (Affymetrix Cytoscan 750K array) revealed presence of trisomy 21.

Fig. 1.

Detection of CMV DNA in maternal and fetal samples. A: Nested PCR (168bp)- Amplified HCMV DNA of 1-mother blood, 2-placenta and of 3-fetus. Ethidium Bromide stained Agarose gel (2%) electrophoresis to detect CMV DNA in maternal blood (lane 1), placental tissue (lane 2), fetal DNA (lane 3), 100 bp ladder (lane 4), blank (lane 5), reaction Control (lane 6), negative control (lane 7), blank (lane 8), positive control (lane 9), positive control (lane 10), B: Primer sequence used for amplification of CMV DNA, C: Nucleotide sequences of CMV amplicons from mother, placenta and fetus tissue and its significant alignments with gene bank.

Table 1.

Investigations performed on maternal blood, nasopharyngeal (NP) swab and products of conception.

Sr No	Maternal investigations	Findings
A
1.	SARS-CoV-2 RT-PCR of Nasopharyngeal swab collected at 10.5 weeks	Positive (E ​= ​23; O ​= ​23)
	SARS-CoV-2 RT-PCR of Nasopharyngeal swab collected at 13 weeks	Positive (E ​= ​35.2; O ​= ​33.59)(performedon COBAS 6800)
2.	COVID-19 Antibody (IgG, CLIA)	Positive (87 BAU/ml).
3.	TORCH (Toxoplasma, ​Rubella, Cytomegalovirus and Herpes simplex virus 1 and 2) serology (ELISA)
	CMV IgM	Negative
	Rubella IgM	Negative
	HSV-1 IgM	Negative
	HSV-2 IgM	Negative
	Toxoplasma IgM	Negative
	CMV IgG	Positive (226.6 AU/ml)
	Rubella IgG	Positive (259 IU/ml)
	HSV-1 IgG	Positive (46.19 AU/ml)
	HSV-2 IgG and Toxoplasma IgG	Non-reactive
	CMV Avidity	High (94.7%)
	Rubella Avidity	High (96.5%)
4.	Immunological markers	Within normal limits
	Antinuclear antibody, Anti-phospholipid (APLA) IgG & IgM and Anti-double strand DNA by ELISA, Cardiolipin (ACL) IgG and IgM testing by fluoro-enzymeimmunoassay
5.	Blood CMV PCR	CMV DNA detected
B	Placenta/POC tissue
1.	SARS-CoV-2 RT-PCR	Negative
2.	CMV nested PCR	CMV DNA detected
3.	Histopathology:	Mild increase in peri-villous fibrin and focal calcification. Occasional villi are degenerated. Suggestive of infection
4.	Genetic test:
	Genomic microarray POC	Trisomy of chromosome 21
	Parental Karyotype	46,XX,9qh+ and 46,XY,9qh+

3. Discussion

Here, we report a case of young primigravida, experiencing first trimester pregnancy loss during active SARS-CoV-2 infection. However, our investigations revealed lack of vertical transmission; instead, we detected CMV DNA in maternal blood, placenta and fetal tissues as well as antibodies against CMV showing high avidity, thereby suggesting transmission of CMV during this period. Moreover, trisomy 21 was detected in fetal tissue. Based on the aforementioned results we were thus able to narrow down the cause of this early pregnancy loss to an infectious etiology and/or trisomy 21. Serological investigations post IUD showed strong maternal IgG positivity and high avidity for CMV and Rubella. Also, an initial PCR screening for TORCH (toxoplasmosis, rubella cytomegalovirus and herpes simplex virus) nucleic acids was negative. However, in light of the CMV infection being the leading cause of congenital infections, a sensitive nested PCR assay was carried out to check presence of CMV DNA [7,8]. Interestingly, CMV DNA was detected in maternal blood, placenta and fetal tissues. Amplicons, upon sequencing were shown to have high homology (<99%) with reference CMV sequences as well as among themselves. These results suggested intra-uterine transmission of CMV. Taken, initially normal USG findings and high avidity of maternal anti-CMV IgG indicated probable reactivation of CMV post SARS-CoV-2 infection [9]. Absence of SARS-CoV-2 RNA in placental and fetal tissues supports reports indicating lack of vertical transmission during early pregnancy [[2], [3], [4]]. Our findings are also congruence with others reports predicting maternal CMV reactivation leading to congenital infection even during early pregnancy [10]. Immunosuppression, required for maintenance of pregnancy, results in increased susceptibility to infection and resulting inflammatory sequelae [11]. SARS-CoV-2 infection, even in mild cases, has been shown to be accompanied by upregulation of systemic inflammatory markers [12]. Considering the role of inflammatory stimuli in CMV reactivation, it is likely that SARS-CoV-2 infection probably resulted in CMV reactivation. However, it is important to note the contribution of other TORCH pathogens to pregnancy losses could not be ruled out completely in the case reported in this study.

4. Conclusion

More studies are required to investigate whether SARS-CoV-2 infection in pregnant women leads to CMV reactivation and thereby contribute to pregnancy losses in some cases. The present study could not delineate the precise cause of pregnancy loss as trisomy 21 was also detected in the abortus of the SARS-CoV-2 infected women. Nonetheless, the study indicates that SARS-CoV-2 infection does not always lead to its vertical transmission. Reactivation of other common viral infections can occur in Covid-19 positive pregnant women. Genetic factors in pregnancy losses should be ruled out in all Covid-19 positive mothers before labeling SARS-CoV-2 virus infection as a cause of fetal demise.

Data availability

The related data is made available in the manuscript.

Code availability

Not applicable.

Consent to participate

Written informed consent was obtained from the patient.

Consent for publication

Written consent was obtained from the patient for publication of the study.

Ethics

The study was approved by the Institutional Ethics Committee of ESI- PGIMSR and Model Hospital, Mumbai, India (Ref.: IEC/Project/I/2021 on 07/06/2021) and IEC of ICMR-NIRRH, Mumbai, India (Ref.: D/ICEC/Sci-165/174/2021 on 23/09/2021). Also written informed consent was taken from the patient.

Authors roles

MG, PG, PS, DB, MS, and AR were involved in, case identification, diagnosis, clinical workup and patient management. SP did the genetic counseling and clinical laboratory investigations and interpreted the data. KM, SP, VB, VP, SB and KS were involved in SARS-CoV-2 RT-PCR. SP, SM, HG, SM and DD performed genetic testing. VP, VB, HP were involved in CMV serology and nested PCR. SP wrote the first draft.GS, KM, VP, and VB edited the manuscript. All the authors approved the final version of the manuscript.

Funding agency

The authors are thankful to ICMR-National Institute for Research in Reproductive Health (ICMR-NIRRH) for financial support

Conflict of interest statement

All authors hereby declares that there is no any financial and personal relationships with other people or organizations that could inappropriately influence their work. There is no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: