Integrated surveillance of human respiratory viruses in addition to SARS-CoV-2 in a public testing facility in the Netherlands.

BACKGROUND: SARS-CoV-2 prevention measures impact the circulation of other respiratory viruses. Surveillance in the network of general practitioners is hampered by widespread testing for SARS-CoV-2 in public testing facilities. OBJECTIVES: To evaluate integrated community surveillance of SARS-CoV-2 and other respiratory viruses and describe epidemiological trends. STUDY DESIGN: Respiratory surveillance was set up within an existing SARS-CoV-2 public testing facility. Community-dwelling (a)symptomatic persons provided consent for completion of a questionnaire and additional testing on residual material from swabs taken for SARS-CoV-2 RT-PCR (Allplex Seegene). Daily, a random subset was tested for sixteen respiratory viruses by multiplex realtime PCRs (Seegene). RESULTS: Between October 6th (week 40) 2021 and April 22nd (week 16) 2022, 3,969 subjects were tested. The weekly median age ranged from 23 to 39 years. The prevalence of respiratory symptoms ranged from 98.5% (week 40) to 27.4% (week 1). The prevalence of detection of any respiratory virus (including SARS-CoV-2), ranged from 19.6% in week 49 to 75.3% in week 14. SARS-CoV-2 prevalence ranged from 2.2% (week 40) to 63.3% (week 14). Overall, SARS-CoV-2 was detected most frequently (27.3%), followed by rhinoviruses (14.6%, range 3.5-47.8%) and seasonal coronaviruses (3.7%, range 0-10.4%, mostly 229E and OC43). Influenzavirus was detected in 3.0% of participants from week 6 onwards. CONCLUSIONS: Integrated respiratory viral surveillance within public testing facilities is feasible and informative. Prevalences may be affected by changes in SARS-CoV-2 prevention and testing policies. Population characteristics help to interpret trends over time. Integrated surveillance may inform policymakers and hospitals for adequate response measures during respiratory seasons.

Hepatitis C virus transmission in a Dutch haemodialysis unit: detailed outbreak investigation using NS5A gene sequencing.

BACKGROUND: Haemodialysis is a risk factor for hepatitis C virus (HCV) transmission. Two patients receiving haemodialysis in a Dutch dialysis unit in The Hague were found to seroconvert to HCV in December 2016 after the yearly routine control for blood-borne viruses. Following the presumed time of infection, three chronically infected HCV patients were identified as possible index cases. AIM: To confirm inter-patient transmission and to identify the source. METHODS: Molecular investigation and review of medical records were performed. FINDINGS: Both of the incident cases and one of the three possible index cases were demonstrated to be infected with HCV genotype 2b based on 5'UTR sequencing. Epidemiological relatedness between these viruses was further investigated by sequencing of the NS5A region. Phylogenetic analysis clearly identified the incident cases and the index case to represent a cluster distinct from unrelated controls with HCV genotype 2b. Detailed review of the medical records identified two possible incidents that might have resulted in the HCV transmission cases: contamination of the venous pressure-sensing port due to high venous pressures or incomplete compliance with infection control precautions of the unit staff during handling of two incidents, that occurred at the same time in a single haemodialysis session with the index patient as well as both incident cases present. CONCLUSION: This study demonstrates that detailed incident recording in combination with state-of-the-art molecular investigations such as sequencing of the NS5A region resulted in unravelling a set of two HCV transmissions that occurred at a haemodialysis unit.

Low serum hyaluronic acid levels associated with spontaneous HBsAg clearance.

PURPOSE: The pathophysiological underlying mechanism of spontaneous HBsAg clearance in hepatitis B virus (HBV) infected patients is largely unknown. However, serum hyaluronic acid (sHA) plays a role in liver fibrosis progression and reversely could serve as a potential biomarker for HBsAg clearance. This study investigates whether low sHA is associated with HBsAg loss in non-Asian HBV patients. METHODS: Non-Asian women living in Amsterdam with known chronic HBV infection between 1990-2003 were invited for a single follow-up visit at the Municipal Health Service Amsterdam between September 2011 to May 2012. Serum hyaluronic acid and liver stiffness measurement together with clinical evaluation, biochemical and virologic blood tests were performed. RESULTS: Of the 160 women, HBsAg loss occurred in 38 (23 %) patients between diagnosis and follow-up. sHA levels were lower in HBsAg negative patients compared to HBsAg positive patients (14.5 [9.4-27.2] ng/mL vs 25.0 [12.3-42.5] ng/mL, p <0.01). A similar distinction in sHA between low and high HBV DNA was noted. sHA had a significant discriminatory ability to differentiate between HBsAg positive and HBsAg negative patients, (AUC 0.65 [95 % CI 0.55-0.75], p < 0.01). In multivariable analysis only sHA level was associated with HBsAg loss (OR 0.4 [0.2-0.9]). Finally, F3-F4 fibrosis (cut-off >8.1 kPa) was diagnosed in 3 % in HBsAg negative patients compared to 10 % in HBsAg positive patients (p = 0.15). CONCLUSION: Serum HA levels are lower in patients who experience spontaneous HBsAg loss compared to HBsAg positive patients.

Historic and current hepatitis B viral DNA and quantitative HBsAg level are not associated with cirrhosis in non-Asian women with chronic hepatitis B.

BACKGROUND: Some studies done in Asian patients have shown that serum levels of hepatitis B virus (HBV) DNA predict the development of cirrhosis. However, it is unclear whether this also applies for non-Asian patients. This study investigated historic and current HBV DNA and quantitative hepatitis B surface antigen (HBsAg) levels as predictors of cirrhosis in non-Asian women with chronic HBV. METHODS: A retrospective cohort study of non-Asian women with chronic HBV was performed. Among other variables, HBV DNA and quantitative HBsAg levels were measured in stored historic serum samples obtained during pregnancy (period 1990-2004) and current serum samples (period 2011-2012) to determine any association with liver cirrhosis by liver stiffness measurement (LSM). RESULTS: One hundred and nineteen asymptomatic, treatment-naïve non-Asian women were included; the median number of years between the historic sample and the current sample was 17 (interquartile range (IQR) 13-20). The median historic log HBV DNA and quantitative log HBsAg levels were 2.5 (IQR 1.9-3.4) IU/ml and 4.2 (IQR 3.6-4.5) IU/ml, respectively. LSM diagnosed 14 patients (12%) with F3-F4 fibrosis, i.e. stiffness >8.1kPa. No association of cirrhosis was found with historic HBV DNA (relative risk (RR) 0.34, 95% confidence interval (CI) 0.05-2.44) or with the quantitative HBsAg level (HBsAg level >1000 IU/ml, RR 0.35, 95% CI 0.11-1.11). Multivariable analysis identified alcohol consumption (odds ratio (OR) 6.4, 95% CI 1.3-30.1), aspartate aminotransferase >0.5 times the upper limit of normal (OR 15.4, 95% CI 1.9-122.6), and prothrombin time (OR 12.0, 95% CI 1.2-120.4), but not HBV DNA or quantitative HBsAg level, to be independent predictors of the presence of cirrhosis. CONCLUSIONS: Neither historic nor current HBV DNA or the quantitative HBsAg level is associated with the development of HBV-related cirrhosis in non-Asian women.

ELF-test less accurately identifies liver cirrhosis diagnosed by liver stiffness measurement in non-Asian women with chronic hepatitis B.

BACKGROUND: The enhanced liver fibrosis test (ELF-test) has been validated for several hepatic diseases. However, its performance in chronic hepatitis B virus (CHB) infected patients is uncertain. OBJECTIVE: This study investigates the diagnostic value of the ELF test for cirrhosis identified by liver stiffness measurement (LSM) in non-Asian women with CHB. STUDY DESIGN: Women of non-Asian origin with perinatally acquired CHB infection, detected during pregnancy in the period 1990-2003, returned to our center between September 2011 and May 2012 for LSM and blood sampling to perform an ELF test and to calculate, APRI and FIB-4 scores. Fibrosis stages were classified by the METAVIR system. RESULTS: A total of 119 women were included in this study with a median age of 43 years, all ALT levels being <2× ULN and all being HBeAg negative. The overall median LSM (IQR) stiffness and ELF test were 5.5kPa (4.0-6.8) and 8.4 (7.8-9.2) respectively. LSM and ELF test classified 14 (12%) and 19 (16%) patients with severe fibrosis to cirrhosis (≥F3, i.e. liver stiffness >8.1kPa), however in only 4 (3%) patients there was an agreement between LSM and ELF test. With LSM as reference, the area under receiver operating characteristic curve (AUROC) for detection of ≥F3 fibrosis was for ELF 0.65 (95% CI 0.51-0.80; p=0.06), APRI 0.66 (0.50-0.82; p=0.07) and FIB-4 0.66 (0.49-0.82; p=0.07). CONCLUSION: The ELF test less accurately discriminates severe fibrosis or cirrhosis when compared to LSM in our cohort of non-Asian women with CHB.

Phylogenetic Diversity of Rhizoctonia solani Associated with Canola and Wheat in Alberta, Manitoba, and Saskatchewan.

Rhizoctonia solani is a damaging soilborne pathogen, which affects most field crops in the Canadian provinces of Alberta, Manitoba, and Saskatchewan. The objective of this study was to conduct a phylogenetic comparison of isolates of R. solani collected from a previous survey in the major canola- and wheat-growing regions of western Canada. A total of 128 multinucleate isolates from a previous survey were identified by internal transcribed spacer (ITS) sequence and compared to anastomosis group (AG) results. The multinucleate isolates of R. solani were grouped into eight distinct clades. Each clade corresponded to a specific AG with the exception of two distinct clades that were observed for isolates classified as AG 2-1 by anastomosis testing. While most isolates of AG 5 clustered together according to ITS sequences, three isolates classified by anastomosis grouping as AG 5 grouped with AG 2-1, AG 4, and a binucleate Rhizoctonia sp. in the phylogenetic analysis. In most instances, the results from AG tests were consistent with ITS sequence, but there were still several cases where isolates were inconsistently classified or failed to undergo anastomosis with any of the tester strains used in this study. This provides support for the use of the ITS region as a valuable tool for rapid identification of R. solani isolates to their respective AGs.

Evaluation of Air Sampling and Detection Methods to Quantify Airborne Ascospores of Sclerotinia sclerotiorum.

Detection and quantification of airborne ascospores as a component of the Sclerotinia rot of carrot (SRC) forecast model is currently accomplished using the blue plate test (BPT), which uses Sclerotinia semiselective medium (SSM). A quantitative polymerase chain reaction (qPCR) assay was developed to reduce the time to specifically quantify ascospores of Sclerotinia sclerotiorum from air samples collected using a Burkard Multi-Vial Cyclone Sampler. The qPCR assay was highly sensitive and detected DNA from 0.5 to 5 × 104 ascospores within a linear range (R2 = 0.99). The qPCR assay was used to quantify ascospores of S. sclerotiorum in air samples collected over three growing seasons. Initial SRC disease was observed 8 and 34 days following detection of 9.5 and 2 ascospores m-3 of air, respectively. Results from air samples collected using an Andersen N6 Sampler and the qPCR assay were compared with the BPT. Ascospore counts from a Burkard Sampler coupled with the qPCR assay and the BPT followed similar trends. In general, fewer ascospores were detected and bioaerosol sampling efficiency was low using an Anderson Sampler. Three days were required to confirm the number of ascospores using SSM in the BPT and with an Andersen Sampler, whereas results from a Burkard Sampler coupled with the qPCR assay can provide results within 5 h of air sampling. The choice of method will depend on the available resources.

Population structure of the butternut canker fungus, Ophiognomonia clavigignenti-juglandacearum, in North American forests.

The occurrence of multiple introduction events, or sudden emergence from a host jump, of forest pathogens may be an important factor in successful establishment in a novel environment or on a new host; however, few studies have focused on the introduction and emergence of fungal pathogens in forest ecosystems. While Ophiognomonia clavigignenti-juglandacearum (Oc-j), the butternut canker fungus, has caused range-wide mortality of butternut trees in North America since its first observation in 1967, the history of its emergence and spread across the United States and Canada remains unresolved. Using 17 single nucleotide polymorphic loci, we investigated the genetic population structure of 101 isolates of Oc-j from across North America. Clustering analysis revealed that the Oc-j population in North America is made up of three differentiated genetic clusters of isolates, and these genetic clusters were found to have a strong clonal structure. These results, in combination with the geographic distribution of the populations, suggest that Oc-j was introduced or has emerged in North America on more than one occasion, and these clonal lineages have since proliferated across much of the range of butternut. No evidence of genetic recombination was observed in the linkage analysis, and conservation of the distinct genetic clusters in regions where isolates from two or more genetic clusters are present, would indicate a very minimal or non-existent role of sexual recombination in populations of Oc-j in North America.

Ribavirin rather than PEG-interferon pharmacodynamics predict nonresponse to antiviral therapy in naive chronic hepatitis C patients.

Twenty to fifty per cent of patients with chronic hepatitis C (CHC) experience nonresponse to current antiviral therapy, which may relate in part to ribavirin or PEG-interferon pharmacodynamics. We evaluated potential relevance of various factors for nonresponse. Two hundred forty-two naive CHC patients who received in a previous trial at least 24 weeks of antiviral therapy, including PEG-interferon alfa-2b and ribavirin, were analysed. Of them, 53% were infected with hepatitis C virus (HCV) genotype 1-4, 71% exhibited high viral load and 32% had severe fibrosis/cirrhosis. After 24 weeks of treatment, 39 patients (16%) were nonresponders. In multivariate analysis, lower serum ribavirin concentrations, HCV genotype 1-4 and higher baseline γ-GT predicted nonresponse. Week-24 ribavirin concentrations (2.2 vs 2.8 mg/L, P < 0.001), average ribavirin doses (14.5 vs 15.2 mg/kg per day, P = 0.03) and week-24 haemoglobin decreases (1.7 vs 2.0 mm, P = 0.02) were lower in nonresponders. Nonresponse rates increased progressively at decreasing ribavirin concentrations: 4%, 11%, 13% and 36% in case of serum ribavirin concentrations ≥4, 3-4, 2-3 and ≤2 mg/L, respectively (P = 0.001). Ribavirin concentrations correlated with both week-24 haemoglobin decreases (r = 0.42, P < 0.001) and ribavirin doses (r = 0.17, P = 0.01). Subgroup analysis in HCV genotype 1-4 patients revealed essentially the same results. Nonresponse was exceptional in HCV genotype 2-3 patients and associated with ribavirin concentrations <2 mg/L. Presumed interferon-related factors (average PEG-interferon doses and decreases in leucocytes, granulocytes, platelets and body weight) did not differ between nonresponders and responders. In conclusion, ribavirin- rather than PEG-interferon-related factors are independent and potentially modifiable predictors of nonresponse in treatment-naive CHC patients.

Discovery of single-nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete Ophiognomonia clavigignenti-juglandacearum from 454 sequence data.

The benefits from recent improvement in sequencing technologies, such as the Roche GS FLX (454) pyrosequencing, may be even more valuable in non-model organisms, such as many plant pathogenic fungi of economic importance. One application of this new sequencing technology is the rapid generation of genomic information to identify putative single-nucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. The focus of this research was to sequence, assemble, discover and validate SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. Genomic DNA from eight isolates of Ophiognomonia clavigignenti-juglandacearum was pooled in one region of a four-region sequencing run on a Roche 454 GS FLX. This yielded 71 million total bases comprising 217,000 reads, 80% of which collapsed into 16,125,754 bases in 30,339 contigs upon assembly. By aligning reads from multiple isolates, we detected 298 SNPs using Roche's GS Mapper. With no reference sequence available, however, it was difficult to distinguish true polymorphisms from sequencing error. Eagleview software was used to manually examine each contig that contained one or more putative SNPs, enabling us to discard all but 45 of the original 298 putative SNPs. Of those 45 SNPs, 13 were validated using standard Sanger sequencing. This research provides a valuable genetic resource for research into the genus Ophiognomonia, demonstrates a framework for the rapid and cost-effective discovery of SNP markers in non-model organisms and should prove especially useful in the case of asexual or clonal fungi with limited genetic variability.

Ophiostoma mitovirus 3a , ascorbic acid, glutathione, and photoperiod affect the development of stromata and apothecia by Sclerotinia homoeocarpa.

Hypovirulence in Sclerotinia homoeocarpa is associated with infection by Ophiostoma mitovirus 3a (OMV3a). OMV3a is also present in asymptomatic isolates, with growth and virulence comparable to that of virus-free isolates. Hypovirulent isolates have impaired mitochondrial function resulting in increased activity of the alternative oxidase pathway, which is implicated in the reduction of reactive oxygen species in other fungi. In this study, hypovirulent, asymptomatic, and virus-free isolates were grown on potato dextrose agar amended with ascorbic acid or glutathione and were incubated under various photoperiods to determine the importance of reactive oxygen species, light, and OMV3a infection for differentiation of stromata and apothecia by S. homoeocarpa. Hypovirulent isolates did not form stromata or apothecia. Glutathione and darkness reduced stromata size and apothecia production by virulent and asymptomatic isolates. Apothecia formed under several different photoperiods, and ascorbic acid increased apothecia production. Ascospores were not detected in these apothecia. The results suggest that hypovirulence, light, and the superoxide radical are important factors in the formation of stromata and apothecia by S. homoeocarpa isolates. This is the first report of sterile apothecia production by North American isolates of S. homoeocarpa and provides a starting point for attempts to produce fertile apothecia.

Reclassification of the butternut canker fungus, Sirococcus clavigignenti-juglandacearum, into the genus Ophiognomonia.

Sirococcus clavigignenti-juglandacearum (Sc-j), which causes a canker disease on butternut, is largely responsible for the decline of this tree in the United States and Canada. The original description of the species was based on anamorphic characters because the teleomorph is unknown. Recent phylogenetic investigations have found that Sc-j is not a member of the genus Sirococcus, and accurate taxonomic classification is required. The objective of this study is to use sequence data to determine the phylogenetic placement of Sc-j within the Gnomoniaceae, Diaporthales. Isolates were recovered from infected Juglans ailantifolia var. cordiformis (heartnut), Juglans cinerea (butternut), and Juglans nigra (black walnut) in Ontario and the eastern United States. The genes coding for ß-tubulin, actin, calmodulin, internal transcribed spacers 1 and 2, and the translation elongation factor 1-alpha from 28 isolates of Sc-j and representatives of the major lineages within the Gnomoniaceae were evaluated. There was no difference in the sequences of the five genes among the isolates of Sc-j studied, indicating a recent introduction followed by asexual reproduction and spread via conidia. The phylogenetic analyses demonstrate this fungus does not belong to the genus Sirococcus, and provides strong support (99% MP and 100% NJ bootstrap values, and 100% Bayesian posterior probabilities) for its inclusion in the genus Ophiognomonia, thereby supporting a reclassification of the butternut canker fungus to Ophiognomonia clavigignenti-juglandacearum.

Impact of new guidelines for blood exposure incidents in The Netherlands.

BACKGROUND: In 2007, a new set of guidelines for blood exposure incidents was introduced in The Netherlands to standardize management and reduce use of hepatitis B immunoglobulin (HBIg). Accidents now have to be assigned into risk categories with the corresponding medical intervention. AIMS: To study the consequences of the guidelines on overall risk assessment and costs of hepatitis B virus (HBV) prevention. METHODS: Incidents (n = 461) from both hospital as well as non-hospital health care workers and others registered by a call centre from the year 2005 were reassessed and reclassified as 'no-risk', 'high-risk' or 'low-risk' according to the corresponding risk categories of the new guidelines. The differences in classification, use of HBV immunoglobulin, source testing and the costs of the HBV prevention strategy were evaluated. RESULTS: Of all incidents, 86% could be reassigned directly into the new risk categories. However, there was a significant shift from 'low-' to 'high-risk' incidents. Overall, administration of HBV vaccination increased and administration of HBIg decreased significantly, although within the group of high-risk incidents, administration of HBIg increased. There was no effect on the frequency of reference serum taken after an incident. While fewer incidents needed intervention, the total costs of HBV prevention still increased by 50%. Total costs increased by 13%, due to a shift in classification. CONCLUSIONS: The use of the new protocol facilitated standardized risk assessment for blood exposure accidents. HBIg administration and source testing decreased. An increased proportion of high-risk classifications resulted in an increase in the associated costs.

New genetic associations detected in a host response study to hepatitis B vaccine.

The immune response to hepatitis B vaccination differs greatly among individuals, with 5-10% of healthy people failing to produce protective levels of antibodies. Several factors have been implicated in determining this response, chiefly individual genetic variation and age. Aiming to identify genes involved in the response to hepatitis B vaccination, a two-stage investigation of 6091 single-nucleotide polymorphisms (SNPs) in 914 immune genes was performed in an Indonesian cohort of 981 individuals showing normal levels of anti-HBs versus 665 individuals displaying undetectable levels of anti-HBs 18 months after initial dose of the vaccine. Of 275 SNPs identified in the first stage (476 normal/372 nonresponders) with P<0.05, significant associations were replicated for 25 polymorphisms in 15 genes (503 normal/295 nonresponders). We validated previous findings (HLA-DRA, rs5000563, P-value combined=5.57 x 10(-10); OR (95%CI)=0.61 (0.52-0.71)). In addition, we detected a new association outside of the human leukocyte antigen loci region that passed correction for multiple testing. This SNP is in the 3' downstream region of FOXP1, a transcription factor involved in B-cell development (P-value combined=9.2 x 10(-6); OR (95%CI)=1.38 (1.2-1.6)).These findings might help to understand the biological reasons behind vaccine failure and other aspects of variation in the immune responses of healthy individuals.

Molecular Diagnostic Assay for Detection of the Butternut Canker Pathogen Sirococcus clavigignenti-juglandacearum.

Butternut canker, caused by the fungal pathogen Sirococcus clavigignenti-juglandacearum, is present throughout the range of butternut (Juglans cinerea) and is the primary cause for its decline. A quick and reliable method for identification of S. clavigignenti-juglandacearum would provide a valuable tool for the detection of the pathogen on propagative material to avoid spread, as well as assist studies targeted at the epidemiology of this pathogen, in particular the dissemination of the pathogen by seeds of the butternut. The objective of this study was to develop a diagnostic assay to detect S. clavigignenti-juglandacearum in butternut plant tissue. The primers were developed using an alignment of internal transcribed spacer (ITS) sequences from isolates of S. clavigignenti-juglandacearum and several closely related species. These primers were tested on J. cinerea, 48 isolates of S. clavigignenti-juglandacearum recovered from diseased trees, and 26 species of other fungi recovered from butternut tissue. The primers amplified a product from the DNA of all isolates of S. clavigignenti-juglandacearum, detected its DNA at a concentration as low as 1 pg/µl, and detected the pathogen at a concentration of 1 × 103 spore/ml. The primers developed in this study will be a valuable tool for the detection of S. clavigignenti-juglandacearum present on butternut seeds, and as a rapid diagnostic tool for early detection of the pathogen on butternut trees.

Plasma HCV-RNA decline in the first 48 h identifies hepatitis C virus mono-infected but not HCV/HIV-coinfected patients with an undetectable HCV viral load at week 4 of peginterferon-alfa-2a/ribavirin therapy.

During peginterferon-alfa-2a/ribavirin therapy, plasma hepatitis C virus (HCV)-RNA decreases with a rapid first phase and a slower second phase. We compared the viral load decrease and slope in the first 48 h in patients with a rapid viral response (RVR, i.e. HCV-RNA < 50 IU/mL at week 4) with patients not achieving an RVR. From 23 HCV-infected (14 mono-infected and nine HCV/HIV-coinfected) genotype 1 or 4 positive peginterferon-alfa-2a/ribavirin-treated patients, plasma HCV-RNA was determined at baseline, 48 h, weeks 1, 2, 4, 8, 12, 48 and 72. The HCV viral load decrease (Delta0-48), the slope (lambda(1)) and the efficiency factor (epsilon) were determined in the first 48 h after the start of therapy. Five (36%) HCV mono-infected patients and three (33%) HIV/HCV-coinfected patients achieved an RVR whereas six (43%) HCV mono-infected patients and five (56%) HIV/HCV-coinfected patients reached a sustained viral response (SVR). In contrast to HIV/HCV-coinfected patients, five HCV mono-infected patients with an RVR showed both a larger Delta0-48 and steeper lambda(1) (-1.77log(10) IU/mL +/- 0.66 and -2.04/day +/- 0.76) compared to nine non-RVR patients (-0.66log(10) IU/mL +/- 0.39; P = 0.019 and -0.76/day +/- 0.41; P = 0.019). When divided by SVR, a greater Delta0-48 and steeper lambda(1) were also seen in both HCV mono-infected and HIV/HCV-coinfected patients. Thus, in the first 48 h after the start of therapy, HCV mono-infected patients with an RVR have a larger viral load decrease, steeper viral slope and a higher efficiency factor as compared with non-RVR patients.

Concurrent selection for microbial suppression of Fusarium graminearum, Fusarium head blight and deoxynivalenol in wheat.

AIM: Identify biological agents that can both control Fusarium head blight (FHB) and reduce deoxynivalenol (DON) production. METHODS AND RESULTS: Concurrent screening methods were used to progressively select soil and food micro-organisms for the ability to suppress Fusarium graminearum, FHB and DON production. The micro-organisms were assessed using up to five assays including: a co-culture and dual-culture assay, an indirect impedance assay, a wheat floret assay, and two assays assessing DON production. Paenibacillus polymyxa W1-14-3 and C1-8-b gave the greatest inhibition of F. graminearum and reduction of DON production in greenhouse evaluations. Compared to a control treatment, they reduced disease severity by 56.5 and 55.4%, F. graminearum colonization of wheat heads by 58.8 and 62.4%, DON production by 84.8 and 89.4%, and increased 100-kernel weights by 56.6 and 66.9%, respectively. CONCLUSIONS: The concurrent selection has resulted in promising antagonists that may possess multiple modes of action, and have the ability to colonize wheat heads in controlled environments. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel concurrent screening method was developed for selection of biocontrol agents for FHB. Two isolates of P. polymyxa were selected and identified. Their potential use as biocontrol agents for FHB is highlighted in this study.

[Inadequate infrastructure for follow-up after needlestick injuries in the Netherlands]. / Versnipperde infrastructuur voor afhandeling van prikaccidenten in Nederland.

OBJECTIVE: To determine how needle-stick injuries are dealt with in the Netherlands. DESIGN: Study using questionnaires. METHOD: In order to study whether victims of needle-stick injuries have access to proper treatment, we sent questionnaires to hospitals (n = 103) and Municipal Health Services (MHS) (n = 36) in the Netherlands. We enquired after the possibilities of risk-estimation and follow-up, the performance of necessary laboratory tests, direct administration of preventive medication and backup facilities. RESULTS: Questionnaires were returned by 113 (81%) institutions. 74% of the hospitals and 71% of the MHS provided follow-up for needle-stick injuries from outside their own institution. Necessary laboratory tests were not always available or sometimes could not be performed on an immediate basis. In addition, essential medication was not always directly available. MHS recognized the advantage of cooperation during followup of needle-stick injuries more than hospitals. CONCLUSION: Based on the results there is no guarantee that victims of needle-stick injuries in the Netherlands have access to appropriate care at any location in the Netherlands on a 24/7 basis. We recommend improvement of the infrastructure and cooperation between health care organizations to guarantee improved follow-up in every region.

A Specific and Sensitive Method for the Detection of Colletotrichum lindemuthianum in Dry Bean Tissue.

To facilitate early diagnosis and improve control of bean anthracnose, a rapid, specific, and sensitive polymerase chain reaction (PCR)-based method was developed to detect the causal agent, Colletotrichum lindemuthianum, in bean (Phaseolus vulgaris) seed. Based on sequence data of the rDNA region consisting of the 5.8S gene and internal transcribed spacers (ITS) 1 and 2 of four C. lindemuthianum races and 17 Colletotrichum species downloaded from GenBank, five forward primers were designed and evaluated for their specificity. Among them, one forward primer was selected for use in combination with ITS4 to specifically detect C. lindemuthianum. A 461-bp specific band was amplified from the genomic DNA template of 16 representative isolates of C. lindemuthianum, but not from 58 representative isolates of 17 other Colletotrichum species or 10 bean pathogens. Moreover, to enhance the sensitivity of detection, nested PCR was applied, which allowed the detection of as little as 10 fg of C. lindemuthianum genomic DNA and 1% infected seed powder, which was mixed with 99% healthy seed powder. The diagnostic analysis can be completed within 24 h, compared with about 2 weeks required for culturing. Furthermore, this method can be performed and interpreted by personnel with no specialized taxonomic expertise.

Hepatitis C: changing genotype distribution with important implications for patient management.

In The Netherlands an estimated 0.1 to 0.4% of the population are chronic hepatitis C (HCV) carriers (15,000 to 60,000 persons). HCV is characterised by genetic heterogeneity and six different genotypes have been identified. The distribution of HCV genotypes is relevant for the clinician, since there are important genotype-specific differences in response to interferon-alpha based treatment regimens. Between 1993 and 2005 a shift was observed in The Netherlands from a dominant prevalence of genotype 1 to a situation in which genotype non-1 is becoming more important.