Case reportDetection of enteroviral RNA on Guthrie card dried blood of a neonate with fatal Coxsackie B3 myocarditis on day 17
Introduction
Enterovirus infection is a common cause of acute viral illness worldwide. In the perinatal period enterovirus infections may vary in clinical presentation from mild non-specific symptoms to severe multisystem disease, including meningoencephalitis, myocarditis, hepatitis and serious generalized sepsis (Abzug, 2001, Bauer et al., 2002, Bendig et al., 2003, Bryant et al., 2004, Sawyer, 1999). Perinatal enterovirus infection can be associated with high morbidity and mortality. Peak incidence is during summer and autumn. Coxsackie virus B3 (CVB3) has been identified as a major causative agent of acute and chronic myocarditis (Jeonghyun et al., 2005).
Most frequently neonates are infected directly from mother to child at delivery. This may result in more serious disease (Bendig et al., 2003, Verboon-Maciolek et al., 2003). The infection can also be acquired postnatally from sources other than the mother. When neonates are symptomatic at birth, in utero transmission is suggested (Bendig et al., 2003). The incubation period is 3–5 days (Bauer et al., 2002).
Diagnosis can be made by serological testing and viral cultures. However, the presence of passively acquired maternal antibodies, the need for specific assays and the delay in the diagnosis render these methods not very useful. Reverse-transcriptase-polymerase chain reaction (RT-PCR) increases the sensitivity and speed of enterovirus diagnosis, and is therefore the method of choice (Bendig et al., 2003, Bryant et al., 2004, Sawyer, 1999).
We describe a neonate with disseminated intravascular coagulopathy and severe cardiomyopathy on day 17 after birth with fatal outcome due to CVB3 infection. In an attempt to determine the time of transmission, RT-PCR on a Guthrie card dried blood spot, collected on day 4 after birth, was used to detect enteroviral RNA. To our knowledge this is the first report on detection of enteroviral RNA in dried blood spots using the Guthrie card.
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Case report
A neonate, born at 39 weeks gestation, was admitted shortly after delivery to a neonatal department because of persistent peripheral cyanosis and cold hands and feet. After a few hours in a warm environment the boy recovered quickly. On day 3 after birth he regained weight. On day 4 he was discharged from the hospital in good health.
On day 17 after birth the child presented in the emergency room with grunting, poor feeding, hypothermia and cardiovascular collaps. While being prepared for
Real-time RT-PCR for the detection of enterovirus RNA
RNA was extracted from NaF-containing and citrate-containing plasma by using the QIAamp® Viral RNA Kit (Qiagen Benelux B.V., The Netherlands). Both samples were obtained while the patient was alive in the neonatal intensive care unit. RNA extraction from paraffin sections of myocardium and frozen myocardial tissue was performed using Rneasy Mini Kit (Qiagen Benelux B.V., The Netherlands). RNA from the Guthrie card was extracted using Rneasy Mini kit after Proteinase K digest (200 μl PBS buffer
Real-time RT-PCR for the detection of enterovirus RNA
Both paraffin section of myocardium and frozen myocardial tissue contained enteroviral RNA. Enteroviral RNA was also detected in Guthrie card dried blood and NaF-containing plasma samples (citrated plasma tested negative).
Diagnostic 5′NCR RT-PCR and amplification of the amino-terminal part of the VP1 segment of enteroviral RNA from plasma, frozen heart tissue, and Guthrie card dried blood
Fragments of 231 bp were seen on the polyacrylamide gel of the 5′NCR RT-PCR for NaF-containing and citrated plasma and frozen myocardial tissue. The degenerative VP1 primers ENTNESS-F/R successfully amplified enterovirus RNA from these samples. The nucleotide sequence of the
Discussion
Enteroviruses cause a wide spectrum of severe manifestations in neonates, including severe, fulminant, sometimes fatal disease with myocarditis. Coxsackie B viruses often infect the heart and in particular CVB3 causes irreversible damage to the cardiomyocytes and is considered a major cause of myocarditis, thereby causing irreversible damage to the cardiomyocytes (Arbustini et al., 2000, Bauer et al., 2002, Dettmeyer et al., 2002, Jeonghyun et al., 2005, Sawyer, 1999).
The evolution of a severe
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