Clinical phenotype and molecular analysis of a homozygous ABCB11 mutation responsible for progressive infantile cholestasis.

The bile salt export pump (BSEP) plays an important role in biliary secretion. Mutations in ABCB11, the gene encoding BSEP, induce progressive familial intrahepatic cholestasis type 2 (PFIC2), which presents with severe jaundice and liver dysfunction. A less severe phenotype, called benign recurrent intrahepatic cholestasis type 2, is also known. About 200 missense mutations in ABCB11 have been reported. However, the phenotype-genotype correlation has not been clarified. Furthermore, the frequencies of ABCB11 mutations differ between Asian and European populations. We report a patient with PFIC2 carrying a homozygous ABCB11 mutation c.386G>A (p.C129Y) that is most frequently reported in Japan. The pathogenicity of BSEPC129Y has not been investigated. In this study, we performed the molecular analysis of this ABCB11 mutation using cells expressing BSEPC129Y. We found that trafficking of BSEPC129Y to the plasma membrane was impaired and that the expression of BSEPC129Y on the cell surface was significantly lower than that in the control. The amount of bile acids transported via BSEPC129Y was also significantly lower than that via BSEPWT. The transport activity of BSEPC129Y may be conserved because the amount of membrane BSEPC129Y corresponded to the uptake of taurocholate into membrane vesicles. In conclusion, we demonstrated that c.386G>A (p.C129Y) in ABCB11 was a causative mutation correlating with the phenotype of patients with PFIC2, impairment of biliary excretion from hepatocytes, and the absence of canalicular BSEP expression in liver histological assessments. Mutational analysis in ABCB11 could facilitate the elucidation of the molecular mechanisms underlying the development of intrahepatic cholestasis.

Combined genetic analyses can achieve efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.

AIM: We evaluated combined genetic analyses with targeted next-generation sequencing (NGS), multiplex ligation probe amplification (MLPA) of Jagged1 (JAG1) genes and microarray comparative genomic hybridisation (CGH) in subjects with Alagille syndrome, incomplete clinical features of Alagille syndrome and biliary atresia. METHODS: Subjects recruited from April 2013 to December 2015 underwent a targeted NGS analysis, including JAG1 and Notch homolog 2 (NOTCH2). If no mutations were detected in JAG1 or NOTCH2, or if copy number variations were suggested by the NGS analysis, we performed an MLPA analysis of JAG1. We also performed a microarray CGH analysis with whole-exon deletion detected by the MLPA analysis. RESULTS: We analysed 30 subjects with Alagille syndrome, nine with incomplete Alagille syndrome and 17 with biliary atresia and detected pathogenic mutations in JAG1 or NOTCH2 in 24/30 subjects with Alagille syndrome and in 4/9 subjects with incomplete Alagille syndrome. No pathogenic mutations were detected in subjects with biliary atresia. The frequency of JAG1 mutations was as follows: single nucleotide variants (51.9%), small insertion or deletion (29.6%) and gross deletion (18.5%). CONCLUSION: Combined genetic analyses achieved efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.

Molecular Genetic Dissection and Neonatal/Infantile Intrahepatic Cholestasis Using Targeted Next-Generation Sequencing.

OBJECTIVES: To ascertain a molecular genetic diagnosis for subjects with neonatal/infantile intrahepatic cholestasis (NIIC) by the use of next-generation sequencing (NGS) and to perform a genotype-phenotype correlation. STUDY DESIGN: We recruited Japanese subjects with NIIC who had no definitive molecular genetic diagnosis. We developed a diagnostic custom panel of 18 genes, and the amplicon library was sequenced via NGS. We then compared clinical data between the molecular genetically confirmed subjects with NIIC. RESULTS: We analyzed 109 patients with NIIC ("genetic cholestasis," 31 subjects; "unknown with complications" such as prematurity, 46 subjects; "unknown without complications," 32 subjects), and a molecular genetic diagnosis was made for 28 subjects (26%). The rate of positive molecular genetic diagnosis in each category was 22 of 31 (71%) for the "genetic cholestasis" group, 2 of 46 (4.3%) for the "unknown with complications" group, and 4 of 32 (12.5%) for the "unknown without complications" group. The grouping of the molecular diagnoses in the group with genetic cholestasis was as follows: 12 with Alagille syndrome, 5 with neonatal Dubin-Johnson syndrome, 5 with neonatal intrahepatic cholestasis caused by citrin deficiency, and 6 with progressive familial intrahepatic cholestasis or benign recurrent intrahepatic cholestasis with low gamma-glutamyl transpeptidase levels. Several clinical datasets, including age of onset, direct bilirubin, and aminotransferases, were significantly different between the disorders confirmed using molecular genetic diagnosis. CONCLUSION: Targeted NGS can be used for molecular genetic diagnosis in subjects with NIIC. Clinical diagnosis should be accordingly redefined in the view of molecular genetic findings.

Use of serial assessment of disease severity and liver biopsy for indication for liver transplantation in pediatric Epstein-Barr virus-induced fulminant hepatic failure.

The decision to perform liver transplantation (LT) in patients with Epstein-Barr virus (EBV)-induced fulminant hepatic failure (FHF) relies on a precise assessment of laboratory and pathological findings. In this study, we analyzed clinical and laboratory data as well as the pathological features of the liver in order to evaluate the pathogenesis and the need for LT in 5 patients with EBV-induced FHF. According to the King's College criteria, the Acute Liver Failure Early Dynamic (ALFED) model, and the Japanese criteria (from the Acute Liver Failure Study Group of Japan), only 1 patient was considered to be a candidate for LT. However, explanted liver tissues in 3 cases exhibited massive hepatocellular necrosis together with diffuse CD8-positive T cell infiltration in both the portal area and the sinusoid. EBV was detected in the liver, plasma, and peripheral blood mononuclear cells (PBMNCs). In 2 cases indicated to be at moderate risk by the ALFED model, liver biopsy showed CD8-positive and EBV-encoded RNA signal-positive lymphocytic infiltration predominantly in the portal area, but massive hepatocellular necrosis was not observed. These patients were treated with immunosuppressants and etoposide under the diagnosis of EBV-induced hemophagocytic lymphohistiocytosis or systemic EBV-positive T cell lymphoproliferative disease of childhood. EBV DNA was detected at a high level in PBMNCs, although it was negative in plasma. On the basis of the pathological analysis of the explanted liver tissues, LT was proposed for the restoration of liver function and the removal of the EBV-infected lymphocytes concentrated in the liver. Detecting EBV DNA by a quantitative polymerase chain reaction in plasma and PBMNCs was informative. An accurate evaluation of the underlying pathogenesis is essential for developing a treatment strategy in patients with EBV-induced FHF.

Stool color card screening for early detection of biliary atresia and long-term native liver survival: a 19-year cohort study in Japan.

OBJECTIVE: To evaluate the sensitivity and specificity of a stool color card used for a mass screening of biliary atresia conducted over 19 years. In addition, the age at Kasai procedure and the long-term probabilities of native liver survival were investigated. STUDY DESIGN: From 1994 to 2011, the stool color card was distributed to all pregnant women in Tochigi Prefecture, Japan. Before or during the postnatal 1-month health checkup, the mothers returned the completed stool color card to the attending pediatrician or obstetrician. All suspected cases of biliary atresia were referred for further examination. Diagnosis was confirmed by laparotomy or operative cholangiography for high-risk cases before the Kasai procedure. Patients with biliary atresia were followed from the date of their Kasai procedure until liver transplantation, death, or October 31, 2013, whichever comes sooner. RESULTS: A total of 313,230 live born infants were screened; 34 patients with biliary atresia were diagnosed. The sensitivity and specificity of stool color card screening at the 1-month check-up was 76.5% (95% CI 62.2-90.7) and 99.9% (95% CI 99.9-100.0), respectively. Mean age at the time of Kasai procedure was 59.7 days. According to Kaplan-Meier analysis, the native liver survival probability at 5, 10, and 15 years was 87.6%, 76.9%, and 48.5%, respectively. CONCLUSIONS: The sensitivity and specificity of the stool color card have been demonstrated by our 19-year cohort study. We found that the timing of Kasai procedure and long-term native liver survival probabilities were improved, suggesting the beneficial effect of stool color card screening.

Nosocomial transmission of varicella to a healthcare provider positive for anti-varicella zoster virus antibodies: nonprotective positivity with an immune adherence hemagglutination assay.

A 24-year-old male healthcare provider, having attended a varicella patient 2 weeks before, developed varicella himself. He had shown a positive result for anti-VZV antibodies measured with an immune adherence hemagglutination assay (1:4) 1 year before. The present case shows that a positive result with this assay does not necessarily indicate protection against VZV infection.

Hypergastrinemia and a duodenal ulcer caused by gastric duplication.

BACKGROUND: Hypergastrinemia and the resultant peptic ulcer related to an enteric duplication has been quite rarely reported in the literature. CASE PRESENTATION: We herein report the case of a 4-year-old girl who presented with hypergastrinemia and a duodenal ulcer at 2 years of age. She had been followed up with a proton pump inhibitor, which resulted in resolution of the ulcer; however, unexplained hypergastrinemia had continued. A cystic lesion at the antrum was discovered at 4 years of age, which we suspected to be a gastric duplication. After we resected the lesion, the hypergastrinemia resolved without recurrence of the duodenal ulcer. The histology was compatible with a gastric duplication, and the lumen was lined with antral mucosa that strongly stained positive for gastrin. We presumed that the antral mucosa inside the duplication in our case had no hydrogen ion feedback inhibition of gastrin release from gastrin cells and increased release of gastrin from the mucosa inside the duplication led to the duodenal ulcer. Only two cases have been reported in the literature that had hypergastrinemia related to enteric duplication. CONCLUSION: Gastric duplication should be included in the differential diagnosis of sustained hypergastrinemia in children.

Surgical site infection in living-donor liver transplant recipients: a prospective study.

BACKGROUND: Infection is a constant threat for the living-donor liver transplantation (LDLT) recipients, although little information is available on the occurrence of infection in such patients. METHODS: One hundred and thirteen consecutive LDLT recipients were prospectively followed for the presence of surgical site infections (SSIs) defined by CDC from April 2001 to March 2002. Risk factors for SSIs were evaluated by univariate and multivariate analysis. RESULTS: Of the 113 LDLT recipients, 42 (37%) developed 57 episodes of SSIs (21 intraabdominal abscess, 20 peritonitis, 8 cholangitis, and 9 wound). Of the 57 episodes, 29 (51%) had secondary bacteremia in 19 patients. Causative pathogens, including 17 episodes of polymicrobial infections, were 37 gram-positive cocci (17 Staphylococcus aureus, 16 Enterococcus spp., and 4 others), 40 gram-negative rods (25 Enterobacteriaceae, 13 Pseudomonas aeruginosa, and 4 others), and 2 Candida albicans. Univariate analysis revealed that ABO incompatibility and repeat surgery were associated with the development of SSIs. Also, univariate analysis revealed that adult recipients, ABO incompatibility, total operation duration, repeat surgery, and NNIS risk index were associated with secondary bacteremia. Multivariate analysis revealed that ABO incompatibility (OR: 14.0; 95% CI, 2.52-77.2) and repeat surgery (OR: 9.29; 95% CI, 2.00-43.1) were independently associated with secondary bacteremia. Eleven of the 42 cases (26%) who developed SSIs died. Of these 11 cases, 5 (45%) developed secondary bacteremia within 30 days before death. CONCLUSION: SSIs occurred in 37% of LDLT recipients. ABO incompatibility and repeat surgery increased the risk of developing SSIs with secondary bacteremia, which correlated with poor prognosis.

Type I biliary atresia without extrahepatic biliary cyst.

Currently, magnetic resonance cholangiography (MRC) is used for the differentiation of biliary atresia (BA) from other causes of infantile cholestasis. The authors present a case of type I BA without an extrahepatic biliary cyst in a 2-month-old girl. MRC clearly visualized the patency of the gallbladder, cystic duct, and hepatic ducts with disappearance of the common bile duct. Intraoperative cholangiography demonstrated a cloudy appearance of the intrahepatic bile ducts, confirming the diagnosis of type I BA. We believe that this is the first reported case of type I BA without an extrahepatic biliary cyst diagnosed by MRC.

Pre-transplant risk factors predicting post-transplant cytomegalovirus infection in liver transplant recipients.

Cytomegalovirus (CMV) infection causes significant morbidity and mortality among transplant recipients. Although it is still not clear if a preemptive strategy is superior to a prophylactic strategy, many transplant programs elect for preemptive treatment for post-transplant CMV infection. In order to improve the preemptive strategy, we analyzed a series of liver recipients by means of quantitative real-time polymerase chain reaction (PCR). Ninety-one liver transplant recipients were monitored by real-time PCR for CMV, and the results were analyzed in terms of preoperative conditions. Multivariate analysis revealed fulminant hepatic failure as an underlying disease (odds ratio, 6.8; 95% CI, 1.2-39.2), while an ABO-incompatible graft (odds ratio, 5.0; 95% CI, 1.3-19.1), and a serological combination of the donor (D) being positive with the recipient (R) being negative for CMV (D+/R-) (odds ratio, 5.8; 95% CI, 1.3-26.0) were independently associated with the development of significant CMV infection. Patients with risk factors had higher peak CMV DNA concentrations than those without, and developed CMV infections faster (P = 0.0002). Screening of recipients according to risk factors and PCR monitoring may result in an optimization of the preemptive strategy.

Prolonged use of dexmedetomidine in an infant with respiratory failure following living donor liver transplantation.

We used dexmedetomidine for more than 2 months in a mechanically ventilated infant without serious adverse effects. An infant with liver cirrhosis of unknown cause underwent living donor liver transplantation at the age of 9 months. Long-term mechanical ventilation was required postoperatively, and midazolam with fentanyl had been used to sedate the patient. They required increase to 1.7 mg.kg(-1).h(-1) and 3.5 microg.kg(-1).h(-1), respectively, which were still inadequate. On postoperative day 29, dexmedetomidine was added. The rate of dexmedetomidine infusion was increased gradually to 1.4 microg.kg(-1).h(-1). It was discontinued temporarily to exclude drug-induced liver dysfunction. However, without dexmedetomidine, adequate sedation level was unattainable. Liver dysfunction was likely to be attributed to cytomegalovirus infection and after restarting dexmedetomidine, the respiratory condition improved. He was extubated 10 weeks after the operation. Dexmedetomidine was successfully tapered off over the following 2 weeks with no signs of withdrawal. Dexmedetomidine was a useful sedative for an infant who required mechanical ventilation for a prolonged period of time.

Isothermal RNA sequence amplification method for rapid antituberculosis drug susceptibility testing of Mycobacterium tuberculosis.

RNA transcript quantification by an isothermal sequence amplification reaction was evaluated for susceptibility testing of 15 Mycobacterium tuberculosis strains. Agreement with the proportion method on Ogawa egg medium and the BACTEC MGIT 960 system was 100 and 87% for rifampin, 93 and 100% for isoniazid, 60 and 53% for ethambutol, and 80 and 80% for streptomycin, respectively.

Can BacT/Alert FA and FN blood culture bottles increase the recovery of microorganisms in the clinical laboratory?

To evaluate whether the use of BacT/Alert FA and FN blood culture bottles increases the yield of microorganisms, we performed a before-after study. BacT/Alert standard aerobic (AE) and anaerobic (AN) bottles were used from January 1999 to May 2001 (before period). FA and FN bottles were used from May 2001 to March 2003 (after period). A total of 7796 AE, 7807 AN, 4798 FA, and 4787 FN bottles were processed. There were 742 (9.5%) AE-, 598 (7.7%) AN-, 521 (10.7%) FA-, and 396 (8.3%) FN-positive bottles. From these positive bottles 776, 631, 585, and 487 microorganisms were isolated, respectively. Among the isolated microorganisms, 58 (7.5%) and 59 (10.1%) Candida species were isolated from AE and FA bottles, respectively, and 17 (2.7%) and 21 (4.3%) obligate anaerobes were isolated from AN and FN bottles, respectively. We conclude that BacT/Alert FA and FN bottles showed a higher percentage of positivity for microorganisms, in particular for Candida species and obligate anaerobes.

National surveillance of species distribution in blood isolates of Candida species in Japan and their susceptibility to six antifungal agents including voriconazole and micafungin.

OBJECTIVES: The aim of this study was to evaluate species distribution and antifungal susceptibility of Candida blood isolates in Japan. METHODS: In a 1 year surveillance programme, 535 Candida blood isolates were collected. Identification of species was followed by examination with the broth microdilution method, as described in NCCLS M27-A2, of antifungal susceptibility to six agents, including voriconazole and micafungin, with readings after 24 and 48 h of incubation. RESULTS: The overall species distribution was: 41% Candida albicans, 23% Candida parapsilosis, 18% Candida glabrata, 12% Candida tropicalis and 2% Candida krusei. The concentrations of fluconazole necessary to inhibit 90% of the isolates (MIC(90)) at 24/48 h were 0.25/1 mg/L for C. albicans, 0.5/2 mg/L for C. parapsilosis, 4/32 mg/L for C. glabrata and 4/>128 mg/L for C. tropicalis. Percentages of fluconazole resistance were 1.8% for C. albicans, 0.8% for C. parapsilosis, 5.2% for C. glabrata and 3.2% for C. tropicalis, taking the tendency of trailing growth of C. tropicalis into account. MIC(90) of voriconazole was 0.5 mg/L, although 35% of isolates less susceptible (>/=16 mg/L) to fluconazole showed resistance (>/=2 mg/L). Micafungin was very active against all species (MIC(90), 0.03 mg/L) except for C. parapsilosis (MIC(90), 2 mg/L). CONCLUSIONS: These data suggest that, in Japan, the species distribution of Candida bloodstream infections and the fluconazole resistance rate are similar to those reported previously in North America and Europe. Voriconazole and micafungin appear to have strong in vitro activity against Candida blood isolates, although continuing surveillance and further clinical research are needed.

Whole-blood counting immunoassay as a short-turnaround test for detection of hepatitis B surface antigen, anti-hepatitis C virus antibodies, and anti-Treponema pallidum antibodies.

Whole-blood samples were used for a counting immunoassay (CIA) with the aim of developing a short- turnaround test. After optimization of the CIA, hepatitis B surface antigen (HBsAg), anti-hepatitis C virus antibodies (anti-HCV), and anti-Treponema pallidum antibodies (anti-TP) were detected as efficiently as by an enzyme immunoassay (EIA) with serum samples. The correlations between whole-blood CIA and serum EIA were 99.8, 97.1, and 99.4% for HBsAg, anti-HCV, and anti-TP, respectively. Whole-blood CIA may be of value when rapid screening of many samples is required.

Biliary bacteria in living related liver transplant recipients: microbiology and rapid detection system using flow cytometry.

Since cholangitis is a major complication in living related liver transplant (LRLT) recipients, rapid detection of biliary bacteria is necessary for the management of patients. We have developed a screening method for the detection of biliary bacteria using flow cytometry (FCM). Two hundred and seventy eight bile samples were obtained from 50 patients with biliary drainage tubes after LRLT at Kyoto University Hospital between July and September 2001. Of the 278 samples, 165 (59.3%) were culture-positive. The most common isolates were Enterococcus species, Pseudomonas species, Staphylococcus species, Klebsiella species, and Candida species. As the original FCM system was inadequate for specifically detecting bacteria in bile samples, we established the most appropriate gate and cut-off value from the particle distribution represented on scattergram of the forward-scattered light and fluorescent light intensity. The 3% cut-off value was most preferably related to the culture results. The FCM system detected biliary bacteria with a sensitivity of 93.9%, specificity of 81.4%, positive predictive value of 88.1%, negative predictive value of 90.2%, false-positive rate of 7.6%, false-negative rate of 3.6%, and percent agreement of 88.9% between FCM and culture. Therefore, FCM can be a useful method in clinical laboratories for the rapid screening for biliary bacteria in LRLT recipients.

Detection of bacteria and fungi in BacT/Alert standard blood-culture bottles.

Incubation periods of aerobic (AE) and anaerobic (AN) blood-culture bottles with the BacT/Alert system were assessed in our laboratory. We reviewed the results of 6229 blood-culture sets collected at Kyoto University Hospital from January 1999 to December 2000. Of these sets, 731 (11.7%) were positive for bacteria or yeast. Excluding 87 sets with growth evidence on arrival, of the 644 positive blood-culture sets from 341 patients, a total of 691 organisms were isolated. Of the 691 organisms, 413 (59.8%) were recovered from both bottles, 206 (29.8%) were recovered only from the AE bottle, and 72 (10.4%) were recovered only from the AN bottle. The AE bottle was significantly superior to the AN bottle in terms of both recovery rate and detection time for overall organisms, but there was no significant difference in detection time for facultative anaerobic bacteria between the two bottles. Of the 691 organisms, 530 (76.7%) were classified as usual pathogens. Of the 530 usual pathogens, 501 (94.5%) were recovered in at least one bottle of each set within the first 3 days, and 523 (98.7%) within the first 5 days of incubation. Twenty-nine organisms initially isolated on day 4 or later were recovered from 19 patients. Of these, chart reviews indicated that 21 organisms recovered from 11 patients were considered clinically significant bacteria, and the reviews also revealed that no patient had a treatment plan altered based on the results of positive blood culture. Seven organisms initially isolated on day 6 or later were recovered from 7 patients. Chart reviews revealed that 5 of these organisms from 5 patients were considered to be clinically significant. In conclusion, if the incubation period had been less than 3 days, 11 patients with clinically significant bacteremia or fungemia, (3.2% of all patients with bacteremia or fungemia) would have been undiagnosed. Similarly, with an incubation period of 5 days, 5 such patients (1.5%) would have been undiagnosed.

Significance of serial real-time PCR monitoring of EBV genome load in living donor liver transplantation.

BACKGROUND: Quantitative analysis of the Epstein-Barr virus (EBV) genome has been recently reported to be helpful for early identification of EBV viremia which could reduce the risk of post-transplantation lymphoproliferative disorder (PTLD). AIM: To demonstrate the significance of serial monitoring of EBV genome load by real-time quantitative polymerase chain reaction (PCR) after living donor liver transplantation. METHODS: From March 1999 to April 2000, the EBV genome load in peripheral blood mononuclear cells (PBMNC) was measured serially in a total of 15 recipients of living donor liver transplantation (LDLT) who had a symptomatic EBV infection. RESULTS: In 15 patients, the mean values of the highest EBV DNA levels from the patients who had fever, URS, diarrhea, ascites, lymphadenopathy and PTLD were 36 232, 16 040, 15 968, 2485, 336 858 and 60 486 copies/microg DNA, respectively. Patients were treated by reduction or discontinuation of immunosuppressives and/or antiviral agents. The EBV DNA levels decreased in all these patients following the recovery from their symptoms. We encountered two cases of PTLD during this study period. One of them was referred to us after the onset of PTLD and one had been undergoing aggressive immunosuppression treatment for severe rejection. Both were successfully treated. CONCLUSIONS: Serial quantitative analysis of the EBV genome load by means of real-time PCR are thought to be useful for preventing PTLD through adjustment of the immunosuppression level in response to the viral genome load following symptomatic EBV infection.