Pulmonary impairment independently determines mortality in critically ill patients with acute-on-chronic liver failure.

BACKGROUND & AIMS: In ACLF patients, an adequate risk stratification is essential, especially for liver transplant allocation, since ACLF is associated with high short-term mortality. The CLIF-C ACLF score is the best prognostic model to predict outcome in ACLF patients. While lung failure is generally regarded as signum malum in ICU care, this study aims to evaluate and quantify the role of pulmonary impairment on outcome in ACLF patients. METHODS: In this retrospective study, 498 patients with liver cirrhosis and admission to IMC/ICU were included. ACLF was defined according to EASL-CLIF criteria. Pulmonary impairment was classified into three groups: unimpaired ventilation, need for mechanical ventilation and defined pulmonary failure. These factors were analysed in different cohorts, including a propensity score-matched ACLF cohort. RESULTS: Mechanical ventilation and pulmonary failure were identified as independent risk factors for increased short-term mortality. In matched ACLF patients, the presence of pulmonary failure showed the highest 28-day mortality (83.7%), whereas mortality rates in ACLF with mechanical ventilation (67.3%) and ACLF without pulmonary impairment (38.8%) were considerably lower (p < .001). Especially in patients with pulmonary impairment, the CLIF-C ACLF score showed poor predictive accuracy. Adjusting the CLIF-C ACLF score for the grade of pulmonary impairment improved the prediction significantly. CONCLUSIONS: This study highlights that not only pulmonary failure but also mechanical ventilation is associated with worse prognosis in ACLF patients. The grade of pulmonary impairment should be considered in the risk assessment in ACLF patients. The new score may be useful in the selection of patients for liver transplantation.

Comparison of short-course antibiotic therapy of 6 or less days with a longer treatment in patients with cholangitis after liver transplantation.

OBJECTIVES: Stenosis of the biliary anastomosis predisposes liver graft recipients to bacterial cholangitis. Antibiotic therapy (AT) is performed according to individual clinical judgment, but duration of AT remains unclear. METHODS: All liver graft recipients with acute cholangitis according to the Tokyo criteria grade 1 and 2 after endoscopic retrograde cholangiography (ERC) were included. Outcome of patients treated with short AT (<7 days) was compared to long AT (>6 days). Recurrent cholangitis (RC) within 28 days was the primary end point. RESULTS: In total, 30 patients were included with a median of 313 (range 34-9849) days after liver transplantation until first proven cholangitis. Among 62 cases in total, 51/62 (82%) were graded as Tokyo-1 and 11/62 (18%) as Tokyo-2. Overall median duration of AT was 6 days (1-14) with 36 cases (58%) receiving short AT and 26 (42%) receiving long AT. RC was observed in 10 (16%) cases, without significant difference in occurrence of RC in short versus long AT cases. CRP and bilirubin were significantly higher in patients with long AT, while low serum albumin and low platelets were associated with risk of RC. CONCLUSION: A shorter antibiotic course than 7 days shows good results in selected, ERC-treated patients for post-transplantation biliary strictures.

Non-invasive assessment of fibrosis regression and portal hypertension in patients with advanced chronic hepatitis C virus (HCV)-associated liver disease and sustained virologic response (SVR): 3 years follow-up of a prospective longitudinal study.

Long-term effects on cirrhosis and portal hypertension of direct antiviral agent (DAA)-based eradication of hepatitis C virus (HCV) are still under debate. We analysed dynamics of liver and spleen elastography to assess potential regression of cirrhosis and portal hypertension 3 years post-treatment. Fifty-four patients with HCV-associated cirrhosis and DAA-induced SVR were included. Liver and spleen stiffness were measured at baseline (BL), end of treatment (EOT), 24 weeks after EOT (FU24) and 1, 2 and 3 (FU144) years post-treatment by transient liver elastography (L-TE) and point shear wave elastography (pSWE) using acoustic radiation force impulse (ARFI) of the liver (L-ARFI) and spleen (S-ARFI). Biochemical, virological and clinical data were also obtained. Liver stiffness assessed by L-TE decreased between BL [median (range), 32.5(9.1-75) kPa] and EOT [21.3(6.7-73.5) kPa; p < .0001] and EOT and FU144 [16(4.1-75) kPa; p = .006]. L-ARFI values improved between EOT [2.5(1.2-4.1) m/s] and FU144 [1.7(0.9-4.1) m/s; p = .001], while spleen stiffness remained unchanged. Overall, L-TE improved in 38 of 54 (70.4%) patients at EOT and 29 of 38 (76.3%) declined further until FU144, whereas L-ARFI values decreased in 30/54 (55.6%) patients at EOT and continued to decrease in 28/30 (93.3%) patients at FU144. Low bilirubin and high albumin levels at BL were associated with improved L-ARFI values (p = .048) at EOT or regression of cirrhosis (<12.5 kPa) by L-TE at FU144 (p = .005), respectively. Liver stiffness, but not spleen stiffness, continued to decline in a considerable proportion of patients with advanced liver disease after HCV eradication.

Quinolone and Multidrug Resistance Predicts Failure of Antibiotic Prophylaxis of Spontaneous Bacterial Peritonitis.

BACKGROUND: The efficacy of antibiotic prophylaxis to prevent spontaneous bacterial peritonitis (SBP) in patients colonized with multidrug-resistant organisms (MDROs) is unknown. We evaluated the effectiveness of fluoroquinolone-based SBP prophylaxis in an era and area of frequent antibiotic resistance. METHODS: This is a prospective observational study in patients with liver cirrhosis and an indication for fluoroquinolone-based prophylaxis of SBP. Patients were recruited and followed in a large German tertiary reference center with comprehensive microbiological and clinical monitoring performed at baseline and after 30, 60, 90, and 180 days of prophylaxis. RESULTS: Overall, 77 patients received antibiotic prophylaxis for an average of 93 days. Baseline prevalence of colonization with MDROs was high (N = 39, 50.6%). At least one de novo MDRO was detected in 27 patients (35.1%) during antibiotic prophylaxis; 33 patients (42.9%) developed secondary infections, including 14 cases (17.9%) of infections with MDROs, and 13 cases (16.9%) of de novo/recurrent SBP. Thirty patients (39.0%) died during follow-up. Significantly higher risks of SBP development during antibiotic prophylaxis were observed for patients with versus without any apparent MDROs (P = .009), vancomycin-resistant enterococci (P = .008), multidrug-resistant gram-negative bacteria (P = .016), or quinolone-resistant gram-negative bacteria (QR-GNB) (P = .015). In competing risk analysis, QR-GNB were independently associated with prophylaxis failure (hazard ratio, 3.39; P = .045) and infections with QR-GNB were independently associated with death before SBP (subdistribution hazard risk, 6.47; P = .034). CONCLUSIONS: Antibiotic prophylaxis of SBP appears to be less efficient in patients with known MDROs. Regular MDRO screening seems to be useful to tailor treatment of secondary infections and re-evaluate antibiotic prophylaxis in case of selection of quinolone resistance.

An update on the routine application of MALDI-TOF MS in clinical microbiology.

Introduction: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has entered clinical diagnostics and is today a generally accepted and integral part of the workflow for microbial identification. MALDI-TOF MS identification systems received approval from national and international institutions, such as the USA-FDA, and are continuously improved and adopted to other fields like veterinary and industrial microbiology. The question is whether MALDI-TOF MS also has the potential to replace other conventional and molecular techniques operated in routine diagnostic laboratories. Areas covered: We give an overview of new advancements of mass spectral analysis in the context of microbial diagnostics. In particular, the expansion of databases to increase the range of readily identifiable bacteria and fungi, the refined discrimination of species complexes, subspecies, and types, the testing for antibiotic resistance or susceptibility, progress in sample preparation including automation, and applications of other mass spectrometry techniques are discussed. Expert opinion: Although many new approaches of MALDI-TOF MS are still in the stage of proof of principle, it is expectable that MALDI-TOF MS will expand its role in the clinical microbiology laboratory of the future. New databases, instruments and analytical software modules will continue to be developed to further improve diagnostic efficacy.

Revisiting traumatic injury risk and agricultural intensification: Postcranial fracture frequency at Cerro Oreja in the Moche Valley of north coastal Peru.

OBJECTIVES: In Lambert and Welker (2017) we explored the association between subsistence economy and postcranial fracture prevalence, finding that low-intensity agriculturalists exhibited significantly lower fracture rates than foragers or high-intensity agriculturalists. Here, we explore the impacts of sampling strategy on fracture rates in a sample of high-intensity agriculturalists from the Moche Valley, Peru, and further test the hypothesis that postcranial fracture risks are higher for intensive agriculture. MATERIALS AND METHODS: The long bones and clavicles of 102 individuals from an Early Intermediate Period cemetery (400 B.C.-A.D. 200) at Cerro Oreja were examined for healed fractures. Sample composition was manipulated in six ways to investigate the effects of age and element completeness on estimates of fracture prevalence. Fracture rates at Cerro Oreja were then compared to those for other high-intensity agriculturalists. RESULTS: Both skeletal element completeness and age composition were found to influence fracture rate estimates, reflecting the greater likelihood of identifying healed fractures on better-preserved bones and the accrual of injuries with age. The fracture rate of 3.4% at Cerro Oreja was the median value among seven high-intensity agriculturalist samples. The fracture distribution at Cerro Oreja was most similar to that observed at Kulubnarti, Sudan (Kilgore et al., 1997). DISCUSSION: Skeletal element completeness and age composition can impact fracture rates estimated for skeletal samples and should be considered when conducting comparative analyses. All rates calculated for Cerro Oreja are within the range of those obtained for other high-intensity agriculturalists and support previous findings that traumatic injury risk is higher for high-intensity agriculturalists. Similarities between Cerro Oreja and Kulubnarti suggest that rugged terrain may exacerbate fracture risk for agriculturalists, illustrating the costs of intensive agriculture in suboptimal environments.

Identification and Characterization of Clostridium difficile Sequence Type 37 Genotype by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Clostridium difficile multilocus sequence type 37 (ST37), which mainly corresponds to ribotype 017, has been a dominant genotype circulating in China. In this study, we report the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to analyze and characterize 204 C. difficile clinical isolates, including 49 ST37 and 155 non-ST37 isolates collected in China and other countries. The distributions of two major protein peaks (m/z 3,242 and 3,286) were significantly different between ST37 and non-ST37 prototype strains and clinical isolates. This difference was reproducible when analysis was performed on different colonies in different runs. This finding was repeated and confirmed by both bioMérieux Vitek MS and Bruker Microflex LT systems on isolates recovered from a variety of geographic regions worldwide. The combination of the two peaks was present in 47 of 49 ST37 isolates, resulting in a sensitivity of 95.9%. In contrast, the peak combination was absent in 153 of 155 non-ST37 isolates, resulting in a specificity of 98.7%. Our results suggest that MALDI-TOF MS is a rapid and reliable tool to identify C. difficile genotype ST37. Work is in progress to characterize the two molecules having peaks at m/z 3,242 and 3,286, which appear to be specific to C. difficile genotype ST37.

Development of a rapid MALDI-TOF MS based epidemiological screening method using MRSA as a model organism.

In this study we present a method using whole cell MALDI-TOF MS and VITEK MS RUO/SARAMIS as a rapid epidemiological screening tool. MRSA was used as a model organism for setting up the screening strategy. A collection of well-characterised MRSA strains representing the 19 most common Pulsed-Field Gel Electrophoresis (PFGE)-types in the region of South-West Sweden for the past 20 years was analysed with MALDI-TOF MS. A total of 111 MRSA strains were used for creating 19 PFGE-specific Superspectra using VITEK MS RUO/SARAMIS. Prior to performing the final analysis, the 19 Superspectra were combined into ten groups displaying similar peak patterns, hereafter named "MALDI-types". Two-hundred fifty-five MRSA strains were analysed to test the constructed Superspectra/MALDI-type database. Matches to the Superspectra above a threshold of 65% (corresponding to the number of matched peaks in the Superspectrum) were considered as positive assignment of a strain to a MALDI-type. The median peak matching value for correct assignment of a strain to a MALDI-type was 78% (range 65.3-100%). In total, 172 strains (67.4%) were assigned to the correct MALDI-type and only 5.5% of the strains were incorrectly assigned to another MALDI-type than the expected based on the PFGE-type of the strain. We envision this methodology as a cost-efficient step to be used as a first screening strategy in the typing scheme of MRSA isolates, to exclude epidemiological relatedness of isolates or to identify the need for further typing.

Low Serum Levels of (Dihydro-)Ceramides Reflect Liver Graft Dysfunction in a Real-World Cohort of Patients Post Liver Transplantation.

Patients after orthopic liver transplantation (OLT) are at risk of developing graft dysfunction. Sphingolipids (SL's) have been identified to play a pivotal role in the regulation of hepatocellular apoptosis, inflammation and immunity. We aimed to investigate the serum SL profile in a prospective real-world cohort of post-OLT patients. From October 2015 until July 2016, 149 well-characterized post-OLT patients were analyzed. SL's were assessed in serum probes via Liquid Chromatography/Tandem Mass Spectrometry. Twenty-nine (20%) patients had a biopsy proven graft rejection with decreased C20-ceramide (Cer) (p = 0.042), C18-dihydroceramide (DHC) (p = 0.022) and C24DHC (p = 0.060) levels. Furthermore, C18DHC (p = 0.044) and C24DHC (p = 0.011) were significantly down-regulated in patients with ischemic type biliary lesions (ITBL; n = 15; 10%). One-hundred and thirty-three patients (89%) have so far received tacrolimus as the main immunosuppressive agent with observed elevations of C14Cer (p = 0.052), C18Cer (p = 0.049) and C18:1Cer (p = 0.024). Hepatocellular carcinoma (HCC) pre-OLT was associated with increases in C24:1Cer (p = 0.024) and C24:1DHC (p = 0.024). In this large prospective cross-sectional study of patients, post-OLT serum levels of (very-)long chain (dihydro-)ceramides associate with graft rejection, ITBL, tacrolimus intake and HCC pre-OLT. Hence, serum SL's may be indicative of graft complications. Further research is necessary to identify their diverse mechanistic role in regulating immunity and inflammation in patients post-OLT.

Traumatic injury risk and agricultural transitions: A view from the American Southeast and beyond.

OBJECTIVES: Bioarchaeological research has documented a general decline in health with the transition from foraging to farming, primarily with respect to changing patterns of morbidity. Less is known about changes in injury risk, an aspect of health more obviously tied to particular landscapes and behaviors associated with different subsistence regimes. The purpose of this research is to evaluate several hypotheses emerging from the ideal free distribution model (Fretwell & Lucas, ) that predict injury risk based on subsistence-specific practices and land use patterns. MATERIALS AND METHODS: Postcranial fracture frequencies for long bones and clavicles in human skeletal remains from three Southeast U.S. regions permit examination of variability in injury risk among low-intensity (floodplain) farmers. Published data on six hunter-gatherer samples, four low-intensity agriculturalist samples, and six high-intensity agriculturalist samples comprise a comparative sample for examining variability in injury risk across three distinct subsistence traditions. Differences are evaluated using Z scores and the Fisher Exact test, Chi-Square test, and Mann-Whitney U test. RESULTS: While statistically significant differences are apparent among low-intensity farming groups in the Southeast sample, in the global comparison postcranial fractures are significantly less common in low-intensity agriculturalists than in hunter-gatherers or high-intensity agriculturalists. DISCUSSION: The results of this study support the hypothesis that, with respect to traumatic injury risk, low-intensity farming is a risk-averse subsistence strategy in comparison with full-time foraging or high-intensity agriculture. These data suggest that it is not agriculture per se that predicts an increase in this health risk, but rather the mode and intensity of agricultural production, findings that have important ramifications for our understanding of the health consequences of major subsistence transitions.

Lactic acidosis in patients with hepatitis C virus cirrhosis and combined ribavirin/sofosbuvir treatment.

BACKGROUND & AIMS: Sofosbuvir (SOF) based interferon-alfa free antiviral therapy has become the treatment of choice for patients with chronic hepatitis C virus (HCV) infection. Little is known about safety of drug combinations using two nucleos(t)ide polymerase inhibitors in patients with HCV associated advanced cirrhosis. Here, we report frequent occurrence of lactic acidosis associated with acute-on-chronic hepatic decompensation during ribavirin (RBV) plus SOF based antiviral therapy. METHODS: Thirty-five patients with chronic hepatitis C and advanced fibrosis, compensated cirrhosis, and decompensated cirrhosis without and after liver transplantation were treated with SOF based antiviral therapy with and without RBV. Adverse events including lactic acidosis (pH <7.35, lactate >20 mg/dl) were recorded 24 weeks before and during (mean ±SD, 18±11 weeks) antiviral therapy. Efficacy was determined by assessment of serum HCV RNA. RESULTS: We observed severe adverse events in 15/35 (43%) patients before (24 weeks) and in 12/35 (34%) patients during antiviral therapy, the majority in association with acute-on-chronic hepatic decompensation. Lactic acidosis occurred in 5/35 (14%) patients during therapy, while no event of lactic acidosis was observed prior to therapy. Lactic acidosis was associated with hepatic decompensation including renal failure and infection, and was severe (pH <7.3) in two patients. CONCLUSIONS: RBV in combination with SOF based antiviral therapy in patients with HCV associated advanced cirrhosis may be associated with the development of lactic acidosis. Impaired renal function, and higher MELD/Child-Pugh scores were identified as potential risk factors.

Clinical value of on-treatment HCV RNA levels during different sofosbuvir-based antiviral regimens.

BACKGROUND & AIMS: The European Association for the Study of the Liver (EASL) guidelines recommend HCV RNA measurements at specific time points during sofosbuvir(SOF)-therapy. However, it remains unclear, how these results should be interpreted. We aimed to analyze whether on-treatment HCV RNA levels predict relapse comparing the CobasAmpliPrep/CobasTaqMan v2.0 (CAP/CTM) and Abbott RealTime HCV (ART) assays. METHODS: Samples were collected from 298 patients (HCV genotypes; GT1-5) at weeks (w) 0, 1, 2, 4, 8, 12, 16, 20 and 24 during SOF-based therapy at two university clinics and tested for HCV RNA level by CAP/CTM and ART. Patients were treated with SOF/ribavirin (RBV) 12/24 w (n=99), pegylated-interferon-alfa (PegIFN)/SOF/RBV 12 w (n=51), SOF/simeprevir (SMV)±RBV 12 w (n=69) or SOF/daclatasvir±RBV 12/24 w (n=79). RESULTS: HCV RNA levels during the first 4weeks of SOF/RBV therapy were significantly lower in GT3 patients who achieved SVR compared with those who relapsed. All GT3 patients with a week 2 result <45IU/ml by CAP/CTM achieved SVR but only 33% of those with ⩾45IU/ml (p=0.0003). Similar results were documented with ART and 60IU/ml as cut-off (SVR: 100% vs. 29%; p=0.0002). In contrast, HCV RNA levels during early treatment phases were not significantly related to relapse in patients treated with other SOF-based regimens. Residual HCV RNA was frequently detected by ART at later stages of therapy. However, SVR rates remained high in these patients. At the end of SOF/SMV±RBV therapy HCV RNA was detectable with ART in 20% of patients, of whom 92% achieved SVR. CONCLUSIONS: HCV RNA levels assessed at week 2 of SOF/RBV therapy can predict relapse in GT3-patients. Detectable HCV RNA results at later stages during SOF-based therapy may occur frequently with the more sensitive ART. However, this should not lead to treatment extension. LAY SUMMARY: We analyzed the predictive value of hepatitis C virus (HCV) RNA levels measured at different time points for treatment efficacy. We found that the level of HCV RNA measured at week 2 of antiviral therapy can be used to predict treatment success in patients with HCV genotype 3 infection treated with sofosbuvir and ribavirin but not in patients treated with other sofosbuvir-based regimens. Low level HCV RNA is frequently detected by the RealTime HCV assay during later stages of antiviral therapy. However, this is not associated with reoccurrence of HCV RNA after the end of treatment.

A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Analysis.

We describe a simple protocol to inactivate the biosafety level 3 (BSL3) pathogens Brucella prior to their analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. This method is also effective for several other bacterial pathogens and allows storage, and eventually shipping, of inactivated samples; therefore, it might be routinely applied to unidentified bacteria, for the safety of laboratory workers.

A possible role of microRNAs as predictive markers for the recurrence of hepatocellular carcinoma after liver transplantation.

With favourable 5-year survival rates up to 75%, liver transplantation (LT) is the treatment of choice for hepatocellular carcinoma (HCC). Nonetheless, tumour recurrence after LT remains a challenge. The aim of this retrospective study was to develop a predictive score for tumour recurrence after LT by combining clinical parameters with HCC biomarkers (microRNA). A microRNA (miRNA) microarray analysis was used to compare miRNA expression patterns in tissue samples of 40 patients with and without HCC recurrence after LT. In a screening cohort (n = 18), the miRNA analysis identified significant differences in the expression of 13 miRNAs in patients with tumour recurrence. Using the most significant miRNAs in this screening cohort, we could develop a predictive score, which combined the expression levels of miR-214, miR-3187 and the Milan criteria, and we could define low- and high-risk groups for tumour recurrence and death. The above score was evaluated in a second and independent cohort (n = 22). In contrast to the Milan criteria alone, this score was significantly associated with tumour recurrence. Our analysis indicated that the use of a specific miRNA expression pattern in combination with a limited tumour burden as defined by the Milan criteria may lead to a more accurate prediction of tumour recurrence.

Microbial typing by matrix-assisted laser desorption ionization-time of flight mass spectrometry: do we need guidance for data interpretation?

The integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology has revolutionized species identification of bacteria, yeasts, and molds. However, beyond straightforward identification, the method has also been suggested to have the potential for subspecies-level or even type-level epidemiological analyses. This minireview explores MALDI-TOF MS-based typing, which has already been performed on many clinically relevant species. We discuss the limits of the method's resolution and we suggest interpretative criteria allowing valid comparison of strain-specific data. We conclude that guidelines for MALDI-TOF MS-based typing can be developed along the same lines as those used for the interpretation of data from pulsed-field gel electrophoresis (PFGE).

Fusobacterium nucleatum subspecies identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

We explored the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification of Fusobacterium nucleatum subspecies. MALDI-TOF MS spectra of five F. nucleatum subspecies (animalis, fusiforme, nucleatum, polymorphum, and vincentii) were analyzed and divided into four distinct clusters, including subsp. animalis, nucleatum, polymorphum, and fusiforme/vincentii. MALDI-TOF MS with the modified SARAMIS database further correctly identified 28 of 34 F. nucleatum clinical isolates to the subspecies level.

Feasibility of global miRNA analysis from fine-needle biopsy FFPE material in patients with hepatocellular carcinoma treated with sorafenib.

Sorafenib is the standard treatment for patients with advanced hepatocellular carcinoma (HCC). However, the median overall survival (OS) benefit is only ~3 months, and sufficient biomarkers predicting treatment response are not available. The aim of the present study was to evaluate miRNA expression patterns from HCC tissue biopsies as potential biomarkers in patients under sorafenib treatment. Nineteen patients with advanced HCC treated with sorafenib were included. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) liver biopsies. miRNA expression profiling of 818 mature miRNAs was performed using GeneChip® miRNA Array 2.0 (Affymetrix). Global miRNA patterns were assessed using unsupervised hierarchical clustering analysis (UCA), and specific miRNAs with correlation with disease control rate (DCR) or good OS were evaluated by pairwise supervised analyses. UCA divided the patients into three distinct groups by their miRNA expression patterns. However, DCR or OS did not correlate with these sub-groups. We have identified several miRNAs that correlated with either DCR or OS (P<0.05). However, with correction for multiple testing, these results did not reach statistical significance in this small cohort. Global miRNA analysis from very low input RNA deriving from liver biopsies showed distinctive clustering of molecular sub-groups in patients with intermediate and advanced HCC. Clinical response including OS under sorafenib did not correlate with global miRNA expression patterns, but we have identified candidate miRNAs for the prediction of DCR and OS to be evaluated in prospective studies and larger patient cohorts.

Telaprevir drug monitoring during antiviral therapy of hepatitis C graft infection after liver transplantation.

BACKGROUND & AIMS: Recurrence of hepatitis C virus (HCV) infection after orthotopical liver transplantation (OLT) is common and associated with reduced graft and patient survival. The protease inhibitor telaprevir may enhance virological response rates in patients after OLT in combination with pegylated interferon-alfa and ribavirin. Pharmacokinetic studies have shown significant drug-drug interactions between telaprevir and immunosuppression (IS), but telaprevir pharmacokinetics in OLT patients with IS are unknown. Aim of the present study was to analyse telaprevir plasma concentrations in patients with HCV genotype 1 infection after OLT in comparison to patients without OLT and IS. METHODS: Five patients with HCV genotype 1 infection after OLT and 37 HCV genotype 1-infected patients patients without prior OLT were treated with telaprevir 2250 mg daily, ribavirin 1000/1200 mg daily and pegylated interferon-alfa-2a 180 µg once weekly (triple therapy). Telaprevir plasma concentrations were analysed by liquid chromatography-electrospray-ionization-tandem mass spectrometry. HCV RNA was assessed by automatized reverse-transcription polymerase chain-reaction. RESULTS: Median (range) telaprevir plasma concentrations of TW 4, 8 and 12 were 3970 (1980-4430) ng/ml and 2520 (1870-8730) ng/ml in patients after OLT and ciclosporin- or tacrolimus-based IS, respectively, as compared to 2790 (1870-3140) in non-OLT patients (P = 0.3). In one patient with tacrolimus-based IS, telaprevir dose had to be adjusted to achieve virological response. Telaprevir plasma concentrations were steady at treatment weeks 4, 8 and 12 in patients with and without IS. CONCLUSIONS: Telaprevir drug monitoring may be necessary in patients with tacrolimus-based IS in patients with HCV graft infection after OLT.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using the Vitek MS system for rapid and accurate identification of dermatophytes on solid cultures.

The objective of this research was to extend the Vitek MS fungal knowledge base version 2.0.0 to allow the robust identification of clinically relevant dermatophytes, using a variety of strains, incubation times, and growth conditions. First, we established a quick and reliable method for sample preparation to obtain a reliable and reproducible identification independently of the growth conditions. The Vitek MS V2.0.0 fungal knowledge base was then expanded using 134 well-characterized strains belonging to 17 species in the genera Epidermophyton, Microsporum, and Trichophyton. Cluster analysis based on mass spectrum similarity indicated good species discrimination independently of the culture conditions. We achieved a good separation of the subpopulations of the Trichophyton anamorph of Arthroderma benhamiae and of anthropophilic and zoophilic strains of Trichophyton interdigitale. Overall, the 1,130 mass spectra obtained for dermatophytes gave an estimated identification performance of 98.4%. The expanded fungal knowledge base was then validated using 131 clinical isolates of dermatophytes belonging to 13 taxa. For 8 taxa all strains were correctly identified, and for 3 the rate of successful identification was >90%; 75% (6/8) of the M. gypseum strains were correctly identified, whereas only 47% (18/38) of the African T. rubrum population (also called T. soudanense) were recognized accurately, with a large quantity of strains misidentified as T. violaceum, demonstrating the close relationship of these two taxa. The method of sample preparation was fast and efficient and the expanded Vitek MS fungal knowledge base reliable and robust, allowing reproducible dermatophyte identifications in the routine laboratory.

Deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis C virus genotype 1-infected patients.

The preeminent mode of action of the broad-spectrum antiviral nucleoside ribavirin in the therapy of chronic hepatitis C is currently unresolved. Particularly under contest are possible mutagenic effects of ribavirin that may lead to viral extinction by lethal mutagenesis of the hepatitis C virus (HCV) genome. We applied ultradeep sequencing to determine ribavirin-induced sequence changes in the HCV coding region (nucleotides [nt] 330 to 9351) of patients treated with 6-week ribavirin monotherapy (n = 6) in comparison to placebo (n = 6). Baseline HCV RNA levels maximally declined on average by -0.8 or -0.1 log10 IU/ml in ribavirin- versus placebo-treated patients. No general increase in rates of nucleotide substitutions in ribavirin-treated patients was observed. However, more HCV genome positions with high G-to-A and C-to-U transition rates were detected between baseline and treatment week 6 in ribavirin-treated patients in comparison to placebo-treated patients (rate of 0.0041 transitions per base pair versus rate of 0.0022 transitions per base pair; P = 0.049). Similarly, the sensitive detection of low-frequency minority variants by statistical filtering indicated significantly more positions with G-to-A and C-to-U transitions in ribavirin-treated patients than in placebo-treated patients (rate of 0.0331 transitions versus rate of 0.0186 transitions per G/C-containing position at baseline; P = 0.018). In contrast, non-ribavirin-associated A-to-G and U-to-C transitions were not enriched in the ribavirin group (P = 0.152). We conclude that ribavirin exerts a mutagenic effect on the virus in patients with chronic hepatitis C by facilitating G-to-A and C-to-U nucleotide transitions.