Published online August 14, 2006
PEDIATRICS Vol. 118 No. 3 September 2006, pp. e929-e933 (doi:10.1542/peds.2006-0554)

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EXPERIENCE & REASON

Probable Intrafamilial Transmission of Coxsackievirus B3 With Vertical Transmission, Severe Early-Onset Neonatal Hepatitis, and Prolonged Viral RNA Shedding

Ling Ling Cheng, MRCPCH^a, Pak Cheung Ng, MD, FRCP (London, Edinburgh)^a, Paul Kay-Sheung Chan, MD, FRCPath, FHKCPath, FHKAM^b, Hiu Lei Wong, MBChB, MRCP (Ireland)^a, Frankie Wai Tsoi Cheng, MRCPCH^a and Julian Wei-Tze Tang, PhD, MRCP, MRCPath, FHKCPath^b

^a Pediatrics
^b Microbiology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, China

ABSTRACT

Here we report a familial cluster of 3 cases of coxsackievirus B3 infection: a recent history of illness in a woman's 3-year-old son with a coxsackievirus B3–positive stool culture indicated that he probably infected his mother at home during her last week of pregnancy. Consequently, she delivered an infected neonate who developed severe hepatitis, disseminated intravascular coagulation, and bilateral intracranial hemorrhage. The neonate remained well for the first 2 days of life. On the third day, he developed fever (39°C) and poor peripheral circulation. On the fourth day, he developed petechiae and bruises over his chest wall and extremities, and prolonged bleeding was observed over venipuncture sites. Investigations revealed severe thrombocytopenia (platelets: 41 x 10⁹/L) and a markedly deranged coagulation profile (prothrombin time: 19 seconds [reference: <10 seconds]; activated partial thromboplastin time: >120 seconds [reference: 24.2–37.0 seconds], serum D-dimers: 6722 ng/mL [reference: <500 ng/mL]), suggestive of disseminated intravascular coagulopathy. Clinical examination revealed yellow sclera, hepatomegaly (5 cm), and splenomegaly (2 cm), consistent with hepatitis. Serial chest radiographs showed bilateral pleural effusions, and an ultrasound of the abdomen demonstrated ascites. An echocardiogram showed normal cardiac structure and good contractility of both ventricles. However, a cranial ultrasound revealed bilateral grade 2 intraventricular hemorrhages. Serum C-reactive protein increased to 33.9 mg/L. Liver-function tests were also markedly deranged at this time, with maximum values for serum alanine transferase, bilirubin, alkaline phosphatase, and ammonia concentration of 1354 IU/L, 258 µmol/L, 189 IU/L, and 147 µmol/L, respectively. Serum glucose levels were normal. Over the next 3 days, his fever subsided, and his liver function and clotting profile normalized by day 13 after onset of illness. A stool sample from the older brother, collected 14 days after his onset of illness at home, was positive for coxsackievirus B3 by both virus culture and enterovirus reverse-transcription polymerase chain reaction. He had neutralizing coxsackievirus B3 antibody titers of 1:2560 and 1:1280 on days 14 and 28 after his onset of illness, respectively. No virus was cultured from the mother's stool sample, collected 5 days after her onset of illness, but the enterovirus polymerase chain reaction was positive and maternal sera neutralized the coxsackievirus B3 isolated from the neonate. The maternal sera also showed a more than fourfold rise in antibody titer from 1:80 to 1:640 on days 5 and 16 after her onset of illness, respectively. Neonatal antibody titers also showed a more than fourfold rise from <1:80 to 1:2560 on days 1 and 21 after his onset of illness, respectively. This demonstrates that both the mother and the neonate had had recent coxsackievirus B3 infections. Serially collected neonatal throat swab and stool samples were culture negative for enterovirus by 4 and 8 days after his onset of illness, respectively. However, enterovirus RNA remained detectable by reverse-transcription polymerase chain reaction in these samples for considerably longer, only becoming undetectable by 16, 23, and 41 days after his onset of illness. We show that even mild household infections may have potentially serious consequences for pregnant women and their infants.

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Key Words: enterovirus • coxsackievirus B3 • neonate • hepatitis • familial transmission • viral shedding

Abbreviations: RT, reverse transcription • PCR, polymerase chain reaction

Enterovirus infection is common in older children and adults but rarely causes severe disease.¹ In contrast, perinatally acquired enterovirus infection can be devastating, especially when associated with concomitant fulminant hepatitis, myocarditis, and meningoencephalitis, for which the case fatality rate can be as high as 42%.^2,3 Severe neonatal hepatitis, with necrosis and failure, may arise with infection by specific members of the enterovirus family, specifically the echovirus and coxsackieviruses A and B.³ The resulting coagulopathy may present as thrombocytopenia, prolonged clotting times, and increased fibrin degradation products with hemorrhagic complications, including intracranial hemorrhage.³

Here we present a case of coxsackievirus B3 infection and shedding in a 3-day-old newborn who presented with high, variable fever, severe hepatitis, and bleeding diathesis. Additional investigation revealed that the mother and older brother had also been recently infected with the same virus, suggestive of intrafamilial transmission.

CASE REPORTS

A 37-year-old mother developed a high fever (39°C) with chills and rigors 6 hours before delivery. She reported that her 3-year-old elder son had also developed a mild febrile illness and sore throat 5 days earlier but that it subsided within 24 hours without additional intervention or hospitalization. A term male infant was born with a birth weight of 3105 g and Apgar scores of 8 and 9, at 1 and 5 minutes, respectively. He was transferred from the labor ward to the neonatal unit for monitoring because of maternal intrapartum fever. There were no other infection risk factors such as prolonged rupture of membranes, maternal group B Streptococcus carriage, or clinical signs suggestive of chorioamnionitis. The infant was completely asymptomatic during the first 48 hours of life. On admission, the sepsis indices were normal, with a total white cell count of 15 x 10⁹/L, neutrophil count of 7.7 x 10⁹/L, platelet count of 306 x 10⁹/L, and serum C-reactive protein level of <1 mg/L.

On the third day of life, he developed high fever (39°C) and poor peripheral circulation. A full sepsis screen was performed, including blood, stool, nasopharyngeal aspirate, throat swab, urine, and cerebrospinal fluid. Intravenous penicillin and gentamicin were commenced to cover the possibility of perinatally acquired bacterial infection.

On the fourth day of life, he developed petechiae and bruises over his chest wall and extremities, and prolonged bleeding was observed over venipuncture sites. Serial hematologic investigations revealed severe thrombocytopenia (platelets: 41 x 10⁹/L) and a markedly deranged coagulation profile (prothrombin time: 19 seconds [reference: <10 seconds]; activated partial thromboplastin time: >120 seconds [reference: 24.2–37.0 seconds]; serum D-dimers: 6722 ng/mL [reference: <500 ng/mL]), suggestive of disseminated intravascular coagulopathy. Clinical examination revealed yellow sclera, hepatomegaly (5 cm), and splenomegaly (2 cm), consistent with hepatitis. Serial chest radiographs showed bilateral pleural effusions, and an ultrasound of the abdomen demonstrated ascites. An echocardiogram showed normal cardiac structure and good contractility of both ventricles. However, a cranial ultrasound revealed bilateral grade 2 intraventricular hemorrhages. His serum C-reactive protein level increased to 33.9 mg/L. Liver-function tests were also markedly deranged at this time, with maximum values for serum alanine transferase, bilirubin, alkaline phosphatase, and ammonia concentration of 1354 IU/L, 258 µmol/L, 189 IU/L, and 147 µmol/L, respectively. Serum glucose levels were normal. His fever gradually subsided over the next 72 hours, and his liver-function and clotting profile returned to normal by the 13th day after onset of neonatal illness.

VIROLOGICAL INVESTIGATIONS AND RESULTS

An in-house nested reverse-transcription polymerase chain reaction (RT-PCR) targeting the 5'-nontranslated region of enteroviruses was used. The protocol was adopted from a previously published method^4,5 using the following primers: forward outer primer (FOP), 5'-CGG CCC CTG AAT GCG GC-3'; reverse outer primer (ROP), 5'-CAC CGG ATG GCC AAT CCA-3'; forward inner primer (FIP), 5'-CCC CTG AAT GCG GCT AAT-3'; and reverse inner primer (RIP), 5'-ATT GTC ACC ATA AGC AGC CA-3'. Briefly, viral RNA was extracted by using the commercial QIAamp viral RNA-extraction kit (Qiagen Ltd, Crawley, United Kingdom) according to manufacturer instructions. A known enterovirus RNA-positive extract was used as a positive control, and double-distilled water was used as a negative control.

The RT and first-round of PCR were conducted in a 1-step single tube with a total reaction volume of 25 µL containing 10 µL of nucleic acid extract (RNA template), 0.1 µM of each primer FOP and ROP, 1 µL of RT/Taq (Superscript III One-Step RT-PCR System with Platinum Taq DNA Polymerase; Invitrogen Corp, Carlsbad, CA), 0.2 mM of each dNTP, and 1.6 mM MgSO₄. The cycling parameters were: RT at 52°C for 30 minutes; initial denaturation at 94°C for 3 minutes; and 40 cycles of denaturation 94°C for 45 seconds, annealing at 60°C for 45 seconds, and extension at 68°C for 45 seconds; and a final extension step of 68°C for 7 minutes. This yielded a first-round PCR product of 191 base pairs (bp).

Second-round amplification was performed in a total volume of 25 µL containing 2 µL of first-round PCR product (DNA template), 0.1 µM of each primer FIP and RIP, 0.625 U of DNA polymerase (HotStar Taq DNA polymerase system, Qiagen), 0.2 mM of each dNTP, and 1.5 mM MgSO₄. Second-round cycling parameters were an initial "hot start" of 95°C for 15 minutes to activate the Taq; 40 cycles of denaturation 94°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 45 seconds; and a final extension step of 72°C for 7 minutes. The final amplicons were 144 bp long and were detected by using agarose gel electrophoresis.

For viral culture, samples were inoculated onto Vero (African green monkey kidney) cell monolayers, which were observed daily for characteristic enterovirus cytopathic effects. Preliminary identification of an enterovirus in the cell culture was performed by using a pan-enterovirus immunofluorescent stain (Chemicon International Inc, Temecula, CA) followed by specific enterovirus serotyping using a Lim pool typing method. Paired neutralization antibody titers were measured by using microneutralization with the coxsackievirus B3 isolated from the infant.

Follow-up sampling and testing of a stool sample from the older brother, collected 14 days after onset of his illness, at home was also positive for coxsackievirus B3 by both culture and PCR. He had similar antibody titers of 1:2560 and 1:1280 on days 14 and 28 after onset of his illness, respectively. No virus was cultured from the mother's stool sample, collected 5 days after onset of her illness, but the enterovirus PCR was positive and maternal sera neutralized the coxsackievirus B3 isolated from the neonate. In addition, the maternal sera showed a greater than fourfold rise in antibody titer, from 1:80 to 1:640 on days 5 and 16 after onset of her illness, respectively. This confirms that the mother had a coxsackievirus B3 infection. For the neonate, his antibody titers also showed a greater than fourfold rise, from <1:80 to 1:2560, between days 1 and 21 after onset of his illness, respectively (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Coxsackievirus B3–Specific Neutralizing Antibody Results Confirming Recent Coxsackievirus B3 Infection Within This Family

 
The infant was kept in isolation and throat swabs, stool, and plasma samples were collected and tested by RT-PCR and virus culture until they were all negative for enterovirus. Throat swab and stool samples were culture negative for enterovirus by 4 and 8 days after his onset of illness, respectively (Table 2). Enterovirus RNA remained detectable for considerably longer in throat swab, stool, and plasma samples, only becoming undetectable by 16, 23, and 41 days after onset of his illness, respectively, by the enterovirus RT-PCR assay (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Duration of Shedding of Coxsackievirus B3 From the Neonate From Throat Swabs, Stool/Rectal Swabs and Plasma, by Virus Culture and RT-PCR) Detection Methods

 

DISCUSSION

We have reported a case of severe neonatal hepatitis with coagulopathy and intracranial hemorrhage resulting from coxsackievirus B3 infection. The coxsackievirus B3–specific neutralizing antibody titers shown in Table 1 demonstrate seroconversion in the neonate and the mother. These titers also demonstrate the absence of transferred specific maternal antibodies to coxsackievirus B3. The timing of the mother's rise in antibody titers (between days 5 and 16 after onset of her illness) supports the diagnosis of an acute coxsackievirus B3 infection at around the time of delivery. The lack of rising antibody titers in the older sibling, together with his earlier history of illness and coxsackievirus B3 culture-positive stool, make it likely that he was the source of the mother's and, ultimately, the neonate's coxsackievirus B3 infections.

Additional epidemiologic evidence that the older sibling was the likely index case was that he had never visited the hospital and therefore did not have any direct contact with the neonate. This also implied that the mother was likely to have been initially infected by the older sibling at home before her hospital admission. Such transmission of enteroviruses within a household is not surprising and was documented recently by Kuramitsu et al.⁶ These authors also noted that siblings are mainly responsible for the introduction of a virus into a family. However, as far as we know, this is the first detailed, documented case of a household, horizontal, intrafamilial transmission resulting in mother-to-child vertical transmission of an enterovirus infection. Nursery outbreaks of nonpolio enteroviruses have been reported, with attack rates of up to 50%,⁷ and it is likely that household attack rates, especially between mothers and their young children, are higher than this.

During pregnancy, the frequency of transmission of enterovirus from an infected mother to her neonate has been reported to be 30% to 50%, usually via maternal secretions during vaginal delivery or other body fluids.⁷ Approximately 60% to 70% of women bearing infected infants have had a febrile illness during the last week of pregnancy.⁸ Infants who develop illness within the first 2 days of life are likely to have acquired their infection transplacentally and be born to mothers who acquired their infection before delivery.^8,9 Although the neonate described here developed illness on the third day of life, transplacental transmission is also possible. Risk factors for severe neonatal disease include early age of onset, prematurity, maternal illness peripartum, absence of neutralizing antibody to the specific virus type, and multisystem involvement.³

Coxsackievirus B3 is an enterovirus that belongs to the picornavirus family. It is usually transmitted via the orofecal route, and its incubation period is estimated to be 2 to 5 days.^1,9 Gastrointestinal infection resulting from enterovirus is common in children and adults, and infected individuals usually have mild symptoms, with the disease running a self-limiting course. In contrast, perinatally acquired enterovirus infection via transplacental transfer or acquisition from inhalation and/or swallowing of contaminated cervical discharge or fecal material during delivery can be devastating.⁹ Echoviruses and coxsackieviruses, in particular, seem to cause severe neonatal hepatic disease with coagulopathy.^3,10 The high case fatality rate of up to 42% reported in neonates² is likely to be associated with the large viral load transferred with rapid bloodstream dissemination, in the absence of neutralizing maternal antibodies, as well as the immature neonatal macrophage response.⁷ More specifically, coxsackievirus B3 infections have been associated with myocarditis,¹¹ meningitis,¹² and varicella-like skin lesions¹³ as well as multiorgan disease,^14,15 in some cases, via perinatally acquired infection.^12,13,15

In addition, data are scarce on the duration of shedding of nonpolio enteroviruses (and there are none for coxsackievirus B3) in perinatally infected neonates. One study reported that nonpolio enteroviruses were detected by virus culture in throat swabs for 1 to 2 weeks (coxsackievirus A16) and from stool for 1 to 11 weeks (coxsackievirus B2, enterovirus 71, echovirus 4, coxsackievirus A16) after infection.¹⁶ Here we also report the shedding duration of coxsackievirus B3 by virus culture in throat swabs and stool/rectal swabs in the first week after onset of neonatal illness. However, using RT-PCR, the duration of detectable enterovirus RNA in neonatal throat swabs and stool/rectal swabs was 16 and 23 days, respectively. These findings are compatible with a previously reported study.¹⁶ For plasma, enterovirus RNA was detectable for up to 41 days, far exceeding that in the stool. The shedding of detectable viral RNA alone, in the absence of culturable virus, implies that the neonate is no longer infectious. However, the long period of detectable viral RNA shedding in the neonatal stool is surprising. This may be a result of incomplete clearance of viral RNA despite the neutralization of viable virus, perhaps because of high viral RNA loads in the neonatal gut during this primary coxsackievirus B3 infection.

CONCLUSIONS

This family cluster of acute coxsackievirus B3 infections included vertical transmission from a peripartum-infected pregnant mother to her neonate, resulting in severe hepatitis with coagulopathy and bilateral intracranial hemorrhage. The finding of prolonged shedding of coxsackievirus B3 in the neonatal throat swab, stool, and plasma samples, most likely because of an absence of maternally transferred specific antibody, further supports this sequence of events. This case report highlights the potential risk to the neonate when a pregnant woman is exposed to even apparently mild illness by a member of her household.

ACKNOWLEDGMENTS

We thank Felix Wong for technical assistance.

FOOTNOTES

Accepted Apr 13, 2006.

Address correspondence to Julian Wei-Tze Tang, PhD, MRCP, MRCPath, FHKCPath, Department of Microbiology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, China. E-mail: julian.tang{at}cuhk.edu.hk

The authors have indicated they have no financial relationships relevant to this article to disclose.

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PEDIATRICS (ISSN 1098-4275). ©2006 by the American Academy of Pediatrics

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My File Cabinet Download to citation manager Request Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Cheng, L. L. Articles by Tang, J. W.-T. Search for Related Content PubMed PubMed Citation Articles by Cheng, L. L. Articles by Tang, J. W.-T. Related Collections Infectious Disease & Immunity Social Bookmarking                         What's this?