Biology, systematics, and clinical manifestations of Zygomycota infections.

Fungi cause opportunistic, nosocomial, and community-acquired infections. Among fungal infections (mycoses) zygomycoses are exceptionally severe, with a mortality rate exceeding 50%. Immunocompromised hosts, transplant recipients, and diabetic patients with uncontrolled keto-acidosis and high iron serum levels are at risk. Zygomycota are capable of infecting hosts immune to other filamentous fungi. The infection often follows a progressive pattern, with angioinvasion and metastases. Moreover, current antifungal therapy frequently has an unfavorable outcome. Zygomycota are resistant to some of the routinely used antifungals, among them azoles (except posaconazole) and echinocandins. The typical treatment consists of surgical debridement of the infected tissues accompanied by amphotericin B administration. The latter has strong nephrotoxic side effects, which make it unsuitable for prophylaxis. Delayed administration of amphotericin and excision of mycelium-containing tissues worsens survival prognoses. More than 30 species of Zygomycota are involved in human infections, among them Mucorales is the most abundant. Prognosis and treatment suggestions differ for each species, which makes fast and reliable diagnosis essential. Serum sample PCR-based identification often gives false-negative results; culture-based identification is time-consuming and not always feasible. With the dawn of Zygomycota sequencing projects significant advancement is expected, as in the case of treatment of Ascomycota infections.

DNA barcoding in Mucorales: an inventory of biodiversity.

The order Mucorales comprises predominantly fast-growing saprotrophic fungi, some of which are used for the fermentation of foodstuffs but it also includes species known to cause infections in patients with severe immune or metabolic impairments. To inventory biodiversity in Mucorales ITS barcodes of 668 strains in 203 taxa were generated covering more than two thirds of the recognised species. Using the ITS sequences, Molecular Operational Taxonomic Units were defined by a similarity threshold of 99 %. An LSU sequence was generated for each unit as well. Analysis of the LSU sequences revealed that conventional phenotypic classifications of the Mucoraceae are highly artificial. The LSU- and ITS-based trees suggest that characters, such as rhizoids and sporangiola, traditionally used in mucoralean taxonomy are plesiomorphic traits. The ITS region turned out to be an appropriate barcoding marker in Mucorales. It could be sequenced directly in 82 % of the strains and its variability was sufficient to resolve most of the morphospecies. Molecular identification turned out to be problematic only for the species complexes of Mucor circinelloides, M. flavus, M. piriformis and Zygorhynchus moelleri. As many as 12 possibly undescribed species were detected. Intraspecific variability differed widely among mucorealean species ranging from 0 % in Backusella circina to 13.3 % in Cunninghamella echinulata. A high proportion of clinical strains was included for molecular identification. Clinical isolates of Cunninghamella elegans were identified molecularly for the first time. As a result of the phylogenetic analyses several taxonomic and nomenclatural changes became necessary. The genus Backusella was emended to include all species with transitorily recurved sporangiophores. Since this matched molecular data all Mucor species possessing this character were transferred to Backusella. The genus Zygorhynchus was shown to be polyphyletic based on ITS and LSU data. Consequently, Zygorhynchus was abandoned and all species were reclassified in Mucor. Our phylogenetic analyses showed, furthermore, that all non-thermophilic Rhizomucor species belong to Mucor. Accordingly, Rhizomucor endophyticus was transferred to Mucor and Rhizomucor chlamydosporus was synonymised with Mucor indicus. Lecto-, epi- or neotypes were designated for several taxa.

Epstein-Barr virus DNA load in peripheral blood mononuclear cells and whole blood from pediatric transplant recipients.

Monitoring of circulating Epstein-Barr virus (EBV) DNA in pediatric transplant patients has been shown to be useful in post-transplant patient management. It still remains unclear which blood sample type is more suitable, and how EBV DNA levels in whole blood (WB) correlate with those in peripheral blood mononuclear cells (PBMCs). The aim of this study was to compare EBV DNA load in WB and PBMCs of pediatric transplant recipients. After liver, kidney, or combined liver-kidney transplantation, 172 matched WB and PBMCs samples were collected from 84 children (130 samples from 42 patients consisted of multiple collections). The EBV DNA level in PBMCs was determined by home-made real-time polymerase chain reaction using TaqMan chemistry. In parallel, the viral load (VL) in WB was measured by a commercial LightCycler EBV Quant Kit. The EBV DNA levels and dynamics of VL changes were assessed and compared between WB and PBMCs. The overall correlation between EBV DNA level in PBMCs and WB was statistically significant and high, r(2) =0.87 (P<0.001). However, the sensitivity of EBV detection was lower in WB (93.9%). Longitudinal analysis of EBV DNA load dynamics in PBMCs and WB indicated that EBV DNA load fluctuations were larger in WB, but the trend of decreases and increases, with minor exceptions, was similar in both sample types. The high correlation of EBV DNA levels, as well as the similar dynamics of EBV DNA changes in both sample types, make WB a good alternative to EBV DNA monitoring in PBMCs of pediatric transplant recipients. However, the subtle increase of the VL may be detected earlier in PBMCs.

Older age of rheumatoid arthritis onset is associated with higher activation status of peripheral blood CD4(+) T cells and disease activity.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, with a clinical manifestation both systemic and in joints. It has been suggested that age at disease onset and/or patients' age have influence on disease activity and clinical outcome. The reasons for the different course of RA in older people are not known; however, the activation status of peripheral blood lymphocytes could be responsible. Our aim was to relate expression of activation markers in peripheral blood CD4(+) T cells of RA patients with patients' age and/or onset age and disease activity measured by DAS28. Seventy RA patients were included into the immunological study. Two separation criteria were performed: based on age of RA onset and on the biological age of patients. We examined different activation markers, CD69, CD25, CD95 and human leucocyte antigen D-related (HLA-DR), on the CD4(+) T cell surface. Division of RA patients in 10-year intervals at 40, 50 and 60 years revealed that RA patients with later disease onset were characterized by higher DAS28. This phenomenon was not limited to the division at 60 years of age but, surprisingly, the major differences were found for the 40-year onset division. Analysis of all four components of DAS28 revealed that disease activity in older disease onset was dependent on all components. Older-onset RA patients had a higher percentage of CD4(+) CD25(+) and CD4(+) CD95(+) T cells. Summarizing the major differences in DAS28 and activation status of CD4(+) T cells observed for onset of disease at 40 years seems to be the most informative about the immunological status of RA patients.

Liver transplantation for severe hepatic graft-versus-host disease in two children after hematopoietic stem cell transplantation.

Chronic graft-versus-host disease (GVHD) is a frequent complication of bone marrow transplantation (BMT). After the skin, the liver is the second, most frequent target of GVHD, which presenting with hyperbilirubinemia, elevated liver enzymes, and coagulopathy. Progressive destruction of small intrahepatic bile ducts causes vanishing bile duct syndrome and leads to end-stage liver disease. We report 2 successful cases of orthotopic liver transplantation performed in children with severe GVHD after hematopoietic stem cell transplantation from a matched unrelated donor (HSCT-MUD).

European biliary atresia registries: summary of a symposium.

Biliary atresia (BA) is a rare but potentially devastating disease. The European Biliary Atresia Registry (EBAR) was set up to improve data collection and to develop a pan-national and interdisciplinary strategy to improve clinical outcomes. From 2001 to 2005, 100 centers from 22 countries registered with EBAR via its website (www.biliary-atresia.com). In June 2006, the first meeting was held to evaluate results and launch further initiatives. During a 5-year period, 60 centers from 19 European countries and Israel sent completed registration forms for a total of 514 BA patients. Assuming the estimated incidence of BA in Europe is 1:18,000 live births, 35% of the expected 1488 patients from all EBAR participating countries were captured, suggesting that reporting arrangements need improvement. At the meeting, the cumulative evaluation of 928 BA patients including patients from other registries with variable follow-up revealed an overall survival of 78% (range from 41% to 92%), of whom 342 patients (37%) have had liver transplants. Survival with native liver ranged from 14% to 75%. There was a marked variance in reported management and outcome by country (e.g., referral patterns, timing of surgery, centralization of surgery). In conclusion, EBAR represents the first attempt at an overall evaluation of the outcome of BA from a pan-European perspective. The natural history and outcome of biliary atresia is of considerable relevance to a European population. It is essential that there is further support for a pan-European registry with coordination of clinical standards, further participation of parent support groups, and implementation of online data entry and multidisciplinary clinical and basic research projects.

Lipid, carbohydrate metabolism, and antioxidant status in children after liver transplantation.

Organ transplantation is a risk factor for atherogenesis that may be related to immunosuppressive therapy. Increased free radical generation may even aggravate atherogenesis. The aim of the study was to assess lipid metabolism in relation to risk factors for atherogenesis as well as carbohydrate metabolism and antioxidant status among children after liver transplantation. We studied 35 children at 3 to 5 years after liver transplant in whom the following parameters were assessed: total cholesterol; triglyceride; high-density lipoprotein cholesterol; low-density lipoprotein cholesterol (LDL-C); very low-density lipoprotein cholesterol; apolipoproteins B, AI, E, lipoprotein (a); vitamin E; glutathione; glucose; insulin; and glutathione peroxidase activity. Three subgroups of patients were assessed according to the immunosuppressive therapy: cyclosporine (CsA), tacrolimus (Tac), or mycophenolate mofetil (MMF) in combination with low-dose CsA or Tac. We observed differences among the subgroups only in total cholesterol (CsA: 131.6 to 285.6; Tac: 144.0 to 181.61; MMF: 132.1 to 181.2) and LDL-C (CsA: 79.4 to 126.9; Tac: 42.2 to 118.8; MMF: 74.2 to 117.3). Lipid metabolism was not significantly disturbed among children after liver transplantation, an observation that does not point to a high risk of atherogenesis. CsA seems to have the strongest untoward effect on cholesterol metabolism. Decreased GSH concentration after liver transplantation may be related to slightly impaired liver function, but GPx activity and vitamin E concentrations remained normal.

Autoimmune hepatitis in transplanted liver.

Liver transplantation is recognized as the appropriate treatment for end-stage liver disease. Four patients undergoing liver transplantation for classical end-stage liver disease developed de novo autoimmune hepatitis (AIH) in the graft. Recurrence of AIH after orthotopic liver transplantation and after reduction in immunosuppressive treatment is reported in one other patient. Markedly elevated serum transaminases were observed, together with an elevated serum IgG and/or globulin fraction and histological feature typical of AIH on liver biopsy.

Surgical treatment of progressive familial intrahepatic cholestasis: comparison of partial external biliary diversion and ileal bypass.

AIMS: Progressive familial intrahepatic cholestasis (PFIC, Byler's disease) is an autosomal recessive disorder resulting in liver fibrosis/cirrhosis and liver insufficiency. Before the 1990s, liver transplantation was the only effective therapy for these children. During the last 12 years, two alternative methods of surgical treatment have been proposed: partial external biliary diversion (PEBD) and ileal bypass procedure (IB), which allow for effective elimination of bile acids accumulated in the body. In this study, we compare the efficacy of these surgical techniques for PFIC. METHODS: During the last 20 years, we have treated 52 children with PFIC. PEBD was done in 21 patients (since 1995), and IB in 5 patients (since 1998), transplantation was performed in 9 patients (since 1990). The efficacy of non-transplantation surgical treatment was assessed by patients' clinical outcome, liver biochemistry, and survival without transplantation during a follow-up period of 12 to 48 months. RESULTS: In 15 out of 21 patients clinical symptoms improved after PEBD and liver function tests normalised (blood bile acids), 1 patient had to be converted to IB due to too high output biliary fistula, 2 patients were transplanted and 3 are considered for transplantation. Out of the 5 children after IB, 4 improved clinically and biochemically, but, after 12 months, symptoms recurred in 3 patients, one patient was converted successfully to PEBD. No significant influence on growth was observed, irrespective of the type of treatment in this group of patients. CONCLUSIONS: PEBD is more effective than IB for the permanent improvement of symptoms of PFIC. Ileal bypass procedure, although initially effective, does not ensure good long-term results in more than 50 % of patients, probably due to intestinal re-absorption of bile acids increasing over time.

Anatomical alteration of the vascular tree observed during living related liver transplantation.

INTRODUCTION: The number of available cadaveric donor organs has reached a plateau. One current solution has been to increase number of living related liver transplantations. MATERIAL AND METHODS: Since October 1999 in the Department of General, Transplant and Liver Surgery, Medical University of Warsaw, 40 living related liver transplantation have been carried out. RESULTS: In 31 (77.5%) cases, a normal arterial supply was observed: the common hepatic artery arose from a celiac trunk. In two cases (5.0%), there was a partial arterial blood supply by the right accessory hepatic artery originating from the superior mesenteric artery. In two cases (5.0%), a right hepatic artery arose completely from the superior mesenteric artery (replaced artery). In one case (2.5%), a common hepatic artery originated from the superior mesenteric artery. In two cases (5.0%), an accessory left segmental artery originated from the left gastric artery. In two cases (5.0%), the function of an absent left hepatic artery was assumed by a replaced left hepatic artery originating from the left gastric artery. In two (5.0%) cases, there were two separate ducts draining the right hemiliver. There were two (5.0%) cases of an accessory duct draining segment IV, originating within the confluence of the right and left hepatic ducts. In one (2.5%) case, the common hepatic duct showed a trifurcation. CONCLUSION: During harvesting from a living donor knowledge of anatomical variants must be used to optomize the liver graft.

Harvesting liver fragments from living-related donors: a single-center experience.

The aim of the study was to estimate the risk of harvesting a liver fragment from a living-related adult donor. Liver fragments were harvested from 44 donors. Liver segments II and III were harvested from 36 donors. Liver segments II, III, IV were harvested from 6 donors, 2 donors gave segments V, VI, VII, and VIII. After preliminary donor selection volumetric assessment of liver segments by computed tomography and arteriography was performed to visualize the cenac trunk and superior mesenteric artery. None of the donors died. No complications were observed during the operation. Only one case, a bile collection, was observed after surgery. We treated this patient with a satisfactory result by sonography-guided drainage. We observed temporary elevation of bilirubin and transaminase levels and a decrease in prothrombin index value. Blood transfusion was not necessary during any of the procedures. Mean hospitalization time after the surgery was 9.4 days. Mean graft weight/recipient weight ratio was 2.54%. The risk of the harvesting liver fragment from a living-related adult donor seems to be minimal.

Hepatobiliary scanning in the diagnosis of biliary atresia.

Early diagnosis is vital in the neonatal cholestasis. The aim of this study was to assess the usefulness of hepatobiliary scanning in the diagnosis of biliary atresia. 33 hepatobiliary scannings performed in 30 children with cholestasis over the last two years were analysed. The mean age at the diagnosis was 6.6 weeks. The investigation was carried out with Multispect camera using intravenous infusion of 99mTc-MBrIDA. In 23 patients there was no passage of the radiolabelled substance into the intestinal tract. In 18 patients biliary atresia was diagnosed. One patient with a clinical suspicion of Alagille syndrome had two scannings performed at the interval of two weeks. In 1 child a common biliary tract cyst with total obstruction of extrahepatic biliary tree was diagnosed. In 18 children with biliary atresia the diagnosis was confirmed during the operation and Kasai procedure was performed. In 2 children the second scanning showed bile drainage. In 3 children intrahepatic cholestasis was diagnosed in addition to the bile passage failure. Hepatobiliary scanning in the diagnosis of neonatal cholestasis was characterised by high sensitivity (100%) but lower specificity (75%). In difficult cases the final diagnosis should be made on a basis of complex clinical, biochemical and radiological techniques and, if necessary, it should be verified by intraoperative cholangiography.

Scintiscanning in diagnostics of hepatopulmonary syndrome in children.

BACKGROUND: Arterial blood oxygenation disturbances accompanying liver cirrhosis are referred to as hepatopulmonary syndrome (HPS). HPS develops due to the formation of intrapulmonary arteriovenous shunts and dilatation of the vascular bed. The aim of the study was to assess the incidence of arteriovenous shunts in children with hepatic cirrhosis and HPS suspected on the basis of clinical signs, qualified for liver transplantation. MATERIAL AND METHODS: The study was carried out in a group of 21 children aged from 1.2 to 17.7 years (mean age 8.4); 8 girls and 13 boys. The patients were diagnosed as follows: biliary cirrhosis due to extrahepatic bile duct impatency--12, post-inflammatory liver cirrhosis due to infection with HVB--2, autoimmune hepatitis--2, fibrosis of the liver--2, alpha 1-antitrypsin deficiency--1, progressive familial intrahepatic cholestasis--1, cystic fibrosis--1. The presence of arteriovenous anastomoses in the lungs was detected by scintiscanning utilizing microspheres (albumin macroaggregates). Under physiologic conditions, technetium-labeled microspheres injected i.v. are accumulated in pulmonary capillaries. Radioactivity detected in other organs (kidneys, brain) indicates the presence of pathologic shunts omitting the alveoli. A percentage ratio of radioactivity detected in the brain to that present in the lungs serves as the index of blood flow through the anastomosis (SI--shunt index). According to Grimon, the mean SI value for healthy children amounts to 0.43%, the values ranging from 1% to 2% are regarded as borderline ones, and those above 2% allow unequivocal diagnosis of HPS. RESULTS: The SI values obtained in the study ranged from 0.06 to 51%. In 7 patients SI exceeded 1%, reaching 1.23% in one, and over 3% in the remaining 6 patients. No correlations were found either between SI value and the etiology of cirrhosis, or between the index and clinical condition of the patient assessed according to Child-Pugh scale. CONCLUSIONS: 1. Scintiscanning revealed the presence of arteriovenous shunts in the lungs of 6/21 (28.6%) investigated patients. 2. No correlation was found between the severity of HPS and the etiology and stage of the underlying disease.

Apolipoprotein E polymorphism in alagille syndrome and progressive familial intrahepatic cholestasis.

The aim of the study was to assess the apolipoprotein E polymorphism (apoE) in two familial cholestatic diseases-Alagille syndrome (AS) and progressive familial intrahepatic cholestasis (PFIC)-and to estimate its association with gallstone formation, cholesterol levels, and response to UDCA treatment. We investigated 16 children with AS age 8.8 +/- 5.7 years (mean +/- SD) and 18 children with PFIC age 6.3 +/- 4.6 years. The frequency of the epsilon-2 allele in AS and PFIC was higher and the frequency of the epsilon-3 allele was lower than in controls. Gallstones were diagnosed in nine children with PFIC and different apoE phenotypes. No association between phenotype and cholesterol levels or response to UDCA therapy was observed in the patients studied. In conclusion, the allele epsilon-2 is overrepresented in AS and PFIC, similar to primary biliary cirrhosis, although this does not seem to contribute to different cholesterol levels, gallstones, and response to UDCA therapy.

Treatment of progressive familial intrahepatic cholestasis: liver transplantation or partial external biliary diversion.

Progressive intrahepatic familial cholestasis (PFIC), previously called Byler's disease, is a syndrome in which children develop severe cholestasis progressing to biliary cirrhosis and chronic liver failure, usually during the first decade of life. Clinical features include jaundice, hepatomegaly, splenomegaly, growth retardation and severe pruritus. Laboratory tests demonstrate elevated bilirubin and bile acids, without an increase in serum gamma-glutamyl-transpeptidase or cholesterol. This study was performed to evaluate our experience with medical therapy as well as two types of surgical treatment used in children with PFIC, particularly partial external biliary diversion (PEBD) as an alternative method of therapy to liver transplantation (OLTx). Between 1979 and 1998 we have treated 46 children with PFIC (27 boys and 19 girls), aged 10 months to 19 yr (at the time of this study). Medical treatment with ursodeoxycholic (UDCA) was used in 39 patients for the period between 6 and 82 months. PEBD (cholecysto-jejuno-cutaneostomy) was performed in 16 patients, OLTx in eight children (including one after unsuccessful PEBD). Retrospective analysis of the clinical course and selected laboratory tests (bilirubin, ASPAT, ALAT, bile acids), and histopathological examinations were performed. Results of treatment were assessed by means of influence of the type of treatment on clinical symptoms, laboratory tests, progress of liver cirrhosis and hepatic failure, as well as physical development and survival. Medical therapy was effective in the long term in four (10%) of the patients resulting in clinical and biochemical normalization. Both surgical methods of therapy of PFIC, PEBD and OLTx, resulted in an 80% success rate and therefore should be used as complementary therapies. In patients before established liver cirrhosis, PEBD should be the first choice of treatment. Patients presenting with cirrhosis or after ineffective PEBD should qualify for OLTx. With this strategy most children with PIFC can be cured.

[Apoptosis and hepatic cell proliferation in autoimmune hepatitis and chronic viral hepatitis C in children: analysis of APO-1/Fas (CD95), bcl-2 and Ki67 expression]. / Apoptoza i proliferacja komórek watrobowych w autoimmunologicznym zapaleniu watroby i przewleklym wirusowym zapaleniu watroby typu C u dzieci: analiza ekspresji APO-1/Fas (CD95), bcl-2 i Ki67.

Hepatocyte damage in autoimmune hepatitis (AIH) and chronic viral hepatitis C (CVH) is attributed to an immune response. We analysed liver biopsy specimens from 4 children with AIH type I, 3 children with AIH type II and 2 children with CVH, using ApopDetek in situ hybridisation method and Mabs anti CD95, Ki67, bcl-2 by means of APAAP technique. The histological appearance of apoptotic bodies in both conditions was similar. The proliferation activity of the hepatocytes was elevated in cases of CVH and less extensive in AIH. Immunohistochemical analysis suggested that the liver damage in AIH and CVH could be mediated by CD95 system as a mechanism of T-cell mediated cytotoxicity.

Diagnosis and monitoring of cytomegalovirus infection after liver transplantation in children.

Cytomegalovirus infection (CMV) complicated the posttransplant course in 9 of 24 children after liver transplantation. We found specific antibodies (IgG and IgM) to be of very low value in diagnosis and monitoring of CMV infection after liver transplantation. Detection of CMV-DNA by PCR method in the blood or urine was very useful for diagnosis, but less for monitoring of the course of disease and its treatment. Measurements of early immediate CMV antigen (IEA), in peripheral blood leucocytes allowed for very early diagnosis of CMV infection and correlated well with the course of disease and response to treatment of the patient.

[Can TNF-alpha and IL-6 be helpful in assessment of chronic hepatitis in children?]. / Czy TNF-alpha i IL-6 moga byc pomocne w ocenie przewleklego zapalenia watroby u dzieci?

The purpose of the work was an assessment of TNF-alpha and Il-6 concentrations in 34 children with diagnosed chronic hepatitis. In all studied patients the values of TNF-alpha and Il-6 concentration were slightly increased. The correlations calculated between TNF-alpha and Il-6 concentrations calculated between TNF-alpha and Il-6 concentrations and laboratory parameters (laboratory indicators of hepatitis activity--AlAT; liver function indicators--prothrombin index, bilirubin concentration, bile acid concentration, alkaline phosphatase activity, anti-pyrin half-life) were non-significant in Spearman non-parametric test (p > 0.005) except for the correlation between albumin and TNF-alpha concentrations. No statistically significant differences of TNF-alpha and Il-6 concentrations were found between groups of patients with active and persistent hepatitis; groups with and without cirrhosis as well as between groups with and without portal hypertension. Normal or slightly increased TNF-alpha and Il-6 concentrations, observed in chronic hepatitis in children should be explained by compensated liver function in such patients.