Are quantitative bacterial wound cultures useful?

Determining if a nonhealing wound is infected can be difficult. The surface of a wound is not sterile and can be colonized with numerous commensal, environmental, and potentially pathogenic microorganisms. Different types of wounds have various clinical presentations, with some signs and symptoms more likely to be present than others depending on the type and location of the wound. Clinicians often order microbiology wound cultures to assist in determining if a nonhealing wound is infected. This minireview briefly summarizes the clinical microbiology of wound cultures, with an emphasis on the history and utility (or lack thereof) of the quantitative wound culture.

Future-generation sequencing and clinical microbiology.

Sequencing technologies are changing the way both laboratory medicine and clinical practice impact patient care. This article focuses on the clinical microbiology laboratory and the potential benefits and limitations of coming generations of sequencing technology. Nucleic acid sequencing technology is rapidly outpacing the infrastructure needed to accurately educate, analyze, and interpret complex massive data sets that are rapidly becoming integrated into clinical practice.

Evaluation of two commercial immunoassays used to screen patients for systemic lupus erythematosus.

Diagnosing systemic lupus erythematosus (SLE) can be challenging as laboratory screening methods, although sensitive, lack specificity. The poor specificity of autoimmune testing produces more false positive results than true positive results. False positive results can cause stress to patients without autoimmune disease and require unnecessary rheumatology consultation to rule out disease. Our objective was to evaluate two screening assays to reduce the number of false positives while maintaining high sensitivity. In this study, we evaluated two immunoassays, the AtheNA Multi-Lyte II ANA System and QUANTA Lite ANA ELISA, to screen patients for SLE. All positive screening results were compared to immunoflourescent ANA testing using theHEp-2000 ANA System. A chart review was performed on all patients tested to determine clinical diagnosis of SLE. The QuantaLite ANA ELISA produced significantly more false positive results than the AtheNA Multi-Lyte II Test System when screening for SLE in our patient population.

Recovery of a catalase-negative Staphylococcus epidermidis strain in blood and urine cultures from a patient with pyelonephritis.

This report describes a 60-year-old patient with bilateral nephrolithiasis. A catalase-negative Staphylococcus epidermidis strain was recovered from both urine and blood cultures. Although rare, isolates of catalase-negative Staphylococcus spp., including Staphylococcus aureus, have been reported. Here, we describe the first report of a catalase-negative S. epidermidis strain.

Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice.

The proportion of the human gut bacterial community that is recalcitrant to culture remains poorly defined. In this report, we combine high-throughput anaerobic culturing techniques with gnotobiotic animal husbandry and metagenomics to show that the human fecal microbiota consists largely of taxa and predicted functions that are represented in its readily cultured members. When transplanted into gnotobiotic mice, complete and cultured communities exhibit similar colonization dynamics, biogeographical distribution, and responses to dietary perturbations. Moreover, gnotobiotic mice can be used to shape these personalized culture collections to enrich for taxa suited to specific diets. We also demonstrate that thousands of isolates from a single donor can be clonally archived and taxonomically mapped in multiwell format to create personalized microbiota collections. Retrieving components of a microbiota that have coexisted in single donors who have physiologic or disease phenotypes of interest and reuniting them in various combinations in gnotobiotic mice should facilitate preclinical studies designed to determine the degree to which tractable bacterial taxa are able to transmit donor traits or influence host biology.

Evaluation of Spectra VRE, a new chromogenic agar medium designed to screen for vancomycin-resistant Enterococcus faecalis and Enterococcus faecium.

Spectra VRE (Remel, Lenexa, KS) is a chromogenic medium designed to recover and differentiate vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (VRE). This medium was compared to bile esculin azide agar (BEAV) and was 98.2% sensitive and 99.3% specific compared to BEAV, which was 87.6% sensitive and 87.1% specific at 24 h.

Evaluation of a chromogenic agar under development to screen for VRE colonization.

BBL CHROMagar VanRE (CVRE) was compared with bile esculin azide agar plus vancomycin to screen for vancomycin-resistant enterococcus (VRE) colonization. CVRE distinguishes Enterococcus faecalis (green colonies) from Enterococcus faecium (mauve colonies) on the basis of chromogenic substrate use. CVRE sensitivity and specificity were 98.6% and 99.1%. Positive and negative predictive values were 95.9% and 99.7%.

Development of a department of defense regional viral respiratory surveillance program.

A continuous viral respiratory surveillance program was established throughout the U.S. Department of Defense beneficiary population living in Europe with a few specimens coming from the Middle East. This program provided influenza rapid antigen test kits, specimen collection kits, detailed instructions, and a questionnaire. Training on specimen collection and testing was provided to health care providers and lab staff. We received 1875 patient specimens (39% active duty, 13% adult beneficiary, and 48% pediatric beneficiary) collected from 36 medical treatment facilities in 10 European and Middle Eastern countries over a 52-week period. Nine hundred and twenty-two questionnaires were received. The greatest activity of viral respiratory infections occurred between weeks 7 to 13. We found the sensitivity of rapid antigen testing compared poorly to both viral culture and PCR; however, the information provided by the rapid testing was utilized locally for guiding patient treatment. Additionally, although 91% of the active duty population received the influenza vaccine, we calculated the vaccine efficacy to be 52%.

Involvement of vacuolar protein sorting pathway in Ebola virus release independent of TSG101 interaction.

Budding of Ebola virus (EBOV) particles from the plasma membrane of infected cells requires viral and host proteins. EBOV virus matrix protein VP40 recruits TSG101, an ESCRT-1 (host cell endosomal sorting complex required for transport-1) complex protein in the vacuolar protein sorting (vps) pathway, to the plasma membrane during budding. Involvement of other vps proteins in EBOV budding has not been established. Therefore, we used VP40 deletion analysis, virus-like particle-release assays, and confocal microscopy to investigate the potential role of ESCRT-1 proteins VPS4, VPS28, and VPS37B in EBOV budding. We found that VP40 could redirect each protein from endosomes to the cell surface independently of TSG101 interaction. A lack of VPS4 adenosine triphosphatase activity reduced budding by up to 80%. Inhibition of VPS4 gene expression by use of phosphorodiamidite morpholino antisense oligonucleotides protected mice from lethal EBOV infection. These data show that EBOV can use vps proteins independently of TSG101 for budding and reveal VPS4 as a potential target for filovirus therapeutics.

Analysis of Ebola virus and VLP release using an immunocapture assay.

Ebola virus (EBOV), an emerging pathogen, is the causative agent of a rapidly progressive hemorrhagic fever with high mortality rates. There are currently no approved vaccines or treatments available for Ebola hemorrhagic fever. Standard plaque assays are currently the only reliable techniques for enumerating the virus. Effective drug-discovery screening as well as target identification and validation require simple and more rapid detection methods. This report describes the development of a rapid ELISA that measures virus release with high sensitivity. This assay detects both Ebola virus and EBOV-like particles (VLPs) directly from cell-culture supernatants with the VP40 matrix protein serving as antigen. Using this assay, the contribution of the EBOV nucleocapsid (NC) proteins in VLP release was determined. These findings indicate that a combination of NC proteins together with the envelope components is optimal for VLP formation and release, a finding that is important for vaccination with Ebola VLPs. Furthermore, this assay can be used in surrogate models in non-biocontainment environment, facilitating both basic research on the mechanism of EBOV assembly and budding as well as drug-discovery research.

Association of ebola virus matrix protein VP40 with microtubules.

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding.

The matrix protein VP40 plays a critical role in Ebola virus assembly and budding, a process that utilizes specialized membrane domains known as lipid rafts. Previous studies with purified protein suggest a role for oligomerization of VP40 in this process. Here, we demonstrate VP40 oligomers in lipid rafts of mammalian cells, virus-like particles, and in the authentic Ebola virus. By mutagenesis, we identify several critical C-terminal sequences that regulate oligomerization at the plasma membrane, association with detergent-resistant membranes, and vesicular release of VP40, directly linking these phenomena. Furthermore, we demonstrate the active recruitment of TSG101 into lipid rafts by VP40. We also report the successful application of the biarsenic fluorophore, FlAsH, combined with a tetracysteine tag for imaging of Ebola VP40 in live cells.

The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively.

We characterized two essential putative GTPases, Nog1p and Lsg1p, that are found associated with free 60S ribosomal subunits affinity purified with the nuclear export adapter Nmd3p. Nog1p and Lsg1p are nucleolar and cytoplasmic, respectively, and are not simultaneously on the same particle, reflecting the path of Nmd3p shuttling in and out of the nucleus. Conditional mutants of both NOG1 and LSG1 are defective in 60S subunit biogenesis and display diminished levels of 60S subunits at restrictive temperature. Mutants of both genes also accumulate the 60S ribosomal reporter Rpl25-eGFP in the nucleolus, suggesting that both proteins are needed for subunit export from the nucleolus. Since Lsg1p is cytoplasmic, its role in nuclear export is likely to be indirect. We suggest that Lsg1p is needed to recycle an export factor(s) that shuttles from the nucleus associated with the nascent 60S subunit.