David R. Hillyard

Community-Acquired Pneumonia Requiring Hospitalization among U.S. Adults

BACKGROUND Community-acquired pneumonia is a leading infectious cause of hospitalization and death among U.S. adults. Incidence estimates of pneumonia confirmed radiographically and with the use of current laboratory diagnostic tests are needed. METHODS We conducted active population-based surveillance for community-acquired pneumonia requiring hospitalization among adults 18 years of age or older in five hospitals in Chicago and Nashville. Patients with recent hospitalization or severe immunosuppression were excluded. Blood, urine, and respiratory specimens were systematically collected for culture, serologic testing, antigen detection, and molecular diagnostic testing. Study radiologists independently reviewed chest radiographs. We calculated population-based incidence rates of community-acquired pneumonia requiring hospitalization according to age and pathogen. RESULTS From January 2010 through June 2012, we enrolled 2488 of 3634 eligible adults (68%). Among 2320 adults with radiographic evidence of pneumonia (93%), the median age of the patients was 57 years (interquartile range, 46 to 71); 498 patients (21%) required intensive care, and 52 (2%) died. Among 2259 patients who had radiographic evidence of pneumonia and specimens available for both bacterial and viral testing, a pathogen was detected in 853 (38%): one or more viruses in 530 (23%), bacteria in 247 (11%), bacterial and viral pathogens in 59 (3%), and a fungal or mycobacterial pathogen in 17 (1%). The most common pathogens were human rhinovirus (in 9% of patients), influenza virus (in 6%), and Streptococcus pneumoniae (in 5%). The annual incidence of pneumonia was 24.8 cases (95% confidence interval, 23.5 to 26.1) per 10,000 adults, with the highest rates among adults 65 to 79 years of age (63.0 cases per 10,000 adults) and those 80 years of age or older (164.3 cases per 10,000 adults). For each pathogen, the incidence increased with age. CONCLUSIONS The incidence of community-acquired pneumonia requiring hospitalization was highest among the oldest adults. Despite current diagnostic tests, no pathogen was detected in the majority of patients. Respiratory viruses were detected more frequently than bacteria. (Funded by the Influenza Division of the National Center for Immunizations and Respiratory Diseases.).

Contulakin-G, an O-glycosylated invertebrate neurotensin

We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide β-d-Galp-(1→3)-α-d-GalpNAc-(1→) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(β→3) GalNAc(α→)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakı́n-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10contulakin-G was approximately an order of magnitude lower than that of neurotensin1–13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.

Identification of Mycobacterium spp. by Using a Commercial 16S Ribosomal DNA Sequencing Kit and Additional Sequencing Libraries

ABSTRACT Current methods for identification of Mycobacterium spp. rely upon time-consuming phenotypic tests, mycolic acid analysis, and narrow-spectrum nucleic acid probes. Newer approaches include PCR and sequencing technologies. We evaluated the MicroSeq 500 16S ribosomal DNA (rDNA) bacterial sequencing kit (Applied Biosystems, Foster City, Calif.) for its ability to identify Mycobacterium isolates. The kit is based on PCR and sequencing of the first 500 bp of the bacterial rRNA gene. One hundred nineteen mycobacterial isolates (94 clinical isolates and 25 reference strains) were identified using traditional phenotypic methods and the MicroSeq system in conjunction with separate databases. The sequencing system gave 87% (104 of 119) concordant results when compared with traditional phenotypic methods. An independent laboratory using a separate database analyzed the sequences of the 15 discordant samples and confirmed the results. The use of 16S rDNA sequencing technology for identification of Mycobacterium spp. provides more rapid and more accurate characterization than do phenotypic methods. The MicroSeq 500 system simplifies the sequencing process but, in its present form, requires use of additional databases such as the Ribosomal Differentiation of Medical Microorganisms (RIDOM) to precisely identify subtypes of type strains and species not currently in the MicroSeq library.

Speciation of Cone Snails and Interspecific Hyperdivergence of Their Venom Peptides: Potential Evolutionary Significance of Intronsa

ABSTRACT: All 500 species of cone snails (Conus) are venomous predators. From a biochemical/genetic perspective, differences among Conus species may be based on the 50‐200 different peptides in the venom of each species. Venom is used for prey capture as well as for interactions with predators and competitors. The venom of every species has its own distinct complement of peptides. Some of the interspecific divergence observed in venom peptides can be explained by differential expression of venom peptide superfamilies in different species and of peptide superfamily branching in various Conus lineages into pharmacologic groups with different targeting specificity. However, the striking interspecific divergence of peptide sequences is the dominant factor in the differences observed between venoms. The small venom peptides (typically 10‐35 amino acids in length) are processed from larger prepropeptide precursors (ca. 100 amino acids). If interspecific comparisons are made between homologous prepropeptides, the three different regions of a Conus peptide precursor (signal sequence, pro‐region, mature peptide) are found to have diverged at remarkably different rates. Analysis of synonymous and nonsynonymous substitution rates for the different segments of a prepropeptide suggests that mutation frequency varies by over an order of magnitude across the segments, with the mature toxin region undergoing the highest rate. The three sections of the prepropeptide which exhibit apparently different mutation rates are separated by introns. This striking segment‐specific rate of divergence of Conus prepropeptides suggests a role for introns in evolution: exons separated by introns have the potential to evolve very different mutation rates. Plausible mechanisms that could underlie differing mutational frequency in the different exons of a gene are discussed.

Constant and hypervariable regions in conotoxin propeptides.

Conotoxins are small cysteine rich peptides found in the venom of the predatory cone snails (Conus) which have prove to be useful high affinity ligands for various receptors and ion channels. The first cloning data for conotoxins, reported here, were obtained for the King‐Kong peptide, a 27 amino acid conotoxin found in the venom of the cloth‐of‐gold cone, Conus textile. Analysis of cDNA clones of the King‐Kong peptide revealed a family of related toxin transcripts. Three different propeptide cDNA sequences were obtained; only one of these encoded sequence for the King‐Kong peptide. The other cDNA sequences encoded two different peptides (KK‐1 and KK‐2). When the predicted propeptide sequences are compared, well defined conserved and hypervariable regions can be identified. The hypervariable regions comprise four regions between Cys residues in the final peptide toxins; the remainder of the propeptide sequences, i.e. the excised N‐terminal regions and the disulfide bonded Cys residues, are highly conserved. We suggest that the conserved regions may direct the formation of a specific disulfide configuration in the King‐Kong family of conotoxins.

μ-Conotoxin PIIIA, a New Peptide for Discriminating among Tetrodotoxin-Sensitive Na Channel Subtypes

We report the characterization of a new sodium channel blocker, μ-conotoxin PIIIA (μ-PIIIA). The peptide has been synthesized chemically and its disulfide bridging pattern determined. The structure of the new peptide is: where Z = pyroglutamate andO = 4-trans-hydroxyproline. We demonstrate that Arginine-14 (Arg14) is a key residue; substitution by alanine significantly decreases affinity and results in a toxin unable to block channel conductance completely. Thus, like all toxins that block at Site I, μ-PIIIA has a critical guanidinium group. This peptide is of exceptional interest because, unlike the previously characterized μ-conotoxin GIIIA (μ-GIIIA), it irreversibly blocks amphibian muscle Na channels, providing a useful tool for synaptic electrophysiology. Furthermore, the discovery of μ-PIIIA permits the resolution of tetrodotoxin-sensitive sodium channels into three categories: (1) sensitive to μ-PIIIA and μ-conotoxin GIIIA, (2) sensitive to μ-PIIIA but not to μ-GIIIA, and (3) resistant to μ-PIIIA and μ-GIIIA (examples in each category are skeletal muscle, rat brain Type II, and many mammalian CNS subtypes, respectively). Thus, μ-conotoxin PIIIA provides a key for further discriminating pharmacologically among different sodium channel subtypes.

Multicenter Comparison of Different Real-Time PCR Assays for Quantitative Detection of Epstein-Barr Virus

ABSTRACT Quantification of Epstein-Barr virus (EBV) in peripheral blood is important for the diagnosis and management of serious EBV diseases, including posttransplant lymphoproliferative disorder. A variety of PCR-based methods are currently in use; however, there is little information on their comparability. This study assessed the relative performance of different quantitative assays. A multicenter comparative study was performed at eight sites using three panels consisting of serial dilutions of quantified EBV DNA and extracts from a total of 19 whole-blood specimens. Samples were distributed and tested blindly. Instrumentation, probe chemistries, amplification targets, and other test-related aspects varied considerably between laboratories. Each laboratorys calibration curve indicated strong evidence of a consistent log-linear relationship between viral load and cycle threshold, suggesting that intralaboratory tracking of a given patient would yield similar relative quantitative trends among the participating test sites. There was strong concordance among laboratories with respect to qualitative test results; however, marked quantitative discordance was seen. For most samples, the across-laboratory interquartile range of the reported viral load (in copies/μl) was roughly 0.6 log-units, and for one sample the overall range was approximately 4.2 log-units. While intralaboratory tracking of patients may yield similar results, these data indicate a need for caution when attempting to compare clinical results obtained at different institutions and suggest the potential value to be gained by more standardized testing methodology.

Diagnosis and outcomes of enterovirus infections in young infants

Background: Enterovirus (EV) infections commonly cause fever in infants younger than 90 days of age. The polymerase chain reaction (PCR) has improved our ability to diagnose EV infections. Objective: To evaluate the utility of blood and cerebrospinal fluid (CSF) specimens for the diagnosis of EV infections by PCR and to describe a large cohort of EV-infected infants. Design/Methods: Febrile infants younger than 90 days of age evaluated for sepsis at Primary Childrens Medical Center in Salt Lake City, UT, were enrolled in a prospective study designed to identify viral infections from December 1996 to June 2002. All patients had bacterial cultures of blood, urine and CSF. Testing for EV was performed by PCR and/or viral cultures. Patients who were positive for EV were identified for this study. Results: Of 1779 febrile infants enrolled, 1061 had EV testing and 214 (20%) were EV-positive. EV infections were diagnosed by PCR of blood, CSF or both in 93% of infants. PCR testing was positive in blood in 57%, and blood was the only positive specimen for 22% of EV infected infants. PCR of CSF was positive in 74%. The mean age of infants with EV infection was 33 days, with 18% younger than 14 days and 5% younger than 7 days. Fifty percent of EV-positive infants had CSF pleocytosis. Of EV PCR-positive infants, 91% were admitted, and 2% required intensive care. Possible serious EV disease was diagnosed in <1%, and there were no deaths. Twelve infants (5.6%) had concomitant urinary tract infection, and 3 (1%) had bacteremia. Conclusions: EV infections are common in febrile infants younger than 90 days. Blood and CSF are equally likely to yield positive results by PCR, but the combination of both specimens improved the diagnostic yield.

Combinatorial peptide libraries in drug design: lessons from venomous cone snails

Many present-day drugs are derived from compounds that are natural products, a traditional source of which is fermentation broths of microorganisms. The venoms of cone snails are a new natural resource of peptides that may have a pharmaceutical potential equivalent to those from traditional sources, particularly for developing drugs that target cell-surface receptors or ion channels. In effect, cone snails have used a combinatorial library strategy to evolve their small, highly bioactive venom peptides. The methods by which the snails have generated thousands of peptides with remarkable specificity and high affinity for their targets may provide important lessons in designing combinatorial libraries for drug development.

Conantokin-G precursor and its role in gamma-carboxylation by a vitamin K-dependent carboxylase from a Conus snail.

Conantokin-G isolated from the marine snailConus geographus is a 17-amino acid γ-carboxyglutamate (Gla)-containing peptide that inhibits theN-methyl-d-aspartate receptor. We describe the cloning and sequence of conantokin-G cDNA and the possible role of the propeptide sequence. The cDNA encodes a 100amino acid peptide. The N-terminal 80 amino acids constitute the prepro-sequence, and the mature peptide is derived from the remaining C-terminal residues after proteolysis, C-terminal amidation, and a unique post-translational modification, γ-carboxylation of glutamate residues to Gla. Mature conantokin-G peptide containing Glu residues (E.Con-G) in place of Gla is a poor substrate for the vitamin K-dependent γ-glutamyl carboxylase (apparentK m = 3.4 mm). Using peptides corresponding to different segments of the propeptide we investigated a potential role for the propeptide sequences in γ-carboxylation. Propeptide segment −20 to −1 covalently linked to E.Con-G or the synthetic pentapeptide FLEEL increased their apparent affinities 2 orders of magnitude. These substrates are not efficiently carboxylated by the bovine microsomal γ-glutamyl carboxylase, suggesting differences in specificities between the Conus and the mammalian enzyme. However, the role of propeptide in enhancing the efficiency of carboxylation is maintained.