Relationship of Multiplex Molecular Pneumonia Panel Results With Hospital Outcomes and Clinical Variables.

BACKGROUND: Antibiotic treatment decisions in severely ill patients must often be made in the absence of microbiologic results. The recently Food and Drug Administration-cleared BioFire FilmArray Pneumonia Panel (PN) detects 15 bacteria semiquantitatively, 3 atypical pneumonia bacteria, 8 viruses, and 7 antimicrobial resistance markers by multiplex PCR in ~1 hour in the laboratory. Previous reports have shown that the PN Panel bacterial detections are highly accurate, even when routine culture had no growth. METHODS: Consecutive bronchoalveolar lavage and endotracheal specimens submitted for culture between June and September 2018 from 270 patients with sufficient clinical and laboratory data were tested with the PN Panel. Patients were divided into 3 groups: (1) both culture and PN Panel positive, (2) PN Panel positive but culture uninformative (no growth or normal flora), and (3) patients with no PN Panel detections. RESULTS: Groups 1 and 2 had significantly higher maximum temperatures on the day of culture (P = .00036, analysis of variance [ANOVA] with Bonferroni correction), higher levels of an inflammatory response as measured by percent polymorphonuclear leukocytes in bronchoalveolar lavage (P = .00025, ANOVA with Bonferroni correction), and gram stain report of white blood cells, as previously reported [1]. CONCLUSIONS: Both group 1 (culture and PN Panel positive), and group 2 (PN Panel positive but culture uninformative) had higher levels of host response inflammatory responses compared with group 3, which had no targets detected, suggesting that PN Panel detections need to be interpreted in the clinical context, even if cultures are discordant. Depending on laboratory turnaround time, there could be opportunities for improved diagnosis and antibiotic stewardship.

Improved methods for the application of random peptide phage libraries to the study of the oligoclonal bands in cerebrospinal fluid of patients with multiple sclerosis.

The target antigens of the oligoclonal bands in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) are unknown but may reflect important autoantigens in MS. One approach to identify candidate antigens is to allow CSF to select peptide motifs from a random phage library. To determine whether selected peptide motifs are related to the pathogenesis of MS, it is important to know if other MS patients and appropriate control patients have antibodies reactive with these sequences either in CSF or sera. Unfortunately, serologic screening of such sequences directly in phage clones gave non-specific reactions. Western blotting was found to obviate the non-specificity problem and together with isoelectric focusing, could also be used to demonstrate the co-migration of antigen specific oligoclonal bands with individual total IgG bands. Using 2D gel electrophoresis, absorption of CSF antibodies by specific peptide sequences selected from the phage library could be demonstrated. These techniques should facilitate the systematic study of the targets of the oligoclonal bands in CSF of patients with MS.

Epstein-Barr virus nuclear antigen-1 (EBNA-1) associated oligoclonal bands in patients with multiple sclerosis.

Oligoclonal bands (OCBs) are frequently observed in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), but the target antigens of these antibodies remain unknown. We used antigen specific immunoblotting to determine whether Epstein Barr virus nuclear antigen-1 (EBNA-1) was a target of the OCBs in the CSF of patients with MS. Antibody indices (AIs) were measured by ELISA and calculated by the formula of Reiber and Lange which includes correction factors for both breakdown of the blood brain barrier and intrathecal polyclonal IgG synthesis. A distinctive oligoclonal antigen specific banding pattern for EBNA-1 was observed in 5/15 MS patients, but 0/12 controls (P=0.037, Fisher's Exact Probability). AIs in this EBNA-l positive subgroup were extremely high, comparable with levels observed in viral CNS infections. In one patient with EBNA-1 specific OCBs, EBNA-1 and a peptide 'equivalent', p62, were able to absorb a component of the total IgG. Our results suggest that in a subset of MS patients, EBNA-1 may be a major target of selected OCBs.

The potential for dissemination of Mycobacterium tuberculosis through the anesthesia breathing circuit.

BACKGROUND: Respiratory pathogens that pass through the anesthesia breathing system potentially can infect other patients. This study was designed to determine if bacteria can pass through contemporary anesthesia breathing systems and if the environment within the machine is hostile to these organisms. METHODS: Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis were nebulized into the expiratory limb of an anesthesia breathing circuit and collected from the inspiratory and expiratory limbs in an impinger system that provided a quantitative determination of the number of organisms entering the circuit and the number that would reach the patient in the inspiratory gas. Bacteria were collected before, during, and after nebulization. A second experiment determined if a saturated solution of soda lime was bactericidal. RESULTS: When the gas flow through the circuit was interrupted for < 1 h following the nebulization period, large numbers of microorganisms (1 x 10(3) to 1 x 10(5), around 100% of the nebulized organisms) were collected from the inspiratory gas. Soda lime itself was not bactericidal for any of the organisms tested, but solutions of this material with a pH of 12 were bactericidal. CONCLUSION: Cross contamination between patients may occur unless the gas flow through the anesthesia breathing system is interrupted for > 1 h.

A comparison of ligase chain reaction to polymerase chain reaction in the detection of Chlamydia trachomatis endocervical infections.

OBJECTIVE: To compare the reliability of ligase chain reaction (LCR) to polymerase chain reaction (PCR) in detecting Chlamydia trachomatis endocervical infections. METHODS: We conducted a prospective study of 486 patients at risk for chlamydial infection of the endocervix. We obtained two endocervical specimens from each patient and used LCR and PCR to detect C. trachomatis. Discrepant results between the two techniques were resolved by repeat testing and by testing for the major outer membrane protein (MOMP) gene, if necessary. We determined the sensitivity, specificity, positive predictive value, and negative predictive value for each test, using concordant results or MOMP gene results as the "gold standard". RESULTS: Of the 486 patients, 42 (8.6%) had evidence of C. trachomatis infection after resolution of discrepant results. Of the 42 true positive specimens, 41 were positive by initial LCR and 38 were positive by initial PCR. Of the 444 true negative specimens, none had a positive initial LCR result, while 2 had a positive initial PCR test. Therefore, compared to the gold standard, LCR had a sensitivity of 97.6% and specificity of 100%, while PCR had a sensitivity of 90% and a specificity of 99.5%. The positive and negative predictive values of LCR were 100% and 99.8%, respectively. PCR had a positive predictive value of 95% and a negative predictive value of 99.1%. The difference in sensitivity of LCR versus PCR was not statistically significant (P = .125). CONCLUSION: LCR and PCR perform equally well in detecting C. trachomatis endocervical infections.

Molecular approach to find target(s) for oligoclonal bands in multiple sclerosis.

OBJECTIVES: Oligoclonal bands are a characteristic finding in the CSF of patients with multiple sclerosis, yet their target antigen(s) remain unknown. The objective was to determine whether a filamentous phage peptide library could be employed to allow the oligoclonal bands to select their own target epitopes. METHODS: CSF IgG antibody from 14 patients with multiple sclerosis and 14 controls was used to select individual phage clones from a bacteriophage library containing approximately 4 x 10(7) different hexamers expressed on its surface pIII protein. The amino acid sequence selected was deduced by sequencing the DNA of the genetically engineered insert. RESULTS: In general, after three rounds of selection, CSF from both patients with multiple sclerosis and controls selected one to two consistent peptide motifs. Five out of 14 patients with multiple sclerosis, and one control, selected the amino acid sequence motif, RRPFF. Given 20 possible amino acids per position, the likelihood of five patients selecting the same linear five amino acid sequence is at most 1.6 x 10(-3), corrected for the number of clones sequenced. A GenBank computer search showed that this sequence is found in the Epstein-Barr Virus nuclear antigen (EBNA-1), and a heat shock protein alphaB crystallin. Human serum antibodies to a synthetic peptide containing RRPFF were virtually exclusively found in patients with prior infection by Epstein-Barr virus. Other studies have suggested a relation between Epstein-Barr virus infection and multiple sclerosis, including nearly 100% Epstein-Barr virus seropositivity among patients with multiple sclerosis and increased concentrations of antibody to EBNA in CSF of patients with multiple sclerosis. By antigen specific immunoblotting, antibodies to the RRPFF motif in the CSF were shown to correspond to a subset of oligoclonal bands in the CSF from the same patient. CONCLUSION: This study shows that phage epitope display libraries may be used to select amino acid motifs which are potentially relevant to the pathogenesis of multiple sclerosis.

Rejection criteria for endotracheal aspirates in neonates. Clinical impact.

It has been suggested that endotracheal suction specimens from adult and pediatric patients be rejected for culture if no organisms are seen on Gram's stain. We reviewed the experience in neonates under three months of age from our neonatal intensive care unit. Of 195 such endotracheal specimens, 86 (44%) had no growth by culture, 83 (43%) grew normal flora only, and 26 (13%) from 22 patients grew a potential pathogen. Detailed chart review was undertaken to determine what the clinical outcome would have been if the culture information were not provided for these patients. Based on this analysis, almost all patients with Gram-negative rods would have empirically received antibiotics on clinical grounds regardless of culture results, and at least one received antibiotics unnecessarily based on a culture result. On the other hand, two patients whose cultures grew Trichosporon and Aspergillus would have been missed.

Determination of free extracellular concentrations of piperacillin by microdialysis.

The tissue penetration and distribution of antibiotics is of great importance, since most of the infections occur in the tissue. At the infection site, the free, unbound fraction of the antibiotic is responsible for the antiinfective effect. These free extracellular concentrations can be measured by microdialysis. It was the aim of the study to correlate free levels of the beta-lactam antibiotic piperacillin in blood with those in tissue. In vivo microdialysis sampling was used to study the tissue distribution patterns of piperacillin in anesthetized rats after single dose iv administration of the drug. The pharmacokinetics of piperacillin in plasma were consistent with a two-compartment body model. Comparisons between calculated free concentrations in the peripheral compartment and measured free extracellular concentrations revealed excellent agreement. Microdialysis is a suitable method to evaluate unbound drug concentrations in the tissues. In case of piperacillin, predictions of the concentration time profiles of free drug in the peripheral compartment can be made on the basis of plasma data.

Pharmacokinetic-pharmacodynamic modeling of the antibiotic effect of piperacillin in vitro.

PURPOSE: It was the aim of the present study to investigate the in vitro antimicrobial effects of the beta-lactam antibiotic piperacillin on Escherichia coli using concentration-time profiles similar to those encountered in vivo. METHODS: An in vitro dilution model was used to expose E. coli to various piperacillin concentration profiles. The antimicrobial effect was evaluated by determination of the number of bacteria over time. RESULTS: A modified Emax-model was found appropriate to describe the pharmacodynamic effect. This model was linked with the respective piperacillin concentrations to provide a suitable pharmacokinetic-pharmacodynamic (PK-PD) model. The average growth half-life in absence of piperacillin was 28 min and the maximum kill half-life was 25 min. The EC50 for the various dosing regimens averaged 5.2 micrograms/mL and was independent of dose. These parameters were used the simulate the bacterial effects of commonly administered doses or dosing regimens in humans. CONCLUSIONS: Based on the in vitro data a more frequent administration of piperacillin will be more efficacious. The proposed PK-PD-model allows a more detailed evaluation of dosing regimens than the use of minimum inhibitory concentrations.

Concentration-dependent synergy and antagonism between cefoperazone and imipenem against methicillin-resistant Staphylococcus aureus.

By the agar dilution checkerboard method, striking synergy between cefoperazone and imipenem was observed with 32 strains of methicillin-resistant Staphylococcus aureus (fractional inhibitory concentration indices, < or = 0.03 to 0.34; mean, 0.12 +/- 0.08). By the double-disk diffusion method, the synergy was confirmed, but about 60% of strains showed antagonism manifest by truncation of the zone of inhibition around cefoperazone. The mechanism may involve auxiliary factors distinct from those essential for the expression of methicillin resistance.

Detection of candidemia by polymerase chain reaction.

Current blood culture methods may not identify all patients with candidemia. We attempted to develop a polymerase chain reaction (PCR)-based method for the detection of candidemia. Several major problems were encountered: use of primers based on single copy or low-copy-number genes lacked sensitivity, while those based on the 18S ribosomal RNA gene often crossreacted with human DNA. One primer set with an internal primer was found to be suitable in a hemi-nested PCR. Sensitivity was in the range of 1 colony forming unit of C. albicans per ml blood in reconstruction experiments. Using stored blood which had been obtained within 12 h of the time a positive blood culture was obtained, PCR was positive in 7/15 (46.7%) patients with culture proven C. albicans candidemia (44% of samples). However, only 1-3 ml blood was available for PCR, while culture results were based on 5-10 ml or more. Two out of 34 (5.9%) unselected outpatient blood samples stored under identical conditions were positive, but both samples were negative when the original DNA was retested. Blood collection and PCR processing equipment appears to be free of contaminating fungal DNA and organisms; however, human skin contains DNA amplifiable by these primers. At present the major limitation is the need for a simple method to recover Candida from 5-10 ml blood while removing haemoglobin and excess white blood cell DNA in a volume suitable for PCR.

Relationship of the polymerase chain reaction for cytomegalovirus to the development of hepatitis in liver transplant recipients.

In a pilot study, the polymerase chain reaction was found to be more sensitive than standard viral culture methods for the detection of cytomegalovirus, particularly from blood and tissues. We therefore applied this technique to 71 serially collected liver biopsies from 16 orthotopic liver transplant patients. All patients were CMV-seropositive (n = 15) or seroconverted (n = 1). Seven patients (9 biopsies) had histologically proved CMV hepatitis, and all these biopsies were CMV PCR-positive. Six of these 7 patients had a prior liver biopsy that was CMV PCR-positive, but culture and histology-negative, an average of 13.2 +/- 6.9 days before the histologically positive biopsy. The 7th patient was not biopsied prior to the diagnostic biopsy. Three patients had 7 liver biopsies that were CMV PCR-positive, but histologically negative for CMV hepatitis. Two of these three had CMV infection confirmed by viral culture of blood or liver biopsy. The remaining 6 patients had a total of 26 liver biopsies that were negative for CMV by PCR, culture, and histology. Among liver transplant patients, CMV PCR performed on liver biopsy specimens correctly identified all histologically proven cases of CMV hepatitis. CMV PCR positivity in liver tissue did not correlate with latent infection and preceded the development of CMV hepatitis or other meaningful CMV infection in 8 of 10 patients.

Reproducibility of the microdilution checkerboard method for antibiotic synergy.

We assessed the reproducibility of the microdilution checkerboard method for measuring antibiotic synergy. Five strains of Pseudomonas aeruginosa were tested with four antibiotic combinations by using 10 replicates each. Twenty-five percent of replicate sets gave discordant classification results (i.e., a 7:3 or worse split in categorization). Determination of the individual MICs of each antibiotic alone was excellent; all 10 replicates were within 1 twofold dilution for 95% of the 80 sets of 10 replicates. The microdilution checkerboard method either should not be used or should be used with at least five replicates per determination, with > or = 80% agreement among the replicates required for classification.

Vancomycin is not an essential component of the initial empiric treatment regimen for febrile neutropenic patients receiving ceftazidime: a randomized prospective study.

The use of vancomycin as part of the initial antibiotic therapy of febrile neutropenic patients has become a controversial issue. Some studies support its incorporation in the initial regimen, and others suggest that vancomycin can be added later. We examined this issue in a prospective, randomized trial. We randomized 127 febrile neutropenic patients to receive either ceftazidime alone or ceftazidime plus vancomycin as the initial empiric antibiotic treatment. We added vancomycin to the ceftazidime arm of the study when fever persisted after 96 h of monotherapy, when new fever occurred after this time, or when a moderately ceftazidime-resistant gram-positive bacterium was isolated. Each of these regimens had similar initial response rates, similar durations of initial fever, similar frequencies of new fever during therapy, similar microbiological cure rates, similar superinfection rates, and similar survival rates. We observed more renal and cutaneous toxicities in patients receiving vancomycin and ceftazidime as initial therapy. We conclude that ceftazidime is appropriate as initial therapy for febrile neutropenic patients and that the addition of vancomycin is appropriate when fever persists after 4 days of monotherapy or when fever recurs following an initial response.

Quantitative tip culture methods and the diagnosis of central venous catheter-related infections.

The diagnostic usefulness of two quantitative catheter culture methods was compared in a prospective study of central venous arterial catheters. The roll-plate method followed by sonication was used to culture 177 catheters from 85 patients, and the sonication method was used to culture 136 catheters from 68 patients. All patients were evaluated for catheter-related infections. Catheter-related infections were associated with greater than or equal to 100 colony-forming units (CFU) isolated from catheter tips by either roll plate (p = 0.01) or sonication (p less than 0.001). The sensitivity, specificity, and positive and negative predictive values of greater than or equal to 10(3) CFU by roll plate for catheter-related septicemia were 56%, 97%, 63%, and 96% compared with 93%, 95%, 76%, and 99%, respectively, for the same level by sonication. For central venous and arterial catheters, the sonication method can distinguish infection from contamination and is superior to the roll-plate method in that it may offer a more sensitive and predictive alternative in the diagnosis of catheter-related septicemia.

Pharmacokinetics of intravenous immunoglobulin (Gammagard) in bone marrow transplant patients.

The pharmacokinetics of an intravenous immunoglobulin (IVIG), Gammagard (Baxter Healthcare Corp., Glendale, CA), were measured in 31 cytomegalovirus (CMV) antibody negative bone marrow transplant (BMT) patients as part of a multicenter efficacy trial of 2 weekly dose regimens. Since all patients lacked antibody to CMV and received only screened CMV negative blood products, the half-life of the exogenous CMV antibody could be measured with an ELISA assay. The CMV antibody titer was related to the immunoglobulin concentration using a standard curve. Compared with the 22-day half-life in normal subjects, the half-life in BMT patients was approximately 6 days for either the 250 mg/kg or 500 mg/kg dose regimen. The half-life did not change over the subsequent 3 weekly doses. Peak concentrations were 3.5 +/- 1.4 and 2.6 +/- 0.7 mg/mL of IVIG in week 1 as well as 5.5 +/- 2.6 and 3.4 +/- 1.2 mg/mL in week 3 after the 250 mg/kg and 500 mg/kg, respectively. Total body clearance of IVIG was 0.61 and 0.46 mL/kg/hr for the 500 mg/kg and 250 mg/kg, respectively.

Multicenter study of a single 500-mg dose of cefotaxime for treatment of uncomplicated gonorrhea.

One hundred thirty-seven evaluable patients with uncomplicated gonorrhea were treated with a single 500-mg intramuscular dose of cefotaxime. All isolates were susceptible to concentrations of cefotaxime less than or equal to 0.1 microgram/ml. The minimum concentration of cefotaxime needed to inhibit 90% of isolates was less than 0.04 microgram/ml. At follow-up, infection was eradicated in 181 of 187 (97%) infection sites. Bacteriologic cures of 100 of 101 (99%), 55 of 56 (98%), 23 of 25 (92%), and 3 of 5 (60%) were attained at the urethral, endocervical, rectal, and oropharyngeal sites, respectively. Side effects were minor, and 90% of patients rated the injection-site pain as absent or mild. A single 500-mg dose of cefotaxime is an effective, economic treatment for uncomplicated gonorrhea.

A psycho-behavioral model of genital herpes recurrence.

To develop a model of how stress and other psychosocial constructs may interact to explain recurrences of genital herpes, assessments of major and minor life stress, locus of control, arousal or stimulation seeking, and social support were given to 153 university students (33% male; 67% female) who were seropositive for genital herpes. Retrospective and concurrent indices of illness vulnerability were evaluated. Serum levels of thymosin-alpha-1, a peptide sensitive to psychosocial stress, were measured at the beginning of the study. A causal model suggested by previous research was not supported by the data. An alternate model showed that psychosocial stress did not affect herpes recurrence directly, but instead predisposed subjects to more generalized illnesses, which in turn mediated recurrences. Social support increased rather than decreased the likelihood of illness vulnerability, thus increasing the risk of recurrence. Higher levels of both arousal seeking and external locus of control increased illness vulnerability but moderated the likelihood of herpes recurrence. Higher levels of thymosin-alpha-1 were related to greater illness vulnerability but this peptide was not associated with psychosocial stress as originally predicted. Additional construct validation of the role of illness vulnerability in increasing the risk of herpes recurrence is recommended.