COINFECTION OF PARVOVIRUS B19 WITH OTHER HEPATITIS VIRUSES LEADING TO FULMINANT HEPATITIS OF UNFAVORABLE OUTCOME IN CHILDREN

Abstract

Fulminant hepatic failure (FHF) associated with parvovirus B19 (B19) infection has a favorable prognosis in children. However, there is no data available to predict outcome in cases of FHF associated with hepatotropic virus coinfection. Clinical characteristics of 3 pediatric groups with FHF were compared and it was observed that B19 coinfection with other viruses adds to the severity of the disease and increases the probability of a fatal outcome.

Acute viral hepatitis caused by common hepatitis viruses A, B, C, D, E, or other hepatotropic viruses including parvovirus B19 accounts for 50% of cases of fulminant hepatic failure (FHF) which has a mortality rate greater than 70%. Association of parvovirus B19 (B19), with acute hepatitis and FHF is described by several authors.^1–4 This is associated with a favorable prognosis in younger children.⁵ However, to the best of our knowledge, there is no study available on prognosis of cases presenting with B19 coinfection with other hepatotropic viruses.

The present study was designed to compare the clinical characteristics and laboratory findings among 3 selected groups presenting with FHF associated with: (i) B19 infection alone; (ii) one or more other hepatotropic viral infection in the absence of B19 infection; and (iii) B19 coinfection with other hepatotropic viruses.

MATERIALS AND METHODS

The study was conducted in a large tertiary care referral center of North India. Pediatric patients, 2 to 12 years of age, admitted to our hospital from January 2003 to December 2007, fulfilling the diagnostic criteria of FHF and laboratory evidence of one or more common hepatotropic viral infection were included in the study. The patients with drug-induced hepatitis, cholestatic jaundice, congestive hepatopathy, and hepatitis resulting from multisystem failure were excluded from the study. Clinical characteristics and results of laboratory investigation previously conducted were recorded at the time of enrolment. Serum samples and liver tissue specimen were obtained from all the patients. All the serum samples as well as liver tissue specimens were analyzed for the presence of B19 genome by PCR based amplification followed by agarose gel electrophoresis. Serum samples were analyzed for the presence IgM antibodies against specific viral antigens by ELISA (Ortho Clinical Diagnostic ELISA Test system 3). We selected B19 genome as viral marker for Parvovirus B19 and IgM antibodies against specific viral antigens as viral markers for other hepatotropic viruses. B19 DNA from serum samples was extracted using from QIA amp DNA mini kit (QIAGEN, Germany) followed by nested PCR as per Abe et al.⁶ Total RNA was extracted from the liver tissues using Qiagen RNeasy kit (QIAGEN, Inc., CA) according to manufacturer’s instructions. The purified RNA obtained from liver tissues was further subjected to digestion by RQI DNase (Promega Co., Madison, WI). The B19 cDNA was synthesized from the pretreated RNA by reaction with 100 units of Moloney murine leukemia virus reverse transcriptase (Fermentas, Germany) and B19 antisense primer. The cDNA obtained was subjected to nested PCR amplification. Serum samples and liver tissues were also obtained from 79 patients with biliary atresia, and were used as controls for B19 genomic tests. Informed consent for participation in this study was obtained from the parents of each child. Institute’s ethical committee approved the work and all parents of all individuals included in the study consented for enrollment.

Statistical calculations were done by Fisher exact test and χ² test taking P < 0.05 as significant. Odds ratios (OR) with a 95% confidence interval limit were calculated from 2 × 2 contingency table.

RESULTS

In the initial phase a total of 48 patients with FHF were included in the study. One or more viral markers were detected in 26 patients either in the serum samples or liver tissue. Of 48 patients B19 genome was present in 19 (39%), of which 13 (27%) were also positive for IgM antibodies against one or more other hepatotropic viruses (HAV in 3 patients, HBV in 6 patients, HCV in 2 patients, and HAV plus HEV in 2 patients). In patients negative for B19 genome (n = 29), 7 (15%) patients showed the presence of IgM antibodies against one hepatotropic virus each (HBV in 3; HCV in 2; and HAV and HEV in one patient each). Among biliary atresia cases B19 genome (DNA) was detected in 11 of 79 cases. PCR-positivity in FHF cases was significantly higher (P = 0.0022) and the risk was increased ∼4-fold (OR = 4.05; 95% CI = 1.71–9.58).

B19 mRNA was detected in 19 (39%) of 48 liver tissue specimens from FHF cases. Presence of B19 mRNA in the liver tissue correlated with the presence of B19 genome in the serum samples. Among the biliary atresia cases B19 mRNA was present in only 8 of 79 (10.1%). Association of B19 mRNA and FHF cases differed significantly with that of biliary atresia cases (P = 0.0002) and the risk was found to be ∼6-fold (OR = 5.82; 95% CI = 2.29–14.77).

Clinical characteristics and laboratory findings of patients in the 3 groups were compared. The groups were found age matched. The main distinctive features of parvovirus B19 and other hepatitis viruses’ coinfection associated FHF were: presence of jaundice, high bilirubin, high alanine aminotransferase or aspartate aminotransferase activity, and unfavorable outcome resulting in death of most of the patients (Table, Supplemental Digital Content 1, https://links.lww.com/A1140). Results of statistical analyses are summarized in the Table 1.

TABLE 1: Comparison of Clinical Characteristics and Outcomes of Patients Having Fulminant Hepatitis Associated With Parvovirus B19 Alone, Parvovirus B19 Coinfection, and Other Hepatitis Viruses

DISCUSSION

Association of fulminant or acute hepatitis with B19 infection has been previously reported.^4,5 The causal association of B19 with FHF was demonstrated by Abe et al⁶ who isolated and characterized erythrovirus B19 genomes from liver tissues of patients with FHF.

Although hepatitis has been attributed to B19 infection by several other groups of investigators,^1–4 the presence of B19 genome in liver tissues has not been proved to be specific for acute infection as it is found in liver tissue of patients with FHF and in control patients.² Because viral genome can persist in tissues, viremia could be a more specific marker of an acute infection.^7,8 Moreover, B19 hepatitis results in high levels of viremia, usually 10¹⁰ virions/mL (our experience). The diagnosis of B19 infection was established by PCR based detection of its genome. In our study B19 DNA was detected in 19 of 48 patients having FHF, which is consistent with the findings of Langnas et al² and Abe et al,⁶ though the prevalence of B19 is higher among our patients.

Abe et al⁶ have detected B19 genome in liver of the patients who were serologically negative for the viral genome. In their study, only one serum sample was found positive for the B19 genome among 8 cases with intrahepatic-B19 DNA. These results suggest that absence of B19 sequence in serum does not rule out the existence of current infection by B19 within the liver. On the other hand, Eis-Hubinger et al⁹ showed that direct detection of viral genomes in liver tissues by a highly sensitive method is as important as serology.

In the present study, the presence of B19 mRNA in the liver tissue was analyzed to detect viral replication. In all of the serum B19-positive cases, B19 mRNA was present in the liver tissue indicating presence of viral replication in all cases with fulminant hepatitis. By contrast, in only a small percentage of cases with biliary atresia (8/79) B19 replication was detected in liver tissues. A significantly high prevalence of B19 infection was found in fulminant hepatitis patients. Low rate of B19 infection has been observed in biliary atresia cases as determined by B19 DNA (in serum) and B19 mRNA (in liver tissues). Similar association has also been observed by a few authors,⁶ but the exact relationship of B19 and biliary atresia is unknown.

The disease severity was significantly greater in patients with B19 coinfection. There is a possibility that B19 causes injury to hepatocytes independently and produces synergistic effect when present along with other hepatitis viruses as a cofactor (like Hepatitis D). In conclusion, pediatric patients with FHF had high prevalence of B19 associated with other hepatitis viruses coinfection, had relatively severe disease and poor outcome.