Progression of hepatitis C in the haemophiliac population in Ireland, after 30 years of infection in the pre-DAA treatment era.

INTRODUCTION: Prior to the introduction of viral inactivation of factor concentrates and screening of blood, 225 people with haemophilia became infected with hepatitis C (HCV) in Ireland. AIM: Our aim was to assess liver disease progression and mortality in this population after 30 years of infection. METHODS: Demographic and clinical data were collected from medical records in five hepatology units and one infectious disease unit retrospectively in 2005, and on four subsequent occasions. RESULTS: The participation rate was 73% (165/225). Eighty three percent of patients, who had been tested for RNA (n = 106/128), developed chronic HCV infection. Thirty four percent were co-infected with HIV. All-cause mortality, after approximately 30 years of infection with chronic HCV, was 44% in HIV positive patients and 29% in HIV negative patients. Liver-related mortality was 12.5% and did not vary significantly by HIV status. Thirty seven percent of patients had developed advanced liver disease, including 20% with cirrhosis and 9% with hepatocellular carcinoma. In the pre-interferon-free direct acting antivirals era, 57% (n = 60/106) of patients were treated for HCV, 65% of whom achieved a sustained virological response. Successfully treated patients had few adverse liver outcomes. CONCLUSION: After 30 years of infection, 40% of the patients who had evidence of chronic HCV had developed advanced liver disease, such as cirrhosis and HCC, or had died from liver-related causes. This proportion is high relative to similar international cohorts despite good anti-HCV treatment uptake and responses.

Long-term follow-up of patients with spontaneous clearance of hepatitis C: does viral clearance mean cure?

Up to 40% of patients with hepatitis C virus (HCV) antibodies are negative for HCV RNA at initial evaluation. If there is a risk of viral re-activation, long term follow-up is required with attendant financial, psychological and medical implications. We investigated the risk of re-activation in the Irish anti-D cohort. Information was obtained from the national hepatitis C database which includes data on patients infected by anti-D immunoglobulin in two large outbreaks, 1977-9 and 1991-94. As part of a screening programme, starting in 1994, 64,907 females exposed to anti-D immunoglobulin were evaluated. Three hundred and forty-seven were found to be antibody positive but HCV RNA negative at initial assessment. 93% had subsequent RNA tests. There was no evidence of HCV recurrence in patients whose infection resolved spontaneously. It appears that two initial sequential negative results for HCV RNA are sufficient to confirm spontaneous viral clearance and probable cure of hepatitis C virus infection.

Reporting of Acute Flaccid Paralysis in Children under 15 years of age: Improving Surveillance, January 2009 - December 2014.

Acute Flaccid Paralysis (AFP) surveillance, including case investigation and specimen collection is a gold standard method for poliomyelitis surveillance. The expected annual non-polio AFP rate <15 years of age in Ireland is = 1/100 000 population. This study reviewed all cases of AFP reported to the Irish Paediatric Surveillance Unit and the Health Protection Surveillance Centre between January 2009 and December 2014 and compared reporting rates with the expected incidence rate annually. We assessed quality of surveillance data in terms of completeness of investigation for each case reported. Forty-three AFP cases in children <15 years were notified; 35 of which were confirmed. Guillain-Barre Syndrome (GBS) accounted for 48.6% (n=17) of AFP notifications. In 2014, the expected annual AFP target rate was reached. This study identified possible under-reporting of AFP paediatric cases in Ireland between 2009-2013. Completeness of investigations has improved over time, but requires further work.

A prolonged outbreak of invasive meningococcal disease in an extended Irish Traveller family across three Health Service Executive (HSE) areas in Ireland, 2010 to 2013.

Between March 2010 and November 2013 eight laboratory-confirmed cases of serogroup B, invasive meningococcal disease (IMD) were identified in an extended Irish Traveller family across three Health Service Executive (HSE) areas of Ireland. Cases were aged between 5 and 46 months, and were either a cousin or sibling of another case. All eight cases survived. Chemoprophylaxis was given to relevant nuclear family members and close contacts on each occasion, but failed to prevent further cases. Neisseria meningitidis isolates from six cases were highly related, belonging to the ST-41/44 clonal complex, and shared the porA designation 7­2,4. In November 2013, the outbreak control team recommended that directly observed ciprofloxacin chemoprophylaxis be administered simultaneously to the extended family, and that the four component meningococcal B (4CMenB) vaccine be administered to family members aged 2 months to 23 years inclusive and relevant close contacts of the eighth case. Subsequently these recommendations were implemented at three regional clinics. Additionally pharyngeal swabs (n=112) were collected to assess carriage rates of N. meningitidis in this extended family. Pharyngeal carriage of N. meningitidis was detected in 15 (13%) family members. From the epidemiological investigation and carriage study overcrowding was the most likely risk factor identified in this outbreak. To date, the combination of directly observed ciprofloxacin chemoprophylaxis and use of 4CMenB vaccine have controlled the outbreak with no further cases diagnosed.

Ultrio and Ultrio Plus non-discriminating reactives: false reactives or not?

BACKGROUND: The low, fluctuating levels of DNA characteristic of occult hepatitis B infection make its detection by nucleic acid testing (NAT) a challenge. METHODS: Four year's routine use of the Ultrio and Ultrio Plus assays in blood donations in New Zealand was analysed. RESULTS: 0·09% of donations tested with Ultrio and Ultrio Plus assays showed reactivity in the multiplex assay, but non-reactivity in all three discriminatory assays and relevant mandatory serological assays (anti-HIV, anti-HCV, HBsAg). These donations were more likely to be anti-HBc reactive (Ultrio, 13%; Ultrio Plus, 57%; random donors, 6·8%). Thirty-four per cent of these anti-HBc-reactive donations were also reactive in either an alternate NAT assay or on repeat multiplex testing. Thirteen per cent of the donors of the discriminatory-negative, anti-HBc-reactive donations who had given other Ultrio- or Ultrio Plus-tested donations had at least one other multiplex reactive donation. CONCLUSION: These findings suggest that their HBV DNA levels are around the assay's limit of detection, that false reactivity cannot be presumed when a donor fails to discriminate and that caution should be applied when deciding whether to continue accepting donations from such donors.

The largest prison outbreak of TB in Western Europe investigated using whole-genome sequencing.

BACKGROUND: In March 2011, the Department of Public Health East in Ireland were notified of two cases of TB in two prisoners sharing a cell. We define the resulting outbreak and highlight the role of public health and laboratory-based molecular epidemiology in mapping and control of a prison outbreak.METHODS: Cases were identified through clinical presentation, contact tracing, case-finding exercise or enhanced laboratory surveillance. Mycobacterium tuberculosis isolates were genotyped and underwent whole-genome sequencing (WGS).RESULTS: Of the 34 cases of TB linked to the outbreak, 27 were prisoners (79%), 4 prison officers (12%) and 3 community cases (9%). M. tuberculosis was isolated from 31 cases (culture positivity: 91%). A maximum of six single-nucleotide polymorphisms separated the isolates, with 22 being identical, suggestive of a highly infectious 'super-spreader´ within the prison. Isolates belonged to the Beijing sub-lineage, and were susceptible to first-line anti-TB agents. A case-finding exercise incidentally detected a prisoner with multidrug-resistant TB. Of the 143 prison officers screened, 52% had latent TB infection. Litigation costs exceeded five million euros.CONCLUSION: This constitutes the largest prison outbreak of TB in Western Europe investigated using WGS. A robust prison entry TB screening and education programme is required to effect better TB control, and prevent future outbreaks and attendant litigation.

Monitoring and treatment of anti-D in pregnancy.

Prophylactic anti-D is a very safe and effective therapy for the suppression of anti-D immunization and thus prevention of haemolytic disease of the foetus and newborn. However, migration from countries with low health standards and substantial cuts in public health expenses have increased the incidence of anti-D immunization in many "developed" countries. Therefore, this forum focuses on prenatal monitoring standards and treatment strategies in pregnancies with anti-D alloimmunization. The following questions were addressed, and a response was obtained from 12 centres, mainly from Europe.

Inventory management.

A critical aspect of blood transfusion is the timely provision of high quality blood products. This task remains a significant challenge for many blood services and blood systems reflecting the difficulty of balancing the recruitment of sufficient donors, the optimal utilization of the donor's gift, the increasing safety related restrictions on blood donation, a growing menu of specialized blood products and an ever-growing imperative to increase the efficiency of blood product provision from a cost perspective. As our industry now faces questions about our standard practices including whether or not the age of blood has a negative impact on recipients, it is timely to take a look at our collective inventory management practices. This International Forum represents an effort to get a snap shot of inventory management practices around the world, and to understand the range of different products provided for patients. In addition to sharing current inventory management practices, this Forum is intended to foster an exchange of ideas around where we see our field moving with respect to various issues including specialty products, new technologies, and reducing recipient risk from blood transfusion products.

RNA polymerase II initiation factor interactions and transcription start site selection.

An RNA polymerase II transcription system was resolved and reconstituted from extracts of Schizosaccharomyces pombe. Exchange with components of a Saccharomyces cerevisiae system was undertaken to reveal the factor or factors responsible for the difference in location of the transcription start site, about 30 base pairs and 40 to 120 base pairs downstream of the TATA box in S. pombe and S. cerevisiae, respectively. Two components, counterparts of human transcription factor IIF (TFIIF) and TFIIH, could be exchanged individually between systems without effect on the start site. Three components, counterparts of human TFIIB, TFIIE, and RNA polymerase II, could not be exchanged individually but could be swapped in the pairs TFIIE-TFIIH and TFIIB-RNA polymerase II, which demonstrates that there are functional interactions between these components. Moreover, exchange of the latter pair shifted the starting position, which shows that TFIIB and RNA polymerase II are solely responsible for determining the start site of transcription.

Initiation by yeast RNA polymerase II at the adenoviral major late promoter in vitro.

Transcription of the yeast CYC1 promoter fused to a sequence lacking guanosine residues provided a rapid, sensitive assay of initiation by RNA polymerase II in yeast extracts. Initiation was enhanced by yeast and mammalian activator proteins. The adenoviral major late promoter fused to the G-minus sequence was transcribed in yeast extracts with an efficiency comparable to that observed in HeLa extracts, showing that promoters as well as transcription factors are functionally interchangeable across species. Initiation occurred at different sites, approximately 30 and 63 to 69 base pairs downstream of the TATA element of the adenoviral promoter in HeLa and yeast extracts, respectively, distances characteristic of initiation in the two systems in vivo. A component of the transcription system and not the promoter sequence determines the distance to the initiation site.

Pathogenesis of mucosal injury in the blind loop syndrome. Brush border enzyme activity and glycoprotein degradation.

The effect of intestinal bacterial over-growth on brush border hydrolases and brush border glycoproteins was studied in nonoperated control rats, control rats with surgically introduced jejunal self-emptying blind loops, and rats with surgically introduced jejunal self-filling blind loops. Data were analyzed from blind loop segments, segments above and below the blind loops, and three corresponding segments in the nonoperated controls. Rats with self-filling blind loops had significantly greater fat excretion than controls and exhibited significantly lower conjugated:free bile salt ratios in all three segments. Maltase, sucrase, and lactase activities were significantly reduced in homogenates and isolated brush borders from the self-filling blind loop, but alkaline phosphatase was not affected. The relative degradation rate of homogenate and brush border glycoproteins was assessed by a double-isotope technique involving the injection of d-[6-(3)H]glucosamine 3 h and d-[U-(14)C]glucosamine 19 h before sacrifice, and recorded as a (3)H:(14)C ratio. The relative degradation rate in both homogenate and brush border fractions was significantly greater in most segments from rats with self-filling blind loops. In the upper and blind loop segments from rats with self-filling blind loops, the (3)H:(14)C ratios were higher in the brush border membrane than in the corresponding homogenates, indicating that the increased rates of degradation primarily involve membrane glycoproteins. Incorporation of d-[6-(3)H]glucosamine by brush border glycoproteins was not reduced in rats with self-filling blind loops, suggesting that glycoprotein synthesis was not affected. Polyacrylamide gel electrophoresis of brush border glycoproteins from the contaminated segments indicated that the large molecular weight glycoproteins, which include many of the surface hydrolases, were degraded most rapidly. Brush border maltase, isolated by immunoprecipitation, had (3)H:(14)C ratios characteristic of the most rapidly degraded glycoproteins. The results indicate that bacteria enhance the destruction of intestinal surface glycoproteins including disaccharidases. Since alkaline phosphatase, a glycoprotein, is not affected, the destruction is selective and presumably involves only the most exposed membrane components.

Rat intestinal phosphodiesterase II. Properties of the highly purified enzyme and its inactivation by iodoacetic acid.

A highly purifed preparation of rat intestinal phosphodiesterase II (oligonucleate 3'-nucleotidohydrolase, EC 3.1.4.18) has been studied using a synthetic substrate, thymidine 3'(2,4-dinitrophenyl) phosphate. The enzyme was most active between pH 6.1 and pH 6.7 and was inhibited by Cu2+ and Zn2+ but unaffected by EDTA, Mg2+, Co2+, and Ni2+. The reaction rate decreased at high levels of enzyme because of competitive inhibition by deoxythymidine 3'-phosphate, a reaction product, which showed a Ki of 2-10(-5) M. The molecular weight of the enzyme by gel-filtration was 150 000-170 000. In electrofocusing experiments multiple peaks of activity were found at pH 3.4, 4.2-4.5and 7.2. Polyacrylamide gel electrophoresis of freshly purified phosphodiesterase II showed up to 10 protein bands in the gels. If the preparations were stored at 4 degrees C for some time only one or two bands appeared. Investigation of the reaction of rat intestinal phosphodiesterase II with a number of possible phosphodiesterase substrates indicated that the enzyme required a nucleoside 3'-phosphoryl residue for the initiation of hydrolysis. Thus compounds such as NAD, ATP, bis-(p-nitrophenyl)phosphate, thymidine 5'-(p-nitrophenyl)phosphate, glycerylphosphorylcholine, guanylyl-(2' leads to 5')-adenosine and 3',5'-cyclic AMP which contain phosphodiester bonds, nevertheless were not substrates for the enzyme. The enzyme was inhibited reverisbly by p-chloromercuribenzoate and p-chloromercuriphenylsulfonate and inactivated irreversibly by iodoacetic acid. Activity of the phosphodiesterase II was reduced to 50% by incubation with 2.0-10(-3)--5.0-10(-3) M iodoacetate for 20--30 min at 24 degrees C at pH 5.0--6.1. Iodoacetamide had no effect. The degree of inactivation by iodoacetate was reduced by the presence of a substrate for the enzyme or, more effectively by deoxythymidine 3'-phosphate, a competitive inhibitor. It is concluded that iodoacetic acid alkylates an essential residue at the active centre of the enzyme.

Evaluation of protein-N-(2-hydroxypropyl)methacrylamide copolymer conjugates as targetable drug carriers. 1. Binding, pinocytic uptake and intracellular distribution of transferrin and anti-transferrin receptor antibody conjugates.

The transferrin receptor of human skin fibroblasts was studied as an in vitro model target antigen receptor for interaction with protein-polymer conjugates having potential for targeted drug delivery. Pinocytic uptake of 125I-labelled N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugated to monoclonal antibody B3/25 (specific for the transferrin receptor) or transferrin was up to 9-fold greater than uptake of the parent HPMA copolymer. The ability of these conjugates to bind specifically was confirmed by Scatchard analysis. Pinocytic internalisation was dependent on the molecular mass of the conjugate. Intracellular routing following internalisation was evaluated using density-gradient centrifugation. Unmodified HPMA copolymer was transferred via the endosomal compartment into secondary lysosomes, where, being resistant to degradation, it accumulated. Although the majority of endocytosed transferrin is recycled via the endosome, it was shown that any transferrin reaching the lysosomes was rapidly degraded and low-molecular-weight degradation products were released. Monoclonal antibody B3/25 showed a subcellular distribution consistent with prolongation on the cell surface, followed by internalisation and subcellular trafficking, via endosomes, into the lysosomal compartment, with subsequent degradation. Conjugation of protein to HPMA copolymer increased lysosomal accumulation of polymer up to 9-fold, with no detectable degradation of conjugate. The data presented here have implications regarding clinical potential of protein-HPMA copolymer conjugates designed for lysosomotropic drug delivery.

Recombination by resolvase is inhibited by lac repressor simultaneously binding operators between res sites.

The Tn3 resolvase requires that the two recombination (res) sites be aligned as direct repeats on the same molecule for efficient recombination to occur. To test whether resolvase must contact the DNA between res sites as predicted by tracking models, we have determined the sensitivity of recombination to protein diffusion blockades. Recombination between two res sites is unaffected either by lac repressor or bacteriophage T7 RNA polymerase being bound between them. Yet recombination is inhibited by lac repressor if the res site is bounded by a lac operator on both sides. We demonstrate that lac repressor will bind to more than one DNA site under the conditions used to assay recombination. This result suggests that lac repressor can inhibit resolvase by forming a DNA loop that isolates a res site topologically. These results do not support a tracking model for resolvase but suggest that the structure and topology of the DNA substrate is important in the formation of a synapse between res sites.

Analysis of inhibitors of the site-specific recombination reaction mediated by Tn3 resolvase.

The Tn3-encoded resolvase protein promotes a site-specific recombination reaction between two directly repeated copies of the recombination site res. Several inhibitors that block this event in vitro have been isolated. In this study four of these inhibitors were tested on various steps in the recombination reaction. Two inhibitors. A9387 and A1062, inhibit resolvase binding to the res site. Further, DNase I footprinting revealed that at certain concentrations of A9387 and A1062, resolvase was preferentially bound to site I of res, the site containing the recombinational crossover point. The two other inhibitors, A20812 and A21960, do not affect resolvase binding and bending of the DNA but inhibit synapse formation between resolvase and two directly repeated res sites.

Crystallization and preliminary X-ray analysis of C-reactive protein from rat.

Crystals of C-reactive protein from rat have been grown both with and without calcium. Two major components of the protein have been resolved on the basis of their calcium-dependent fine specificity for monophosphate esters. Crystals grown without calcium are tetragonal, space group P42(1)2 with unit cell parameters a = b = 163.81(9)A and c = 125.21(6)A, and diffract X-rays to 3.0 A resolution. The rotation function specifies a molecular 5-fold symmetry axis at 24 degrees away from c in the (110) plane.