Hepatobiliary Quiz-7 (2013)

1. Correct answers: 1, 3 and 4

HEV is a single stranded positive sense 7.2 kilo base pair ribonucleic acid (RNA) virus consisting of three overlapping open reading frames ORF1, ORF2 and ORF3.¹ The largest of these, ORF1 forms the 5′ distal end of the genome and codes for the non-structural replication proteins including the RNA dependent RNA polymerase (RdRp). The 3′ ORF2 codes for an arginine rich major structural protein.^2-4 ORF3 codes for a small protein thought to be a minor structural protein which has a role in viral egress and association into virons.⁵ The 5′ untranslated region (UTR) at the end of the HEV genome is responsible for binding the ORF2 protein and this interaction may play a role in viral encapsidation.⁶ The 3′ UTR of HEV has been shown to form cis-active stem-loop structures which localized the viral RdRp henceforth implicated in the initiation of virus replication.^7,8 The poly adenosine tail at the 3′ end is approximately 150-200 nucleotides long and is crucial for RdRp binding to the 3′ UTR. HEV replication takes place via a negative-sense RNA intermediate like most other positive sense RNA viruses.⁹ Replication takes place mostly in the cytoplasm.

2. Correct answers: 3 and 4

HEV RNA by reverse transcriptase PCR can be detected in feces and sera from most patients within 6 days after exposure to the virus and is the first sign of infection and disappears with the increase in ALT levels and appearance of anti-HEV antibodies.^10,11 Protracted viremia ranging from 4 to 16 weeks has also been reported in approximately 10% of the patients who do not show seroconversion. IgM anti-HEV antibodies are detectable as early as 1-4 days after onset of disease and decline within six months.^12,13 IgG anti-HEV antibody is detected during the peak of aminotransferase rise and persists for several months to years.¹⁴ Liver injury during HEV infection is immune mediated. A strong HEV specific cellular immune response occurs against capsid protein, HEV 239 (368-606) that decreases along with the decreasing IgM anti-HEV antibody titer and normalization of liver function. The decrease in HEV specific T-cell responses with convalescence suggests the involvement of T-cell responses in the pathogenesis of acute hepatitis E and recovery.¹⁵ Patients of HEV related acute liver failure have significantly higher levels of both Th1 (IFN-gamma, IL-2 and TNF-alpha) and Th2 (IL-10) cytokines compared to patients with mild acute HEV infection suggesting that status of host immune response plays a role in outcome.¹⁶ The delayed appearance of anti-HEV, after pathologic changes have occurred suggests that antibody is not necessary for initiating the pathologic process.

3. Correct answers: 1, 4 and 5

HEV infection can evolve to chronic hepatitis in immunosuppressed patients, i.e., patients with a solid-organ transplant (SOT), patients with hematological diseases, and human immunodeficiency virus (HIV)-positive patients. Chronic HEV has been described only in relation to genotype 3 infection. Seroprevalence of HEV in SOT recipients ranges from 2.3 to 43.9% and the prevalence of infection in SOT recipients as assessed by HEV RNA positivity ranges from 0.9 to 3.5% in various series. HEV RNA positivity is only 0-1.3% in HIV positive individuals.¹⁷ Out of 85 SOT patients in one series, 34% had spontaneous clearance of HEV within the first 6 months after diagnosis of HEV infection with no HEV reactivation later on, whereas 66% developed chronic hepatitis. Compared to frequent symptoms and icterus in immunocompetent patients, only 32% of the patients were symptomatic with fatigue being the main symptom. Only one patient was icteric and liver enzyme levels were increased to around only 300 IU/L, much lower than those usually observed in immunocompetent patients (≈3000-5000 IU/L). Nine (9.4%) developed cirrhosis on follow-up.¹⁸ Use of tacrolimus and higher degree of immunosuppression in the form of lower CD2, CD3, and CD4 T-cell subsets and lower serum concentrations of the IL-1 receptor antagonist and the IL-2 receptor during acute phases of HEV infection has been associated with chronic HEV infection. A large heterogeneity of quasispecies after acute infection has been observed in patients with persistent HEV compared to patients with resolving hepatitis.^18,19

4. Correct answer: 2 and 3

Main side effects of calcineurin inhibitors (CNIs) include hypertension, diabetes, hyperlipidemia, nephrotoxicity and neurotoxicity. Diabetes and neurotoxicity are more common with tacrolimus, whereas hypertension and hyperlipidemia are more common with cyclosporine. Gingival hyperplasia and hypertrichosis are typically caused by cyclosporine only. Both tacrolimus and cyclosporine are metabolized by cytochrome p450 system in liver, so their serum levels are affected by various drugs acting on cytochrome p450 system.²⁰ Mycophenolate inhibits inosine 5′monophosphate dehydrogenase (rate limiting enzyme in the de-novo synthesis of guanosine nucleotides) thus resulting in blockade of lymphocyte proliferation. Main advantage of mycophenolate is lack of nephrotoxicity. It is used in combination with CNIs, permitting lower doses of CNIs and thus less renal dysfunction.^21,22 Mycophenolate is not preferred without CNIs as the rate of rejection is higher when it is used alone. Adverse events related to mycophenolate are gastrointestinal intolerance and bone marrow suppression.²³ Sirolimus and everolimus are inhibitors of mammalian target of rapamycin (mTOR) and interfere with IL-2 signaling to T-cells. Side effects include hyperlipidemia, metabolic syndrome, myelosuppression, proteinuria, poor wound healing, pneumonitis and skin rash.^24,25 Initial reports of increased incidence of hepatic artery thrombosis have not been substantiated.^26,27 Sirolimus has additional anti-tumor effects and has been shown to decrease recurrence of hepatocellular carcinoma (HCC) and increase survival after liver transplantation for HCC.²⁷ Basiliximab and daclizumab are IL-2 receptor (CD25) blocking antibodies that have been used as induction agents to delay introduction of CNIs in patients with renal dysfunction.^28,29 One theoretical concern with their use is elimination of T regulatory cells which promote tolerance as these cells also express CD25.³⁰

5. Correct answers: 2, 4 and 5

The liver is involved commonly in sarcoidosis with elevated serum alkaline phosphate seen in about one third of patients^31,32 and liver biopsy showing granulomas in as many as 80-95% of cases.^33,34 Clinically hepatomegaly is found in about 19% of patients of sarcoidosis. Hepatic sarcoidosis can occur without pulmonary involvement in a quarter of cases.³⁵ Symptoms due to liver involvement are rare.³⁶ Only rarely do patients with hepatic sarcoidosis have cholestatic symptoms or portal hypertension. Portal hypertension was seen in only 1.2% patients in an Indian series³⁶ and in 3% in a Western series.³⁷ Cirrhosis can be seen on biopsy in upto 6% and significant fibrosis in another 15% of patients with clinical liver disease.³⁷ Of patients with portal hypertension 75% have esophageal varices which can bleed on follow-up and initial presentation of sarcoidosis with variceal bleeding has been reported.³⁸

6. Correct answers: 1 and 3

Insulin resistance, a key metabolic determinant of non-alcoholic fatty liver disease (NAFLD) is more common in South Asians. South Asians have lower adiponectin and higher leptin, and for the same body mass they have lower lean body mass and greater visceral adiposity which is proportionately related to insulin resistance.^39,40 Young, lean healthy Indians have four fold higher increased insulin resistance compared to weight matched individuals of other communities like Blacks, Hispanics, Caucasians or even East Asians.⁴¹ Asian countries have a high burden of NAFLD which is increasing with time due to this genetic susceptibility to metabolic syndrome as well as socio-economic changes causes increasing westernisation of diet and lifestyle. Prevalence of NAFLD in China is 10% in rural regions compared to 32% in urban Hong Kong. Similarly in India the prevalence is 9% in rural areas and upto 32% in urban areas. India and Sri Lanka (33%) have the highest reported prevalence of NAFLD in Asia.^42,43 Prevalence of NAFLD in Shanghai, China increased from 4 to 14% between 1995 and 2002.⁴⁴

Of the many genes studied in relationship to NAFLD, the strongest evidence is for the PNPLA3 gene. A recent meta-analysis has confirmed not only the role of this polymorphism in the development of NAFLD across all ethnicities, but also its association with more severe disease with 3.24 times higher risk of high necro-inflammatory scores and liver fibrosis in GG compared with CC genotype.⁴⁵ The role of APO C3 gene is controversial with one study demonstrating a high risk of NAFLD in Asian Indians with polymorphisms in the APO C3 gene compared to other ethnicities.⁴⁶ However, the role of this gene has not been validated in subsequent studies in other ethnicities.⁴⁷

7. Correct answers: 2 and 3

Recurrence of NAFLD after liver transplantation for NASH related cirrhosis is very common and ranges from 60% after 4 months to almost 100% after 5 years of liver transplantation with prevalence of NASH being 11%-33% after 2 years. These figures are much higher than the development of de-novo NAFLD after transplantation for other etiologies of cirrhosis.^48,49 However, overall survival is not inferior although the cause for post-transplant mortality is more likely to be due to cardiovascular events (21% versus 14%) rather than liver-related deaths (0.7% versus 10%) in patients transplanted for NASH compared to other etiologies of cirrhosis.⁵⁰ Cardiovascular events are correlated to the high frequency of development of metabolic risk factors due to the side effects of immunosuppressive agents even if they were absent prior to transplantation. Prevalence of metabolic syndrome is 52% after 6 years of transplantation compared to 5% pre-transplantation in all patients of cirrhosis, and a pre-transplant diagnosis of cryptogenic cirrhosis is an independent predictor of development of metabolic syndrome after transplantation.⁵¹ Similarly, patients with NASH cirrhosis are also more likely to have significant renal dysfunction in the form of stage 3 or 4 chronic kidney injury (71%) compared to other etiologies of cirrhosis (50%) after 2 years of transplantation, even after matching for pre-transplant renal function.⁵²

8. Correct answers: 2, 3 and 5

Resting energy expenditure is higher in patients with cirrhosis due to poor glycogen stores resulting in lipolysis and gluconeogenesis during even short periods of fasting.^53,54 Gluconeogenesis is an energy-expensive process and also leads to breakdown of muscle protein, increased ammonia generation and negative nitrogen balance. To counteract this phenomenon, small frequent meals along with a late evening snack containing about 50 g of complex carbohydrate is advisable. A meta-analysis has shown that a late evening snack may improve health related quality of life, increase survival and reduce the frequency and severity of hepatic encephalopathy.⁵⁵ Protein restriction should be avoided and 1.2-1.5 g/kg ideal body weight of protein is recommended per day.⁵⁶ Normoproteinemic diet is well tolerated and is beneficial even in patients with hepatic encephalopathy.⁵⁷ Vegetable proteins are theoretically superior to animal proteins as they contain significantly more dietary fiber that has favorable prebiotic properties for ammonia excretion in stool. Also, vegetable proteins are poor in the sulfated amino acids that are the precursors of mercaptans and indole/oxindole compounds implicated in the pathogenesis of hepatic encephalopathy and rich in ornithine and arginine which promote ammonia metabolism through the urea cycle.^58,59 Branched chain amino acids (valine, leucine and isoleucine) are deficient in patients with cirrhosis and supplementation has been shown to improve event-free survival.^60,61

9. Correct answers: 1, 3 and 4

Vasodilatory effect of nitric oxide is counteracted by vasoconstrictors like vasopressin, angiotensin, and aldosterone to maintain blood pressure in patients with cirrhosis.⁶² Agents that counteract the effects of these vasopressors like angiotensin converting enzyme inhibitors and angiotensin receptor blockers or decrease cardiac output like β blockers should be avoided or used with caution in patients with cirrhosis and ascites.⁶³ Arterial pressure independently predicts survival in patients with cirrhosis; those with a mean arterial pressure>82 mmHg have a 1 year survival of 70% compared to 40% for those with pressure <82 mmHg.⁶⁴

Standard medical therapy in the form of sodium restriction to below 2 g per day along with combination diuretic therapy (upto 160 mg of furosemide and 400 mg of spironolactone per day) is successful in controlling ascites in 90% of cases.^65,66 Refractory ascites is failure to lose weight with inadequate natriuresis or development of significant complications of diuretics with this regimen. Oral midodrine 7.5 mg three times daily has been shown to increase urine volume, urine sodium, mean arterial pressure, and survival.⁶⁷ Addition of midodrine to diuretics to increase blood pressure can theoretically convert diuretic-resistant patients to diuretic-sensitive. Patients who remain refractory to medical therapy even after discontinuing beta blockers and adding midodrine can be treated with serial therapeutic paracenteses with albumin infusions or transjugular intrahepatic portasystemic shunt and should be referred for liver transplantation at the earliest. Peritoneovenous shunts have poor long-term patency, excessive complications, and no survival advantage^65,68 and have nearly been abandoned now except for cases who are not candidates for transplant or TIPS, and who are not candidates for serial paracenteses because of multiple abdominal scars or logistic difficulties for regular paracentesis due to distance from health care centers.

10. Correct answers: 2, 3 and 4

Culture-negative neutrocytic ascites is defined as an absolute neutrophil count of >250/cc of ascitic fluid with negative fluid cultures in the absence of other causes of raised cells like hemorrhagic ascites, peritoneal carcinomatosis, pancreatitis, or peritoneal tuberculosis.⁶⁹ Patients with culture-negative neutrocytic ascites have similar clinical features and outcome as patients with spontaneous bacterial peritonitis (SBP) and hence warrant empiric antibiotic treatment similar to SBP.⁷⁰ Ascitic fluid culture positivity in the absence of neutrophilic ascitic fluid is called monomicrobial nonneutrocytic bacterascites. Of these 62% cases will resolves spontaneously without antibiotics and patients that do progress to SBP will have signs and symptoms of infection at the time of the paracentesis.⁷¹ Therefore patients of ascites and cirrhosis who have clinical features suggestive of SBP warrant empirical antibiotics until culture reports are available even in absence of neutrophilic ascites. In conjunction to antibiotic therapy, albumin infusion 1.5 g/kg body weight within 6 h of diagnosis and 1 g/kg on day 3 has been shown to decrease development of renal failure and improve survival in SBP.⁷² However, another study found benefit of albumin only in patients whose creatinine was >1 mg/dL, blood urea nitrogen>30 mg/dL, or total bilirubin>4 mg/dL.⁷³ Long-term antibiotic prophylaxis for SBP is indicated in patients with prior history of SBP, low ascitic fluid protein concentration (<1.5 g/dl), liver failure (Child score ≥9 and bilirubin ≥3 mg/dl) or impaired renal function (creatinine >1.2 mg/dl, blood urea nitrogen >25 mg/dl or serum sodium <130mEq/dl).⁷⁴ Short-term prophylaxis for 1 week in patients with gastrointestinal bleeding has been shown to reduce infection by 32% as well as decrease mortality by 9.1%.⁷⁵ Indiscriminate prophylactic antibiotics should be avoided to prevent colonization and infections with resistant bacteria.