Routine Isolation Research Articles

Experiencing neutropenia: Quality of life interviews with adult cancer patients

BackgroundNeutropenia is a common toxicity in chemotherapy but detailed information about how neutropenia is associated with changes in patients' quality of life is not readily available. This prospective study interviewed patients with grade 4 neutropenia to provide qualitative information on patients' experience of developing and coping with grade 4 neutropenia during a cycle of chemotherapy.MethodsA sample of 34 patients who developed grade 4 neutropenia during the first cycle of chemotherapy completed a total of 100 structured clinical interviews. Interviews were transcribed, and 2 raters inductively developed 5 broad categories comprising 80 specific complaint domains nominated by patients. Thirty-five patient-nominated problems were mentioned in 5% or more of the interviews.ResultsFatigue was the most common physical symptom. Interference in daily routine, negative self-evaluation, negative emotion, and social isolation were other common complaints associated with neutropenia.ConclusionNeutropenia is associated with a number of negative experiences among cancer patients undergoing chemotherapy, and these negative experiences have an adverse effect on the patient's quality of life. Oncology nurses can play a key role in helping patients manage adverse effects to maintain their quality of life.

Reflectance confocal microscopy for characterization of mammary ductal structures and development of neoplasia in genetically engineered mouse models of breast cancer

The earliest steps of breast cancer begin with aberrations in mammary ductal structure. Techniques that enable an investigator to image in situ and then analyze the same tissue using biochemical tools facilitates identification of genetic networks and signaling pathways active in the imaged structure. Cellular confocal microscopy (VivaCell-TiBa, Rochester, New York) is used to image mammary ductal structures and surrounding vasculature in situ in intact wild-type and genetically engineered mice that develop ER alpha-initiated ductal carcinoma in situ (DCIS) and ER alpha-driven invasive mammary cancer. In wild-type mice, normal mammary ductal structures that appear from puberty through lactation are visualized and serially sectioned optically, and a developmental atlas is created. Altering tissue preparation enabled visualization of the vasculature surrounding the ductal structures. In the genetically engineered mice, aberrant mammary ductal structures and cancers are imaged and compared to corresponding normal structures. Different preparation techniques are able to preserve tissue for routine histological analyses and RNA isolation. Comparative studies demonstrate that reflectance confocal imaging provides more cellular detail than carmine-alum-stained mammary gland whole mounts and equivalent detail with hematoxylin and eosin stained tissue sections. In summary, reflectance confocal microscopy is a tool that can be used to rapidly and accurately analyze mammary gland structure.

Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8 + T cells in order to express HPV16E7 11–20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E7 11–20-specific TCR into blood-derived, CD8 + recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E7 11–20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-γ secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E7 11–20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.

Diabetes mellitus in itself is not a risk factor for antibiotic resistance in Escherichia coli isolated from patients with bacteriuria.

To investigate whether diabetes mellitus is a risk factor for resistance in Escherichia coli isolated from patients with bacteriuria. Data were obtained from a multicentre study. A clean-voided midstream urine culture was collected from 636 women with diabetes, who were between 18 and 75-years-old, attended an out-patient department and had no symptoms of a urinary tract infection. The resistance of E. coli was determined for different antimicrobials. The results were compared with resistance data from routine isolates of E. coli, obtained from women in the same age category, time period and location. A total of 135 E. coli were isolated from women with diabetes mellitus (mean age 57 +/- 14 years) and compared with 5907 routine isolates of E. coli obtained from female patients visiting an out-patient department (mean age 52 +/- 17 years). The resistance rates of E. coli isolated from diabetic patients and the routine isolates of E. coli to trimethoprim-sulfamethoxazole were 19% and 23%, respectively, to amoxicillin 16% and 32%, to nitrofurantoin 1% and 3%, to ciprofloxacin 0% and 4%, to ofloxacin 0% and 5%, and to norfloxacin 1% and 4%. The resistance of uropathogenic E. coli in non-hospitalized women with diabetes mellitus is not higher than that seen in routine isolates of E. coli. This suggests that diabetes in itself is not a risk factor for resistance.

Cross-talk between phosphatidylinositol 3-kinase/AKT and c-jun NH2-terminal kinase mediates survival of isolated human islets.

Therapeutic strategies aimed at the inhibition of specific cell death mechanisms may increase islet yield and improve cell viability and function after routine isolation. The aim of the current study was to explore the possibility of AKT-JNK cross-talk in islets after isolation and the relevance of c-jun NH2-terminal kinases (JNK) suppression on islet survival. After routine isolation, increased AKT activity correlated with suppression of JNK activation, suggesting that they may be related events. Indeed, the increase in AKT activation after isolation correlated with suppression of apoptosis signal-regulating kinase 1 (ASK1), a kinase acting upstream of JNK, by phosphorylation at Ser83. We therefore examined whether modulators of phosphatidylinositol 3-kinase (PI3K)/AKT signaling affected JNK activation. PI3K inhibition led to increased JNK phosphorylation and islet cell death, which could be reversed by the specific JNK inhibitor SP600125. In addition, IGF-I suppressed cytokine-mediated JNK activation in a PI3K-dependent manner. We also demonstrate that inhibition of PI3K rendered islets more susceptible to cytokine-mediated cell death. SP600125 transiently protected islets from cytokine-mediated cell death, suggesting that JNK may not be necessary for cytokine-induced cell death. When administered immediately after isolation, SP600125 improved islet survival and function, even 48 h after removal of SP600125, suggesting that JNK inhibition by SP600125 may be a viable strategy for improving isolated islet survival. Taken together, these results demonstrate that PI3K/AKT suppresses the JNK pathway in islets, and this cross-talk represents an important antiapoptotic consequence of PI3K/AKT activation.

Effects of duplicate and screening isolates on surveillance of community and hospital antibiotic resistance.

To investigate common contentions that duplicate and screening isolates consistently show marked excess resistance, and that inclusion of such isolates significantly distorts regional resistance estimates. Two Welsh surveys of antibiotic resistance for routine diagnostic isolates were analysed, comprising 309,129 isolates of six common community pathogens and 85,061 ward isolates of 11 common hospital pathogens. Duplicate isolates were defined as isolates from the same patient of the same pathogen with an indistinguishable susceptibility pattern, excluding the initial isolate. Significance was assessed from 95% confidence limits of the difference between resistance estimates. Duplicate isolates comprised approximately 20% of total isolates. For the 195 antibiotic-pathogen combinations investigated, differences in resistance between duplicate and non-duplicate isolates were statistically significant for 93. Only 54 combinations showed significantly increased resistance amongst duplicates, and only 30 of these showed a difference >5%. Comparisons of de-duplicated with un-de-duplicated regional resistance estimates showed significant differences for only 18 of 195 antibiotic-pathogen combinations; none were sufficient to alter judgement on clinical use. Screening isolates produced little disturbance of resistance estimates for Staphylococcus aureus, with the exception of flucloxacillin resistance, where inclusion of screening and duplicate isolates resulted in an increase of 4.4% for both community and hospital resistance estimates. The contentions were incorrect for these regional surveys. However, the proportion (and so effects) of screening and duplicate isolates may be greater in surveys of units with frequent repetitive sampling practice (burns, ITU, cystic fibrosis), or pathogens subjected to unusually intensive infection control sampling.

Recent Innovations and Analytical Applications of Phage Display Libraries

Filamentous phages are used widely for research and analytical technology development. This is due to the unique biology of the f-phages including its single stranded DNA genome and ability to display proteins on the surface of the intact phage particle, which have advanced the fields of recombinant DNA technology and molecular diversity technologies, respectively. Herein we review recent trends in phage display technologies that allow the evolution of selected binders into optimized molecules. Using this technology large polypeptide libraries are presented on the surface of filamentous phages. The selection of peptides and antibodies from phage display libraries has proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications.

Surveillance for respiratory syncytial virus in infants hospitalized for acute lower respiratory infection in Chile (1989 to 2000).

Hospitalized infants (4,618) were studied for lower respiratory infections from 1989 through 2000 by routine immunofluorescence assay and viral isolation. The hospitalization rate for respiratory syncytial virus (RSV) averaged 2% per year. The fatality rate was 0.1%. Monthly RSV detection varied from 14 to 88%, and epidemics lasted 3.5 to 6 months. From 1994 high-early versus low-late epidemic patterns alternately were observed, the first influenced by a group B strain.

Isolation of bovine retinal pigment epithelial cells using adhesion to agarose: demonstration of cellular and regional heterogeneity.

The retinal pigment epithelium (RPE) shows cell heterogeneity in morphology and enzymatic activity. Routine isolation procedures for RPE cells may reduce enzymatic activity and prevent the quantification of regional enzymatic differences in vivo. We developed a new technique for the isolation of RPE cells based on adhesion of the cells to agarose. The morphology of the isolated cells resembled that of RPE cells in vivo. The cells were viable in the dye exclusion test and showed a histochemical staining pattern as RPE cells in vivo. With this technique, quantitative regional differences in the enzymatic activities were detected.

Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord.

Mesenchymal stem cells (MSCs) have the capability for renewal and differentiation into various lineages of mesenchymal tissues. These features of MSCs attract a lot of attention from investigators in the context of cell-based therapies of several human diseases. Despite the fact that bone marrow represents the main available source of MSCs, the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferative/differentiation capacity with age. Thus, the search for possible alternative MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells and does not contain mesenchymal progenitors. However, MSCs circulate in the blood of preterm fetuses and may be successfully isolated and expanded. Where these cells home at the end of gestation is not clear. In this investigation, we have made an attempt to isolate MSCs from the subendothelial layer of umbilical cord vein using two standard methodological approaches: the routine isolation of human umbilical vein endothelial cell protocol and culture of isolated cells under conditions appropriate for bone-marrow-derived MSCs. Our results suggest that cord vasculature contains a high number of MSC-like elements forming colonies of fibroblastoid cells that may be successfully expanded in culture. These MSC-like cells contain no endothelium- or leukocyte-specific antigens but express alpha-smooth muscle actin and several mesenchymal cell markers. Therefore, umbilical cord/placenta stroma could be regarded as an alternative source of MSCs for experimental and clinical needs.

Evaluation of the Vitek 2 system for rapid identification of clinical isolates of gram-negative bacilli and members of the family Streptococcaceae.

Accuracy of the Vitek 2 automated system (bioMérieux Vitek, USA) for rapid identification of bacteria was evaluated using a collection of 858 epidemiologically unrelated gram-negative and 99 gram-positive clinical isolates. Isolates were tested after subculturing to ensure purity. Conventional agar-based biochemical tests (Steers replicator) were used as a reference method of identification. Gram-negative bacteria were identified to the species level with 95.3% accuracy by the system ( Enterobacteriaceae, 95.9%; and non- Enterobacteriaceae, 92.5%), and gram-positive isolates with 72% accuracy. Although Vitek 2 identified routine clinical isolates of gram-negative bacilli and Enterococcus faecalis and Enterococcus faecium reliably, rapidly, and reproducibly, improvement is required in the identification of less common species of enterococci and viridans group streptococci.

Application of actinomycetes to soil to ameliorate water repellency.

The aim of this study was to develop a novel isolation technique using a mixture of Bacillus and Streptomyces phages to selectively isolate wax-utilizing non-streptomycete actinomycetes effective in ameliorating water repellency in a problem soil. Phages added to a soil suspension reduced the dominance of Bacillus and Streptomyces isolates and significantly increased the number of non-streptomycete actinomycetes on isolation plates. Promising isolates, grown on a medium containing beeswax as sole carbon source, were selected for application to water repellent soil. Their addition significantly reduced water repellency. Phage application significantly increased the isolation of non-streptomycete actinomycetes. Wax-utilizing isolates were found to significantly reduce water repellency in a problem soil. The phage technique can be used for the routine isolation of non-streptomycete actinomycetes. Beeswax medium can be used to selectively isolate wax-utilizing micro-organisms with the potential to ameliorate water repellency in soil.

Culturing and characterization of astrocytes isolated from juvenile rainbow trout ( Oncorhynchus mykiss)

An access to brain cell cultures from fish would enable screening of possible neurotoxic chemicals contaminating the aquatic environment. In the present study, a protocol for a successful routine isolation and culturing of brain cells from juvenile rainbow trout was worked out. The coating material was shown to be of importance for cell proliferation. Cells grow better on a surface coated with laminin than on those coated with poly- l-lysine (PLL), poly- d-lysin (PDL) or poly- l-ornithine (PLO). The best cell growth was obtained on double-coated surfaces (PLL, PDL or PLO plus laminin). On such a culture substrate and with a seeding density of 1×10 7 cells/cm 2 confluence was obtained within 3–4 weeks at an incubation temperature of 18 °C. Approximately 95% of the cells were identified as astrocytes on the basis of a positive staining with antibodies against the astrocyte specific glial protein (GFAP). No oligodendrocytes or fibroblasts were identified in the cultures, and despite several efforts, neurons did not grow under the culture conditions used. When challenged with ligands known to awake a calcium transient in mammalian astrocytes, 44% of the cells responded to ATP with an increase in [Ca 2+] i , 38% to norepinephrine, 27% to 5-hydroxytryptamine, 7% to histamine and 6% to glutamate. Kainate, quisqualate and γ-aminobutyric acid did not awake a calcium transient in the cells. Using a proper protocol, it is thus quite easy to get an almost pure culture of astrocyte, whereas neurones proved to very difficult to culture.

Comparative evaluation of Candi Select test and conventional methods for identification of Candida albicans in routine clinical isolates

The Candi Select test (Sanofi Diagnostics, Pasteur, Marnes-La-Coquette, France) is a new yeast-selective medium for the identification of Candida albicans in the clinical laboratory. The performance of this test was compared with the conventional methods of chlamydospore formation, germ tube formation and carbohydrate fermentation. Four hundred and twenty clinical yeast isolates from 412 fresh clinical specimens, including 283 C. albicans, 59 C. tropicalis, 39 Trichosporon spp., 19 C. glabrata, 11 Cryptococcus neoformans and 9 other yeasts, were evaluated. Colonies of C. albicans produced a blue-green colour on the Candi Select media which could be distinguished from the other yeasts with the naked eye within 24-48 h. The sensitivity and specificity of the Candi Select test for the identification of C. albicans were 99.65% and 97.08%, respectively. The blue-green colonies of C. albicans were easy to identify and recognize in mixed cultures and did not need detailed microscopic examination.

MRSA: risk assessment and flexible management.

MRSA is an increasing problem in hospitals, particularly among older patients. The author discusses prevention of cross-infection, with an emphasis on the importance of risk assessment and good infection control procedures, rather than the routine isolation of infected patients.

Characterization of Shiga toxin-producing Escherichia coli O26 strains and establishment of selective isolation media for these strains.

We characterized the carbohydrate-fermenting ability of 31 strains of Shiga toxin-producing Escherichia coli (STEC) O26 isolated from diarrhea patients in Aichi Prefecture, Japan, in order to establish selective isolation media for these strains. None of the 31 STEC O26 strains (24 O26:H11, 7 O26:H-) fermented rhamnose, whereas all of the other 108 STEC strains (100 O157, 8 O111) and all of the non-STEC strains except one (i.e., 58 of 59) fermented rhamnose. The great majority of the STEC O26 strains (96.8% [30 of 31]) showed very high resistance to potassium tellurite (MIC > or = 50 microg/ml), whereas the majority of the non-STEC strains (72.9% [43 of 59]) showed very high sensitivity (MIC < or = 1.56 microg/ml) to this compound. Accordingly, we developed a rhamnose-MacConkey (RMAC) medium in which the lactose in MacConkey medium was replaced by rhamnose, and cefixime-tellurite-RMAC (CT-RMAC) medium in which potassium tellurite (2.5 mg/liter) and cefixime (0.05 mg/liter) were added to RMAC. All of the STEC O26 strains generated colorless (rhamnose-nonfermented) colonies on both media; the vast majority of selected E. coli strains (95.7% [89 of 93; including 26 STEC O157, 8 STEC O111]), other than STEC O26, generated red colonies on RMAC, and most of the non-STEC strains (84.7% [50 of 59]) did not grow on CT-RMAC. We demonstrate that both the RMAC and the CT-RMAC media can be used for the isolation of STEC O26 and that CT-RMAC has better specificity for the routine isolation for STEC O26 in a laboratory.

The control of H5 or H7 mildly pathogenic avian influenza: A role for inactivated vaccine

Biosecurity is the first line of defence in the prevention and control of mildly pathogenic avian influenza (MPAI). Its use has been highly successful in keeping avian influenza (AI) out of commercial poultry worldwide. However, sometimes AI becomes introduced into poultry populations and, when that occurs, biosecurity again is the primary means of controlling the disease. There is agreement that routine serological monitoring, disease reporting, isolation or quarantine of affected flocks, application of strict measures to prevent the contamination of and movement of people and equipment, and changing flock schedules are necessities for controlling AI. There is disagreement as to the disposition of MPAI-infected flocks: some advocate their destruction and others advocate controlled marketing. Sometimes biosecurity is not enough to stop the spread of MPAI. In general, influenza virus requires a dense population of susceptible hosts to maintain itself. When there is a large population of susceptible poultry in an area, use of an inactivated AI vaccine can contribute to AI control by reducing the susceptibility of the population. Does use of inactivated vaccine assist, complicate or interfere with AI control and eradication? Yes, it assists MPAI control (which may reduce the risk of highly pathogenic AI (HPAI)) but, unless steps are taken to prevent it, vaccination may interfere with sero-epidemiology in the case of an HPAI outbreak. Does lack of vaccine assist, complicate or interfere with AI control and eradication? Yes, it assists in identification of sero-positive (convalescent) flocks in a HPAI eradication program, but it interferes with MPAI control (which in turn may increase the risk of emergence of HPAI). A number of hypothetical concerns have been raised about the use of inactivated AI vaccines. Infection of vaccinated flocks, serology complications and spreading of virus by vaccine crews are some of the hypothetical concerns. The discussion of these concerns should take place in a scientific framework and should recognize that control of MPAI reduces the risk of HPAI. That inactivated vaccines have reduced a flock's susceptibility to AI infection, have reduced the quantity of virus shed post-challenge, have reduced transmission and have markedly reduced disease losses, are scientific facts. The current regulations preventing vaccination against H5 or H7 MPAI have had the effect of promoting circulation of MPAI virus in commercial poultry and live poultry markets. In the absence of highly pathogenic avian influenza, there is no justification for forbidding the use of inactivated vaccine.

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1.

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 x 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

Antibacterial prescribing and antibacterial resistance in English general practice: cross sectional study.

Objective: To quantify the relation between community based antibacterial prescribing and antibacterial resistance in community acquired disease.Design: Cross sectional study of antibacterial prescribing and antibacterial resistance of routine isolates within...

Molecular techniques should not now replace cell culture in diagnostic virology laboratories.

The value of molecular techniques for virology is not in dispute; the issue debated here is whether or not to abandon virus isolation altogether. Modern clinical virology relies on rapid virus detection for timely infection control and antiviral therapy. The role of virus isolation, inevitably a slower process as it involves replication in cell cultures, is most significant in providing epidemiological data, in the diagnosis of new or unexpected infection, and in yielding infectious virus for further study. Examples include identification of enterovirus serotypes in outbreaks, diagnosis of atypical virus infections, and provision of virus isolates for phenotypic antiviral susceptibility assays. Many viruses can be detected after overnight culture using the centrifugation-enhanced (shell vial) technique. In contrast to this established track record, the commercial development of molecular assays has been concentrated on blood-borne viruses, and standardisation of procedures for other viruses is lacking. Accreditation of molecular techniques is just beginning, and few external quality assurance schemes are available yet. In my view, it is premature to abandon routine virus isolation, although as molecular diagnosis expands, the facilities for cell culture and isolation work may become more centralised to retain expertise and to provide the range and quality of service required.