Detecting bacteria in platelet concentrates by use of reagent strips.

BACKGROUND: Iatrogenic infection of immunosuppressed or immunocompromised hosts secondary to receipt of blood components containing bacteria may result in serious adverse outcomes. Measurement of pH and glucose by use of inexpensive reagent strips has been proposed as a practical means of screening for bacteria in platelet concentrate (PC) units. STUDY DESIGN AND METHODS: Glucose and pH were measured in 3093 PC units by use of reagent strips (Multistix, Bayer Corp.) to screen for bacterial content. Any PC classified by the reagent strip method as containing bacteria was subsequently cultured to confirm the presence and quantity of bacteria present. RESULTS: Thirty of 3093 PC units were classified as containing bacteria by the reagent strip method. Two of the 30 PC units positive by the reagent strip method were also positive by standard bacterial culture technique. Bacillus cereus was isolated from both units. CONCLUSION: Screening PC units by the reagent strip method resulted in 9.7 units per 1000 being wasted, but prevented two patients from receiving a PC unit containing B. cereus.

Rapid detection of microorganisms in blood cultures of newborn infants utilizing an automated blood culture system.

BACKGROUND: Neonatal sepsis is a low incidence, high-risk disease with many sepsis work-ups performed to detect a single case. Seventy-two hours of antibiotic therapy have been traditionally recommended pending negative culture results. Improved culture media and new technology integrated into blood culture systems could shorten incubation time required to detect positive culture results. This would then change the length of antibiotic therapy in the management of the newborn infant with suspected sepsis. In addition, previous data supporting the 72-hour recommendation were retrospectively acquired, utilized nonautomated systems, and reported in an era with a different population of microorganisms cultured in special care nurseries. OBJECTIVE: Evaluate the time of incubation to detect positive blood cultures from newborn infants with suspected sepsis using a computer-assisted, automated blood culture system, ESP (Trek Diagnostic Systems, Inc, Westlake, OH). DESIGN: Prospective, observational study. PATIENTS AND SETTING: All positive blood culture results that were obtained from term and preterm newborn infants born from November 1993 through June 1997 at a publicly funded hospital with over 6000 live births per year. METHODS: As positive blood culture results were identified, data were prospectively obtained from the patient's medical record. The computer algorithm in the automated blood culture system determined the time to positivity. Time to positivity was determined for blood cultures obtained before the initiation antimicrobial therapy and compared with those cultures obtained after beginning therapy. Time to positivity was also evaluated for clinically important Gram-positive and Gram-negative bacteria and yeast. RESULTS: Four hundred fifty-five positive blood culture results were obtained from 222 patients. Gram-positive organisms accounted for 80% (366/455) of the positive culture results, Gram-negative organisms accounted for 11% (48/455), and yeast for 9% (41/455). Virtually all cultures growing clinically significant Gram-positive and Gram-negative organisms were positive by 24 to 36 hours of incubation. Cultures growing Staphylococcus epidermidis were virtually all positive after 36 to 48 hours of incubation. Of cultures growing yeast, 88% (36/41) were positive by 48 hours of incubation. There was no difference in time to positivity in pretherapy or posttherapy obtained positive blood cultures. Prenatally administered antibiotics did not affect time to positivity in positive cultures drawn on the first day of life. In a selected group of microorganisms that are the frequent cause of bacteremia in term infants, 97% and 99% of cultures were positive by 24 to 36 hours of incubation when only pretherapy cultures are evaluated. CONCLUSIONS: The ESP blood culture system identified 77%, 89% and 94% of all microorganisms at 24, 36, and 48 hours of incubation in aerobic cultures obtained from both term and preterm infants. Introduction of antimicrobial therapy did not affect time to positivity. Reducing duration of antibiotic therapy to 24 to 36 hours should be considered in term, asymptomatic newborn infants undergoing evaluation for suspected sepsis for maternal indications. Confirmation of similar rapidity of detection using other blood culture systems should be undertaken.

Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis.

Ethambutol (EMB) is a central component of drug regimens used worldwide for the treatment of tuberculosis. To gain insight into the molecular genetic basis of EMB resistance, approximately 2 Mb of five chromosomal regions with 12 genes in 75 epidemiologically unassociated EMB-resistant and 33 EMB-susceptible Mycobacterium tuberculosis strains isolated from human patients were sequenced. Seventy-six percent of EMB-resistant organisms had an amino acid replacement or other molecular change not found in EMB-susceptible strains. Thirty-eight (51%) EMB-resistant isolates had a resistance-associated mutation in only 1 of the 12 genes sequenced. Nineteen EMB-resistant isolates had resistance-associated nucleotide changes that conferred amino acid replacements or upstream potential regulatory region mutations in two or more genes. Most isolates (68%) with resistance-associated mutations in a single gene had nucleotide changes in embB, a gene encoding an arabinosyltransferase involved in cell wall biosynthesis. The majority of these mutations resulted in amino acid replacements at position 306 or 406 of EmbB. Resistance-associated mutations were also identified in several genes recently shown to be upregulated in response to exposure of M. tuberculosis to EMB in vitro, including genes in the iniA operon. Approximately one-fourth of the organisms studied lacked mutations inferred to participate in EMB resistance, a result indicating that one or more genes that mediate resistance to this drug remain to be discovered. Taken together, the results indicate that there are multiple molecular pathways to the EMB resistance phenotype.

Cross-modal transfer of shape is difficult to demonstrate in one-month-olds.

We tested 1-month-olds for cross-modal transfer of shape between touch and vision using a procedure described by Meltzoff and Borton, but including controls for side bias and stimulus preference. In Experiment 1 (N = 48), infants' looking times to smooth and nubby visual stimuli were not influenced by previous oral exposure to one of the shapes during the preceding 90 s, except for an effect on the first test trial in one group; this effect could have been due to limited cross-modal transfer, to Type 1 error, or to side bias, possibly interacting with a small stimulus preference. The failure of that effect to replicate in a group (N = 16) with less side bias (Experiment 2) suggests that it was not due to cross-modal transfer. Experiment 3 (N = 32), an exact replication of Meltzoff and Borton's experiment, also failed to yield evidence of cross-modal transfer. Overall, there is not good evidence that 1-month-olds can transfer information about these shapes from touch to vision. Future studies exploring the ability to transfer information about other shapes will be easier to interpret if they include controls for side bias and stimulus preference.

Intensity of infection in AIDS-related intestinal microsporidiosis.

To quantify intensity of infection in AIDS-related microsporidiosis, 20 patients with known microsporidiosis submitted stools for quantitative spore counts after staining with a calcofluor white stain. Nine patients collected stools for 24 h, for assessment of daily spore excretion, stool-to-stool variation in spore excretion, and patient-to-patient variation in intensity of infection. The number of organisms seen in small bowel biopsy specimens from 7 patients was compared with quantitative fecal spore excretion. Fecal spore concentration in 20 patients ranged from 4.5x105 to 4.4x108 spores/mL of stool. There was a strong correlation between fecal spore excretion and duodenal biopsy spore counts (r=.82; P<.024). Microsporidium infections in AIDS patients can be quantified by counting spores in stool and by small bowel biopsy. Variations in intensity of infection from patient to patient are great and are similar to those in AIDS-related Cryptosporidium infection.

Acquisition of word-object associations by 14-month-old infants.

The following experiments were designed to determine the age at which infants can first readily learn word--object pairings with only minimal exposure and without social or contextual support. To address this question, 8- to 14-month-old infants were tested on their ability to form word--object associations in a "switch" design. Infants were habituated to 2 word--object pairings and then tested with 1 trial that maintained a familiar word--object pairing and 1 that involved a familiar word and object in a new combination. Across 6 experiments, only 14-month-old infants formed word--object associations under these controlled testing conditions but appeared to do so only when the objects were moving. Although 8- to 12-month-olds did not form the associations, they appeared to process both the word and the object information. These studies provide strong evidence that 14-month-old infants can rapidly learn arbitrary associations between words and objects, that this ability appears to develop at about 14 months of age, and that the Switch design is a useful method for assessing word--object learning in infancy.

Effects of requiring prior authorization for selected antimicrobials: expenditures, susceptibilities, and clinical outcomes.

Antimicrobial control programs are widely used to decrease drug expenditures, but effects on antimicrobial resistance and outcomes for patients are unknown. When a requirement for prior authorization for selected parenteral antimicrobial agents was initiated at our urban, county teaching hospital, total parenteral antimicrobial expenditures decreased by 32%. Susceptibilities to all beta-lactam and quinolone antibiotics increased, with dramatic increased susceptibilities in isolates recovered in intensive care units, increased susceptibilities in isolates recovered in other inpatient sites, and little change in susceptibilities in isolates recovered in outpatient sites despite no change in infection control practices. For patients with bacteremia due to gram-negative organisms, overall survival did not change with restrictions. No differences occurred in the median time from initial positive blood culture to receipt of an appropriate antibiotic or in the median time from positive blood culture to discharge from the hospital. Thus, requiring preapproval for selected parenteral agents can decrease antimicrobial expenditures and improve susceptibilities to antibiotics without compromising patient outcomes or length of hospital stay.

Infants listen for more phonetic detail in speech perception than in word-learning tasks.

Infants aged 4-6 months discriminate the fine phonetic differences that distinguish syllables in both their native and unfamiliar languages, but by 10-12 months their perceptual sensitivities are reorganized so that they discriminate only the phonetic variations that are used to distinguish meaning in their native language. It would seem, then, that infants apply their well honed phonetic sensitivities as they advance and begin to associate words with objects, but the question of how speech perception sensitivities are used in early word learning has not yet been answered. Here we use a recently developed technique to show that when they are required to pair words with objects, infants of 14 months fail to use the fine phonetic detail they detect in syllable discrimination tasks. In contrast, infants of 8 months--who are not yet readily learning words--successfully discriminate phonetic detail in the same task in which infants aged 14 months fail. Taken together, these results suggest a second reorganization in infants's use of phonetic detail as they move from listening to syllables to learning words.

Detection of Pneumocystis carinii in tracheal aspirates of intubated patients using calcofluor-white (Fungi-Fluor) and immunofluorescence antibody (Genetic Systems) stains.

OBJECTIVE: To compare the detection rate of Pneumocystis carinii in endotracheal aspirates with that rate in bronchoalveolar lavage fluid, using calcofluor-white (Fungi-Fluor) and immunofluorescence antibody (Genetic Systems) staining methods. DESIGN: Prospective, consecutive cases. SETTING: Medical intensive care unit at Ben Taub General Hospital. PATIENTS: Thirty-one intubated patients admitted with respiratory failure and suspected P. carinii pneumonia. INTERVENTIONS: An endotracheal aspirate specimen was obtained after maximally advancing a closed-system suction catheter, instilling aliquot portions of saline, and suctioning the lavage fluid. This procedure was followed within 30 mins by fiberoptic bronchoscopy and bronchoalveolar lavage. MEASUREMENTS AND MAIN RESULTS: Endotracheal aspirate and bronchoalveolar lavage specimens from each patient were mixed with Saccomano's fixative, blended, and centrifuged. Using a modified method for P. carinii cysts, the sediment was stained with the test calcofluor-white stain Solution A and the test antibody stain. The test antibody stain on the bronchoalveolar lavage specimens was positive for P. carinii for 13 patients and was used as the standard for comparison. In the endotracheal aspirate specimens, the test antibody stain detected 12 (92%) P. carinii-positive patients while the test calcofluor-white stain detected ten (77%) P. carinii-positive patients. CONCLUSIONS: We described a simple method for obtaining, processing, and staining endotracheal aspirate specimens for P. carinii. Obtaining an endotracheal aspirate specimen did not require specially trained personnel or a specialized and more expensive catheter, and was not associated with any complications.

A rapid and inexpensive method for processing induced sputum for detection of Pneumocystis carinii.

The authors describe a method to process induced sputum specimens for detection of Pneumocystis carinii which is simple, rapid and inexpensive. Induced sputum and bronchoalveolar lavage (BAL) were obtained within a 24-hour period from 41 patients who were HIV-positive and had pulmonary symptoms suspicious for P carinii pneumonia. Induced sputum or BAL fluid was placed into Saccomanno's fixative, blended, and centrifuged. The sediment was stained for P carinii cysts by a modified method with Fungi-Fluor Solution A (Polysciences, Warington, PA) and the Genetic Systems Pneumocystis carinii Immunofluorescence Antibody (Genetic Systems, Seattle, WA). The Genetic Systems stain on the BAL specimen was positive in 35 patients and was the standard for comparison. With a single induced sputum, the Genetic Systems stain detected 31 (89%) positive patients, whereas the Fungi-Fluor stain detected 21 (60%). The sensitivity for detecting P carinii cysts in induced sputum was significantly greater (P < 0.05) for the Genetic Systems stain.

Rapid identification of common human pathogens by high-resolution proton magnetic resonance spectroscopy.

Routine procedures for recovery of bacteria from clinical specimens involve culturing the latter on various nonselective and selective agar media. The bacteria are then identified by means of biochemical and immunological test procedures. Reduction of the time required to identify the bacteria is highly desirable for rapid clinical diagnosis. Towards this end the potential of proton nuclear magnetic resonance (NMR) spectroscopy for providing a "fingerprint" within the proton spectrum of five bacterial genera, reflecting their characteristic cell wall constituents, has been investigated. Establishing a database of high-resolution proton NMR spectra of a large number of bacterial species is a prerequisite for attaining this objective. A database has been established for five common human pathogens: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. On the basis of the presence of characteristic resonances in their spectra, a simple algorithm has been developed to differentiate and identify these microorganisms. The NMR spectra of E. coli and S. aureus showed no dependency on the type of growth medium, growth density, or incubation time.

Modification of the fungi-fluor and the genetic systems fluorescent antibody methods for detection of Pneumocystis carinii in bronchoalveolar lavage specimens.

Four hundred forty-five bronchoalveolar lavage specimens from patients with the human immunodeficiency virus were preserved in Saccomano's fixative and stained for Pneumocystis carinii cysts by a modified method with Fungi-Fluor Solution A (Polysciences, Warrington, Pa) and the Genetic Systems Pneumocystis carinii Immunofluorescence Antibody (Genetic Systems Corp, Seattle, Wash). The majority of patients had been treated for suspected P carinii pneumonia for a few days prior to collection of bronchoalveolar lavage specimens. P carinii cysts were detected in 194 (43.6%) specimens. Both stains identified P carinii in 166 (37.3%) specimens and were negative in 251 (56.4%), yielding a concordance rate of 93.7%. P carinii cysts were detected in 25 (5.6%) specimens by the Genetic Systems stain only, and in 3 (0.7%) specimens by the Fungi-Fluor stain only. The sensitivity for detecting cysts of P carinii was significantly greater with the Genetic Systems stain (P < .01).

Visual alignment from the midline: a declining developmental trend in normal, strabismic, and monocularly enucleated children.

Children, 1.8 to 5.0 years of age, were asked to sight through a tube at targets. There were three groups tested: children with normal binocular vision, children with strabismus, and children with one eye enucleated. The younger normal and strabismic patients placed the tube midway between the two eyes. Surprisingly, the younger enucleated children also placed the tube at the midline. This "Cyclops effect" diminished as the children grew older, with a transition to sighting monocularly by the age of 4 years. The tendency to align with the midline by the younger children, regardless of the degree of their binocular vision, presumably is a natural response to a cyclopean projection center in the midline. As children mature, they learn to meet the demands of monocular preference tasks by aligning objects in front of one eye.

Automated systems for identification of microorganisms.

Automated instruments for the identification of microorganisms were introduced into clinical microbiology laboratories in the 1970s. During the past two decades, the capabilities and performance characteristics of automated identification systems have steadily progressed and improved. This article explores the development of the various automated identification systems available in the United States and reviews their performance for identification of microorganisms. Observations regarding deficiencies and suggested improvements for these systems are provided.

Pulmonary coccidioidal pseudomycetoma.

Coccidiomycosis is rarely associated with a pulmonary mycetoma. We report a patient with progressive cavitary coccidiomycosis, whose initial radiographic and clinical appearance simulated a mycetoma. Examination of the surgically resected lung showed necrotizing Coccidioides immitis granulomas with spherules and arthroconidialike structures, but no evidence of a mycetoma. We propose the term pulmonary coccidioidal pseudomycetoma as the best descriptor for this patient's clinical, radiographic, pathologic, and microbiologic presentation.

Role of solid media when used in conjunction with the BACTEC system for mycobacterial isolation and identification.

This study evaluated the necessity and the contribution of solid media when used in conjunction with radiometric Middlebrook 7H12 (BACTEC 12B; Becton Dickinson, Towson, Md.) medium for recovery and complete identification of mycobacteria. Each of 1,184 digested, decontaminated respiratory specimens was inoculated into one BACTEC 12B vial, one 7H11 plate, and two Lowenstein-Jensen (LJ) slants. When the 12B vial was smear positive for acid-fast bacilli, the organisms were subcultured onto LJ slants and the BACTEC p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test system was inoculated with the organisms. Niacin tests were performed by using the growth from the original LJ slants and organisms from the LJ slants subcultured from 12B or 7H11 medium. The times to achieve definitive NAP and niacin test results were recorded. Recovery of all 143 isolates found in this study could not be achieved with a single medium. Among the three media, the highest percentage (92.8%) of Mycobacterium tuberculosis isolate recovered was with BACTEC 12B. The use of either 7H11 medium or LJ slants along with a 12B vial increased by 4 to 6% the total percentage of M. tuberculosis organisms that were isolated. Isolation of the M. tuberculosis complex and NAP differentiation in 12B medium were completed in an average of 17 days. On average, isolation and definitive niacin test results for M. tuberculosis cultures were obtained in 39.3 days by a conventional procedure and in 36.3 days when 12B subcultures were used. These results support the conclusion that LJ slants contribute 4 to 6% increased recovery of M. tuberculosis when used in conjunction with 12B medium. Additionally, a subculture onto LJ slants from 12B medium yielded sufficient growth for niacin testing earlier than an original LJ slant did.

Evaluation of the FiltraCheck-UTI for detection of bacteriuria.

The FiltraCheck-UT1 (FC) is a disposable colorimetric urine screen for bacteriuria that requires less than 1 min to perform. The results of the FC and two other urine screens, the Bac-T-Screen (BTS) and the Chemstrip LN test strip (LN), were compared with quantitative culture method. A total of 551 urine specimens were tested. The sensitivity of FC, BTS, and LN for probable pathogens at greater than or equal to 10(5) CFU/ml was 94.8%, 97.4%, and 77.9% respectively. These values for specimens with probable pathogens at greater than or equal to 10(4) CFU/ml were 91.1%, 92.1%, and 74.3%, respectively. When the LN was combined with the FC or BTS as a urine screen, the sensitivity for probable pathogens was improved.

Rapid presumptive identification of gram-negative rods directly from blood cultures by simple enzymatic tests.

Gram-negative rods were presumptively identified directly from blood cultures within 15 min as Escherichia coli, a member of the Klebsiella-Enterobacter group, or oxidase positive. Samples of artificially seeded blood cultures (193 cultures) and patient blood cultures (78 cultures) were filtered into a Dynadepth test card with the Bac-T-Screen instrument (Vitek, Inc., Hazelwood, Mo.). Triton X-100 was then filtered into the test card to lyse the blood cells but not the entrapped bacteria, and either methylumbelliferone-labeled substrates or oxidase reagent was applied to the filter surface. The oxidase test was read within 30 s, and the methylumbelliferone and indole tests were read after a 10-min incubation at room temperature. Positive beta-galactosidase, beta-glucuronidase, and indole test results predicted the identification of E. coli with a 96 to 100% sensitivity and a 99 to 100% specificity. Positive beta-xylosidase and beta-galactosidase test results and negative oxidase and beta-glucuronidase test results were 85 to 93% sensitive and 100% specific for a Klebsiella-Enterobacter organism. A positive oxidase test result and negative beta-glucuronidase, beta-xylosidase, and indole test results were highly predictive of Pseudomonas aeruginosa (sensitivity, 100%; specificity, 99%). The procedures described are rapid and simple and provide a direct presumptive identification of the gram-negative rods most commonly found in blood cultures.

Identification of Mycobacterium tuberculosis in sputum by direct inoculation of the BACTEC NAP vial.

The Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, and M. africanum) can be differentiated from mycobacteria other than M. tuberculosis (MOTT bacilli) with the BACTEC NAP test (Johnston Laboratories, Becton, Dickinson & Co., Towson, MD), by selectively inhibiting their growth with p-nitro-alpha-acetyl-amino-beta-hydroxypropriophenone (NAP). The BACTEC NAP test is recommended for isolated mycobacterial cultures. In this report, a direct NAP test is performed by immediate inoculation of BACTEC NAP vials with processed sputum specimens that stain acid-fast positive. Seventy-six of 80 M. tuberculosis were correctly identified and all of the MOTT bacilli (nine M. avium complex and one M. kanasii) were correctly classified. The average time required for identification of M. tuberculosis with the direct BACTEC NAP test described here is more convenient than the recommended indirect test, and it is an accurate and rapid method to differentiate the M. tuberculosis complex from MOTT bacilli.