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 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.

Recovery of Malassezia pachydermatis from eight infants in a neonatal intensive care nursery: clinical and laboratory features.

A 15-month retrospective survey of 507 admissions to a neonatal intensive care unit revealed 8 patients from whom Malassezia pachydermatis was isolated from one or more clinical specimens. The fungus was cultured from blood (four patients), central venous catheter tips (three patients), urine (four patients), cerebrospinal fluid (one patient), eye discharge (one patient), ear discharge (one patient) and tracheal aspirate (one patient). Seven of the eight infants displayed an episode of one or more of the following symptoms: apnea, bradycardia, temperature instability and hepatosplenomegaly. These episodes were temporally related to recovery of M. pachydermatis from clinical specimens. The seven symptomatic infants had received multiple antibiotics as well as long term hyperalimentation, including lipids, by infusion through deep vein catheters; the single asymptomatic infant did not. These data suggest an association between M. pachydermatis and the febrile systemic syndrome of neonates recently described for extracutaneous infections due to Malassezia furfur.

Reactivity of serologic tests for the detection of antibody specific to cytomegalovirus. II. Latex agglutination and enzyme-linked immunosorbent assay.

Two hundred seven sera were assayed for antibody-specific for cytomegalovirus (CMV) by two enzyme immunoassays (EIAs; Bioenzabody, Litton Bionetics, and Abbott CMV Total Antibody EIA, Abbott Laboratories) and latex agglutination (LA; CMVScan, Hynson, Westcott and Dunning). The overall accuracy of the LA, Litton EIA, and Abbott EIA was 95.8%, 86.2%, and 88.6%, respectively. Although the Abbott EIA had a sensitivity of 98.8%, the specificity was only 35.5%. The positive predictive values of the LA, Litton EIA, and Abbott EIA were 99.4%, 95.9%, and 88.9%, respectively, while the negative predictive values of each of these tests were 81.1%, 56.2%, and 84.6%, respectively.

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.

Multiple drug-resistance in Shigella flexneri isolated from a patient with human immunodeficiency virus.

Persistent shigellosis, due to Shigella flexneri resistant to multiple antibiotics, developed in a 40-yr-old homosexual man with human immunodeficiency virus infection. The Shigella strain demonstrated resistance to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole. Although Shigella flexneri isolates resistant to trimethoprim-sulfamethoxazole are uncommon in the United States, laboratories should monitor resistance patterns through routine in vitro susceptibility testing.

Mycobacterium fortuitum peritonitis in a patient undergoing chronic peritoneal dialysis.

Peritonitis, due to Mycobacterium fortuitum, developed in a 15-yr-old young man undergoing chronic peritoneal dialysis. Although of low pathogenic potential, this rapidly growing non-tuberculous mycobacterium does cause human disease particularly in the compromised host and should be considered as a potential cause of peritonitis in the chronic peritoneal dialysis patient.

Antimicrobial susceptibility of clinical isolates of Haemophilus influenzae to ampicillin-sulbactam.

A total of 1092 clinical isolates of Haemophilus influenzae (306 type b; 786 non-type-b), from five medical centers were obtained during 1987 and 1988. Disk diffusion antimicrobial susceptibilities were obtained for all isolates, and broth microdilution susceptibilities were obtained for 502 isolates. Beta-lactamase was produced by 34.3% of type-b and 22.1% of non-type-b isolates, with some geographic variations. Using disk diffusion antimicrobial susceptibility testing, all isolates were susceptible to ampicillin-sulbactam, ceftriaxone, cefuroxime, and rifampin; two isolates were resistant to chloramphenicol. Whether tested using a fixed ratio of ampicillin to sulbactam of 2:1 or a fixed concentration of sulbactam, the ampicillin-sulbactam combination demonstrated good activity against clinical isolates of H. influenzae. Only 8 of the 1092 isolates did not produce beta-lactamase but demonstrated MICs of greater than or equal to 2 micrograms/ml for ampicillin.

The adherence of verocytotoxin-producing Escherichia coli to rabbit intestinal cells.

Verocytotoxin-producing Escherichia coli (VTEC) have been recognised recently as an important cause of human disease. The adherence of VTEC to rabbit intestinal tract and the relationship between adherence and other virulence traits were studied. Twenty clinical isolates of VTEC (O157:H7 and other serotypes) and a control, commensal E. coli strain, were examined. Bacteria were evaluated for the presence of surface fimbriae, plasmid profile and hybridisation with a 3.4 kb DNA probe derived from the 60-MDa plasmid of such strains. Adherence was determined by electronmicroscopy and quantitatively with radio-labelled bacteria. Of the VTEC strains, 12 (60%) had surface fimbriae; all O157:H7 and 10 (70%) of 14 of the non-O157:H7 strains hybridised with the probe. No isolate was negative for both of these virulence traits and there was no correlation between their presence. The plasmid profiles varied among the strains, with no correlation to virulence traits. The adherence of VTEC strains differed significantly, ranging from 0.3 to 34.0 bacteria/intestinal cell. The mean adherence of fimbriate strains was greater than that of non-fimbriate strains (3.9 versus 2.7 bacteria/cell), although marked variability was noted in both groups. This study showed that VTEC strains differed markedly in their adherence capability and that neither the presence of fimbriae nor hybridisation with the 3.4-kb probe was essential for adherence. Several distinct mechanisms probably play a role in VTEC adherence.

Bacterial flora of the gastrointestinal tract of opossums.

The bacterial flora of the stomach, small intestine, cecum and bile from 20 healthy opossums (Didelphis virginiana) captured from the wild was studied. Results showed that their gastrointestinal flora was similar to that found in other small mammals but, in addition, opossums are heavily colonized by Salmonella spp., which might adversely affect their adequacy as laboratory animals for some experimental protocols.

Evaluation of computer case simulations for teaching clinical pathology to second-year medical students.

A computer-assisted learning program for teaching clinical pathology to second year medical students has been developed and evaluated. These programs are designed to be used as supplements to formal lectures, laboratory exercises, and small group discussions. Students are given case histories and asked to select differential diagnoses, order and interpret laboratory and diagnostic tests, and make final diagnostic conclusions. In some cases, laboratory monitoring of treatment, e.g., drug therapy, is emphasized. The performance of the student is objectively evaluated during each stage. In addition, the amount spent for each workup is recorded with penalties given for excess or inappropriate test ordering. Separate evaluations are performed to assess the effectiveness of these programs as an alternative teaching format to (1) formal lectures and reading assignments, and (2) faculty-directed small group discussions. It is concluded that the computer-assisted learning method is equivalent to lectures and group discussions and is a format that is well accepted by students.

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.

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.

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.

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.

Detection of piluslike structures on clinical and environmental isolates of Vibrio vulnificus.

Twenty clinical isolates of Vibrio vulnificus were compared with 10 environmental strains by using electron microscopy and agglutination assays with human erythrocytes, guinea pig erythrocytes, and Saccharomyces cerevisiae. In addition, the isolates were tested for ability to adhere to the human epithelial cell lines HEp-2 and A549. When examined by electron microscopy, 16 (80%) of the 20 clinical isolates demonstrated the presence of piluslike structures; the composition of the bacterial populations ranged from 0 to 68% piliated cells. In contrast, only 3 (30%) of the 10 environmental isolates were piliated, with a range from 0 to 16% piliated cells. A significant association between the presence of piliated cells and the isolate source was found (P less than 0.05). None of the 30 strains agglutinated erythrocytes or yeast cells. V. vulnificus adherence results obtained with HEp-2 cells showed 10 (50%) of 20 clinical isolates and 0 (0%) of 10 environmental isolates with averages of greater than 10 adherent bacteria per cell, demonstrating a correlation between attachment and the isolate source (P less than 0.05). Selected strains were tested to determine whether methyl alpha-D-mannopyranoside, fructose, or alpha-L-(-)-fucose would inhibit bacterial adherence to HEp-2 cells. Multiple patterns of adherence inhibition were observed. Adherence to A549 cells showed 8 (40%) of 20 clinical isolates and 0 (0%) of 10 environmental strains with averages of greater than 10 adherent bacteria per cell. A statistical association between attachment and the isolate source was demonstrated (P less than 0.05). These data suggest that the presence of piluslike structures and the ability to adhere to human epithelial cell lines may be more closely associated with V. vulnificus isolates from clinical specimens than with environmental strains.