Disparities in the Timeliness of Addressing Patient-Initiated Telephone Calls in a Primary Care Clinic: The Impact of Quality Improvement Interventions.

A timely response to patient-initiated telephone calls can affect many aspects of patient health, including quality of care and health equity. Historically, at a family medicine residency clinic, at least 1 out of 4 patient calls remained unresolved three days after the call was placed. We sought to explore whether there were differential delays in resolution of patient concerns for certain groups and how these were affected by quality improvement interventions to increase responsiveness to patient calls. A multidisciplinary team at a primary care residency clinic applied Lean education and tools to improve the timeliness of addressing telephone encounters. Telephone encounter data were obtained for one year before and nine months after the intervention. Data were stratified by race, ethnicity, preferred language, sex, online portal activation status, age category, zip code, patient risk category, and reason for call. Stratified data revealed consistently worse performance on telephone encounter closure by 72 hours for Black/African American patients compared to Hispanic and non-Hispanic White patients pre-intervention. Interventions resulted in statistically significant overall improvement, with an OR of 2.9 (95% CI: 2.62 to 3.21). Though interventions did not target a specific population, pre-intervention differences based on race and ethnicity resolved post-intervention. Telephone calls serve as an important means of patient communication with care teams. General interventions to improve the timeliness of addressing telephone encounters can lead to sustainable improvement in a primary care academic clinic and may also alleviate disparities.

SIG-DB: Leveraging homomorphic encryption to securely interrogate privately held genomic databases.

Genomic data are becoming increasingly valuable as we develop methods to utilize the information at scale and gain a greater understanding of how genetic information relates to biological function. Advances in synthetic biology and the decreased cost of sequencing are increasing the amount of privately held genomic data. As the quantity and value of private genomic data grows, so does the incentive to acquire and protect such data, which creates a need to store and process these data securely. We present an algorithm for the Secure Interrogation of Genomic DataBases (SIG-DB). The SIG-DB algorithm enables databases of genomic sequences to be searched with an encrypted query sequence without revealing the query sequence to the Database Owner or any of the database sequences to the Querier. SIG-DB is the first application of its kind to take advantage of locality-sensitive hashing and homomorphic encryption to allow generalized sequence-to-sequence comparisons of genomic data.

Fecal Impaction With Multisystem Organ Involvement.

Fecal impactions are a common complaint in the emergency department (ED) population. The potential for significant derangement in physiologic processes of other organ systems is often underappreciated. A 19-year-old male, previously healthy, presented to the ED at our institution with complaint of abdominal pain, which was found to be secondary to severe fecal impaction. In the search for alternative diagnoses, imaging was performed, which revealed effects on multiple other organ systems. This case illustrates the secondary effects of a severe fecal impaction. The emergency physician must be aware of these consequences, as the opportunity to review labs and imaging is not often provided in the standard workup of these patients.

A strain of Yersinia pestis with a mutator phenotype from the Republic of Georgia.

We describe here a strain of Yersinia pestis, G1670A, which exhibits a baseline mutation rate elevated 250-fold over wild-type Y. pestis. The responsible mutation, a C to T substitution in the mutS gene, results in the transition of a highly conserved leucine at position 689 to arginine (mutS(L689R)). When the MutSL 689R protein of G1670A was expressed in a ΔmutS derivative of Y. pestis strain EV76, mutation rates observed were equivalent to those observed in G1670A, consistent with a causal association between the mutS mutation and the mutator phenotype. The observation of a mutator allele in Yersinia pestis has potential implications for the study of evolution of this and other especially dangerous pathogens.

Population-level variation of the preproricin gene contradicts expectation of neutral equilibrium for generalist plant defense toxins.

The preproricin gene encodes ricin, the highly toxic, type II ribosome-inactivating protein of castor bean (Ricinus communis L.). As a generalist plant defense gene, preproricin is expected to exhibit population-level variation consistent with the neutral equilibrium model and to comprise few functionally different alleles. We first test the hypothesis that the preproricin gene family should comprise six to eight members by searching the publicly available draft genome sequence of R. communis and analyzing its ricin-like loci. We then test the neutral equilibrium expectation for the preproricin gene by characterizing its allelic variation among 25 geographically diverse castor bean plants. We confirm the presence of six ricin-like loci that share with the preproricin gene 62.9-96.3% nucleotide identity and intact A-chains. DNA sequence variation among the preproricin haplotypes significantly rejects tests of the neutral equilibrium model. Replacement mutations preserve the 12 amino acids known to affect catalytic and electrostatic interactions of the native protein toxin, which suggests functional divergence among alleles has been minimal. Nucleotide polymorphism is maintained by purifying selection (omega < 0.3) yet includes an excess of rare silent mutations greater than predicted by the neutral equilibrium model. Development of robust detection methods for ricin contamination must account for the presence of these other ricin-like molecules and should leverage the specificity provided by the numerous single nucleotide polymorphisms in the preproricin gene.

Characterization of ricin toxin family members from Ricinus communis.

Ricin inhibits translation by removal of a specific adenine from 28S RNA. The Ricinus communis genome encodes seven full-length ricin family members. All encoded proteins have the ability of hydrolyzing adenine in 28S rRNA. As expected, these proteins also inhibited an in vitro transcription/translation system. These data show that the ricin gene family contains at least seven members that have the ability to inhibit translation and that may contribute to the toxicity of R. communis.

Recombinant protein production in insect larvae: host choice, tissue distribution, and heterologous gene instability.

The expression of the fluorescent protein, DsRed, facilitates the optimization of protein production in orally-infected whole larvae. Trichoplusia ni was shown to be a much better host for recombinant AcMNPV compared to four other noctuid Lepidopteran species achieving 100% infectivity at the minimal tested dose. The highest density of marker protein was found in endothelial and tracheal cells, fat body, and hemocytes. Trichoplusia ni larvae possessed visually detected color over sequential passages of oral infection until the sixth round. Western blot analysis confirmed the progressive decrease of both tetramer and monomer forms of DsRed. The intact DsRed gene and promoter region was present in late passages, but viral population carrying the heterologous gene had dropped more than 2-logs after the fifth round while the amount of total viral DNA remained unchanged over sequential passages.

Production of a recombinant antibody fragment in whole insect larvae.

Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.

Real-time fluorogenic PCR assays for the detection of entA, the gene encoding staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) is among the most potent of the growing list of known enterotoxins produced by Staphylococcus aureus. SEA, a 27 kDa monomeric protein, is encoded by the entA gene. We have developed two real-time fluorogenic PCR assays for the detection of nucleic acid sequences in entA. The assays are useful in detecting and identifying strains of S. aureus that produce SEA and can serve a confirmatory role in determining the presence of SEA in food samples. The assays were tested in two real-time PCR formats, using either dye-labeled DNA probes corresponding to each primer set that are degraded by the 5' exonuclease activity of Taq polymerase, or a PCR master mix that contains the DNA-binding dye SYBR Green. In both formats the assays have a limit of detection of between 1 and 13 copies of a S. aureus genome that contains a copy of entA. Neither assay cross-reacted with genomic DNA isolated from other strains of S. aureus or other species.

Real-time fluorogenic reverse transcription-PCR assays for detection of bacteriophage MS2.

Bacteriophage MS2 is used in place of pathogenic viruses in a wide variety of studies that range from testing of compounds for disinfecting surfaces to studying environmental transport and fate of pathogenic viruses in groundwater. MS2 is also used as a pathogen simulant in the research, development, and testing (including open air tests) of methods, systems, and devices for the detection of pathogens in both the battlefield and homeland defense settings. PCR is often used as either an integral part of such detection systems or as a reference method to assess the sensitivity and specificity of microbial detection. To facilitate the detection of MS2 by PCR, we describe here a set of real-time fluorogenic reverse transcription-PCR assays. The sensitivity of the assays (performed with primer pairs and corresponding dye-labeled probes) ranged from 0.4 to 40 fg of MS2 genomic RNA (200 to 20,000 genome equivalents). We also demonstrate the usefulness of the primer pairs in assays without dye-labeled probe that included the DNA-binding dye SYBR green. None of the assays gave false-positive results when tested against 400 pg of several non-MS2 nucleic acid targets.

A laboratory-based study to assess the performance of surgical gloves.

The inherent tear resistance and elasticity of latex and the touch sensitivity it provides has made it the traditional material of choice for surgical gloves, protecting both health care workers and patients from the transmission of bloodborne infections. Although increased incidence of latex allergy has led to increased use of nonlatex surgical gloves, the effectiveness of these gloves as a barrier to infection has not been examined thoroughly. This laboratory-based study compared the performance of latex and nonlatex surgical gloves in a simulated stress protocol. The propensity of surgical gloves to fail was dependent on glove material, manufacturer, and stress. Nonlatex neoprene and nitrile gloves were comparable to latex and can provide a good alternative to latex for allergic patients and health care workers. In this study, isoprene was found to be inferior to latex and other nonlatex materials. The presence or absence of glove powder had no significant influence on the probability of glove failure.

Regulation of Sinorhizobium meliloti 1021 rrnA-reporter gene fusions in response to cold shock.

We previously reported that mutants of Sinorhizobium meliloti 1021 carrying luxAB insertions in each of the three 16S rRNA genes exhibited a dramatic (> or = 28-fold) increase in luminescence following a temperature downshift from 30 to 15 degrees C. These results raised the possibility that the rRNA operons (rrn) of S. meliloti were cold shock loci. In testing this possibility, we found that fusion of the S. meliloti 1021 rrnA promoter to two different reporter genes, luxAB and uidA, resulted in hybrid genes that were transiently upregulated (as measured by transcript accumulation) about four- to sixfold in response to a temperature downshift. These results are consistent with the hypothesis that the rrn promoters are transiently upregulated in response to cold shock. However, much of the apparent cold shock regulation of the initial luxAB insertions was due to an unexpected mechanism: an apparent temperature-dependent inhibition of translation. Specifically, the rrnA sequences from +1 to +172 (relative to the start of transcription) were found to greatly decrease the ability of S. meliloti to translate hybrid rrn-luxAB transcripts into active protein at 30 degrees C. This effect, however, was largely eliminated at 15 degrees C. Possible mechanisms for the apparent transient increase in rrnA promoter activity and temperature-dependent inhibition of translation are discussed.

PCR-based method for detecting viral penetration of medical exam gloves.

The test approved by the U.S. Food and Drug Administration for assessment of the barrier quality of medical exam gloves includes visual inspection and a water leak test. Neither method tests directly the ability of gloves to prevent penetration by microorganisms. Methods that use microorganisms (viruses and bacteria) to test gloves have been developed but require classical culturing of the organism to detect it. We have developed a PCR assay for bacteriophage phiX174 that allows the rapid detection of penetration of gloves by this virus. The method is suitable for use with both latex and synthetic gloves. The presence of glove powder on either latex or synthetic gloves had no effect on the ability of the PCR assay to detect bacteriophage DNA. The assay is rapid, sensitive, and inexpensive; requires only small sample volumes; and can be automated.

Issues in preparedness for biologic terrorism: a perspective for critical care nursing.

Although the use of microorganisms as weapons is as old a practice as war itself, the sense of our collective vulnerability to these agents has seldom been as great. The events of late 2001 demonstrated that the United States is vulnerable to terrorist attack carried out by highly motivated, organized, funded, and trained individuals. It is our collective good fortune that the perpetrator of the anthrax mailings was not bent on destruction of the scale witnessed on September 11, 2001. Because acute care and critical care nurses are on the forefront of community disease surveillance, they must be aware of the signs and symptoms of illness that may indicate that a biological attack has taken place. Many symptoms of infection or intoxication by biological warfare agents (bacterial, viral, and toxic) are nonspecific and flulike in nature, at least early in the disease process. The essential details of the presentation, diagnosis, treatment, and prophylaxis of the biological warfare agents that merit greatest concern are provided, and five biological warfare agents of particular interest are described in detail: anthrax, ricin (castor bean) toxin, smallpox, plague, and tularemia. Recommendations are given for additional Web-based resources to allow further study.

Performance of latex and nonlatex medical examination gloves during simulated use.

BACKGROUND: In response to the rise in latex allergies, gloves made from a variety of nonlatex materials have been introduced into the health care environment. To date, at least 1 study, by Rego and Roley (1999), has reported that both latex and nitrile medical examination gloves provide comparable barrier protective qualities. The purpose of our study was to determine the effects of glove stress, type of material (vinyl, nitrile, copolymer, latex), and manufacturer on the barrier effectiveness of medical examination gloves. METHOD: A total of 5510 medical examination gloves (1464 nitrile, 1052 latex, 1006 copolymer, and 1988 vinyl) were divided into 2 groups: stressed and unstressed. Unstressed gloves were visually inspected and water-tested according to the Food and Drug Administration water-testing standards. Stressed gloves were manipulated according to a designated stress protocol, visually inspected, and then subjected to the same Food and Drug Administration water-testing standards. RESULTS: Our limited sample size demonstrated that nitrile gloves had the lowest failure rate (1.3%), followed by latex (2.2%); vinyl and copolymer gloves had the highest failure rate (both 8.2%). With use of a logistic regression analysis adjusting for manufacturer and stress, latex examination gloves were found to be 3 times more likely to fail than nitrile gloves (odds ratio, 3.2; 95% CI, 1.37-7.50). Nitrile gloves were also found to fail significantly less often than vinyl or copolymer gloves (odds ratio, 12.60; 95% CI, 5.80-27.40). CONCLUSIONS: Nitrile examination gloves are a suitable alternative to latex, whereas vinyl and copolymer examination gloves were found to be less effective barriers. Further research is indicated to determine whether nitrile gloves can provide effective barrier qualities during clinical use versus laboratory simulations.

Mutants of Rhizobium tropici strain CIAT899 that do not induce chlorosis in plants.

Type B strains of Rhizobium tropici induce severe foliar chlorosis when applied at planting to seeds of symbiotic host and non-host dicotyledonous plants. A Tn5-induced mutant, designated CT4812, or R. tropici strain CIAT899 that was unable to induce chlorosis was isolated. Cloning and sequencing of the DNA flanking the transposon in CT4812 revealed that the Tn5 insertion is located in a gene similar to glnD, which encodes uridylyltransferase/uridylyl-removing enzyme in enteric bacteria. Two marker-exchange mutants with insertions in glnD also failed to induce chlorosis in bean (Phaseolus vulgaris) plants. The 5'-most insertion in glnD (in mutant strain ME330) abolished the ability of R. tropici to utilize nitrate as a sole carbon source, whereas a mutation in glnD further downstream (in mutant strain ME245) did not have an obvious effect on nitrate utilization. A gene similar to the Salmonella typhimurium virulence gene mviN overlaps the 3' end of the R. tropici glnD homologue. A mutation in mviN had no effect on the ability of CIAT899 to induce chlorosis in bean plants. Therefore the glnD homologue, but not mviN, appears to be required for induction of chlorosis in plants by R. tropici strain CIAT899. A high nitrogen: carbon ratio in the rhizosphere of bean plants also prevented R. tropici from inducing chlorosis in bean plants. Mutations in either the glnD homologue or mviN had no significant effect on root nodule formation or acetylene reduction activity. A mutation in mviN eliminated motility in R. tropici. The sequence data, the inability of the glnD mutant to utilize nitrate, and the role of the R. tropici glnD gene in chlorosis induction in plants, a process that is nitrogen regulated, suggest that glnD plays a role in nitrogen sensing in R. tropici as its homologues do in other organisms.