Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares.

The first U.S. multicenter clinical trial to assess the performance of the Cepheid Xpert MRSA assay (Xpert MRSA) was conducted. The assay is a qualitative test designed for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from nares swabs. This novel test combines integrated nucleic acid extraction and automated real-time PCR for the detection of a MRSA-specific signature sequence. A total of 1,077 nares specimens were collected from seven geographically distinct health care sites across the United States with prevalence rates ranging from 5.2% to 44%. Nares specimens were tested by (i) the Xpert MRSA assay, (ii) direct culture on CHROMagar MRSA medium (direct CM culture), and (iii) broth-enriched culture (Trypticase soy broth with 6.5% sodium chloride) followed by plating onto CHROMagar MRSA medium (broth-enriched CM culture). When direct CM culture was designated the reference method, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MRSA assay were 94.3%, 93.2%, 73.0%, and 98.8%, respectively. When broth-enriched CM culture was used as the reference method, the clinical sensitivity, specificity, PPV, and NPV of the Xpert MRSA assay were 86.3%, 94.9%, 80.5%, and 96.6%, respectively. The BD GeneOhm MRSA (BDGO) assay was performed as a comparative molecular method. No statistical performance differences were observed between the Xpert MRSA and BDGO assays when they were compared to culture methods. From this large-scale, multicenter clinical comparison, we conclude that the Xpert MRSA assay is a simple, rapid, and accurate method for performing active surveillance for MRSA in a variety of health care populations.

Ultrahigh-power semiconductor diode laser arrays.

The power available from transistor-sized semiconductor diode lasers has increased rapidly in recent years, more than doubling every year. Continuous outputs of several watts and pulsed powers in the kilowatt range with 50 percent overall efficiency are now possible with these compact devices. These developments may signal the start of a technological advancement in optics comparable to the solid-state revolution in electronics 20 years ago.

Rochalimaea henselae causes bacillary angiomatosis and peliosis hepatis.

BACKGROUND: Recent studies have demonstrated that a newly described agent of persistent bacteremia, Rochalimaea henselae, and the agent of bacillary angiomatosis are both closely related to Rochalimaea quintana. Bacillary peliosis hepatis seemed likely to have the same etiologic agent as bacillary angiomatosis. We sought these pathologic changes in patients from whom R henselae was cultivated. METHODS: For two patients whose histopathologic findings we reviewed, additional light and electron microscopy were performed. Their bacterial isolates were compared by electrophoretic patterns of outer membrane proteins, restriction endonuclease digestion patterns of DNA, and reaction with murine antiserum. RESULTS: A previously reported human immunodeficiency virus-infected man with persistent bacteremia due to R henselae was found to have bacillary peliosis hepatis. Rochalimaea henselae was also isolated from the spleen of a woman receiving immunosuppressive therapy after allogeneic renal transplantation. She had developed fever, liver and spleen nodules, and periaortic lymphadenopathy. Bacillary peliosis of her liver and spleen, as well as bacillary angiomatosis of liver, spleen, and a lymph node, were found. The bacterial isolates had comparable electrophoretic patterns of outer membrane proteins and of restriction endonuclease-digested DNA, which differed from the respective patterns of R quintana. Murine antisera raised to the first isolate reacted strongly with the second by means of immunoblot and immunofluorescence techniques, while reacting only weakly with R quintana. CONCLUSION: Rochalimaea henselae, recently recognized to cause persistent fever and bacteremia in immunocompetent and immunocompromised persons, also causes bacillary angiomatosis and parenchymal bacillary peliosis.

Immunocytochemical identification of Rochalimaea henselae in bacillary (epithelioid) angiomatosis, parenchymal bacillary peliosis, and persistent fever with bacteremia.

We report the immunocytochemical identification of Rochalimaea henselae, a newly recognized fastidious, Gram-negative, Warthin-Starry-positive organism, as the common pathogen in bacillary angiomatosis (BA), bacillary peliosis (BP) of the liver and spleen, and persistent fever with bacteremia in immunocompromised patients. Immunogenic proteins of the R. henselae strain isolated from the blood of a febrile immunocompromised patient with BP of the liver were used to produce primary immune serum in rabbits. Using immunocytochemical procedures, the polyclonal antiserum reacted strongly not only with the immunizing strain of the bacteria, but also with other blood isolates of R. henselae (five cases) from both immunocompromised and immunocompetent patients and with the organisms present in the tissue lesions of cutaneous BA (five cases) and BP of the liver (two cases) and spleen (one case). The blood isolates and BA and BP tissue samples were obtained from widely separated geographic areas. The antiserum was weakly cross-reactive with cultures of Rochalimaea quintana, an organism closely related to R. henselae, but this reactivity was eliminated by specific adsorption. The antiserum did not cross-react with the Warthin-Starry-positive organisms associated with cat scratch disease (Afipia felis), syphilis (Treponema pallidum), Lyme disease (Borrelia burgdorferi) or chronic active gastritis (Helicobacter pylori). Likewise, the antiserum did not identify organisms in eight cases of Kaposi's sarcoma, a disorder of immunocompromised patients that is clinically similar to BA. Further studies are needed to determine the prevalence of this newly recognized organism as well as its possible involvement in other angioproliferative diseases.

Evaluation of the automated Bact-Alert system for pediatric blood culturing.

The Organon Teknika BacT/Alert (Organon Teknika, Durham, NC), using the Pedi-BacT 20 mL aerobic bottle (BPBCS) was compared to the Wampole Isolator (WI) 1.5 Microbial tube (Wampole Laboratories, Cranbury, NJ), for detection and recovery of pediatric pathogens. The BPBCS continuously monitors culture bottles for changes in CO2 concentrations, while WI cultures are examined twice daily for appearance of colonial growth on agar media. Of 5,175 paired blood cultures, 383 pathogens were recovered from 606 positive cultures. There were 272 pathogens recovered by both systems, 64 from BPBCS only, and 47 from WI only. Overall recovery rates were 88% for BPBCS and 83% for WI. There was no significant difference between the two systems in detection or times to positivity of staphylococci, Enterobacteriaceae, or pseudomonads. Trends toward better recovery of streptococci (20 vs. 10) and fastidious microaerophiles (3 vs. 0) were found with BPBCS, whereas more slowly growing pathogens (Rochalimaea henselae [1], Mycobacterium avium-intracellulare [1]) were recovered by WI only, but because of their lower frequency did not achieve statistical significance. Detection of Haemophilus influenzae (14.9 hours in WI vs. 45.4 hours in BPBCS) was faster with WI. False positive plus contaminant cultures were detected in 5.9% BPBCS versus 1.5% WI. BPBCS offers detection of bacteremia at a rate comparable to WI with advantages of automation.

Comparative evaluation of selective and nonselective culture techniques for isolation of group A beta-hemolytic streptococci.

A new selective blood agar medium, Strep A Isolation Agar (SI) from Remel (Lenexa, KS), was compared with Becton Dickinson's Streptococcus Selective Agar (SA) (Becton Dickinson Microbiology Systems, Cockeysville, MD) and with a nonselective Columbia Blood Agar (CB) (Difco, Detroit, MI). Throat swabs from patients with acute pharyngitis were cultured with the use of a single swab to inoculate each of the three plates in a specific order, rotating in three-week cycles. Plates were examined (each medium by a different technologist) after 24 and 48 hours of incubation at 35 degrees C in 5% carbon dioxide, and beta-hemolytic streptococci were serogrouped with the use of coagglutination. The positivity rate was significantly greater for SI (25%) and SA (26%) than for CB (18%) (P less than 0.001). The respective rates of Group A streptococcal detection by SI, SA, and CB were 91%, 95%, and 67%, respectively. However, a feature associated with the use of SI or SA, in contrast to CB, was delayed identification of isolates by 24-48 hours because of small colony size, slower growth rate, and inability to serogroup colonies taken directly from primary culture plates. Recovery of non-Group A beta-hemolytic streptococci occurred with CB (12%) greater than SI (8%) greater than SA (6%). SI is superior to a nonselective medium, such as CB, and is equal to SA for recovery of Group A streptococci from throat cultures.

Catalase-negative listeria monocytogenes causing meningitis in an adult. Clinical and laboratory features.

A 63-year-old previously healthy woman presented with acute meningitis. Cultures of the cerebrospinal fluid yielded a serotype 1/2a isolate of Listeria monocytogenes that was biochemically typical in all respects, other than the reproducible lack of catalase production. During therapy, the patient developed oculomotor dysfunction that was attributed to an abscess in the internal capsule. This case report documents the existence of catalase-negative L. monocytogenes indicating that catalase production should not be a strict criterion for identification of Listeria. Furthermore, this clinical experience extends in vitro and experimental animal studies indicating that catalase production is not a necessary virulence factor for invasion by Listeria.

Morphologically variable bacilli of cat scratch disease are identified by immunocytochemical labeling with antibodies to Rochalimaea henselae.

The identification of the causative organisms of cat scratch disease (CSD) has been elusive. The demonstration of Warthin-Starry stain-positive pleomorphic bacilli in lymph nodes of patients with CSD and recent serologic and epidemiologic data suggest an etiologic role of Rochalimaea henselae in CSD. The authors studied lymph node biopsy specimens of 46 patients with illnesses clinically consistent with CSD and found pleomorphic bacilli in 15 (33%). The organisms were labeled by polyclonal rabbit antibodies induced by outer surface proteins of R henselae. This finding further supports the possibility of an important role of R henselae in the pathogenesis of CSD.

Bactericidal activity of cefoperazone in comparison with other beta-lactam antibiotics and gentamicin.

Killing curves were employed to determine the in vitro bactericidal activity of cefoperazone against several aerobic bacterial species of clinical origin. Cefoperazone was bactericidal at concentrations equal to, or greater than, the minimal inhibitory concentration (MIC) but allowed regrowth of the organisms after four to six hours when tested in concentrations of one-fourth or one-half the MIC. Comparative studies with gentamicin, LY127935 (moxalactam), piperacillin, cefamandole, and cephalothin indicate that, in general, cefoperazone demonstrated similar killing activity as other beta-lactam compounds, with some strain differences noted. Gentamicin, as the representative aminoglycoside, demonstrated a greater rate of bactericidal activity than the beta-lactam compounds studied.

Aeromonas species in aquaria: a reservoir of gastrointestinal infections?

To assess the role of aquaria in the epidemiology of aeromonas associated gastroenteritis, the prevalence and antibiotic susceptibility of aeromonads in ornamental aquaria were determined and compared to that of isolates obtained from patients with gastroenteritis. Thirty-eight (76%) of 50 aquaria, including those located in our hospital, yielded 61 isolates: 24 (39.3%) Aeromonas sorbria, 18 (29.0%) A. hydrophila, 15 (24.6%) A. caviae, and four (6.5%) which could not be speciated. In comparison, 41 (65%) of 63 faecal isolates were A. caviae. The aquarium isolates, in contrast to enteric isolates, were generally resistant to chloramphenicol, tetracycline, and trimethoprim/sulphamethoxazole. These findings indicate aquaria are unlikely sources of aeromonas-associated gastroenteritis, but their role as possible reservoirs for non-enteric infections with aeromonads needs to be defined.

Immune response of neonatal specific pathogen-free cats to experimental infection with Bartonella henselae.

The purpose of this study was to determine whether neonatal cats develop and maintain a persistent bacteremia for longer than do adult cats with a normal mature immune system, and whether neonatal cats are susceptible to infection with Bartonella henselae by oral inoculation. Neonatal specific pathogen-free (SPF) cats were inoculated with B. henselae intradermally (n = 4) or orally (n = 5) or with 0.9% NaCl (n = 2). Blood was collected periodically through 16 weeks post-inoculation (PI) for serology, bacteriology and complete blood count. Cats inoculated orally or intradermally at 3-5 days of age were bacteremic through 12-16 weeks PI, similar to what is documented for adult cats inoculated intradermally or intravenously. One cat inoculated at age 2 weeks was bacteremic through 10 weeks PI; the other was not bacteremic. Intradermally inoculated neonatal cats produced serum IgG antibodies to B. henselae but orally inoculated neonatal cats did not. Infected cats with and without serum IgG antibodies to B. henselae became blood-culture negative simultaneously, suggesting that IgG is not required to clear bacteremia.

Evidence of reproductive failure and lack of perinatal transmission of Bartonella henselae in experimentally infected cats.

Five female specific pathogen-free (SPF) cats inoculated intradermally with B. henselae and bacteremic for 4 weeks, and one cat inoculated with 0.9% NaCl, were bred with uninfected SPF male cats. The uninfected female became pregnant with one breeding, while three infected cats became pregnant 1-12 weeks later, after repeated breedings. Two infected females either did not become pregnant or maintain pregnancies despite repeated breedings. Infected cats produced anti-B. henselae IgM and IgG antibodies. Fetuses and kittens of infected cats were not infected and did not produce anti-B. henselae antibodies. Male cats bred with infected females did not become infected or seroconvert. Maternal anti-B. henselae IgG antibodies detected in sera of kittens 2 weeks post-partum were no longer detectable 10 weeks post-partum. These findings suggest that B. henselae causes reproductive failure in female cats, but is not transmitted transplacentally, in colostrum or milk, or venereally. Infected cats immunosuppressed with methylprednisolone acetate after their kittens were weaned had no detectable bacteria in tissues, suggesting that they were no longer infected.

Campylobacter enteritis. A 3-year experience.

Campylobacter jejuni was isolated from 135 infants and children seen at the Oklahoma Children's Memorial Hospital over a 3-year period. The comparative frequency of isolation of C. jejuni, Salmonella, and Shigella were 1.5 percent, 2.2 percent, and 3.1 percent, respectively. Campylobacter enteritis was most prevalent during the warm months from May to October, peaking in July. Seventy percent of the afflicted children were 2 years old or younger; only 13 percent were older than 5 years. There were the usual clinical presentations (acute onset of diarrhea, fever, abdominal pain, and bloody stools) of Campylobacter enteritis, but other, less common, patterns also were seen. These included chronic diarrhea without significant systemic manifestations; asymptomatic bloody stools, particularly in neonates; and fever and abdominal pain without diarrhea. Severe complications included hemolytic-uremic syndrome, sepsis associated with septic arthritis and osteomyelitis, and failure to thrive.

Applications of cellular fatty acid analysis.

More than ever, new technology is having an impact on the tools of clinical microbiologists. The analysis of cellular fatty acids by gas-liquid chromatography (GLC) has become markedly more practical with the advent of the fused-silica capillary column, computer-controlled chromatography and data analysis, simplified sample preparation, and a commercially available GLC system dedicated to microbiological applications. Experience with applications in diagnostic microbiology ranges from substantial success in work with mycobacteria, legionellae, and nonfermentative gram-negative bacilli to minimal involvement with fungi and other nonbacterial agents. GLC is a good alternative to other means for the identification of mycobacteria or legionellae because it is rapid, specific, and independent of other specialized testing, e.g., DNA hybridization. Nonfermenters show features in their cellular fatty acid content that are useful in identifying species and, in some cases, subspecies. Less frequently encountered nonfermenters, including those belonging to unclassified groups, can ideally be characterized by GLC. Information is just beginning to materialize on the usefulness of cellular fatty acids for the identification of gram-positive bacteria and anaerobes, despite the traditional role of GLC in detecting metabolic products as an aid to identification of anaerobes. When species identification of coagulase-negative staphylococci is called for, GLC may offer an alternative to biochemical testing. Methods for direct analysis of clinical material have been developed, but in practical and economic terms they are not yet ready for use in the clinical laboratory. Direct analysis holds promise for detecting markers of infection due to an uncultivable agent or in clinical specimens that presently require cultures and prolonged incubation to yield an etiologic agent.

Comparison of lysis-direct plating and broth methods for pediatric blood cultures: clinical relevance and cost effectiveness.

To determine the clinical significance and therapeutic impact of pediatric blood cultures, we analyzed 1650 cultures comparing conventional broth and direct plating methods. The rate of positive cultures was 5.8%. Of 96 positive cultures 68 were deemed clinically significant. The mean time to detection was 23.8 and 33.7 hours, and the sensitivity was 89 and 83% in the Isolator and broth systems, respectively. The overall rate of insignificant isolates was 1.7% and was similar for both methods. Clinical interventions based on blood culture results occurred in 39 patients, primarily those with pneumococcal and staphylococcal bacteremia. Only six of 28 clinically insignificant isolates had negative impact. Use of the pediatric Isolator permitted earlier institution of appropriate therapies and shorter duration of unneeded or toxic therapies without frequent negative impact. Since direct costs were comparable the Isolator system may be a clinically valuable and cost effective alternative to conventional methodology.

Subgrouping of Pseudomonas cepacia by cellular fatty acid composition.

The cellular fatty acid compositions were determined for 42 strains of Pseudomonas cepacia from five cystic fibrosis centers in North America. All isolates contained significant (20%) amounts of hexadecanoic (C16:0), and cis-9 hexadecenoic (C16:1 cis9) acids and an isomer of octadecenoic acid (C18:1). None had hydroxy acids containing fewer than 14 carbon atoms. The quantitative data from the fatty acid analysis were highly reproducible and provided a basis for numerical analysis. Five subgroups comprising all the strains were obtained by cluster analysis and further characterized by principal-component analysis. With minor exceptions, the predominant subgroup identified in each center was different from that identified in other centers and accounted for one-half of the isolates within each center. Cellular fatty acid composition is a useful adjunct to biochemical characterization for the identification of P. cepacia isolated from cystic fibrosis patients. Numerical analysis of the fatty acid data can separate P. cepacia into subgroups, which may provide useful epidemiologic information or a basis for further analysis by more complex techniques such as DNA probe analysis.