In vitro activity of levofloxacin against contemporary clinical isolates of Legionella pneumophila, Mycoplasma pneumoniae and Chlamydia pneumoniae from North America and Europe.

OBJECTIVE: To assess the activities of levofloxacin and the comparator agents erythromycin, clarithromycin, azithromycin and doxycycline against atypical respiratory pathogens. METHODS: One hundred and forty-six Legionella pneumophila, 41 Mycoplasma pneumoniae and nine Chlamydia pneumoniae isolates were procured from various culture collections in North America and Europe and tested for susceptibility to the above agents by broth microdilution. The isolates came primarily from clinical sources and were collected from patients between 1995 and 1999. RESULTS: Against L. pneumophila, levofloxacin was the most active agent, with an MIC(90) of 0.03 mg/L, twofold more active than clarithromycin (0.06 mg/L), 16-fold more active than erythromycin and azithromycin (0.5 mg/L) and 64-fold more active than doxycycline. Against M. pneumoniae, azithromycin (MIC(90) < or = 0.0005 mg/L) was the most active agent. However, two isolates of M. pneumoniae, one from the USA and one from Finland, were macrolide resistant (MIC > or = 4 mg/L), but levofloxacin susceptible (MIC 0.25 mg/L). The geographic origin of L. pneumophila and M. pneumoniae did not affect the MIC range for any antimicrobial agent tested. Against C. pneumoniae, clarithromycin was the most active agent, with an MIC range of < or =0.008-0.03 mg/L. CONCLUSIONS: Levofloxacin had comparable activity to the other agents tested against the atypical respiratory pathogens, confirming its potential as an alternative for empirical therapy of community-acquired pneumonia.

Rapid detection of Mycobacterium tuberculosis in contaminated BACTEC 12B broth cultures by testing with Amplified Mycobacterium Tuberculosis Direct Test.

Contamination of broth cultures of acid-fast bacilli (AFB) by bacterial species other than Mycobacterium species frequently occurs. Many of these contaminated cultures require redecontamination and reincubation before the appropriate tests can be performed for identification, significantly affecting the turnaround time for reporting culture results. In this study, the Amplified Mycobacterium Tuberculosis Direct Test (MTD; Gen-Probe) was performed to detect the Mycobacterium tuberculosis complex (MTBC) in 125 BACTEC 12B broth cultures with positive growth indices. Among these, 41 grew non-AFB bacteria only, and all 41 were negative by the MTD. The remaining 84 bottles contained contaminated cultures that grew both AFB and other bacteria or yeasts. Repeat decontamination and reincubation of these specimens required a mean time of 13 days (range, 3 to 40 days). The MTD results were positive for 10 samples, 9 of which were MTBC culture positive and 1 of which grew Myobacterium celatum, a species known to cross-react in the MTD. All cultures growing other mycobacterial species were negative by the MTD. The results of this study demonstrate that the MTD is both sensitive and specific in detecting MTBC in contaminated broth cultures and that, when used selectively, the MTD can potentially rule in or out a diagnosis of MTBC as much as 12 days earlier than using nonamplified DNA probe testing alone can.

Benchmarking the in vitro activities of moxifloxacin and comparator agents against recent respiratory isolates from 377 medical centers throughout the United States.

To benchmark the activity of moxifloxacin (a newer fluoroquinolone), a U.S. study comprising 16,141 contemporary isolates of Streptococcus pneumoniae (5,640), Haemophilus influenzae (6,583), and Moraxella catarrhalis (3,648) referred from 377 institutions during 1998 is described. For S. pneumoniae the modal MIC and MIC at which 90% of the isolates were inhibited (MIC(90)) for moxifloxacin were 0.12 and 0.25 microg/ml, respectively, independent of susceptibility to other drug classes, geography, or site of infection. Eleven isolates were intermediate or resistant to levofloxacin and grepafloxacin; of these isolates, 1 remained susceptible to sparfloxacin, 2 remained susceptible to moxifloxacin, and 4 remained susceptible to trovafloxacin. All 11 isolates possessed classic mutations in gyrA and/or parC known to confer reduced susceptibility to fluoroquinolones. Four isolates (originating from four separate states) belonging to a multidrug-resistant, fluoroquinolone-resistant clone were identified by pulsed-field gel electrophoresis. For moxifloxacin and trovafloxacin, at least 87% of isolates demonstrated MICs > or =3 twofold concentrations below the susceptibility breakpoints, in contrast to no more than 15% for levofloxacin, grepafloxacin, and sparfloxacin. Of the isolates that were multidrug resistant (7.4%), >98% remained susceptible to moxifloxacin. The modal MIC and MIC(90) for M. catarrhalis (both 0.06 microg/ml) and for H. influenzae (both 0.03 microg/ml) were independent of beta-lactamase production. These data demonstrate the in vitro activity of moxifloxacin and establish a baseline for future studies.

Benchmarking the in vitro activity of moxifloxacin against recent isolates of Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae. A European multi-centre study.

To benchmark the activity of moxifloxacin, a European study comprising 900 Streptococcus pneumoniae, 1051 Haemophilus influenzae, and 226 Moraxella catarrhalis referred from 30 institutions during 1998 is described. For S. pneumoniae, moxifloxacin and trovafloxacin MIC(90) and modal MICs values were 0.12 microg/ml and independent of susceptibility to other drug classes, geography, or site of infection. MIC(90)/modal MICs were, respectively, 0.25/0.12 microg/ml for grepafloxacin, 0.25/0.25 microg/ml for sparfloxacin, and 1.0/0.5 microg/ml for levofloxacin. The moxifloxacin C(max):MIC ratio of 20.8-26.3 is higher than comparator fluoroquinolones. Five isolates were intermediate or resistant to grepafloxacin, sparfloxacin, or levofloxacin of which four and three remained susceptible to trovafloxacin and moxifloxacin, respectively. For moxifloxacin, > 90% of S. pneumoniae isolates demonstrated MICs > or =3 dilutions below the susceptibility breakpoint used. Modal MICs and MIC(90) for M. catarrhalis (both 0.03 microg/ml) and H. influenzae (0.03 microg/ml and 0.06 microg/ml) were independent of beta-lactamase production. These data demonstrate the in vitro activity of moxifloxacin and establish a baseline for future surveillance studies that will be important for detecting and tracking any trends in changing activity of this fluoroquinolone.

Evaluation of two rapid assays for detection of Clostridium difficile toxin A in stool specimens.

Rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is highly desirable in the setting of hospital cost containment. We tested 654 stool specimens to compare the performance of two assays for rapid detection of toxin A, the Immunocard Toxin A test (Meridian Diagnostics, Inc.) and the Culturette Brand Toxin CD enzyme immunoassay (EIA) (Becton Dickinson Microbiology Systems), with a cytotoxin assay (Cytotoxi Test; Advanced Clinical Diagnostics) and culture on cycloserine-cefoxitin-fructose agar followed by determination of the production of toxins A and B. A chart review was performed for patients whose stool specimens provided positive results on one to three of the assays. With the "gold standard" of all four assays positive or chart review evidence of CDAD, 97 (14.8%) stool specimens were positive by one or more assays and 557 (85.2%) were negative by all methods. Total agreement for all assays was 90.5% (592 of 654). The sensitivity, specificity, positive predictive value, and negative predictive value for toxigenic culture were 94.7, 98.6, 87.1, and 99.5%, respectively, for toxigenic culture; 87.7, 98.6, 86.2, and 98.8%, respectively, for the cytotoxin assay; 71.9, 99.3, 91.1, and 97.3%, respectively, for the Immunocard; and 68.4, 99.1, 88.6, and 96.9%, respectively, for the Culturette EIA. While easy to perform and highly specific, these rapid assays do not appear to be sufficient for accurate diagnosis of CDAD.

Laboratory diagnosis of respiratory virus infections in 24 hours by utilizing shell vial cultures.

Immunofluorescence staining of centrifugation-enhanced shell vial (SV) cultures for respiratory viruses (RV) after 24 h of incubation, rather than the more commonly prescribed times of 48 h and 5 days, allowed for the detection of 77% of the RV-positive specimens that would ordinarily not have been detected as positive until 48 h. Staining SVs at 24 h also permitted earlier detection of viruses that were missed by rapid antigen detection methods.

Cytomegalovirus infection in children with human immunodeficiency virus infection.

OBJECTIVES: To determine retrospectively the prevalence of positive cytomegalovirus (CMV) cultures in pediatric patients with human immunodeficiency virus infection. METHODS: We reviewed the records of 273 children with human immunodeficiency virus infection referred to the Pediatric Branch of the National Cancer Institute for whom CMV cultures were performed between January, 1991, and October, 1994. RESULTS: Of this group 189 patients (69%) had negative CMV cultures and 84 (31%) had positive cultures. The prevalence of CMV-related disease was 9% for the entire group, including 4 (2.1%) patients with negative CMV cultures. Among the 84 patients with positive CMV cultures, 21 (25%) had evidence of CMV disease. Patients with positive CMV cultures had a statistically significant decrease in survival in the presence of severe immunocompromise defined as an age-corrected CD4 count of < 21%. Nine of 35 (26%) autopsies performed demonstrated evidence of CMV disease, including 7 patients with disseminated CMV disease. CONCLUSIONS: Although CMV disease appears to be less frequent in children than adults, CMV infection still contributes significantly to morbidity and mortality in this population, especially when combined with severe immunosuppression.

Rapid identification of respiratory viruses: impact on isolation practices and transmission among immunocompromised pediatric patients.

OBJECTIVE: To determine whether empiric isolation of patients with acute respiratory virus infection symptoms could be discontinued when preliminary shell vial cultures were negative, and the impact of this approach on hospital resources. DESIGN: In 1993, we retrospectively reviewed respiratory virus test results from 1992 to 1993 and extended data collection prospectively through the 1993 to 1994 season. The rapid test and 48-hour shell vial results were compared to a standard of rapid test plus 5-day shell vial culture results to determine the sensitivity and specificity of these "preliminary" results. SETTING: A 400-bed tertiary referral research hospital. PATIENTS: Patients from any inpatient unit or clinic with acute respiratory virus infection symptoms who had a specimen submitted for respiratory virus culture. Patients were placed on empiric respiratory isolation pending culture results. RESULTS: The overall sensitivity of the combined rapid and 48-hour culture results in adults and children was 97%. All 15 pediatric patients with respiratory syncytial virus infection who had specimens submitted on first suspicion of respiratory virus infection were positive by rapid test. Culture results were positive within 48 hours for 100% of patients with influenza A (15 patients), influenza B (6), and parainfluenza (18) viruses. Of 59 pediatric inpatients who were isolated empirically awaiting 5-day culture results, 31 (52%) ultimately were determined to be culture negative. CONCLUSIONS: Empiric isolation of symptomatic children can be discontinued at 48 hours when both the rapid test and the early culture results are negative. Our institution would have saved 93 days of unnecessary isolation over 2 years had such a policy been in place.

Clinical evaluation of the BacT/Alert and isolator aerobic blood culture systems.

The BacT/Alert (BTA) (Organon Teknika, Durham, NC) and Isolator 10 (ISO) (Wampole Laboratories, Cranbury, NJ) blood culture systems were evaluated for their ability to detect aerobic and facultatively anaerobic microorganisms in blood of adult patients. For each culture 8 mL of blood was inoculated into both the aerobic standard BTA bottle and the ISO tube. Of 7,259 paired culture sets, 1,168 organisms were recovered, and 667 (57.1%) of these were considered clinically significant. This represented 540 clinically significant positive cultures from 266 patients. Of the significant isolates, 410 were recovered by both systems, 108 by BTA only and 149 by ISO only (P <.025). Overall, the BTA detected 77.7% of the significant isolates, whereas ISO detected 83.8%. The ISO recovered significantly more isolates of Staphylococcus aureus (P = .0001), coagulase-negative Staphylococcus spp (P <.01), and non-Enterobacteriaceae gram-negative rod species (P <.0025), whereas the BTA detected significantly more isolates of Streptococcus spp (P <.0025). Growth of S aureus (P <.0025), Enterococcus spp (P <.0025), and Streptococcus spp (P <.0075) was detected earlier by the BTA when laboratory coverage was available during the first shift only (7:30 AM to 4:00 PM), and additionally of Enterobacteriaceae (P <.0005) and other gram-negative rod species (P <.0001) if coverage was extended to 12:00 AM. Yeasts were detected more rapidly by the ISO (P <.0025). The ISO contamination rate (5.9%) was six times that of the BTA. Taking into account its ability to rapidly detect most organisms, its automated and thus labor-saving features, and the minimal contamination rate associated with its use, the BTA appears to be a reliable alternative to the ISO as a blood culturing system, although improvement in detection of staphylococci and non-Enterobacteriaceae gram-negative rods would be desirable.

Effects of sonication of clinical specimens on detection of cytomegalovirus in shell vial cultures.

BACKGROUND: The centrifugation-enhanced shell vial (SV) method of virus culturing has decreased the time to laboratory detection of many viruses, and has become the principal method for the rapid detection of cytomegalovirus (CMV) in clinical specimens. OBJECTIVES: To examine the use of sonication and centrifugation of clinical specimens as a means of optimizing the sensitivity of SV detection of CMV, decreasing the toxicity of specimens to the SV monolayer, and facilitating the examination and interpretation of SV monolayers. STUDY DESIGN: A total of 350 clinical specimens submitted for CMV culture were processed and then divided in half, with one-half sonicated for 1 min in a cup-horn-equipped sonicator, and the other half left unsonicated. Sonicated specimens were centrifuged to recover a cell-free supernatant. SVs containing MRC-5 fibroblast monolayers were inoculated with either the unsonicated whole specimen or the cell-free supernatant, and were stained with monoclonal antibodies directed against the immediate-early antigen of CMV after 24 and 48 h of incubation. RESULTS: While no significant difference was observed in the overall number of specimens in which CMV was detected following sonication, sonication did afford a 31% increase in the number of CMV-positive specimens detected at 24 h. A significant reduction in toxicity of all specimens except for blood was observed for the sonicated specimens, although sonication of blood increased the number of blood specimens toxic to the monolayer by 40%. Use of the cell-free inoculum following sonication facilitated microscopic examination and interpretation of SV monolayers without adversely affecting the sensitivity of the culture. CONCLUSIONS: Sonication of clinical specimens prior to shell vial culturing for CMV is beneficial and can help to reduce specimen toxicity, facilitate interpretation of monolayers, and allow the earlier detection of a positive specimen.

Gut bacteria provide precursors of benzodiazepine receptor ligands in a rat model of hepatic encephalopathy.

Benzodiazepine receptor (BZR) ligands are elevated in animals and humans with fulminant hepatic failure (FHF) and contribute to the pathogenesis of the associated hepatic encephalopathy (HE). As gut factors are proposed to play a role in the pathogenesis of HE, we investigated gut flora as a source of BZR ligands. Rats received daily oral neomycin, vancomycin and metronidazole (AB +) or saline (AB -) before and concurrent with the induction of FHF with thioacetamide. BZR ligands were extracted from brain and plasma and quantified using radiometric techniques. Plasma BZR ligand concentrations in AB(+) and AB(-) rats with HE were higher than AB(+) and AB(-) control rats. Brain BZR ligand concentrations increased in AB(+) and AB(-) rats with HE. Stool cultures from antibiotic treated rats with HE indicated the presence of multidrug resistant Acinetobacter lwoffii. Although no significant concentrations of BZR ligands were detected in culture media inoculated with A. lwoffii, administering A. lwoffii to normal rats significantly elevated BZR ligand levels in brain, but not plasma. Thus, antibiotic treatment of rats is associated with the growth of a resistant strain of bacterium which produces an inactive BZR ligand precursor. BZR ligands may be synthesized from these precursors in the brain and efficiently cleared by a normal liver following brain-to-plasma transfer. Impairment of this clearance process in FHF facilitates their accumulation, enabling agonist BZR ligands to contribute to the pathogenesis of HE by further enhancing GABAergic neurotransmission.

Effect of potential interference factors on performance of enzyme immunoassay and latex agglutination assay for cryptococcal antigen.

The PREMIER Cryptococcal Antigen Enzyme Immunoassay (Meridian Diagnostics) did not give discrepant results with rheumatoid factor, syneresis fluid, or serum macroglobulins from systemic lupus erythematosis patients. The Cryptococcal Antigen Latex Agglutination System (Meridian Diagnostics) did cross-react with syneresis fluid but not with the other serum factors tested.

Effect of centrifuging shell vials at 3,500 x g on detection of viruses in clinical specimens.

An increase in shell vial centrifugation force to 3,500 x g and a concomitant reduction in spin time to 15 min did not decrease the sensitivity of detecting viruses in clinical specimens compared with the accepted practice of using 700 x g for 40 min. No damage to the cell monolayer (ML) at the higher g force was observed. Toxicity to the ML is decreased with the shorter spin, probably because of reduced time of contact between the specimen and the ML.

The production of a bactericidal monoglyceride in staphylococcal abscesses.

The treatment of abscess homogenates with calcium ionophores stimulated the production of a bactericidal lipid with properties indistinguishable from those of a previously unidentified bactericidal lipid that had been detected in staphylococcal abscesses. The lipid was identified as a monoglyceride by thin layer chromatography. It resembled the unidentified lipid in that it had a high specific activity, exhibited differential activity, was inhibited by Staphylococcus aureus delta toxin, lecithin and Ca++, and its activity was reduced by oxidation. Stimulation of monoglyceride production by calcium ionophore requires the joint presence of components from the sedimented and supernatant fractions of abscess homogenates, and was not produced if boiled homogenate was used. The addition of verapamil interfered with the production of monoglyceride in homogenates treated with calcium ionophore. Monoglyceride was produced only in abscess homogenates and not in homogenates of other normal tissues or tissues taken from mice infected with S. aureus. Calcium ionophore could be replaced by inositol triphosphate, suggesting that monoglyceride production involved the release of calcium from intracellular stores. The 2-monoglyceride was the form originally produced in abscess homogenates, but this spontaneously isomerized to the 1-monoglyceride. The fatty-acid moiety of the monoglyceride consisted primarily of 16:0 and 16:1 fatty acids.

Bacteremia and subcutaneous abscess caused by Proteus penneri in a neutropenic host.

Proteus penneri bacteremia and concomitant subcutaneous infection developed in a neutropenic patient with acute lymphocytic leukemia. The skin infection occurred while the patient was being treated empirically with cefoperazone and metronidazole. This case demonstrates the invasive potential of this microorganism in the proper setting.

Mycobacterium chelonei infection of a Broviac catheter insertion site.

Mycobacterium chelonei infection developed at the insertion site of an indwelling Broviac catheter in a child with erythroleukemia. Direct adherence to and colonization of the intra- and extra-luminal surfaces of the catheter, with extension to the adjacent subcutaneous tissue, by this rapidly growing mycobacterium may have been the primary factor underscoring the infection. Nontuberculous mycobacteria such as Mycobacterium chelonei grow readily on routine bacteriologic media and resemble Corynebacterium spp. (diphtheroids) in their Gram staining and microscopic characteristics. The persistence of the infectious process and a diphtheroid-like microorganism despite antimicrobial therapy should raise the suspicion for a mycobacterial species.

Legionella pneumophila: initial detection through evaluation of Giemsa-stained smears of a bronchial aspirate.

The presence of Legionella species in a bronchial aspirate was strongly suspected by the visualization on Giemsa-stained smears of slender violaceous intra- and extracellular bacilli despite the absence of fluorescence on smears stained with L. pneumophila serogroup 1 conjugate. Subsequently, cultures of the aspirate grew L. pneumophila reactive with serogroup 6 fluorescein-conjugated antibody.