Qualitative and quantitative detection of SARS-CoV-2 RNA in untreated wastewater in Western Cape Province, South Africa.

Recent studies have shown that the detection of SARS-CoV-2 genetic material in wastewater may provide the basis for a surveillance system to track the environmental dissemination of this virus in communities. An effective wastewater-based epidemiology (WBE) system may prove critical in South Africa (SA), where health systems infrastructure, testing capacity, personal protective equipment and human resource capacity are constrained. In this proof-of-concept study, we investigated the potential of SARS-CoV-2 RNA surveillance in untreated wastewater as the basis for a system to monitor COVID-19 prevalence in the population, an early warning system for increased transmission, and a monitoring system to assess the effectiveness of interventions. The laboratory confirmed the presence (qualitative analysis) and determined the RNA copy number of SARS-CoV-2 viral RNA by reverse transcription polymerase chain reaction (quantitative) analysis from 24-hour composite samples collected on 18 June 2020 from five wastewater treatment plants in Western Cape Province, SA. The study has shown that a WBE system for monitoring the status and trends of COVID-19 mass infection in SA is viable, and its development and implementation may facilitate the rapid identification of hotspots for evidence-informed interventions.

IP-10 in chronic hepatitis C patients treated with high-dose interferon.

INTRODUCTION: Interferon-g-inducible protein-10 (IP-10) serum levels are associated with IL28B genotype and may predict response to interferon÷ribavirin-based therapy in chronic hepatitis C patients. Our aim was to relate IP-10 levels before and during treatment to treatment outcome, viral HCV-RNA kinetics and IL28B genotype. PATIENTS AND METHODS: A cohort of chronic hepatitis C patients was treated with high-dose interferon for six weeks, followed by standard peginterferon÷ ribavirin for 24 or 48 weeks. IP-10 and HCV-RNA levels were frequently determined before, during and after treatment. RESULTS: IP-10 levels increased from log2.56 pg÷ml at baseline to log3.48 pg÷ml at Day 1 and gradually diminished thereafter. IP-10 levels at any time point were not statistically different between patients with or without sustained viral response (SVR). Patients with IL28B CC genotype had significantly lower baseline IP-10 levels (p = 0.019) and a higher increase of IP-10 levels from baseline to Day 1 than patients with IL28B non-CC genotypes (p = 0.015). Patients with HCV-RNA decline ≥ 2.28log10 at Day 1 had significantly lower baseline IP-10 levels (p = 0.016) and a higher increase of IP-10 levels from baseline to Day1 (p = 0.047) than patients with HCV-RNA decline of < 2.28log10 at Day 1. CONCLUSIONS: In patients treated with high induction dose interferon, IP-10 levels at any time point were not predictive for SVR. Low baseline IP-10 levels and a higher increase of IP-10 levels from baseline to Day 1 were associated with IL28B CC genotype and HCV-RNA decline ≥ 2.28log10 at Day 1. This suggests that, in our cohort, for prediction of SVR the added value of IP-10 to IL28B genotype and early viral kinetics is limited.

Treatment of chronic hepatitis B virus infection - Dutch national guidelines.

The development of this guideline was initiated and coordinated by the Netherlands Association of Gastroenterologists and Hepatologists (Nederlandse Vereniging van Maag-Darm-Leverartsen). The aim is the establishment of national standards in the evaluation and antiviral treatment of patients with chronic hepatitis B virus (HBV) infection. This includes recommendations on the initial evaluation of patients, choice and duration of antiviral therapy, follow-up after antiviral therapy and monitoring of patients not currently requiring antiviral therapy. The initial evaluation of chronic HBV-infected patients should include testing of liver biochemistry, virus serology and abdominal imaging. In patients without cirrhosis, antiviral treatment is recommended for those with a serum HBV DNA of at least 1.0 x 105 c/ml (>or=2.0 x 10(4) IU/ml) in combination with: a) elevation of serum alanine aminotransferase (ALAT) level above twice the upper limit of normal during at least three months, and/or b) histological evidence of porto-portal septa or interface hepatitis on liver histology. In patients with cirrhosis, antiviral treatment is recommended if serum HBV DNA is 1.0 x 10(4)c/ml (>or=2.0 x 10(3) IU/ml) or higher, independent of ALAT levels or histological findings. If the patient has decompensated cirrhosis, antiviral treatment is recommended if serum HBV DNA is 1000 c/ml (>or=200 IU/ml) or higher. Patients who do not have an indication for antiviral treatment should be monitored because there is a risk of (re)activation of disease activity. Monitoring every three to six months is recommended for HBeAg-positive and HBeAg-negative patients with high viraemia (HBV DNA >or=1.0 x 10(5) c/ml or >or=2.0 x 10(4) IU/ml) and normal ALAT levels. For patients with serum HBV DNA below 1.0 x 10(5) c/ml (<2.0 x 10(4) IU/ml) the recommended frequency of monitoring is every three to six months for HBeAg-positive patients and every six to 12 months for HBeAg-negative patients. Peginterferon (PEG-IF N) therapy should be considered as initial therapy in both HBeAg-positive and HBeAg-negative patients without contraindications for treatment with this drug because of the higher chance of achieving sustained response compared with nucleos(t)ide analogue therapy. In patients starting nucleos(t)ide analogue therapy, the use of lamivudine is not preferred if long-term antiviral treatment is expected due to the high risk of antiviral resistance against this drug. Of the currently licensed nucleos(t)ide analogues, entecavir has the lowest risk of antiviral resistance (compared with lamivudine, adefovir and telbivudine), while suppression of viral replication seems most profound with either entecavir or telbivudine. The recommended duration of treatment with PEG-IF N is one year for both HBeAg-positive and HBeAg-negative patients. In HBeAg-positive patents, nucleos(t)ide analogue therapy should at least be continued until HBeAg seroconversion and a decline in HBV DNA to below 400 c/ml (80 IU/ml) has been achieved and maintained for six months during therapy. Whether nucleos(t)ide analogue therapy can be safely discontinued in HBeAg-negative patients is unknown; usually prolonged or indefinite antiviral treatment is necessary. Patients receiving PEG-IF N should be monitored once a month, while three monthly monitoring suffices for those receiving nucleos(t)ide analogues. Genotypic analysis of the HBV polymerase is indicated if an increase in serum HBV DNA of at least 1 log(10) c/ml (IU/ml) compared with the nadir value is observed during nucleos(t)ide analogue therapy. Antiviral therapy should be changed as soon as possible in case of confirmed genotypic resistance. Adding a second antiviral agent seems beneficial over switching to another agent. With the availability of multiple new antiviral drugs for the treatment of chronic hepatitis B, effective treatment is now possible for more patients and for longer periods. However, the complexity of HBV therapy has also increased. Nowadays, virtually all chronic HBV-infected patients can be effectively managed, either by inducing sustained off-treatment response or by maintaining an on-treatment response.

Treatment of chronic hepatitis C virus infection - Dutch national guidelines.

The development of this guideline was initiated and coordinated by the Netherlands Association of Gastroenterologists and Hepatologists (Nederlandse Vereniging van Maag-Darm-Leverartsen). The aim is the establishment of practical guidelines in the evaluation and antiviral treatment of patients with chronic hepatitis C virus (HCV) infection. This includes recommendations for the initial evaluation of patients, the choice and duration of antiviral therapy and the follow-up after antiviral therapy. Hepatitis C is a slowly progressive disease. The initial evaluation of chronically HCV-infected patients should include liver biochemistry testing, virological testing and abdominal ultrasound imaging. Liver biopsy is no longer a routine procedure. Antiviral treatment should be considered for all HCV-infected patients. Current antiviral treatment is a long-term process and is associated with substantial side effects. When deciding whether to start treatment or not, the chance of successful treatment (80% with hepatitis C genotype 2 and 3 and 50% with hepatitis C genotype 1 and 4), the fibrosis stage, the expected side effects and the compliance of the patient should be taken into consideration. In the absence of significant fibrosis and necroinflammation in liver biopsy, postponing treatment is an option. Current antiviral treatment is contraindicated in patients with Child-Pugh-class B or C cirrhosis. The possibility of a liver transplantation should be investigated in these patients. Significant comorbidity with a limited life expectancy is an absolute contraindication for antiviral treatment Treatment of chronic hepatitis C consists of administration of peginterferon and ribavirin for 24 or 48 weeks. Patients with hepatitis C genotype 1 or 4 are treated for 48 weeks. Patients with hepatitis C genotype 2 or 3 are treated for 24 weeks. In patients with undetectable HCV RNA after four weeks (28 days) of treatment, a shorter treatment is equally effective (12 to 16 weeks for hepatitis C genotype 2 or 3; 24 weeks for hepatitis C genotype 1 or 4). Outpatient clinic visits are recommended at the start and after 2, 4, 8, and 12 weeks of treatment, and thereafter every four to six weeks until the end of treatment. It is recommended to stop treatment if the HCV RNA level has not decreased by at least 2 log10 IU/ml (c/ml) after 12 weeks of treatment or when HCV RNA is still detectable after 24 weeks of treatment. The recommended frequency of outpatient clinic visits for patients who are not being treated is once every six months in patients with cirrhosis, otherwise every 12 months. It is expected that new anti-HCV-medication (STAT-C, specifically targeted antiviral therapy for HCV) will become available in the near future. Therefore treatment of chronic HCV infection will probably be more effective in the future.

Continuous or intermittent vascular clamping during hemihepatectomy in pigs: hyaluronic acid kinetics in the assessment of early microvascular liver damage.

OBJECTIVE: To assess the uptake of hyaluronic acid (HA) as a marker of microvascular damage in a model of hemihepatectomy in pigs having continuous or intermittent vascular inflow occlusion. DESIGN: Prospective, animal study. SETTING: Laboratory for experimental surgery, University hospital, The Netherlands. INTERVENTIONS: Total liver ischaemia was achieved during 90 minutes by continuous (n = 5) or intermittent (n = 5) occlusion of the portal vein and hepatic artery followed by 120 minutes of reperfusion. In a second series of pigs (n = 8) a left hemihepatectomy was added to the protocol. MAIN OUTCOME MEASURES: Uptake of exogenous HA was assessed before ischaemia and after 120 minutes of reperfusion, together with the galactose elimination capacity. Plasma activities of aspartate aminotransferase (AST), alanine amino transferase, and lactate dehydrogenase were measured and specimens of liver were obtained for histopathological examination. RESULTS: HA uptake was slightly reduced after reperfusion in unresected livers compared with uptake before ischaemia. After hemihepatectomy HA uptake after reperfusion was significantly reduced after both continuous and intermittent occlusion, but more HA was taken up after continuous occlusion (p = 0.02). Release of AST after reperfusion was increased only after hemihepatectomy. CONCLUSIONS: Microvascular damage, as assessed by HA uptake capacity, significantly contributed to normothermic ischaemia and reperfusion injury in porcine liver. Vascular inflow occlusion during 90 minutes in combination with hemihepatectomy resulted in less liver damage when vascular occlusion was continuous rather than intermittent.

Prolonged continuous or intermittent vascular inflow occlusion during hemihepatectomy in pigs.

OBJECTIVE: To assess ischemia and reperfusion (I/R) injury in a hemihepatectomy model in pigs after prolonged continuous or intermittent vascular inflow occlusion in the liver. SUMMARY BACKGROUND DATA: Massive intraoperative blood loss during liver resections can be prevented by temporary vascular inflow occlusion, consequently leading to ischemia and reperfusion injury in the remnant liver. Previously, in a pig liver resection model in which only limited I/R injury was induced during brief (90 min) vascular inflow occlusion, the authors demonstrated reduced I/R injury after continuous (CNT) occlusion, compared to intermittent (INT). This liver resection study on pigs was undertaken to assess I/R injury after prolonged (120 min) CNT or INT occlusion. METHODS: In pigs (37.0 +/- 1.5 kg), liver ischemia during 2 hours was CNT (n = 6) or INT (n = 6) (eight subsequent periods of 12 min ischemia and 3 min recirculation), followed by 6 hours of reperfusion. A left hemihepatectomy (45.5% +/- 1.4%) was performed within the first 12 minutes of ischemia. No hepatic pedicle clamping or liver resection was performed in control experiments (n = 6). Microvascular damage was assessed by hyaluronic acid (HA) uptake capacity of the liver (parameter of early sinusoidal endothelial cell damage) and restoration of intrahepatic tissue pO2 during reperfusion. Hepatocellular damage was tested by plasma concentrations of aspartate aminotransferase (AST), alanine aminotransferase, and lactate dehydrogenase (LDH). RESULTS: Hyaluronic acid uptake after 6 hours of reperfusion, compared to preischemic uptake, was unaltered in the control group, but was significantly reduced in both resection groups. However, more HA was taken up after INT occlusion, compared to CNT (60.4% +/- 5.6% and 39.5% +/- 3.7%, respectively; ANOVA: p = 0.001). Intrahepatic tissue pO2 distribution after 6 hours of reperfusion more closely returned to preischemic configuration in the INT group than in the CNT group, indicating reduced microcirculatory disturbances after INT occlusion. Release of AST and LDH after 6 hours of reperfusion was significantly increased in both CNT and INT groups. Lower AST levels, however, were found after INT occlusion than after CNT occlusion (267.0 +/- 74.7 U/l and 603.3 +/- 132.4 U/l, respectively; p = 0.06). CONCLUSIONS: Intermittent hepatic vascular inflow occlusion during prolonged liver ischemia in pigs resulted in less microcirculatory and hepatocellular injury, compared to continuous occlusion. Intermittent clamping is preferable when prolonged periods of vascular inflow occlusion are applied during liver resections.

Warm flush at 37 degrees C following cold storage attenuates reperfusion injury in preserved rat livers.

Pretransplant rinse solutions have been shown to reduce reperfusion injury in cold-stored liver grafts, especially at the nonparenchymal level in sinusoidal endothelial cells (SEC). In this study, different rinse temperatures were tested in a rat liver preservation model. Livers were washed out in situ via the portal vein with cold (4 degrees C) University of Wisconsin (UW) solution, and after hepatectomy (t0), were stored for 8, 16, or 24 h of cold ischemia time (CIT). After storage, livers were flushed with UW solution at either 4 degrees C, 20 degrees C, or 37 degrees C and reperfused for 90 min (37 degrees C). Control livers were reperfused at t0 without preflush. Levels of hyaluronic acid (HA), purine nucleoside phosphorylase (PNP), AST, and LDH were measured in the reperfusion medium. Bile production was monitored during reperfusion. At the end of reperfusion, liver biopsies were taken for enzyme hystochemistry (5'-nucleotidase and LDH). After 8-h CIT and a flush at 4 degrees C, a release of endogenous HA (-7%) was observed, whereas uptake of exogenous HA occurred after the 20 degrees C flush (2%, P = NS) and after the 37 degrees C flush (24%, p < 0.001). HA release occurred at all three preflush temperatures after the 16-h CIT but was significantly lower when flushed at 37 degrees C (-10%) that at 4 degrees C and 20 degrees C (-64% and -17%, respectively, p = 0.05). After the 24-h CIT, the release of endogenous HA increased in the 4 degrees C and 20 degrees C preflush groups, but not in the 37 degrees C group. Levels of PNP and AST increased until the 24-h CIT in all groups but were significantly lower after preflush at 37 degrees C. Release of LDH did not increase with increasing periods of cold storage in any of the flush series. Compared to control livers, mean bile production during reperfusion was significantly decreased following preflush at 4 degrees C or 37 degrees C after all periods of CIT. No differences in mean bile production could be demonstrated in the three preflush groups after any period of CIT. LDH activity in liver tissue was best preserved after the 8 and 16-h CIT in combination with the 37 degrees C preflush, indicating less hepatocellular damage. In conclusion, in cold stored rat livers flushed at 37 degrees C before reperfusion, SEC and hepatocellular damage is attenuated.