Bocavirus infection following paediatric liver transplantation.

HBoV is an emergent virus, which is frequently detected as a co-infective agent. However, it can cause disease on its own. It is associated with respiratory and diarrhoeal illness in children and adults, whether immunocompetent or immunocompromised. We report HBoV infection in a child post-liver transplantation, who presented with persistent fever and mild tachypnea, 3 weeks after a successful transplant. She recovered spontaneously with no graft dysfunction.

Unusual shunt for symptomatic portal vein thrombosis after liver transplantation - Clatworthy revisited.

PV thrombosis is not an uncommon occurrence following pediatric LT. Symptomatic PHT following PV thrombosis is treated medically, surgical portosystemic shunting (mesorex, splenorenal, and mesocaval) being reserved for refractory cases. A 10-yr-old boy suffered recurrent malena and hemorrhagic shock because of chronic PV thrombosis following LT nine yr ago (1999). Extensive work-up failed to localize the bleeding source. The liver function remained normal. Initial attempts at surgical shunts failed owing to thrombosis (mesocaval 2001, splenorenal, inferior mesenteric-left renal vein, splenic-left external iliac vein 2008). In this situation, we performed a Clatworthy shunt by anastomosing the divided lower end of the LCIV to the side of SMV. There was a single, large caliber anastomosis. Post-operatively, the malena stopped completely, and clinically, there was no lower limb edema or encephalopathy. Doppler USG revealed persistence of hepatopetal flow within the portal collaterals. Follow-up at two yr reveals stable hepatic function with a patent shunt. To the best of our knowledge, we are not aware of a Clatworthy shunt being performed in a transplant setting. We reviewed the literature pertaining to this shunt in non-transplant patients with PHT.

Novel multiplex oligonucleotide-conjugated bead suspension array for rapid identification of enterovirus 71 subgenogroups.

A high-throughput multiplex bead suspension array was developed for the rapid subgenogrouping of EV71 strains, based on single nucleotide polymorphisms observed within the VP1 region with a high sensitivity as low as 1 PFU. Of 33 viral isolates and 55 clinical samples, all EV71 strains were successfully detected and correctly subgenogrouped.

Rates and determinants of antibiotics and probiotics prescription to children in Asia-Pacific countries.

Antibiotic therapy may have important side effects. Guidelines recommend the administration of specific probiotics to reduce the risk of antibiotic-associated diarrhoea (AAD). The rates and determinants of antibiotics and co-prescription of probiotics in children remain poorly known in Asia-Pacific countries, which are very heterogenous in terms of economic development, health care organization and health policies. A survey among general practitioners (GPs) and paediatricians was performed in seven countries of the Asia-Pacific area (Australia, Japan, Indonesia, India, China, Singapore, and South Korea). Physicians completed an online questionnaire that explored their current habits and the determinants for prescribing antibiotics and probiotics. For the 731 physicians who completed the questionnaire (390 paediatricians and 341 GPs), 37% of all consultations for a child led to the prescription of antibiotics (ranging from 17% in Australia to 47% in India). A large majority of physicians (84%) agreed that antibiotics disrupted gut microbiota and considered probiotics an effective intervention to prevent AAD (68%). However, only 33% co-prescribed probiotics with antibiotics (ranging from 13% in Japan to 60% in South Korea). The main reasons for prescribing probiotics were previous episodes of AAD (61%), presence of diarrhoea (55%), prolonged antibiotic treatment (54%) or amoxicillin-clavulanic acid therapy (54%). Although current local guidelines recommend the use of selected probiotics in children receiving antibiotics in Asia-Pacific area, the rates of antibiotics and probiotics prescription significantly vary among countries and are deeply affected by country-related cultural and organisational issues.

Seroprevalence and risk factors for the presence of ruminant pestiviruses in the Dutch swine population.

Swine can be infected with classical swine fever virus (CSFV), as well as ruminant pestiviruses: bovine viral diarrhoea virus (BVDV), and Border disease virus (BDV). Cross-reactions between pestiviruses occur, both regarding protective immunity and in diagnostic tests. The presence of BVDV and BDV in a swine population may thus affect the transmission of CSFV, but also the diagnosis of a CSFV infection. In this study, the seroprevalence against BVDV and BDV in two categories of swine, sows and finishing pigs, in the Netherlands was determined. Furthermore, several risk factors, associated with the presence of swine and ruminants on the same farm or in the immediate surroundings, were evaluated. In sows, the seroprevalence against BVDV was 2.5% on the animal level, and 11.0% on herd level. In finishing pigs these prevalences were 0.42% and 3.2%, respectively. Antibodies against BDV were found in three sows only. Risk factors, associated with a BVDV-seropositive status in breeding pigs, were the presence of cattle on the same premises and a high density of sheep and/or goats herds in a radius of 3km. While BVDV and BDV hardly pose any threat to the swine population themselves, knowledge, and therefore regular monitoring, on the presence of these viruses in the swine population is important with respect to CSF eradication. It will allow for a better interpretation of diagnostic test results, both in terms of possible false positives and false negatives, but may also bring about additional measures or surveillance protocols in times of CSF outbreaks to avoid surprises caused by cross-reactivity with ruminant pestiviruses.

Transmission of African Swine Fever Virus via carrier (survivor) pigs does occur.

We investigated whether ASF carrier pigs that had completely recovered from an acute infection with ASFV Netherlands '86, could transmit the disease to naive pigs by direct contact transmission. For this, we used pigs that had survived an ASFV infection, had recovered from disease, and had become carriers of ASFV. These clinically healthy carriers were put together one-by-one with naive contact pigs. Two of the twelve contact pigs developed an acute ASFV infection. Using the results of the experiment we quantified the transmission parameters ßcarrier (0.039/day) and Tcarrier (25.4 days). With the survival rate of 0.3 for our ASFV isolate, these parameter values translate into the contribution of carriers to R0 in groups of pigs being 0.3. Further, we placed naive contact pigs in an ASFV contaminated environment. Here, no contact infections were observed. Our findings show that clinically healthy carriers can be a source of acute new infections, which can contribute to the persistence of ASFV in swine populations. The estimates that we provide can be used for modelling of transmission in domestic pigs and, in part, for modelling transmission in wild boar.

Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in Western blots.

A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis. Oligonucleotides corresponding to the amino-acid sequence of synthetic peptide SP32 were cloned and over-expressed in Escherichia coli as a recombinant glutathione-S-transferase (GST)-SP32 fusion protein. In ELISAs, this protein did not react with human anti-EV71 IgG antibodies, but there was significant immunoreactivity according to western blot analysis. The amino-acid sequence of SP32 was highly specific for detecting EV71 strains in western blot analysis, and showed no immunoreactivity with monoclonal antibodies raised against other enteroviruses, e.g., CA9 and Echo 6.

Limited BVDV transmission and full protection against CSFV transmission in pigs experimentally infected with BVDV type 1b.

Bovine viral diarrhea virus (BVDV) in pigs may interfere with the detection and epidemiology of classical swine fever virus (CSFV). To investigate the importance of BVDV infections in pigs, first we studied the transmission dynamics of a recent BVDV field isolate. Subsequently, the protection of BVD antibodies against transmission and clinical disease of CSF virus was studied. Only limited transmission of BVDV occurred (R = 0.20), while no CSFV transmission occurred in pigs with BVDV antibodies. We concluded that BVDV transmission among pigs is possible, but seems to be limited and thus the virus should disappear from a population if no new introductions occur. Furthermore, the presence of BVD antibodies may completely prevent the transmission of CSFV and therefore could protect pigs against classical swine fever. It was also noticed that double infections with BVDV and CSFV were incorrectly diagnosed using the neutralization peroxidase linked assay (NPLA), which is the golden standard for antibody detection. This might hamper the diagnosis of CSF in herds with a high BVD prevalence.

Outcome of liver transplantation for children with liver disease.

INTRODUCTION: The advent of liver transplantation has revolutionised the outcome of children with both acute liver failure and chronic end-stage liver disease. The aim of this study was to review the outcome of all paediatric liver transplants performed since the National Liver Transplant Programme began in 1990. METHODS: A retrospective review of all paediatric liver transplants from 1990 to December 2004 was performed. RESULTS: 46 liver transplants were performed in 43 children, of whom 23 (53.3 percent) were female. Median age at transplant was 21 months (range 11 months to 14 years). The most common indication for liver transplant was biliary atresia (71.7 percent). Living-related transplants accounted for 63 percent (29). Re-transplant rate was 6.5 percent with allograft loss as a result of hepatic artery thrombosis (two) and hepatic vein thrombosis (one). Tacrolimus was the primary immunosuppressive agent used in 89 percent of patients, with a 19.6 percent incidence of acute allograft rejection within the first six months. There were nine deaths. They were related to portal vein thrombosis (three), chronic rejection (one), sepsis (two), post-transplant lymphoproliferative disease (two) and primary graft non-function (one). Overall actuarial one- and five-year survival rate was 85.7 percent and 81.8 percent, respectively. CONCLUSION: Liver transplantation is an established form of intervention for end-stage liver disease and a variety of liver-related metabolic disease. Our results are comparable to those of well-established liver transplant centres.

Liver transplantation in Singapore 1990-2004.

INTRODUCTION: Liver transplantation is the accepted standard of care for patients with hepatocellular carcinoma, decompensated liver cirrhosis, and acute liver failure. Since the first liver transplant done in Singapore in 1990, results have been improving. We review the overall results of liver transplantation over the last 15 years. METHODS: All transplant cases from 1990 to 2004 were reviewed retrospectively. RESULTS: 100 liver transplants were performed over the last 15 years; four in the first five years and 96 in the subsequent ten years. Overall one- and five-year survival rates were 80 percent and 78 percent, respectively. 44 were paediatric transplants, of which biliary atresia was the commonest indication for paediatric transplant. 56 were adult transplants of which hepatocellular carcinoma and decompensated hepatitis B cirrhosis were the commonest indications for adult transplant. Infection remained the commonest cause of mortality. CONCLUSION: The number of transplants carried out per year was small due to the low cadaveric donation rate, but the survival of liver transplant patients was comparable to well-established liver transplant centres.

Referral patterns and waiting times for liver transplantation in Singapore.

INTRODUCTION: Referral patterns, waiting times, waiting list, and mortality provide information on how effectively a transplant programme deals with referred patients. This paper aims to review these parameters in the Singapore National Liver Transplant Programme. METHODS: Data of all patients referred to the Singapore National Liver Transplant Programme since its inception were captured and outcomes were retrieved and described. RESULTS: 562 patients were referred for liver transplant evaluation from 1990-2004, consisting of 457 adults and 105 children. The main indications for referral were hepatitis B liver disease and hepatocellular carcinoma in adults, and biliary atresia in children. Most patients were of United Network of Organ Sharing (UNOS) status 3 or 4 at the time of referral. 114 (20.28 percent) patients had transplants, consisting of 66 adults (14.44 percent) and 48 (45.71 percent) children. 138 adults and ten children were rejected for transplant, mainly for the reason of being "too early". The median waiting time for adults who had transplants was 3.3 months while adults still on the waiting list had been waiting for 16.2 months. The overall waiting list mortality was 44.3 percent, being 52.5 percent in adults and 23.2 percent in children. CONCLUSION: The overall transplantation rate is low and the waiting list mortality is high as a result of low availability of organs, particularly in adults. Paediatric liver transplant appears to have been better at dealing with referred patients but this is probably due to availability of living-related liver transplant. Improvement in these may result from the Human Organ Transplant Act.

A rotavirus vaccine for infants: the Asian experience.

INTRODUCTION: Severe rotavirus gastroenteritis in children causes significant morbidity worldwide and substantial deaths in developing countries. Hence, a live attenuated vaccine Rotarix was developed with human strain RIX4414 of G1P1A P[8] specificity. RIX4414 trials in infants have begun in developed and developing countries worldwide. An overview of RIX4414 in developed and developing countries and prospects with this vaccine in Asia are presented. METHODS: Completed RIX4414 trials have been reviewed. RESULTS: Two oral doses of RIX4414 were well tolerated with a reactogenicity profile similar to placebo. RIX4414 was also highly immunogenic, e.g., in a dose-ranging study conducted in Singapore, 98.8% to 100% of infants had a vaccine take after 2 doses. RIX4414 did not affect the immune response of simultaneously administered routine infant vaccines. RIX4414 significantly reduced severe rotavirus gastroenteritis in settings where multiple serotypes including the emerging G9 type co-circulated. CONCLUSION: These encouraging results warrant further evaluation of the vaccine worldwide and especially in developing countries with the highest need. Therefore, evaluation of the Rotarix vaccine is continuing in large phase III trials in Asia and worldwide.

Paediatrics in Singapore: the early days.

Paediatric services in Singapore started at the end of the 19th century. Because of inadequate housing, poor sanitation and inadequate health services, malnutrition and infections were the main problems. Infant mortality was in excess of 300/1000. There were many prominent doctors who worked in extremely demanding environments in order to better the health of children at that time. Over the years, there has been a steady improvement of healthcare in Singapore and this has been mainly due to the foresight and hard work of the pioneers. With excellent primary healthcare and specialised paediatric centres, paediatric services in Singapore are among the best in the world.

Two consecutive partial liver transplants in a patient with Classic Maple Syrup Urine Disease.

Maple syrup urine disease is caused by a deficiency in the branched chain ketoacid dehydrogenase (BCKAD) complex. This results in the accumulation of branched chain amino acids (BCAA) and branched chain ketoacids in the body. Even when aggressively treated with dietary restriction of BCAA, patients experience long term cognitive, neurological and psychosocial problems. Liver transplantation from deceased donors has been shown to be an effective modality in introducing adequate BCKAD activity, attaining a metabolic cure for patients. Here, we report the clinical course of the first known patient with classic MSUD who received two consecutive partial liver grafts from two different living non-carrier donors and his five year outcome posttransplant. We also show that despite the failure of the first liver graft, and initial acute cellular rejection of the second liver graft in our patient, his metabolic control remained good without metabolic decompensation.

Use of soy-protein formulas and soyfood for feeding infants and children in Asia.

Soybeans have been cultivated and consumed in Asia for many centuries. Soy products can be found in all households in Asian countries, and Asian children begin to consume soy formulas and soy products at a very young age. In a study of soy exposure in a group of healthy Singaporean children < 10 y of age, 70% had consumed soy products and of those > 95% had consumed soy products before the age of 18 mo. Soy products are commonly used as food flavorings and for weaning Asian children with lactose intolerance or allergy to cow-milk protein. The widespread use of soy formulas and soy products by Asian children is mainly due to the high nutritive value and palatability of these products.

PIP: Soybean cultivation and consumption in Asia dates back to 2838 BC with a documented evidence of soy farming between the 7th and 11th century. In most Asian countries soy products have become staples, which include soy cooking oil, soy flour, soy sauce, soy beverages, various forms of soybean curd (tofu) and soy infant formulas. A study conducted that evaluates the daily lactose intake among Singaporean children found that approximately 10% of the children were consuming soy formulas. This study examines the use of soy-protein formulas and soyfood for feeding infants and children in Asia. Results confirm the consumption of soy products by 90% of healthy Asian children, with 95% of these children consuming soyfood before 18 months of age. The use of tofu during weaning was more preferred by many Asian mothers because of its availability, soft consistency, high palatability, and high nutritional value. On the other hand, the use of soy formulas has been proven effective in treating children with lactose intolerance compared to cow-milk protein. Furthermore, the use of soy formulas was found to significantly reduce the prevalence of atopic diseases in the first 6 months of life, as well as for children with infantile atopic dermatitis, recurrent bronchiolitis, and bronchial asthma.

The nature of small bowel luminal fluid lactase.

The evidence indicating a mucosal source for the jejunal fluid lactase activity of children is so far inconclusive. Samples of jejunal mucosa and the adjacent fluid were obtained simultaneously from 15 children. Lactase activity was measured at pH 5.9 in mucosa and fluid. Fluid activities showed a significant positive correlation with the activity of the corresponding mucosal homogenate but a stronger correlation was found with an enterocyte microvillous membrane fraction prepared from the same homogenate (r = 0.807 and 0.889, respectively). Kinetic and pH optima studies were consistent with a microvillous membrane origin. Fluid activity and pH optimum were not changed detectably when measured in the presence of an enterocyte lysosomal acid lactase inhibitor. Jejunal fluid lactase activity and its properties closely reflect the microvillous membrane enzyme. Lysosomal acid lactase does not contribute measurably to the total lactase activity of jejunal fluid.

Comparative study on sixteen enzyme-linked immunosorbent assays for the detection of antibodies to bovine herpesvirus 1 in cattle.

Sixteen commercial or non-commercial enzyme-linked immunosorbent assays (ELISAs) for bovine herpesvirus 1 (BHV1)-specific antibody detection in serum were compared using 41 bovine sera of well defined origin. All ELISAs were able to detect correctly most of the antibody negative sera (specificity > or = 92%). The ability, however, to detect specific antibodies varied considerably between ELISAs. Sensitivity, estimated by testing 18 positive sera, ranged between 50% and 100%. Sera with titers of at least 64, as measured by the 24 h virus neutralisation test, were identified as being positive by all ELISAs. Most assays were unable to detect specific IgM antibodies present in sera collected 9 days after experimental infection. Only one assay, an indirect ELISA using undiluted test serum, showed a sensitivity of 100%. This ELISA was found to be 8 times more sensitive than the 24 h neutralisation test and had the unique property of showing a weak consistently positive response with some sera collected from breeding bulls. The findings of this study indicate the need for international standardisation of tests to detect BHV1-specific antibodies in cattle.

A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk.

To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-structural peptide NS3 of pestiviruses. The test can be performed at high reproducibility using undiluted samples. In testing 1000 field serum samples, the ELISA showed a specificity and a sensitivity relative to the virus neutralization test of 99% and 98%, respectively. The blocking ELISA is able to detect specific antibodies in serum obtained 12 days after an acute infection and in serum of vaccinated and challenged animals. A frequency distribution diagram, obtained by testing almost 1800 random Dutch field serum samples, showed a clear separation between a negative population (maximum frequency of the % inhibition at -5%) and a positive population (maximum frequency of the % inhibition at 95%). Based on these data, the prevalence of seropositive animals was 65% (95% confidence interval: 63%-67%). By exchanging plasma- and bulk milk samples between two laboratories (The Netherlands and Denmark), a good overall agreement was found between results obtained with the Ceditest and those obtained with the Danish blocking ELISA as used in the Danish BVDV eradication programme. The results indicate that BVDV infections can reliably be diagnosed by the Ceditest ELISA and that the test is suitable for use in large scale screening and eradication programmes.

Role of glucose homeostatic mechanisms in viral infections.

Though normoglycaemia is thought to indicate adequacy of cellular glucose content, ketoacidosis occurs in viral infections even in the presence of normo- and hyperglycaemia. These wide variations in glucose concentrations may reflect the patients' adaptive abilities in situations where glucose transport is impaired by viruses. Hypoglycaemia would suggest poor adaptation and hyperglycaemia good adaptation. Increased free fatty acid (FFA) concentrations and enzyme activity are probable adaptive mechanisms. If so, they should decrease with hyperglycaemia-producing infusions. Profiles of glucose, FFA, enzymes, and fever over 19 days in 24 children with viral infections are reported. On admission 87.5% were normo- or hyperglycaemic with increased FFA, AST, LDH, and fever (P less than 0.005) when compared with values 19 days later. With infusions that produced hyperglycaemia, there was clinical recovery with a decrease in FFA and enzyme activity. The hyperglycaemia observed in 56.5% therefore points to glucose homeostatic mechanisms being geared to maintain the intracellular milieu. Hence normoglycaemia does not always indicate cellular glucose adequacy.

Successful listing of patients for liver transplant was related to participation of referring doctor in the transplant program.

INTRODUCTION: Experience with liver transplantation is limited in many parts of Asia. Therefore, patients from nontransplant centers may not be referred in a timely fashion for transplants. Our aim was to evaluate the pattern of referral for liver transplantation and their outcomes in Singapore. METHODS: Consecutive patients referred from 1990 to 2001 were reviewed. Patients from any hospital in Singapore (or the region) could be referred to the program. They were discussed at the weekly meetings. Appropriate patients were placed on the waiting list. "Pending" indicated that the disease was early or there were unsettled medical or social issues. Unsuitable patients were "rejected" for transplant. RESULTS: There were 385 patients referred over a 12-year period. Hepatitis B cirrhosis and hepatocellular carcinoma (HCC) were the most common indications among adults, whereas biliary atresia was the most common for children. Pediatric patients were more likely than adult patients to be listed for transplant (53/76 vs 106/309, P < .001). Patients referred by regular attendees of the program were more likely to be accepted than nonattendees (38% vs 25%, P = .04). "Disease too early", "advanced HCC", and "refusal by family members" were the most common reasons for rejection. CONCLUSION: Members of the Liver Transplant Program were more likely to refer suitable patients for transplant at the appropriate time. Better interaction between gastroenterologists inside and outside the transplant program would help to improve the timing of referrals for liver transplantation, and hence, patient survival.