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.

Mixed-culture cytopathogenicity between KC and XC oncornavirus indicator lines and "virus-free" human choriocarcinoma cells.

When rat (XC) cells transformed by Rous sarcoma virus and human (KC) cells were cocultivated with appropriate infected cells, cell fusion was extensive and rapid. Many human cell lines were screened, with negative results; however, several hormone-secreting human choriocarcinoma cells fused extensively with both KC and XC cells. No evidence of a virus involvement in this interaction was found by direct examination, transmission, or immunologic tests.

Daily stress and recurrence of genital herpes simplex.

It is widely believed that emotional stress triggers recurrences of both oral and genital herpes simplex virus. On a daily basis, we studied 64 subjects prospectively for 1 to 3 months; all subjects had at least one culture-proved recurrence of genital herpes simplex virus type 2 before or during the study. All subjects filled out a questionnaire that measured psychological/emotional stress in six areas: physical health, relations with friends, relations with family, relations with sex partner(s), financial, and vocation/education. These questionnaires were mailed back to us daily. Data were analyzed by General Linear Models analysis of variance to compare stress reported on each of the 6 days before a recurrence with that on days during or after a recurrence, as well as days not temporally related to recurrence. Under these conditions, in which retrospective bias was minimized, there was no significant increase in psychological/emotional stress on any day in any area preceding a recurrence of genital herpes simplex virus.

Antibodies to herpes simplex type 1 in patients with active duodenal ulcer.

One hundred seventy-two patients requiring upper gastrointestinal tract endoscopy were examined prospectively for evidence of herpes simplex virus type 1 (HSV-1) infection. No viruses were recovered from active ulcers (11 with duodenal and eight with gastric ulcers). Using both enzyme-linked immunosorbent assay (ELISA) and standard complement fixation methods, patients with endoscopically proved active duodenal ulcer had significantly higher mean serum antibody levels to HSV-1 (but not to cytomegalovirus) than those without evidence of peptic ulcer. Neither patients with a history of or evidence of past peptic ulcer nor those with active gastric ulcer had higher serum antibody levels to HSV-1 compared with subjects without ulcers. These data provide support for an association between active duodenal ulcer and HSV-1 infection, the nature of which is not defined by these studies.

Specific cell-mediated immunity and infections with herpes viruses in cardiac transplant recipients.

Immune responses and infections with herpes viruses were studied prospectively in 36 cardiac transplant recipients. Specific lymphocyte transformation and interferon production in response to viral antigens, viral culture results, antibody levels, responses to phytohemagglutinin, and T-cell numbers were determined. Responses to phytohemagglutinin and T-cell numbers were depressed for six to 12 weeks. Cytomegalovirus infection occurred in 100 percent of seropositive patients and in 62 percent of seronegative patients. Primary infection was more frequently symptomatic. Heart implantation from a seropositive patient wwas significantly correlated with subsequent infection in seronegative patients. Depression of transformation in response to cytomegalovirus correlated with prolonged shedding. Herpes simplex infection occurred in 95 percent of seropositive patients but decreased after 12 weeks. Asymptomatic shedding was rare, and primary infection did not occur. Return of transformation in response to herpes simplex was associated with decreased infection. Herpes zoster occurred in 22 percent during the first year, and transformation responses to varicella-zoster returned thereafter. Depression of interferon production in response to viruses did not correlate with infection as well as did lymphocyte transformation.

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.

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.

Pharmacokinetics of cytomegalovirus specific IgG antibody following intravenous immunoglobulin in bone marrow transplant patients.

Cytomegalovirus (CMV) associated interstitial pneumonitis is a major cause of death among bone marrow transplant patients. A variety of intravenous immunoglobulin (IVIg) preparations have shown some promise in preventing this complication. As part of a multicenter trial of Sandoglobulin, the pharmacokinetics of CMV specific IgG was measured in order to guide future dosing schedules. A dose of 500 mg/kg was administered weekly beginning 1 week before transplant and continuing until day 98 following transplant. The half-life of CMV specific IgG was measured by an ELISA method after the first, third and fifth doses of IVIg. CMV seronegative patients received only screened CMV negative blood products, which permitted assessment of the half-life of IVIg CMV antibody. Peak titers achieved were comparable to those of the CMV seropositive patients averaging 1:2702 (range 1:596-1:10 514). Total IgG levels rose to a peak of about 75% above baseline. After the first dose of IVIg, the half-life of CMV IgG antibody was 3.4 +/- 2.0 (SD) days, although it lengthened to 6.1 +/- 5.1 days after the fifth dose of IVIg. The half-life of total IgG was estimated to be between 5 and 10 days, depending on the assumptions made regarding endogenous production. If high levels of IVIg are necessary for protection from CMV associated interstitial pneumonitis, weekly dosing will be important in order to maintain sufficient levels to be protective.

False positive indirect fluorescent antibody (IFA) tests for antibody to herpes simplex virus 1. Comparison of four commercially available methods.

Four commercially available herpes simplex virus 1 (HSV-1) indirect fluorescent antibody (IFA) kits were compared with the use of sera selected because they were negative for HSV antibody by complement fixation (CF less than 1:8) and by ELISA (less than 1:100). However, 14 of 24 (58.3%) of these HSV-1 antibody-negative sera were positive at greater than or equal to 1:10 with the use of the HSV-1 IFA kit from Electronucleonics, 15 of 24 (62.5%) were positive with the Clinical Sciences HSV-1 IFA kit, 4 of 24 (16.7%) were positive with Zeus Scientific, and 4 of 18 (22.2%) were positive with the Gull Laboratories product. HSV-1 induces Fc receptors that commonly cause false positive IFA tests for HSV antibody. Therefore, further studies were undertaken to determine whether Fc receptors accounted for these false positive results. Staphylococcal protein A (SPA) is known to bind to the Fc portion of human IgG and therefore could be used to distinguish between the binding of an antibody by its Fab or its Fc portion. Thus, when fluorescein isothiocyanate conjugated (FITC) SPA was used as conjugate instead of FITC antibody to human IgG, true HSV-1 antibody-containing sera remained positive, but the false positives identified in the commercial IFA kits did not. The authors conclude that HSV-1-induced Fc receptors are responsible for most of the problem of these false positives and that HSV-1 serology probably should not be done by this type of IFA method until this problem is corrected.

Rotazyme assay in neonates without diarrhea. Results of screening survey and preliminary analysis of false positive specimens.

One hundred forty-seven stool specimens from 93 infants younger than four months of age in a Neonatal Intensive Care Unit were tested for rotavirus by the Rotazyme ELISA method (Abbott Laboratories, North Chicago, IL). None of the infants had diarrhea at the time of the testing. Ten of 147 (6.8%) specimens were either low or suspect positive. None had rotavirus by electron microscopy. Excluding the suspect positives, which were negative on retesting, the false positive rate was only 6 of 147 (4.1%). Of five specimens with sufficient material and repeatedly positive tests, heat to 56 degrees C for one-half hour eliminated the binding to the Rotazyme bead but had no effect on the rotavirus positive control. One patient was found to have an extremely high positive Rotazyme test, independently of the survey. No virus was found in this specimen by electron microscopy, and the material responsible for the false positive result was not removed by centrifugation (100,000 X g for one hour), heating to 56 degrees C for one-half hour, trypsin, ether/beta-mercaptoethanol, or dialysis. Thus, false positives were encountered, but the overall rate was acceptably low. Such false positives are likely to result from more than one cause and, depending on results of further study, may be confirmed as false positives by loss of reactivity at 56 degrees C for one-half hour and perhaps lack of binding to a control bead.

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.

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.

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.

Trifluorothymidine: potential non-invasive diagnosis of herpes simplex infection using 19F nuclear magnetic resonance in a murine hepatitis model.

Trifluorothymidine (TFT) is known to be concentrated in herpes simplex virus (HSV) infected cells in vitro in the form of phosphorylated derivatives. We studied a murine hepatitis model of HSV infection to determine whether this in vitro observation would also be demonstrable in vivo. Following i.v. injection of 100 or 160 mg/kg TFT, TFT was found in significantly higher concentrations in the livers of HSV-2 infected mice than in the livers of uninfected mice, mice infected with murine hepatitis virus (MHV-A59) or mice with hepatitis from carbon tetrachloride treatment. Neither altered renal function, nor altered pharmacokinetics could account for this difference. 19F Nuclear Magnetic Resonance spectroscopy readily detected the 19F from TFT in both liver extracts and whole livers, particularly at higher tissue levels, i.e. greater than 50 micrograms/g tissue. If further studies with living animals support these preliminary observations, clinical application could be pursued.

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.

Medical management of antimicrobial-associated diarrhea and colitis.

Gastrointestinal complications, including diarrhea, may occur with virtually all antimicrobial agents. Such diarrhea may represent either a common, nonspecific adverse effect, or it may be one of the manifestations of antimicrobial-associated colitis (AAC), a potentially fatal complication. Clostridium difficile and a cytotoxin neutralized by Clostridium sordellii antitoxin has been isolated from the stools of nearly all patients with antibiotic-associated pseudomembranous colitis, many patients with AAC, and approximately 20% of those with antimicrobial-induced diarrhea. Demonstration that C. difficile is responsible for cytotoxin production has allowed for specific therapy for these disorders. General treatment measures include discontinuation of the causative antimicrobial agent(s), bowel rest, and supportive care with fluids, electrolytes and colloids, if necessary. Antiperistaltic agents and corticosteroids are not recommended. Various antimicrobials demonstrate potential efficacy in treating AAC in humans. Oral vancomycin is the most widely tested and is currently the treatment of choice. It achieves high concentrations in the feces and is very active against C. difficile in doses of 125-500 mg by mouth every six hours. Other potentially useful but inadequately tested antimicrobials include metronidazole (500 mg by mouth every eight hours) and bacitracin (25,000 units by mouth every six hours). Tetracycline has been employed with some success in nonspecific antibiotic-associated diarrhea, although it is as yet untested in humans with AAC and may induce diarrhea itself. Both miconazole and rifampin are highly effective against C. difficile in vitro but have not been evaluated in AAC. Anion-exchange resins bind the cytotoxin found in stools of patients with AAC. Cholestyramine has been used with variable response in oral doses of 4 g every six to eight hours. Since these resins may also bind vancomycin, resulting in lowered vancomycin concentrations in the stool, combination therapy should be used cautiously. With specific therapy directed against the toxin and aggressive supportive therapy, surgical intervention is rarely necessary. More recently, investigations have been directed at using bacterial preparations to suppress C. difficile by restoring the normal flora. The development of immunological agents (i.e., vaccines, toxoids, antitoxins) for the prevention or treatment of AAC would be a significant advance in therapy.

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.

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.