Fulminant hepatitis B virus (HBV) infection in an infant following mother-to-child transmission of an e-minus HBV mutant: Time to relook at HBV prophylaxis in South African infants.
The current HBV prophylaxis in SA falls woefully short of current international recommendations, namely that all infants should receive a birth dose of HBV vaccine (as recommended by the World Health Organization ), pregnant women should be screened for hepatitis B surface antigen (HBsAg), and HBV VL should be performed on positive patients. Lastly, mothers with HBV VLs >200 000 IU/mL (American Association for the Study of the Liver and European Association for the Study of the Liver) or >6 log IU/mL (Asian Pacific Association for the Study of the Liver) should receive antiviral prophylaxis with lamivudine, telbivudine or tenofovir in the second and third trimesters of pregnancy. 
A 10-week-old male infant was admitted to Red Cross War Memorial Children's Hospital in Cape Town, SA, on 25 November 2016 with fever and poor feeding of 3 days' duration. He was receiving breast and formula milk feeds. He had received an intramuscular dose of a combination vaccine (HBV, Haemophilus influenzae type B, diphtheria, tetanus, acellular pertussis and inactivated polio) 4 days prior to admission. There was no history of diarrhoea, vomiting, cough, seizures, jaundice or recent travel. His mother was from the Democratic Republic of Congo (DRC). She had arrived in Cape Town while pregnant and had a normal vaginal delivery in Cape Town at 39 weeks' gestation. The baby's birth weight was 3 660 g, he had good Apgar scores, birth RPR for syphilis was negative, his immunisation status was up to date, and there was no history of contact with individuals with tuberculosis. He had not received any medication prior to presentation.
At presentation, his temperature was 38.8[degrees]C, he was active and not jaundiced, the findings on abdominal examination were normal, and there was no clinically evident focus of infection. Dipstick examination of the urine was negative, cerebrospinal fluid examination was not suggestive of meningitis, and a chest radiograph showed patchy infiltrates in the left lung field. Because neonatal sepsis was suspected he was commenced on intravenous cefotaxime and ampicillin pending the results of blood culture, which ultimately demonstrated no microbial growth.
On the 4th day of admission, marked jaundice was observed and the results of liver function tests and a coagulation profile were markedly deranged: total bilirubin (TB) 170 mmol/L (normal 5-21 mmol/L), conjugated bilirubin (CB) 98 mmol/L (normal 0-6 mmol/L), alanine aminotransferase (ALT) 1 880 U/L (normal 4-35 U/L), aspartate aminotransferase (AST) 3 424 U/L (normal 0-65 U/L), alkaline phosphatase (ALP) 1 481 U/L (normal 82-383 U/L), gamma-glutamyl transpeptidase (GGT) 145 U/L (normal 12-122 U/L), lactate dehydrogenase (LDH) 626 U/L (normal 180-430 U/L), ammonia 105 mmol/L (normal 40-80 mmol/L), international normalised ratio (INR) 8.7 (normal 2.0-3.0), prothrombin time (PT) 115.6 seconds (control 14.7 seconds), activated partial thromboplastin time (PTT) 119.3 seconds (control 30.0-40.0 seconds), fibrinogen 0.9 g/L (normal 2.0-4.0 g/L) and factor V 7.5% (normal 48-132%). He had microcytic anaemia, a sickle test was normal, and his C-reactive protein (CRP) level was 21 mg/dL (normal <10 mg/dL).
Further laboratory results were as follows: negative hepatitis A immunoglobulin M (IgM), low-positive HBsAg (signal close to the cut-off for the assay), positive hepatitis B surface antibody (antiHBs) of 80.7 mIU/mL and negative hepatitis B core IgM. Hepatitis C antibody, hepatitis E IgM, herpes simplex virus 1 and 2 polymerase chain reaction (PCR), HIV DNA PCR, parvovirus B19 PCR, rubella IgM and Treponema pallidum antibody tests were negative. The cytomegalovirus VL was 730 IU/mL, the Epstein-Barr virus VL was lower than the detectable level, the thyroid-stimulating hormone level was 0.86 mIU/L (normal 0.72-11.0 mIU/L) and the thyroxine level was 21.2 pmol/L (normal 11.5-28.3 pmol/L). Galactosaemia and MPV17-related hepatocerebral mitochondrial DNA depletion syndrome were considered, but the common c.404C >T (p.5135L) and c.C106T (P.Q36X) mutations in the galactose-1-phosphate uridyltransferase (GALT) and MPV17 genes, respectively, were not detected. An abdominal ultrasound scan revealed a non-enlarged liver with coarse echotexture. The mother tested positive for HBsAg and hepatitis B e antibody (anti-HBe), but was HBeAg-negative. Her HBV VL was detectable at 760 IU/mL.
The clinical manifestations together with the laboratory findings suggested a diagnosis of fulminant hepatic failure secondary to vertical HBV infection. Treatment included vitamin K, oral lactulose and N-acetylcysteine. The infant received lamivudine and his antibiotics were changed to piperacillin/tazobactam and amikacin. He remained critically ill, requiring several platelet, cryoprecipitate and fresh frozen plasma infusions, and continued to deteriorate, becoming hypoglycaemic on day 10 of admission.
Table 1 shows evolution of the HBV markers. Repeat liver function tests and a repeat coagulation profile on day 15 of admission showed persistent derangements, i.e. TB 369 mmol/L, CB 93 mmol/L, ALT 51 U/L, AST 55 U/L, ALP 3 227 U/L, GGT 32 U/L, LD 436 U/L, INR 7.54, PT 86.3 seconds (control 12.6 seconds), PTT 109.8 seconds (control 29.5 seconds) and fibrinogen 0.7 g/L (normal 2.0 - 4.0 g/L). The infant remained critically ill, his liver transaminases continued to decline, the coagulopathy persisted, the jaundice worsened, and he continued to experience hypoglycaemic spells. He subsequently developed melaena and grade II encephalopathy. He continued to deteriorate and died on day 45 of admission.
Genetic analysis of HBV from mother and infant
Total nucleic acid was extracted from serum samples of mother and infant using the MagNA Pure LC automated extraction method as per the manufacturer's instructions (Roche Diagnostics GmbH, Germany). For HBV genotype assignment, a semi-nested PCR was used to amplify a region of the surface and overlapping polymerase (S/pol) gene: primers P6F and P2R  in the first round and P7F  and P2R in the second. The PCR protocol from Smuts et al.  was followed with minor modifications.
The basal core promoter (BCP), pre-core/core region (PC) was amplified using a nested PCR protocol.  The entire surface antigen gene (preS1, preS2 and S) was amplified using primers and protocol from Chook et al. 
Bidirectional Sanger sequencing of the S/pol, BCP/PC and S amplicons was performed using the BigDye terminator cycle sequencing kit (Applied Biosystems, USA). The HBV genotype was determined using the web-based geno2pheno program (http:// www.geno2pheno.org/index.php).
To determine the heterogeneity of the BCP/PC and S genes, amplicons from both mother and infant were cloned into a pGEM-T vector according to the manufacturer's instructions (Promega Corp., USA). Multiple clones were subjected to Sanger sequencing and aligned using BioEdit version 22.214.171.124 (Softpedia, Romania).
We present this case because it highlights the need to improve HBV prophylaxis to infected mothers and their infants. The case is unusual because the mother was anti-HBe-positive and had a low VL, only 760 IU/mL, but still transmitted infection to her infant. Also, the infant developed a fulminant infection that led to his death. The commonest scenario associated with MTCT of HBV is when the mother is HBeAg-positive and has a high HBV VL (>6 log IU/mL). This was previously reported to be relatively uncommon in Africa and accounted for the decision to omit a birth dose of HBV vaccine from the infant immunisation schedule in SA. However, more recent studies have found that between 18% and 37% of HBV-positive pregnant women in SA are HBeAg-positive.[9,10,20] Many HBV-positive individuals are co-infected with HIV, and this could influence the HBeAg status of infected individuals.
Was this MTCT?
Genetic analysis of the virus in mother and infant samples showed that both were infected with HBV genotype E. The mother was from the DRC, and genotype E is a predominant HBV genotype found in that country.  Sequence analysis of the BCP/ PC genomic region and the preS1, preS2 and S open reading frames (ORFs) of HBV revealed that the mother's and infant's viruses had multiple signatures in common, providing evidence that their infections were indeed epidemiologically linked. Common motifs included a near-identical deletion in the preS2 region (position 399-420 in the mother and position 402-420 in the infant), a 3-nucleotide insertion at position 598-600 in the small S ORF, and multiple singlenucleotide polymorphisms (Fig. 1, A and B).
The HBeAg-negative phenotype and its significance
HBV infection in infants and young children is usually associated with subclinical infection. Liver damage/hepatitis in HBV infection is immune mediated (due to cell-mediated immune attack on HBV-infected liver cells). The milder clinical illness in the very young has been attributed to the tolerising effect of HBeAg. This protein is a potent immune modulator and immune evasion molecule, acting at multiple levels to induce immune tolerance: it blocks cellular responses to type 1 and 3 interferons,  interferes with toll-like receptor signalling and induces the expression of PD-1 (an exhaustion marker) on CD8+ cells, compromising their antiviral activity.  The effect of this is to attenuate the antiviral immune response (and severity of clinical hepatitis) and enable the virus to establish persistent infection in the host.
Both the mother's and the infant's virus had the common G1896A mutation in the BCP/PC region. This mutation is frequently selected for during the immune-active phase of chronic HBV infection and probably became the predominant species in the mother's blood years before. This mutation introduces a stop codon that truncates the HBeAg protein at the level of translation. In addition, the mother's virus had further mutations in the BCP region (1762T, 1764A), known to reduce the synthesis of the pre-C mRNA transcript from which the HBeAg is synthesised. Transmission of HBV lacking functional HBeAg prevents the establishment of chronic infection but predisposes to more severe clinical hepatitis (due to the vigorous immune attack on infected liver cells). The fact that HBsAg was negative in the second blood sample from the infant is evidence that he was indeed clearing the infection. In a study of infants infected with HBV through MTCT, HBeAg-negative status in mothers was more likely to be associated with fulminant hepatitis in their infants, while infants of HBeAg-positive mothers were more likely to have mild hepatitis followed by chronic infection. 
When did transmission happen?
MTCT of HBV can occur in utero, intrapartum or after birth (in breast-fed infants). As for HIV, the greatest risk of HBV transmission is during birth and this is when transmission probably occurred in this case.  The infant received his first dose of HBV vaccine at 6 weeks of age and the second, just days before the clinical presentation, too late to prevent transmission. Viruses from breakthrough infections that occur in infants who have received HBV immunoglobulin or vaccine at birth frequently have mutations that alter the antigenicity of the major neutralising domain of the viral HBsAg, in the so-called 'a' determinant. Most common is a glycineto-arginine change at amino acid position 145, first described by Carman et al.  This mutation arises because the presence of neutralising antibody early on selects for viral variants with altered HBsAg protein that is not neutralised by the antibody. No changes were present in this region in either the mother's or the infant's virus, suggesting that the infection was already well established before the first dose of vaccine.
While this is a rare complication of HBV infection, it highlights the fact that vertical infections in infants do occur in SA despite current prophylaxis. Most transmissions from HBeAg-positive mothers are probably missed, as the infants do not present with a typical clinical illness, and it is the rare fulminant presentations that draw attention. Current HBV prophylaxis in SA is out of step with current guidelines.  Introduction of a birth dose of vaccine alone would improve efficacy, but given the prevalence of HBV infection among local pregnant women, we consider that prenatal screening is needed. Screening could be done at booking using a rapid test for HBsAg. The most cost-effective approach would be for women who test positive to receive tenofovir prophylaxis in the second to third trimesters. A small field study has already demonstrated the feasibility of this approach in the Western Cape Province.  Also, the safety and acceptability of this drug has already been established for HIV-infected pregnant women.
* Mother-to-child HBV transmission may occur even when the maternal VL is low (<3 log IU/mL).
* Fulminant hepatitis in this infant was due to transmission of an HBV strain unable to express the e-antigen.
* This case provides evidence of failure of the current infant HBV prophylaxis programme in SA.
Author contributions. DRH and SK conceived the study and wrote part of the article. OB, BE and RdL provided clinical management of the case and wrote the clinical part of the case report. HS performed molecular testing and analysis and gave input into writing the article.
Conflicts of interest. None.
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Accepted 19 December 2017.
OBabatunde, (1) MBBS, MSCI, FMCPaed; H Smuts, (2,3) PhD; B Eley, (1) MB ChB, FCP (SAPaed), BSc Hons; S Korsman, (2,3) MB ChB, FC Path (SA) Viro, MMed; R de Lacy, (4) MB ChB, FCP (SAPaed); D R Hardie, (2,3) MB ChB, MMed
(1) Paediatric Infectious Diseases Unit, Red Cross War Memorial Children's Hospital, Cape Town, South Africa; and Department of Paediatrics and Child Health, Faculty of Health Sciences, University of Cape Town, South Africa
(2) National Health Laboratory Service, Groote Schuur Hospital, Cape Town, South Africa
(3) Division of Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, South Africa
(4) Gastroenterology Unit, Red Cross War Memorial Children's Hospital, Cape Town, South Africa; and Department of Paediatrics and Child Health, University of Cape Town, South Africa
Corresponding author: D R Hardie (firstname.lastname@example.org)
Table 1. Plasma markers of HBV infection in mother and infant Infant, day 4 Infant, day HBV marker Mother of admission 17 of admission HBsAg Positive Low positive Negative Anti-HBs (mlU/mL) Negative 80 126 HB core IgM Negative Negative Low positive (1.21 S/CO) HB e antigen Negative ND * ND * Anti-HBe Positive ND ND HBV VL (IU/mL) 760 465 209 Hepatitis D PCR ND Negative ND HBV = hepatitis B virus; HBsAg = hepatitis B surface antigen; anti/ HBs = hepatitis B surface antibody; IgM = immunoglobulin M; anti/HBe = hepatitis B e antibody; S/CO = signal/cut-off; ND = not done; VL = viral load; PCR = polymerase chain reaction. * Insufficient sample available to perform e-antigen testing. Sequence analysis of infant's virus indicated the e-minus phenotype. Fig. 1. Genetic features of HBV present in mother and infant blood. (A) Sequence analysis of the BCP and PC region revealed that immune escape mutations A1762T and G1764A were present in the mother's virus, but absent from the infant's virus. Both contained the G1896A mutation, which truncates the HBeAg protein and confers the e-minus phenotype. (B) In the preS/S genes, the preS2 start codon was mutated in the infant's virus, but intact in the mother's. Both had large deletions in the preS2 gene, and there was a 21nt deletion from 399 to 420 in the mother's virus and an 18nt deletion from 402 to 420 in the infant's virus. A 3nt insertion was present in the small S ORF in both the mother's and the infant's virus. The 'a' determinant was unchanged. (BCP = basal core promoter; Pre-C = pre-core; ORF = open reading frame; PC = pre-core-core; s Ag = surface antigen; nt = nucleotide; aa = amino acid. Mutations and deletions in the HBV genome in mother and infant blood are indicated in red.) A BCP BCP/PC A1762T G1764A C1766T T1768A KOZAC Mother A C A GCAC Infant A G G A GCAC Pre-C ORF PC start BCP/PC cod on T1858C G1862T G1896A G1899A 1814--16 Mother ATG T G A G Infant ATG T G A A B preS2 preS1 start preS2 open reading frame Mother -- ATG 21nt del 399-420 (in frame 7 aa deletion) Infant -- ATC 18nt del 402-420 (in frame 6 aa deletion) s ag start s ag open reading frame 'a' determinant Mother ATG 3nt insertion at position Wild type 598-600 (aa = L, 200) Infant ATG 3nt insertion at position Wild type 598-600 (aa = L, 200)
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|Title Annotation:||CASE REPORT|
|Author:||Babatunde, O.; Smuts, H.; Eley, B.; Korsman, S.; de Lacy, R.; Hardie, D.R.|
|Publication:||SAMJ South African Medical Journal|
|Article Type:||Case study|
|Date:||May 1, 2018|
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