A controlled comparison of the BacT/ALERT® 3D and VIRTUO™ microbial detection systems.

The performance of the next-generation BacT/ALERT® VIRTUO™ Microbial Detection System (VIRTUO™, bioMérieux Inc., Hazelwood, MO) was compared to the BacT/ALERT® 3D Microbial Detection System (3D, bioMérieux Inc., Durham, NC) using BacT/ALERT® FA Plus (FA Plus), BacT/ALERT® PF Plus (PF Plus), BacT/ALERT® FN Plus (FN Plus), BacT/ALERT® Standard Aerobic (SA), and BacT/ALERT® Standard Anaerobic (SN) blood culture bottles (bioMérieux Inc., Durham, NC). A seeded limit of detection (LoD) study was performed for each bottle type in both systems. The LoD studies demonstrated that both systems were capable of detecting organisms at nearly identical levels [<10 colony-forming units (CFU) per bottle], with no significant difference. Following LoD determination, a seeded study was performed to compare the time to detection (TTD) between the systems using a panel of clinically relevant microorganisms inoculated at or near the LoD with 0, 4, or 10 mL of healthy human blood. VIRTUO™ exhibited a faster TTD by an average of 3.5 h, as well as demonstrated a significantly improved detection rate of 99.9% compared to 98.8% with 3D (p-value <0.05).

Antimicrobial binding and growth kinetics in BacT/ALERT® FA Plus and BACTEC® Aerobic/F Plus blood culture media.

The capacity of absorbent beads in BacT/ALERT® FA Plus and BACTEC® Aerobic/F Plus blood culture bottles to bind and neutralize antibiotics was compared. Binding was established using reverse-phase HPLC, and inactivation was based on the recovery of susceptible test stains from simulated blood cultures. The FA Plus medium demonstrated more rapid and better overall binding kinetics for each drug tested, resulting in significantly better overall recovery rates. Differences in time to detection favored the FA Plus medium for three drug/organism combinations and Aerobic/F Plus for two.

Next-generation and whole-genome sequencing in the diagnostic clinical microbiology laboratory.

The identification and/or prediction of the antimicrobial resistance of microorganisms in clinical materials solely by molecular means in the diagnostic microbiology laboratory is not novel. However, the ability to sequence multitudes of bacterial genomes and deliver and interpret the resultant sequence information in near "real-time" is the basis of next-generation sequencing (NGS) technologies. There have been numerous applications and successes of NGS applications in the clinical and public health domain. However, none have, as yet, delivered perhaps the most sought after application, i.e., the generation of microbial sequence data for "real-time" patient management. In this review, we discuss the use of NGS and whole-genome sequencing (WGS) of microorganisms as a logical next step for the routine diagnosis of infection and the prediction of antimicrobial susceptibility in the clinical microbiology laboratory.

Peripheral intravenous catheter complications in critically ill children: a prospective study.

Six hundred fifty-four peripheral Teflon catheters in 303 pediatric intensive care unit patients were examined to determine complication rates and associated risk factors. Phlebitis, extravasation, and bacterial colonization occurred at rates of 13%, 28%, and 11%, respectively. Logistic regression of factors that increased phlebitis risk revealed infusion of hyperalimentation (odds ratio 2.9) or lorazepam (odds ratio 2.2) and catheter location (odds ratio 2.9) as the most important determinants of phlebitis risk. Age (less than or equal to 1 year, odds ratio 2.0), catheter time in situ (less than or equal to 72 hours, odds ratio 2.1), and infusion of antiepileptics (odds ratio 2.1) were the most important determinants of extravasation. Catheters were colonized most frequently with coagulase-negative Staphylococcus (51/54). Sepsis attributable to catheter colonization occurred in 1 patient. Duration of catheter placement (greater than or equal to 144 hours, odds ratio 5.8) was an important determinant of catheter colonization. Colonization risk increased from 11% in catheters that were in situ for 48 to 144 hours to 34% for catheters that were in for longer than 144 hours. Infusion of diazepam (odds ratio 11.0) or lipid emulsions (odds ratio 2.5) and age (less than or equal to 1 year, odds ratio 2.2) were also important determinants of colonization risk. Replacing catheters in critically ill children every 72 hours would not decrease phlebitis, bacterial colonization, or catheter-induced sepsis and could increase extravasation risk. Catheters can be safely maintained with adequate monitoring for up to 144 hours in critically ill children.

Assessing the need for anaerobic medium for the recovery of clinically significant blood culture isolates in children.

We examined the sensitivity of several BACTEC media options for the detection of bacteremia and fungemia in children at two pediatric health care facilities. At one institution a single aerobic bottle containing PEDS Plus medium was as sensitive in identifying positive blood cultures as the combination of aerobic and anaerobic media (77% vs. 80%; P = 1.0). When data from both facilities were combined, a blood culture set containing both aerobic and anaerobic media detected significantly more positive blood cultures than any single aerobic medium. However, the aerobic/anaerobic bottle combination was not significantly better than a blood culture set containing two aerobic media and, in absolute terms, the latter identified more cases of bacteremia. Of the 116 clinically significant episodes of bacteremia identified in this study, only 1 was caused by an anaerobic bacterium. We conclude that the routine use of anaerobic media for blood cultures of pediatric patients might be unnecessary but that the use of two different aerobic media could increase the sensitivity of the BACTEC blood culture system.

Epidemiologic markers of pediatric infections caused by coagulase-negative staphylococci.

Several epidemiologic markers (species, slime production and antimicrobial susceptibility) were examined for 256 isolates of coagulase-negative staphylococci (C-S) obtained from clinical specimens in a 1-year period. The medical records of the 169 pediatric patients from whom the C-S were obtained were reviewed and divided into infected (N = 11) and uninfected (N = 158) groups. The phenotypic traits of strains associated with infection included: (1) slime production (P = 0.014); (2) slime-positive Staphylococcus epidermidis (P = 0.002); and (3) resistance to penicillin (P = 0.03), oxacillin (P less than 0.001), clindamycin (P = 0.003), chloramphenicol (P less than 0.001) and trimethoprim/sulfamethoxazole (P less than 0.001). Infected patients were significantly older (P = 0.006) than uninfected patients. Simultaneous isolation of the same strain of C-S from the aerobic and anaerobic bottles of a single blood culture increased the probability of sepsis (P = 0.004). The combination of these patient and laboratory data may be useful in determining the clinical significance of C-S recovered from pediatric patients.

Clinical significance of fungi isolated from cerebrospinal fluid in children.

We reviewed the isolation of fungi from cerebrospinal fluid (CSF) cultures at Texas Children's Hospital during the past 6 years to evaluate the significance of a positive culture and to identify potential risk factors. Thirty-seven fungal isolates were recovered from 23 patients representing 2% of all 1498 positive CSF cultures for the study period. Candida species accounted for 94.5% of all fungal isolates. Nine of the 23 patients were newborns and 8 of these were very low birth weight premature neonates. C. albicans was recovered from the CSF of all newborns. Eleven patients were children 4 months to 14 years old. Three patients had positive cultures of CSF obtained on postmortem examination. Leading potential risk factors for positive CSF cultures from neonates included antimicrobial therapy, prematurity, very low birth weight, umbilical catheterization, total parenteral nutrition, intubation and respiratory distress syndrome. For children beyond the newborn period, potential risk factors were antimicrobial therapy for concurrent bacterial infection, chronic systemic or central nervous system disease and central venous cathterization. Disseminated fungal infection was documented in 40% of all patients with positive CSF cultures. Fungi recovered from 7 (35%) of 20 live patients were considered contaminants. We conclude that true fungal meningitis in children is accompanied by multiple positive cultures from CSF or CSF and a second site. A single positive CSF culture for fungi should be considered significant when both CSF findings compatible with meningitis and associated risk factors are present. The isolation of fungi from a single CSF culture can be considered insignificant when CSF findings are within normal limits despite the presence of potential risk factors or vice versa.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral toxoplasmosis in an immunocompromised host. A precise and rapid diagnosis by electron microscopy.

In immunocompromised patients with cerebral toxoplasmosis, the tachyzoite forms rather than cystic and bradyzoite forms of the protozoon are commonly seen. These tachyzoites are minute, scattered among cellular debris, sometimes lodged inside macrophages and neutrophils, and difficult to visualize by light microscopy, even with special stains. Immunodiagnostic tests may be falsely negative due to inability of the host to produce appropriate antibodies. Isolation of the organism is dangerous because Toxoplasma gondii is highly infective. In this situation, transmission electron microscopy (EM) may be a diagnostic tool of choice. It demonstrates the fine definitive features of the protozoon and can be expedited to give results in five hours. Further evaluation of EM for diagnosing possible toxoplasmosis in immunocompromised patients is indicated.

Mixed morphotype susceptibility testing of Pseudomonas aeruginosa from patients with cystic fibrosis.

Disk diffusion antibiograms were determined for mixtures of Pseudomonas aeruginosa morphotypes isolated from the sputum of patients with cystic fibrosis (CF). The results were compared with the predicted susceptibility patterns derived from the antibiograms of individual morphotypes within the mixture. Fifty separate cultures, each yielding two, three, or four distinct morphotypes of Pseudomonas aeruginosa were evaluated. Overall, the correlation between observed and predicted results was 92.2% with only 2.9% of all observations leading to major disagreements in susceptibility. These data suggest that mixed morphotype susceptibility is potentially a useful method to monitor the collective resistance of colonizing strains of Pseudomonas aeruginosa from the respiratory tract of patients with CF.

Antimicrobial activity of merocyanine 540: a photosensitizing dye.

The antimicrobial activity of merocyanine 540 (MC 540), a photosensitizing dye previously used to purge malignant cells from autologous bone marrow grafts, was evaluated against a panel of Gram-positive and Gram-negative bacteria and Candida albicans in the presence and absence of light. In the absence of light, MC 540 demonstrated no antibacterial activity against any of the organisms tested. When combined with increasing intervals of photoillumination, growth inhibition was observed with all Gram-positive organisms tested except Mycobacterium fortuitum. Photosensitizing growth inhibition was also observed with Moraxella catarrhalis but not with any other Gram-negative bacilli including members of the Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophila, or Burkhoderia cepacia. These results suggested that differences in cell wall structure confer resistance to the photodamaging effects of the dye. MC 540 exhibited no antimicrobial activity against C. albicans in the presence or absence of light.

Evaluation of two rapid modifications of the 4-nitrophenyl-beta-D-glucopyranosiduronic acid (PGUA) assay for the identification of Escherichia coli from urine.

Two rapid modifications of a tube assay for the detection of beta-glucuronidase activity (PGUA assay) were evaluated for the identification of Escherichia coli from urine cultures. A microwell and filter paper adaptation of the PGUA assay were tested using 1,234 oxidase-negative, Gram-negative rods isolated from urine on MacConkey agar in clinically significant numbers. There was perfect correlation between both methods and 676 of 797 E. coli isolates were PGUA-positive within 2 h while all of remaining isolates were PGUA-negative (sensitivity = 85%; specificity = 100%). We conclude that either modified format of the PGUA assay provides a useful, inexpensive, and rapid alternative spot test for the definitive identification of E. coli from urine because of the high degree of specificity.

Neisseria elongata subsp. elongata, as a cause of human endocarditis.

Neisseria elongata subsp. elongata, previously considered nonpathogenic, is a potential agent of human endocarditis. We report the second case of human endocarditis caused by this organism. The patient was successfully treated with Ceftriaxone alone for a total of six weeks.

"Campylobacter upsaliensis" sepsis in a boy with acquired hypogammaglobulinemia.

A 16-year-old boy with lupus erythematosus and hypogammaglobulinemia developed bacteremia with "Campylobacter upsalinesis," a recently described Campylobacter species with minimal catalase activity. Because "C. upsaliensis" Gram stains poorly and because it is susceptible to antibiotics in standard selective media, it may be overlooked in routine handling of blood cultures.

Osteomyelitis and septic arthritis caused by Haemophilus influenzae, type f, in a young girl.

A 4-year-old girl with Legg-Calve Perthes' disease and immunoglobin G1 subclass deficiency developed osteomyelitis of the proximal femur and septic arthritis of the hip secondary to Haemophilus influenzae, type f. This microorganism is a rare cause of invasive infections in children, primarily of the central nervous system (CNS) and respiratory track. It has not previously been associated with bone and joint infections.

Propionibacterium avidum as the etiologic agent of splenic abscess.

Propionibacterium avidum was isolated from an intrasplenic abscess in a patient recovering from coronary artery bypass surgery. This organism has not previously been reported as an etiologic agent of splenic abscess nor has splenic abscess been described as a complication of coronary bypass surgery. This report emphasizes the potential pathogenicity of normal microbial flora following surgical manipulation.

Unexpected laboratory diagnosis of latent urogenital coccidioidomycosis in a nonendemic area.

Dissemination of coccidioidomycosis beyond the respiratory tract is not uncommon, but involvement of the urogenital tract is rarely observed. Most, if not all, reported cases of urogenital infections caused by Coccidioides immitis have been diagnosed in areas considered endemic for the fungus, where suspicion of this illness and its extrapulmonary manifestations is high. In nonendemic regions, however, C immitis is not likely to be considered as a possible cause of genitourinary tract infections unless the organism is fortuitously identified by histologic, cultural, or serologic studies. We report the unexpected isolation of C immitis from routine bacterial cultures of a draining urethral-scrotal fistula from a patient residing in a nonendemic area of the United States. We also discuss the difficulties and potential hazards encountered during the laboratory evaluation of this case.