Clinical Factors and Viral Load Influencing Severity of Acute Hepatitis A

Background and Aims

Clinical manifestations of hepatitis A virus (HAV) infection vary from mild to fulminant hepatic failure (FHF) in adults. We investigated the relationship between laboratory findings, including viral load, and clinical outcomes in patients with acute hepatitis A (AHA) and evaluated predictive factors for severe acute hepatitis (s-AH).

Methods

We analyzed the clinical manifestations of AHA in 770 patients. Patients with a prothrombin time (PT) of less than 40% of normal were classified as s-AH and included 4 patients with FHF, 11 patients with acute renal failure, and 3 patients with prolonged jaundice (n = 128). Other patients were defined as mild acute hepatitis (m-AH) (n = 642). Serum samples were obtained from 48 patients with acute hepatitis A. Among them, 20 with s-AH, and 28 with m-AH, were tested for HAV RNA titer.

Results

In a multivariate analysis, age (HR = 1.042, P = 0.041), peak creatinine (HR = 4.014, P = 0.001), bilirubin (HR = 1.153, P = 0.003), alanine aminotransferase (ALT) (HR = 1.001, P<0.001), initial lactate dehydrogenase (LDH) (HR = 1.000, P = 0.045) and total cholesterol (HR = 0.978, P<0.001) were independent factors for s-AH. Serum HAV RNA was detected in 20/20 (100%) patients with s-AH and 22/28 (78.6%) patients with m-AH. In a multivariate analysis of the 48 patients who were tested for HAV RNA, peak ALT (HR = 1.001, P = 0.004) and HAV RNA titer (HR = 2.076, P = 0.012) were independent factors for s-AH.

Conclusions

Clinical factors including age, peak creatinine, bilirubin, ALT, initial LDH and total cholesterol were independent factors for s-AH in a multivariate analysis. In particular, HAV load strongly correlated with the severity of hepatitis A.     

Nocardia vulneris sp. nov., isolated from wounds of human patients in North America

Nocardia species are ubiquitous in the environment with an increasing number of species isolated from clinical sources. From 2005 to 2009, eight isolates (W9042, W9247, W9290, W9319, W9846, W9851^T, W9865, and W9908) were obtained from eight patients from three states in the United States and Canada; all were from males ranging in age from 47 to 81 years old; and all were obtained from finger (n = 5) or leg (n = 3) wounds. Isolates were characterized by polyphasic analysis using molecular, phenotypic, morphologic and chemotaxonomic methods. Sequence analysis of 16S rRNA gene sequences showed the eight isolates are 100 % identical to each other and belong in the genus Nocardia. The nearest phylogenetically related neighbours were found to be the type strains for Nocardia altamirensis (99.33 % sequence similarity), Nocardia brasiliensis (99.37 %), Nocardia iowensis (98.95 %) and Nocardia tenerifensis (98.44 %). The G+C content of isolate W9851^T was determined to be 68.4 mol %. The DNA–DNA relatedness between strain W9851^T and the N. brasiliensis type strain was 72.8 % and 65.8 % when measured in the laboratory and in silico from genome sequences, respectively, and 95.6 % ANI. Whole-cell peptidoglycan was found to contain meso-diaminopimelic acid; MK-8-(H₄)_ω-cyc was identified as the major menaquinone; the major fatty acids were identified as C_16:0, 10 Me C_18:0, and C_{18:1 w9c}, the predominant phospholipids were found to include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides; whole-cell sugars detected were arabinose and galactose; and mycolic acids ranging from 38 to 60 carbon atoms were found to be present. These chemotaxonomic analyses are consistent with assignment of the isolates to the genus Nocardia. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra of the clinical isolates showed genus and species level profiles that were different from other Nocardia species. All isolates were resistant to ciprofloxacin, clarithromycin and imipenem but were susceptible to amikacin, amoxicillin/clavulanate, linezolid and trimethoprim/sulfamethoxazole. The results of our polyphasic analysis suggest the new isolates obtained from wound infections represent a novel species within the genus Nocardia, for which the name Nocardia vulneris sp. nov. is proposed, with strain W9851^T (= DSM 45737^T = CCUG 62683^T = NBRC 108936^T) as the type strain.     

Recognition intensities of submolecular structures,mammalian glyco-structural units,ligand cluster and polyvalency in abrin-a-carbohydrate interactions

Abrin-a is the most toxic fraction of lectins isolated from Abrus precatorius seeds and belongs to the family of type 2 ribosome inactivating proteins (RIP). This toxin may act as a defense molecule in plants against viruses, fungi and insects, where attachment of abrin-a to the exposed glycans on the surface of target cells is the crucial and initial step of its cytotoxicity. Although it has been studied for over four decades, the recognition factors involved in abrin-a-carbohydrate interaction remains to be clarified. In this study, roles of mammalian glyco-structural units, ligand clusters and polyvalency in abrin-a recognition were comprehensively analyzed by enzyme-linked lectinosorbent binding and inhibition assays. The results indicate that: (i) this toxin prefers oligosaccharides having α-anomer of galactose (Gal) at the non-reducing terminal than the corresponding β-anomer; (ii) Galα1-3Galα1- (B_α), Galα1-4Gal (E), Galβ1-3GalNAc (T) and Galβ1-3/4GlcNAc (I/II) related oligosaccharides were the active glyco-structural units; (iii) tri-antennary II_β, prepared from N-glycan of asialo fetuin, played a dominant role in recognition; (iv) many high-density polyvalent I_β/II_β and E_β glycotopes enhanced the reactivity; (v) the carbohydrate recognition domain of abrin-a is proposed to be a combination of a small cavity type of Gal as major site and a groove type of additional one to tetrasaccharides as subsites with a preference of α1-3/4/6Gal, β1-3GalNAc, β1-3/4/6GlcNAc, β1-4/6Glc, β1-3DAra and β1-4Man as subterminal sugars; (vi) size of the carbohydrate recognition domain may be as large enough to accommodate a linear pentasaccharide and complementary to Galα1-3Galβ1-4GlcNAc β1-3Galβ1-4Glc (gailipenta) sequence. A comparison of the recognition factors and combining sites of abrin-a with ricin, another highly toxic lectin, was also performed to further understand the differences in recognition factors between these two type 2 RIPs.     

Role of hydrogen generation by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in the oral cavity

Some gastrointestinal bacteria synthesize hydrogen (H₂) by fermentation. Despite the presence of bactericidal factors in human saliva, a large number of bacteria also live in the oral cavity. It has never been shown that oral bacteria also produce H₂ or what role H₂ might play in the oral cavity. It was found that a significant amount of H₂ is synthesized in the oral cavity of healthy human subjects, and that its generation is enhanced by the presence of glucose but inhibited by either teeth brushing or sterilization with povidone iodine. These observations suggest the presence of H₂-generating bacteria in the oral cavity. The screening of commensal bacteria in the oral cavity revealed that a variety of anaerobic bacteria generate H₂. Among them, Klebsiella pneumoniae (K. pneumoniae) generated significantly large amounts of H₂ in the presence of glucose. Biochemical analysis revealed that various proteins in K. pneumoniae are carbonylated under standard culture conditions, and that oxidative stress induced by the presence of Fe⁺⁺ and H₂O₂ increases the number of carbonylated proteins, particularly when their hydrogenase activity is inhibited by KCN. Inhibition of H₂ generation markedly suppresses the growth of K. pneumoniae. These observations suggest that H₂ generation and/or the reduction of oxidative stress is important for the survival and growth of K. pneumoniae in the oral cavity.     

Effects of Manduca sexta allatostatin and an analogue on the peach-potato aphid Myzus persicae (hemiptera: aphididae) and degradation by enzymes in the aphid gut

The oral toxicity of the C‐type allatostatin, Manduca sexta allatostatin (Manse‐AS) and the analogue δR³δR⁵Manse‐AS, where R residues were replaced by their D‐isomers, were tested against the peach‐potato aphid Myzus persicae by incorporation into an artificial diet. Both peptides had significant dose‐dependent effects on mortality, growth, and fecundity compared with control insects. The analogue, δR³δR⁵Manse‐AS, had an estimated LC₅₀ of 0.31 µg/µl diet and was more potent than Manse‐AS (estimated LC₅₀ of 0.58 µg/µl diet). At a dose of 0.35 µg δR³δR⁵Manse‐AS/µl diet, 76% of the aphids were dead after 6 days and all were dead after 10 days. In comparison, three times the dose of Manse‐AS was required to achieve 74% mortality after 8 days and 98% mortality after 16 days. The degradation of both peptides by extracts prepared from the gut of M. persicae was investigated. The estimated half‐life of Manse‐AS, when incubated with the gut extract from M. persicae, was 31 min. Degradation was due to a cathepsin L‐like cysteine protease, carboxypeptidase‐like activity, endoprotease activity with glutamine specificity, pyroglutamate aminopeptidase activity, and possibly trypsin‐like proteases. The half‐life of the δR³δR⁵ Manse‐AS analogue was enhanced (73 min) with the D‐isomers of R appearing to prevent cleavage around the R residues by cathepsin L‐like cysteine proteases or from trypsin‐like proteases. The greater stability of the analogue may explain its increased potency in M. persicae. This work demonstrates the potential use of Manse‐AS and analogues, with greater resistance to enzymatic attack, in aphid control strategies. © 2010 Wiley Periodicals, Inc.     

Oxidative damage in Parkinson disease: Measurement using accurate biomarkers

Oxidative damage has been implicated in the pathogenesis of Parkinson disease (PD) but the literature data are confusing. Using products of lipid and DNA oxidation measured by accurate methods, we assessed the extent of oxidative damage in PD patients. The levels of plasma F₂-isoprostanes (F₂-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), cholesterol oxidation products, neuroprostanes (F₄-NPs), phospholipase A₂ (PLA₂) and platelet activating factor–acetylhydrolase (PAF-AH) activities, urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), and serum high-sensitivity C-reactive protein were compared in 61 PD patients and 61 age-matched controls. The levels of plasma F₂-IsoPs, HETEs, 7β-and 27-hydroxycholesterol, 7-ketocholesterol, F₄-NPs, and urinary 8-OHdG were elevated, whereas the levels of plasma PLA₂ and PAF-AH activities were lower, in PD patients compared to controls (p <  0.05). The levels of plasma F₂-IsoPs, HETEs, and urinary 8-OHdG were higher in the early stages of PD (p trend <  0.05). There was a significant negative correlation between the cumulative intake of levodopa and urinary 8-OHdG (r =  ?0.305, p =  0.023) and plasma total HETEs (r =  ?0.285, p =  0.043). Oxidative damage markers are systemically elevated in PD, which may give clues about the relation of oxidative damage to the onset and progression of PD.     

The ribonucleases J1 and J2 are essential for growth and have independent roles in mRNA decay in Streptococcus pyogenes

The paralogous ribonucleases J1 and J2, recently identified in Bacillus subtilis, have both endoribonucleolytic and 5′‐to‐3′ exoribonucleolytic activities and participate in degradation and regulatory processing of mRNA. RNases J1 and J2 have partially overlapping target specificities, but only RNase J1 is essential for B. subtilis growth. Because mRNA decay is important in regulation of virulence factors of Streptococcus pyogenes (the group A streptococcus, GAS), we investigated the role of these newly described RNases in GAS. We found that conditional mutants for both RNases J1 and J2 require induction for growth, so we conclude that, unlike the case in B. subtilis, both of these RNases are essential for GAS growth, and therefore their functions are not redundant. We compared decay of representatives of the two classes of messages we had previously identified: Class I, which decay rapidly in exponential and stationary phase of growth (hasA and gyrA), and Class II, which are stable in stationary phase and exhibit a biphasic decay curve in exponential phase (sagA and sda). We report that RNases J1 and J2 affect the rate of decay of Class I messages and the length of the first phase in decay of Class II messages.     

Development and Application of a New Method for Specific and Sensitive Enumeration of Spores of Nonproteolytic Clostridium botulinum Types B,E, and F in Foods and Food Materials

The highly potent botulinum neurotoxins are responsible for botulism, a severe neuroparalytic disease. Strains of nonproteolytic Clostridium botulinum form neurotoxins of types B, E, and F and are the main hazard associated with minimally heated refrigerated foods. Recent developments in quantitative microbiological risk assessment (QMRA) and food safety objectives (FSO) have made food safety more quantitative and include, as inputs, probability distributions for the contamination of food materials and foods. A new method that combines a selective enrichment culture with multiplex PCR has been developed and validated to enumerate specifically the spores of nonproteolytic C. botulinum. Key features of this new method include the following: (i) it is specific for nonproteolytic C. botulinum (and does not detect proteolytic C. botulinum), (ii) the detection limit has been determined for each food tested (using carefully structured control samples), and (iii) a low detection limit has been achieved by the use of selective enrichment and large test samples. The method has been used to enumerate spores of nonproteolytic C. botulinum in 637 samples of 19 food materials included in pasta-based minimally heated refrigerated foods and in 7 complete foods. A total of 32 samples (5 egg pastas and 27 scallops) contained spores of nonproteolytic C. botulinum type B or F. The majority of samples contained <100 spores/kg, but one sample of scallops contained 444 spores/kg. Nonproteolytic C. botulinum type E was not detected. Importantly, for QMRA and FSO, the construction of probability distributions will enable the frequency of packs containing particular levels of contamination to be determined.Food-borne botulism is a severe and deadly intoxication caused by the consumption of food containing as little as 30 to 100 ng of preformed botulinum neurotoxin (45). More than 2,500 cases of botulism were reported in Europe in 1999 and 2000, with the majority of cases in the east of the continent (44). Currently, 25 to 50 food-borne botulism cases are diagnosed annually in the United States (27). There are seven distinct botulinum neurotoxins (types A to G) and a number of subtypes (6, 26, 45). In view of the potency of the botulinum neurotoxin and the severity of botulism, four phylogenetically distinct bacteria are grouped together as the Clostridium botulinum species, solely on the basis of their ability to form botulinum neurotoxin. The divergence between these four distinct bacteria is strong enough to merit their classification as distinct species and in some cases is significantly greater than that between bacteria belonging to different genera, e.g., Bacillus subtilis and Staphylococcus aureus (7). Two of these bacteria (proteolytic C. botulinum and nonproteolytic C. botulinum) are responsible for the majority of cases of food-borne botulism. Strains of proteolytic C. botulinum produce neurotoxins of type A, B, or F, form spores of high heat resistance, and have a minimum growth temperature of approximately 12°C (39). Strains of nonproteolytic C. botulinum produce neurotoxins of type B, E, or F, form spores of moderate heat resistance, and are able to grow and form toxin at 3°C (18, 48) and are recognized as the major hazard associated with minimally heated refrigerated foods (4, 37, 43, 44, 48). These new foods meet consumer demand for high-quality, convenient foods that are low in preservatives, and sales are presently increasing by about 10% per annum in many countries (3, 47).Quantitative microbiological risk assessment (QMRA) is now established as an important microbiology food safety tool (42). Process risk models have been used to assess the safety of specific foods with respect to nonproteolytic C. botulinum and the food-borne botulism hazard (e.g., 2, 41). These process risk models benefit from high-quality information, including that on the incidence of spores of nonproteolytic C. botulinum spores in food materials. The implementation of food safety objectives (FSO) also benefits from the availability of high-quality information on the microbial contamination of foods and food materials (24). This information is most effective in the form of probability distributions rather than as average spore concentrations or other statistics.The difficulty with enumerating nonproteolytic C. botulinum in foods is that there is no effective selective culture medium available. Surveys of the extent of contamination of foods and food materials have used a nonselective enrichment followed by either testing for neurotoxin using a mouse test or enzyme-linked immunosorbent assay (ELISA) or testing for the presence of neurotoxin genes using a PCR test (3, 10, 13, 35, 38, 39). This approach, however, is not optimized for nonproteolytic C. botulinum or proteolytic C. botulinum (therefore potentially failing to recover all spores of either organism) and may also not distinguish nonproteolytic C. botulinum from proteolytic C. botulinum. Heating at 80°C for 10 min followed by incubation at 35°C (54) may be reasonably selective for proteolytic C. botulinum, but there is no similar approach for nonproteolytic C. botulinum, although incubation at 28°C (54) may offer an element of selection. It is necessary, therefore, to develop a method to enumerate spores of nonproteolytic C. botulinum in food materials that is robust and optimized, as well as sensitive and specific for this particular pathogen (and does not also detect proteolytic C. botulinum). When enumerating bacteria in foods, it is essential to demonstrate the efficiency of the method by verifying that small concentrations (in the present study, spores of nonproteolytic C. botulinum) can be detected following addition to test samples.This paper describes the development, validation, and application of a new method to enumerate spores of nonproteolytic C. botulinum in foods and in food materials. This method has been designed to generate data for the construction of probability distributions that can be used in QMRA and FSO settings. Most of the effort has been dedicated to the development and evaluation of the enrichment procedure rather than the PCR test, as the PCR test has received much attention from others (e.g., 3, 10, 16, 36, 38). A low-temperature selective-enrichment procedure is described that has been optimized specifically for nonproteolytic C. botulinum over proteolytic C. botulinum and other bacteria. In order to detect low concentrations of spores, large quantities (200 g) of food materials and foods have been tested. Specific detection of neurotoxin genes is achieved by the use of an established multiplex PCR (36), with an internal amplification control now included (25). By the use of a set of control samples inoculated with defined concentrations of spores of nonproteolytic C. botulinum, the detection limit has been estimated for each food material and food tested. The method has been used in an extensive survey of raw materials intended for use in pasta ready meals, as well as the final meals themselves. The implications for risk assessment and risk management of chilled foods are discussed.