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Etiology of DNA Virus Infections in Liver Transplant Recipients With Neonatal Hepatitis
Etiology of DNA Virus Infections in Liver Transplant Recipients With Neonatal Hepatitis R. Yaghobi, S. Zamani, B. Gramizadeh, and M. Rahsaz ABSTRACT Neonatal hepatitis is a syndrome of symptoms associated with a history that includes any type of infectious, genetic, toxic, or metabolic causation. Various infectious agents have been implicated in hepatic inflammation in neonates including bacterial and viral pathogens, especially DNA viruses. We used molecular and antigenic methods to evaluate the role of DNA viruses, such as hepatitis type B viruses (HBV), human cytomegalovirus (HCMV), herpes simplex virus (HSV), and adenovirus, in neonatal hepatitis complications. Twenty-six paraffin-embedded biopsy and autopsy tissues obtained between 1996 and 2007 from 22 infants with neonatal hepatitis were studied retrospectively. The genome prevalence of HBV, HCMV, HSV, and adenovirus were analysed using qualitative polymerase chain reaction (PCR) protocols. The antigenic presentation of HSV-1, HSV-2, HBV, HCMV, and adenovirus were evaluated using immunohistochemistry (IHC) methods. The HCMV genome was detected separately in 1 of 22 (4.5%) paraffin-embedded autopsy and biopsy tissues. Also 3/22 (13.6%) samples were infected with HBV and HSV genomes. HBV and HSV-1 antigens were present in 1/26 (4.5%) neonatal samples and HSV-2 antigens in 5/26 (22.7%) by IHC protocols, but adenovirus and HCMV antigens were not detected among samples from infants with neonatal hepatitis. Detection of separate co-infections of HSV, HCMV, and HBV genomes in autopsy and biopsy tissues of HBV and HSV-1 or HSV-2 antigens in these patients, showed the importance of these viral infections in clinical neonatal hepatitis. MONG the numerous possible causes of neonatal hepatitis, a heterogeneous group of inflamatory disorders of liver that occur in infants younger than 3 months of age, viral infections are important.1,2 The prevalence and role of some of the hepatotropic DNA viruses that induce neonatal hepatitis are not well understood.1,2 Human cytomegalovirus (HCMV) can cause an uncommon mild form of neonatal liver failure. A few neonates develop hepatic fibrosis or noncirrhotic portal hypertension and/or rarely display cirrhosis with chronic cholestasis.1,2 HCMV is also an important cause of giant-cell hepatitis and intrahepatic calcification in neonates.2 Herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2) strains usually cause severe multisystem disorders in the newborn. Neonatal liver failure with an acute pattern of injury is typical.1,2 Liver biopsy specimen shows areas of necrosis with viral inclusions in intact hepatocytes; however, profound coagulopathy may preclude biopsy.1,2 Hepatitis B virus (HBV) infection in neonates does not cause jaundice unless an acute pattern of neonatal liver failure or severe hepatitis occurs
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© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 42, 837– 838 (2010)
after a typical incubation period. Vertical HBV infection is generally subclinical in the neonatal period; Neonatal liver failure may rarely occur with hepatitis B in neonates.1,2 Adenoviruses are well known to cause approximately 5% of acute respiratory diseases in children younger than 5 years of age. Apart from the respiratory tract, adenoviruses can replicate in various organs including the liver with rare prevalence in neonates. Some case reports in the literature have described hepatitis and disseminated intravascular coagulation with a mortality rate approaching 80%, most commonly occurring in the first 2 weeks of life.1,2 Although the pathogenic mechanisms of DNA viral infections in neonatal hepatitis are unclear, we studied the molecular and antigenic tissue prevalence of HCMV, HSV, HBV, and From the Shiraz Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. Address reprint requests to R. Yaghobi, Shiraz Transplant Research Center, Namazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran. E-mail: [email protected] 0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.03.023 837
838
adenovirus among infants with neonatal hepatitis who underwent orthotopic liver transplantation (OLT).
YAGHOBI, ZAMANI, GRAMIZADEH ET AL
We retrospectively studied 22 paraffin-embedded biopsy and autopsy tissues of infants with neonatal hepatitis who were admitted over 12 years from 1996 –2007, for OLT. HBV, HCMV, HSV, and adenovirus DNA was extracted from paraffin-embedded liver tissues using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The HCMV genome was amplified by an in-house nested polymerase chain reaction (PCR) protocol, which was optimized previously.3 Adenovirus DNA was diagnosed in these samples by an in-house qualitative PCR method. The mixture conditions of PCR reactions in 50 L were as follows: 5 L of 10⫻ PCR buffer; 1 L MgCl2 (50 mmol/L); 1.5 L dNTP (10 mmol/L); 1 L of each primer (20 pmol); 0.5 L Taq (2.5 U); and 5 L sample DNA. The thermocycling condition included 94°C for 3 minutes followed by a program of 35 cycles of 1 minute at 94°C, 1 minute at 62°C and 1 minute at 72°C, and, finally, 1 cycle of 5 minutes at 72°C. HBV-DNA was identified using a HBV qualitative PCR Kit (Cinnagen, Tehran, Iran). HSV-1 and HSV-2 genomes were detected using a HSV-PCR Kit (Sacace, Como, Italy). Also, the presence of antigens of HSV-1 or HSV-2, HBV, HCMV, or adenovirus in liver tissues of patients with neonatal hepatitis was evaluated using immunohistochemistry (IHC) methods.
Prenatal and postnatal viral infections may induce acute or chronic hepatitis in neonates.2,5 In the present research HBV molecular and antigenic presentation was confirmed in 3 and 1 of liver biopsy and autopsy specimens of newborns with hepatitis, respectively. But Amer et al have shown the absence of HBsAg in the serum of neonates with cholestasis, which may result in a misdiagnosis of HBV infection among unused PCR or IHC materials of liver tissues.6 In agreement with other studies, genomic and antigenic evidences of infection with HSV-1 and HSV-2 were confirmed in liver tissues of hepatitis affected neonates using PCR (13.6%) and IHC (22.7%) techniques, respectively.2,4,6 Despite common congenital HCMV infection affecting 1%–2% of newborns, the neonatal liver complications due to HCMV infection have been noted to be uncommon, possibly due to fail of prior detection of HCMV antigens in liver biopsy and autopsy tissues of neonates suffering from hepatitis. But compared with other research studies, HCMV-DNA was noted using PCR in liver tissues of infants with neonatal cholestasis.2,7,8 However, detection of HBV, HCMV, and HSV infections in liver tissues of neonates with hepatitis, has shown in this study the decisive importance of these viruses in clinical patterns of neonatal hepatitis.
RESULTS
REFERENCES
The molecular prevalence of studied DNA viruses was as follows. HBV and HSV genome was separately detected in 3/22 (13.6%) liver biopsy and autopsy tissues. HCMV-DNA was detected in 1/22 (4.5%) paraffin-embedded liver tissues. Co-infection of HCMV and HSV genomes was detected in 2/22 (9.1%) neonate samples. But an adenovirus genome was not detected in these samples. HSV-1 Ag was detected in 1/22 (4.5%) patients with neonatal hepatitis and HSV-2 Ag in 5/22 (22.7%) liver autopsy and biopsy materials of neonatal hepatitis patients. The HBsAg was detected in 1/22 (4.5%) paraffin-embedded liver tissues. Adenovirus and HCMV antigens were not detected in liver autopsy or biopsy samples of neonatal hepatitis patients.
1. McKiernan PJ: Neonatal cholestasis. Semin Neonatol 7:153, 2002 2. Roberts EA: Neonatal hepatitis syndrome. Semin Neonatol 8:357, 2003 3. Banan AA, Yaghobi R, Ramzi M, et al: Impact of human cytomegalovirus infection UL55 nested polymerase chain reaction method in hematopoietic stem cell transplant donors and recipients. Transplant Proc 41:2898, 2009 4. Aanpreung P, Laohapansang M, Ruangtrakool R, et al: Neonatal cholestasis in Thai infants. J Med Assoc Thai 88:S9, 2005 5. Ozkan TB, Mistik R, Dikici B, et al: Antiviral therapy in neonatal cholestatic cytomegalovirus hepatitis. BMC Gastroenterol 7:9, 2007 6. Amer OT, Abd El-Rahma HA, Sherief LM, et al: Role of some viral infections in neonatal cholestasis. Egypt J Immunol 11:149, 2004 7. Oliveira NLG, Kanawaty FR3, Costa SCB, et al: Infection by cytomegalovirus in patients with neonatal cholestasis. Arq Gastroenterol 39:132, 2002 8. Fischler B, Papadogiannakis N, Nemeth A: Etiological factors in neonatal cholestasis. Acta Paediatr 90:88, 2001
MATERIALS AND METHODS
DISCUSSION
More than 40% of infantile cholestasis is due to neonatal hepatitis with an incidence of 1 in 4800 –9000 live births.1,4
A
© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 42, 837– 838 (2010)
after a typical incubation period. Vertical HBV infection is generally subclinical in the neonatal period; Neonatal liver failure may rarely occur with hepatitis B in neonates.1,2 Adenoviruses are well known to cause approximately 5% of acute respiratory diseases in children younger than 5 years of age. Apart from the respiratory tract, adenoviruses can replicate in various organs including the liver with rare prevalence in neonates. Some case reports in the literature have described hepatitis and disseminated intravascular coagulation with a mortality rate approaching 80%, most commonly occurring in the first 2 weeks of life.1,2 Although the pathogenic mechanisms of DNA viral infections in neonatal hepatitis are unclear, we studied the molecular and antigenic tissue prevalence of HCMV, HSV, HBV, and From the Shiraz Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. Address reprint requests to R. Yaghobi, Shiraz Transplant Research Center, Namazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran. E-mail: [email protected] 0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.03.023 837
838
adenovirus among infants with neonatal hepatitis who underwent orthotopic liver transplantation (OLT).
YAGHOBI, ZAMANI, GRAMIZADEH ET AL
We retrospectively studied 22 paraffin-embedded biopsy and autopsy tissues of infants with neonatal hepatitis who were admitted over 12 years from 1996 –2007, for OLT. HBV, HCMV, HSV, and adenovirus DNA was extracted from paraffin-embedded liver tissues using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The HCMV genome was amplified by an in-house nested polymerase chain reaction (PCR) protocol, which was optimized previously.3 Adenovirus DNA was diagnosed in these samples by an in-house qualitative PCR method. The mixture conditions of PCR reactions in 50 L were as follows: 5 L of 10⫻ PCR buffer; 1 L MgCl2 (50 mmol/L); 1.5 L dNTP (10 mmol/L); 1 L of each primer (20 pmol); 0.5 L Taq (2.5 U); and 5 L sample DNA. The thermocycling condition included 94°C for 3 minutes followed by a program of 35 cycles of 1 minute at 94°C, 1 minute at 62°C and 1 minute at 72°C, and, finally, 1 cycle of 5 minutes at 72°C. HBV-DNA was identified using a HBV qualitative PCR Kit (Cinnagen, Tehran, Iran). HSV-1 and HSV-2 genomes were detected using a HSV-PCR Kit (Sacace, Como, Italy). Also, the presence of antigens of HSV-1 or HSV-2, HBV, HCMV, or adenovirus in liver tissues of patients with neonatal hepatitis was evaluated using immunohistochemistry (IHC) methods.
Prenatal and postnatal viral infections may induce acute or chronic hepatitis in neonates.2,5 In the present research HBV molecular and antigenic presentation was confirmed in 3 and 1 of liver biopsy and autopsy specimens of newborns with hepatitis, respectively. But Amer et al have shown the absence of HBsAg in the serum of neonates with cholestasis, which may result in a misdiagnosis of HBV infection among unused PCR or IHC materials of liver tissues.6 In agreement with other studies, genomic and antigenic evidences of infection with HSV-1 and HSV-2 were confirmed in liver tissues of hepatitis affected neonates using PCR (13.6%) and IHC (22.7%) techniques, respectively.2,4,6 Despite common congenital HCMV infection affecting 1%–2% of newborns, the neonatal liver complications due to HCMV infection have been noted to be uncommon, possibly due to fail of prior detection of HCMV antigens in liver biopsy and autopsy tissues of neonates suffering from hepatitis. But compared with other research studies, HCMV-DNA was noted using PCR in liver tissues of infants with neonatal cholestasis.2,7,8 However, detection of HBV, HCMV, and HSV infections in liver tissues of neonates with hepatitis, has shown in this study the decisive importance of these viruses in clinical patterns of neonatal hepatitis.
RESULTS
REFERENCES
The molecular prevalence of studied DNA viruses was as follows. HBV and HSV genome was separately detected in 3/22 (13.6%) liver biopsy and autopsy tissues. HCMV-DNA was detected in 1/22 (4.5%) paraffin-embedded liver tissues. Co-infection of HCMV and HSV genomes was detected in 2/22 (9.1%) neonate samples. But an adenovirus genome was not detected in these samples. HSV-1 Ag was detected in 1/22 (4.5%) patients with neonatal hepatitis and HSV-2 Ag in 5/22 (22.7%) liver autopsy and biopsy materials of neonatal hepatitis patients. The HBsAg was detected in 1/22 (4.5%) paraffin-embedded liver tissues. Adenovirus and HCMV antigens were not detected in liver autopsy or biopsy samples of neonatal hepatitis patients.
1. McKiernan PJ: Neonatal cholestasis. Semin Neonatol 7:153, 2002 2. Roberts EA: Neonatal hepatitis syndrome. Semin Neonatol 8:357, 2003 3. Banan AA, Yaghobi R, Ramzi M, et al: Impact of human cytomegalovirus infection UL55 nested polymerase chain reaction method in hematopoietic stem cell transplant donors and recipients. Transplant Proc 41:2898, 2009 4. Aanpreung P, Laohapansang M, Ruangtrakool R, et al: Neonatal cholestasis in Thai infants. J Med Assoc Thai 88:S9, 2005 5. Ozkan TB, Mistik R, Dikici B, et al: Antiviral therapy in neonatal cholestatic cytomegalovirus hepatitis. BMC Gastroenterol 7:9, 2007 6. Amer OT, Abd El-Rahma HA, Sherief LM, et al: Role of some viral infections in neonatal cholestasis. Egypt J Immunol 11:149, 2004 7. Oliveira NLG, Kanawaty FR3, Costa SCB, et al: Infection by cytomegalovirus in patients with neonatal cholestasis. Arq Gastroenterol 39:132, 2002 8. Fischler B, Papadogiannakis N, Nemeth A: Etiological factors in neonatal cholestasis. Acta Paediatr 90:88, 2001
MATERIALS AND METHODS
DISCUSSION
More than 40% of infantile cholestasis is due to neonatal hepatitis with an incidence of 1 in 4800 –9000 live births.1,4