Optimal use of Rapid Diagnostics in Infection Control and Prevention

Clinical laboratories have implemented rapid diagnostic tests (RDTs) for the identification of bacterial pathogens, with subsequent improvements in antimicrobial stewardship, but these tests may also have a role in infection prevention. Early identification of pathogens by RDTs should allow faster implementation of infection prevention strategies with the goal of reducing transmission. In this review, we assess the use of RDTs as an infection control tool by exploring their role in screening, as well as diagnosis, of methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant gram-negative organisms, Clostridium difficile, and Mycobacterium tuberculosis. This assessment highlights the fact that implementation of RDTs to improve infection prevention will require ongoing collaboration between clinical laboratory personnel, infection prevention departments, and clinicians.     

ISSag1 in streptococcal strains of human and animal origin

The chromosomal region of Streptococcus agalactiae harboring the C5a peptidase and the lmb genes displays the structure of a composite transposon. Its presence in a streptococcal strain is associated with the origin of this strain from a human host. In S. agalactiae it is flanked by two copies of the insertion element ISSag2, and the nucleotide sequence for a third IS element (ISSag1) can be found in this region. Based on amino acid sequence similarity of the deduced transposase ISSag1 belongs to the IS3 family. It is 1251 bp long and flanked by 37 bp imperfect inverted repeats. Horizontal gene transfer among different bacterial species is facilitated by mobile genetic elements. To investigate if ISSag1 homologues are also present in other streptococcal species, various species of pyogenic streptococci from animal and human origin were analyzed by Southern blot hybridization and PCR. Among the different streptococcal species, multiple copies of an ISSag1 homologue could only be detected in S. dysgalactiae subsp. dysgalactiae strains of animal origin. All of the S. agalactiae strains harbored only a single copy, that was always found in strains with the scpB-lmb composite transposon. A single copy of an ISSag1 homologue could also be detected in some of the S. pyogenes and S. dysgalactiae subsp. equisimilis strains. Nucleotide sequencing of the IS element in S. dysgalactiae subsp. dysgalactiae strains revealed several different variants. One of the variants showed the features of a regular IS3 element. The other two variants that were observed displayed a 500-bp deletion and a mosaic structure composed of ISSag1 and ISSag2 homologues. This mosaic structure suggests that recombination and horizontal gene transfer events in S. dysgalactiae strains of bovine origin could have played a role in the assembly of the scpB-lmb composite transposon structure.     

Clinical Impact of Multiplex Syndromic Panels in the Diagnosis of Bloodstream,Gastrointestinal, Respiratory,and Central Nervous System Infections

Recent technological advances are revolutionizing clinical microbiology. In place of the traditional diagnostic approach in which a clinician suspecting an infectious disease is required to test for the most likely etiology, perform follow-up testing for rarer causes, and then perhaps be left with a diagnosis of exclusion, many laboratories are now offering multiplex syndromic panels to provide a comprehensive diagnostic approach to bloodstream, gastrointestinal, respiratory, and central nervous system infections. In addition to simplifying testing and laboratory workflow, multiplex panels can improve patient care, promote antimicrobial stewardship, and assist with infection control and prevention. This review discusses the growing body of evidence demonstrating the clinical impact and cost-effectiveness of multiplex panels.     

Come in Out of the Cold: Alternatives to Freezing for Microbial Biorepositories

Biorepositories are “libraries” in which biospecimens, bacteria, or DNA and RNA extracts are stored for either clinical or research purposes. Such specimens enable modern molecular-based research and could support method verification, validation, quality control, and, in some cases, proficiency testing in clinical laboratories. Cryopreservation of extracted nucleic acids ensures the stability and longevity of DNA and RNA from patient samples, with the most common methods used for long-term storage of samples being the use of ?80°C freezers or liquid nitrogen. Frozen biospecimens are crucial for translational research as they contain well-preserved nucleic acids and protein; however, traditional ?80°C freezers consume both energy and space, with costs of maintenance and repairs reaching thousands of dollars annually to freeze and protect biospecimens. Additionally, liquid nitrogen is hazardous to work with, and failure to maintain adequate levels in storage containers can result in loss of specimens. Recently, new room temperature, or “green,” technology has been developed for dry storage of nucleic acids, ultimately reducing costs in terms of energy output and carbon footprint. This review compares and contrasts the use of dry-storage infrastructure with that of freezing samples, in terms of its use in clinical microbiology and highlights considerations to be made if implementing the technologies. Storage alternatives to freezers that equal or exceed their performance with regard to sample preservation and protection against degradation while at the same time reducing space requirements, costs, and energy consumption could be a financial and operational benefit if properly deployed and characterized. Perhaps it is time for laboratories to consider getting molecular quality control material and remnant extracted samples in from the cold and to evaluate the use of dry, room temperature storage technology for use in our microbial biorepositories.     

Trends in Laboratory Utilization

Laboratory utilization is definitely a hot topic in clinical laboratory medicine, but what's all the buzz about? Laboratory utilization encompasses a number of efforts aimed at managing laboratory resources and improving patient care. Many laboratory utilization programs have proven to dramatically decrease clinical laboratory spending. Likewise, many have proven to benefit patient care by decreasing blood loss due to phlebotomy, reducing the risk of spurious results, and helping to get the right test for the right patient at the right time. This article reviews examples of laboratory utilization trends, including laboratory utilization committees, laboratory test formularies, electronic tools, and test approval systems.     

The ICF syndrome, a DNA methyltransferase 3B deficiency and immunodeficiency disease

Only one human disease that involves Mendelian inheritance of immunodeficiency and aberrant DNA methylation has been identified. This is a rare chromosome breakage disease called the immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF). Its diagnostic characteristics are agammaglobulinemia with B cells as well as DNA rearrangements targeted to the centromere-adjacent heterochromatic region (qh) of chromosomes 1, 16, and sometimes 9 in mitogen-stimulated lymphocytes. These rearrangement-prone regions show DNA hypomethylation in all examined ICF cell populations. This review summarizes our knowledge about the immunological symptoms of ICF; the nature of DNMT3B mutations in ICF patients; the phenotypes of DNA hypomethylation mutants in humans, mice, and Arabidopsis; the epigenetics of ICF; and ICF-specific RNA expression and cell-surface antigen expression in lymphoblastoid cell lines. Comparisons of ICF and control lymphoblastoid cell lines and ICF patients' symptoms suggest an involvement of DNA methylation in the late stages of lymphocyte maturation.     

The flotillins are integral membrane proteins in lipid rafts that contain TCR-associated signaling components: implications for T-cell activation

Lipid rafts play an important role in signal integration and cellular activation by the T-cell antigen receptor (TCR). We demonstrate that flotillin-1 and flotillin-2 are important structural raft components, which redistribute to the site of TCR engagement. An antibody to flotillin-1 was able to immobilize other TCR-associated raft components. Although rafts purified from unstimulated cells demonstrated abundant Lck but inabundant LAT, rafts from stimulated cells include an abundance of both components. This suggests dynamic changes in lipid raft composition during CD3/CD28 costimulation. Stimulation of primary human CD4(+) T cells leads to increased GM1 and flotillin-1 expression in the surface membrane, where these components colocalize. This may reconstitute new signaling complexes required for T-cell activation. Altered lipid raft composition and function may play a role in the decline of antigen responsiveness in senescent T cells. In this regard, we observed an increase in the raft-associated gangliolipid, GM1, in resting human CD4(+) and CD8(+) lymphocytes with aging.     

Mammalian DNA (cytosine-5) methyltransferases and their expression

Two classes of functional DNA (cytosine-5) methyltransferases have been discovered in mammals to date. One class methylates the unmodified DNA and is designated as the de novo enzyme, whereas the other maintains the methylation status of the daughter strand during DNA replication and thus is referred to as a maintenance DNA methyltransferase. Each enzyme catalyzes methyl group transfer from S-adenosyl-L-methionine to cytosine bases in DNA. During methylation the enzyme flips its target base out of the DNA duplex into a typically concave catalytic pocket. This flipped cytosine base is then a substrate for the enzyme-catalyzed reaction. The newly formed 5-methylcytosine confers epigenetic information on the parental genome without altering nucleotide sequences. This epigenetic information is inherited during DNA replication and cell division. In mammals, DNA methylation participates in gene expression, protection of the genome against selfish DNA, parental imprinting, mammalian X chromosome inactivation, developmental regulation, T cell development, and various diseases.     

Determinants of HIV-specific CD8 T-cell responses in HIV-infected pediatric patients and enhancement of HIV-gag-specific responses with exogenous IL-15

Cellular immune responses play a central role in controlling HIV-1 infection. HIV-specific IFN-gamma production by CD8 T cells was evaluated in 17 HLA-A2+ HIV-infected pediatric patients (age range 1 month to 16 years) in an ELISPOT assay. Most patients (15/17) exhibited responses to HIV-gag, followed by responses to envelope gp120, gp41, and V3 loop. Only 7 patients responded to all four antigenic peptides. Treatment-related immune reconstitution of CD4 T cells was associated with increase in gag-specific responses, but these declined with prolonged viral suppression. Exogenous IL-15 resulted in augmentation of HIV-gag-specific response in 71% of patients, while IL-2 and IL-7 had variable effects, augmenting responses in 25% patients. Thus, HIV-specific CD8 T-cell responses are dependent on both CD4 T-cell help and antigenic stimulation. The cytokine IL-15 may be a useful modality as adjunctive therapy to augment HIV-specific memory CD8 T cells.     

IL-4 and many roads to lupuslike autoimmunity

Interleukin-4 (IL-4) is a multifunctional cytokine. Although most studies have focused on the B-cell stimulatory and Th2 promoting properties of IL-4 in the development of autoantibodies and autoantibody-mediated diseases, a few reports suggest a T-cell suppressor role for this cytokine in lupus. Since these properties of IL-4 may sometimes result in opposing outcomes, amplifying or inhibitory, on overall B-cell functions, it is not surprising that a few studies have found no role for IL-4 in the development of autoantibodies and lupus. Evidence for a more novel role for IL-4 in the development of lupus nephritis comes from recent studies, which suggests that IL-4 may directly promote extracellular matrix deposition in the glomeruli. Consistent with this idea, blockade of IL-4 by antibody treatment or of its signaling by inactivation of the Stat6 gene ameliorates glomerulosclerosis and delays or even prevents the development of end-stage renal disease, despite the presence of high levels of IgG anti-dsDNA Antibodies. Thus, IL-4 may serve multiple roles in the development of lupus: it may enhance autoantibody production via its direct B-cell effects, protect against autoimmunity via its T-cell suppressor effect, or perpetuate tissue damage via its direct effects on target organs.     

Carotid body thin slices: responses of glomus cells to hypoxia and K(+)-channel blockers

We describe the rat carotid body thin slice preparation, which allows to perform patch-clamp recording of membrane ionic currents and to monitor catecholamine secretion by amperometry in single glomus cells under direct visual control. We observed several electrophysiologically distinct cell classes within the same glomerulus. A voltage- and Ca(2+)-dependent component of the whole cell K(+) current was reversibly inhibited by low P(O(2)) (20 mmHg). Exposure of the cells to hypoxia elicited the appearance of spike-like exocytotic events. This response to hypoxia was reversible and required extracellular Ca(2+) influx. Addition of tetraethylammonium (TEA, 2-5 mM) to the extracellular solution induced in most (>95%) cells tested a secretory response similar to that elicited by low P(O(2)). Cells non-responsive to hypoxia but activated by exposure to high external K(+) were also stimulated by TEA. A secretory response similar to that of hypoxia or TEA was also observed after treatment of the cells with iberiotoxin to block selectively maxi-K(+) channels. Our data further support the view that membrane ion channels are critically involved in sensory transduction in the carotid body. We demonstrate that in intact glomus cells inhibition of voltage-dependent K(+) channels can contribute to initiate the secretory response to low P(O(2)).     

Statistics for Method Verification of Qualitative Assays in Clinical Microbiology

Despite improvement over the last decade, inadequate method verification strategies and documentation are still common laboratory inspection deficiencies. Some of the gaps occur because microbiologists may not use scientific experiment concepts, such as experimental design, data validation, biostatistics, and electronic documentation, that can improve their verification strategies for new test methods. Other gaps occur because laboratory resources are often limited. Clinical laboratories face tough choices when they consider implementing a new test method, and this review summarizes the basic statistical concepts that microbiologists need to critically plan and review qualitative method verification with the aim of determining whether a new diagnostic test is fit for patient testing.     

Self-Collected Specimens for Infectious Disease Testing

Self-collected specimens for infectious disease testing are becoming more commonplace. There are multiple published studies demonstrating that self-collected vaginal swabs for detection of sexually transmitted pathogens are as accurate as physician-collected endocervical swabs. Similarly, self-collected penile-meatal swabs are also acceptable for detecting sexually transmitted pathogens; however, unlike self-collected vaginal swabs, penile-meatal swabs are not considered an “on-label” specimen for U.S. FDA-cleared in vitro diagnostic products. Data on the accuracy of self-collected nasal specimens for respiratory tract infections are encouraging, but studies also show that patients do not always follow instructions when mailing samples back to the laboratory. Unfortunately, there are only a few reports of self-collected specimens for detecting enteric pathogens, such as Salmonella, Shigella, or Campylobacter. Microbiologists need to be proactive in making sure that training materials developed for self-collection (such as laminated cards, videos, and other resources) are accurate and easy to understand (which includes being available in multiple languages) and provide clear instructions on how to handle a specimen once it has been collected.     

Arterial blood gas control in the upright versus recumbent Asian elephant

In the elephant, there is concern that lateral recumbency (LR) impairs respiratory muscle and lung function resulting in clinically significant arterial hypoxemia. Using healthy adult female Asian elephants (Elephas maximus, n=6), the hypothesis was tested that, given the O(2) binding characteristics of elephant blood, substantial reductions in arterial O(2) pressure (Pa(O(2))) in LR could be tolerated without lowering arterial O(2) content appreciably. Fifteen minutes of LR decreased Pa(O(2)) from 103+/-2 (upright, U) to 77+/-4 mmHg (P<0.05) and hemoglobin O(2) saturation (U, 97.8+/-0.1, LR, 95.3+/-0.5%, P<0.05). However, due to a recumbency-induced hemoconcentration, arterial O(2) content was unchanged (U, 18.2+/-2.4, LR, 18.3+/-2.1 ml O(2) per 100 ml). In addition, there was a mild hyperventilation in LR that reduced arterial CO(2) pressure (P(CO(2))) from 39.4+/-0.3 to 37.1+/-1.0 mmHg (P<0.05). These data indicate that the Asian elephant can endure at least short periods of LR without lowering arterial O(2) content.