An adventitious agent-free clonal cell line that is highly susceptible to foot -and-mouth disease virus.

Porcine LFBKαVß6 cells have been successfully used for diagnostics and propagation of all FMDV serotypes/subtypes. Unfortunately, after initial characterization, these cells showed contamination with bovine viral diarrhea virus (BVDV), a non-cytopathic adventitious agent. Persistent infection with BVDV could interfere with diagnostic tests and, also prevent consideration for other uses, i.e., vaccine production. In this study, we developed a three-prong methodology to completely remove BVDV from LFBKαVß6 cells. Combined treatment with siRNA against BVDV NS5A, porcine interferon alpha and ribavirin resulted in the elimination of BVDV, as determined by immunohistochemistry analysis, quantitative RT-PCR and RNA sequencing. Importantly, elimination of BVDV from LFBKαVß6 did not affect FMDV growth and plaque phenotype from different serotypes isolated and propagated in the clean cell line, newly named MGPK αVß6-C5. Additionally, isolation of FMDV from field oro-pharyngeal samples, was successful at the same sensitivity as in BVDV-contaminated LFBKαVß6 cells. Our results identified a direct method to efficiently eliminate BVDV from porcine cells without altering FMDV permissiveness, diagnostic value, or potential for use in vaccine production. Furthermore, these cells may provide an improved platform for diagnostics and propagation of other viruses of interest in the veterinary field and the virology community at large.

Genetic diversity and comparison of diagnostic tests for characterization of foot-and-mouth disease virus strains from Pakistan 2008-2012.

We report the laboratory analysis of 125 clinical samples from suspected cases of foot-and-mouth disease (FMD) in cattle and Asian buffalo collected in Pakistan between 2008 and 2012. Of these samples, 89 were found to contain viral RNA by rRT-PCR, of which 88 were also found to contain infectious FMD virus (FMDV) by virus isolation (VI), with strong correlation between these tests (κ = 0.96). Samples that were VI-positive were serotyped by antigen detection ELISA (Ag-ELISA) and VP1 sequence acquisition and analysis. Sequence data identified FMDV serotypes A (n = 13), O (n = 36) and Asia-1 (n = 41), including three samples from which both serotypes Asia-1 and O were detected. Serotype A viruses were classified within three different Iran-05 sublineages: HER-10, FAR-11 and ESF-10. All serotype Asia-1 were within Group VII (Sindh-08 lineage), in a genetic clade that differs from viruses isolated prior to 2010. All serotypes O were classified as PanAsia-2 within two different sublineages: ANT-10 and BAL-09. Using VP1 sequencing as the gold standard for serotype determination, the overall sensitivity of Ag-ELISA to correctly determine serotype was 74%, and serotype-specific sensitivity was 8% for serotype A, 88% for Asia-1 and 89% for O. Serotype-specific specificity was 100% for serotype A, 93% for Asia-1 and 94% for O. Interestingly, 12 of 13 serotype A viruses were not detected by Ag-ELISA. This study confirms earlier accounts of regional genetic diversity of FMDV in Pakistan and highlights the importance of continued validation of diagnostic tests for rapidly evolving pathogens such as FMDV.

Prevalence of type III secretion protein exoenzymes and antimicrobial susceptibility patterns from bloodstream isolates of patients with Pseudomonas aeruginosa bacteremia.

The purpose of this study was to determine the prevalence of two type III secretion effector proteins, exoU and exoS from bloodstream isolates of hospitalized patients with Pseudomonas aeruginosa (PSA) bacteremia, to characterize antimicrobial susceptibility patterns, and to compare mortality rates. PSA bloodstream isolates and antibiotic susceptibility profiles were collected from a university-affiliated hospital. ExoS and exoU genes were detected by polymerase chain reaction. Hospital mortality was assessed by medical chart review. 119 of 122 (97.5%) PSA bloodstream isolates contained either the exoS or exoU genes. ExoS was the most prevalent (n=86; 70.5%) followed by exoU (n=31; 25.4%), both genes (n=2; 1.6%) or neither gene (n=3; 2.5%). Isolates containing the exoU gene were significantly more likely to be resistant to cefepime, ceftazidime, piperacillintazobactam, carbapenems, fluoroquinolones, and gentamicin (p<0.05 for all). Mortality was high in patients with PSA bacteremia and did not differ among patients infected with the exoS isolates (n=37; 43%) or exoU isolates (n=11; 35%). One of two type III secretion effector proteins were almost universally present in PSA bloodstream isolates. Isolates containing the exoU gene were more likely to be resistant to multiple antibiotics.

Prevalence of AmpC over-expression in bloodstream isolates of Pseudomonas aeruginosa.

This study examined the contribution of AmpC over-expression to beta-lactam resistance in clinical isolates of Pseudomonas aeruginosa obtained from a hospital in Houston, TX, USA. Seventy-six non-repeat bloodstream isolates obtained during 2003 were screened for ceftazidime resistance in the presence and absence of clavulanic acid 4 mg/L. AmpC was identified by isoelectric focusing (with and without cloxacillin inhibition); stable derepression was ascertained phenotypically by a spectrophotometric assay (with and without preceding induction by imipenem) using nitrocefin as the substrate, and was confirmed subsequently by quantitative RT-PCR of the ampC gene. The clonal relatedness of the AmpC-over-expressing isolates was assessed by pulsed-field gel electrophoresis. In addition, the ampC and ampR gene sequences were determined by PCR and sequencing. For comparison, two standard wild-type strains (PAO1 and ATCC 27853) and three multidrug-susceptible isolates were used as controls. AmpC over-expression was confirmed in 14 ceftazidime-resistant isolates (overall prevalence rate, 18.4%), belonging to seven distinct clones. The most prevalent point mutations in ampC were G27D, V205L and G391A. Point mutations in ampR were also detected in eight ceftazidime-resistant isolates. AmpC over-expression appears to be a significant mechanism of beta-lactam resistance in P. aeruginosa. Understanding the prevalence and mechanisms of beta-lactam resistance in P. aeruginosa may guide the choice of empirical therapy for nosocomial infections in hospitals.

Comparison of risk factors for candidemia versus bacteremia in hospitalized patients.

BACKGROUND: Classic risk factors for candidemia include use of total parenteral nutrition (TPN), hospital location, use of central venous catheter, and others. Unfortunately, most of these variables are now also risk factors for antibiotic-resistant bacteria. Thus, use of these risk factors to identify patients at high risk for candidemia is difficult. The purpose of this study was to compare these classic risk factors for candidemia in patients with bloodstream infections to determine the relative strength of these predictors in differentiating patients with candidemia and bacteremia. METHODS: Clinical data were collected from the medical charts of patients who had been hospitalized between 2002 and 2004. Patients with their first episode of candidemia or bacteremia during their hospital stays were included. Risk factors were assessed using a multivariate logistic regression model and internally validated using a bootstrap analysis. A p-value < 0.05 was considered significant. RESULTS: A total of 164 patients (82 with candidemia) were evaluated. According to the logistic analysis, patients who had stayed in the intensive care unit (ICU) (OR = 6.24; 95% CI: 2.58-15.09) or had been using TPN (OR = 4.69; 95% CI: 1.76-12.48) were more likely to have candidemia than bacteremia. While patients with pulmonary (OR = 0.15; 95% CI: 0.055-0.39) or cardiac disease (OR = 0.21; 95% CI: 0.086-0.51) had a greater chance to have bacteremia than candidemia (p < 0.01 for all variables). These results were further validated using bootstrap analysis. CONCLUSION: Among classic risk factors for candidemia, the ICU location at the time of culture and TPN use were most predictive of candidemia while certain medical disorders predicted patients at the highest risk for bacteremia. These results can be used to help identify patients most likely to benefit from empiric antifungal therapy.

Sampling variability in the microbiological evaluation of expectorated sputa and endotracheal aspirates.

A five-center study was conducted with the aim of determining how reproducibly expectorated sputa and tracheal aspirates could be sampled when preparing Gram-stained smears and inoculating cultures. With both specimen types, excessive variation was noted among Gram stain results obtained from replicate smears. Less variation was noted among culture results, especially with tracheal aspirates.

Evaluation of an enzyme-linked viral inducible system for the rapid detection of Herpes simplex virus.

A commercial enzyme-linked viral inducible system (ELVIS HSV; BioWhittaker, USA) was evaluated in comparison with the spin-amplified tube cell culture (SATCC) method for the rapid detection of herpes simplex virus in 1007 clinical specimens. A total of 91 (9%) specimens were positive by SATCC. The sensitivity, specificity, positive predictive value, and negative predictive value of ELVIS was 88%, >99%, 99%, and 99%, respectively. Herpes simplex virus was detected sooner by ELVIS than by SATCC in 34 of 80 (42%) specimens. Preincubated ELVIS shell vials held at room temperature for 24 h prior to reincubation and inoculation produced results similar to those of freshly preincubated shell vials, with no reduction in either the number or the staining intensity of the infected cells. The results of this study indicate that ELVIS HSV is an accurate method for the rapid detection of herpes simplex virus in a wide variety of clinical specimens.

Destructive native valve endocarditis caused by Staphylococcus lugdunensis.

Coagulase-negative staphylococci are uncommon causes of native valve endocarditis, and the clinical course after valvular infection with these organisms is variable. In clinical practice, species identification is frequently not done, and possible differences in the pathogenicity of various species may be unrecognized. We report a case of Staphylococcus lugdunensis native valve endocarditis associated with valve leaflet perforation and cerebral embolization. This recently described species appears to be more virulent when infecting native cardiac valves than other species of coagulase-negative staphylococci. We review S lugdunensis native valve endocarditis.

Infection in the bone marrow transplant recipient and role of the microbiology laboratory in clinical transplantation.

Over the past quarter century, tremendous technological advances have been made in bone marrow and solid organ transplantation. Despite these advances, an enduring problem for the transplant recipient is infection. As immunosuppressive regimens have become more systematic, it is apparent that different pathogens affect the transplant recipient at different time points in the posttransplantation course, since they are influenced by multiple intrinsic and extrinsic factors. An understanding of this evolving risk for infection is essential to the management of the patient following transplantation and is a key to the early diagnosis and treatment of infection. Likewise, diagnosis of infection is dependent upon the quality of laboratory support, and services provided by the clinical microbiology laboratory play an important role in all phases of clinical transplantation. These include the prescreening of donors and recipients for evidence of active or latent infection, the timely and accurate microbiologic evaluation of the transplant patient with suspected infection, and the surveillance of asymptomatic allograft recipients for infection. Expert services in bacteriology, mycology, parasitology, virology, and serology are needed and communication between the laboratory and the transplantation team is paramount for providing clinically relevant, cost-effective diagnostic testing.

The presence of total quality management and continuous quality improvement processes in California public health clinics.

Total quality management (TQM) and continuous quality improvement (CQI) processes have not been fully integrated into public health practice. Current levels of participation and interest in TQM/CQI were assessed in California's 62 county departments of health services. Survey results indicated that only 18.5 percent of the 54 respondents were using TQM/CQI. Of those not using TQM/CQI, 75 percent were interested in these activities. Improvement of public health clinic ability to compete and to survive in a rapidly changing health care environment requires fostering this interest through public health decision-maker support, increased TQM/CQI training opportunities, and demonstration of TQM/CQI cost-effectiveness in public health.

Microdilution antifungal susceptibility testing of Candida albicans and Cryptococcus neoformans with and without agitation: an eight-center collaborative study.

The growth patterns observed in the trailing wells when fluconazole is being tested may give rise to readings that suggest resistance or increased MICs for known susceptible strains. We conducted a multicenter study to evaluate the intralaboratory and interlaboratory reproducibilities of a method that uses agitation to disperse these types of growth. Ten strains of Candida albicans and five strains of Cryptococcus neoformans were tested against fluconazole, flucytosine, and amphotericin B by using a microdilution adaptation of the proposed reference method of the National Committee for Clinical Laboratory Standards for yeasts (M27-T). The endpoint criterion used before agitation was consistent with the M27-T recommendation, while a criterion of 50% or more reduction of growth compared with the control was used after agitation. The results of this study showed that use of agitation and the modified endpoint criterion both improved intralaboratory and inter-laboratory agreement and increased the frequency of interpretable MICs. The MICs obtained by this method were comparable to those obtained by the broth macrodilution M27-T method. Like M27-T, this method was not able to definitely distinguish amphotericin B-susceptible from -resistant strains, although the MICs for the resistant strains were consistently higher than those for the susceptible ones. The findings imply that agitation should be seriously considered when antifungal agents, particularly fluconazole, are tested in a microdilution format.

A blocking ELISA for detection of antibody to a subgroup-reactive epitope of African horsesickness viral protein 7 (VP7) using a novel gamma-irradiated antigen.

A novel gamma irradiated inactivated cell culture derived African horsesickness viral (AHSV) antigen was used in a blocking ELISA (B-ELISA) for detecting antibody to a subgroup-reactive epitope of AHSV. A monoclonal antibody (MAB), class IgM, against an epitope on African horsesickness (AHS) viral protein 7 (VP7) was developed in BALBc mice and used in the B-ELISA. The MAB, designated F9H, was blocked by 69 serums from equidae with antibody to AHS, but its binding activity was not appreciably affected by 301 serums that did not contain antibodies to AHS virus. An ELISA protocol using a blocking format is described.

Effect of threonine-to-methionine substitution at position 265 on structure and function of TEM-1 beta-lactamase.

TEM beta-lactamase variants with the amino acid substitutions R164S, E104K, G238S, and E240K (ABL numbering) display increased activity toward extended-spectrum cephalosporins. The T265M substitution is frequently found to be associated with the above substitutions in extended-spectrum beta-lactamases. However, the residue is located away from the active site in the three-dimensional structure and has been assumed to have no effect on catalysis. To examine the effect of the substitution on the structure and function of TEM beta-lactamase we constructed the following mutants: G238S, T265M, T265M:G238S, and T265M:G238S:E240K. Each enzyme was purified to homogeneity and the kinetic parameters kcat, Km and kcat/Km were determined for cefotaxime, ceftazidime, cephaloridine, and ampicillin. The results indicate that the T265M mutation has little effect on hydrolysis. In addition, we used immunoblotting to show that the substitution has little or no effect on the in vivo steady-state levels of beta-lactamase.

Characterization of TEM-1 beta-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime.

Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins such as cefotaxime and ceftazidime. To identify other amino acid substitutions that alter the activity of TEM-1 toward extended-spectrum cephalosporins, a random library was constructed that contained all possible amino acid substitutions over the 3-residue window of 238-241 (ABL numbering). Mutants were selected for 100-fold greater ceftazidime resistance than wild-type. All mutants had a serine substitution at position 238, a lysine or arginine at position 240, and a small amino acid at position 241. The role of each substitution was investigated by constructing individual G238S, E240K, and R241G substitutions as well as the G238S:E240K double mutant. Each enzyme was purified to homogeneity and the kinetic parameters kcat and Km were determined using several substrates. The G238S substitution increases catalytic efficiency for both ceftazidime and cefotaxime. However, to achieve large increases in catalytic efficiency, both G238S and the E240K substitutions are required. The R241G substitution results in a small increase in catalytic efficiency for only ceftazidime. The contribution of each residue to the transition-state stabilization energy was found to be additive indicating that the substitutions act independently to change the catalytic properties of the enzyme.

Fluconazole susceptibilities of Candida species and distribution of species recovered from blood cultures over a 5-year period.

The distribution and fluconazole susceptibilities of Candida species isolated over a 5-year period were investigated. Susceptibilities were determined by using a new microtiter procedure and the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard. The new method correlated well with the NCCLS proposed standard and gave very clear end points. Results indicate that there are species-related differences in MICs as reflected in the MICs for 90% of species tested. Candida albicans is most susceptible to fluconazole, while Candida glabrata is among the least susceptible. These findings coincided with the observation of a shift in distribution of yeast species recovered from blood cultures from 1987 to 1992. C. albicans was the predominant species (87%) in the pre- or early fluconazole years but decreased to only 31% of the isolates in 1992. Thus, Candida species for which MICs of fluconazole were higher have become more prominent in recent years. Significantly, throughout this period, MICs for each species did not change appreciably.

Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta-lactamase.

In order to understand how TEM-1 beta-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161-170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified. Characterization of altered beta-lactamase enzymes indicated that while their catalytic efficiency (kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 beta-lactamase.