Practical Guidance for Clinical Microbiology Laboratories: Viruses Causing Acute Respiratory Tract Infections.

Respiratory viral infections are associated with a wide range of acute syndromes and infectious disease processes in children and adults worldwide. Many viruses are implicated in these infections, and these viruses are spread largely via respiratory means between humans but also occasionally from animals to humans. This article is an American Society for Microbiology (ASM)-sponsored Practical Guidance for Clinical Microbiology (PGCM) document identifying best practices for diagnosis and characterization of viruses that cause acute respiratory infections and replaces the most recent prior version of the ASM-sponsored Cumitech 21 document, Laboratory Diagnosis of Viral Respiratory Disease, published in 1986. The scope of the original document was quite broad, with an emphasis on clinical diagnosis of a wide variety of infectious agents and laboratory focus on antigen detection and viral culture. The new PGCM document is designed to be used by laboratorians in a wide variety of diagnostic and public health microbiology/virology laboratory settings worldwide. The article provides guidance to a rapidly changing field of diagnostics and outlines the epidemiology and clinical impact of acute respiratory viral infections, including preferred methods of specimen collection and current methods for diagnosis and characterization of viral pathogens causing acute respiratory tract infections. Compared to the case in 1986, molecular techniques are now the preferred diagnostic approaches for the detection of acute respiratory viruses, and they allow for automation, high-throughput workflows, and near-patient testing. These changes require quality assurance programs to prevent laboratory contamination as well as strong preanalytical screening approaches to utilize laboratory resources appropriately. Appropriate guidance from laboratorians to stakeholders will allow for appropriate specimen collection, as well as correct test ordering that will quickly identify highly transmissible emerging pathogens.

Regional antibiotic delivery for the treatment of experimental prosthetic graft infections.

OBJECTIVES: To evaluate the efficacy of antibiotic-impregnated polymethylmethacrylate (PMMA) beads in eradication of an arterial prosthetic graft methicillin-resistant Staphylococcus aureus (MRSA) biofilm in an experimental animal model. METHODS: Forty rats underwent subcutaneous implantation of a MRSA-colonized arterial polytetrafluoroethylene (PTFE) 1 x 1 cm wafer on the back. The effect of regional antibiosis produced by antibiotic PMMA bead placement adjacent to the infected PTFE wafer was determined using four 10-animal study groups: control (no antibiotic), PMMA bead with no antibiotic, PMMA bead with 10% vancomycin, and PMMA bead with 10% daptomycin. After 3 d, the PTFE wafers were explanted and quantitative biofilm cultures, expressed as colony-forming units (CFU) per graft wafer, performed using real-time polymerase chain reaction to assess MRSA eradication. No systemic antibiotic was administered. Bioassays of antibiotic bead bacteriocidal were performed by measuring zone of inhibition diameters on MRSA colonized agar culture plates prior to and following graft explantation. RESULTS: All animal tolerated implantation of the MRSA-infected PTFE wafer and survived the 3 d until graft explantation. Quantitative biofilm cultures demonstrated a significant decrease (P < 0.01) in MRSA CFUs present on the PTFE wafer surfaces in the presence of both the vancomycin- and daptomycin-impregnated beads compared to controls and plain PMMA beads. Both vancomycin and daptomycin PMMA beads retained antibacterial activity after 3 d of implantation with decrease in zones of inhibition of 15% and 45%, respectively. CONCLUSIONS: Regional antibiotic delivery using an antibiotic-impregnated PMMA bead reduced the bacterial biofilm concentration in experimental subcutaneous pocket model of vascular surgical site infection. The delivery of antibiotics via a PMMA bead may be a useful adjunct in the treatment of vascular surgical site infection.

Comparison of DNA pyrosequencing with alternative methods for identification of mycobacteria.

Identification of mycobacterial clinical isolates by pyrosequencing within the hypervariable A region of the 16S rRNA gene was compared to other identification methods. For >90% of isolates, these identifications correlated to the level of complex or species. For identification of many mycobacteria, pyrosequencing offers an inexpensive alternative to traditional sequencing.

Evaluation of two TaqMan PCR assays for the detection of HIV-1 proviral DNA in blood samples.

This study compared two published TaqMan PCR assays targeting different regions of the HIV-1 genome for detection of HIV-1 proviral DNA. The gag specific PCR demonstrated a lower sensitivity than the assay targeting the LTR region. The LTR assay is a highly reproducible and specific technique for HIV-1 proviral DNA detection.

Identification of transcription start sites and preferential expression of select CB2 transcripts in mouse and human B lymphocytes.

Marijuana cannabinoids, the endocannabinoids, and cannabinoid cell receptors have been shown to play important roles in immune regulation particularly as potent modulators of anti-inflammatory cytokines. The predominant cannabinoid receptor involved in this immune regulation is cannabinoid receptor 2 (CB(2)), which is predominantly expressed in B lymphocytes. However, the promoter region and mechanisms of CB(2) gene regulation are unknown in this immune cell type. Utilizing a combination of bioinformatics, 5' rapid amplification of cDNA ends (5' RACE), real-time reverse transcription-polymerase chain reaction, DNA sequencing, and luciferase reporter assays, we show that human B cells express one CB(2) transcript while mouse B cells express three CB(2) transcripts, with specific transcript selection occurring during B cell activation by lipopolysaccharide. Alignment of our sequenced RACE products to either the mouse or human genome, along with the GenBank submitted mRNA sequences, revealed that the transcripts we isolated contained previously unidentified transcriptional start sites (TSS). In addition, expression construct testing of the genomic region containing the TSSs of the mouse CB(2) exon 1 transcripts showed an eightfold increase of promoter activity over baseline. These data show for the first time that human B cells use only one TSS for CB(2) while mouse B cells use multiple TSSs and that the mouse TSSs are in a genomic area with promoter activity, thus suggesting the location of the gene promoter region. Defining these TSSs also provides clues to the various gene regulatory factors involved in the expression of CB(2) during B cell activation.

Cannabinoid receptor 2 (CB2) mediates immunoglobulin class switching from IgM to IgE in cultures of murine-purified B lymphocytes.

Marijuana cannabinoid treatment increases Th2 activity, and previous reports showed that B cells express the highest level of CB(2) mRNA relative to other immune cells, suggesting that cannabinoids play a critical role in B cell activation and maturation. We previously reported evidence of Th2 biasing and class switching in cannabinoid-treated and antigen-challenged mice. We now explore the possibility that cannabinoids directly influence B cell antibody class switching. Mouse splenic B cells were purified by negative selection and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective cannabinoid agonist, CP55940, or the CB(1) selective cannabinoid agonist, methanandamide, and analyzed at different days by flow cytometry for surface expression of either IgM or IgE. Cells treated with CP55940 showed an increase in expression of IgE by day 5 in culture; methanandamide had no effect. CP55940 also induced an increase in secreted IgE in culture supernatants as analyzed by ELISA. In addition, CB(2) receptors were increased on B cells after stimulation with IL-4 and anti-CD40, and the class switching effect of CP55940 was attenuated by the CB(2) antagonist, SR144528. These results suggest that cannabinoids bias toward Th2-type immunity by directly inducing B cell class switching from IgM to IgE through a mechanism involving CB(2) receptors.

Staphylococcal cassette chromosome mec and Panton-Valentine leukocidin characterization of methicillin-resistant Staphylococcus aureus clones.

Staphylococcal cassette chromosome mec (SCCmec) types and Panton-Valentine leukocidin (PVL) gene carriage were compared among suspected community-associated methicillin-resistant Staphylococcus aureus MRSA (CA-MRSA) and health care-associated MRSA (HA-MRSA) isolates. CA-MRSA isolates carried the SCCmec type IV complex, and most were PVL positive. The HA-MRSA isolates carried the SCCmec type II complex and did not harbor the PVL genes.

Legionella pneumophila infection up-regulates dendritic cell Toll-like receptor 2 (TLR2)/TLR4 expression and key maturation markers.

Dendritic cells (DCs) have a critical role in linking innate to adaptive immunity, and this transition is regulated by the up-regulation of costimulatory and major histocompatibility complex (MHC) molecules as well as Toll-like receptors. These changes in DCs have been observed to occur following microbial infection, and in the present study, we examined the effect of Legionella pneumophila infection on the expression of these DC markers. We showed that bone marrow-derived DC cultures from BALB/c mice infected with live L. pneumophila resulted in the up-regulation of Toll-like receptors 2 and 4 and the activation of CD40, CD86, and MHC class I/II molecules.

Chlamydia pneumoniae infection induces differentiation of monocytes into macrophages.

Migration and differentiation of monocytes to the intima of blood vessels may be a crucial first step in the development of atherosclerosis associated with Chlamydia (Chlamydophila) pneumoniae. However, the involvement of C. pneumoniae infection in such steps is not clear. In the present study, therefore, the differentiation-inducing activity of C. pneumoniae to monocytes was examined. Human THP-1 monocytic cell line cells were infected with C. pneumoniae, and the differentiation of monocytes to macrophages was assessed by cell morphology, phagocytic activity, and expression of a cell surface adhesion molecule. The monocytic cells infected with viable bacteria markedly differentiated into macrophages associated with diffused cell morphology, increased uptake of polystyrene beads and increased ICAM-1 (intercellular adhesion molecule 1) expression on the cell surfaces. Heat-killed bacteria did not induce any morphological changes or increase of phagocytosis, but they did induce an increase of cell surface ICAM-1 expressions in THP-1 monocytic cells. The antibiotic minocycline treatment of infected cells resulted in marked inhibition of the cell differentiation as well as C. pneumoniae growth in the cells, but not ICAM-1 expression. In addition, the experiments with human peripheral blood monocytes infected with C. pneumoniae also showed the differentiation of macrophages assessed by morphological change and phagocytic activity. These results indicate that C. pneumoniae infection may directly induce the differentiation of monocytes to macrophages. However, antigenic stimulation of monocytes with bacteria may not be sufficient for a full macrophage differentiation.